Biochemistry II Pouria Farsani 2013 1 BIOCHEMISTRY II EXAMINATION QUESTIONS 2013 POURIA FARSANI Biochemistry II Pouria Farsani 2013 2 1.Factors influencing results of laboratory examination (three phases of examination, biological and analytical factors, sample collection and handling of samples, interpretation of results, reference interval and its calculation, critical difference) Three phases of examination 1. Pre-analytical phase 2. Analytical phase (For more detailed information about analytical factors see Biochemistry II, Practicals, ED. 2008, p. 22-24) 3. Post-analytical phase 1. Pre-analytical phase – preparation of the patient, collection of the biological material, transport of sample to the laboratory, preparation of sample for processing. -1/3-2/3 of the errors occur in this phase -Patient identification is very important – sample identification -Sample collection and handling of samples for testing may affect the laboratory test value -Physiological factors, non-controllable and controllable, also affect the laboratory test value • Not controllable biological factors -Race, ethnicity – even though all humans are 99.9% genetically identical, various race or ethnic populations differ in the amount of skeleton, skeletal muscles, physiological levels of some analytes and distribution of some genes -Gender – before puberty this factor plays little importance – few differences in laboratory data. After puberty, concentration of numerous components may be greater in men than in women due to different distribution of hormones. Higher concentrations of enzymes originating from skeletal muscles are greater in men due to men having a greater muscle mass -Age – the degree of sexual maturity and the amount of skeletal mass are factors which depend on age. It is common for that many of the laboratory parameters are lower in children than in adults. During adolescence increased osteoblastic activity (bone development) leads to increased values (ALP) -Pregnancy – change in hormonal production and their actions, influence of placenta, transfer of analytes from amniotic fluid etc. -Biorhythms – these may be linear (age) or cyclic such as in changes of metabolism due to action of hypothalamus and/or pituitary hormones (e.g. circadian rhythms, menstrual cycle). Climatic or seasonal influences such as dietary changes, physical activity, exposure to sunlight, temperature – magnitude of these changes. Cyclical changes may be predicted with some probability Biochemistry II Pouria Farsani 2013 3 • Controllable biological factors -Body habitus – body weight may change distribution of volumes of some analytes thus affecting their concentration. Obesity => ↑ cholesterol, LDL-cholesterol, TAG, uric acid, insulin, cortisol -Physical activity – duration and intensity of the activity together with physical condition of the patient are the factors upon which exercise affects blood composition. Muscular activity has both transient and longer lasting effects. Acute strenuous and exhausting exercise => anaerobic catabolism ↑. Prolonged endurance exercise => aerobic anabolism ↑ Exercise => cellular ATP↓ => cellular permeability ↑ => serum activities of enzymes and metabolites originating from skeletal muscles ↑. When activity is stopped the levels of substances return to normal except for enzymes which may remain elevated for 24 hours or more. -Diet/fasting/starvation – amount and composition of ingested food and beverages affect the hormones and enzymes which are released before and during food intake Proteins ↑ => phosphates, uric acid, and urea ↑ Lipids ↑ => ratio of nitrogen compounds ↓ (e.g. uric acid) Vegetarian food => LDL-cholesterol TAG ↓ total bilirubin pH of urine ↑ Some metabolic pathway may be affected by some foods and beverages Caffeine => catecholamines, glucose, FFA ↑ Dehydration => difficulties in withdrawing blood. Serum protein, concentration of other compounds, hematocrit ↑ -Smoking – nicotine may affect levels of many analytes. Smoking affects metabolism of glucose. Furthermore smoking increases concentration of cholesterol and TAG -Alcohol – analytes are dependent on whether the abuse is chronic or acute. In general it affects the metabolism of glucose and TAG. It also increases liver enzymes in blood. One single dose (small and medium) minimally affects a test result. Long term abuse however => hypoglycemia, keto- and lactoacidosis, serum uric acid ↑ -Drugs – induction/inhibition of liver enzymes, affection of bonding capacity of transport proteins, cytotoxicit, interference with determination of analytes -Stress – change of hormone production which leads to a change in metabolism of many compounds. Severe illness or during blood collection are situation associated with stress. Fear/anxiety => catecholamines ↑ hyperventilation => acid-base imbalance => lactate ↑ Biochemistry II Pouria Farsani 2013 4 -Environmental factors – altitude > 3000 m, ambient temperature, geographic localization (country side or city). Travel across several time zones affects the circadian rhythm => change of analytes, retention of sodium ions and water – values regress within 2 days after return. -Mechanical action – muscle trauma, intramuscular injection => ALT, AST, CK myoglobin ↑. Increased pressure on uterus during pregnancy => ALT ↑. Digital investigation of the prostate => PSA ↑ • Handling of samples -Sample collection – patients should not eat 10-12 hours before blood collection – fat food and alcohol should be excluded. The patient should drink a glass of water or bland tea in the morning before the blood collection. Incompliance may cause distorted findings in parameters. Special tests have recommended precautions regarding e.g. diet. Functional tests also have recommendations (PSA can be positive after riding a bicycle). Blood should not be withdrawn from the arm that has an infusion running into it (haemodilution) There are different test tubes for different tests. -Time of the collection – concentrations of some substances may vary throughout the day. Morning hours are preferred -Posture – supine to sitting position may decrease the plasma volume as water is filtered from tine intravascular environment into the interstitium. This leads to the increase of non-filterable elements, proteins and substances, which are bound to proteins (tot. protein, albumin, enzymes, lipids, calcium, iron) by 5-15%. Equalization of fluid shift takes about 20-30 min. Sitting is the standard posture -Tourniquet application – should not be used more than one minute. It should be released before the withdrawal begins. Pumping of fist before venipuncture should be avoided. In the case of prolonged usage of tourniquet, concentration of on-filterable elements may increase (proteins). This is due to the hydrostatic pressure, which is increased with the usage of tourniquet, causing a filtration of water and filterable elements. Haemoconcentration may be a result of prolonged tourniquet application. Also keep in mind that it is uncomfortable for the patient! -Compression of the vein/finger – influences the concentration of blood gases, lactate and pH. -Hemolysis – visible if the concentration of hemoglobin > 0.2 g/l. This results in the increased concentration of analytes, whose concentration is high in erythrocytes and vice versa. Cause of haemolysis: *use of very big or small needle *rapid evacuation of syringe *heavy shaking of blood in the test tube Biochemistry II Pouria Farsani 2013 5 *moisture in the collection set or test tube *detergents in the test tube *wrong proportion of anticoagulant *keeping blood in the fridge, in the sun, above the radiator *centrifugation at higher speeds of rotation • Transport of samples – serum/plasma is preferred rather than to transport whole blood due to the risk of haemolysis. During the transport the blood sample should be kept at zero degrees. • Stabilization and storage of sample – depending on the determined analyte, lower temperature (4, -20, -80), protection against sunlight, correction of pH of the sample, addition of stabilisator. Interpretation of results For this topic, distinguish between: reference value, reference interval, reference individual and reference group. -Reference interval and its calculation – a comparison of results with the reference interval is most frequently carried out in biochemical examinations. Reference values are needed for all tests performed in the clinical laboratory, not only from healthy individuals but also from patients with relevant diseases The reference values are obtained from a chosen group of individuals, so called reference individuals (age, sex, race) with defined state of health. Reference values replace the term normal values. The reference individual – selected as a basis for comparison with individuals under clinical investigation. The reference individual must meet a selection of criteria – selection criteria. These criteria include statements describing the source population, specific criteria for health or the disease of interest. The group of reference individuals is ideally a random sample of all individuals in the parent population who fulfill the selection criteria. The following are examples of exclusion criteria for health-associated reference values: *pregnancy *excessive exercise *stress *intake of pharmacologically active agents such as alcohol, tobacco, oral contraceptives, drug treatment for disease *genetically determined risk *obesity *hypertension The values which are obtained from the reference individuals are analyzed statistically to determine an interval of values that includes a specified percentage of all the values (reference interval) from the sample group. Biochemistry II Pouria Farsani 2013 6 The reference interval includes 95% of results of the reference group. The other 5% is not included (2.5% of the highest values and 2.5% of the lowest values). Using parametric or non-parametric statistical methods, the reference interval can be estimated. Which one of these that is used, is dependent on the distribution of reference values. To estimate the reference intervals, it is necessary to exclude outlying values, to test values (Gaussiam or non-Gaussian distribution), to estimate reference interval using parametric or non-parametric method, to test influence of age/sex. The parametric method is used for data which has a Gaussian distribution (normal distribution). The mean and standard deviation (SD) are calculated from reference values. reference interval = 95 % of data = mean ± 2 SD The non-parametric method can also be used for estimation of reference interval. The basic assumption is that the values do not fit a Gaussian distribution. This method is based on sorting the reference values in ascending order of magnitude where the reference range can be calculated by ranking the values and deleting the lowest and highest 2.5%. The advantage is that it requires no assumptions to be made about the characteristics of the distribution and it does not require any transformation of the data to be made. The major disadvantage however is that it cannot be applied to small sets of reference values. The recommended minimum number of reference values is 120. For a graphical representation of parametric and non-parametric methods, see: Biochemistry II Practicals, 2008, p. 26. Regarding the usage of 95% – it is the expression of the probability as well. A healthy person has 95% probability that his test result will lie in the given reference interval. The remaining 5% represent the probability, that the result will be lower (2.5%) or higher (2.5%) than the reference interval. Decision making analysis Instead of using reference values (“from-to”) a cut-off value (decision limit) is used in some cases. These values allow us to describe the determined result as the positive test in case the obtained value is higher than the cut-off value. In case the value obtained equals or is lower than the cut-off it is considered as negative. Critical difference Clinical laboratory tests are mostly used for monitoring purposes. A change of results may be due to analytical imprecision and intra-individual biological variation – change does not always reflect improving or worsening of the patient’s condition. Therefore, in order for a change to be significant, it must exceed to so-called critical difference (CD). CD a.k.a. reference change value (RCV) is expressed as statistically significant difference between the two results of the given laboratory test that was measured in an individual between a given time interval. Biochemistry II Pouria Farsani 2013 7 The CD can be calculated if the data on analytical imprecision (analytical variation CVa (repeatability between days), and intra-individual biological variation CVi are known. CD= 2.77 × √ + If the difference of two results of the given laboratory test in an individual is measured between some definite time interval, is higher than the calculated CD, it can be expected that the results are different. These results differ with a certain probability (usually 95%). This means that the differing results are not caused by the analytical or biological variability, but they reflect the change of the clinical state of the patient. In case two different measurements are lower than the acceptable CD, the arithmetic mean is calculated for the given parameter. If the CD is crossed, a third examination is carried out after some time. Example: In a 50y. man, serum LDL-cholesterol was determined 2.8mmol/l, after one month, the analysis was repeated with the result 3.2 mmol/l. CD of LDL-cholesterol is 26%. Decide whether the values are different. Do they reflect the change of clinical status? Solution: add and subtract 26% to/from 2.8 mmol/l. The two different values that will be obtained when performing this calculation refer to a range – values within that range do not exceed the critical difference. In case 3.2 mmol/l will lie within that range, the clinical status has not changed. Diagnostic accuracy This refers to the probability that a patient has a disease based upon the given test results We want to distinguish between whether or not the patient is actually sick. Test results can be: TN – true negative FN – false negative TP – true positive FP – false positive Sensitivity = proportion of people with disease who have positive test result. Sensitivity = TP/ TP + FN (x100%) Specificity = proportion of people without disease who have a negative test result Specificity = TN/ TN + FP (x100%) Test result Diseased Not diseased Totals Positive TP FP TP + FP Negative test FN TN TN + FN Totals TP + FN TN + FP TP + TN + FP + FN Biochemistry II Pouria Farsani 2013 8 2. The significance of (both functional and non-functional) enzyme assays in blood serum. Isoenzymes – multiple forms of LD and CK It is the catalytic concentration of enzymes that facilitates their detection. The significance of enzyme assays is that it aids diagnosis and prognosis. It is the analysis of the presence and distribution of enzymes and isoenzymes – whose expression is normally tissue-, time-, or circumstance specific – often aids diagnosis. Myocardial infarction is an example – detection of CK (see q. 20 for more detailed info). -Furthermore, the use of enzymes in clinical diagnosis can be summarized as: *detection of damage of particular tissues *identification of imminent damage to a tissue *determination of the damage extent *estimation of the severity of cell damage *diagnosis of basic diseases *differential diagnosis for diseases within the damaged organ -The information suitable for more accurate diagnosis is obtained: *catalytic concentration (damage => increased activity of respective enzyme(s)) *spectrum of enzymes present in blood (e.g. necrosis of liver cells => LD > AST > ALT) *calculation of the ratio of enzyme activities (see q. 43 for details regarding liver pathology) *monitoring of enzyme activity (usage of the shape of kinetic curves to derive time period for which the diseases is present or the phase of the disease) *determination of isoenzymes Enzyme assays • Enzyme-linked immunoassays (ELISA) ELISA is used to detect proteins that lack catalytic activity. It uses antibodies covalently linked to a “reporter enzyme” such as alkaline phosphate or horseradish peroxidase whose products are readily detected, generally by the absorbance of light or by fluorescence. (This information was gathered from Harper’s illustrated BCH. For more detailed information about the formation of the “sandwich complex” by which ELISA functions, see Biochemistry II, Practicals, ED. 2008, p. 76) • NAD(P)+ -Dependent dehydrogenases – spectrophotometrical assay The ability of a substrate/product to absorb light is exploited. NADH and NADPH (NAD(P)H) are the reduced coenzymes. When these are reduced they absorb light at 340 nm. If they are oxidized however they do not! When NAD(P)+ is reduced, the absorbance at 340 nm increases proportionally hence the presence/quantity of the enzyme is proportional to the quantity of NAD(P)H produced. Biochemistry II Pouria Farsani 2013 9 Conversely, for a dehydrogenase that catalyzes the oxidation of NAD(P)H, a decrease in absorbance at 340 nm will be observed – proportional to the quantity of enzyme present. • Coupling to a dehydrogenase This assay is used for enzymes whose reactions are not accompanied by a change in absorbance of fluorescence – the detection of these enzymes is more difficult. Methods used in this case are: -Transformation of product or substrate into a more readily detected compound -Separation of reaction product prior to measurement -Devise a synthetic substrate whose product absorbs light or fluoresces -“Coupled assay”, a dehydrogenase whose substrate is the product of the enzyme of interest is added in catalytic excess. The rate of appearance or disappearance of NAD(P)H then depends on the rate of the enzyme reaction to which the dehydrogenase has been coupled Nonfunctional plasma enzymes aid diagnosis and prognosis Enzymes in blood plasma can be divided into two types: -Functional plasma enzymes – these are normally present and perform a physiologic function in the blood. These enzymes include, cholinesterase, enzymes of blood clotting cascade, and lipoprotein lipase. The majority of these enzymes are synthesized and secreted by the liver -Nonfunctional plasma enzymes – enzymes released from various tissue cells. These perform no known physiologic function; their concentrations are negligible under physiological conditions. These enzymes arise from the routine normal destruction of erythrocytes, leukocytes and other cells. Tissue damage or necrosis resulting from injury or disease is generally accompanied by their increased levels in plasma The reasons for why intracellular enzymes in extracellular fluid have increased activities are: -Increased permeability of cell membrane/cell damage -Physiologically increased synthesis (osteoblasts – ALP) -Induction of synthesis by certain drugs/alcohol -Increased concentration of bile satls (tnesides) => liberation of membrane bound enzymes (ALP, GMT) -Renal failure – non-filtering of small enzymes (e.g. amylase) -Antibodies against some enzymes. The complex formed (Ab-E) – macroenzymes has a longer half-life Biochemistry II Pouria Farsani 2013 10 The following table summarizes these enzymes and the significance of their detection: Biochemistry II Pouria Farsani 2013 11 Isoenzymes We summarize what iosenzymes are by the following points: -They have genetically determined differences in primary structure -Catalyze the same reaction -May have different subcellular distribution (cytoplasm × mitochondria) -May have different tissue distribution -May be combined from more subunits (quarternary structure) -May differ in kinetic properties (KM) -Elevated blood values – specific markers of tissue damages Isoenzymes of LD and CK Biochemistry II Pouria Farsani 2013 12 3. Provision of glucose in different states, the factors increasing susceptibility of glucose (glucagon, adrenalin, cortisol). Glucosuria In healthy individuals the glucose level in blood is kept within a very narrow range. I postresorption phase the range is 3.9-5.6 mmol/l. There is an intra-individual variation, coefficient of 1-2% and an inter-individual variation coefficient of 5%. Absorption phase This phase is the ongoing phase, for 4 hours, during and after a meal. There is of course a time data for each phase depending on how much food that was ingested, the amount of energy reserves and other factors. 0.5-1 hour after a meal containing saccharides the glucose level in blood increases to 8- 10mmol/l. Glucose serves as the major energy source for most tissues and in the liver it is stored as glycogen. As the glucose is being consumed by its catabolism and storage, the level begins to decrease ~1 hour after the meal. After 2-4 hours => normal glycaemia. At this stage glycogenolysis begins. Post-resorption phase Over-night fasting is a typical example of this phase. (12-14 hours of no food => short-time starvation). The glycogenolysis releases glucose from the liver into the blood. When the glycogen storage begins to deplete, degradation of lipids in adipose tissue by HSL starts to take place, releasing FA and glycerol into blood. The FA are used as an alternative fuel for certain tissues. They are utilized by some tissues for energy (beta-oxidation) and in the liver for production of KB which is another alternative fuel source. Glycerol is used for the gluconeogenesis in the liver. Biochemistry II Pouria Farsani 2013 13 The glycogenolysis and gluconeogenesis are the main processes which provide glucose to the body during fasting. If starvation for more than 30 hours proceeds, the glycogen pool will be practically exhausted and the gluconeogenesis will be the only source of glucose. Insulin and glucagon level changes regulate the metabolism of glucose in the different phases. (for more detailed information about glucose provision in different phases, see questions regarding integration of metabolism) For blood Glc origin, see graph below: Biochemistry II Pouria Farsani 2013 14 Factors increasing susceptibility of glucose -Glucagon – is synthesized in the pancreatic alpha-cells as a part of precursor protein – progulcagon. The proglucagon is produced in the alpha-cells and L-cells of the small intestine. The polypeptide is similar to glycentin, glucagon, glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 Glucagon is proteolytically cleaved from proglucagon in the pancreas. Low blood glucose level and high levels of certain AA stimulates this process. After a meal, GLP-1 is released from the small intestine; it binds to receptors in the pancreatic beta-cells, inducing insulin release. When glucose levels is low however, glucagon which is released, binds to beta- and deltacells of the pancreas. The delta-cells release somatostatin which binds to the beta-cells, inhibiting the release of insulin. The highest concentration of glucagon is found in the portal blood, since it acts mostly in the liver. Glucagon also acts on adipose tissue activating lipases. It has no effect on metabolism of muscles, due to muscles not having any receptors for glucagon. Glucagon has a half-life of 3-6 min and is cleared by the liver and kidneys. Content of ingested food determines the ratio of insulin and glucagon. A meal rich in protein increases both of these hormones. Insulin stimulates the AA uptake for proteosynthesis and glucagon increases the gluconeogenesis from the AA. Catecholamines Adrenaline and noradrenaline are the major stress hormones which regulate the blood glucose level. Noradrenaline is the neurotransmitter of postganglionic sympathetic neurons. Both noradrenalin and adrenaline are released from the adrenal medulla after nerve stimuli. They are released during the conditions which burden the body – such as cold, physical exertion and physiological stress. They act on aplha1-adrenergic receptors in the liver whilst in muscles and adipocytes they act on beta-receptors. Adrenaline acts through second messengers cAMP and Ca2+ . Biochemistry II Pouria Farsani 2013 15 The following effects are given: The most important objective is to mobilize glycogen and lipid reserves for their utilization in muscles. Glycolysis in muscles is activated whilst in the liver it is inactivated by the action of catecholamines. The catecholamines have an antagonistic effect to insulin. Under normal conditions they are not important regulators of glycaemia. In hypoglycemia however, their effects are potentiated, thus the signs of hypoglycemia indicate sympathetic activation. Glucocorticoids (cortisol) Chronic stress => release of cortisol from adrenal cortex. Even though glucocorticoids act in synergy with adrenalin, they act as gene regulators instead – changing the speed of enzyme synthesis. Hence the effects of glucocorticoids appear after several hours or days. The objective of glucocorticoids is to prepare the body for the effects of adrenalin. They: -stimulate synthesis of HSL -promote proteolysis -induce phosphoenolpyruvate carboxykinase (PEPCK) in the liver -support glycogen synthesis by increasing the glucose availability Biochemistry II Pouria Farsani 2013 16 Glucosuria Only a small amount of glucose and other saccharides (galactose, fructose, pentoses, lactose and maltose) are found in urine in normal conditions. This is of course depending on the food which was ingested. The physiological amount of glucose in urine is not big enough to be detected in by routine tests. In case we find a concentration >0.8 mmol/l of glucose in urine we determine it as glucosuria. This means that the maximal reabsorption of the cells in the proximal tubules is exceeded – this happens in hyperglycemia – glucose that cannot be reabsorbed is then excreted into urine. This is actually where the word-meaning of Diabetes Mellitus stems from. Since diabetes means “running through”, referring to the excessive amounts of urine, and Mellitus means “honey sweet” referring to the sweet taste of urine (diagnosis method back in the days, tasting the urine). We speak about a renal threshold which is 10 mmol/l of glucose concentration in blood. However, it may vary from 2.8-18 mmol/l. In rare cases, when glycaemia values are normal we can still detect glucosuria – glucose is found in urine even though the blood levels are normal. This would be due to a disruption in the tubular reabsorption. In order to detect glucosuria we use a non-specific test with Benedict’s reagent, however these tests are used exceptionally. More commonly, as a specific test, we use glukoPHAN test strips. The test strip contains glucose oxidase and peroxidase enzymes together with a chormogenous substrate which is oxidized in the presence of glucose forming peroxide. The result is the change of color of the test strip. The result is clearly positive when glucose concentration in urine is 2 mmol/l. The test is specific; it does not react with any other saccharides. Biochemistry II Pouria Farsani 2013 17 4. The basic metabolic disorder in diabetes mellitus: Hypoglycemia and hyperglycemia, the cause of ketoacidosis or of hyperosmolar coma. Long term complications of diabetes Diabetes as one of the most common diseases of our civilization is characterized by an absolute or relative insulin deficiency. According to the WHO definition: -Concentration of fasting capillary blood glucose ≥ 7mmol/l or ≥ 11 mmol/l any time after a meal, together with clinical symptoms of diabetes: *polyuria *polydipsia *ketonuria *weight loss -It is cause by insufficient effect of insulin, (absolute deficit due to defective secretion or relative deficit due to a failure of its action in peripheral tissues – insulin resistance) when a higher dose of insulin is needed to induce the “normal” quantitative response – when normal blood glucose or hyperglycemia is associated with hyperinsulinemia -The metabolic impact results in an impaired metabolism of glucose but also proteins and lipids Biochemistry II Pouria Farsani 2013 18 -The clinical findings in chronic hyperglycemia are due to: *reduced insulin-dependent glucose transport (GLUT 4) *decrease in glucose utilization in the liver (glycolysis) *increased gluconeogenesis in liver *increased hepatic glycogenolysis in liver **there are other types of DM: • Gestational DM and impaired glucose tolerance – 2% prevalent in pregnant women. It causes complications for the fetus and it increases the risk for development of type 2 diabetes later in life for the woman • Other specific types of DM – caused by genetic defects or beta-cells or insulin receptor, infection, induction by drugs or chemicals, endocrinopathy, autoimmune DM Metabolic disorders in diabetes (see the scheme above) Absence of insulin => reduced uptake and metabolism of glucose in tissues Absence of insulin => gluconeogenesis in liver ↑ + lipolysis in adipose tissue ↑ Lipolysis ↑ => higher amounts of fatty acids (FA) are released than the tissues can consume FA are degraded by the beta-oxidation in the liver instead => acetyl-CoA ↑ => excess of acetyl-CoA => ketoneogenesis ↑ Biochemistry II Pouria Farsani 2013 19 Increased ketoneogenesis is due to the fact that oxaloacetate which is required for the processing of acetyl-CoA in the CAC is used for the gluconeogenesis. That is why acetyl-CoA is used for the ketoneogenesis instead FA can also be incorporated in VLDL in the form of triglycerides which may lead to hypertriglyceridemia Ketoneogenesis ↑ => metabolic acidosis => transfer of K+ ions into blood => hyperkalemia => excretion of K+ in urine => osmotic diuresis => hyponatremia As acidosis is treated, K+ return back into the cell => hypokalemia Loss of water is characterized by high levels of sodium and glucose in plasma Ketoacidosis The causes for ketoacidosis are explained in the points above. Ketoacidosis is characterized by blood pH < 7.36 (glycaemia is increased 2.5-6 times above the physiological range) The patient’s breath smells like acetone and Kussmaul breathing is observed. Hyperosmolarity Osmolarity > 310 mmol/l: more frequent in type 2 DM (blood glucose is increased 4-45 times above the physiological range) Loss of fluid => reduction in blood volume => osmotic diuresis => dry skin, low blood pressure, rapid heart rate. Hyperosmolarity => confusion A combination of ketoacidosis and hyperosmolarity can lead to hyperglycemic ketoacidotic coma A non-ketone hyperosmomlar coma occurs in patients with DM2. It is due to hyperosmolarity without ketoacidosis where the major symptom is severe dehydration. Hypoglycemia in treated DM Hypoglycemia is when plasma glucose is < 2.5 mmol/l. An insulin overdose or reduced food intake at an unchanged dose of insulin is the most common reason for hypoglycemia. The symptoms are as follows: -Hunger -Pallor -Tremor of the hands -Sweating -Nervousness -Weakness -Palpations -Stiffness around the mouth -Drunken behavior -Blurred vision -Severe cases => loss of consciousness and convulsions Biochemistry II Pouria Farsani 2013 20 -Untreated hypoglycemia is extremely dangerous and may lead to coma and permanent brain damage A patient with diabetes and frequently repeated hypoglycemia incidents during the course of the disease has attenuated responses and signs of hypoglycemia – glucagon and adrenaline are released slowly. This results in the syndrome of impaired awareness of hypoglycemia this can lead to hypoglycemic coma. First aid is to inject glucagon. Administration of glucose per os or IV is also of great importance! Long-term complications of diabetes – uncompensated DM -Non-enzymatic glycation of proteins (Mailard reaction) – the long-term elevated glucose levels result in increased glycation of proteins. Glycated hemoglobin or glycated albumin is used for obtaining information on the glycation process for a certain period of time The serious impact however lies in the production of other glycated proteins with a longer half-life. The glycation leads to a modification of their structure and thus a change in their properties such as: *inactivation of enzymes *inhibition of regulatory molecule production *reduced sensitivity to proteolysis *abnormalities in the function *increased immunogenicity *cross-linking of glycoproteins Biochemistry II Pouria Farsani 2013 21 Oxidative stress The hydroxyl group in the glucose molecule can also participate in electron transfer, contributing to the formation of reactive radicals. Furthermore, glucose molecules and fructose (lesser) may be subject to auto-oxidation => superoxide radicals => hydrogen peroxide from which hydroxyl radicals are formed. Reactive dicarbonyl compounds that can participate in the polypeptide chain fragmentation are the final products of glucose auto-oxidation. Additionally, the glycated proteins may also take part in the formation of reactive forms of oxygen. AGE = advanced glycation end-products – these have serious consequences in the development of the cardiovascular complications of diabetes (common long term effect!) Binding of AGE on macrophages or endothelial cells in blood vessels => release of cytokines (interleukin-1, -6 and -18 and tumor necrosis factor alpha) => may act on platelets => affect fibrinolytic system. The cytokines can also induce synthesis of cytoadhesive molecules (E-selectin and adhesion molecules for leukocytes) => influence of interaction of arterial wall with circulating elements. -Activation of the glucitol pathway Neuropathy and cataract are also two very common long term complications of DM! -Lipid metabolism disorders – the long-term hyperlipidemia => development of atherosclerosis Biochemistry II Pouria Farsani 2013 22 5. Lipids in blood plasma and the major classes of lipoproteins (differences in the lipid and apolipoprotein content, in size, in properties and in electrophoretic mobility, the origin in enterocytes and hepatocytes) The transport of lipids in the blood includes: -Transport of FA from adipocytes into other tissues -Transport of ingested lipids from the small intestine into tissues through lymph -Transport of endogenously synthesized lipids from the liver into other tissues -Reverse transport of cholesterol from extrahepatic tissues to the liver The major classes of lipoproteins -Chylomicrons – these transport the ingested lipids (~100 g/day) from the small intestine into other tissues. They can only be found in blood after a meal. Their main component is TAG. Furthermore they contain ingested cholesterol and lipophilic vitamins. Half-life: 5-15 min -VLDL – formed in the liver. They contain TAG which are synthesized in the liver (20-50 g /day). TAG is also the main component. VLDL also contain cholesterol for transport into other tissues. Half-life: 2h -IDL – produced by VLDL metabolism in the plasma. Major component is cholesterol. Halflife: 2h -LDL – formed from IDL particles in the plasma. Major component is esterified cholesterol which is provided from extrahepatic tissues. Half-life: 2-4 days Biochemistry II Pouria Farsani 2013 23 -HDL – Several subtypes which differ by the proportion of different lipids. Nascent HDL are released from the liver and intestinal mucosa. Their content is only proteins and phospholipids. HDL also serve to reverse the transport of cholesterol from extrahepatic tissues to the liver. Furthermore, mature HDL also contains cholesterol. Half-life 10h Differences in lipid and apolipoprotein content Before we start to explain the lipoproteins in more detail, we need to explain the general schematic structure of a lipoprotein. -The core is non-polar containing non-polar TAG and esterified cholesterol -The polar surface layer of amphiphathic phospholipids, free cholesterol (non-esterified) and apoproteins The composition of the lipoproteins differ where the most important differences are the greatest differences in composition which is illustrated in the scheme => Each lipoprotein also has a different composition of apoproteins. These proteins can be both peripheral and integral components of lipoproteins. The apoproteins have a structural significance, but they also have roles in protein metabolism. Furthermore some of them serve as enzyme cofactors, activators, or inhibitors, while others identify lipoproteins for specific reactions with receptors. Biochemistry II Pouria Farsani 2013 24 We can summarize the main protein content of the lipoproteins in the table below: CM nascent / CM remnants ApoB-48 and ApoA-I / ApoB-48 and ApoE VLDL nascent ApoB-100 and ApoE IDL ApoB-100 and ApoE LDL ApoB-100 HDL nascent and HDL2-3 ApoA-I and ApoA-II (ApoC and ApoE) -Apo(lipo)proteins – different functions and different compositions in lipoproteins. Some of them also have roles in proteins metabolism. They function, among many other functions as, enzyme cofactors, activators or inhibitors, while other identify lipoproteins for specific reactions with receptors. The most significant apolipoproteins and their functions are summarized below: Difference in size See table below. Note that the values regarding composition have their most important representation in the scheme on the previous page – all values are not needed to be memorized. Biochemistry II Pouria Farsani 2013 25 Electrophoretic mobility There are many methods by which separation of lipoproteins can be made; ultracentrifugation and electrophoresis are among them. -Ultracentrifugation – Lipoproteins are classified based on their behavior during density gradient ultracentrifugation. The ratio of proteins and lipids determine this density where the higher amounts of proteins, the higher the density. Density of each lipoprotein can be seen in the table above. Note that VLDL = very low density lipoprotein, IDL = intermediate, LDL = low, HDL = high. -Electrophoresis – the same methods are applied as for electrophoresis of blood plasma proteins. The difference is in the usage of agents to stain the different zones. HDL are the most mobile fraction, hence they are alphalipoproteins found in the alpha fraction. LDL – Beta, VLDL – between alpha and beta. Chylomicron are only present in blood after meals, hence they are not electrophoretically mobile. Chylomicrons also consist mainly of TAG, therefore they do not have a charge, which is also why they do not move in the electrophoretic filed. Biochemistry II Pouria Farsani 2013 26 Origin in enterocytes and hepatocytes -Enterocytes – CM together with HDL origin from the enterocytes. Firstly, mixed micelles of FA and monoacylglycerols are passively diffused via the brush border microvilli into the enterocytes. Inside the enterocytes the assembly of CM takes place, accordingly to the scheme => SCFA stands for short chain FA. Lipophilic vitamins are also assembled into the CM. Most HDL in plasma are spherical particles (see also q. 7), formed from discoid precursors called lipid-poor or lipid free particles. These precursors contain mainly ApoA-I and phospholipids and are synthesized in the liver and enterocytes. The precursor HDL receive cholesterol and phospholipids and become discoid HDL. The action of LCAT enzyme then converts the discoid HDL to spherical HDL. -Liver – VLDL and HDL VLDL are produced in the hepatocytes. They include: *TAG which are synthesized in the liver *cholesterol *phospholipids *proteins (ApoB-100 being the major one) VLDL gets into the blood directly via the fenestrated endothelium in the liver sinusoids by exocytosis as nascent particles. Origin of TAG in the liver: -FA synehtesized from acetyl-CoA -Acetyl-CoA originates mainly from the metabolism of saccharides (after meal) -FA can be taken up from blood as well during starvation -TAG are synthesized from FA Biochemistry II Pouria Farsani 2013 27 6. Transformation of chylomicrons and VLDL Chylomicrons As mentioned in q.5, the nascent CM are synthesized in the enterocytes. The major protein is ApoB-48 – a protein which is structurally and genetically related to ApoB-100 which is synthesis in the liver and included in the VLDL structure. Both these proteins are encoded by the same gene. In the enterocytes, the primary transcript is edited and a stop codon is inserted into mRNA, which stops the translation of the 2153 AA residue. That is why ApoB-48 only has 48% of the ApoB-100 length. ApoB-48 is essential for CM synthesis. That is why, if ApoB-48 is not synthesized, the CM cannot be synthesized either – the disorder is known as abetalipoproteinemia – lipid and lipophilic vitamins malabsorption. CM are secreted into the chyle of the lymphatic system by exocytosis and reach the blood via the thoracic duct approx. 1-2 hours after the beginning of a meal. CM continues to get to the blood several hours after the meals. The degradation process is more rapid however. It takes less than an hour from entry into plasma to uptake. The peak of CM are found 3-6 hours after a meal. After 9 hours the degradation is complete. Nascent CM + ApoE and ApoC-II from HDL => mature CM LPL which is anchored by heparan sulfate on the luminal surface of capillary endothelial wall breaks down CM TAG. The action of heparin can release LPL => TAG in blood ↑ LPL catalyzes the hydrolysis of TAG in the lipoproteins (TAG => glycerol + 3 FA) ApoC-II activates LPL (LPL is produced by adipocytes, skeletal- and cardiac muscle cells and lactating mammary gland) LPL isoform produced in adipocytes has a higher KM than the isoform produced by muscles hence it is mainly active after meals resulting in high CM levels Insulin also stimulates the release of LPL in adipocytes Entrance of FA released from CM into adipose tissue used for the synthesis of reverse TAG FA can also enter muscles cells where they are oxidized A small portion of FA are released into blood and bound to albumin Glycerol is released from CM => liver => TAG synthesis Breakdown of TAG => CM ↓ => cholesterol becomes their major component ApoC-II is transferred back to HDL => CM is now known as CM remnants (ApoB-48 + ApoE) which are taken up by receptors on the hepatocyte membrane by endocytosis Biochemistry II Pouria Farsani 2013 28 The receptors on the hepatocyte membrane react specifically with ApoE. There are two types of receptors: LDL receptors (ApoB/E) and LRP (LDL receptor/related protein). CM remnants are highly atherogenic. Furthermore they are cytotoxic. High levels of remnants increase the coagulation activity of factor VII. Under physiological conditions however, the remnants have a very short half-life, thus not interfering much with the process of atherosclerosis. Retardation of CM metabolism => remnants particles more cholesterol and less TAG increase => risk for cardiovascular disease ↑. This can be seen in persons with prolonged postprandial lipidemia VLDL As mentioned in q. 5 VLDL are synthesized in the liver. Similar to the case of CM, VLDL receives apoproteins from HDL. Cholesterol is also transferred from HDL to VLDL – a process which involves cholesterol ester transfer protein (CETP). LPL action on VLDL (similar action as for CM) When most TAG are removed, ApoC-II is transferred back to HDL The majority of ApoE remain part of the modified VLDL – these particles are known as IDL Biochemistry II Pouria Farsani 2013 29 ½ of IDL are taken up by hepatocytes (ApoB/E receptor) The other ½ loses another portion of TAG thorough the action of hepatic lipase which is anchored to the sinusoidal surface of hepatocytes by heparan sulfate (can also be released by the action of heparin) Hepatic lipase unlike LPL is not activated by ApoC-II and it does not break down TAG in CM and VLDL Hepatic lipase hydrolyses TAG in IDL and HDL Removal of TAG from IDL => LDL particles which are rich in cholesterol and its esters (~70% of total plasma cholesterol) ~ 2/3 of LDL are transported to the liver and taken up by ApoB/E receptors (ApoB-100 is the ligand of this receptor) The other 1/3 is transported by the blood to extrahepatic tissues, taken up by ApoB/E as well LDL can vary in size, density and composition. There are large LDL1, medium LDL2 and small LDL3. LDL3 are products of atypical large VLDL catabolism which are found in the Biochemistry II Pouria Farsani 2013 30 liver at high levels of TAG. These can easily penetrate the vascular endothelium, they are susceptible to oxidative modification – they are not taken up by LDL receptors. Macrophages which use the scavenger LDL receptors take up these LDL particles, accelerating the progression of atherosclerosis. Uptake and degradation of LDL in the cell The LDL-receptor is a single-chain glycoprotein, passing through the membrane with its Cterminus on the cytoplasmic side. ApoB/E binds to the N-terminus on the extracellular side – binding occurs in clathrin-coated pits. Binding => internalization of ligand receptor complexes in the form of clathrin-coated vesicles (receptor mediated endocytosis). Vesicles in the cell loses clathrin and fuse with lysosomes to form endolysosomes LDL receptor separates from its ligand and return to plasma membrane LDL components are hydrolyzed by the action of lysosomal enzymes Diffusion of free cholesterol into the cytoplasm Cholesterol inhibits HMG-CoA reductase => suppression of cholesterol synthesis de novo Cholesterol activates Acetyl-CoA cholesterol acyltransferase (ACAT) in the ER => cholesterol is esterified with FA through the action of ACAT Cholesterol in cell ↑ => inhibition of LDL receptor replenishment (reduction/downregulation in their expression) => blockage of further uptake of cholesterol from blood Membrane-bound transcription factors called SREBP (sterol regulatory element binding protein) provide feedback Biochemistry II Pouria Farsani 2013 31 -Metabolism of LDL is slower – it can circulate in plasma up to 3 days -Since not all LDL particles are captured using LDL receptors, macrophages and certain endothelial cells can take these up by scavenger receptor (SR) with a lower affinity for LDL than ApoB receptors, hence they participate in the LDL uptake, especially when plasma concentrations are high -The problem is that SR have a high affinity for damaged and chemically modified LDL particles – irreversible => accumulation of cholesterol => foam cell formation -The longer half-life of LDL increases their risk for undergoing damage and chemical modification -Familial hypercholesterolemia – due to numerous of mutations in the gene for LDL receptor => patients with this condition (~0.2 % of population) suffer from progressive atherosclerosis -LDL-receptor production may be reduced or completely suppressed -Disorders results in reduced or completely suppressed LDL particle uptake => no inhibition of cholesterol synthesis in the cell => raise of cholesterol levels in blood -LDL which are not taken up due to above mentioned diseases are subject to modifications and are taken up by scavenger receptors -Xanthomas in tendons, increased risk for heart attack -Difficult therapy, diet restriction does not help -Insulin and triiodothyronin increases the uptake of LDL by the liver yet vice versa for glucocorticoids. This explains why diabetes and hyperthyroidism are associated with risk of hypercholesterolemia and atherosclerosis Biochemistry II Pouria Farsani 2013 32 7. Metabolism of high-density lipoproteins HDL which are the smallest and thickest lipoproteins in blood plasma are important for the reverse transport of cholesterol – transport of excess of cholesterol from peripheral tissue cells to the liver. HDL exists in several modifications by differing in size, shape, content of lipids, apoproteins electrophoretic mobility. The main subfractions are HDL2 and HDL3 HDL particles in plasma are mostly spherical which are formed from discoid precursors known as lipid-poor or lipid-free particles. These precursors contain mainly ApoA-I and phospholipids – they are synthesized in the liver and in enterocytes. Furthermore they can be formed from VLDL and CM as well by removing surface portions during LPL action. As the precursors HDL receives cholesterol and phospholipids they become discoid HDL. Action of LCAT converts discoid HDL to spherical HDL. LCAT which is synthesized in the liver is attached to the HDL surface and activated by ApoA-I in HDL. The esterification of surface HDL cholesterol is catalyzed by LCAT – cholesteryl ester is the product which is non-polar and moves to the core of the particle. -HDL3 – Smaller with a higher density -HDL2 – larger with lower density Both of these HDL types can reversibly convert to each other in the HDL cycle. There are two mechanisms by which cholesterol is transferred from peripheral tissues and macrophages 1. Formation of new HDL from apoproteins (lipid-free and lipid-poor particles). Firstly discoid HDL are formed, then HDL3. Cholesterol and phospholipids are transported into the particles – transport is mediated by ABC transporters A1. Deficiency of ABCA1 => Tangier disease – caused by mutation in the gene for ABCA1 2. Nonspecific efflux of cholesterol from the cell membrane surface of already formed small discoid HDL. Biochemistry II Pouria Farsani 2013 33 HDL attaches to the cell curface using SR-B1 receptor, then ABCG4 (ATP-binding cassette protein G4) participates in the transfer of cholesterol. A cholesterol concentration gradient is needed for this transfer; it is made by LCAT on the HDL surface. HDL3 + cholesterol + lipids + phospholipids => HDL2 There are also two ways by which the peripheral cholesterol which was captured in HDL and esterified by LCAT can be transported to the liver 1. HDL particles can serve as donors of cholesteryl esters for VLDL, IDL and CM remnants. Cholesterol-transfer protein (CETP) mediates the transfer – TAG transport in the opposite direction accompanies the transfer. The remnant lipproteins are taken up by the liver 2. Direct uptake of HDL-cholesterol in the liver and steroidogenic tissues: binding of HDL to scavenger receptor SR-B1. In this interaction with the receptor the particles are not endocytosed, only the cholesteryl esters are transferred from the HDL core to the cell membrane The dual role of SR-B1 in the reverse transport of cholesterol: -Involved in the beginning, during the efflux of cholesterol from peripheral cells to HDL -In the final phase during the transport of cholesterol from HDL to the liver The HDL particles serve as a mobile reservoir of apoprotein (ApoE and ApoC-II). These apoproteins are provided to CM and VLDL particles. After CM and VLDL lose most of their TAG => reduction in size => return of ApoC-II and a part of ApoE to HDL HDL particles in blood plasma are associated with enzyme paraoxonase 1 (PON1) – a glycoprotein synthesized in the liver. PON1 is an esterase and it can hydrolytically cleave organophosphates – broad substrate specificity. In the metabolism of phospholipids it hydrolytically cleaves oxidized phospholipids thus inhibiting lipid peroxidation in LDL (unknown mechanism). PON1 ↓ => independent predictor of coronary disease Lecitin cholesterol acyltransferase – LCAT -Transfers a FA from lecitine (phosphatidylcholine) in cholesterol -It is a plasmatic enzyme acting on the surface of HDL, it is activated by ApoA-I Biochemistry II Pouria Farsani 2013 34 It functions by: -Transferring acyl of FA to OH group of cholesterol -Non-esterified cholesterol is converted to esterified => it is less polar and more voluminous – it is sequestered to the core of HDL Summary of HDL metabolism: Biochemistry II Pouria Farsani 2013 35 8. The movements of cholesterol and its elimination. The balance of sterols and the bile acids transformation. Drugs affecting level of cholesterol Cholesterol is both ingested and synthesized by almost any cell (mostly in hepatocytes, nerve cells and enterocytes). The synthesis in cells during physiological conditions is regulated by a reserve of cholesterol in the cell. When sufficient amount (sufficient dietary intake) the synthesis is inhibited. Synthesis is activated when cholesterol is depleted. The steroid skeleton of cholesterol cannot be degraded in the human body hence it must be excreted – excretion via bile being the only way. The bile contains bile acids formed from cholesterol in the liver and also the native cholesterol Biochemistry II Pouria Farsani 2013 36 -The ingested cholesterol is mostly in a non-esterified form. Approx. 10-15% of it is esterified -Degradation takes place by the action of hydrolase in the small intestine -Ingested cholesterol mixes together with cholesterol coming via the bile. A portion of this (~0.5g/d) is excreted in the stool. This portion is partially modified by bacteria to coprostanol and cholestanol -The cholesterol that remains will be reabsorbed by enterocytes together with approx. 98% of the bile acids (returns back into the entereohepatic circulation) -The transmembrane protein NPC1L1 (Niemann-Pick C1 like 1 protein) mediates the reabsorption of cholesterol – this transport can be inhibited by ezetimibe – a drug used to lower blood cholesterol levels. It is usually used together with inhibitors of endogenous cholesterol synthesis (statins) – both processes which may elevate blood cholesterol synthesis (statins) and intake (ezetimibe) is inhibited -Reduction of bile acid reabsorption. This needs a special diet to be applied (adequate amount of dietary fibers and phytosterols) or the usage of certain drugs based on resins (cholestyramine) -Nicotinic acid reduces the secretion of VLDL from the liver hence affection cholesterol levels Biochemistry II Pouria Farsani 2013 37 Biochemistry II Pouria Farsani 2013 38 9. The metabolic interrelationships among body organs predominating in a well feed state (absorptive phase) See also Lippincott’s 5th ED p. 328 In general, we can describe the absorptive phase as: -After substantial meal -All nutrients are available in sufficient amounts -Chemical energy is stored in the form of glycogen and lipds -Principal hormonal regulation – insulin The phase lasts about 2-4 hrs after indigestion. If we want to look at the interrelationships, we need to look at the pathways of saccharides, lipids and amino acids individually. Saccharides: Biochemistry II Pouria Farsani 2013 39 Liver: After a meal containing carbohydrates, the liver becomes the net consumer of glucose, retaining about 60% of the glucose which it receives via the portal system. This consummation of glucose is due to the formation of the other products from glucose in the liver. Glucose enters the liver independently on insulin; however, it only enters the liver when the concentration in the portal vein is 10-40 mmol/l, or when the concentration in the hepatic artery is above 5 mmol/l. This is because the KM value for the GLUT 2 transporter in the liver is high (about 10 mmol/l). Glucose in the liver, after a meal, is metabolized in the hepatocytes to glucose-6-phosphate (phosphorylation of glucose by the action of glucokinase, which is induced by insulin). -Glc => glycogen (glycogen synthase) -Glc => pyruvate => acetyl-CoA => CAC => energy -Glc => pyruvate => acetyl-CoA => FA => TAG (VLDL) -About 40% of the glucose passes through the liver into the blood stream -A small amount is converted into specialized products (pentoses, NADPH, galactose, glucuronate) Biochemistry II Pouria Farsani 2013 40 -An excess of glucose => lipids (VLDL) => blood => adipose tissue => obesity Extrahepatic tissues: -Erythrocytes have glucose as the only fuel source, utilizing it to produce lactate (anaerobic glycolysis). -Brain has glucose as an exclusive fuel source in the well-fed state, utilizing it to form CO2 + H2O (aerobic glycolysis). There are no stores of glycogen in the brain, thus it is highly dependent on the availability of blood glucose. Glucose is the only fuel, able to pass the blood brain barrier. The glucose is transported by the insulin-insensitive GLUT 3 transporter, located on neuron membrane for entry of glucose from ECF into nerve cells and GLUT 1 located on the glial cells -Muscles which are at rest use glucose as their source of energy, utilizing it to produce CO2 (aerobic glycolysis) and substrate for muscle glycogen (limited capacity). The glucose enters the muscles via GLUT 4 transporters and the intake increases 10-20 times -Myocardium obtains energy from both metabolism of glucose and oxidation of FA. These two pathways are in balance, depending on the availability of the substrates. -In adipose tissue glucose (GLUT 4) is a source of energy for the TAG synthesis where: Glc => => glyceraldehyde-3-P + dihydroxyacetone-P => glycerol-3-P Glucose is also used in the pentose cycle to form NADPH+H+ Lipids: Biochemistry II Pouria Farsani 2013 41 After a meal the lipids: -Exogenous TAG (CM) and endogenous lipids (VLDL) supply mainly the adipose tissue increasing the synthesis of FA and TAG. Because the adipose tissues lack glycerol kinase, glycerol-3-P is used in the TAG synthesis which comes from the metabolism of glucose. This is the reason for why TAG storage is favored in a well fed state Muscles, myocard and kidney are supplied in a lesser extent. -FA are released from the TAG by the action of LPL which is attached to the endothelial walls. -The FA are substrates for the TAG synthesis in the adipose tissue -LPL is activated in the adipose tissue, mainly by insulin (not very much in muscles however) and. Exogenous lipids (CM) are directed to the adipose tissue. -The FA are a secondary fuel source for the muscles. Glucose serves as the primary fuel source. FA => acetyl-CoA => CAC => CO2 + energy In the liver the FA synthesis increases (de novo). The TAG synthesis increases as well because of the availability of acyl-CoA. Amino acids: -Partially, they are metabolized in the enterocytes (Gln), providing the direct source of energy for these cells -Some AA are utilized in the liver to produce proteins (protesynthesis) and other nitrogencontaining molecules. The branched amino acids (leucine, isoleucine and valine) are not degraded in the liver however (lack of amino transferases). Instead they pass through the liver unchanged, in order to be metabolized in the muscles Biochemistry II Pouria Farsani 2013 42 After a meal, insulin is released by the beta cells of the pancreas. Its functions and characteristics are: -Anabolic peptide hormone, membrane receptor -Second messenger (kinase/phosphatase cascade) -It decreases blood by four processes: 1.Supports glucose entry into muscles and adipocytes 2.Stimulates glycogen synthesis in liver and muscles 3.Inhibitis glycogenolysis + gluconeogenesis 4.Supports glycolysis in the liver, muscles and other tissues -Stimulates TAG synthesis in adipocytes and liver. Stimulates proteosynthesis in muscles In other words, insulin stimulates the synthesis of the three main energy stores as well the cellular utilization of glucose. Insulin induces the synthesis of the key enzymes of glycolysis (glucokinase, phosphofructokinase, pyruvate kinase) and glycogenesis (glycogen synthase). Biochemistry II Pouria Farsani 2013 43 10. The metabolic interrelationships among body organs predominating after a brief fast (post-absorptive phase) and during prolonged fasting (starvation) -In fasting, when the first feelings of hunger are felt -Starts 4-16 hours after a meal -It continues to starvation, (long-term or short term) -Glucagon is the influencing hormone, with rising levels (x2-3), whilst insulin levels drop The decreased insulin to glucagon ratio, plus the lack of circulating substrates results in a catabolic period which is characterized by a degradation of TAG, glycogen and proteins (only about 1/3 can be used for energy). This catabolic period serves for two main purposes: 1. Maintaining the glucose level in plasma in order to sustain energy metabolism for the brain, RBC’s and other glucose requiring tissues. 2. Mobilizing FA for the synthesis and release of ketone bodies from the liver in order to supply energy to all the other tissues Biochemistry II Pouria Farsani 2013 44 Saccharides and proteins: Liver: As mentioned, plasma glucose levels are crucial be maintained. Two processes perform this maintenance: 1. Liver glycogenolysis (phosphorolysis) (Glc)n + Pi => (Glc)n-1 + Glc-1-P => Glc-6-P => free glucose (Phosphorylase is activated by glucagon and adrenaline) 2. Liver gluconeogenesis from: Alanine, lactate, glycerol … recycling three C atoms (saving Glc) and other glucogenic AA. The glucagon induces synthesis of three key enzymes: *phosphoenolpyruvate carboxykinase (PEPCK) *fructose-1-6-bisphosphate *glucose-6-phosphatase The consumption of glucose in other tissues is reduced, thus most tissues begin to use FA as their energy source. Biochemistry II Pouria Farsani 2013 45 Muscles: Proteolysis of the muscles protein begins by the stimulation of cortisol. This provides AA (Ala, Gln most important) that are used by the liver for the gluconeogenesis. After several weeks the proteolysis decreases, as the need for glucose as fuel source for the brain decreases since the brain has started to utilize KB. Lipids: In the post-resorption phase the following happens with lipids: -Lipolysis in adipocytes -HSL is activated by glucagon -FA are transported in ECF with the aid of albumin -KB are firstly utilized in the muscles, further on in CNS Adipose tissue: The hormonal stimulation by glucagon and adrenalin activates the HSL, thus releasing the FA and glycerol into blood (mostly from abdominal fat). A few hours of starvation leads to an increased utilization of FA by the muscles. Lower amount of insulin => LPL is not activates => decreased uptake of FA Biochemistry II Pouria Farsani 2013 46 Liver: - The increased level of FA in the blood supplies the increased FA oxidation – this is the major source of energy in the hepatocytes -Oxidation of FA leads to an increased amount of acetyl-CoA in the liver thus leading to an increased synthesis of ketone bodies (acetyl-CoA does not enter the CAC since the CAC is not proceeding due to lack of OAA which is being used in the gluconeogenesis). Muscles, myocardium and CNS start to utilize the KB after a prolonged period of starvation The beta-oxidation is facilitated by glucagon activating carnitine palmitoyl acyltransferase which transmits acyls into mitochondria (activated via cAMP). FA also participates in this activation via peroxisome proliferator/activated receptor (PPAR-alpha). Acyl-CoA carboxylase is inactivated (inhibition of malonyl-CoA synthesis => inhibition of FA synthesis). Pyruvate is spared for the gluconeogenesis, as it is in the muscles. Muscles: The resting muscles and myocardium use FA as their major fuel source (exercising muscles initially use glycogen stores as their source of energy), this is during the first 2 weeks of fasting (KB are also utilized). After about 3 weeks, the KB utilization decreases and FA are used as fuel source almost exclusively. This also leads to the increase of KB in blood. … Glucagon is the antagonist of insulin: -Peptide hormone with membrane receptor -cAMP is the second messenger -It stimulates the degradation of energy stores (Glycogen, TAG and proteins) -Supports gluconeogenesis from lactate and AA -Inhibits the synthesis of glycogen and TAG -It acts on liver and adipocytes, not on muscles Biochemistry II Pouria Farsani 2013 47 Starvation See also Lippincott’s 5th ED. p. 334-335 When we speak about starvation, we distinguish between long term and short term starvation. In general, starvation results in the increase of metabolic features which are typical for the post-resorption phase. Firstly the aim is to maintain the glucose level in blood, however, during prolonged starvation saving proteins is necessary as well. Saccharides: In short term (few days) starvation the glycogen stores in the liver run out after about 24 hours. Glucose levels are maintained by gluconeogenesis. Ala, Gln and 2-oxoacids released from the muscles are taken up by the liver. Up to 75g of protein per day is degraded during the first three to five days of starvation, thus the muscle proteolysis is increased. In long term (weeks) starvation the: -Brain utilizes glucose less (anaerobic glycolysis in order to recycle 3C) -Liver gluconeogenesis decreases in order to save glucose -Renal gluconeogenesis performs 50% of the gluconeogenesis -Muscle proteolysis decreases in order to save proteins -Erythrocytes consume glucose at a constant rate (36g/d), consuming up to 45% of the total glucose production in long term starvation Biochemistry II Pouria Farsani 2013 48 Lipids: In short term starvation: -Lipolysis in adipose tissues proceeds -Approximately 60% of the FA released from adipose tissue are taken up in the liver and reesterified back to TAG, becoming a part of VLDL, being transported back into the adipose cells. This cycle is known as the TAG/FA cycle which ensures a sufficient level of FA in the blood -Synthesis of KB and their utilization increases. The lack of OAA which is used for gluconeogenesis increases the KB synthesis -Glycerol for the synthesis of triglycerides is synthesized in the liver and adipose tissue by the glycerogenesis which is a shortened pathway for gluconeogenesis from OAA. It ends by dihydroxyacetone phosphate and its reduction to glycerol phosphate In the long term starvation: -Levels of KB in blood increases considerably (acidosis, due to excess of acyl-CoA, which cannot enter the CAC since OAA is used for gluconeogenesis, thus acetyl-CoA is utilized for the KB production). Cause of KB increase is partially because of increase of transcription of gene for HMG-CoA synthesis -KB is excreted by urine -KB is mainly utilized by the brain and in a lesser extent by muscles and myocardium (leads to increase of KB in blood) -The acidosis is compensated in the liver at the expense of urea Kidney compensation for acidosis: The glutamine which is released from muscles is taken up by the kidneys and converted to 2OG and ammonia by glutaminase and glutamate DH. This takes places in the renal tubular cells. As ammonia passes into urine it binds the protons which originate from the dissociation of KB, thus it is converted to NH4 + - removing the protons out of the body. … Biochemistry II Pouria Farsani 2013 49 The main priorities in starvation is to save glucose (utilization of KB in brain) and to save proteins (the KB save gluconeogenesis from AA) Biochemistry II Pouria Farsani 2013 50 11. The main features of metabolism at over-nutrition and obesity (production of adipokines, changes in metabolism of lipids and saccharides, insulin resistance, consequences) In general we characterize obesity as an imbalance between energy input and output. Over-eating is the main cause for this but also low physical activity and genetic predisposition. Obesity as a disorder of body weight regulation is characterized by changes in the metabolism. -Hypertrophy and hyperplasia of adipocytes (abdomen mainly). The adipocytes have an insufficient supply of oxygen, thus releasing FA since they lose their ability to store fat. As adipocytes grow they undergo lipolysis after reaching a certain size, resulting in an increase of plasma FA as well -Hypertrophy and hyperplasia of adipocytes results in an increased release of adipokines (hormones: leptin, resistin, angiotensinogen. Cytokines: TNF-alpha, IL-6. Enzymes: adipsin, ACE, CETP) (see below) Simultaneously, adiponectin production is decreased, which seems to normally increase the response to insulin. -The hypothalamus becomes less sensitive to leptin Biochemistry II Pouria Farsani 2013 51 -Storage of fat in other organs: muscles, heart, pancreas, liver (steatosis) – ectopic fat accumulation. -Increased production of MCP + TNF-alpha => pro-inflammatory => insulin resistance => atherosclerosis => metabolic syndrome (see below, insulin resistance) Adipokines The adipose tissue is the largest endocrine active organ of the body, producing and secreting adipokines. They assist in: -Regulation of apatite -Body energy expenditure (thermogenesis) and insulin sensitivity -Affect inflammation and the immune system -Act on vascular endothelium and on metabolism of steroid hormones -Affect growth and proliferation of cells or promote their survival >Leptin: polypeptide of 167 AA, product of the obese (ob) gene on the 7th chromosome. Produced by adipocytes after increase of TAG in them. Leptin => hypothalamus => release of neuropeptides, anorexigenic factors => satiety signal, reduction of appetite. The amount of leptin is directly proportional to the amount of adipose tissue. Lack of leptin leads to obesity, however in obese people it is in abundance thus resistance to leptin is a part of obesity syndrome. High levels of leptin => resistance => no loss of appetite => increase imbalance between energy input and output. Slight drop of leptin in people with high levels => misinterpretation of hypothalamus as a lack of nutrients => hunger sensation increases. Leptin also: -Increases beta-oxidation in hepatocytes protecting the liver from excessive accumulation of TAG. The effect is similar in muscles -Activates glycogenolysis in hepatocytes thus inhibiting excessive accumulation of liver glycogen >Adiponectin secreted more from adipocytes in subcutaneous fat than visceral fat. It is the most abundant hormone secreted of the hormones secreted by adipocytes, circulating in blood with relative high concentrations. In patients with obesity and atherosclerosis its levels are decreased. Its functions are involved in regulation of glucose and lipid metabolism – insulin sensitizer in muscles and liver. Therefore it suppresses the glucose output from the liver into the circulation => stimulation of FA oxidation. In myocardium, skeletal muscles and CNS two receptors are found AdipoR1 and AdipoR2. As adiponectin attaches to these receptors AMP-dependent protein kinase (AMPK) and PPAR-alpha are activated => increase of FA oxidation in liver and glucose uptake into skeletal muscles Biochemistry II Pouria Farsani 2013 52 >Resistin see picture below. >Visfatin produced by visceral adipose tissue. It has similar effects as insulin and it acts as an insulin sensitizer. It induces signal molecule to phosphorylation in the insulin receptor pathway. Visfatin contributes to the defense against insulin resistance. >Tumor necrosis factor-alpha (TNF-alpha) levels increase in obese people and decrease with weight loss. Insulin resistance It is assumed that it is the role of FA or one of the agents secreted by adipose tissue causes the increased insulin resistance in obesity. Insulin resistance is characterized by the uncontrolled hepatic glucose production, and decreased glucose uptake by muscles and adipose tissue. It causes all the effects of insulin in the adipose tissue to be attenuated resulting in: -Increased activity of HSL => FA elevation in blood -LPL is not activated => reduction of FA uptake. Loss of adipocytes ability to store TAG -High levels of FA => liver and muscles => re-synthesis of TAG => incorporation into VLDL together with cholesterol which is transported back into blood => cholesterol and TAG elevation in blood -TAG storage in liver and skeletal muscles, ectopic fat accumulation -High FA levels in blood => increased insulin resistance in muscles and liver => reduction of intake and oxidation of glucose -Gluconeogenesis increases in liver whilst glycogenolysis decreases => increased glycogen storage Biochemistry II Pouria Farsani 2013 53 -Increased sensitivity to catecholamines in hypertrophied adipose tissues in the abdominal cavity => increased lipolysis => increased FA transport to the liver -Insulin resistance => decreased usage of insulin with continuous production => hyperinsulinemia => stimulates sympathetic effects, retaining sodium and water with subsequent vasoconstriction => hypertension Biochemistry II Pouria Farsani 2013 54 12. The main features of metabolism at stress and metabolic stress We define simple stress during simple situations, such as in physical activity or in psychological stress. The hormones affecting the metabolism in this situation are catecholamines; noradrenaline and adrenalin released from the adrenal medulla. They increase intracellular cAMP when acting on beta-adrenergic receptors (skeletal muscles and adipocytes) and increase Ca2+ when acting on alpha1-adrenergic receptors (mainly liver). The reaction of the catecholamines is very quick, within a few seconds, reacting with “fight and flight”. Where the effects are the following: -Increased glycogenolysis in the liver in order to increase the glucose level in blood. The glycolysis is on the other hand inhibited since the catecholamines are antagonists to insulin -Increased glycogenolysis and glycolysis in muscles in order to provide quick energy -Increased lipolysis in adipose tissue. The mobilization of lipid and glycogen reserves for their utilization is the most important process in simple stress. In chronic stress however, glucocorticoids (cortisol) are released instead. They do not act via second messengers as catecholamines do. Instead they act as gene regulators, changing the speed of enzyme synthesis. Thus their effects are manifested after several hours or days. The main objective is to prepare the body for the effect of adrenaline. Biochemistry II Pouria Farsani 2013 55 By increasing the glucose availability the glycogen synthesis is promoted as well (inducing glycogen synthase). In metabolic stress there is a reaction of the system to trauma, sepsis, severe disease etc. The adaptation is influenced by stress hormones as well as neuroendocrine agents and cytokines associated with damage to the body. Diseases related to severe eating disorders or problems with indigestion results in malnutrition which leads to metabolic stress. Stress starvation develops which leads to increased levels of: -Cortisol -Glucagon -Catecholamines -Growth hormone The stress response includes the decreased synthesis and degradation of albumin and other short-term dispensable proteins, providing the necessary amino acid in order to increase the production of proteins needed for damaged tissue repair and to stop bleeding. Increased catabolism of adipose tissue and especially muscle tissue provides lactate, alanine and glycerol – basic substrates for gluconeogenesis and beta oxidation. TNF-alpha and interleukines-1 and 6 which are produced by macrophages and lymphocytes affect the metabolism as well. Where TNF-alpha: -Inhibits TAG synthesis in adipocytes -Inhibits LPL -Stimulate lipolysis -Inhibits the release of insulin -Cause insulin resistance -(ketogenesis is not increased) An increase in temperature is induced by cytokines as well. Biochemistry II Pouria Farsani 2013 56 The decrease in albumin and the cytokine-induced increased permeability of the vascular wall leads to an increased movement of water, electrolytes and proteins into the extravascular space => intravascular hypovolemia => hypotension. Replacement of fluid => further dilution of the serum proteins => further leakage into interstitial space => edema. In general, stress starvation in the course of disease has a much faster progression than simple starvation. Biochemistry II Pouria Farsani 2013 57 13. The main features of metabolism of adipose tissue at various conditions (after the meal, starvation, obesity). Main hormones of adipose tissue See. schemes for q. 9-10. After the meal, carbohydrate and fat metabolism (see. fig 24.5 Lippincott’s 5th ed. p. 325) -Increased level of insulin activates LPL mainly in adipocytes whilst LPL in the muscles is not active. This results in a routing of lipids from CM into adipose tissue, increasing the intake of FA. -Insulin also stimulates the increased intake of glucose into the adipose tissue (GLUT 4) where glucose is used for glycerol phosphate production in the glycolysis (glycolysis is increased) -Glycerol-P + FA = TAG (thus TAG reserves are increased in adipocytes after a meal) -An increased activity in HMP is also seen where adipose tissue metabolizes glucose by the means of HMP => producing NADPH which is essential for FA synthesis. However, in humans, de novo (from acyl-CoA) synthesis is not a major source of FA in adipose tissue -The elevated insulin also favors the dephosphorylated (inactive) form of HSL thus decreasing TAG degradation. Conclusion: -Increased glucose intake -Increased glycolysis -Increased activity in HMP -Increased FA synthesis -Increased TAG synthesis -Decreased TAG degradation Starvation: carbohydrate and fat metabolism (see. fig 24.13 Lippincott’s 5th ed. p. 331) -Decreased levels of insulin => depression of glucose intake by GLUT 4 thus the FA and TAG syntheses are decreased. LPL activity is also depressed thus FA uptake is decreased. -Increased levels of glucagon and noradrenalin increases the activity if HSL, thus increasing the TAG degradation => increased release of FA and glycerol into blood During simple and prolonged starvation the TAG/FA cycle and glycerogenesis starts (see q. 10). Biochemistry II Pouria Farsani 2013 58 Obesity In the development of obesity, a higher energy input together with a lower energy output results in an increased storage of TAG in the adipocytes. Adipocytes undergo hypertrophy and hyperplasia, increasing the release of adipokines and decreasing the release of adiponectin. Insulin resistance follows (see q. 11). Not only subcutaneous adipocytes undergo hypertrophy and hyperplasia, but also visceral adipocytes. The visceral adipocytes are much more metabolically active. They have a larger influence on metabolic dysfunction in obesity, since their release of cytokines and FA enter the portal vein directly. Therefore they have a direct access to the liver, where they are taken up. This may lead to insulin resistance. It leads to an increased synthesis of TAG, which are released as VLDL contributing to the hypertriglyceridemia. FA from subcutaneous adipose depots, enter the general circulation where they are oxidized by muscles, thus reaching the liver in lower concentrations. Main hormones of adipose tissue (See q.11) Biochemistry II Pouria Farsani 2013 59 14. Proteins in human nutrition, parameters of quality, nitrogen balance and assessment of catabolic state Proteins in human nutrition With a standard every day diet, about 1-2% of the proteins are degraded every day. Daily food intake contains about 100g of protein, providing needed amino acids for the amino acid pool. Concurrently with the degradation of proteins, the amino acid pool in the blood supplies amino acids for the synthesis of proteins (300-600g/d), neurotransmitters and other nitrogen compounds. Parameters of quality We determine the quality of a protein by investigating the representation of all nine essential amino acids and their suitable ratios together with the digestibility of the protein. The biological value or net protein utilization are parameters used previously. Today, we use the amino acid score which reflects the desired intake of essential amino acids, corrected by digestibility of the protein. The parameters are described as followed: -True digestibility (TD) – relative amount of nitrogen (%) absorbed from the food, in relation to the total amount of nitrogen intake (absorption in relation to intake) -Biological value (BV) – relative amount of nitrogen (%) used for synthesis of endogenous proteins out of the total nitrogen absorbed into the body from food (synthesis in relation to absorption) -Net protein utilization (NPU) = TD × BV Biochemistry II Pouria Farsani 2013 60 -Protein digestibility-corrected amino acid score (PDCAAS) – relative amount of limiting amino acid in the tested protein related to the amount of the same amino acid in the reference protein x TD. Limiting amino acid – the least represented essential amino acid in a protein compared to its representation in the reference protein. Reference protein – idealized protein with a content of essential amino acids corresponding to the required intake (for infants, it is the representation in breast milk, for other age groups it is the recommended intake of essential amino acids for the age group 2-5 years). Proteins of cow’s milk or eggs are usually used since they contain an appropriate representation of all essential amino acids. They are also easily digestible. The minimal intake of quality protein is approx. 0.4-0.5 g/kg/day, assuming the physical activity is low. This intake should cover the basal losses arising from the metabolic processes in the body. Adding normal physical activity to that, about 0.8 g/kg/day is needed (with sufficient energy intake). The following factors affect the intake of proteins: -Digestibility of foods -Rate of proteosynthesis in the body -Proportion of fats and carbohydrates in the diet -Stress -Drugs -Severe metabolic disorders There is an upper limit of protein intake, 1.3-2.0 g/kg/day in: -Children -Pregnant and lactating women -Convalescence -Patients treated with metabolically demanding methods of treatment (e.g. dialysis) -Active athletes Vegetarians should make sure to have a sufficient intake of quality proteins with egg and milk whilst vegans need to respect food composition corresponding complementarity of proteins. Biochemistry II Pouria Farsani 2013 61 Complementary proteins must be consumes simultaneously. Vegan diet is not recommended for people in growth phase, patients with unbalanced metabolic status or pregnant women. Assessment of catabolic state By determining plasma protein concentration the nutritious state can be assessed. The catabolic state is given for the last 1-4 weeks due to the biological half-life of proteins. Thus the course of changes can be monitored. The disadvantage of protein markers is in their dependence on the synthetic function of the liver. -Albumin (RI 35-53 g/l t ½ = 19 days) -Transferrin (RI 2.0-3.6 g/l t ½ = 7 days) -Prealbumin (RI 0.2-0.4 g/l t ½ = 2 days) There is a specific catabolic index of muscle contractile proteins which is the excretion of 3methylhistidine. The ratio of 3-methylhistidine/creatinine is investigated (physiological values 0.16-0.30). This ratio is independent on gender and age. The disadvantage of using this marker is that its amount is dependent on muscle activity and intake of meat. Nitrogen balance The relationship between quantity of nitrogen consumed as proteins or amino acids and the quantity of the nitrogen excreted is termed as the nitrogen balance. -Calculation of intake: NIN = mass of protein intake (g) × 0.16 (g/24h) 0.16, is explained by the fact that proteins contain 16% of nitrogen on an average. -Calculation of excretion – we express this quantity as the amount which is excreted in urine. 1.0g/day is added to the amount in order to account for other losses (stool, sweat). We calculate the mount of nitrogen excreted into urine from the value of urea which is excreted. This is based on the presumption that urea compromises 84% of the total nitrogen excreted in urine – 1 mmol of urea contains 0.028g of nitrogen thus => NOUT = (cUREA)URINE × VU × 100/84 × 0.028 + 1 (g/24h) *(cUREA)URINE = concentration of urea in urine in mmol/l Biochemistry II Pouria Farsani 2013 62 * VU = 24 hour diuresis in L *In the case of massive proteinuria, it is necessary to add the mass of nitrogen in the urine protein which is expressed as: “(mass of protein in urine in g/l) × 0.16 × VU” to the result of nitrogen excretion (calculation above). -Balanced (neutral) nitrogen balance – intake = excretion. Everything is O.K -Positive nitrogen balance – intake > excretion. This means that nitrogen is retained in the body, (new proteins are synthesized). This occurs during the growth of children, in pregnancy and during the repair of damaged tissues (as in convalescence). It is also desirable in patients which chronic renal insufficiencies, other diseases or injuries -Negative nitrogen balance – excretion > intake. This is seen in severe situations (starvation, malnutrition, severe infections disease, after trauma, burns or surgery). The excretion per day is increased as the body may use its own stores of protein during the above mentioned situations. This catabolic period is usually characterized by a considerable decrease of body weight – the loss of 1 g of nitrogen is equivalent to 6.25 g of proteins and about 25-30 g of muscle tissue (muscles contains about 75-80% of water and 20-25% of proteins) Biochemistry II Pouria Farsani 2013 63 15. Metabolism of amino acids at various phases of metabolism (resorption phase, starvation, high protein diet, hypercatabolic state, lack of dietary proteins) Resorption phase Amino acids are produced by the cleavage of proteins in the GIT (see q. 39) to then be transported into cells of intestinal mucosa. Proteins are cleaved by (pepsinogen + HCL =>) pepsin in the stomach providing polypeptides and AA. In the small intestine further cleavage is carried out by trypsin, chymotrypsin, elastase and carboxy peptidase and amino peptidase providing oligopeptides, di- and tri peptides and AA. AA enter the enterocyte with Na+ co-transport (seven transport systems). Di- and tri peptides enter enterocytes with H+ linked transport. Newborns have the ability to absorb intact proteins in order to receive immunoglobulins from the colostrum. After a meal we can see an increase of AA in blood of 30-100% (average conc. in postresorption phase is 3.2 mmol/l). Glutamate and glutamine are utilized for energy by the enterocytes. They divide rapidly, thus they need a constant supply of AA in order to synthesize proteins, purines and pyrimidines. The other AA travel via the portal blood to the liver. Approx. 20% of the amino acids in the portal blood are BCAA, these are not utilized by hepatocytes, due to the lack of the specific amino transferases. Almost all of the other AA are completely captured by the hepatocytes. Anabolism of AA takes place rather than catabolism due to higher KM values for catabolic enzymes. This ensures that when all the needed AA are present, protein synthesis (supported Biochemistry II Pouria Farsani 2013 64 by insulin) can take place. Only an excess of AA leads to their catabolism. They can be oxidized to urea and CO2 (CAC), or the resulting intermediates can be used for lipogenesis. Approx. 1/3 of the AA absorbed gets into the systematic circulation of which 70% are BCAA (to be utilized by muscles). If protein synthesis is needed in muscles, essential AA will be released into the systematic circulation again. In the liver: -Increased AA degradation – surplus of AA are either released into the blood or they are deaminated with resulting carbon skeleton being degraded by the liver to pyruvate, acetylCoA or CAC intermediates. -Increased protein synthesis – proteins cannot be stored like TAG or glycogen. The increased synthesis aims to replace any proteins that were degraded in the previous posresorptive phase. In skeletal muscles: -Increased protein synthesis – in order to replace proteins that were degraded since the previous meal -Increased uptake of BCAA – for proteosynthesis and energy production Post-resorption and starvation The protein synthesis is limited. Degradation of labile proteins is initiated in muscles. This is stimulated by deceased insulin and increased cortisol levels. Cortisol stimulates the production of ubiquitin and ubiquitin-dependent proteolysis. The increased level of glucagon stimulates the enzymes (PEP carboxykinase) for gluconeogenesis. Fructose 1,6-bisphosphatase is also induced by the drop of its inhibitor fructose 2,6-bisphosphatase. Concurrently glucagon and glucocorticoids stimulate the AA uptake in the liver for the gluconeogenesis and ureagenesis. The substrates for the gluconeogenesis are supplied mainly from the glucogenic AA, which are released into blood due to muscle proteolysis. These are mainly Alanine 50% of all released AA and glutamine 25%. The gluconeogenesis in the liver utilizes mainly pyruvate formed from the transamination of alanine. Other amino acids are usually partially metabolized in muscles to intermediate products (pyruvate and 2-OG) that provide alanine and glutamine by receiving amino group. The oxoacids which are formed by transamination of BCAA reach the liver via the blood. Oxo-acids of: -Valine => glucose -Leucine => ketones -Iso-leucine => glucose and ketones Most amino acids are transaminated to produce glutamate and oxo-acids which can be used for the gluconeogenesis as well. Biochemistry II Pouria Farsani 2013 65 The formation of glucose in the liver during starvation is directly linked with the synthesis of urea. With several weeks of starvation, proteolysis is decreased in order to save protein. Energy is supplied to the brain in the form of KB instead. High protein diet with low carbohydrate and lipid content As in the other metabolic phases, glutamine and glutamate are taken up by enterocytes in order to be utilized for energy. Their concentration in portal blood is very low. 60-70% of the amino acids are taken up by the liver; most of them are utilized for the production of glucose. If the meal consists purely of proteins, the release of glucagon is stimulated, increasing the AA uptake as well as the gluconeogenesis. The release of insulin is lower than usual. It does not inhibit the liver gluconeogenesis but it still aids in the uptake of BCAA by muscles to start protein synthesis. Higher carbohydrate intake => higher insulin/glucagon ratio => increased shift in use of AA from gluconeogenesis to protein synthesis in the liver. The Atkins protein diet: high protein, low in carbohydrates =>low levels of insulin => reduced storing of reserves. Glucagon increases => glucagon/insulin ratio is favored =>mobilization of reserves (release of FA from adipocytes). These metabolic circumstances lead to an increased level of KB for energy supply to the brain. Biochemistry II Pouria Farsani 2013 66 Hypercatabolic state -Surgery -Trauma -Burns -Sepsis This leads to an increased utilization of fuel and negative nitrogen balance. All reserves, proteins, fat and carbohydrates are mobilized in order to maintain normal tissue function when the food intake is limited. This mobilization is also to provide energy as well as needed amino acids for immune response (main role of released AA), wound healing and acute phase proteins (synthesized in the liver). In the acute phase response, synthesis of plasma proteins, such as albumin is reduced. Naturally these processes lead to a decreased synthesis of proteins and an increased degradation in muscles. The increased degradation is accompanied by increased synthesis of urea. Cortisol and some cytokines (TNF and IL-1) are the mediators, where cortisol stimulates ubiquitin-mediate proteolysis (like in long term starvation). The increased availability of AA together with the increased levels of cortisol stimulates the gluconeogenesis, providing glucose for the glucose-dependent immune cells (e.g. lymphocytes). Oxidation of BCAA => glutamine production increases. Glutamine levels are also increased when synthesis of glutamine synthase is induced resulting in the increased release of AA and glutamine from muscle cells. The increased glutamine efflux from skeletal muscles serves as an energy source for rapidly dividing cells, a source of nitrogen for a number of syntheses and other processes essential for growth and division of cells. The action of glutamine in kidneys aids in the suppression of acidosis that may result as a consequence of stress. Lack of dietary protein Two conditions are of interest: -Marasmus (energy deficiency > protein deficiency). The condition is found in children when the demand for protein and energy is increased. Deficiency of all nutrients causes this disease resulting in: -Reduced amount of body fat -Muscle atrophy (autophagy) -Extremely low weight -(no signs of edema or changes in plasma proteins) Anorexia nervosa could be a cause of this. -Kwashiorkor (protein deficiency > energy deficiency). Cause by long term diet with deficiency of quality proteins and a relative shortage of energy (energy intake consists mainly Biochemistry II Pouria Farsani 2013 67 of carbohydrates). In other words: when the protein deprivation is relatively greater than the reduction in total calories. The symptoms are: -Edema -Muscle atrophy -Low overall weight -(a layer of fat is normal, while psychomotor changes are often the main symptom) … Some pathological processes may result in a low protein intake, causing secondary protein deficiency (kwashiorkor-like malnutrition, protein malnutrition). The pathological processes: -Proteins are used as an energy source within a low intake of carbohydrates (gluconeogenesis) -In hepatic impairment with decreased ability to synthesize proteins (especially albumin) -Increased protein losses by urine in kidney disease (nephrotic syndrome) -Inability to absorb proteins due to malfunction of reabsorption in the digestive tract (malabsorption syndrome) Glutamine has not been discussed properly in this question. See q. 16. Biochemistry II Pouria Farsani 2013 68 16. Ammonium transport, the glutamine cycle and the glucose-alanine cycle See schemes for q. 15. Metabolism of various compounds in all tissues produces ammonia. The levels of ammonia in the blood must be kept low however, due to its slight increase (hyperammonemia) being toxic for the CNS. Ultimately, ammonia is disposed as urea in the liver. We focus on the transport mechanism, from peripheral tissues to the liver. Sources of ammonia (See also Lippincott’s 5th ed. p. 256-257) -Main source: proteins in diet => AA => transdeamidation in the liver (linking of aminotransferase and glutamate dehydrogenase reactions) => ammonia. -From glutamine: in kidneys by the action of glutaminase and glutamate dehydrogenase, excretion in urine as NH4 + (acid-base balance significance). Intestinal glutaminase produce ammonia by hydrolysis of glutamine -From bacterial action in the intestine -From amines -From purines and pyrimidines – catabolism of -From BCAA in muscles – glu + NH4 + + ATP => Glutamine (glutamine synthetase) Transport of ammonia -Urea – formation of urea in the liver => kidneys => passing through glomerular filter => urine -Glutamine – nontoxic storage and transport of ammonia kept in the amide of glutamic acid. Glutaminase in liver and kidneys removes glutamine by deamination. In the liver it is converted to urea (NH4 + which is not used in the urea synthesis, is used for the formation of glutamine in the perivenous liver area which returns to the blood stream); whilst in the kidneys it is used in excretion of protons. In the brain BCAA can be used as an energy source as well – ammonia is removed with the formation of glutamine in astrocytes. It is either released into the blood or transported into nerve cells where it serves a precursor for glutamate and GABA. Glutamine cycle Glutamine is the main amino acid of the plasma (30-35% of amino nitrogen in plasma). The glutamine pool in the blood serves to provide a number of essential processes Biochemistry II Pouria Farsani 2013 69 The cells which use glutamine as an energy source also need it to support cellular proliferation. The kidneys use glutamine as an energy source partially under normal conditions, but mostly during starvation and metabolic stress. Gln => glu => (GD) => 2-OG => CO2 + Glucose (or serine or alanine). In CAC Gln => OAA => gluconeogenesis. -Intestinal mucosa – takes up glutamine from dietary intake and from glutamine available in blood -Muscle – releases glutamine after utilization of BCAA which produced NH4 + (this process is increased in the hypercatabolic state in order to provide energy for rapidly dividing cells) -Brain – takes up glutamine from blood for glutamate and GABA production. It releases glutamine into the blood after utilization of AA -Liver – transforms glutamine to urea for its excretion. The excess of NH4 + is again converted to glutamine which is released into blood. -Kidneys – transforms glutamine to ammonia which is excreted in urine, taking up protons reducing acidity, or, it utilizes glutamine for energy production. Biochemistry II Pouria Farsani 2013 70 Glucose – alanine cycle The purpose of this cycle is to provide glucose for muscles, nontoxic transport of ammonia and to eliminate the ammonia obtained by the proteolysis in muscles. It takes place in the phases where proteolysis in muscles is active. The transamination of alanine in the liver provides pyruvate and glutamate. The glutamate is then used in the formation of urea. Biochemistry II Pouria Farsani 2013 71 17. Degradation of hemoglobin, formation of bile pigments. Metabolism and secretion of bile pigments. The main types of hyperbilirubinaemia There are many schemes in this question, some of them are repeated. Approximately 300-400 mg of heme is degraded every day; about 80% comes from hemoglobin. After about 120 days, changes in the membranes of erythrocytes result in the phagocytosis of them by cells of spleen bone marrow and liver (Kupffer celles). The globin chain is cleaved off, denatured and broken down into AA. Heme is released into cytoplasm, primarily degraded in the ER when O2 and NADPH are present. The heme oxygenase has two isoforms, one substrate-inducible (type I), and one constitutive (type II). The enzyme catalyzes cleavage of the alpha-methane bridge between the A and B rings. The methane group is cleaved off and oxidized to CO. The product is biliverdin which is converted by biliverdin reductase to bilirubin (unconjugated). Bilirubin is bound to albumin and transported to the liver, passing into the hepatocytes by facilitated diffusion. In the hepatocytes bilirubin binds to ligandin (and other proteins). In the ER conjugation with glucuronic acid takes place, producing bilirubinglucosiduronate and bilirubinbisglucosiduronate (O-glycoside of ester type). The latter one is the predominant form of conjugated bilirubin. Conjugated bilirubin which is much more soluble than unconjugated bilirubin is exported into bile by ABC-transporter MRP2 (ABCC2) De-conjugation and hydrogenation takes place in the small intestine providing colorless urobilinogen by the action of bacteria. The brown color of stool is due to a portion of the urobilinogens being oxidized to dipyrrolmethens which can condense to give the colored bilifuscins. Another portion of urobilinogens are reabsorbed in the terminal part of the small intestine and returned back to the liver, where the urobilinogen is transported back into the bile. <4 mg/day is excreted into urine. The urobilinogens which are excreted in the feces condense in air being oxidized to dark brown fecal urobilins. Biochemistry II Pouria Farsani 2013 72 Biochemistry II Pouria Farsani 2013 73 Biochemistry II Pouria Farsani 2013 74 Main types of hyperbilirubinaemia Elevated amounts of bilirubin in blood causes icterus (jaundice) (serum bilirubin > 40 mmol/l). We distinguish between three major types of hyperbilirubinaemia, each with a difference in the increase of unconjugated, conjugated or both types of bilirubin. In order to distinguish between the different types of hyperbilirubinaemia, the ratio of the unconj. and conj. bilirubin must be assessed. A long term increase of conj. bilirubin leads to the formation of delta-bilirubin which represents the portion of bilirubin which has undergone non-enzymatic reaction with albumin forming a covalent peptide bond. Reaction with other plasma proteins to form delta-bilirubin occurs in a lesser extent. Assessing the value of delta-bilirubin will indicate a previous persistence of conjugated hyperbilirbuinaemia. Its degradation reflects that of albumin, 19 days half-life. This complex does not normally penetrate the glomerular membrane. These are the main types of hyperbilirubinaemia (serum bilirubim > 20 mmol/l): 1. Prehepatic (hemolytic – increased levels of unconjugated bilirubin) An increased production of bilirubin 2. Hepatic / hepatocellular (mixed – both forms of bilirubin are increased depending on the disorder phase) Biochemistry II Pouria Farsani 2013 75 3. Posthepatic (obstructive – conjugated bilirubin is elevated) Unconjugated bilirubin is very non-polar, thus it can easily pass the blood-brain barrier into CNS causing encephalopathy (accumulation of unconj. bilirubin in the basal ganglia, kernicterus). This is especially dangerous in newborns, due to the immaturity of the bilirubinmetabolizing system. In other words, a reduced uptake of unconj. bilirubin, low conjugation activity and low levels of intracellular UDP-glucuronate. The bacterial conversion of bilirubin is not sufficient in this case. Phototherapy is given: Biochemistry II Pouria Farsani 2013 76 -Type I Crigler-Najjar syndrome – complete deficiency of UDP transferase. Fatal outcome a few days after birth. -Type II Crigler-Najjar syndrome – partially preserved UDP-transferase activity -Gilbert syndrome – impaired conjugation. 30% functional UDP-transferase. Mutation in enzyme gene which limits detection of a promoter site. Constant slight increase of bilirubin in blood, no therapy is needed -Destruction of RBC’s directly intravascularly – the hemoglobin which is released (can cause oxidative damage to lipids and proteins) is taken up by haptoglobin and transported into phagocytic cells for elimination. Free heme and hematin, are bound by hemopexin and transported to the liver where further metabolism takes place by heme oxygenase – this prevents the loss of iron from the body Biochemistry II Pouria Farsani 2013 77 18. Structure of contractile elements of skeletal muscle fibers (sarcomere, the proteins of thick and thin filaments, functions). Cycle of contraction and relaxation of skeletal muscle Skeletal muscle cells are giant multinucleated cylindrical cells known as muscle fibers or rhabdomyocytes. Numerous of ovoid nuclei are found in each fiber – located peripherally just under the sarcolemma. The contractile fibrils of the muscle are the myofibrils. Looking at the myofibrils in a longitudinal section (see picture =>) we can distinguish different bands (in cross section they appear as dots – see picture below): -A-band – the dark band (the area of myofilaments – thick filaments overlapping thin filaments) -I-band – the light band (containing only thin filaments) -Z-disc – the thin dark band intersecting the I-band (acting-anchoring structures for thin filaments of adjacent cells) -H-band – a thin light band intersecting the A-band -M-line – found within the H-band Sacromere The segment of myofibril which extends between two Z-discs is known as the sacromere. Biochemistry II Pouria Farsani 2013 78 Proteins of thick and thin filaments The myofibrils are composed of more slender filamentous units known as myofilaments – of which we distinguish between two different sets composed of different proteins: -Thick filaments – contain myosin – the A-band. In their centers, the myosin filaments are slightly thickened, giving rise to the M-band. In the cross section we can see these thick filaments arranged in a regular hexagonal pattern -Thin filaments – contain actin – they constitute the I-band as they extend in both directions, even for a small distance into the adjacent A-bands – overlapping of ends of filaments which are connected by cross bridges The H-zone is the part of the A-band that is free of actin filaments. (mistake in the colored picture above, actin and myosin should exchange positions) As we can see in the right picture above, six thin actin filaments permanently surround one thick myosin filament. The thick myosin filament is an asymmetrical protein consisting of two heads and a tail. It has two heavy chains and four light chains. The globular head of myosin II (similar to myosin but found in cardiomyocytes) is ATPase, activated by actin. Biochemistry II Pouria Farsani 2013 79 The thin filaments are actin and protein complexes composed of troponin and tropomysin Functions The mechanism of contraction is explained by the sliding filament theory of Huxley: The thin and thick filaments react by sliding past each other during contraction – their length does not change. The thick myosin filaments in the A-band are relatively stationary. The acting filaments however, attached to the Z-discs extend further into the A-band causing the Z-discs to be drawn toward each other => compression of the sarcomere => shortening of the myofibril => contraction. In other words, there is not shortening of actin and myosin occurring, but an increase in the overlap between them! Biochemistry II Pouria Farsani 2013 80 Cycle of contraction and relaxation of skeletal muscle The energy which is which is needed for contraction is provided by the hydrolysis of ATP by ATPase localized in the cross bridges that interconnect the actin and myosin filaments. A) Troponin-I inhibits the binding of myosin to actin. The spreading of an action potential opens calcium channels, Ca2+ enters the cell as well as it is released from the sarcoplasmic reticulum. B) Calcium binds to the troponin-tropomyosin complex (troponin-C) => actin activates myosin’s ATPase => hydrolysis of ATP (ATP =>ADP + Pi) => myosin-ADP-Picomplex binds to actin C) The previous scenario resulted in the lifting of the myosin head and release of TpI between actin and myosin due to a conformational change of the troponintropomyosin complex – increasing the myosin-actin formation by four powers of ten. This results in the release of Pi which leads to an even increased tilt of the myosin heads causing the subsequent release of ADP. The release of Pi causes the sliding of actin and myosin past each other – first step of the power stroke The subsequent release of ADP results in further sliding, ultimately resulting in the final positioning of the myosin head – second part of the power stroke D) After maximal positioning of the myosin head, the complex is known as the rigorcomplex which is stable. Binding of ATP weakens this bond – dissociation of the actin-myosin complex (Mg2+ makes a chelate). Calcium released from troponin-C allowing troponin-I to bind to the myosin head again, together with subsequent hydrolysis of ATP results in relaxation. Biochemistry II Pouria Farsani 2013 81 19. Provision of energy for muscle contraction and relaxation (substrates and pathways depending on the intensity as well as duration of muscular work or exercise) Biochemistry II Pouria Farsani 2013 82 Explanation of the graph: -ATP is firstly used as the source of energy for the muscles – 3s -Creatinine phosphate (mistake in the graph!) is utilized during the first ~10s (mistake in the graph, it says creatine) -Energy is then obtained after 30 seconds from the anaerobic glycolysis which yields lactate + 2ATP -Aerobic oxidation of glucose proceeds after 10 min which yields 2 pyruvate => 2 acyl-CoA => 38 ATP -Oxidation of FA is used lastly, after ~2 hours where: *Stearic acid => 146 ATP *Palmitic acid => 129 ATP Supply to myocardium Fatty acids are the primary source of energy for the myocardium in both fed and fasting states. Ketone bodies are utilized during starvation. Due to the fact that the myocardium is in constant need of oxygen, it cannot obtain energy from anaerobic glycolysis. Biochemistry II Pouria Farsani 2013 83 20. Differences in the mechanisms of cardiac and smooth muscle contraction. Biochemical markers of myocardial damage We start by individually explaining the mechanisms of contraction of heart- and smooth muscles – a summary table will follow in the end: -Cardiac muscle The contraction of the heart is induced by itself (cardiac autonomic nervous system), not by external stimuli although the nervous system may have an effect. The heart rate is controlled by the SA-node – spontaneous depolarization during diastole. The AV- node and Purkinje cells are the backup systems operating at a slower rhythm. -Smooth muscle We distinguish between multiunit- and visceral smooth muscle where the multiunit is only controlled by the autonomic nervous system (each cell has separate innervation – does not possess automaticity). The visceral smooth muscles have the ability of automaticity – besides the ANS, the can also by controlled by means of hormones, substance effects, and reflexes. -Cardiac muscle Contain myosin, actin and troponin in the contractile apparatus. Intercalated discs separate one cell from another (for information about contraction action of striated muscle see q. 18) -Smooth muscle Does not contain troponin – only actin and myosin. Visceral smooth muscles are spindle-shaped with the ends laid one on another – gap junctions and mechanical purposes (continuity of the contractile system). Myosin of smooth muscle differs from striated muscle myosin both genetically and functionally: *ATPase activity is only 10% of the activity of striated muscle *interaction with actin occurs only when one of the light chains (MLC, myosin light chains) is phosphorylated on a specific serine residue *it forms thick filaments with cross-bridges that are not arranged with the same regularity, but they are distributed along the whole length *phosphorylation of the light chain is catalyzed by the myosin light chain kinase (MLCK) activiated by Ca2+ -calmodulin (phosphorylation of myosin light chains in skeletal muscle probably changes the pulling intensity that the muscle develops) Biochemistry II Pouria Farsani 2013 84 -Cardiac muscle (for function of the calcium channels. See q. 75) The main source of calcium for the cardiomyocytes comes from the SR RELAXATION: rapid reduction of sarcoplasmic Ca2+ - performed by Na+ /Ca2+ -exchanger and Ca2+ -ATPase (SERCA) in the sarcoplasmic membrane. The Na+ /Ca2+ -exchanger is the major route of transport of Ca2+ ions out of the cells – facilitation of relaxation. Sometimes however, it can proceed in the opposite direction and increase the Ca2+ uptake in the SR. Ca2+ -ATPase also participates in the uptake of Ca2+ into SR – its activity is regulated by the level of calcium and by phospholamban. Both these systems are activated by phosphorylation (calmodulin-dependent protein kinase II). Biochemistry II Pouria Farsani 2013 85 The Na+ concentration determines in which directions the Na+ /Ca2+ -exchanger proceeds. When Na+ ↑ => transport proceeds in to opposite direction => Ca2+ enters the cell. That is why => all factors which increase Na+ in ICF => Ca2+ in ICF ↑ => intensification of contraction – positive inotropic effect – such as digitalis (drug) for instance which inhibits coupled Na+ /K+ /ATPase. Cardiotonics are substances which increase the myocardial contraction. Biochemistry II Pouria Farsani 2013 86 -Smooth muscle The concentration of Ca2+ in smooth muscle varies with the plasma membrane permeability which is, unlike skeletal muscle, controlled by the autonomic nervous system. An increase of intracellular concentration (~10 umol/l) activates muscles contractions; when it drops to 0.1umol/l as a result of the activity of Ca2+ -ATPase, MLCK is inactivated, MLC is dephosphorylated by the effect of MLC phosphatase, which is followed by RELAXATION. -Cardiac muscle Adrenergic nerves innervate the conduction system of the atrial and ventricular fibrils – they are predominantly terminated by beta-receptors – they can bind adrenaline and noradrenaline. Binding of catecholamine to beta1-recetptor => adenylate cyclase => cAMP ↑ => activation of intracellular protein kinases => phosphorylation of various proteins => activation of these proteins => heart rate and contractility ↑ Biochemistry II Pouria Farsani 2013 87 -Smooth muscle Smooth muscle responds to hormonal stimuli. Adrenaline => adenylate cyclase => cAMP ↑ => activation of protein kinase A => phosphorylation of MLCK => phosphorylated MLCK is released from the bond with Ca2+ calmodulin => loss of MLCK activity Prevailing of myosin dephosphorylation => relaxation There is a structural and functional similarity between calmodulin and troponin C (TnC). TnC is a kind of calmodulin which evolved in skeletal muscles in order to achieve rapid response to a change in Ca2+ concentration. Several mechanisms regulate smooth muscle contraction: -Ca2+ -calmodulin -cAMP -Diacylglycerol -Caldesmon Biochemistry II Pouria Farsani 2013 88 The text above has explained in detail what is summarized in the table below: Biochemical markers of myocardial damage Myocardial damage is associated with a decreased blood flow to the myocardium which results in anaerobic glycolysis. This then results in the increase of lactate, permeability of the cell membrane decreases and proteins stored in the cytoplasm can be released. If the ischemia lasts for a longer period, glycolysis will stop because of acidosis. This will results in the irreversible damage of the cells and their necrosis (infarction). It is under these conditions when contractile and mitochondrial proteins (especially troponin T and mitochondrial fractions of creatine kinase and AST) are released into the blood. Biochemistry II Pouria Farsani 2013 89 A combination of three biochemical tests, together with ECG and clinical symptoms are used in the follow-up and diagnosis of myocardial infarction (MI). The biochemical tests are: -Myoglobin -CK-MB mass -Cardiac troponin (T or I) Creatine kinase Creatin kinase (CK) is a cytoplasmic enzyme which catalyzes the phosphoarylation of creatine. There are three forms, each of them consisting of two subunits, wither of B type (brain) and/or M type (muscle). -CK-MM isoform is found mainly in skeletal and cardiac muscles -CK-MB forms about 40% of the total CK in the myocardium, but it is also found skeletal muscles, forming about 1-2% of the whole amount of CK -CK-BB is included in higher concentrations in the brain. It is also produced by the placenta and other tissues The reference values of CK are: -0.41-3.16 ukat/l in men -0.41-2.83 ukat/l in women 3-6 hours after MI elevated levels of CK are found, the maximal value of which is reached after 24 hours. An increase may also be a result of enzyme release from skeletal muscle due to physical strain, convulsions, muscle injury, intramuscular injection etc. is recently considered as a non-specific indicator and of little importance. CK-MB This is a specific marker of MI. it has a time course similar to that of total CK. It is important to know that even though this is considered as a specific marker, its levels may increase due to extensive skeletal muscle damage, considering that skeletal muscles also have a small proportion of CK-MB. We determine the specificity of damage by estimating the ratio, where in case CK-MB/tot CK is > 6% we can conclude high probability of myocardial damage. We can also determine the CK-MB mass by immunoanalytical procedures. In this test, degraded molecules which has lost their enzymatic activity, react with a specific antibody. The determination of CK-MB mass is more specific considering that its elevated values in blood are found sooner compared to the activity of CK-MB, also the test is more sensitive – the upper reference limit for CK-MB mass is up to 5ug/l. The CK-MB test is recommended in case evaluations of troponins cannot be carried out Biochemistry II Pouria Farsani 2013 90 Troponin T and I (TnT, TnI) Troponins are proteins that participate in the regulation of striated muscle contraction. Ischemia => release of cytoplasmic TnT in the course of 4-6 hours. Irreversible ischemia => release of TnT bound in tropomyosin complex with peak values on the 4th day. Increased levels persists from 10 days to two weeks. Immunochromatographic method is used to detect TnT in blood (two lines = positive). No TnT levels should be found in the blood of healthy individuals. During Acute MI the levels may rise up 300 times. TnT can be found sometimes in dialyzed patients as well. TnI detection is of similar use as TnT in the diagnostics of AMI. Dialysed patents do not have have an incrase of TnI as frequently as TnT. The role of both troponins is in diagnosis if AMI is interchangeable. Their determination is significantly more sensitive and only this method is able to prove any ever so tiny damage. Myoglobin Myoglobin is found both in skeletal muscles and myocardium. It is released early during myocardial ischemia – usually between the first and second hour. Because the myoglobin molecule is small, it is excreted easily in the course of 12-24 hours. The physiological range is dependent on individual factors such as body mass, age sex. Cut off values are: -60ug/l in women -70ug/l in men Myoglobin values are not only elevated during myocardial damage, but also during some myopathies and muscle injuries. Its levels can also rise due to renal insufficiency. Its determination has to be combined with the other laboratory indications. Myoglobin has a negative predictive value – if it is not elevated 4hours after the onset of pain, myocardial infarction can be excluded! AST Previously this was also used as a marker of myocardial damage. Today the most important markers are the combinations of: -CK-MB -TnT -Myoglobin Together with ECG and presence of symptoms. Biochemistry II Pouria Farsani 2013 91 21. Synthesis of NO, isoenzymes of NO synthase, function of NO in organism, receptors of NO Synthesis of NO No is synthesized by nitric oxide synthase (NOS) from arginine in endothelial cells (activation e.g. acetylcholine). Some exogenous compounds known as NO donors such as: glyceryl trinitrate, nitroprusside, etc. form NO as well in the body. NO readily permeates membranes, it can be produced by one type of cell and rapidly diffuse into neighboring cell types. Isoenzymes of NO synthase (see also q. 68) Biochemistry II Pouria Farsani 2013 92 Function of NO in organism and receptors of NO Receptors with guanylate cyclase activity convert GTP to cGMP after the binding of ligand where cGMP is the second messenger. It activates protein kinase G. Two types of receptors: -Membrane-associated -Soluble (cytoplasmic) – binds to NO -Protein kinase G *sensitive to cGMP *expressed widely in many cells *phosphorylates various proteins (enzymes, transportation proteins etc.) -Effect of PKG in smooth muscle – phosphorylation of proteins *inactivation of proteins => attenuation of Ca2+ release from ER => drop in Ca2+ *activation of MLC phosphatase => repression of actin-myosin interaction *decrease of K+ -channels activity => decrease of hyperpolarization => increased influx of Ca2+ into the cell =>=>=> relaxation of smooth muscle The receptors for NO are the soluble receptors in the cytoplasm with guanylate cyclase activity. Soluble guanylate cyclase is a dimeric molecule which contains a heme. NO binds to the heme => increase of catalytic activity of guanylate cyclase => increased production of second messenger cGMP => muscle relaxation (see reaction involving activation of PKG and Ca2+ above) The ultimate effect of NO is therefore muscle relaxation. However, inactivation of phosphodiesterase also increases cGMP this prolonging muscle relaxation – potentiation of the NO effect. There are several types of phosphodiesterases known, depending on the type of the cell. Biochemistry II Pouria Farsani 2013 93 Viagra Sildenafil (Viagra) acts as a selective inhibitor of phosphodiesterase 5 (PDE5) which is highly expressed in vascular smooth muscle (corpora cavernosa penis). Sexual stimulation => release of NO in corpora cavernosa => increased level of cGMP => prevention of cGMP decomposition by Sildenafil => remaining of dilation of blood vessels in erectile tissue => increased blood flow => erection Viagra is concurrently 80-4000 fold less potent as an inhibitor of other PDE isoforms (including PDE3 which is expressed in heart). This reduces the risk of interference of Viagra with functions in the body. Viagra itself dos not generate cGMP, and therefore cannot create an erection by itself without prior sexual stimulation. Biochemistry II Pouria Farsani 2013 94 22. Nervous tissue – main types of cells and their function, myelin membrane, specific properties of endothelial lining of vessels in brain, provision of energy and nutrient requirements, relationship of neurotransmitters to amino acids (a survey) • ~2.4% of adult body weight. 83% of which is the brain • Large consumption of energy – 60% of the glucose • Consumption of 20% of the O2 • Content of specific complex lipids • Rapid turnover of proteins • Cell types: -CNS: *neurons *astrocytes *oligodendrocytes *ependymal cells *microglia -PNS: *neurons *Schwann cells *satellite cells Endothelial lining of vessels in brain This lining is what forms the so called blood-brain barrier (BBB) where free transport of substrates from blood is restricted. All hydrophilic substrates require transport systems. The only substrates which can pass the blood-brain barrier freely are water and small lipophilic molecules (O2, CO2, NH3, ethanol). In peripheral tissues, free diffusion through intercellular space, pinocytosis and transporters allow the entrance and passage in/through the cells. The capillary bed in the CNS however has a continuous basement membrane where: -Numerous of tight junctions limit free diffusion -No pinocytosis occurs -The basement membrane is highly consistent -GLUT 1 transporters have low efficiency Protective function of the endothelial cells in brain plays a significant role as well: -Prevents chemical fluctuations which occur in the blood to affect the delicate brain – limitation of exchange between blood and brain -Minimizes the ability of harmful substances to enter the brain Biochemistry II Pouria Farsani 2013 95 -It has many drug-metabolizing enzyme systems -It has active P-glycoprotein pumps that actively pump hydrophobic drugs that could penetrate the barrier (this limits the use of drugs for treatment of brain and spinal cord) -Transport of molecules through BBB *monocarboxylic acids (lactate, acetate, pyruvate, ketone bodies) – specific transporters *essential AA rapidly enter CFS via a single amino acids transporter *non-essential AA – restricted transport – their synthesis takes place in the brain *vitamins – specific transporters *some proteins (e.g. insulin, transferrin, IGF) – cross BBB by receptor mediated endocytosis (protein binds to membrane receptor => endocytosis of complex => release on the other side of the membrane) Insulin receptors are found in certain parts of the brain (mostly in neurons). Insulin => initiation of signal => regulation of both the structure and function of neural circuits Neuroglobin It is a new type of globin which is expressed mainly in the brain – monomeric structure, hemoprotein. Its function is probably to increase the availability of O2 in the brain Neurons -Dendrites – with receptors of neurotransmitters -Perikaryon – metabolic structure with intensive proteosynthesis.it is highly susceptible to low supply of O2. In this part metabolism of glucose, as well as synthesis of neurotransmitters and lipids take place -Axon – active transport of Na+ and K+ across the axolemma. Voltage operated ion channels enables inception and spreading of action potentials *axonal transport – anterograde (from body – kinesin) and retrograde (to body – dynein) – provides shift of proteins, mitochondria (meeting changes of energy needs) and synaptic vesicles between perikaryon and synaptic terminals. The axonal transport occurs along microtubules *myelin sheath – rapid salutatory conduction of nerve impulses -Axonal terminals – synapses – neurotransmitters are released from the synaptic vesicles into the synaptic cleft by exocytosis Biochemistry II Pouria Farsani 2013 96 Astrocytes Roles: • Connected by gap junctions – syncytium • Support the migration of neurons – forms the matrix that keeps them in place • Synthesis and degradation of glycogen • Glycolysis => lactate and its transport to neurons • Uptake and metabolism of neurotransmitters such as GABA and glutamate • Control of the extracellular environment of the brain (Na+ /K+ -pump, “uptake” of K+ , Na+ -HCO3 - co-transport, transport of gases) • Secretion of neurotransmitters (NO, VIP – vasoactive intestinal peptide, PGE2) In astrocytes, glutamine is recycled, where glutamate and GABA excreted by neurons into synaptic clefts are taken up by astrocytes and converted back to glutamine. Ammonia is metabolized in the recycling of glutamine. It is a pathway to decrease levels of ammonia if they are too high by incorporation into Glu and Gln (glutamate DH, glutamine synthetase) Depletion of 2-OG for CAC => depletion of ATP => accumulation of Gln and Glu in astrocytes => failure to transport Glu from synaptic cleft => damage of neurons by exitotoxic effects of Glu. Glu accumulation + Damage of osmotic regulation in astrocyte => increased water intake => edema of brain Biochemistry II Pouria Farsani 2013 97 Myelin Myelin is formed when protruding parts of plasma membranes of glial cells wraps around axons. -CNS: Oligodendrocytes -PNS: Schwann cells Myelin consists of: -30% cholesterol -16% cerebrosides -Structural proteins – main ones: *porteolipidic protein *basic protein of myelin (encephalitogen) *high molecular-weight protein known as Wolfram’s protein -Myelin damage *demyelination disease – loss or damage of myelin, both in CNS and PNS *multiple Sclerosis – autoimmune. Demyelination of white matter and multifocal inflammation => damaged nerve conduction Biochemistry II Pouria Farsani 2013 98 Provision of energy and nutrient requirements Energy in the brain is provided by the following means: • Glucose metabolism (~ 100g/day) • Utilization of ketone bodies (starvation => 50% of energy) • CAC • Aerobic metabolism – consumption of 20% O2 • If O2 supply is interrupted for more than 4-5 min or glucose is cut off for more than 10-15 min =>=>=> brain damage! -Glucose transports in brain *endothelial membranes – GLUT 1 *glial cells – GLUT 1 *neurons membranes – GLUT 3 *glucos intake rate is limited by KM for GLUT 1 The transport through the capillary walls is much less efficient due to the BBB Low levels of glucose (< 3mmol/l) => insufficient transport (KM) => hypoglycemic symptoms -Energy from ketone bodies -Lipids in brain *FA and other lipids cannot penetrate the BBB – they need to be synthesized in the brain *essential FA (linolenic and linoleic) have specific transporters Biochemistry II Pouria Farsani 2013 99 *in order to produce myelin, very long FA are synthesized in the brain as well – DHA (docosahexaenoic) is the most abundant (22:6 (n-3)) *beta-oxidation, peroxisomal FA oxidation occur in brain *cholesterol synthesis Relationship of neurotransmitters to AA -GABA (~50%) – obtained by decarboxylation of glutamate – glutamate decarboxylase -Serotonin – obtained by hydroxylation and decarboxylation of tryptophan – tryptophan hydroxylase, aromatic amino acid decarboxylase -Dopamine – obtained by hydroxylation and decarboxylation of tyrosine – tyrosine hydroxylase, DOPA decarboxylase Aromatic amino acid decarboxylase -Histamine – decarboxylation of histidine Biochemistry II Pouria Farsani 2013 100 23. Cerebrospinal fluid – origin and composition The CSF is a transcellular fluid which surrounds the brain both internally and externally. Internally it is located in the ventricles. CSF is formed in the choroid plexus and it has a volume of 120-180 ml in adult people. -It protects the brain and spine from injury -It provides basic mechanical and immunological protection for the brains. The blood brainbarrier and blood-CSF barrier prevents the passage of most substances except for CO2 and O2, water and lipophilic substances -CSF also serves a vital function in vertebral autoregulation of cerebral blood flow and as a pH buffer -CSF is a clear and colorless liquid. Its composition can be seen in the table below: Routine in CFS examination -Firstly the appearance is checked, color and clarity -Determination of glucose, protein, lactate -Spectrophotometry (detection of oxyhemoglobine, bilirubine, methemoglobine) -Albumine -Cytology examinations (white blood cells, red blood cells) CSF is analyzed in order to diagnose medical disorders that affect the CNS. These conditions are: -Meningitis and encephalitis (bacterial and viral) -Fungal or parasitic infections Biochemistry II Pouria Farsani 2013 101 -Metastatic tumors (e.g. leukemia) -Tumors of CNS that shed cells into the CSF -Bleeding in the brains and spinal cord -Multiple sclerosis -Gullian-Barré syndrome – a demyelinating disease involving peripheral sensory and motor nerves -Injury In case CSF would be detected in biological material we speak about CSF leakage: -Beta-trace protein (BTP) *enzyme synthesized in the choroidal plexus *conc. in CFS is 20-30x higher than in plasma *immunonephlometric method -Beta-2 transferrin (asialotransferrine) *formed from transferrin by action of enzyme neuraminidase *not present in plasma Biochemistry II Pouria Farsani 2013 102 24. Membrane potential of a neuron, depolarization and the action potential propagation. Voltage-operated and receptor-operated (ligand gated) ion channels – examples Nerve impulse -The neurons are irritable cells -The lipid bilayer is practically impermeable to the unevenly distributed Na+ and K+ ions -There is a resting membrane potential – -70mV on the inner side of the plasma membrane which is maintained by Na+ -K+ -ATPase -The channels which are present in the membrane are ligand-gated Na+ /K+ channels: *voltage-operated Na+ channel *voltage-operated K+ channel -A stimulus triggers a depolarization. In case this depolarization reaches a “threshold value” => opening of voltage-operated Na+ channels => massive entrance of Na+ into the cell => depolarization takes place => triggering of the slow opening K+ channels, Na+ channels will slowly close => influx of K+ => repolarization takes place. -Na+ /K+ pumps restore the concentration gradients disrupted by the action potential -Action potential is propagated to the axon terminals -It is the changes of membrane potential during an action potential which propagated the nerve impulse along the membrane! Biochemistry II Pouria Farsani 2013 103 As the action potential reaches the axon terminal either neurotransmitters or neurohormones are released. -Neurotransmitters – released into the synaptic cleft to influence neighboring cells. They have a short range and short life span -Neurohormones – released into the blood in order to be transported a longer distance e.g. hormones from hypothalamus, hypophysis, (e.g. oxytocin, ADH). Adrenaline is another example. -Some neurotransmitters can function as neurohormones as well. Synaptic transmission Neurotransmitters act as chemical signals between nerve cells or between nerve cells and the target cells. -Ionotropic – electrical signal -Metabotropic – chemical signal Biochemistry II Pouria Farsani 2013 104 Voltage-gated channels They are opened/closed by changes in membrane potential -Activation of gate of Na+ channel – opened by depolarization = nerve membrane is permeable to Na+ -Inactivation gate of Na+ channel – closed by depolarization = membrane is impermeable to Na+ (e.g. repolarization phase) Receptor operated (ligand gated) channels They are opened/closed by hormones, second messengers or neurotransmitters -Acetylcholine nicotinic receptor – Na+ /K+ channel at motor end plate, neuromuscular junctions, autonomic ganglia and adrenal medulla It opens when 2 molecules of acetylcholine binds to it => activation It is an asymmetric pentamer of four kinds of membrane spanning subunits – 2 alpha, beta, gamma and delta. Acetylcholine binds to both alpha subunits => opening of channel => large influx of Na+ and smaller efflux of K+ => depolarization The change in conformation: the channels undergo frequent transmission between opened and closed states in a few milliseconds. When it is closed it is the resting state. -Excitatory receptors: *acetylcholine (Na+ /K+ ) *glutamate *aspartate (Na+ , K+ , Ca2+ ) -Inhibitory receptors: *GABA (Cl- ) *Glycine (Cl- ) Do not confuse glycine with glutamate! Biochemistry II Pouria Farsani 2013 105 25. Adrenergic synapse (release and inactivation of the transmitter, the types of adrenergic receptors, signal transduction) Adrenergic and Cholinergic systems Noradrenaline – sympathetic, thus: Adrenergic system Acetylcholine – parasympathetic, thus: Cholinergic system Exceptions: -All preganglionic sympathetic fibers are cholinergic (they then synapse with noradrenergic neurons in their respective ganglia) -Only postganglionic fibers of the sympathetic system are adrenergic. However! The postganglionic sympathetic fibers which innervate sweat glands are cholinergic. In relation to the topic of this question, we conclude that the neurotransmitter of most postganglionic sympathetic neurons is noradrenaline. Release and inactivation of the transmitter Catecholamines are produced and stored in the adrenal medulla. When needed the catecholamines are released into blood in order to reach their target cell, we speak about neurohormones. Neurohormones which are released into the blood move a long distance; they could be hormones of the hypothalamus or hypophysis (oxytocin and vasopressin). In this case we focus on the hormones produced by the adrenal medulla – catecholamines. Sometimes, neurotransmitters (see q. 26) can act as neurohormones simultaneously. Biochemistry II Pouria Farsani 2013 106 -Synthesis and storage of catecholamines *tyrosine => DOPA => Dopamine in cytosol => transported into vesicles (ATP dependent) => beta-hydroxylation of noradrenaline in vesicles => transport of vesicle to nerve terminals In the adrenal medulla, the chromafin cells are modified sympathetic neurons that do not have axonal fibers – they release their chemical transmitter (95% adrenaline) directly into the circulation. As mentioned, catecholamines are stored in chromafin granules with cotransmitters (neuropeptides), ATP and chromagranine. Cholinergic neurons in the adrenal medulla stimulate the secretion – nicotine receptors. Biochemistry II Pouria Farsani 2013 107 Types of adrenergic receptors Biochemistry II Pouria Farsani 2013 108 Biochemistry II Pouria Farsani 2013 109 26. Cholinergic synapse (biosynthesis of the neurotransmitter and the release of it, two principal types of acetylcholine receptors and mechanisms of their function) Cholinergic synapses, biosynthesis of the neurotransmitter The neurotransmitter of cholinergic synapses is acetylcholine. Besides being active in parasympathetic postganglionic nerve endings, it is also active in all preganglionic fibers of the autonomic nervous system and as well sympathetic postganglionic nerve endings for sweat glands. Acetylcholine is synthesized in the cytoplasm of presynaptic nerve terminals. Acetyl-CoA is synthesized in mitochondria; it reacts with choline in a reaction catalyzed by choline acetyltransferase in the soma before it reaches the nerve terminals by axoplasmic transport. Choline on the other hand is provided by the hydrolysis of acetylcholine by acetylcholine esterase. It must be taken up from the ECF by the way of a carrier – the rate limiting step of acetylcholine synthesis. Choline is also taken in from food and also given from serine. Release of acetylcholine During the propagation of a depolarization wave, the increased influx of Ca2+ (voltage-gated Ca2+ which open upon spreading of action potential) resulting in the increased intracellular concentration => activation of Ca2+ -calmodulin-dependent protein kinase => it phosphorylates synapsin-1 => interaction of synapsin-1 with membrane of synaptic vesicles => initiation of their fusion with the presynaptic membrane => exocytosis of neurotransmitter => recycling of membrane vesicles. Arrival of a nerve impulse releases ~300 vesicles at the neuromuscular junction (~40 000 acetylcholine molecules in each vesicle) => increase of acetylcholine concentration in the synaptic cleft more than 10 000 times. Biochemistry II Pouria Farsani 2013 110 Two principal types of acetylcholine receptors and mechanisms of their function -Nicotinic type acetylcholine receptor It is located at neuromuscular junction of skeletal muscles and in dendrites of almost all peripheral efferent neurons. Five subunits which penetrate the membrane – see scheme. Acetylcholine binds to the two alpha-subunits => conformation change and influx of Na+ and efflux of K+ => change of potential => activation of another sequence => cell response. Stop of acetylcholine secretion => decreased concentration => acetylcholine ceases binding to the receptors. -Myastenia gravis – T- and B-cells produce antibodies against acetylcholine receptors in skeletal muscles. Antibodies bind to receptors forming receptor-antibody complex => removal of complex by endocytosis and degradation in lysosomes => decreased ability of receptors to respond to acetylcholine and reduction of receptors => => => muscle weakness. Therapy is administration of reversible inhibitors of acetylcholine esterase => increase of acetylcholine effects. Biochemistry II Pouria Farsani 2013 111 -Muscarine recepeptors Biochemistry II Pouria Farsani 2013 112 27. Acetylcholine esterase and its inhibitors (examples of organophosphates insecticides, typical signs of toxic effects, the first aid – the counteractive alkaloid) Acetylcholine esterase is bound on the surface of the postsynaptic membrane. Its function is to break down acetyl choline by hydrolysis to acetate and choline. The choline esterase belongs to a group of serine hydrolases. The enzyme has two sites for biding of substrate, forming an ES complex. These two sites are called anionic and esteratic. Substrate binding => decreased of activation energy needed for acetylcholine breakdown. Hydrolysis of acetylcholine => acetyl group binds to OH group of serine => temporary acetylation of enzyme => acetate + choline Inhibitors of acetylcholineesterase The inhibitors may bind to the active sites of the enzyme. We distinguish between two different kinds of inhibitors: a) Reversible: carbamates (fysostigmine, rivastigmine, neostigmine) These inhibitors bind to both sites of the enzyme. The carbamoyl group temporarily binds to the enzyme => hydrolysis of this bond takes longer (30-60 min) than in the case of acetylated enzyme => decrease of active acetylcholinesterase => increase of endogenous acetylcholine. In therapy these effects are used in the treatments of myestema gravis, prevention of digestive and urinary tracts, post-surgery agony, induction of miosis and reduction of intraocular pressure in glaucoma. b) Irreversible: organophosphates (diisopropyl fluorophosphates, soman, sarin, insecticides) These toxic inhibitors bind only to the esteratic site. The binding occurs in two phases: Biochemistry II Pouria Farsani 2013 113 1. Reversible phase – may be affected by substances known as reactivators 2. Irreversible phase – formation of a covalent bond between organophosphate and enzyme The reactivators are derivatives of pyridine, oximes (obidooxime). The reactivators are administered in combination with spasmolytics (atropine) in the case of intoxication with inhibitors of acetylcholinesterase. The drugs mentioned in the table, affect the channels Na+ /K+ channels shown below: Biochemistry II Pouria Farsani 2013 114 Signs off toxic effects (SLUDGE (M) syndrome) S – Salivation L – Lacrimation U – Urinary incontinence D – Defecation G – GIT irritation/diarrhea E – Emesis (vomiting) M – Miosis (pupil constriction) / muscle spasm First Aid Anitcholinergics must be administered in order to block the parasympathetic nervous system. Atropine is used for this. Atropine also blocks both vagal effects on the heart by blocking acetylcholine action at muscarine receptors. Together with Atropine, Pralidoxime chloride is also administered. Biochemistry II Pouria Farsani 2013 115 28. Inhibitory GABAergic synapse (GABAA receptors, the effect of benzodiazepines and other ligands) GABA (gamma-aminobutyric acid) is the major inhibitory neurotransmitter in the CNS. The GABAergic synapses represent about 60% of all synapses within the brain. The GABAA receptor consists of three subunit types: α2β2γ. The binding site for GABA is located between subunits α and β. Binding of GABA or agonist => opening of transmembrane Cl=> inflow of Cl- => hyperpolarization of postsynaptic membrane => depolarization (formation of action potential) disabling. The modulatory sites on the GABAA receptor Besides the binding site for GABA, the GABAA receptor has at least eleven allosteric modulatory sites for compounds that enhance the response to endogenous GABA. Biochemistry II Pouria Farsani 2013 116 -One important site is for benzodiazepines (between subunits α and γ. Binding of diazepine => increased affinity of binding site for GABA => increased frequency of channel opening => increased Clinflux => deepening of postsynaptic inhibition. The effects of benzodiazepines are therefore: -Sedative -Hypnotic -Anxiolytic -Myorelaxant -Barbiturates binding site is also of significance. Lower concentration of barbiturate which binds to the receptor => prolonged time for opening of Cl- . High concentrations which bind to GABAA => direct opening of Clchannels without involvement of GABA. -Neruosides are involved in the dynamic modulation of GABAA receptor functions *prognenolone sulphate => decreases response of the GABAA receptors *allopregnanolone and Tetrahydrodeoxycorticosterone => potentiate response resulting from GABAA receptors, increasing the efficiency of the inhibitory synaptic transmission in various brain areas *endogenous nerupeptide endozepine (diazepam-binding inhibitor, DBI) affects binding diazepam to GABAA receptor *glycine in the brainstem and spinal cord has similar function as GABA in the brain – alkaloid strychnine blocks the inhibitory effects of glycine => seizure position Biochemistry II Pouria Farsani 2013 117 29. Composition and function of extracellular matrix. Fibrilar and interfibrilar components. Markers of ECM remodelation Composition See scheme below: The extracellular matrix (ECM) is the main component of the connective tissue. The connective tissue forms and maintains the shape of the body. When we speak about soft tissues, we speak about the liver, brain or epithelia where their main components are cells. It is the cytoskeleton (of the cells) and adhesion between these cells that determine the mechanical properties of the soft tissues. On the other hand, when we speak about connective tissue it is the ECM that determines the mechanical properties. The cells themselves in connective tissue have a relatively small contribution. Functions of the ECM • Fills the spaces between cells • Provides an environment for the organization and differentiation of cells • Provides an environment for the exchange and transport of substances • Provides an environment for linking tissue cells (binds cells and tissues together) • Maintains a 3D shape during morphogenesis and would healing Biochemistry II Pouria Farsani 2013 118 • Acts as a barrier against microorganisms • Involved in dell division, adhesion, motion, growth and regeneration of tissues Fibrilar and interfibrilar components • Fibrilar components – collagen fibers, connective tissue and elastin • Interfibrilar components – (amorphous) proteoglycans, hyaluronic acid, glycoproteins, fibronectin, laminin. Bones also include insoluble minerals Markers of ECM remodelation Dynamic remodeling of the extracellular matrix (ECM) is essential for development, wound healing and normal organ homeostasis. During ECM we have some specific markers that we can pay closer attention to: • Serum markers of collagen synthesis -Amino-terminal propeptide of procollagen type I or III (PINP, PIIINP) -Carboxy-terminal propeptide of procollagen type I or III (PICP, PIIICP) • Serum markers of collagen degradation -Carboxyterminal telopeptide of collagen I (CITP) -Matrix-Metalloproteinases (MMP-1, MMP-2, MMP-9) • Serum markers of inhibition of degradation -Tissue inhibitors of Matrix Metalloproteinases (TIMP-1) ECM is important in many pathological conditions such as fibrosis or cancer. Also, a publication by the biochemistry department of Masaryk University has the following abstract: -Recent data suggest that inflammation, oxidative stress and remodeling of extracellular matrix may be associated with the development of heart failure. We assessed the association between circulating levels of soluble intercellular adhesion molecule-1 (sICAM-1), tumor necrosis factor alpha, malondialdehyde (MDA), metalloproteinase-2 and 9 (MMP) and their tissue inhibitor (TIMP-1) in patients with acute myocardial infarction Also, in seminar 15, the last pages regard fibronectin and laminin, for more detailed insight, view those pages. The corresponding lecture has some information as well. Biochemistry II Pouria Farsani 2013 119 30. Collagen and elastin (structural features, biosynthesis of collagen). Lysine – the role of lysyl residues in connective tissue (covalent crosslinks of collagen fibrils, desmosine of elatin) Collagen -Collagen is the most abundant protein in the body, constituting about 25 % proteins in adults and 15-20% of proteins in children -It is present in all connective tissues with a different distribution (see table) -There are about 30 types of collagen known. These differ by structure, function and location. Not all of them are fibrilar, only types I, II, III, V, VI, and XI -A triple helix is present in all collagens, at least partially. A triple helix forms tropocollagen molecules – the basic structural units of collagen fibers Structural features -Amino acids in collagen – the fibrilar collagens have α-chains which are a polymer of repeats of GLY-X-Y where: *X: proline, 3-hydrtoxyproline, Glu, His, Leu, Phe *Y: 4-hydroxyproline, Thr, Lys, Arg *in ever third position there is a glycine *on average, every 4th AA is proline or hydroxyproline *the content of glycine is approx. 30% *the content of hydroxyproline and hydroxylysine is approx. 25% *hydroxyl groups of hydroxylysine can be enzymatically glycosylated by glucose or galactose – this is known as posttranslational modification. This is also the small carbohydrate component of collagen (0.5-1% in fibrilar collagen) *glycosylation occurs before the triple helix formation (see collagen synthesis) -Collagen contains very few essential AA, hence it has a low nutritional value – denatured collagen is called gelatin Tendons 80-90 % Skin 50-70 % Cartilage 50-70 % Arteries 10-25 % Lungs 10 % Liver 4 % Biochemistry II Pouria Farsani 2013 120 -Secondary structure of collagen The high content of proline and hydroxyproline forms a polyproline type of helix. A regular α-helix is stabilized by H-bonds between C=O and NH groups of the same polypeptide chain. This is not the case of polyproline which is stabilized by the formation of trimer: -The secondary structure of collagen is an extended helix with three amino acids per turn. It is stabilized by formation of trimers at which three helical peptides are bound around each other – the triple helix. The structure is held together by H-bonds between the chains (between glycyl and carbonyl residues) -The types of collagen • Fibril-forming types, I, II, III, V, XI -Type I – the most common type of collagen. It has a rather unusual AA composition with 33% of glycine and 10% of proline. It also contains hydroxlyated proline and lysine. It is strong, slightly glycosylated (~1%) and it has no cysteinyl residues. Collagen type I is found in: *skin *bones *tendons *dentin Collagen type I comprises two α1(I) chains and one α2 (I) chains. Each of the three chains consists of a left-handed helix of polyproline type II. -Type II – it is mainly found in hyaline cartilage of joints. It has a high degree of glycosylation. It has no cysteinyl residues -Type III – is found in the skin, aorta and uterus. It is an elastic type, forming thin reticuline fibrils, the glycosylation is very low. Cysteinyl residues are present and also a small amount of disulfide bridges • Network-forming types, IV, VIII, X -Type VI – flexible collagen which is the main component of basement membranes such as in renal glomeruli, capsules of the eye lens The saccharidic component is about 15%. Also, cysteinyl residues and disulfide bridges are present. It does not form fibrils – its flexible triple helices include some non-helical segments, at the C-ends globular domains are located. The chains associate into irregular twodimensional networks which is the basis of the basement membranes. See scheme below: Biochemistry II Pouria Farsani 2013 121 • FACIT, fibril-associated collagens with interrupted triple-helices • Transmembrane domains – consists of anchoring fibers which link the extracellular matrix with the connective tissue • Other types Biosynthesis of collagen The biosynthesis is a complex process which takes place in fibroblasts, chondroblasts and osteoblasts. We divide the processes of synthesis into intracellular- and extracellular processes. -Intracellular process: Synthesis of polypeptide on rough ER => pre-procollagen (contains 1050 AA of tropocollagen and C- and N-terminal propeptides. The N-terminal contains a signal sequence which is used for the insertion of the polypeptide chain into the ER) => removal of signal sequence on N-terminal by signal peptidase from pre-procollagen => formation of procollagen Formation of intermolecular disulfide bridges in the C-terminal Biochemistry II Pouria Farsani 2013 122 Hydroxylation of Pro/Lys forming 4-hydroxyproline, 3-hydroxyproline and 5hydroxylysine. This requires ascorbic acid as a cofactor (see “hydroxylation of proline and lysine residues” below) Glycosylation of 5-hydroxylysine by UDP-glucose and UDP-galactose in the ER Formation of a triple helix by linling the cains in the C=>N direction Secretion of procollagen by exocytosis -Extracellular process: Removal of N- and C-terminal propeptides (non-helical) by the action of proteases => formation of tropocollagen (short non-helical telopeptides remain at its ends) Aggregation of tropocollagen units => formation of protofibrils Interaction of protofibril with proteoglycans => formation of microfibrils Maturation – oxidation of lysine side chains to allysine, formation of intermolecular cross-links (these cross-links are vital for the production of solid fibrils and for the proper function of collagen. First they are reducible, but during collagen maturation they are converted to irreducible, stable bonds. Fibrils are adjoined in parallel. The units are shifted against each other by ¼ of length Biochemistry II Pouria Farsani 2013 123 Illustration of biosynthesis of collagen (the main and most important steps were explained above. Below follows the same explanations with corresponding illustrations) -Inicitation of triple helix formation – we speak about extension propeptides – sequences of 20-35 AA on both amino- and carboxy-terminals with high content of cysteine residues The formation of procollagen triple-helix is initiated by formation of interchain disulfide bridges on both terminal propeptides => self-assembly Winding of the procollagen middle parts results in the triple helix Biochemistry II Pouria Farsani 2013 124 Procollagen is converted to tropocollagen – specific procollagen peptidases catalyze the removal of globular N- and C-terminal propeptides by hydrolysis within the nonhelical segments. In clinical biochemistry, the abbreviations PINP and PICP are used for those propeptides Microfibrils are formed by interactions with proteoglycans Biochemistry II Pouria Farsani 2013 125 Lysine – the role of lysyl residues in connective tissue Some lysyl and hydroxylysyl residues in telopeptides are oxidized by lysyl oxidase (enzyme requiring Cu2+ ) to allysine and hydroxyallysine. The newly formed aldehyde groups react non-enzymatically with other aldehyde groups, unmodified lysyl and hydroxylysyl or histidyl residues form cross-links. These cross-links are vital for the production of solid fibrils and for the proper function of collagen. Firstly they are reducible, but during collagen maturation, they are converted to irreducible, stable bonds. Fibrils are adjoined in parallel. The units are shiftet against each other by ¼ of length, so transverse striation of protofibrils can be seen in the electron-optical image. Covalent crosslinks of collagen fibrils The maturation is the formation of these covalent crosslinks (intra-chain and inter-chain) Covalent cross-links are made non-enzymatically – non-catalyzed reactions between side chains of allysine and lysine. There are also different types of interchain covalent bridges: -Aldimine bridge – is formed when the aldehyde group of allysine reacts with the ε-amino group of lysine. The products is an aldimine (Shiff base) The reaction is rapid, but the product is unstable. It is stabilized very slowly by hydrogenation; the crosslink is then called lysinonorleucine bridge. Biochemistry II Pouria Farsani 2013 126 The aldimine bridges can react with the aldehyde of another allysine side chain. This closes a heterocyclic pyridine ring which covalently binds three different chains of collagen fibril – an intermolecular cross-bridge of hydroxypiridine type is formed. The hydroxypyridinium ring is very stable. It can be determined even after hydrolysis of all peptide bonds in collagen fragments which are excreted into urine as deoxypyridinoline or pyridinoline. -Aldol type – produced when two aldehyde groups of allysine react with each other (aldol condensation). One aldehyde group remains free in the bridge so that it can take part in another reaction. The resulting aldol is unstable, but it can be stabilized by elimination of water (a double bond is formed) to the dehydro-allysine-aldehyde bridge. -Structure formula summary of cross-links in collagen can be seen below: Collagen degradation -Molecules of collagen are metabolically very stable; the half-life may be up to several years -Connective tissue is however constantly remodeled in response to growth or injury – starvation and inflammatory diseases increase the breakdown -Degradation is carried out by collagenases which belong to the family of matrix metalloproteinases (MMP) containing Zn, which is essential for their proteolytic function -In vitro denaturation => gelatin (free of tropocollagen units) Biochemistry II Pouria Farsani 2013 127 Metabolism of collagen – collagen and diseases The metabolism of collagen changes with increasing age. In the older collagen, more crosslinks are formed and also the amount of collagen in the connective tissue increases with age. Injury => collagen synthesis ↑ => fibroblasts are pushed to the edges of the wound into the blood clot and produce collagen => formation of scar (collagen I and III) -Fibroses – similar to the latter process. Overproduction of collagen during necrosis if parenchymal cells in other tissues => lung fibrosis, liver cirrhosis, atherosclerosis -Osteogenesis (brittle bones) – mutation in the gene for α1 and α2 chains of collagen I – impaired formation of triple helices of collagen => collagen is easily digested by intracellular proteases -Ethlers-Danols syndrome – characterized by stretchy skin and loose joints – impairments of collagen synthesis due to mutation of genes -Bacterial infection – synthesis of collagen is stimulated in order to prevent the spread of infection – encapsulation of the bacteria in the form of on abscess. Some pathogenic bacteria secrete collagenase which degrades tropocollagen Elastin Elastin is the major protein of elastic connective tissue and elastic cartilage. It has an amorphous structure with a large number of cross-links and it is insoluble. Elastin is found in: -Arterial walls, Pulmonary alveoli, Skin, Nuchal ligament -Unlike collagen, there is probably only one type of elastin -Elastin is surrounded by a microfibrilar sheath of fibrillin and fibromodulin. During the formation of elastic fibers, these two proteins are arranged in the form of oxytalan fibrils, which serve as a mold that is then filled by tropoelastin, and in which tropoelastin is crosslinked to mature elastin Furthermore elastin: • Produced by smooth muscle cells, fibroblasts and chondrocytes • Main AA are: -Glycine (~13%) -Proline (~11%) -Valine -Alanine (~22%) -Hydroxyproline (~1%) • No hydroxylysine • No glycosylation, the chains do not have a regular secondary structure • Tropoelastin is the precursor for its synthesis Biochemistry II Pouria Farsani 2013 128 Synthesis of elastin Secretion of tropoelastin from the cell => oxidative deamination of some lysine residues of tropoelastin (same enzyme as in the metabolism of collagen) => formation of cross-links between chains (simple aldol or aldimine bonds and more complicated bridges binding more chains which are closed in a heterocycle). After maturation of the crosslinks, elastin becomes highly insoluble. It is extremely stable and has a very low turnover state. Desmosine of elastin The main cyclic cross-link formed in elastin is desmosine. As explained above there are simple cross-links, these are known as merodesmosine – three side chains (the reaction aldol with the lysine side chain) – acyclic. Four side chains of allysine and lysine form a tetrafunctional crosslink (reaction of merodesmosine with allysine) with the stable pyridinium structure known as desmosine or isodesmosine. Below follows a summary illustration of elastin synthesis: Biochemistry II Pouria Farsani 2013 129 Elasticity of collagen It is assumed that in the relaxed state, elastin is maintained in a compact disorganized state in which hydrophobic interactions are applied between the side chains of amino acids of hydrophobic interactions. Stretching => cancellation of these hydrophobic interactions while the elastin network is still held together by covalent crosslinks Degradation of elastin The half-life of elastin very long – in the aorta it is referred to about 40 years! After birth, elastin synthesis is distinctly reduced, if possible at all. -The loss of tissue elasticity – This takes place namely in skin, but also in large vessles and in the lungs. This is followed by aging. In the skin it is exhibited as wrinkles – a decrease of elasticity by 2-5% per decade (extrapolated to neonatal period) -The loss of vessel wall elasticity – in aging it does not quite depend on the low elastin content, but rather on elastin modification due to partial degradation cause by activity of elastase (released mostly by polymorphonuclear leukocytes), supported by the interactions of elastin fibres with LDL or bile acids -The loss of lung tissue elasticity – associated with direct degradation of elastin in the walls of alveoli. Elastase is relased from circulating neutrophils but, under normal conditions, elastase is almost completely inhibited by α1-antitrypsin. Inherited deficit of α1-antitrypsin or factors that diminish its effect (smoking, ozone in the ground smog) distinctly increase the risk of pulmonary emphysema. Fibrilin Fibrilin is a large glycoprotein which is the main component of microfibrils in many tissues. It is secreted by fibroblasts. Marfan syndrome is due to a mutation in the fibrilin gene. When fibrilin is defective it leads to abnormal connective tissues. It affects the: -Eyes (dislocation of the lens) -Skeletal system (most patients are tall, exhibit long limbs) -Cardiovascular system (weakness of the aortic media and dilation of ascending aorta) Biochemistry II Pouria Farsani 2013 130 Biochemistry II Pouria Farsani 2013 131 31. The bone – mineralization and remodelation, hormonal control. Biochemical markers of bone formation and bone resorption The bones have the highest content of organic matrix, it is a metabolically active tissue and it contains about 1 kg of calcium. Furthermore, the bones are composed of: -Inorganic (mineral) compounds – 45% *hydroxylapatite *octacalcium phosphate *amorphous calcium phosphate *minor components: CaCO3, CaF2, CaCl2, Mg3(PO4)2 -Organic compounds – 30% *collagen type I *proteoglycans *sialoprotein *osteonectin *osteopontin *osteoprotegrin *osteocalcin – contains γ-carboxyglutamate residues for binding calcium protein-glutamate + CO2 + O2 + vit Kred → protein-γ-carboxyglutamate posttranslational carboxylation of glutamate residue osteocalcin / coagulation factors (hemostasis) -Water – 25% Bone is dynamic tissue with high metabolic activity Cell types in bone tissue -Osteoblasts • Fibrocyte type, mononucleated with a big ER • Occurs on the surface of growing or remodeling bones • They synthesize osteoid (extracellular matrix) and deposit bone minerals • Successively settle and transform into osteocytes Biochemistry II Pouria Farsani 2013 132 • The surface of osteoblasts binds alkaline phosphate (bALP) that supports mineralization of the matrix • They have receptors for parathormone, calcitriol and prolactin • Produce a number of growth factors and other signal molecules -Osteoclasts • Modified macrophages • Polynucleated cells (10-12 nuclei) • Specialized for bone resorption • Occur at sites of bone resorption (resorption pits) • The membrane is repeatedly folded into ruffled borders = equivanlent of lysosome • Include many lysosomal hydrolases (protase, collagenase, sulfatase etc.) • High activity of acidic phosphatase (bACP) • Produce H+ ions: carbonic anhydrase (CA II) + membrane H+ -K+ -ATPase • Osteoclasts digest collagen fragments through phagocytosis • They have receptors for calcitonin -Osteocytes • Formed from osteoblasts incorporated into bone matrix • Prevailing cellular elements of mature bone • They are dispersed in lacunas and form a cellular net by contacs between their projections • Their physiological role is poorly understood • Serve as sensors of mechanical loading – produce signals for bone re/modeling • Their metabolic activity is low, they have a reduced ER and GA • The life span is estimated to about 25 years, extinct ells initiate bone resorption Biochemistry II Pouria Farsani 2013 133 Mineralization and remodelation Hormonal control See PTH, calcitriol and calcitonin in q. 79. The paradox about PTH claims that: PTH activated osteoclasts and bone resorption. However, receptors for PTH are on osteoblasts. Biochemistry II Pouria Farsani 2013 134 Biochemical markers of bone formation and bone resorption The detection of these markers is useful in clinical biochemistry in tests for osteoporosis and other metabolic osteopathies -Biomarkers of bone formation: *the catalytic concentration of the isoenzyme of alkaline phosphate (bALP) in serum is the marker osteoblast activity *concentration of osteocalcin (bone Gla protein BGP) in serum. Osteocalcin is a modulator of bone remodelation, secreted by osteoblasts. Its production is induced by clacitriol. Osteocalcin is the main non-collagen protein in the extracellular matrix of bones *concentration of N- or C-terminal propeptides of procollagen I in serum (PINP and PICP, see q. 30) -Biomarkers of bone resorption: *catalytic concentration of the bone isoenzyme (one of six) of acid phosphatase (bACP) in serum *concentration of C-terminal telopeptide of collagen I (ICTP) in serum or urine. C-terminal telopeptides are terminal nonhelical sequences of tropocollagen I, without cross-links *excretion of N-terminal telopeptide of collagen I (INTP) into urine – N-terminal nonhelical sequences of tropocollagen I *determination of (deoxy)pyridinoline and galactosyl hydroxylysine in urine Biochemistry II Pouria Farsani 2013 135 32. Retinol and its derivatives – the biological role, biochemistry of visual excitation (activation of transducin, consequences in decrease of cGMP with hyperpolarization and in decreased Ca2+ stimulating guanylate cyclase) Retinol and its derivatives There are two groups of compounds that have vitamin A activity: • Retinoids include: -Retinol -Retinaldehyde (retinal) – from oxidation of retinol catalyzed by retinol dehydrogenease (reversible reaction!) -Retinoic acid – from oxidation of retinaldehyde (irreversible!). It cannot be reduced in the body; hence it cannot give retinol or retinaldehyde. These are preformed vitamin A, found only in foods of animal origin (liver, kidney, butter, egg yolk). • Carotenoids – found in plants. These are composed of carotenes and related compounds, many of which are precursors of vitamin A. The precursors can be cleaved to yield retinaldehyde, then retinol and retinoic acid. The most important (quantitatively) provitamin A carotenoids are α-, β- (majority), and γ-carotenes and cryptoxanthin. The conversion of these to retinol is inefficient in humans. β-Carotene and other provitamin A carotenoids are cleaved in the intestinal mucosa by carotene dioxygenase => retinaldehyde which undergoes reduction => retinol. Retinol is then esterified and secreted in chylomicrons together with esters formed from dietary retinol. Sources are yellow and dark green vegetables and fruits – carrots are a very good source! They provide β-Carotene which is converted to retinol in the body (see steps of this process above) The biological role Light-absorbing visual pigments and a variety of enzymes and transmitters in the rods and cones mediate the conversion of light stimuli into electrical stimuli: this is known as photoelectric transduction. -In rods rhodopsin is found -In cones iodopsin is found All-trans-retinol in the pigment epithelium of the retina is isomerized to 11-cis-retionol and oxidized to 11-cis-retinaldehyde. Biochemistry II Pouria Farsani 2013 136 Rhodopsin consists of opsin and 11-cis-retinal (bound to lysine residue of opsin) Absorption of light by rhodopsin leads to an isomerization of 11-cis-retinal to alltrans-retinal and a conformational change of opsin Release of retinaldehyde from opsin Initiation of nerve impulse Another biological role is that retinoic acid has a role in the regulation of gene expression and tissue differentiation. Visual excitation -Activation of transducin – the formation of the initial excited form of rhodopsin (see above), bathorhodopsin, occurs within picoseconds of illumination, there is then a series of conformational changes leading to the formation of metarhodopsin II, which reacts with a Gs protein called transducin. The activation of transducin initiates a guanine nucleotide amplification cascade and then a nerve impulse – the brain detects light. For details regarding the amplification cascade see q. 71. Transducin activation ultimately results in the decrease of cytosolic cGMP (see cont. of process below). In darkness however, the rods are not exposed to light, thus the cytosolic cGMP (synthesis by guanylate cyclase) concentration is higher: cGMP is bound to cation channels (Na+ , Ca2+ ) in the outer segment of the photosensor Channels are kept open Ca2+ is immediately pumped out by 3Na+ /Ca2+ exchanger, hence cytosolic Ca2+ concentration is kept constant in darkness Influx of Na+ and Ca2+ => depolarization => release of glutamate (neurotransmitter) -Consequence in decrease of cGMP with hyperpolarization – in darkness the cGMP concentration decreases in response to a light stimulus – transducin activation: Exchange from GDP-transducin => GTP-transducin (active) α-GTP separates from the rest of the molecule Disinhibition of cGMP phosphodiesterase => hydrolysis of cGMP to GMP Reduction of cytosolic cGMP cGMP dissociates from cation channels => channels close 3Na+ /Ca2+ continues to pump out Ca2+ => hyperpolarization Inhibition of glutamate release Biochemistry II Pouria Farsani 2013 137 -Decreased Ca2+ stimulating guanylate cyclase – since the 3Na+ /Ca2+ exchanger continues to function after the closure of the channels: Cytosolic Ca2+ continues to decrease. When a threshold for this decrease is reached, guanylate cyclase-activating protein loses its 4 Ca2+ and thus stimulates gunaylate cyclase Acceleration of cGMP synthesis Rise of cGMP concentration Re-opening of cation channels The sensor is ready for a new light stimulus This Ca2+ cycle therefore mediates a negative feedback loop for cGMP production. Biochemistry II Pouria Farsani 2013 138 33. Calciferols (calciols) – structure, sources, transformations, effects, mechanism of action See also scheme (photographed scheme, important!) and text in q. 58 Biochemistry II Pouria Farsani 2013 139 Biochemistry II Pouria Farsani 2013 140 For more detailed information about synthesis and effects of mechanism, see q. 58 and 79. Biochemistry II Pouria Farsani 2013 141 34. Vitamin K – the biochemical function, significance for blood clotting, structural analogs and significance (It is advisable to read q. 63 and 64 before proceeding with this question) Biochemical function and significance for blood clotting Vitamin K is required for the synthesis of blood clotting proteins (II, VII, IX and X). It functions as a coenzyme in the formation of gamma-carboxylglutamate in enzymes of blood clotting and bone matrix. Deficiency of this vitamin leads to impaired blood clotting and hemorrhagic disease. Three compounds have the biological activity of vitamin K: 1. Phylloquinone – the normal dietary source found in green vegetables 2. Menaquinones – synthesized by intestinal bacteria 3. Menadione and menadiol diacetate – synthetic compounds that can be metabolized to phylloquinone -Vitamin K acts as a cofactor at carboxylation of N-terminal glutamyl residues to γcarboxyglutamate (Gla) (post-translational modification) Biochemistry II Pouria Farsani 2013 142 Prothrombin together with several other proteins of the blood clotting system (VII, IX and X proteins C and S) contain 4-6-γ-carboxyglutamate residues. γ-Carboxyglutamate chelates calcium ions, permitting the binding of the blood clotting proteins to the membranes. During vitamin K deficiency, or in the presence of warfarin, an abnormal precursor of prothrombin (preprothrombin) that contains little or no γ-carboxyglutamte, and unable of chelating calcium, is released into the blood – impairment of clotting. Structural analogs and significance The enzyme, hydrogenase (warfarin-sensitive reductase) reduces the inactive oxidized form of vitamin K to the reduced, active form (vitamin K recycling). Warfarin acts upon this enzyme preventing oxidized vitamin K to be reduced. The oxidized form accumulates and is then excreted. -We speak about coumarin as the anticoagulant, which inhibit the vitamin K cycle in the liver -The coumarin anticoagulants are: *Dikumarol (Pelentan) *Monokumarin (Warfarin) -The principle of the action of these drugs is that they are similar to vitamin K In comparison to heparin which has an immediate effect as an anticoagulant, coumarin has a slower effect. Biochemistry II Pouria Farsani 2013 143 35. Digestion in the mouth and in the stomach (constituents of the saliva, the gastric secretion, secretion of HCl, humoral control of hydrochloric acid output) Digestion in the mouth – constituents of the saliva Digestion of food begins in the mouth, both mechanically by mastication and chemically by the constituents of saliva: -Mucins – lubrication of food – easier swallowing – facilitation of mastication -Low NaCl content, hypotonic – suitable for rinsing the taste buds (NaCl) while eating -α-Amylase – digestion of starch (see also q. 37) -Immunoglobulin A and lysozyme – part of the immune defense system. Lysozyme is a murein cleaving enzyme which attacks bacterial cell walls -High HCO3 – pH around 7 which is optimal for α-amylase-catalyzed digestion -Lipases – hydrolysis of TAG => 1,2-diacylglycerol + FA -Secretion: about 1-1.5 l/day Other functions of saliva involve: -Saliva dissolves compounds in food which is a prerequisite for taste buds stimulation and for dental and oral hygiene -Infants need saliva to seal the lips when suckling -Swallowed saliva is also important for buffering the acidic juices refluxed into the esophagus -Secretion of profuse amounts before vomiting also prevents gastric acid from damaging the enamel -Saliva secretion is very dependent on the body water content. A low content results in decreased saliva secretion – the mouth and throat become dry, thereby evoking the sensation of thirst. This is an important mechanism for maintain the fluid balance Digestion in the stomach – gastric secretion About 3-4 liters of gastric juice is secreted each day by different cells: -Chief cells – pepsinogen and lipases -Parietal cells – HCl and intrinsic factor (a glycoprotein needed for the absorption of vit. B12) -Mucous neck cells + mucous cells on the surface of gastric mucosa – mucins and HCO3 -Pepsins function as endopeptidases in protein digestion. They are split from pepsinogens exocytosed from chief cells at a pH of <6. Acetylcholine released locally in response to H+ (and thus indirectly to also to gastrin) is the chief activator in this reaction Biochemistry II Pouria Farsani 2013 144 -Gastric acid – the pH of gastric acid drops to ~0.8 during peak HCl secretion. Swallowed food buffers it to a pH of 2-4, which is optimal for most pepsins and gastric lipases. The low pH contributes to the denaturation of dietary proteins and has a bactericidal effect Secretion of HCl H+ -K+ -ATPase in the luminal membrane of parietal cells drives H+ ions into the glandular lumen in exchange for K+ (primary active transport) H+ concentration in the lumen is increased by a factor of ~ 107 . Concurrently, K+ taken up in the process circulates back to the lumen via luminal K+ channels For every H+ ion secreted, one HCO3 (obtained from CO2 + OHin a reaction catalyzed by carbonic anhydrase) leaves on the blood side and is exchanged for a Clion via an anion antiporter Intracellular Cl- increases Cldiffuses out into the lumen via Cl- channels Ultimately, one Clion reaches the lumen for each H+ ion secreted Humoral control of hydrochloric acid output – stimulation The ultimate goal of activation is to increase H+ secretion by H+ -K+ -ATPase (activation of parietal cells). The secretion of HCl is stimulated in phases by the following factors: -Neural -Local gastric -Intestinal factors Biochemistry II Pouria Farsani 2013 145 -Vagal stimulation – food intake and lack of glucose in brain triggers the reflex (conditioned) for secretion. *afferent fibers: optic, gustatory and olfactory nerves *efferent fibers: vagus nerve -The direct effects of the vagal reflex: Acetylcholine as the neurotransmitter directly activates parietal cells in the fundus (M3 cholinoreceptors, DAG IP3/Ca2+ as second messengers) H+ -K+ -ATPase activation H+ secretion ↑ -The indirect effects of the vagal reflex: Vagus nerve releases gastrin-releasing peptide (GRP) GRP stimulates gastrin secretion from G-cells in the antrum -Stimulation by gastrin – released as a result of vagal stimulation Gastrin released into the systematic circulation activates the parietal cells via gastrinreceptors (CCKB) H+ secretion ↑ Furthermore, G-cells which release gastrin are also activated by food intake – this is a local gastric and intestinal factor -Histamine – the glands in the fundus contain H-cells which are activated by gastrin, acetylcholine – they release histamine, which has a paracrine effect on neighboring parietal cells Humoral control of hydrochloric acid output – inhibition The following factors inhibit gastric juice secretion:” pH < 3 in inhibits the G-cells (negative feedback) and activated D-cells. D-cells are also activated by calcitonin gene-related peptide CGRP which is released by neurons. D-cells secrete somatostatin (SIH) which in turn has a paracrine effect SIH inhibits both H-cells and G-cells H+ secretion ↓ Biochemistry II Pouria Farsani 2013 146 Biochemistry II Pouria Farsani 2013 147 36. The bile – formation, composition, functions of the constituents Bile acids are synthesized from cholesterol in hepatocytes. The primary bile acids are cholic- (prevailing) and chenodeoxycholic acid. These are secreted as conjugates with glycine and taurine. In the ileum they are deconjugated and dehydroxylated by bacteria at position C7 to secondary bile acids. The secondary bile acids are less soluble in water thus more difficult to reabsorb from the small intestine. The least soluble acid (only one OH at C3), which has excretion as its pathway is litocholic acid. More than 95% of bile acids are reabsorbed by cotransport with Na+ (ASBT, apical sodiumdependent bile acid transporter) in the ileum and transported back to the liver. Formation of bile acids can be summarized in the following scheme. Under physiological conditions, the bile acids are mainly taken up from sinusoids in periportal hepatocytes of zone 1. During increased levels of bile acids in blood (after meal, in obstruction), the perivenous hepatocytes of zone 3 are involved as well. Uptake of bile acids is ATP-independent. Biochemistry II Pouria Farsani 2013 148 Bile is produced when active ATP-dependent ABC transporters, transport substances which are taken up on sinusoidal (basolateral) side of hepatocyte to be secreted on the canalicular side, into bile ducts. Transport systems are summarized in the following table. Transport of, into bile ducts: -Bile acids – by ABCB11 -Phospholipids – by MRP2 transporter (ABCC3) -Cholesterol and neutral sterols – by ABCG5 and ABCG8 -Water influxes into the primary bile as an osmotic gradient is created by the active secretion of substances. Biochemistry II Pouria Farsani 2013 149 The primary bile which is produced by hepatocytes is further modified in the bile ducts. Water is reabsorbed by the epithelial cells of the bile ducts and hormones (secretin, vasoactive intestinal peptide VIP, somatostatin) stimulate the release of some other substances (HCO3 primarily). Bile is released into the duodenum within 30 min after food ingestion. Composition of bile A stable mixture of bile contains mainly: -80 % bile acids -15 % phosphatidylcholine -5 % cholesterol In total however, bile consists of 95% water. There are also low amounts of AA, glutathione, steroids, enzymes, porphyrins, vitamins, heavy metals and xenobiotics. Function of bile -Tenside actions – action of bile acids emulsify lipids and fat-soluble vitamins in the intestine. The high concentrations of bile acids and phospholipids stabilize the micellar dispersion of cholesterol in the bile (crystallization of cholesterol => cholesterol gall-stones) -Excretion – of cholesterol and bile acids is the major way of removing cholesterol from the body. The bile also removes lipophilic metabolites, drugs, toxins and some metals such as zink, mercury and copper. Lipophilic substances are difficult to excrete by the kidneys, both exogenous (xenobiotics) and endogenous (bilirubin) -Neutralization – of the acidic chyme in conjugation with HCO3 from pancreatic secretion Biochemistry II Pouria Farsani 2013 150 37. Digestion and absorption of saccharides (amylases and intestinal brush-border enzymes) Amylase Two forms exist, salivary and pancreatic. They hydrolyze starch at α (1→4) glycoside bonds forming oligosaccharides which are then further digested to mono and disaccharides Digestion Carbohydrates provide half to two-thirds of the energy requirement. At least 50% of dietary carbohydrates consist of starch (amylose and amylopectin) which is a polysaccharide. Other important dietary carbohydrates are cane sugar and lactose. -In the mouth Start of carbohydrate digestion Ptyalin (an α-amylase) in saliva breaks starches down to oligosaccharides (maltose, maltotriose,α-limit dextrins) in a neutral pH environment -In the proximal stomach – the above mentioned process continues -In the distal stomach Interruption of the above mentioned process as the food is mixed with acidic gastric juices A pancreatic α-amylase, with a pH optimum of 8 is mixed into the chyme in the duodenum, thus: Continuation of polysaccharide digestion can proceed to the final oligosaccharide stage mentioned above Absorption and intestinal brush border enzymes The carbohydrates can only be absorbed in the form on monosaccharaides. Therefore, enzymes integrated the luminal brush border membrane of enterocytes breaks down maltose, maltotriose and α-limit dextrins into glucose as the final product which is absorbed. These acting enzymes are: -Maltase -Isomaltase The hydrolysis of saccharose, lactose and trehalose is catalyzed by other burs border enzymes. This releases, in addition to glucose, galactose (from lactose) and fructose. Acting enzymes are: -Lactase -Saccharase (sucrase) -Trehalase Biochemistry II Pouria Farsani 2013 151 -Absorption *glucose and galactose are first actively taken up by the Na+ symport carrier SGLT1 into the mucosal cells before passively diffusing into the portal circulation via GLUT2, the glucose uniport carrier (facilitated diffusion) *fructose crosses the enterocytes by passive uniporters, GLUT5 in the luminal- and GLUT2 in the basolateral membrane Lactase deficiency (intolerance) – lactose cannot be broken down and absorbed unless sufficient lactase is available. Lactase deficiencies lead to diarrhea, firstly because water is retained in the intestinal lumen due to osmotic mechanisms, and secondly because intestinal bacteria convert the lactose into toxic substances Biochemistry II Pouria Farsani 2013 152 38. Digestion and absorption of lipids from the GIT (incl. chylomicrons, the fate of them) The western diet contains approx. 40% lipids or more. About 90% of that is TAG. The rest is phospholipids (PL), cholesterolesters (CE), glycolipids (GL), and lipophilic vitamins (LV). The action of lipases, esterases and bile acids in the GIT provide primary products: -Free FA -2-monoacylglycerols -Lysophospholipids -Cholestrol -Lipophilic vitamins These primary products are absorbed into enterocytes in form of micelles (particles < 20 nm). In further detail we explain stepwise action of enzymes as they cleave lipids: Mouth and stomach -Lingual and gastric lipase hydrolyze a small amount of the TAG. Further mechanical action of the stomach converts dietary lipids into an emulsion containing droplets about 1 µm in diameter Small intestine -Pancreatic lipase: TAG => 2-monoacylglycerol + 2 FA (< ¼ of TAG => glycerol + FA) -Phospholipase A2 PL => lysophospholipid + FA -Cholestrol esterase CE => cholesterol + FA -Emulsification of lipids – this is a condition for effective digestion of the hydrophobic lipids. Emulsification increases the effective surface between oil and water thus facilitating the contact with enzymes. The emulsification is accomplished by action of detergents and mechanical mixing (peristalsis). Emulsification in the small intestine produces: -Salts of bile acid -Phospholipids -Salts of FA Biochemistry II Pouria Farsani 2013 153 -Colipase – a protein secreted from the pancreases which binds the lipase at a 1:1 ratio causing it to be more active. It anchors lipase to bile acids on the surface of emulsified lipid droplets -Absorption of lipids by enterocytes *FA and monoacylglycerols are absorbed by passive diffusion *short chain FA (up to 10C) do not enter micelles, they are absorbed directly *bile acids which remain in the intestine are extensively absorbed in the ileum *transport of cholesterol is mediated by NPC1L1 (Nieman-Pick C1 like 1) -Hormones affecting digestion of lipids *Secretin – the intestinal “S-cells” produce secretin upon stimulation of H+ entering the lumen. Secretin is then released into the blood; it stimulates the release of secretes containing HCO3 from the gallbladder and pancreas *Cholecystokinin (CCK) – the intestinal “I-cells” produce CCK upon stimulation of small peptides and lipids. CCK is then released into blood; it stimulates secretion of amylase, lipase and proteases from exocrine cells of pancreas CCK also potentiates effect of secretin on excretion of HCO3 and stimulates the secretion of bile from the gallbladder. -Steatorrhoea (lipid malabsorption) – loss of lipids by feces (normally ~98% of lipids in food are absorbed). The possible causes could be: *insufficient supply of bile due to damage of liver of obstruction of bile duct *disturbed function of pancreas *disturbed function of intestinal mucosa This condition results in the insufficient uptake of lipophilic vitamins. Biochemistry II Pouria Farsani 2013 154 -Resynthesis of lipids within the mucosal cells 1. FA -FA + CoA + ATP => Acyl-CoA =AMP + PPi 2. TAG -Acyl-CoA + Monoacylglycerol => Diacylglycerol + CoA -Acyl-CoA + Diacylglycerol => Triacylglycerol + CoA 3. Resynthesis of phospholipids from lysophospholipids 4. Resynthesis of cholesterolesters These processes are located in the ER! FA with short chain and free glycerol do not take part in these processes as they are transported directly into the portal vein. Chylomicrons, the fate of them (see also q. 5) Chylomicrons metabolism and assembly: • They are formed in intestinal mucosal cells • The carry TAG, CH, LV which are ingested in food • Their main apoprotein is apo-B-48. The minor apoprotein component is apo-A (the other cannot be synthesized in intestinal cells). The synthesis of apo-B-48 limits formation of CM • They are released by exocytosis into the lacteals (lymphatic vessles originating in the villi of the small intestine) – chyle lymph • They follow lymphatic vessels and enter the blood in the thoracic duct -Metabolism of CM in blood • They enter the blood 1-2 hours after the meal as nascent CM Biochemistry II Pouria Farsani 2013 155 • Modification of CM: in blood, apo-E and apo-C-II are transferred from circulating HDL to chylomicrons • In capillaries of most peripheral tissues, CM are degraded by LPL -Lipoprotein lipase (LPL) • Negatively charged enzyme on the surface of endothelial cells in capillaries (anchored by heparansulfate to the capillary walls) • Predominantly in adipose tissue, and skeletal- and cardiac muscle • It is activated by apo-C-II • LPL can be released by heparin • Synthesis of isoenzyme in adipose tissue is stimulated by insulin • Deficit of LPL results in triacylglycerolemia • LPL catalyzes the hydrolysis of TG contained in circulating lipoproteins providing glycerol + 3 FA • More than 90% of TAG in CM is degraded by LPL. CM then decreases in size and its density increases (CM-remnant) • CM-remnant is rapidly removed from the circulation by the liver (apo-E-receptor) • Chylomicrons in blood are removed during approx. 30 min. (cholesterol taken in food is transported to the liver by the removal of remnants from blood) • FA which were released by the action of LPL enter the beta-oxidation in muscle and myocardium. They are also deposited as TAG in adipose tissue The text regarding CM written above together with the following schemes below are merely a partial repetition of q. 5. However, repetition is the mother of wisdom… ;-) Biochemistry II Pouria Farsani 2013 156 Biochemistry II Pouria Farsani 2013 157 39. Proteolytic enzymes of the digestive tract (secretion, activation, specificity), absorption of amino acids and peptides In order for the proteolytic enzymes to be able to catalyze the hydrolysis of peptide bonds in dietary proteins, denaturation is needed (by heat in cooking and by the action of gastric acid). This is because the bonds are not accessible for the protelytic enzymes prior to the denaturation. Proteolytic enzymes – secretion, activation, specificity Proteases are the proteolytic digestive enzymes. The specificity differs for the amino acids forming the peptide bond to be hydrolyzed. We distinguish the two main classes with different specificity: • Endopeptidases – hydrolyze peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act yielding smaller fragments. These enzymes follow below: -Pepsin in the gastric juice (HCL + pepsinogen => pepsin). It is inactivated in the small intestine (pH 7-8) -Trypsin -Chymotrypsin -Elastase The latter three are secreted by the pancreas, see below for further explanation. • Exopepsidases – catalyze they hydrolysis of peptide bonds, one at a time, from the ends of peptides. These enzymes follow below: -Carboxypeptidases – secreted in the pancreatic juice, release amino acids from the free carboxyl terminal -Aminopeptidases – secreted by the intestinal mucosal cells, release amino acids from the amino terminal -Dipeptidases – in the brush border of intestinal mucosal cells, catalyze the hydrolysis of dipeptides, which are not substrates for amino- and carboxypeptidases -Proteases from pancreas – these are firstly secreted in their inactive form – as proenzymes: *trypsinogen *chymotrypsinogen *proelastase *procarboxypeptidase They are activated only when they reach the intestine, where an enteropeptidase, secreted by duodenal epithelial cells, first converts trypsinogen to trypsin. Trypsin then converts chymotrypsinogen to active chymotrypsin. Trypsin also activates many other pancreatic Biochemistry II Pouria Farsani 2013 158 proenzymes including proelastases and procarboxypeptidases. Besides these pancreatic enzymes, trypsin also activates intestinal proaminopeptidase to aminopeptidase. Pathological activation of the proenzymes within the pancreas causes the organ to digest itself => acute pancreatic necrosis – very life-threatening! -Activation and secretion – the proteases are secreted as inactive zymogens. The active site of the enzyme is masked by a small region of the peptide chain that is removed by hydrolysis of a specific peptide bond. Pepsinogen is activated to pepsin by gastric acid and also by pepsin itself (autocatalysis). Activation of pancreatic enzymes, see above. Absorption of amino acids and peptides The end product of the action of endopeptidases and exopeptidases is a mixture of free amino acids, di- and tripeptides, and oligopeptides, all of which are absorbed. -Amino acids – There are several different amino acid transporters. These transporters have specificity for the nature of the amino acid side chain (large, or small, neutral, acidic or basic) *neutral (use several different transporters) and acidic are absorbed across the intestinal mucosa by Na+ symporters (secondary active transport). From the mucosal cells they then passively diffuse with carriers into the blood Amino acids like glutamate and aspartate which for the most part are broken down in the mucosal cells, also have their own (Na+ and K+ dependent) carrier systems. *basic amino acids (arginine, lysine, ornithine) are partly taken up into enterocytes by Na+ independent mechanisms, as the membrane potential is a driving force for their uptake AA absorption disorders can be congenital and affect various amino acid groups. These disorders are often associated with defects of renal tubular reabsorption (renal aminoaciduria, e.g. cystinuria) -Dipeptides and tripeptides – enter the brush border of the intestinal mucosal cells, where they are hydrolyzed to free amino acids, which are then transported into the hepatic portal vein. Relatively large peptides may be absorbed intact, either by transcellular transport (symport carrier, driven by an H+ gradient) or paracellular transport. Many of such peptides are large enough to stimulate antibody formation – this is the basis of allergic reactions to foods. Biochemistry II Pouria Farsani 2013 159 Biochemistry II Pouria Farsani 2013 160 40. Biochemistry of small and large intestine, endocrine function Small intestine The main function of the small intestine is to finish digesting the food and to absorb the accumulated breakdown products as well as water, electrolytes and vitamins. -Goblet cells – interspersed between the resorbing enterocytes. They secrete mucus which acts as a protective coat and lubricant. -Intestinal glands (crypts of Lieberkün) – located at the base of the villi. They contain: *undifferentiated- and mitotic cells that differentiate into villous cells *mucous cells *endocrine- and paracrine cells receive information about the composition of chyme from chemosensor cells. The chyme composition triggers the secretion of endocrine hormones and of paracrine mediators (see endocrine function below) *immune cells *duodenal glands (Brunner’s glands) secrete a HCO3 -rich fluid containing urogastrone (human epidermal growth factor), an important stimulator of epithelial cell proliferation The tips of the villi are continually shed and replaced by new cells from the crypts of Lieberkün. Thereby, the entire small intestine is renewed every 3-6 days. The dead cells disintegrate in the lumen, thereby releasing enzymes, stored iron etc. Bacteria also deconjugates and dehydroxylate primary bile acids to secondary bile acids See also q. 36-39 for more detailed information of biochemical functions. Large intestine The large intestinal mucosa has characteristic pits (crypts), most of which are lined with mucus-forming cells (goblet cells). Some of the surface cells are equipped with a bursh border membrane and reabsorb ions and water. The large intestine has two main functions: 1. Serves as a reservoir for intestinal content (caecum, ascending colon, rectum) 2. Absorbs water and electrolytes – the 0.5-1.5 l of chyme that reaches the large intestine is reduced to 0.1-0.2 l. The chyme picks up cellular debris together with other waste products. Once all nutrients have been absorbed, the remaining waste products pass to the rectum where they are stored as fecal matter before defecation. Biochemistry II Pouria Farsani 2013 161 Furthermore, there is a bacterial action taking place in the large intestine: The large intestine of a healthy adult contains 1011 to 1012 bacteria per ml of intestinal content. Their metabolic activity plays an important role for the host: -Vitamin K (Menaquinones) and biotin synthesis -Conversion of indigestible substances (e.g. cellulose and partially digested saccharides (e.g. lactose) into absorbable short chain fatty acids and gases (methane, H2, and CO2) -Conversion of conjugated bilirubin to urobilinogen (most of which is reabsorbed again and transported back to the liver see. q. 17) -Modification of cholesterolexcreted in stool to coprostanol and cholestanol -Production of ammonia Biochemistry II Pouria Farsani 2013 162 Endocrine function The two pictures will explain the main ideas: -CCK = cholecystokinin -GIP = glucose-dependent insulinotropic peptide Biochemistry II Pouria Farsani 2013 163 41. The specific functions of the liver in metabolism of nutrients and proteosynthesis. Metabolic zonation of liver Specific functions The major metabolic functions of the liver include the uptake of most nutrients from GIT and their intensive intermediary metabolism as well as conversion. The liver is an organ of storage as well for glycogen, iron and vitamins. It controls the supply of essential compounds such as glucose, VLDL, KB, plasma proteins etc. Ureosynthesis and biotransformation of xenobiotics belongs to its detoxification role. Furthermore the liver is an organ of excretion of cholesterol, bilirubin, hydrophobic compounds and some metals. -Saccharides: the liver produces more glucose than it consumes. Furthermore the liver: • The primary regulator of glucose level in blood • Synthesizes glycogen in the postprandial state • In fasting/starvation it supplies the body with glucose via the glycogenolysis and gluconeogenesis • Glucose => galactose => plasma glycoproteins • Glucose =>glucuronic acid => conjugation reactions • Glucose => pentose cycle => NADPH => FA/TAG/VLDL; cholesterol • Fructose => acyl-CoA => FA => TAG => VLDL -Lipids: The liver utilizes FA for energy supply, not from glycolysis. This is because glucose and glycogen are spared for the extrahepatic tissues. • Short chain FA (C4 – C12) are taken up directly from the portal blood whilst other FA enter the liver via CM • Completion and secretion of VLDL and HDL • Production of KB (cannot be utilized in the liver) • Secretion of cholesterol and bile acids into bile is the major way of cholesterol elimination from the body -Nitrogen compounds (AA, purines, bilirubin) • Deamination of AA that are in the excess of requirements • Intensive proteosynthesis of major plasma proteins and blood-clotting factors • Uptake of ammonium and ureosynthesis Biochemistry II Pouria Farsani 2013 164 • Formation of uric acid by the catabolism of purine bases • Capturing, conjugating and excreting bilirubin -Biotransformation of xenobiotics • Detoxification of some drugs and toxins • Excretion of some metals -Vitamins • Hydroxylation of calciols to calcidiols • Splitting beta-carotene to retinol • Storage of lipophilic vitamins and cobalamin (B12) -Iron and copper metabolism • Synthesis of transferrin; • Ceruloplasmin • Ferritin stores • Excretion of copper (bile) -Production and inactivation of hormones *production of: • IGF-1, angiotensinogen (precursor), thrombopoetin, hepcidin • Transport proteins for thyroid gland and steroid hormones • Dehydrogenation of cholesterol to 7-dehydrocholesterol, 25-hydroxylation of calciol *inactivation of: • Steroid hormones – hydrogenation, hydroxylation, conjugation • Inactivation of insulin and glucagon (insulinase) • Inactivation of iodothyronines – deiodation, deamidation, conjugation Metabolic zonation of the liver Enzymatic equipment, number of cells and arrangement of cellular organelles of hepatocytes differ in different areas of the liver, this determines their metabolic capacity. The acinus is the functional unit of the liver. Its axis is formed by the terminal hepatic arterioles, terminal portal venule, bile duct and lymphatic blood vessels. It is located between two or more central veins, which are located at the periphery of the acinus. Biochemistry II Pouria Farsani 2013 165 We divide the acinus into three zones which correspond to the distance from the arterial blood supply: -1st zone, periportal – closest to the blood supplying vessels, best O2 supply, the mitochondria are larger and more numerous here as well. Predominance of oxidative processes occurs here: *proteosynthesis *ureosynthesis *beta-oxidation *catabolism of AA *ammonia detoxification by urea formation *gluconeogenesis *bile production *cholesterol synthesis The activity of the following enzymes is higher: -succinate DH -malate DH -cytochrome oxidase -Glutathione (antioxidant) -Glutathione reductase The latter two mentioned gives a higher resistance against active forms of oxygen radicals, compared to the 3rd zone, which is less resistant. In fasting => increase of gluconeogenic zone + activity of gluconeogenic enzymes. Decrease in activity of glycolytic enzymes -2nd zone – the middle, transient zone -3rd zone, perivenous – at the acinus periphery. Lowest O2 supply. High ER activity, (cyt P450, detoxification). Activity of hydrolytic enzymes. Predominance of biotransformation processes: *glutamie synthesis *pentose phosphate pathway *uptake of glucose *glycogen synthesis (storage) *glycolysis *lipogenesis (storage) *ketogenesis *conversion of ammonia that has not been detoxified in the periportal area to glutamine Biochemistry II Pouria Farsani 2013 166 Biochemistry II Pouria Farsani 2013 167 42. Function of liver in excretion This question is a combination of the main ideas from q. 17, 36, 41, 86 and 87. The first three are the most relevant. The liver detoxifies and excretes many mostly lipophilic substances, which are either generated during metabolism (e.g. bilirubin or steroid hormones) or come from the intestinal tract (e.g. antibiotics). This requires a process of biotransformation of the substances (see q. 86 and 87). The lipophilic substances are then transformed into water-soluble substances which can be excreted into urine or into feces (bile excretion). Substances excreted into urine can be further processed in the kidneys. The main pathways functions in liver excretion are summarized below: -Bile formation see q. 36 – excretion of cholesterol, steroid hormones, certain vitamins and drugs -Bilirubin excretion (see also q. 17) – change of the decomposed hemoglobin which comes from breakdown of RBC. 200-250 mg of bilirubin is excreted in the bile each day. ~90% of this is excreted in the feces -Elimination of proteins and amino acids by urea formation. Ammonia is formed in the deamination of amino acids. Due to its toxicity, it is transported in the form of urea -Detoxification and excretion of lipophilic substances Biochemistry II Pouria Farsani 2013 168 43. Biochemical tests used for identification of liver injuries (detection of cell damage, cholestasis, reduced proteosynthetic capacity etc.) No “hepatic tests” are specific for a certain type of hepatic damage. Results of common laboratory examinations can also be altered by other non-liver diseases. However, a combination of selected laboratory examinations together with anamnesis and other examining methods will give a much better insight in ongoing pathology of the liver. We divide the basic biochemical tests assigned for the examination of the function of the liver and bile tract into several groups: -Hepatocyte damage (membrane) – AST, ALT and GLD -Cholestase parameters – ALP, GGT -Tests determining the liver capacity for the transport of the organic anions and for the elimination of the endogenic and exogenic substances out of the circulation – bilirubin and bile acids -Tests measuring the liver capacity for the metabolism of xenobiotics – aminopyrine and bromosulphophtalein -Tests determining the synthetic activity of the liver – albumin, cholinesterase (CHE), transthyretin (prealbumin, half-life 2 days) and coagulating factors -Non-specific tests, which help to specify the diagnosis – serologic tests for hepatitis, the level of immunoglobulins and specific antibodies. -Tests of major metabolic functions (not very decisive): *saccharide metabolism: low glucose tolerance (in oGT test) *lipid metabolism: increase in VLDL (TAG) and LDL (cholesterol) *protein catabolism: decreased urea, ammonium increase (in the final stage of liver failure, hepatic coma) • Determination of ALT catalytic concentration in serum (0.15-0.9 ukat/l) – cytoplasm Increase indicates defective integrity of hepatocytes caused by various agents (acute chronic hepatitis, toxic liver damage etc.) • Determination of AST catalytic concentration in serum (0.05-0.72 ukat/l) – cAST 30%, mAST 70% Increase indicated the disorganization of the cytoplasmic membranes in tissues with high AST activity, especially in myocardium and hepatic parenchyma. Biochemistry II Pouria Farsani 2013 169 • Significance of the ALT and AST determination in diagnosis of hepatobiliary damage As mentioned before, the single test of aminotransferases are not specific for hepatic diseases. In case the values are physiological, the presence of hepatobiliary disease can be eliminated. >20x increase of aminotransferases => acute viral hepatitis, acute toxic hepatitis and states accompanying hepatic hypo-perfusion respectively hypoxia or congestion. -Inflammation: AST/ALT ratio <1 -Hepatocyte necrosis => increase in mAST: ratio >1 (also found in myocardial infarction -Specific test for alcoholic hepatic diseases: ratio >2 • Determination of ALP catalytic concentration in serum (0.66-2.2 ukat/l. Children up to 8 ukat/l) – cell membranes of the biliary tract endothelium -Cholestasis => mechanical damage of membranes and by the surfactant effect of bile acids -High levels of ALP with the hepatic isoenzyme predominance is characteristic for cholestasis and obstructive icterus -Increase could in rare cases be accompanied with infectious mononucleosis -Slight increase: indication of number of hepatic diseases without more distinct cholestatic factor -Increased intestinal isoenzyme of ALP is found in some patient with cirrhosis due to the reduced trapping in the liver -Levels of ALP should always be judged in context with other obstructive indicators, esp. with GGT. • Lactate dehydrogenase (LD) and its LD5 (M4) isoenzyme (3.5-7.7 ukat/l) Normal distribution of LD5 is 6-16% of the catalytic concentration. High increase of serum LD is accompanied in early stages of acute viral hepatitis and the states connected with the hepatic hypo-perfusion or hypoxia. Besides hepatic disease, increased level of LD5 is associated with skeletal muscle’s disease, some tumors and infectious mononucleosis. Biochemistry II Pouria Farsani 2013 170 • Gamma-glutamyl transferase, GGT. (<0.84 ukat/l) – microsomal fraction of hepatocytes and in the cell membranes of the biliary tract endothelium GGT is present in all tissues. It transfers the gamma-glutamyl residue on the amino groups of some L-amino acids. The reaction represents the first step in the transformation of the conjugates of some xenobiotics with glutathione into Ssubstituted N-acetylcysteine. The majority of GGT is found in liver, kidneys, pancreas and intestine. GGT is sensitive but it is not very specific as a marker of hepatic disease. >x10 increased level is accompanied by cholestasis and primary metastatic tumors of the liver. Isolated increase in GGT occurs in alcoholics without signs of hepatic damage. Acute pancreatitis can also result in increase of GGT • Glutamate dehydrogenase, GLD Mitochondrial enzyme present in all tissues. Measurable increase in serum occur in hepatocellular necrosis (sever toxic damage, hepatodystrophy). Irritation of the biliary tract endothelium by cholestasis can result in the increase of GLD as well. • 5-Nucleotidase, NTS Catalyzes the hydrolysis of nucleotides by release of inorganic phosphate from the 5th position of the pentose ring. It is common in the intestine, brain, heart, blood vessels, pancreas and liver. Elevation is found in hepatic damage – it is a highly specific indicator of hepatic damage. Its levels correlated with the activity of ALP and GGT. In order to diagnose damage in childhood where the activity of ALP was physiologically increased, NTS is determination is significant. • Determination of total bilirubin in serum (5-20 umol/l) (conjugated <5 umol/l) Unconjugated is calculated as the difference between the total and conjugated bilirubin Unconjugated levels between 17-70 umol/l together with normal results of the other examinations are characteristic for chronic hemolysis (hemolytic anemia) and for benign hyperbilirubinaemia of the Gilbert type. Postnatal hyperbilirbinaemia: up to 135 umol/l of unconjugated bilirubin and up to 170 umol/l of all forms of bilirubin in the serum. Biochemistry II Pouria Farsani 2013 171 Bilirubin is used as an important test for chronic hepatic diseases, especially for those combined with cholestasis. Damage of hepatocytes => disruption of capturing, conjugating and secreting of bilirubin into bile. Urobilinogen is found in urine in this case due to the hepatocytes being unable to capture entire urobilinogen from the portal blood. Obstruction of bile duct => 170-500 umol/l bilirubin. Complete blockage => bilirubin cannot get into intestine => no formation of urobilinogen => acholic stool and negative urobilnogen in urine. There are different types of hyperbilirubinaemia, see q. 17. • Test for bilirubin in urine (<0.5 umol/l) Fresh urine must be used. Positive result if conjugated bilirubin in serum exceeds 30 umol/l. Unconjugated bilirubin in plasma is bound to albumin and does therefore not pass through the glomeruli. Positive result of bilirubin in urine indicates either a defect in the ability of hepatic cells to excrete it into bile, or a sign of mechanical blockage of the bile ducts. We speak about hepatocellular or obstructive hyperbilirubinaemia. Persisting disorder => formation of delta-bilirubin bilirubin => cannot be detected in urine. • Test for urobilinogens in urine (4 umol /day) Bile => intestine, reduction by enzymes of intestinal bacteria => colorless products (chromogens) called urobilinogens => mostly reabsorbed in the intestine => elimination in the liver => small amount excreted in feces and urine Usage of fresh and cooled urine within 2 hours after voiding. Positive result on test strip indicates values > 17umol/l Increase is a very early symptom of functional overload or insufficient liver functions – inability to take up and eliminate the required quantity which is reabsorbed. Positive test may indicate: -Overloading of liver’s capacity by: *enormous physical activity *single heavy alcohol consumption *increased hemolysis Biochemistry II Pouria Farsani 2013 172 -Hepatocellular damage caused by virus hepatitis, toxic substances or tumors Absence is due to complete biliary obstruction (clay-colored stool) or absence of intestinal bacteria (intensely colored stool by bilirubin) or severe diarrhea. • Determination of albumin in serum (35-53 g/l) Albumin main functions is: -Maintain oncotic pressure -Buffering capacity by binding Ca2+ -Transport of FA, bilirubin, steroid and thyroid hormones and many drugs Ca. 14g/day is released into plasma from hepatocytes. Its half-life is 19 days. Increase is rare and usually due to dehydration. Hypoalbuminaemia is due to decreased proteosynthesis in the hepatocytes (pathology or insufficient intake in food) or due to excessive losses of albumin (in urine, excessive burns or chronic inflammation of the digestive system) which cannot be compensated by increased protesynthesis of the liver. It leads to a decreased oncotic pressure which leads to edema. Escape of albumin into ascites is another factor affecting the albumin level. Chronic hepatic disease => slow decrease of albumin accompanied by increase of gamma-globulins. Advanced cholestasis => decrease in albumin, increase in beta-globulins Alcohol cirrhosis is associated with defect in nutrition and the unfavorable effect of alcohol on proteosynthesis => decreased albumin level. • Cholinesterase, CHE (65-200 ukat/l) Present in many organs, made by several isoenzymes. Slight increase is seen during acute viral or toxic hepatitis. A decrease is seen during: -Chronic hepatitis -Cirrhosis -Hepatic tumors -Infiltration processes The levels if CHE is monitored during the above mentioned conditions. A sudden or considerable decrease would be a negative sign. Biochemistry II Pouria Farsani 2013 173 • Coagulating factors They have a short half-life, thus their changes are rapid and important in the diagnosis of hepatic diseases. The Quick’s test (prothrombin time) is most commonly used. It represents the activity of external coagulating systems. Repeated examinations are used in the monitoring of the course of chronic hepatic diseases. It is an indicator of the severity of damage and prognosis. Results are expressed in INR (international normalized ratio) with normal values around 1. Low levels can also be a result of vitamin K deficiency. Biochemistry II Pouria Farsani 2013 174 44. Distribution of body water, factors influencing the distribution of body water and its excretion (ADH, aldosterone, natriuretic peptides), consequences of retention or of dehydration The distribution of body water is divided into compartments as seen in the schemes above. Biochemistry II Pouria Farsani 2013 175 -Intracellular fluid (ICF) – 30-35% of TBWgt is in soft tissues, especially muscles. The rest, 8-10% of TBWgt, is in the connective tissues, cartilage and bone -Extracellular fluid (ECF) – this volume can change very quickly in critically ill patients. It is divided into intravascular and interstitial fluid: *interstitial fluid (ISF) – 10-15% of TBWgt. It provides the exchange of substances between cells and external environment. It is a similar ionic composition to that of plasma with the exception of low protein concentration and greater concentration of Cl*intravascular fluid (IVF) – plasma contains water representing 3.5-5% of TBWgt. It is separated from the interstitial fluid by the vascular wall -Transcellular fluid – present in specific body cavities where it is secreted by specific cells. It cannot be counted as either of the components of ECF. Normally it does not exceed 500 ml but it rises to 2-3 liters after a meal. It includes: *cerebrospinal fluid (CSF) *synovial fluids *ocular fluids *fluid secreted in the digestive tract *pericardial fluid *peritoneal fluid *renal tubular fluid -Third space – pathological space. It cannot be classified as either ECF or ICF. Third space develops in situations such as: *accumulation of fluid in ileus (bowel obstruction) when 8-10 liters of fluid can accumulate in the lumen of the intestine *peritonitis – 5-8 liters of fluid that may be accumulated in the peritoneum *traumatic edema *ascites Factors influencing the distribution of body water and its excretion It is important to know, that isovolemia, isotonia and isoionia (volume, osmolality and ionic composition) is highly dependent on the intake and excretion of body fluids – the water balance Biochemistry II Pouria Farsani 2013 176 There are some factors which may affect the water balance – factors that may affect the intake or the output. It can be seen in the table below: It is also important not to only know the changes of distribution, but to also know the composition of the fluids received or lost that ultimately changed the water distribution. In vomiting for instances there is a loss of H+ . However, changes in intake and loss of water are not the only influencing factors, but also the transfer between the body compartments, such as in transfer from plasma into the interstitium in hyponatremia. Another factor which affects the TBW is age (the differences are due to different body fat content – a factor dependent on age and sex): Biochemistry II Pouria Farsani 2013 177 Furthermore we speak about regulating hormones: ADH – antidiuretic hormone, Vasopressin The main determinant of ECF volume is the amount of sodium in the body. Around 8-15g of NaCl is absorbed each day. The kidneys have to excrete the same amount over time to maintain the sodium and ECF homeostasis. Since sodium is the major extracellular ion, changes in total body sodium content lead to changes in ECF volume. Many factors are involved in this regulation, ADH being one of them. The other factors are: *rate of glomerular filtration (GFR) *renin-angiotensin system *intrarenal hydrostatic pressure *sympathetic nerves in the kidneys ADH is a nonapeptide which induces water retention in the renal collecting duct and induces vasoconstriction by stimulating the secretion of endothelin-1. ADH secretion is stimulated by increased plasma and cerebrospinal fluid osmolality and the Gauer-Henry reflex which occurs when a decrease (>10%) in ECF volume is communicated to the hypothalamus. ADH secreting stimulating factors detection: -Osmoreceptors in the hypothalamus (also contribute to trigger thirst) -Changes of volume which is recorded by low-volume baroreceptors in large veins and right atrium. High-volume receptors are found in sinus caroticus and aorta -Reduced renal blood flow => release of renin => increased production of angiotensin II => brain => secretion of ADH Biochemistry II Pouria Farsani 2013 178 ADH => kidneys => V2 receptors in the distal tubule and connecting segment => activation of aquaporins => increased water reabsorption and urine osmolality. Osmolality decrease with 1% => four-fold increase in the ADH output. Other signals which stimulates ADH release is decrease of volemia (<10-20%) and decrease of blood pressure > 5% ADH secretion increases continuously with increasing plasma osmolality with the maximum secretion occurring at 296-298 mmol/kg – no more increase after that. Thirst arises with plasma osmolality of about 290 mmol/kg. Aldosterone Aldosterone is the main mineralocorticoid hormone synthesized in zona glomerulosa of the adrenal cortex. As with other steroid hormones, aldosterone is not stored, but synthesized when needed. Its principal function is to regulate Na+ and K+ transport in the kidney, gut and other organs. The secretion is stimulated by a drop in blood volume and blood pressure. Furthermore, the secretion is mediated by angiotensin II and by hyperkalemia. Atriopeptin inhibits its synthesis. Stimuli for its secretion also involve: -Increase in K+ or decrease in Na+ -RAAS activation Biochemistry II Pouria Farsani 2013 179 It functions by: -Increasing the retention and absorption of Na+ => increase of water retention => increase in extracellular volume -Increased K+ excretion -Action of aldosterone on gene expression level – it increases the number of Na+ -K+ ATPase molecules and synthesis of Na+ -K+ -channels in the target cells In detail: The increase in Na+ reabsorption is achieved by increased production of transport proteins, called aldosterone-induced proteins (AIPs). This is a genome-mediated effect that begins approx. 30 min to 1 hour after aldosterone administration or secretion. The maximum effects are observed after several hours. Aldosterone increases Na+ reabsorption, thereby depolarizing the liminal cell membrane. Consequently, it increases the driving force for K+ secretion and increases K+ conductance by increasing the pH of the cell. Both effects lead to increased K+ secretion. Aldosterone has a very rapid non-genomic effect on the cell. Biochemistry II Pouria Farsani 2013 180 Natriuretic peptides The natriuretic peptides increase the sodium excretion. -Atrial natriuretic peptide (ANP) – synthesized and stored in atrial myocytes. It is released during pressure and volume overload of the heart -Brain natriuretic peptide (BNP) – similar to ANP. Synthesized and stored in ventricular myocytes The two above mentioned natriuretic peptides acts as antagonists of RAAS and have the following effects: *diuretic and natriuretic => decrease in volemia *increase of Na+ excretion *increase in glomerular blood flow and filtration rate *inhibiting renin & aldosterone & ADH release – inhibition of Na+ reabsorption. *peripheral vasodilation => decreased blood pressure -Urodilatin – similar to ANP. Produced by the cells of the distal renal tubule and secreted into the urine Biochemistry II Pouria Farsani 2013 181 -Prostaglandins E2 and I2 – increase the excretion of Na+ as well The mechanisms of the hormones mentioned above act by protecting against hypervolemia and vasoconstriction during a high dietary sodium intake. Consequences of retention or of dehydration Biochemistry II Pouria Farsani 2013 182 Biochemistry II Pouria Farsani 2013 183 Biochemistry II Pouria Farsani 2013 184 45. Osmotic and oncotic pressure of blood plasma, plasma osmolality (values of the main parameters, empirical relations for a rough estimate of plasma osmolality) and osmolality regulation Osmotic and oncotic pressure The osmotic (oncotic) pressure (~ 3 kPa) is created as plasma proteins cannot leak out of the vasculature and bind a certain amount of water. The pressure is consistent over the entire length of the same capillary, it acts against the pressure of blood which is greater at the beginning of the capillary bed (~ 4.5 kPa) than the oncotic pressure and vice versa at the end of the capillaries (~ 2 kPa). This effect ensures that a proportion of fluid is filtered from the blood to the interstitium at the beginning of the capillary bed of the tissue – the opposite effect is carried out at the end sections of the capillaries where most interstitial fluid is “sucked” back into the bloodstream. The portion which is not sucked into the blood stream proceeds to the lymph instead. Biochemistry II Pouria Farsani 2013 185 The osmotic pressure is given in the calculation above. Summary of oncotic pressure is given below. Biochemistry II Pouria Farsani 2013 186 Plasma osmolality (isotonia) Osmolality (mmol/kg H2O) is the activity of omsotically active particles – osmolarity on the other hand expresses the concentration of these particles. -Osmolarity Furthermore the osmolarity (mmol/l) refers to ALL the particles in a solution. Effective osmolarity is approx. equal to the tonicity (mmol/l): *concentration of particles impermeable through semipermeable membrane ONLY (ions, glucose, mannitol) *responsible for water movement between compartments *effective osmolarity < theoretical osmolarity -Osmolality Osmolality is measured by cryoscopy and it is estimated by the following formulas below: -Values of the main parameters • Blood plasma osmolality: ~ 300 mmol/kg H2O • Critical value: > 350 mmol/kg H2O -Osmolal gap – is used as a screening tool to identify unmeasured solutes such as xenobiotics, glycerol, acetone, AA, etc. *normal value: < 10 mmol/kg H2O *expressed as: OsmGap = Osmmeas – Osmcalc Biochemistry II Pouria Farsani 2013 187 Regulation of osmolality The osmolality regulation is carried out by water excretion through the kidneys, and water intake when feeling thirsty. ADH is the main hormone of this regulation (from the supraoptic and paraventricular nuclei). The secretory granules near the pituitary, where ADH is stored, secrete ADH based upon different stimuli – the main ones of which are a change in osmolality or plasma volume. The ADH response to the osmolality changes is fast. A decrease of plasma osmolality (hypotonic fluid intake) stops the release of ADH => reduction of water reabsorption => excretion of diluted urine. For more detailed description of ADH function and osmolality regulation, see q. 44. Biochemistry II Pouria Farsani 2013 188 46. Electrolyte status of blood plasma. Relation of ion concentration to acid-base balance (buffer base and strong ion difference, anion gap) Biochemistry II Pouria Farsani 2013 189 The ionic composition of plasma and ISF is almost the same. ISF is a plasma ultrafiltrate with minimal proteins. In order to maintain ISF electroneutrality in the absence of proteins, a new equilibrium must be established (Gibbs-Donnan equilibrium). Therefore, the concentration of anions (Cl, HCO3 ) in the ISF is higher than in plasma. On the other hand, Na+ is slightly lower. Relation of ion concentration to acid base balance We assess the acid-base balance disturbances based on the principle of electrical neutrality of plasma: cations = anions [Na+ ] + [K+ ] + [Ca2+ ] + [Mg2+ ] = [Cl] + [HCO3 ] + [Albx] + [Piy] + [UA- ] where Albx− is albumin, Piyphosphates, and UA− unmeasured anions of organic and inorganic acids. Biochemistry II Pouria Farsani 2013 190 Strong ion difference (SID) SIDeff = [Na+ ] + [K+ ] + [Ca2+ ] + [Mg2+ ] – [Cl] – [UA- ] The formula above gives the SID. Comparing SID with the ionogram it shows that SID is the anion column filled with hydrogen carbonate, plasma proteins and inorganic phosphates. An exact measurement of the SID is complicated by difficulties in determining UA. Therefore, an empirical relationship is used: SIDeff = [HCO3 ] + 0.28×[albumin] + 1.8×[Pi] (39 ± 1 mmol/l) Where concentrations of HCO3and Pi are in mmol/l, concentration of albumin in g/l; coefficient 0.28 corresponds to the number of negative charges of 1 gram of albumin; coefficient 1.8 represents the number of negative charges of 1 mmol of inorganic phosphate in 1 l at pH 7.4. Changes in SID value is influenced particularly by changes in the concentrations of Na+ , Cland UA. Anionic gap (AG) (12–18 mmol/l) AG = [Na+ ] + [K+ ] – [Cl] – [HCO3 - ] According to the ionogram, the AG is primarily determined by plasma proteins and UA, less by phosphate. AG is increased if the following are elevated: -Lactate -Acetoacetate -Beta-hydroxybutyrate -Phosphate in uremia -Exogenous anions during intoxications (salicylates, formate from methanol) AG cannot be used to determine the contribution of protein to the potential disturbance of ABB. During hypoalbuminemia, AG may be normal even though the concentration of lactate may be increased – this is a disadvantage of the AG value. Due to this, AG is corrected to the concentration of serum albumin: AGcorr = AG + 0.25 × ([Alb]ref − [Alb]determ) where [Alb]ref represents the reference value of albumin concentration, generally 40 g/l. The value of AGcorr can with higher probability indicate pathological presence of UA. Buffer bases Looking at SID we said that the SID is the anion column filled with: -Hydrogen carbonate -Plasma proteins -Inorganic phosphates Biochemistry II Pouria Farsani 2013 191 These are the base buffers which will be explained in further detail, each, below. -Hydrogen carbonate (bicarbonate) buffer – the most important buffer in ECF which can be easily regenerated. It is in equilibrium with other systems, it is capable of exchanging H+ with these systems. Carbonic acid (H2CO3) and HCO3 constitutes this buffer. Its pH is defined by the HendersonHasselbalch equation: Biochemistry II Pouria Farsani 2013 192 *Partial pressure of carbon dioxide (pCO2) – it is termed as the respiratory component in the Henderson-Hasselblach equation. The balance between the amount of CO2 produced in tissues and amount of CO2 that is exhaled from the body by the lungs is expressed in the pCO2. As pCO2 passes through the pulmonary capillaries, equilibrium between pCO2 in alveolar air and blood takes place so that the arterial blood is virtually identical to pCO2 in the alveolar air. Hypercapnia = pCO2 > 5.8 kPa Hypocapnia = pCO2 < 4.8 kPa *Hydrogen carbonate ion (HCO3 ) – is referred to as the metabolic component of the HendersonHaselblach equation. Its concentration in blood depends on the function of the the kidneys and the law of electrical neutrality and/or on the concentration of strong cations and anions and weak non-volatile acids. -Protein buffer – It is mainly albumin, hemoglobin in erythrocytes and intracellular proteins which act as the protein buffers. Almost all proteins are in a pH range higher than their isoelectric point in the physiological pH => hence their negative charge and ability to bind H+ , behaving as a strong conjugate base. H-proteinn− H+ + protein(n+1)− *Albumin – Depending on the amount of FA bound, albumins isoelectric point ranges between 4.7-5.5. Its buffering effect is affected by its concentration. Dissociation of albumin is changed with the pH of plasma – its charge will affect the concentration of other buffer bases. Biochemistry II Pouria Farsani 2013 193 *Hemoglobin buffer – oxygenated hemoglobin behaves as a stronger acid that splits off the proton while deoxygenated hemoglobin behaves a weaker acid, or as a stronger conjugate base, and accepts proton. -Hydrogen phosphate buffer – the charge of the two major phosphate species in the plasma is their sum. Like albumin, this charge affects the concentration of other anions in ECF. The hydrogen phosphate buffer works in more in ICF but also in bones and teeth. It is one of the possible buffers which are involved in acid-base regulation in the kidney. -Weak non-volatile acids (Atot) – expresses the sum of concentrations of albumin and phosphate [Atot] = [Albx−] + [Piy−] [Atot] expresses the sum of concentrations of negative charges of albumin and phosphate Unmeasured anions (UA) It expresses the proportion of other anions except those which are included in the electroneutrality equation such as lactate, ketone bodies, glycolate in ethylene glycol poisoning, formate in methanol poisoning, salicylate etc. Increased expense of other ions, usually HCO3 results in an increased amount of UA in plasma [UA] = [Na+] + [K+] + [Ca2+] + [Mg2+] – [Cl−] – [HCO3−] – [Albx−] – [Piy−] (6–10 mmol/l) Biochemistry II Pouria Farsani 2013 194 47. Alkali cations – distribution in various compartments, approx. daily intake and output, control of the excretion (angiotensin-aldosterone, natriuretic peptides), consequences of retention or of heavy losses of electrolytes For ionogram regarding distribution in ISF, see q.46 Looking at the ionograms – the alkali cation distributions in various compartments (plasma/ECF and ICF) are given in the cation (+) columns. Approx. daily intake and output Biochemistry II Pouria Farsani 2013 195 Biochemistry II Pouria Farsani 2013 196 Control of the excretion (angiotensin-aldosterone, natriuretic peptides) See q. 44 and 58. Biochemistry II Pouria Farsani 2013 197 Consequences of retention or of heavy losses of electrolytes -Hypernatremia => coma -Hyponateremia => muscle weakness -Hyperkalemia => palpation, muscle weakness, arrhythmia, cardiac fibrillation, death -Hypoerkalemia => vomiting, arrhythmia, death -Hypercalcemia => vomiting, coma, arrhythmia, death -Hypocalcemia => muscle cramps, convulsions -Hypermagnesemia – occurs in kidney failure. The symptoms are weakness, impaired breathing, decreased respirations, hypotension, hypercalcemia, arrhythmia/asystole, decreased or absence of tendon reflexes, bradycardia. Asystole and arrhythmia are possible cardiac complications. Magnesium acts as a physiologic calcium blocker which results in electrical conduction abnormalities. -Hypomagnesemia – serum levels are below 0.7 mmol/l. Hypomagnesemia is not the same as magnesium deficiency, as we are only referring to a disturbance in the electrolyte balance in this topic. Many conditions may lead to hypomagnesemia, such as diarrhea, malabsorption, alcoholism, chronic stress, diuretics, etc. The symptoms are cardiac arrhythmia, increased irritability of the nervous system with tremors, athetosis (slow, involuntary, convoluted, writhing movements of the fingers, hands, toes, and feet and in some cases, arms, legs, neck and tongue), nystagmus and an extensor plantar reflex. Furthermore, confusion, disorientation, hallucinations, depression, epileptic fits, hypertension, tachycardia and tetany may follow as symptoms as well. -Losses of sodium and potassium – by urine: *diuretics (furosemide, thiazides) *osmotic diuresis (hyperglycemia in DM) *renal failure *low/high production of aldosterone (=> loss of Na+ /K+ ) –by digestion juices: *vomiting *diarrhea –by sweat: *Excessive physical activity => loss of Na+ Biochemistry II Pouria Farsani 2013 198 There is also an interconnection of water and ion balance where water intake/output causes a temporary change in ECF concentration of electrolytes. This induces changes in the organism, where water is transferred between ECF and ICF and where hormonal response regulates the intake/output of water (ADH). The electrolyte input/output causes a temporary change in solutes concentration or in volemia which induces the following changes in the organism: -Fluid transfer -Change in absorption/secretion of ions (aldosterone) -Adjustment of water input/output (ADH and aldosterone) Biochemistry II Pouria Farsani 2013 199 48. Transport of CO2 in blood: pCO2 in arterial and venous blood, [[[[HCO3 - ]]]], carbaminohaemoglobin, physically dissolved CO2, the ratio [[[[HCO3 - ]]]]/[[[[CO2+H2CO3]]]]) When reading the text below, keep in mind that solutes and molecules always proceed from higher pressure to lower pressure. CO2 which is produced in the tissues diffuse into the blood, since the pCO2 is greater in the tissues than in the blood – hence diffusion is possible. The blood which is rich in pCO2 proceeds to the lungs. In the lungs, the pCO2 in the alveoli is lower than in the blood – hence CO2 diffuses from the blood into the air in the lungs. Concurrently the opposite occurs in the transfer of O2 – the pO2 is greater in the alveoli than in the blood – hence O2 diffuses into the blood from the capillaries. The blood which comes from the lungs, rich in O2 proceeds to the tissues. The pO2 in the tissues is lower than in the blood – hence diffusion of O2 from blood to tissues takes place. The following text below is graphically explained in the scheme which follows it: As CO2 diffuses into the blood from the tissues, the pCO2 in blood increases from 5.3 kPa to 6.3 kPa. Most of this CO2 is diffused into erythrocytes where carbonic anhydrase (CA) converts a part of the CO2 to H2CO3 producing HCO3 by dissociation => increased HCO3 concentration in erythrocytes in comparison to plasma => exchange with chloride ions in plasma (HCO3 - /Clexchanger in erythrocyte membrane). The shift of chloride ions is to maintain electroneutrality – Hamburgers shift. The other portion of CO2 reacts with hemoglobin producing carbamate. H2CO3 dissociation => formation of HCO3 => release of H+ which are buffered by binding to deoxygenated hemoglobin. Hemoglobin oxygenation => release of the H+ => acceleration of conversion of HCO3 - to H2CO3 => concurrent diffusion of Clback into erythrocytes. Biochemistry II Pouria Farsani 2013 200 CO2 can be transported in different forms: Carbaminohemoglobin – carbamate (Hb + CO2) Hemoglobin does not only transport O2 from the lungs to the peripheral tissues, it transports CO2 and protons from peripheral tissues to the lungs as well. Hemoglobin carries CO2 as carbamates formed with the amino terminal nitrogens of the polypeptide chains CO2 + Hb-NH3 + ↔ 2H+ Hb-NH-COOCarbamates change the charge on amino acid terminals from positive to negative, favoring salt bond formation between the alpha and beta chains. Furthermore: ~5 % of CO2 from tissues are carried as carbamate -CO2 is covalently bound to the N-terminal of heme – not to the iron! -The reaction is reversible -CO2 can also bind to amino groups on the polypeptide chains of plasma proteins Physically dissolved CO2 This is the proportion (~5 %) which is not chemically bound – it is simply dissolved in blood. Biochemistry II Pouria Farsani 2013 201 The [HCO3 ]/[CO2 + H2CO3] ratio A concentration of a buffer base is 20 times higher than the concentration of a buffer acid hence 20 times more resistant to acids. The ratio is thus 20:1 [CO2 + H2CO3] = 0.22 pCO2 (0.22 is the solubility coefficient of CO2) The Henderson-Hasselbalchs equation for hydrogen carbonate system in plasma gives: Where at physiological pH 7.4 and physiological pCO2, HCO3 concentration can be calculated accordingly: 7.4 = 6.1 + log [HCO3 ] / 0.22 × 5.3 1.3 = log [HCO3 ] / 1.2 101.3 = [HCO3 ] / 1.2 20 = [HCO3 ] / 1.2 [HCO3 ] = 24 mmol/l The ratio is calculated at 6.1pKa of plasma 37C ̊ 7.4 = 6.1 + log x x = log-1.3 x = 20 Ratio = 20:1 Biochemistry II Pouria Farsani 2013 202 49. Metabolic acidosis – causes and principle of compensation -Findings: ↓ pH, ↓BE, ↓ HCO3 - -Causes: 1. Increased production of H+ -Lactic acidosis *Hypoxia *Intensive muscular work *Anemia *Intoxication by salicylates, methanol or ethylene glycol *Thiamine deficiency *Alcoholism *Inherited metabolic disorders -Ketoacidosis in starvation and/or decompensated DM -Acidosis from retention of endogenous non-volatile acids in renal failure 2. Exogenous supply of H+ -Salicylate/penicillin overdosing -Excessive administration of diluted HCl solutions, NH4Cl, Arg. HCl in treatment of metabolic alkalosis. 3. Loss of HCO3 - -Diarrhea -Burns -Renal tubular disorders -Diuretics 4. Excessive dilution of HCO3 -Excessive infusions of isotonic solutions (HCO3 is diluted in blood faster than it can be replenished by the metabolism) In the 1st and 2nd case AG and UA are increased. This is due to the buffering reaction concentration of [HCO3 ] event. Albxa Piyis decreased. Concentration of chlorides is not changed – normochloremic acidosis Biochemistry II Pouria Farsani 2013 203 In the 3rd and 4th case AG and UA remain unchanged. The loss of HCO3 is replaced by Cl– hyperchloric acidosis. SID is also decreased. Correction and compensation -Buffering effects – first phase of the disorder. Buffering effect of bicarbonate is applied. H+ + HCO3 → H2CO3 → H2O + CO2 The H+ which are released from the tissues are buffered by reaction with HCO3 => HCO3 - ↓↓↓↓ in plasma. After a few hours or days H+ starts to transfer into the cells. Here a reaction with intracellular buffers (hemoglobin mainly but also proteins and phosphates) takes place – up to 60% of the increased H+ may be buffered by intracellular systems. 1 20 22,0.2 3 < − pCO HCO When the H+ are transferred into ICF, K+ is secreted => hyperkalemia and depletion of intracellular K+ -Respiratory compensation – increase of pulmonary ventilation – second phase of the disorder. The main purpose is to decrease pCO2 pH ↓ => stimulation of peripheral arterial chemoreceptors => start of hyperventilation (Kussmaul breathing) within 12-24 hours => pCO2 ↓ => altering pH based on the HendersonHasselbalch equation. pH approaches 7.4 but concentrations of HCO3 and pCO2 are non-physiological -Renal correction – third phase of the disorder – correction within 2-3 days – excretion of acidic urine. H+ secretion is increased, accompanied by secretion of the respective anion (lactate, acetoacetate, 3-hydroxybutyrate) => increased reabsorption of HCO3 . Concurrently, Na+ ions which passed to the ultrafiltrate with acid anions are reabsorbed. 1 20 22,0.2 3 ≈ − pCO HCO Biochemistry II Pouria Farsani 2013 204 Biochemistry II Pouria Farsani 2013 205 50. Respiratory acidosis – causes and principle of compensation -Findings: ↓pH, ↑pCO2, HCO3 is normal or slightly elevated -Causes – CO2 production in the body exceeds its elimination. • Hypoventilation -Respiratory center depression *Opiates *Sedatives *Narcotics *High pCO2 -Disorders of ventilation, diffusion perfusion *Lung diseases *Chest/diaphragm injuries -Gas transport disorders *Anemia *Circulatory insufficiency *CO poisoning -Disorders of gas exchange between blood and tissue *Cyanide poisoning -Disorders of neuromuscular transmission *Drugs *Nicotine *Botulin poisoning -Inappropriate artificial ventilation As respiratory acidosis may lead to hypoxia, lactic acidosis may be a result (anaerobic glycolysis ↑). Correction and compensation -Reaction of buffer bases in respiratory acidosis – pCO2 is increased (and therefore also [HC2O3]), the reaction H2CO3 H+ + HCO3 proceeds with the increased concentration of HCO3 in the ECF. The H+ ion which is produced by dissociation of H2CO3 in ECF immediately reacts with other buffering systems. Biochemistry II Pouria Farsani 2013 206 The reason for why HCO3 is normal or only slightly elevated is due to part of it diffusing from the ECF into the ICF where it reacts with cytoplasmic buffering systems. Therefore, the total buffer base content in ECF is reduced hence concentration is unchanged or slightly increased. Due to this, HCO3 concentration is a poor indicator of the total amount of bases in plasma. -Renal compensation – within 1-2 days – kidneys begin to secrete more NH4 + and H+ ions. HCO3 reabsorption increases, concurrently excretion of chlorides in the urine increases as well. -Respiratory compensation – correction through the lungs is very limited since the ABB disorder is due to malfunction of the respiratory system itself. Ventilation support (pCO2 > 8kPa) is needed in case therapy for respiratory malfunction is not helping or incase improvement in spontaneous ventilation of the patient is not expected. Biochemistry II Pouria Farsani 2013 207 51. Metabolic alkalosis – causes and principle of compensation -Findings: ↑pH, ↑HCO3 , ↑BE -Causes: 1. Loss of H+ and Clfrom GIT *Vomiting *Aspiration of gastric contents 2. HCO3 - supply *Excessive administration of NaHCO3 solution in treatment of acute metabolic acidosis 3. Loss of Cland K+ by urine due to diuretics 4. Hypoalbuminemia In the 1-3 disorders SID is increased (decreased in chlorides) – hypochloremic alkalosis. In the 4th disorder a drop of total anions is seen (decrease of albumin by 1g/l leads to a rise in the concentration of HCO3 by 0.25-0.3 mmol/l) – normocholermic alkalosis. Correction and compensation (not as efficiently compensated as metabolic acidosis) -Buffering effect – base excess (during loss of HCl) reacts with the acidic component of buffer systems which leads to the formation of H2CO3 which breaks down to CO2 and water. -Respiratory compensation – ↑ pH => inhibition of respiratory center => hypoventilation => retention of CO2 (hypercapnia) => H2CO3 ↑ => pCO2 ↑. Therefore the ratio of HCO3 - /pCO2 which was raised normalizes but their concentrations are non-physiological. Hypoventilation also leads to a decrease of pO2. This is the limiting factor of compensation since hypoxemia stimulates the respiratory center resulting in an impossible further increase of pCO2 as the increase of pCO2 is dependent on the inhibition of the respiratory center. -Renal correction – reduced exchange of Na+ for H+ , decreased production of NH4 + and decreased HCO3 - reabsorption. Biochemistry II Pouria Farsani 2013 208 52. Respiratory alkalosis – causes and principle of compensation -Findings: ↑ pH, ↓ pCO2, HCO3 unchanged or slightly lowered -Causes – exhalation of CO2 from the lungs dominates over its production in tissues • Hyperventilation -Improper controlled breathing -Increased irritation of the respiratory center *Impulses from the CNS (hysteria, anxiety, infection) *Intoxication by drugs (salicylates) *Irritation of the thermoregulatory center (fever, physical work) -Normal pregnancy -Respiratory center disorders (ictus, trauma, tumor, inflammation) Correction and compensation -Reaction of buffer bases in respiratory alkalosis – ↑ pH => ↓ of acidic component of the buffer system in ECF. A significant increase in pH may put vital functions at risk (arrhythmias, convulsions); therefore the reaction of buffer bases is fast. Due to CO2 exhalation exceeding the production in tissues, CO2 loss will be partially replaced by new formation of H2CO3 from bicarbonate and protons bound to other bases. This leads to a slight decrease of HCO3 concentration but an increase of the negative charge on the other buffer bases. -Renal compensation – starts within a few days and it is the opposite reaction of respiratory acidosis compensation (see q. 50). Proton retention is increased whilst bicarbonate reabsorption is decreased => ↑ bicarbonate in urine => ↑ urine pH > 6.5 -Respiratory compensation – elimination of cause of respiratory alkalosis => lower pCO2 will stimulate hypoventilation => ↑ pCO2 Biochemistry II Pouria Farsani 2013 209 53. Buffering systems in blood, blood plasma (components, concentrations), the main buffer bases in interstitial and intracellular fluids. Blood acid-base parameters (reference values, changes of the values in acute disturbances and in the course of their compensation) See also q. 46-52. Looking at the table above, we can see the different buffer systems in blood and blood plasma. For detailed description of the different buffers see q. 46. Biochemistry II Pouria Farsani 2013 210 Buffer capacity Buffer capacity is a measure of the buffering power, of a buffer system. It corresponds to the number of added H+ and OHions per unit volume that change the pH by one unit. It depends firstly on the concentration of both components and secondly on ratio of both components. Components and concentrations The total Ve+ charge = total Ve- charge = 154 COMPONENT CONCENTRATION (mmol/l) Na+ 140 K+ 4.5 Ca2+ 5 Mg2+ 2 Cl- 103 HCO3 - 25 Proteins 18 HPO4 - 2 SO4 2- 1 Organic anions 5 The main buffer bases in interstitial and intracellular fluids -Interstitial fluid – hydrogencarbonate (HCO3 ) is the main buffer here -Intracellular fluid – protein and hydrogenphosphate (HPO4 2) are the main buffers here Blood acid-base parameters We distinguish between parameters which are measured in arterial blood and parameters which are calculated. -Measured in arterial blood – arterial blood is collected from arteries into heparinized tubes or capillary tubes – the sample should not contain any air bubbles. The parameters in brackets are referred to as supporting data. Biochemistry II Pouria Farsani 2013 211 -Calculated parameters – besides the parameters mentioned in the table below, we also take total concentration of buffer-bases of blood and plasma shown in the table in the beginning of this question. Changes of the values in acute disturbances and in the course of their compensation Biochemistry II Pouria Farsani 2013 212 Metabolic acidosis Parameter Physiol. st. Ac. change Compensation Correction [HCO3 ] 24 mmol/l ↓↓↓↓ - →→→→ N pCO2 5.3 kPa N ↓↓↓↓ - [A] / [HA] 20 : 1 < 20 : 1 ≤ 20:1 ~20:1 pH 7.40 ± 0.04 < 7.36 ≤ 7.4 ~7.4 System Lungs Kidney Process Hyperventilation ↑ HCO3 - reabsorption ↑ NH4 + / H2PO4 - excretion Metabolic alkalosis Parameter Physiol. st. Ac. change Compensation Correction [HCO3 ] 24 mmol/l ↑↑↑↑ - →→→→ N pCO2 5.3 kPa N ↑↑↑↑ [A] / [HA] 20 : 1 > 20 : 1 ~20:1 pH 7.40 ± 0.04 > 7.44 ~7.4 System Lungs Kidney Process Hypoventilation ↑ HCO3 - excretion Reduced exchange Na+ H+ . NH4 + production ↓ Biochemistry II Pouria Farsani 2013 213 Respiratory acidosis Parameter Physiol. st. Ac. change Compensation Correction [HCO3 ] 24 mmol/l N, ↑↑↑↑ ↑↑↑↑ pCO2 5.3 kPa ↑↑↑↑ →→→→ N [A] / [HA] 20 : 1 < 20:1 ~20:1 pH 7.40 ± 0.04 < 7.4 ~7.4 System Kidney Lungs Process HCO3 - reabsorption NH4 + / H2PO4 - excr. Hyperventilation Respiratory alkalosis Parameter Physiol. st. Ac. change Compensation Correction [HCO3 ] 24 mmol/l N, ↓↓↓↓ ↓↓↓↓ pCO2 5.3 kPa ↓↓↓↓ - →→→→N [A] / [HA] 20 : 1 > 20:1 pH 7.40 ± 0.04 > 7.4 System Kidney Lungs Process Excretion of HCO3 - Hypoventilation (if possible) Biochemistry II Pouria Farsani 2013 214 54. The role of the kidney and liver in acid-base balance Kidneys The kidneys function by three mechanisms in the acid-base balance: -Secretion and excretion of H+ -Resorption and excretion of HCO3 -Secretion of ammonia from tubular cells HCO3 is freely filtered into the glomerular filtrate. A small loss of HCO3 however into the urine can cause serious disturbance in the acid-base balance. Due to this, the major portion of HCO3 is reabsorbed (90% in the proximal tubule). The filtered HCO3 combines with H+ ions which are secreted into the tubules in exchange for Na+ (or by H+ -ATPase) to form H2CO3 => carbonic anhydrase on the brush border catalyzes the formation of CO2 which diffuses along the concentration gradient into the cells. Once in the cell a second pool of carbonic anhydrase (cytoplasmic) catalyzes the formation of HCO3 which is transported out of the cell on the basolateral side in the exchange for Na+ . We can shortly say that the kidneys assist in the acid-base balance – regulating pH fluctuations by either retaining or excreting protons which are generated by the dissociation of H2CO3. This occurs mainly in the proximal tubule. The final adjustment of urine pH however takes place by a similar process in the collecting duct. The carbonic acid is formed in the cell from CO2 which: a) Diffuses into the cell from the lumen of tubules b) Is formed in the tubular cells by metabolism c) Diffuses into the cell from plasma during acidosis -H+ secretion in proximal tubules: *H+ -ATPase *Antiport with Na+ -H+ secretion in distal tubules and collecting duct – type A, intercalated cells *Active secretion of H+ into urine – H+ -ATPase, H+ -K+ -ATPase *HCO3 - reabsorption -H+ secretion in distal tubules and collecting duct – type B, intercalated cells *HCO3 - secretion *H+ reabsorption -Secretion of H+ from three sources: a) CO2 from plasma b) CO2 from tubular fluid c) CO2 produced within the tubular cell Biochemistry II Pouria Farsani 2013 215 Normal pH: slightly higher amount of H+ excreted into lumen than HCO3 is filtered into the tubular fluid Most protons are used for the reabsorption of HCO3 Small daily excretion of H+ which is bound to NH3 (50 mmol/d) and HPO4 - (20 mmol/d) Acidosis => increased secretion of H+ from tubular cells and decreased HCO3 is filtered => increased availability of protons improves the efficiency of HCO3 - reabsorption. Acidosis is also corrected by concurrent increased production of NH3 from glutamine and glutamate in the tubular cells. The NH3 which passes into the urine buffers H+ ions in the urine => increased concentration of NH4 + in urine by up to 10 times. Decreased efficiency of H+ transport into the tubular fluid occurs when pH 4.5 is reached – the transport of H+ against the concentration gradient has resulted in the tubular fluid becoming 800 times more acidic than plasma. This is related to the fact that at this point, H+ cannot be transported from the tubular cells since the concentration gradient is too great for the secretory process to continue. This is why the pH of urine cannot be lower than 4.5. It is HPO4 (filtered from blood) and NH3 (Secreted from tubular cells) which buffers the H+ ions. Biochemistry II Pouria Farsani 2013 216 If more buffer base is available in the urine, more H+ can be secreted before the limiting gradient is reached. Even though kidneys may need hours to days in order to compensate for changes in body fluid pH, in comparison to the immediate response of body buffers with a few minutes reaction time, the kidneys are still the most potent acid-base regulator system. Alkalosis => decreased H+ secretion from tubular cells and concurrent increase of HCO3 filtering. The lack of H+ in order to reabsorb the HCO3 results in the increase of HCO3 in urinary excretion. SUMMARY OF RENAL RESPONSE TO ACIDOSIS Biochemistry II Pouria Farsani 2013 217 Liver contribution to maintenance of acid-base balance The liver eliminates NH3 in two ways: 1. Urea synthesis – release of 2H+ => acidifying process 2. Glutamine synthesis – no release of H+ Acidosis => synthesis of glutamine is favored in the urea detoxification in order decrease the production of H+ which happens in the urea synthesis. Liver failure => stagnation of both ways of ammonia detoxification => metabolic alkalosis Biochemistry II Pouria Farsani 2013 218 55. Filtration rate of the plasma through the glomeruli (composition and permeability of the filtration medium, glomerular filtration rate – creatinine clearance, glomerular proteinuria) Since questions 55-58 will discuss the functions of the kidneys in greater detail, we start by briefly explaining the main ideas regarding kidneys and their functions (even though it is not specifically asked for in this question). The kidneys are mainly organs of homeostasis their functions can be summarized as followed below: • Excretion of catabolites and xenobiotics – urea, creatinine, urates, metabolites of hormones etc. • Regulatory function – homeostatic functions: -Osmolality – maintaining volemia and electrolyte equilibrium -Arterial blood pressure – production of renin and maintenance of natremia and volemia -Acid-base balance – ammoniogenesis, H+ secretion, HCO3 - reabsorption/secretion -Erythropoiesis -Calcium and phosphate homeostasis • Metabolic functions: -Gluconeogenesis -Arginine production -Production and elimination of NH3 • Endocrine functions: -Erythropoietin -Calcitriol -Urodilatin -Prostaglandins E2 & I2 -Enzyme renin Biochemistry II Pouria Farsani 2013 219 The kidney is divided into the cortical part (cortex) and medullary part (medulla). -Cortical part – consist mainly of: *glomeruli *distal tubules *convoluted portion of proximal tubules The blood flow decreases from the cortex to the papilla (400-500ml/min/100g of cortex tissue to 125ml/min/100g of outer medulla and 15ml/min/100g of the inner medulla) therefore oxidative processes take place in it – obtaining energy. Anaerobic processes takes place in the medulla. See scheme below. -Medullary part – contains: *straight portion of proximal tubules *thick ascending limb of loop of Henle Biochemistry II Pouria Farsani 2013 220 There are two types of nephrons: -Cortical – glomeruli in the outer part of the cortex close to the surface of the kidney. The loop of Henle is short extends into the cortex and partially into the medulla. Processes here serve for common concentration of urine. -Juxtamedullary – glomeruli is found deep in the cortex, near the medulla. The loop of Henle is longer and embedded in the medulla. Processes here serve for powerful concentration of urine – countercurrent multiplier system. These nephrons make up about one fifth of all nephrons. Biochemistry II Pouria Farsani 2013 221 Glomerular filtration – composition and permeability of the filtration medium The glomerular filtration medium consists of three layers: 1. Fenestrated endothelium (lamina fenestra) 50-100 nm pores which do not prevent penetration of proteins 2. Basement membrane – fine fibrils embedded in a gelatinous matrix with a thickness of 300-350 nm. Sub- layers: *lamina rara interna *lamina densa *lamina rara externa The ultrastructure of the basement membrane proteins involves: *type IV collagen *laminin *heparin sulfate proteoglycan *entactin *fibronectin 3. Epithelial cells (podocytes) – visceral layer of Bowman’s capsule. Protrusions of the podocytes known as pedicles are intertwined. A slit diaphragm is found among these protrusions, the main protein of which is nephrin. The pores (5 nm) of this membrane represent the last barrier for the filtration of plasma proteins. -When we speak about filtration through these layers, we take size and charge of the molecules into consideration as the main factors upon which passage is dependent on. *Molecules with Mr < 10 000 are freely filterable, if not bound to plasma proteins *Molecules with Mr > 100 000 are not filtered at all – nor are cellular elements -Shape of the molecules is another factor which influences permeability – elongated molecules are filtered more easily compared to spherical ones -The glomerular endothelial cells and podocytes have negatively charged glycocalyx and the basement membrane also contains negatively charged sialic acids, eialoproteins and heparin sulfate – the density of the charge increases towards the epithelial layer. This negative charge results in significantly decreased ability of large negatively charged molecules to be filtered compared to uncharged molecules. Albumin is one example which under physiological circumstances only is filtered in small quantities. -Furthermore mesangial stellate cells of the glomeruli represent the phagocytotic action of large particles from plasma such as immune complexes. Biochemistry II Pouria Farsani 2013 222 The scheme below illustrates the glomerular filtration: Glomerular filtration rate (GFR) The GFR is measured indirectly as renal clearance – theoretical volume of blood plasma from which a substance is cleared (completely eliminated) by renal function per unit of time – a substance which passes freely into the primary urine during ultrafiltration in the glomeruli. It should neither be secreted nor reabsorbed. Substances used for measuring clearance will be discussed below. GFR is nonselective (Mr < 10 kDa) and passive. It measures to approx. 2mL/s/1.73m2 which equals approx. 20% of renal plasma flow and it depends on: -Net filtration pressure -Glomerular membrane permeability (hydraulic conductivity) -Size of the filtering area Effective filtration pressure is determined by the blood pressure in the glomerular capillaries minus the pressure in Bowman’s capsule and oncotic pressure in plasma. As mentioned above clearance estimation is used to determine GFR. Substances used are either endogenous or exogenous which is administered in an adequate dose. Biochemistry II Pouria Farsani 2013 223 -Inulin clearance is the gold standard. It is a plant polysaccharide which is soluble in water – it meets the above criteria for determining clearance. The method involves inulin administration intravenously, collection of urine during hourly periods and measuring amount of inulin in each sample. Creatinine clearance Usage of creatinine to estimate GFR is very convenient since the amount produced endogenously in muscle tissue is relatively constant and directly proportional to the body surface area. Creatinine also meets up to the criteria for a substance used to measure clearance. A small amount of creatinine however (~10%) in the final urine is derived from tubular secretion. Another important fact is that plasma creatinine concentration is very stable in normal subjects. The principle of calculation of creatinine clearance is that glomerular filtrate is the same as its concentration in uncleared plasma, cp. Furthermore, the volume of formed filtrate should be the same as the volume of plasma which is completely cleared from creatinine Vp. Substance amount of cleared creatinine = cp × Vp. The principle claims that the same amount (as above) of creatinine is excreted into the final urine per second, thus it simultaneously equals the product of the concentration of creatinine in final urine cu and volume of urine excreted per second Vu. The principle is summarized as follows: cp Vp = cu Vu Biochemistry II Pouria Farsani 2013 224 If we determine the concentration of creatinine in plasma/serum (umol/l) and in urine (mmol/l) (24 hrs) we can calculate the GFR. Meatless diet for 3 days is necessary before the examination is carried out. The volume of urine excreted per second Vu (mL/s) is calculated from the volume of urine excreted over a specific time. Formula which is used for this calculation is: Vp = Vu GFR The GFR obtained in this formula however needs to be corrected to the standard body surface area 1.73 m2 (S) which is calculated as: S = 0.167 (m2) Finally corrected GFR is given as: GFRcorr = GFR . The creatinine clearance is associated with some inaccuracies however. Creatinine is secreted in the proximal tubules as well, hence its value in healthy humans exceeds GFR by 10-20 % – it has little relevance at normal filtering speed. In advanced renal failure however, the contribution of creatinine secretion is significant and thus affecting the evaluation of GFR. A common reason for error in common determination is an inaccurate urine collection. Accurate correction can be straining on both patient and nursing staff. That is why the use of computational methods has increased where accurate urine collections are not needed. MRDR (Modification of Diet in Chronic Renal Disease) is used which only requires serum creatinine concentration as the measured parameter. Serum creatinine is then, together with age, sex and ethnicity used in a specific formula to calculate GFR. Biochemistry II Pouria Farsani 2013 225 Looking at the green table above, we can see that formation = filtration = excretion in healthy individuals. In case of renal pathology, where formation = excretion but not filtration, we can see that creatinine in serum will increase due to the inability of the kidneys to eliminate it – serious indicator! Cystatin C Serum cystatin C is another, newer, parameter used to estimate GFR. Cystatin C is a basic protein (Mr 13 000) belonging to the families of cysteine protease inhibitors. It is constantly produced by all nuclear cells of the proximal tubule. Its concentration is not affected by diet, infections, liver functions, malignancies or myopathies hence. Throughout the day its concentration in serum is almost constant. Although the disadvantage of its usage due to it being measured by immunochemical methods; cystatin C is considered a sensitive indicator of a slight decline in GFR. Glomerular proteinuria There is a selective and a non-selective glomerular proteinuria. -Selective – caused by the affection of the outer part of the basal membrane and podocytes. Common findings in urine: *albumin (Mr 68000) *transferrin (Mr 89000) Proteins with greater molar mass, especially immunoglobulins, cannot be detected in urine. -Non-selective – caused by damage of the inner part of the basal membrane and mesangia. Common findings in urine: *proteins with greater molar mass, especially immunoglobin G *albumin *transferrin Biochemistry II Pouria Farsani 2013 226 56. Reabsorption and secretion in the renal tubules (water, electrolytes – natriuresis, low molecular compounds – glucose, amino acids, tubular proteinuria), the term fractional excretion E/F See scheme for main functions of nephron in q. 55 for understanding difference between, reabsorption, filtration and excretion (=filtration – reabsorption + secretion). Transport processes in the proximal tubule The fluid in the beginning of the proximal tubule has the same composition as that of plasma, except for the lack of proteins. About 70% all substances and water will be reabsorbed during the flow through the proximal tubules => change of concentration of substances. The primary urine osmolality remains practically constant and is almost equal to the plasma osmolality. Proximal tubule epithelial cells have relatively permeable tight junctions – water and small ions can pass (paracellular transport) through these. In case there is an increase in entrance of non-reabsorbable substances into the proximal tubule => proportional decrease in water reabsorption => increase in osmotic diuresis. Therapeutically, mannitol is used for this purpose. We summarize the functions of the proximal convoluted tubule before viewing the processes in detail: • Obligatory reabsorption -Water (~70%) -Most of Na+ (65%), K+ (55%), Cl(50%) and HCO3 - (90%) -Almost all AAs, Glc, lactate, vitamins -Urea ~50 % -Some solutes “renal threshold” *concentration of a solute dissolved in the plasma ultrafiltrate above which kidneys fail to reabsorb all and this spill solute into urine • Secretion -organic ions, creatinine, drugs, etc. A general description of a tubular cell is needed in order to discuss its detailed actions, see scheme => Biochemistry II Pouria Farsani 2013 227 In proximal tubular cells: -Reabsorption – non-regulated (exception for Ca2+ , Mg2+ , Pi) -Paracellular transport – easy -Surface area – high -Content of mitochondria – high -Na+ – The reabsorption of Na+ is the main driving force for the reabsorption of various substances. Na+ ions are actively pumped by Na+ /K+ -ATPase in order to maintain a low concentration of sodium ions in the tubular cells => difference between concentration of sodium ions and potential between the tubular fluid and cell. Entrance of sodium into the cell occurs by cotransport and antiport mechanisms. Luminal membrane sodium cotransport (secondary active transport) with sodium takes place with: -Glucose -AA (“AK” in the scheme) -Phosphates -Other molecules using specific cotransporters The sodium ion concentration is the driving force for the transfer of cotransported molecules. Furthermore, there is a cotransport of sodium across the luminal membrane in the exchange for H+ . This is also a secondary active transport system which is important in the acidification of urine. About 65% of K+ is reabsorbed mainly by paracellular transport where K+ that are pumped into the cell at the basolateral membrane diffuse back via K+ channels. Moreover, glucose, AA and phosphates leave the cell by facilitated diffusion at the basolateral side, independent of Na+ . Aquaporins and paracellular transport stand for the free passage of water through the proximal tubule epithelium. Biochemistry II Pouria Farsani 2013 228 -HCO3 – see also q. 54. A small loss of hydrogen carbonate by urine may cause serious disturbances of the acid-base balance hence ~ 90% of HCO3 is reabsorbed by processes occurring in the proximal tubule. Filtered HCO3 + H+ (which are secreted into the lumen in the exchange for Na+ ) =>H2CO3. Action of carbonic anhydrase (CA) on the brush border => release of H2O from H2CO3 in order to form CO2. CO2 diffuses along concentration gradient into the cells where cytoplasmic CA catalyzes the formation of H2CO3 which dissociates to produce HCO3 - . Cotransport with Na+ is used, transporting hydrogen carbonate out of the cell at the basolateral side. CA inhibitors (acetazolamide and dorzolamide) act as weak proximal diuretics -Chlorides – ~50 % are reabsorbed in the PT. Chlorides pass from primary urine, into the tubular cells by: *antiport with other anions (formats, oxalates) – partially *paracellular transport – significantly At the beginning of the proximal tubule LNTP (lumen-negative transepithelial potential) is the driving force. It is created when sodium ions are transported from the lumen into the tubular cells and then into the blood, giving rise to an excess of positive charge in the peritubular capillaries thus puling chloride along the electrical gradient. Biochemistry II Pouria Farsani 2013 229 The driving force for chloride ion transport in the middle and late parts of the proximal tubule is carried out by paracellular diffusion along the chemical gradient. The lumen-positive transepithelial potential, LPTP is created as subsequent Na+ and water reabsorption in the early part of the tubule creates a concentration difference of chloride ions in the lumen and interstitium, or in a vessel. Lastly, substances and water reabsorption from the proximal tubules is the transfer from interstitium, back into the peritubular capillaries. The proteins which remained in the blood create the oncotic pressure, which is the main driving force for this reverse transport! -Organic anions – secreted in the PT – mainly carboxylic acids and sulfonic acids such as *oxalate *urate *hippurate *barbiturate *penicillin The transport is mediated by organic anion transporter (OAT) at the basolateral membrane. As organic anions accumulate in the tubular cell, they pass into the urine along the concentration gradient. ABC transporters pump some organic anions into urine as well. -Organic cations – secreted similarly as org. anions. They include: *amines *ammonium Biochemistry II Pouria Farsani 2013 230 Transport into the tubular cell from through the basolateral side occurs via facilitated diffusion (organic cation transporter OCT) Transport across luminal membrane into urine is mediated by cation/H+ exchanger – its driving force is the H+ gradient between and the cell which is maintained by Na+ /H+ exchange. Endogenous substances such as, adrenaline, choline, serotonin and other substances are excreted via this way. There are many OAR/OCT transporters on the basolateral membrane. They have a broad specificity and allow secretion of a large amount of chemically diverse compounds. Ions may compete for binding to a transporter; this can affect the rate of elimination of some drugs by the kidneys. Some uncharged organic compounds pass from the blood through the tubular epithelium by simple diffusion. In case organic acids or amines occur in the form or neutral uncharged molecules under the given pH, they will be subjects of this simple diffusion. Biochemistry II Pouria Farsani 2013 231 -Uric acid – belongs to the organic cations/anions that can be actively reabsorbed, it is secreted in a lesser extent. -Glucose and AA – reabsorption is carried out with Na+ symport (secondary active transport). The sodium ion concentration is the driving force for the transfer of cotransported molecules. *Sodium is transported along the gradient -Proteins – most of which overcome the glomerular filter are reabsorbed in the tubules. Albumin is reabsorbed in the PT via two systems: 1. Specific albumin receptors – found on the surface of the bursh border of PT cells. It is an energy requiring system, which after binding of albumin => endocytosis. Under normal conditions this system is almost saturated this serving in the uptake of albumin at the normal speed of its filtration. 2. Non-specific with high capacity – non-selective endocytosis of a considerable amount of albumin, it can reabsorb up to 2.6 g of filtered albumin daily. This mechanism will also reabsorb other proteins with higher Mr which passed through the filter. Reabsorbed proteins are degraded by lysosomal enzymes. Smaller proteins (Mr < 69 000) will be completely reabsorbed under normal circumstances by another system in the PT. Tubular proteinuria (proteinuria > 150 mg or proteins excreted /day) Tubular proteinuria occurs when reabsorption of low molecular proteins in the proximal tubule is disrupted – tubular damage. This could be due to toxic damage of tubules during pyelonephritis. Biochemistry II Pouria Farsani 2013 232 We speak about complete or incomplete -Complete – almost all proteins with molar mass between 10 000 – 70 000 are excreted. This regards: *alpha1-antitrypisn *alpha1-acidic glycoprotein *retinol binding protein *beta2-microglobulin The proteins mentioned above are assessed as markers for tubular proteinuria. -Incomplete – excretion of microproteins – proteins with molar mass from 10 000 – 40 000. This regards: *alpha1-acidic glycoprotein *beta2-microglobulin Methods of electrophoresis with agarose or polyacrylamide gel are mainly used to determine the spectrum of proteins present in proteinuria (glomerular as well which was discussed in q. 55) Biochemistry II Pouria Farsani 2013 233 Fractional excretion, E/F The difference between the speed of the glomerular filtration and volume of the final urine formed in one second (Vu) is equal to the volume of water, which is reabsorbed in one second mainly in the PT (obligatory), but also in the terminal segments of nephrons (variable reabsorption). It is expressed by the portion of the total volume glomerular filtrate – the portion reabsorbed from the glomerular filtrate so called fractional reabsorption of water (tubular reabsorption of water). It is expressed as follows: FR(H2O) = = 1 – It is also expressed as a portion of water excreted into urine, so called, fraction excretion of water: EF(H2O) = = It is evident that FR(H2O) + FR(H2O) = 1 In healthy people the values of renal fractional reabsorption are within the range 0.985 – 0.997. Decrease of these values is typical for a failure of the regulation of water reabsorption by vasopressin such as in diabetes insipidus. A minute change in this value may result in a remarkable change in urine volume. Biochemistry II Pouria Farsani 2013 234 The graphs below show the process of reabsorption/secretion of glucose in PT and the overall reabsorption/secretion processes of different solutes summarized: Biochemistry II Pouria Farsani 2013 235 57. Transport processes in loop of Henle, distal tubulus and collecting duct. Main types of diuretics and principle of their effect Processes in the loop of Henle (LH) aim to form a hyperosmotic environment in the surrounding renal medulla (approx. 900-1400 mmol/kg compared to plasma osmolality of 280-290 mmol/kg) There is an osmotic gradient formed in the LH which is used to concentrate the urine in the collecting ducts. -Descending limb of LH – highly permeable to water but relatively impermeable to ions such as Na+ and Cl. In the urea recirculation, urea can diffuse from the interstitium back into the primary urine. -Ascending limb – ion reabsorption => interstitial osmolality in the renal medulla ↑ => suction of water from the descending limb => concentration of the filtrate. Na+ /K+ /2Cl- transporter on the luminal membrane mediates the transport of the ions into the cell. This transport is electroneutral – loop diuretics inhibit it specifically (furosemide, bumetanide). Furthermore, we can find on the basolateral membrane Na+ /K+ -ATPase which transports Na+ while K+ is recycled back into the lumen by the K+ -channel in the luminal membrane. Clleaves the cell at the basolateral side via the Clchannel or K+ /Clcotransport. The luminal cell membrane is most permeable to K+ while the basolateral membrane is permeable to Cl- . When these two ions diffuse out of the cell, a potential difference is created between the lumen and the interstitial fluid surrounding the tubules. The potential is more positive in the lumen by +6mV => lumen-positive transepithelial potential (LPTP) – the driving force for paracellular transport of small cations (Na+ , K+ , Ca2+ , Mg2+ , NH4 + ). The epithelial membrane is extremely impermeable to water in this section (ascending limb). Biochemistry II Pouria Farsani 2013 236 Countercurrent multiplication system Due to the fact that the ascending and descending limbs of LH have different permeability, a countercurrent multiplication system is created – active transport of solutes in thick ascending limb. A countercurrent exchange is also created (vasa recta). -Countercurrent system: *parallel flow, which changes to opposite direction *the outlet is close to the inlet *occurs between the LH and vasa recta Biochemistry II Pouria Farsani 2013 237 Distal nephron transport processes The distal part of the nephron is the: -Distal convoluted tubule -Connecting segment -Collecting duct Significantly less salt and water is reabsorbed than in the proximal part. Approx. 9 % of filtered Na+ and 19 % of filtered water is reabsorbed in the distal nephron. The distal nephron is also able to create a steep concentration gradient between the urine and plasma. Furthermore, the difference is that the epithelium in the distal nephron is much less permeable than the epithelium in the proximal tubule hence permeability to water is lower and variable. The fluid which enters the convoluted tubule is hypotonic when compared to plasma. Distal convoluted tubule Na+ /Clcotransporter is found in the cell membrane of the epithelial cells of this portion. This transporter can be inhibited by thiazide diuretics. Na+ is again pumped by Na+ /K+ ATPase at the basolateral side. The permeability to water is low and ADH has no effect on this portion. Biochemistry II Pouria Farsani 2013 238 Connecting segment – cortical collecting duct The collecting duct is an important site for the regulation of K+ excretion. The cortical collecting duct secretes most of the secreted K+ , mainly by the principal cells. K+ enters the cells on the basolateral membrane via Na+ /K+ ATPase and passes through the K+ -channels on the luminal membrane. Na+ on the other hand enters the cell by diffusion via the Na+ -channel (ENaC – epithelial Na channel). Aldosterone activates the ENaCand Na+ /K+ /ATPase channels, increasing their synthesis. Therefore, aldosterone action results in the increase of Na+ reabsorption and K+ secretion! The antagonist of aldosterone, spironolactone, block the effects of it => ↓ Na+ reabsorption and ↓ K+ excretion. Spironolactone acts as potassiumsparing diuretics. The intercalated discs between the principal cells of the collecting ducts are responsible for acid-base transport. -Alpha-intercalated cells – secrete H+ ions using K+ /H+ /ATPase and vacuolar H+ -ATPase on the luminal membrane while the Cl- /HCO3 exchanger resides on the basolateral membrane. At this sight, HCO3 is reabsorbed in a similar manner as in the proximal tubule. Furthermore: blood pH ↓ => engagement of H+ -pumps in the luminal membrane of alpha-cells => ↑ H+ secretion. Biochemistry II Pouria Farsani 2013 239 -Beta-intercalated cells – opposite polarity. They secrete HCO3 in exchange for Cl- and reabsorb H+ . Blood pH ↑ => HCO3 secretion by beta-cells increases. Collecting duct ADH influences the epithelial cells in the cortical and medullary collecting ducts. In case ADH is absent the cells will be impermeable to water => reabsorption of ions in such circumstances => osmotically dilute urine. When ADH is present => permeability of cells to water ↑ => concentration of urine ↑. As the osmolality of urine increases the ADH level in plasma rises as well. This hormone binds to a specific V2 receptor in the basolateral membrane => activation of Gs protein => activation of adenylate cyclase => ↑ cAMP => activation of protein kinases => activation of phosphorylation of other proteins => movement of intracellular vesicles containing aquaporin-2 (AQP2) towards the liminal membrane and integration of aquaporins into the membrane => ↑ luminal permeability to water. At the basolateral side water leaves the cell via AQ3 and AQ4 => concentrating urine in the collecting duct. This response takes minutes. ADH however, has a delayed effect on the collecting duct; it increases the transcription of the gene for AQ2. Biochemistry II Pouria Farsani 2013 240 There is a difference in permeability to urea between the cortical and medullary portions where the cortical portion is impermeable to urea whereas the medullary portion is permeable. Urea escapes into the interstitial medulla from the medullary collecting duct and from there recycled back into the descending limb of LH. Biochemistry II Pouria Farsani 2013 241 The capillaries which make up the renal countercurrent system run along as loops with LH are freely permeable to water and solutes. However due to the exchange between the capillaries of the ascending and descending limbs the osmotic gradient is not disrupted. In case the capillaries would run directly (not in the counterflow arrangement) the medullary osmotic gradient would disappear. Aquaporins These are membrane channels for water which are critical for water content in the cells. They are small hydrophobic integral membrane proteins which are impermeable to charged molecules including protons. In the membranes they are arranged in tetramers, each of the monomers (six membrane alphahelical domains) is a channel for the water molecules. Water molecules pass through the aquaporins individually bidirectionally depending on the osmolality of the environment. There are 13 isoforms of aquaporins, 8 of which are found in the kidneys. Some of the aquaporins are adjustable, some are kept open. Diuretics Biochemistry II Pouria Farsani 2013 242 Summary schemes for potassium and transport processes regarding questions 55-57 This table is important! Biochemistry II Pouria Farsani 2013 243 58. Endocrine functions of the kidney (erythropoietin, renin-angiotensin system, aldosterone, calcitriol) Erythropoietin Erythropoietin promotes the proliferation and maturation of red blood cells. It is secreted by the liver in the fetus and chiefly by the kidney (90%) in the postnatal life. When O2 ↓ (high altitude, hemolysis, etc.) => production of heterodimer HIF (hypoxia inducible factor) => gene expression => erythropoietin production ↑ => RBCs and fraction of reticulocytes ↑ In other words we summarize erythropoietin with the following points: • 166 AA • It is synthesized by fibroblasts of the renal cortex (liver) • It is the most important regulator of erythropoiesis by: -Increasing cell differentiation and proliferation -Increasing gene expression for: *transferrin *globins *enzymes for heme synthesis Biochemistry II Pouria Farsani 2013 244 Renin-angiotensin system See also q. 44 Renin is a proteolytic enzyme which is secreted by juxtaglomerular cells in the renal afferent arterioles. Remember: Afferent => Glomerulus => Efferent (A.G.E) Stimuli: -A decrease in blood flow (below 90 mmHg) is the main stimuli. It will trigger the renal baroreceptors in order to release renin (various kinds of hypotension, renal artery stenosis) -Reduced NaCl intake in the macula densa of the distal tubule => activation of beta1adrenoreceptor in juxtaglomerular cells. These pathways are activated and strengthened by each other where a: -Decrease in effective arterial volume due to bleeding, diarrhea, low salt intake, heavy sweating etc. => renin ↑ -Increase in effective arterial volume => inhibition of renin release Renin functions as follows: Renin => catalyzes cleavage of angiotensin I from angiotensinogen (released from the liver) => angiotensin-converting-enzyme (ACE) produced in lungs, etc. which is bound to the surface of endothelial cells, cleaves two amino acids from angiotensin I producing => angiotensin II – this occurs approx. 30-60 min after the drop in blood pressure. Biochemistry II Pouria Farsani 2013 245 The effects of angiotensin II on the balance of Na+ and water are: Production and secretion of aldosterone ↑ (from zona glomerulosa of adrenal cortex) Na+ reabsorption in proximal tubule ↑ (remember that water “loves” to follow sodium…) ADH ↑ => thirst ↑ -Furthermore, angiotensin II => vasoconstriction ↑ (during bleeding for instance in order to maintain blood pressure) -Inhibition of ACE => treatment for hypertension Aldosterone See q.44 Biochemistry II Pouria Farsani 2013 246 Calcitriol (1.25-dihydroxyvitamin D, t1/2 ~ 4-6h) Calcitriol (lipophilic secosteroid) which is also known as vitamin D is secreted from the cells of proximal tubules. It is synthesized as follows: Cholocalciferol (Vit. D3) is produced from hepatic 7-dehydrocholesterol in the skin via an intermediate product (previtamin D) in response to UV light. Cholocalciferol binds to Dbinding protein (with higher affinity than previt. D) => liver => transformation to calcidiol (25-hydroxycalciferol), the form which vitamin D is stored as in the liver. In the kidneys calcidiol is transformed into calcitriol by the action of 1-alpha-hydroxylase – the activating enzyme. 24-hydroxylase on the other hand deactivates calcitriol. The following situations stimulates the activity of 1-alpha-hydroxylase • ↓ Serum Ca2+ • ↑ Parathormone levels (PTH levels rises with hypocalcaemia) • ↓ Serum phosphate The actions of clacitriol: -Provides Ca2+ and phosphate to ECF for bone mineralization -In children vitamin D deficiency causes rickets whilst in adults it causes osteomalacia Biochemistry II Pouria Farsani 2013 247 The scheme below will summarize the most important roles of calcitriol: Biochemistry II Pouria Farsani 2013 248 59. Nitrogenous low-molecular constituents of blood plasma and urine (alanine, glutamine, urea, creatinine, uric acid, NH4 + ) – origin, main factors affecting their plasmatic concentration and excretion Nitrogenous compounds Origin Factors affecting conc. + excr. Excretion/d ay (mmol) Alanine Proteolysis. Glucose-alanine cycle (proteolysis in muscle) Intake of prot. Elimination of nitrogen from muscles during proteolysis (proteolytic activity in muscles ↑) Glutamine Proteolysis and Glutamate + NH4 + . Reaction is catalyzed by glutamine synthase. Origin is mostly from muscle tissue but also liver and brain Intake of prot. Acidosis (synthesis in liver ↑) (converted to NH4 + in kidneys) Urea Detox. of NH3 by the liver Intake of prot. Pregnancy, catabolism ↑ (hemorrhage in GIT, intense workout), defects in renal function (GFR <30 %), liver disease (conc. ↓) acidosis (synth. In liver ↓) 330-600 Creatinine Breakdown of creatine phosphate in muscles Nephropathies (conc. ↑), skeletal muscle damage 9-16 Uric acid Catabolism of purine bases Purine content in diet, Metabolic disorder (excessive purine synthesis), increased breakdown of nucleic acids (leukemia, cytostatics, radiotherapy), renal disease (decreased excr. 2.4-3.5 NH4 + See: “sources of ammonia” in q. 16 Acidosis (excr. ↑), hypercatabolic state, 20-50 Approx. 4-14 mmol of free AA are excreted per day. See also “nitrogen balance”, in q. 14 It is important to have an integrative knowledge in this question regarding the pathway of each compound! This is found in the previous questions, especially in questions regarding metabolism. Biochemistry II Pouria Farsani 2013 249 60. Major fractions of the plasma proteins, main components in electrophoretic fractions (albumin, haptoglobin, transferrin, examples of the acute phase proteins, their origin and natural functions, principles of appreciating the alterations in individual plasma proteins) The plasma is the liquid component of blood constituting approx. 54% of the total blood volume. In plasma, fibrinogen and other clotting factors are present. Plasma is obtained by collecting blood into test tubes that contain anticoagulants. Composition of plasma: -Water (90%) -Ions -Molecular non-electrolytes -Proteins Division of plasma proteins into functional groups: -Transport prot. -Acute phase prot. -Proteins protecting against infection – defense -Coagulation factors – clotting and fibrinolysis -Enzymes and their inhibitors -Antioxidant prot. -Maintenance of oncotic pressure Structural types of proteins: -Simple polypeptides -Glycoproteins -Lipoproteins (complex) The plasma proteins keep the colloid-osmotic pressure, the acid-base balance and they serve as an amino acid pool The reference range of total proteins in serum for adults is 65-85 g/l. -Most important plasma proteins: • Transport proteins -Albumin -Transferrin -Ceruloplasmin -Haptoglobin -Hemopexin -Prealbumin -RBG (retinol binding globulin) -TBG (thyroid binding globulin) Biochemistry II Pouria Farsani 2013 250 -Transcortin -SHBG (sex hormone binding globulin) -Transcobalamines • Defense proteins -Immunoglobulins -C-reactive protein, CRP • Proteins of inflammation -CRP, C3, C4, C1, INA -Alpha-1-antitrypsine -Alpha-1-antichymotrypsine -Alpha-1-acidic glycoprotein -Haptoglobin -Ceruloplasmin -Fibrinogen • Clotting factors -Hypoproteinaemia – due to decreased proteosynthesis, insufficient intake in food or severe acute infectious disease. Plasma dilutions when giving large infusions or blockage of kidney excretion can also result in hypoproteinaemia -Hyperproteinaemia – mostly due to dehydration. It is rarer than hypoproteinaemia however. Increased synthesis of immunoglobulins (gammopathy) can also be a reason. Main components in electrophoretic fractions Separation and quantitative analysis of main protein fractions in serum. Migration of proteins is dependent on the mobility of proteins (net electrical charge, isoelectric point, molecular mass and size of proteins) and properties of the support. About 10 000 proteins are estimated from which 22 high abundance proteins represent approx. 99% of total protein in human plasma (see table below). Separation takes place at pH 8.6 in agarose gel and stained with amido black. Biochemistry II Pouria Farsani 2013 251 -Acute inflammatory disease: increase of alpha1- and alpha2-globulin bands (acute phase proteins). Later the gamma- globulins are increased and albumin is decreased -Chronic inflammation: increase in the gamma-globulin fraction Albumin (see also q. 43) Albumin is the only fraction which is formed by one protein; the others are mixtures in electrophoresis. Albumin which is the major plasma protein (35-53 g/l) is produced in the amount of 10-12 g/day. It has a biological half-life of 19-20 days. The important functions of albumin are: -Maintaining oncotic pressure in capillaries -Acting as a buffer base – because of its negative charge (~12mmol/l) it acts as a buffer base and binds Ca2+ (~50% of total calcium) -Transport – of FFA, bilirubin, steroid and thyroid hormones (weakly and non-specifically) and drugs (e.g. salicylates, penicillins, sulfonamides and barbiturates) Hypoalbuminemia – occurs in liver disease, nutritional depletion, loss of albumin in renal diseases, chronic intestinal inflammations, vast burns, hyperhydration. The result of this condition =>plasma oncotic pressure ↓ => edema Haptoglobin (0.4-2.1 g/l) Haptoglobin is produced mostly by hepatocytes but also by other tissues: e.g., skin, lung, and kidney. It is a plasma alpha2-sialoglycoprotein which binds extracorpuscular hemoglobin. Doing so it prevents both iron loss and kidney damage during hemolysis. It then transports then hemoglobin into phagocytic cells (reticuloendothelial system) where elimination takes place. The haptoglobin-hemoglobin complex is too large to pass through the glomerular filter thus haptoglobin function to prevent loss of free hemoglobin into the kidney, conserving valuable iron and preventing kidney damage. Biochemistry II Pouria Farsani 2013 252 Hemolytic anemia results in the decrease of its level. This is due to the hemoglobinhaptoglobin complex having a lower half-life than haptoglobin itself, thus hemolytic anemia (constant release of hemoglobin) would result in an increased amount of complexes with an increased rate in elimination. Haptoglobin is also an acute phase protein where an increased level in plasma is seen in a variety of inflammatory states. Haptoglobin exists in three polymorphic forms: -Hp 1-1 -Hp 2-1 -Hp 2-2 Furthermore, haptoglobin also protects against free radicals in hemolysis and exhibits an antiinflammatory action by inhibition of prostaglandin synthesis. Transferrin It is a plasma glycoprotein (beta1-globulin fraction) with a concentration of 2.5-4.5 g/l (30- 50umol/l) in serum. Its half-life is 8.5 days. It has two binding sites for Fe3+ and is has a total iron binding capacity > 60umol/l The serum Fe3+ is the transferrin-Fe3+ and its concentration is about 10-20umol/l -14-26 umol/l for men -11-22 umol/l for women The saturation of transferrin with Fe3+ equals usually about 1/3 of the total iron binding capacity. During iron deficiency, transferrin biosynthesis is increase. At the same time the plasma iron concentration decreases. Therefore we can see a decrease in saturation of transferrin In chronic alcoholics the glycosylation of transferrin is impaired this carbohydrate-deficient transferrin (CDT) is followed. It is a marker of chronic alcohol abuse. -Increase: iron deficit -Decrease: iron overload, acute inflammations (negative acute-phase protein), impaired liver proteosynthesis. Acute phase proteins (APP) These proteins are elevated as the body responds to inflammation. They can increase from 50% up to 1000-fold in the case of CRP. Chronic inflammation and cancer are other are also accompanied with an elevation of the acute phase proteins. When we speak about APP we distinguish between positive APP and negative APP. Biochemistry II Pouria Farsani 2013 253 -Positive APP – increased values are found as their hepatic synthesis is induced by numerous of cytokines that enter the circulation as products of e.g. macrophages, epithelial cells and fibrocytes. There are two types of APP: -Negative APP – decreased values are found as their synthesis in the liver is decreased in the catabolic state. The negative APP are (followed by their response time): *Transthyretin (prealbumin) - <24 hrs. *Transferin – 24-48 hrs. *Albumin - > 48 hrs. Below, the different APP, both positive and negative, are going to be discussed in detail -Alpha1-antitrypsin: (2-4g/l) it is the major component of the alpha1-band. It is synthesized by hepatocytes and macrophages and it acts as the principal serine protease inhibitor, inhibiting the proteinase released from leukocytes (mainly elastase) as well as other proteases that may occur in plasma. These proteinases attack the elastin between alveoli in the lung, which is why deficiency is observed in certain cases of emphysema -Ceruloplasmin: alpha2-band. (150-600 mg/l) This protein binds copper (90% of copper plasma). However, it does not take part in Cu2+ transport. Its biological function is the ferroxidase activity that prevents the occurrence Fe2+ ions and possible Fenton reaction. Ceruloplasmin is thus considered as one of the endogenous antioxidants Low levels of it is associated with Wilson Disease. It is a blue protein which firmly binds 8 Cu2+ ions. It contains 90% of Cu2+ plasma content. The other 10% is carried by albumin. Ceruloplasmin exhibits a copper dependent oxidase activity. It oxidizes Fe2+ in transferrin to Fe3+ . -Haptoglobin: see previous two pages -Albumin, prealbumin and transferrin: decreased levels, negative acute-phase proteins -Ferritin: positive acute-phase protein whose level is elevated in acute inflammations -C-reactive protein (CRP): this protein has an ability to precipitate C-polysaccharide of pneumococci in the presence of Ca2+ ions. It plays a significant role in the non-specific immune response which appears in the initial phase after the penetration of antigens into the organism. CRP is the dominant acute phase protein with rapid response Biochemistry II Pouria Farsani 2013 254 *Elevation: after 6h, max. after 48h. Physiological values are < 6 mg/l, values above that are counted as positive. CRP has the most rapid and highest increase in its concentration. A bacterial inflammation is accompanied by a marked and early increase. Viral inflammation => small increase. CRP not only gives a good indication whether antibiotics should be applied or not – it is also used to monitor the effectiveness of the therapy. Biochemistry II Pouria Farsani 2013 255 61. Metabolism of iron (absorption, transfer and distribution in the body, functions, iron balance) Absorption Iron absorption occurs mainly in the duodenum and it varies according to the need of iron. About 3 to 15% of iron ingested is absorbed. However, it may increase to 25% during iron deficit. Because there is no physiological pathway for iron excretion, its losses are controlled by this regulated absorption. Values regarding losses and absorption will be discussed in “iron balance” below. Iron is ingested either as heme (hemoglobin, myoglobin found in meat and fish) or non-heme iron: -Absorption of heme iron – this form of iron is absorbed relatively efficiently by heme transporter. Inside the cell heme oxidase releases Fe2+ from the heme. It then either enters the bloodstream or is converted to Fe3+ and bound by ferritin in order to remain in the mucosa. The Fe3+ which is bound by ferritin returns to the gut lumen during cell turnover -Absorption of non-heme iron – this form of iron can only be absorbed as Fe2+ . Ferric reductase which is present on the surface of enterocytes converts Fe3+ to Fe2+ . Furthermore, ascorbic acid also aids in this conversion. The iron is then transferred from the apical surface of the enterocytes into the cell by the divalent metal transporter DMT1 (secondary active transport). This transporter is not specific for iron, as it can transport a wide variety of divalent cations. Once inside the cell iron is either stored as ferritin or it is transferred across the basolateral membrane into the plasma. Transport across the basolateral membrane is carried out by ferroportin. This protein may interact with the copper-containing protein hephestin Biochemistry II Pouria Farsani 2013 256 (membrane-bound homologue of the ferroxidase protein ceruloplasmin) with ferroxidase activity. It converts Fe2+ back to Fe3+ , the form in which it is transported in the plasma by transferrin. The ferroxidase activity is important in the release of iron from the cells. Gastroferrin is a component of gastric secretion. It is a glycoprotein that binds Fe3+ , maintaining it soluble. -Regulation of absorption – hepcidin plays a key role. There are however two ways by which iron uptake is regulated: 1) Iron deficit => aconitase (iron-regulating protein) in the cytosol binds with ferritin mRNA, thus inhibiting mucosal ferritin translation => larger quantities of absorbed Fe can enter the bloodstream. 2) Hepcidin (polypeptide 25 AA) diminishes the density of hephestin in the plasma membrane of mucosal cells (hepatocytes and macrophages as well). Secretion of hepcidin from the liver decreases during iron deficit. This results in the increase of Fe3+ release into the blood. Iron overload stimulates the biosynthesis of hepcidin and vice versa. The effects of hepcidin can be summarized accordingly: -Reduces Fe2+ absorption in duodenum -Prevents the release of recyclable Fe from macrophages -Inhibits Fe transport across the placenta -Diminishes the accessibility of Fe for invading pathogens Biochemistry II Pouria Farsani 2013 257 Transfer and distribution -Transferrin – a plasma glycoprotein found in the β1-globulin fraction. *plasma concentration: 2.5 – 4.0 g/l (30 – 50 µmol/l) *transferrin molecule has two binding sites for Fe3+ ions. Its total iron binding capacity (TIBC) for Fe ions is higher than 60 µmol/l *serum Fe3+ (i.e. transferrin-Fe3+ ) concentration is about 10-20 µmol/l, 14 – 26 µmol/l ♂ 11 – 22 µmol/l ♀ *saturation of transferrin with Fe3+ equals usually about 1/3 *biosynthesis of transferrin is stimulated during iron deficiency (decrease of plasma iron concentration) hence a decrease in the saturation of transferrin is observed *carbohydrate-deficient transferrin (CDT) is monitored in alcoholics – marker of abstinence Biochemistry II Pouria Farsani 2013 258 -Uptake of iron by cells – this takes place by specific receptor-mediated endocytosis. Some receptors are released from the plasmatic membranes. Increase in serum concentration of those soluble transferrin receptors is the earliest marker or iron deficit -Lactoferrin – in milk tears and saliva. It is very similar to transferrin. It is has a bactericidal effect by binding all iron needed for growth of bacteria -Ferritin – occurs in most tissues (liver, spleen, bone-marrow, enterocytes). The protein is apoferritin when it has not bound any iron. One molecule can bind up to 4500 Fe3+ ions. Minute amounts of ferritin is released into blood plasma from extinct cells. Plasma ferritin concentration (25-300 µg/l) is proportional to the ferritin stored in tissues, unless the liver is impaired (increased release from the liver) In case the loading of ferriting is excessive, ferritin aggregates into its degraded form, hemosiderin, in which the mass fraction of Fe3+ can reach 35%. Biochemistry II Pouria Farsani 2013 259 Functions Iron functions as a prosthetic group in enzymes in order for their proper function. Iron is also the prosthetic group of heme proteins, hemoglobin and myoglobin. This is essential for their ability to bind and transport oxygen. Furthermore iron is important for: -Connective tissue synthesis -Proper function of the immune system -Synthesis of DNA -Component of neurotransmitters Iron balance The balance together with a summary of iron is given in the table below: Biochemistry II Pouria Farsani 2013 260 62. Antigens and antibodies (the terms complete antigen – immunogen, hapten, sequential and conformational immunogenic determinants – epitopes, classifications of the human immunoglobulins and their molecular structures, specific roles of different Ig domains). The antibody-antigen reaction (formation of an immune complex, precipitation and agglutination as secondary reactions) We explain some basal terms before treating the subjects mentioned above -Immunity – the body’s ability to react to the presences of a foreign protein or heteropolysaccharide (an antigen) with a useful immune response as to eliminate antigens (microorganisms, transplants, tumor cells) in order to retain the molecular integrity and individuality of its own -Immune response – the complex of reactions mediated through lymphoreticular system that follows an invasion of the foreign antigen into the body -Lymphoreticular system (lymphoid organs or tissues) *central, primary – thymus and equivalents of bursa of Fabricius (present in birds) *peripheral, autonomic – spleen, lymphatic nodules, bone marrow, tonsils, Peyer’s patches (small intestine), T- and B lymphocytes Immunogen Immunogens a.k.a. complete antigens are mostly molecular substances. After these compounds invade the body they are recognized as foreign compounds by immunocompetent cells. This initiates a specific immune response where antibodies are produced. Hapents Hapents are small organic molecules such as short peptides or certain drugs. Upon invasion, they are also recognized as foreign compounds. However, they do not initiate an immune response. Hapetns may also provoke formation of antibodies, provided that they are attached to a macromolecular carrier, which can be quite neutral from the immunological point of view. This means that an immunogen does originate, as a carrier which possesses a haptenic determinant. -Antigens from the chemical point of view are: *proteins and polypeptides *saccharidic components of glycolipids and glycoproteins *bacterial heteropolysaccharides *peptidoglycans *lipopolysaccharides *some nucleic acids can act as immunogens *currently, some phospholipids are also mentioned as immunogens Biochemistry II Pouria Farsani 2013 261 Epitope And epitope or antigen determinant is the part of immunogen molecule that initiates the specific immune response (it can be very small) We distinguish between two types of determinants on the surface of native protein molecules: Sequential – (3-8 amino acid residues) Or Conformational – (up to 20 amino acid residues) There are also saccharidic determinants which are mostly short oligosaccharides (1-5 monosaccharide units) at the non-reducing end. Specific roles of different Ig domains -Immunoglobulins • Glycoproteins with different content of saccharide component • Present in blood and tissues • Synthesized as a defense against invasion of the body by foreign organism • Each antibody molecule contains at minimum two identical light chains and two identical heavy chains (see scheme) -Light and heavy chains • The light chains, kappa (κ) and lambda (λ), have their structural differences in the CL regions • The heavy chains, gamma (γ), alpha (α), my (µ), delta (δ) a epsilon (ε), have their structural differences in the CH region • The immunoglobulin class is determined by the type of heavy chain Biochemistry II Pouria Farsani 2013 262 • The variable region of the immunoglobuline consists of variable domains: VH domain and VL domain • There are no two variable regions with identical sequences • Variable regions are comprised of relatively invariable regions and other hypervariable regions (about 5 AA): light chain has 3 hypervariable regions whilst the heavy chain has 4 hypervariable regions • Hypervariable regions comprise the antigen binding site -Functions of immunoglobulin domains – variable domains • Variable domains VL and VH are responsible for the distinctive function of immunoglobulins. Together they form a binding site for a specific antigenic determinant • The specificity of the binding sites is high. It depends on the amino acid sequence of hypervariable loops (complementarity-determining regions. There are three in VL and four in VH) • Each antigen binding site can bind non-covalently on antigenic determinant or one hapten. The strength of this interaction is called affinity • Binding sites exhibit high affinity for only a limited number of similar determinants – this is considered as a rule. With increasing strength of interactions, the number of such “cross-reacting” determinants increases. Numerous determinants are bound very weakly, however, these weak interactions are not significant practically Biochemistry II Pouria Farsani 2013 263 -Functions of immunoglobulin domains – constant domains – these mediate biological functions known as effector functions: • Interaction of variable domains with the antigen initiate the process which results in antigen elimination • Domains CL and CH1 are connected by a disulfide bond. The change in conformation evoked by the interaction with antigen induces conformational changes of all the remote constant domains. In the complement cascade, CH1 domain binds the complement component C4b • The hinge region joins both heavy chains. In the heavy chains of IgM, the hinge is substituted by a special domain, CH2 • Domains CH2 of immunoglobulins IgG and IgM are binding sites for the first complement component C1q or certain immunomodulating peptides • Domains CH3 (in IgM CH4) enable, together with domain CH2, cytotropic reactions – binding to Fc-receptors of phagocytes and B- or T cells, which initiates readily either phagocytosis of immunocomplexes, or formation of the complex with the cell exposing an antigen – a signal for extinguishment of the cell Classification of the human immunoglobulins and their molecular structures Biochemistry II Pouria Farsani 2013 264 -IgG – immunoglobulins class G -IgA – immunoglobulins class A Biochemistry II Pouria Farsani 2013 265 -IgM – immunoglobulins class M Biochemistry II Pouria Farsani 2013 266 Antibody-antigen reaction – formation of an immune complex • The primary event is the formation of an antibody-antigen complex – binding of the specific immunoglobulin to the corresponding antigen. This binding usually results in marked conformational changes • Antigens are either soluble (colloid particles), or corpuscular (antigenic determinants on the surface of cells or other insoluble particles). Soluble Ab complexes are called immunocomplexes • Two stages of the formation of immunocomplexes can be distinguished: -The binding itself that is relatively fast (formation of non-covalent interactions, the most important of which are the hydrophobic) -Complex transformation, which can take longer time (the complex is stabilized through formation of more interactions) Antibody-antigen reaction – precipitation and agglutination as secondary reactions The secondary processes are associated with formation of Ag-Ab complexes -Immunopercipitation – immunoglobulin molecules include two antigen-binding sites, so that they can cross-link soluble multivalent antigens at a certain limit concentration (and a proper concentration ratio of both). These three-dimensional networks are insoluble and visible as turbidities or precipitates. An excess of both antibody and antigen inhibits precipitate formation -Agglutination of cells or other particles – a similar process: Immunoglobulins act as cross-links between antigenic determinants of multivalent corpuscular antigens (cells, bacteria, generally agglutinogens). Aggregates of particles (agglutinates) are easily distinguishable from sediments of particles that are not agglutinated … -Cytotropic reactions – Fc receptors bind immunocomplexes, the result may be either phagocytosis of the immunocomplex or (mediated by cytotoxic T-cells) extinguishment of the antigen-exposing cell -Triggering of the complement cascade (the classical pathway of activation of complement components) is a process that leads to the lysis of foreign target cell Biochemistry II Pouria Farsani 2013 267 63.Blood clotting cascade. Fibrinogen, transformation to fibrin, and fribinolysis Platelets • Small fragments from megakaryocytes • They have: no nuclei, few mitochondria, cytosolic enzymes which can generate energy, granules with specific substances • They have high content of actin and myosin – ability to contract • They remain functional for an average of 10 days -The action platelets during thrombosis or hemostasis – normally the platelets do not stick to the smooth endothelial wall of blood vessels. However, disruption of the lining due to an injury/damage of the vessels results in the adhesion of platelets to the exposed collagen. The platelets are bound by means of specific membrane proteins (GP Ib/IX) and the von Wilebrand factor which circulates in plasma. The platelets are activated when binding to collagen, they then quickly reorganize their cytoskeleton (actine polymerization), change their shape which helps them to adhere to the collagen – formation of pseudopodia. Together with changing chape upon activation, release of granule content (see table below) and aggregation takes place as well. | Biochemistry II Pouria Farsani 2013 268 -Activation of platelets – stimulation of the phosphatidylinositide pathwy: • Thrombin binds to receptors on the surface of platelets => activation of Gq protein => phospholipase Cβ => hydrolysis of PIP2 to IP3 and diacylglycerol (DG) DAG => activation of protein kinase C => phosphorylates protein pleckstrin => aggregation of platelets and release of storage granules: -Vasoconstriction (serotonin, PDGF, TXA2, PAF) -Adhesion (vWf) -Activation of other platelets (ADP, TXA2, PAF) • When the platelet binds thrombin it activates the change of the shape of platelet as well as phospholipid scrambling. This exposes the negatively charged phospholipis on the outer cell surface • Arachidonic acid mobilizes and synthesis of thromboxane takes place • The exposure of glycoproteins GP IIb/IIIa on the surface of platelets supports the platelet-platelet interaction Biochemistry II Pouria Farsani 2013 269 Hemostasis – arrest of bleeding • A processes that follows after injury of a smaller vessel or by trauma • It is composed of a series of interactions between the vessel wall, blood platelets, coagulating factors and the fibrinolytic system • The ultimate goal is to stop bleeding during several minutes We distinguish four phases of hemostasis: 1. Vasoconstriction 2. Formation of a primary platelet plug (white thrombus) 3. Formation of a fibrin network and a secondary platelet-fibrin plug 4. Activation of the fibrinolytic system – the digestion of the fibrin plug The endothelium damage triggers the secretion of von Willebrand factor from endothelial cells Subendothelial collagen is Adhesion of platelets to collagen by means of von Willebrand factor Collagen activates the bound platelets Biochemistry II Pouria Farsani 2013 270 Aggregation is supported by ADP, fibronectin, TXA2 and PAF from thrombocytes 1. Vasoconstriction – the vascular spasm reduce the blood flow by a reflex which is stimulated by products from the platelets: -Serotonin -PDGF -Tromboxan A2 in this manner the bleeding from small vessels can be stopped 2. Platelet aggregation – compounds stimulate aggregation of platelets which leads to the formation of the primary platelet plug. The compounds are: -Fibronectin – from platelets -vWF – from platelets -TXA2 – from platelets -Fibrinogen – formed in the coagulation cascade -Thrombin – formed in the coagulation cascade 3. Blood coagulation The two systems cooperate – both activate plasmatic factor X that converts prothrombin to thrombin. Thrombin then activates fibrinogen to fibrin. There are two systems of blood coagulations where the coagulation cascade of secondary hemostasis has two pathways: 1. The contact activation pathway (intrinsic pathway) – accessory system of coagulation Biochemistry II Pouria Farsani 2013 271 2. The tissue factor pathway (extrinsic pathway) that leads to the fibrin formation – the primary pathway for the initiation of blood coagulation The two systems above cannot function separately: -The primary system is essential for incitation of coagulation -The accessory system is not essential for inicitation of coagulation -Coagulating factors – these are mostly synthesized in the liver and released into the blood in the form of inactive precursors (zymogens) *Factor VIII (and probably IX and XII) are also synthesized in the spleen *von Willebrand factor is synthesized I endothelial cells *The coagulating factors bind to the negatively charged surface of phospholipids membranes of platelets, erythrocytes, leukocytes and endothelial cells *Ca2+ is important for the binding -Activation of coagulation factors – most of the factors circulate as inactive zymogens (inactive precursors) of serine proteases (XII, XI, IX, VII, X, II, thrombin) -These are activated by hydrolytic cleavage of a part of the molecule at a specific site => zymogen becomes active protease. Amplification of the effect is the followed -Thrombin – this is a serine protease which is the key enzyme of hemostasis. It affects: *Coagulation and anticoagulation processes *Fibrinolytic and antifibrinolytic processes The interplay of all components of hemostasis is ensured by various sensitivity to thrombin! Biochemistry II Pouria Farsani 2013 272 -Initiation of coagulation: Damage of blood vessel => release of endothelium tissue factor (TF) TF reacts with factor VII that circulates in blood Factor VII is activated to VIIa TF functions as a cofactor of proteolytic activity of factor VIIa and this activates factor X to Xa Factor Xa together with factor Va, platelets, phospholipids, PI and Ca2+ from prothrombinase Prothrombinase converts a small amount of prothrombin to thrombin – the reaction occurs on the surface of platelets Concurrently, tissue factor pathway (TFPI) is activated an inhibits VIIa-TF complex Tissue factor and factor VIIa also activates factor IX in the intrinsic pathway and enhance the activation of prothrombinase Biochemistry II Pouria Farsani 2013 273 -Amplification of coagulation: The small amount of thrombin that was formed by the initiation activates the accessory (intrinsic) system Activation of factors Xi, IX and cofactors VIII and V Factor IX with cofactor VIIIa and platelets phospholipids and Ca2+ form the intrinsic tenase complex Factor Xa similarly as in the initiation, generates Va, PI and Ca2+ prothrombinase complex and activates the formation of thrombin in a sufficient amount for cleavage of fibrinogen Biochemistry II Pouria Farsani 2013 274 -The role of calcium cations – factors II, VII IX and X. IX contains γ-carboxylglutamate residues (gla). Their conversion to active forms with protease activity requires the presence of Ca2+ . The Ca2+ ions bind gla and PI, a PS from membrane of platelets Fibrinogen -The structure of fibrinogen involves two triple helices of polypeptide chains – Aαααα, Bββββ and γγγγ - dimer (AααααBββββ γγγγ)2 -It is a soluble glycoprotein, 330 kDa, with a plasma concentration of 1.5-4g/l (plasma beta2globulin fraction) Biochemistry II Pouria Farsani 2013 275 Transformation to fibrin (see scheme above as well!) Thrombin hydrolyses the bonds between the fibrinopeptides and the α and β portions of A α and B β chains of fibrinogen Fibrinomonomer (αβ γ)2 without negatively charged fragments, A,B is formed Fibrin monomers spontaneously associates by formation of non-covalent bonds => formation of fibrin clot Thrombin concurrently converts factor XIII to factor XIIIa Factor XIIIa is specific the transglutaminase – it catalyzes formation of peptide bonds between -CONH2 of glutamine and ε-NH3-of lysine The covalent bond stabilize the fibrin cloth (cross-links) – a more stable fibrin clot is formed Biochemistry II Pouria Farsani 2013 276 -Structure illustration of formation of cross-links -Control of clotting – levels of circulating thrombin must be carefully controlled in order to prevent further fibrin formation or platelet activation. At each point of coagulation cascade, a feedback mechanism must keep the balance between activation and inhibition Fibrinolysis Fibrionlysis is triggered by the proteolytic effect of plasmin Plasmin is formed from plasminogen Plasminogen is circulating as plasmatic zymogen Activation of plasminogen is triggered by proteolytic action of tissue plasminogen activator (tPA) released from vascular endothelium tPA is catalytically inactive unless bound to fibrin Plasminogen and tPA binds to fibrin => plasminogen is activated to plasmin => plasmin digests fibrin network to soluble degradation products Plasminogen can be activated by urokinase as well – it is synthesized in monocytes, macrophages, fibroblasts… -Streptokinase (SK) – a protein secreted by several species of streptococci. It can bind and activate human plasminogen. Therefore, SK is used as an effective and inexpensive clotdissolving drug in some cases of myocardial infarction and pulmonary embolism -Tissue-type plasminogen activator (t-PA, alteplase) and other thrombolytic drugs (streplase, saruplase) are produced by recombinant gene therapy Biochemistry II Pouria Farsani 2013 277 Biochemistry II Pouria Farsani 2013 278 64. Inhibitors of blood clotting – anticoagulants in vivo (function of heparin and coumarin derivatives) and in vitro (prevention of blood clotting, blood plasma) Inhibitors of blood clotting • Anti-thrombin – inhibits thrombin • Protein C system • Tissue factor inhibitor (TFPI) In vitro – prevention of blood clotting, blood plasma -Inhibition of clotting by the action of anti-thrombin III (ATIII) – ATIII is a plasmatic protein produced by the liver. It belongs among the serpins (inhibitors of serine proteases) When ATIII forms a complex with thrombin and other factors (IXa, Xa, Xia, and XIIa) it inhibits their proteolytic function. The affinity of ATIII to thrombin is potentiated by heparin (~1000x) (natural from mast cells or administered) => anticoagulation effect of heparin. An inherited deficiency of ATIII can cause thrombosis! -Control of clotting by the action of thrombmodulin (TM) – a glycoprotein receptor on the surface of intact endothelial cells. It prevents the propagation of clotting into the area where the intact endothel is (follow the scheme below). Biochemistry II Pouria Farsani 2013 279 -Control of clotting by TFPI – the inhibitor of tissue factor. Its synthesis in endothelial cells is triggered by the increased level of factor Xa. It inactivates factors VII, IX and X -Inhibition of clotting by prostcyclins – the endothelial cells produces the prostacyclins (PGI2) which inhibits aggregation of platelets In vivo function of heparin and coumarin derivatives -Heparin – an acidic glycosaminoglycan produced by mast cells: *D-glucosamine L-iduronic acid *D-glucosamine L-glucuronic acid Sulfated. Heparin is obtained from animal courses where: *UH (UHF) – infractioned heparin has non-selective effects (MH 3-40 000) *LMWH – low molecular heparin – obtained by fractionation (MH 4.5-6 000) The pentasccharide chain of heparin is the binding site for ATIII. Specific positions of sulfate groups are decisive for activation of ATIII Biochemistry II Pouria Farsani 2013 280 -Coumarin anticoagulants – inhibits vitamin K cycle in the liver. Its derivative drugs are Dikumarol (Pelentan) and Monokumarin (Warfarin). Warfarin, dikumarol has the principle of its action by being similar to vitamin K. The following coagulating factors are involved: II, VII, IX, X, II, Prot. C and S Biochemistry II Pouria Farsani 2013 281 65. Endothelium – function and special substances produced by endothelial cells Endothelia -The endothelium is a single layer of specialized epithelial cells -The total surface layer is estimated to ~1000 m2 and the mass is ~1% of the total body mass Types of endothelial cells: • Continuous endothelial cells without fenestrae with tight junctions located in CNS – they have pinocytar activity • Continuous endothelial cells without fenestrae with tight junctions located in heart and muscle tissue – they have pinocytar activity • Fenestrated endothelial cells located in: -Gastrointestinal villi -Glomeruli of kidneys -Endocrine glands • Discontinuous endothelium with broad fenestrae without basal membrane located in: -Sinusoids in the liver -Bone marrow -Spleen Functions of endothelial cells • Regulation of blood and tonus of vessel walls • Activation of platelets • Influence in formation of platelet plugs, clotting and clot dissolution • Secretion of many substances • Modification of lipoproteins • Sight of location of many receptor on their surface • A healthy functioning endothelium is crucial for remodeling of blood vessels through the angiogenesis and vasculogenesis during growth and repair of tissues Biochemistry II Pouria Farsani 2013 282 Special substances produced by endothelial cells The glycocalix with negative charge is the carbohydrate –rich layer which lines the vascular endothelium. It is found on the surface of endothelial cells. The composition of the glycocalix can be seen in the picture below: Biochemistry II Pouria Farsani 2013 283 66. Red blood cells (metabolism, membrane, blood group determining structures) Red blood cells are produced in red bone marrow from progenitor cells. The proliferation and differentiation takes several days, up to a week. The production is regulated by erythropoietin which is a glycoprotein: -Produced in the kidneys (liver, bone marrow) -Signal for its synthesis is hypoxia in kidneys -It binds to membrane receptor of progenitor cells and trigger proliferation and differentiation (see also q. 58) Red blood cells (RBC, erythrocytes) are biconcave with a diameter of 8 µm. their deformation is possible. The high surface-to-volume ratio facilitates their role in gas exchange. RBC’s do not have a nucleus or cellular organelles, they have cytoskeletal components. The content of hemoglobin is 28-36pg Hb/erc Reticulocytes still containing ribosomes and elements of ER are released into the circulation where they transform into adult RBC’s – their lifespan is 120 days. Membrane RBC’s have an extra membrane and cytoskeleton proteins that allows the red cell to be stretched and deformed as the it passes through narrow vascular beds. On the cytoplasmic side of the membrane, proteins form a two-dimensional lattice that gives the cell its flexibility. Cytoskeletal proteins are fixed to the inner surface of the membrane and help determine the shape and flexibility of the RBC. The composition of the erc membrane is: -52% proteins -40% lipids -8% saccharides Biochemistry II Pouria Farsani 2013 284 -Anion exchanging protein (band 3) – is a transmembrane protein extending several times across the membrane. Through this protein: *exchange of chlorides for HCO3 (Hamburger effect) -Glycoproteins A,B,C • Transmembrane glycoproteins – single pass type • Its aminoterminal end extrudes out from the surface of the RBC • It is highly sialylated and represents the major part of the glycocalyx on the outer surface. The negative electric charges prevent agglutination of RBC • Polymorphism of glycophorin A in its amino acid sequence denotes the MN blood groups of individuals’ erythrocytes -Cytoskeletal proteins – they are fixed to the inner surface of the membrane and help determine the shape and flexibility of the RBC -Spectrin • The major cytoskeletal protein • Consists of two long polypeptide chains that form a loosely coiled dimer; two dimers form a tetramer • Binding sites for other cytoskeletal and membrane proteins (ankyrin, actin, protein 4.1) • Forms 25% of membrane protein mass and 75% of total cytoskeleton mass Dimer of spectrin Biochemistry II Pouria Farsani 2013 285 -Pathological formations of RBC • Spherocytosis – autosomal dominant hereditary disease – abnormalities in the amount or structure of spectrin Spherical erc are present in blood. They are not deformable and more susceptible to osmotic lysis than normal erc are. This results in hemolytic anemia. • Eliptocytosis – similar to hereditary spherocytosis. However, the red cells assume an elliptic shape. This condition is also due to abnormalities in spectrin Metabolism The main features of erc metabolism are summarized as follows below: • The energy source is anaerobic glycolysis (this is the only way of energy utilization as RBC’s lack mitochondria. Lactate is the product) • Transport of glucose – GLUT-1 • Transport of water – Aquaporin-1 • The pentose phosphate pathway is efficient, it metabolizes up to 10% of the total flux of glucose. NADPH produced is required for the reduction of oxidized glutathione and methaemoglobin • Production of 2,3-BPG • The iron of Hb must be maintained in ferrous state • Glutathione is maintained in reduced from • Certain enzymes of nucleotide metabolism are present • Variety of transporters that maintain ionic and water balance • No catabolism of fatty acids and ketone bodies • Some lipids (e.g. phospholipids, cholesterol) from the red cell membrane can exchange with corresponding lipids of plasma lipoproteins • No oxidative phosphorylation, synthesis of fatty acids, proteins, nucleic acids, cholesterol, lipids Biochemistry II Pouria Farsani 2013 286 -Erythrocytes and oxidative stress – high pO2 and the presence of Fe2+ in hemoglobin represents a menace to processes and structures within the erc. Efficient antioxidants protect the erc from damage caused by oxidative stress: • Superoxide dismutase and catalase – decompose superoxide anion and hydrogen peroxide • Glutathione peroxidase – catalyzes reduction of hydrogen peroxides by GSH (reduced glutathione). GSH is regenerated by NADPH in the reaction catalyzed by glutathione reductase -NADPH is required – for regeneration of glutathione to its reduced form GSH. NADPH is generated in two reactions of the pentose phosphate pathway catalyzed by glucose-6-p dehydrogenase and 6-phosphogluconate dehydrogenase -Deficiency of glucose-6- dehydrogenase – the cause of hemolytic anemia NADHP is not formed in the pentose-phosphate pathway => hydrogen peroxide is not eliminated by glutathione peroxidase Hydrogen peroxide accumulated within the cell Products of peroxidation are formed Erythrocyte membrane is damaged Hemolysis *this is a hereditary diseases caused by point mutations within the gene located in chromosome X *it is very extremely frequent in some regions of the world (tropical Africa, the Mediterranean, certain parts of Asia, and for example among Afro-Americans with 11% of incidence) *the deficiency is relatively benign in the absence of oxidative stress. However, an exposure to oxidants (e.g. drugs – antimalarial pamaquine, sulfonamides, chemicals – naphthalene consumption of fava beans, some infections) may result in a severe attack of hemolytic Biochemistry II Pouria Farsani 2013 287 anemia. This is due to RBC being sensitive to the increase in production of oxygen radicals and peroxides *this enzyme deficiency protects against falciparum malaria. The parasites causing this disease require reduced glutathione and the products of the pentose phosphate for optimal growth *Heinz bodies are present in erc with G-6-P-dehydrogenease deficiency. This is because when reduced glutathione is deficient proteins may be partially denatured. Non soluble proteins are bound to membrane forming Heinz bodies. The erc are rigid and not flexile – they are removed by macrophages in liver and spleen Blood group determining structures -AB0- system – specific enzymes (transferases) are responsible for attachment of specific antigens to the H substance on the surface of the erc. These antigens are either antigen A or antigen B. In the case of recessives, no antigen is bound to the H substance (0-group). In the membrane of erc – oligosaccharides base (H substance or H antigen) – serves as the precursor to which A or B antigen can be attached • Group A – attachment of N-acetyl-D-galactosamine (A-antigen) • Group B – attachment of D-galactose (B-antigen) Biochemistry II Pouria Farsani 2013 288 Biochemistry II Pouria Farsani 2013 289 -Rh blood group system (“Rehsus factor”) – more than 50 antigens of on the RBC surface among which 5 antigens (D, C, c, E, e) are the most important. The commonly used terms Rh factor, Rh positive/negative refer to the D antigen only *existence or absence of the D antigen on the erc surface is determined by allels D/d that are in a fully dominant relationship *Rh+ individuals are dominant DD or heterozygotes Dd *Rh- individuals are recessive homozygotes (dd) with no D antigen in the membrane of the erc *in case an individual has no D antigen on the erc, anti-D antibodies are produced -MN antigen system – polymorphism of glycophorin A in its amino acid sequence denotes the MN blood groups of individuals’ erythrocytes Biochemistry II Pouria Farsani 2013 290 67. Methemoglobin in normal blood (origin, reduction to Hb), inherited and acquired methaemoglobinaemia (nitrates and nitrites in the environment) Origin, reduction to Hb Methemoglobin is a form of hemoglobin, in which the iron in the heme group is in the Fe3+ (ferric) state, not the Fe2+ (ferrous) of normal hemoglobin. Methemoglobin cannot bind oxygen, unlike oxyhemoglobin. It is bluish chocolate-brown in color. In human blood a trace amount of methemoglobin is normally produced spontaneously. But when it is present in excess the blood becomes abnormally dark bluish brown. The NADH-dependent enzyme methemoglobin reductase is responsible for converting methemoglobin back to hemoglobin. -Formation and reduction of methemoglobin – catalyzed by methemoglobin reductase (MetHb) In the blood of healthy individuals, less than 1% of total hemoglobin is present in the form of methemoglobin. Inherited methaemoglobinaemia An inherited deficiency of MetHb redcutase or point mutation of the hemoglobin gene (hemoglobin M) Aquired methaemoglobinaemia This occurs after ingestion of certain drugs (e.g. sulfonamides) or chemicals (e.g. aniline, nitrites, in sucklings also nintrates). Evident cyanosis appears usually when more than 10% of total hemoglobin is oxidized to methaemoglobin … -Megaloblastic (macrocytic) anemia • Anemia connected with production of enlarged erythrocytes (macrocytes) in blood and bone marrow • The cause is diminished DNA synthesis in the precursors of erc, triggered by the lack of vit. B12 or folic acid • Both of these vitamins are needed for synthesis of purine and pyrimidine nucleotides (FH4 transfers one-carbon fragments needed for synthesis, B12 ensures the availability Biochemistry II Pouria Farsani 2013 291 of FH4 – folate trap) • Insufficient DNA synthesis => inability of erc to produce DNA quickly enough to divide at the right time as they grow, and thus grow without mitosis => macrocytes • Causes of megaloblastic anemia: -Folate deficiency *limited intake *malabsorption at damage of intestine (sprue) *anticancer drugs (folate reductase inhibitors) -B12 deficiency *limited intake *lack of intrinsic factor in the stomach (gastrectomia, antibodies against intrinsic factor, or antibodies against parietal cells of gastric mucosa (percinous anemia – originating from autoimmune processes. It causes atrophy of gastric mucosa)) Nitrates and nitrites in the environment -Nitrates (NO3 − ): • Nitrate salts are found naturally on earth as large deposits, particularly of Chile saltpeter a major source of sodium nitrate • Nitrates are mainly produced for use as fertilizers in agriculture High levels of nitrate fertilization also contribute to elevated levels of nitrate in the harvested plant • High nitrate drinking water • Vegetables containing high levels of nitrate. Lettuce may contain elevated nitrate under growth conditions such as reduced sunlight, undersupply of the essential micronutrients molybdenum (Mo) and iron (Fe), or high concentrations of nitrate due to reduced assimilation of nitrate in the plant. -Nitrites (NO2 − ) – sodium nitrite is used for the curing of meat because it prevents bacterial growth and, as it is a reducing agent, in a reaction with the meat's myoglobin, gives the product a desirable pink-red 'fresh' color, such as with corned beef. Because of the relatively high toxicity of nitrite (the lethal dose in humans is about 22 milligrams per kilogram of body weight), the maximum allowed nitrite concentration in meat products is 200 ppm. Under certain conditions – especially during cooking – nitrites in meat can react with degradation products of amino acids, forming nitrosamines, which are known carcinogens. Biochemistry II Pouria Farsani 2013 292 The tables below do not directly relate to the content of the question 67. They are merely a pathological general overview regarding the topics of RBC and disorders. Biochemistry II Pouria Farsani 2013 293 Biochemistry II Pouria Farsani 2013 294 68. Leukocytes – characteristic metabolic processes. Neutrophil granulocytes – interaction with endothelium, production of special proteins, selectins, secretion granules, phagocytosis, respiratory burst Leukocytes Leukocytes are cells of the immune system. They defend the organism against/by: -Pathogens -Identification and elimination of cancer cells -Removal of worn-out cells and tissue debris Biochemistry II Pouria Farsani 2013 295 -Polymorphonuclear leukocytes (PMN) Neutrophils • The most numerous circulating leukocytes (50 – 70%) • Formed in the bone marrow • They have an important role in non-specific defense mechanisms • The actively invade microorganism by means of phagocytosis – they are microphages • The half-life in blood is 6-7 h, in connective tissue it is 1-4 days • They contain three types of granules (primary lysosomes and specific granules) • They contain CD11/CD18 integrins and further adhesive molecules in the plasma membrane • They contain receptors for chemokines on the plasma membrane Characteristic metabolic processes -The main metabolic features of neutrophils • Active glycolysis and synthesis of glycogen • Active pentose phosphate pathway • There is only a slight activity of the citric acid cycle and oxidative phosphorylation due to the low number of mitochondria • They can survive even under anaerobic conditions • The proteosynthetic apparatus is developed less perfectly than in other cells • They contain certain unique enzymes and proteins (myeloperoxidase, NADPHoxidase, cytokines, lactoferrin) Neutrophil granulocytes – interaction with endothelium -The role of neutrophils in antibacterial defense A bacterial invasion into a tissue results in the migration of neutrophils from capillaries to the sight of infection Their movements are initiated and directed by chemotaxis. Leukotactic substances (attractants) are for example: -Various complement components -Small bacterial peptide fragments -Eicosanoids, namely LTB4 Neutrophils then adhere to the endothelial cells of the capillary wall. This process is known as migration of neutrophils and is supported by membrane proteins, integrins and selectins. The neutrophils then penetrate through the capillary wall to the sight of infection – diapedesis Biochemistry II Pouria Farsani 2013 296 -Migration of neutrophils - the migration of neutrophils to endothelial cells occurs in four stages: 1. Slowing or rolling of the neutrophils within the vessel (venule) mediated by selectins e.g. selectin L that interacts with glycoporteins CD3 and glyCAM on the endothelial surface 2. Activation. It results in neutrophils firmly adhering to the surface of endothelial cells, they flatten as well. Adhesion molecules e.g. LFA-1 and Mac-1 (integrins) on neutrophils and ICAM-1 and ICAM-2 (integrin ligands) on endothelial cells take part in adhesion 3. Potentiation of the activation is carried out by other molecules like TNF, interleukins, PAF, leukotriens and others 4. Diapededis – neutrophils migrate through the junctions of endothelial cells into the interstitial tissue. This requires involvement of adhesion molecule PECAM-1. Chemotaxis is also involved in this stage Selectins Selectins are transmembrane peptides of the lectine type. They are adhesion molecules that bind specific oligosaccharide sequences of glycoproteins and glycolipids. One of the ligands is e.g. oligosaccharide sequence named sialyl-Lewis-x antigen. L-selectins are on leukocyte membranes and their ligands ore on endothelial cells. E-selectins is on endotehelial membranes and it has ligands on leukocytes. Biochemistry II Pouria Farsani 2013 297 Production of special proteins Phagocytosis Phagocytosis is triggered by the activation of neutrophils in tissues by the following steps: Specific receptors on the membrane of neutrophils react with bacteria, chemotactctic factors or antigen-antibody complexes Activation of the phosphatidylinositol system Increase of Ca2+ in cytoplasm Activation of formation of pseudopodias Triggering of phagocytosis There are some products produced when neutrophils are activated => Biochemistry II Pouria Farsani 2013 298 -Process of phagocytosis The invading pathogen is surrounded by pseudopodias Neutrophils then engulf the invading pathogen by phagocytosis – formation of a phagosome Phagosome fuses with lysosomes and specific granuels forming a phagolysosome. H+ -ATPase maintain the content at a pH about 4 and hydrolases catalyze the digestion of organic components The process of phagocytosis is illustrated in the following scheme: Biochemistry II Pouria Farsani 2013 299 Secretion granules These are important proteins in neutrophils Respiratory burst The respiratory burst in the sole profitable utilization of reactive oxygen species production – it helps kill bacteria engulfed by phagocytic cells. There are three ways of microorganism destruction: 1) NADPH-oxidase activation 2) NO-production 3) Hydrolytic enzyme action (proteinases in neutrophils) -NADPH-oxidase activation – interaction of neutrophils with bacteria, binding of chemotactic factors or immunocomplexes onto specific receptors in plasma membrane activates motility of neutrophils, secretion of granules, and the activity of NADHP-oxidase (a flavoprotein) that initiate the respiratory burst. NADPH –oxidase is present in plasmatic membrane of neutrophils. It is activated by contact with various ligands. Activation is carried out via the phosphatidylinositol system. Increased level of then Ca2+ activates respiratory burst. Biochemistry II Pouria Farsani 2013 300 Formation of superoxide anion-radical NADPH-oxidase 2O2 + NADPH →→→→ 2O2 .- + NADP + H+ The consumption of oxygen rises – respiratory burst The superoxide anion-radical is discharged to the outside of the cell or into phagolysosomes. Inadequate activation of neutrophils and release of high amount of reactive oxygen radicals can result in damage of surrounding tissue Spontaneous dismutation of suproxide results in formation of hydrogen peroxide: O2.- + O2.- →→→→ H2O2 + O2 Hydrogen peroxide can damage a pathogene directly or it can be converted to OH•: H2O2 + M+ → OH• + OH- + M2+ (M = metal) Enzyme myeloperoxidase produce HClO: H2O2 + X- + H+ →→→→ HXO + H2O (Xis most often Cl- ) HClO is a strong oxidant with strong microbicidal effects! *NADPH-oxidase deficiency: • Chronic granulomatous disease – immunodeficiency • No oxygen radicals are formed • Decreased defense of organism against bacterial infections • The disease is characterized by recurrent infections and widespread granulomas and frequent purulent infections -Production of NO in neutrophils (this topic is also related to q. 21) • Mainly by inducible NO-synthase (iNOS) NOS Arginin → citrulin + NO • NOS is induced by cytokines (INF-γ, TNF) or bacterial lipopolysaccharide • NO• can kill bacteria directly (e.g. by inhibition of respiratory chain) or indirectly – by reaction with O2 forming ONOO-(peroxynitrite) that attaches Fe-S proteins and -SH groups, nitrates proteins, inactivates enzymes Biochemistry II Pouria Farsani 2013 301 *Three types of NO synthase in the organism 1. eNOS (endothelim) 2. nNOS (nervous tissue) 3. iNOS (hepatocytes, macrophages, monocytes, neutrophils) • eNOS and nNOS are constitutive enzymes • iNOS is inducible (induction by TNF-α, bacterial endotoxins, anti-inflammatory cytokines) -Proteinases in neutrophils • Lysosomas enzymes of neutrophils can be released into normal tissues, the amount of which is markedly increased during inflammation • The activity of hydrolytic enzymes is normally kept in check by a number of antiproteinases in plasma and extracellular fluid. When the balance is violated considerable tissue damage can develop The different types of leukocytes -Eosinophilic granulocytes • 1-4% of leukocytes • Their primary function is defense against parasites • Are also migrating and display respiratory burst • Are involved in allergic reactions Biochemistry II Pouria Farsani 2013 302 -Basophils • 0.25-0.5 % of leukocytes (similar to mast cells) • Release heparin and histamine • Synthesize prostaglandins and leukotriens -Monocytes • 2-6% of leukocytes • Migrate into the tissue where they mature • Are transformed into macrophages • They have the ability to engulf bacteria and digest them (like neutrophils) -B-lymphocytes • Are transformed to plasmatic cells that produce: -Antibodies – bind to bacteria and mark them for destruction -Humoral activity -T-lymphocytes • Several types • They contribute to immune defense in two major ways 1. Some of them: directly, regulating immune response 2. Some other directly attack infected cancerous cells Differentiation into the different types of cells Biochemistry II Pouria Farsani 2013 303 69. Principles of metabolism control (control of enzyme activity and of protein synthesis, control of transport across membranes, extracellular signals) In general we describe three formal levels in which the control of metabolism is achieved: 1. Regulation of metabolic events within particular compartments (organelles) – depends on interactions between molecules in the compartment 2. Regulation that occurs within complete cells without any regard to extracellular signals. Here, proteosynthesis and transport across membranes have important roles 3. Regulations that are consequences of communication between cells in particular tissues. This depends on extracellular signals (neurotransmitters, hormones, cytokines etc.) Rate limiting steps are check-points by which methabolic pathways are controlled. We describe some important factors in control of metabolism: -Equipment of cells with enzymes and other proteins -Specific receptors -Existence of multiple enzyme forms (isoenzymes) – control of particular reactions in different by different mechanisms in various tissues/compartments in various time periods -Accessibility of nutrients – energetic state of the cell depends on this Below follows the major mechanisms that provide control of metabolism: Regulation of the amount of enzymes (proteosynthesis & regulation of enzyme degradation) • Regulation of proteosynthesis The expression of some genes occurs at a nearly constant rate (synthesis of constitutive enzymes). Regulatory signals cause a respond by which genes are expressed or silenced. The enzymes that are controlled in this way are adaptable enzymes – mostly inducible Regulation of proteosynthesis may occur at: -Level of gene amplification -Transcription -Posttranscriptional hnRNA processing (alternate mRNA spicing) -Export of mRNA from nucleus -Degradation of mRNA -Translation -Posttranslational modification Biochemistry II Pouria Farsani 2013 304 Expression of genes can be induced by binding of signal molecules on membrane receptors or by interactions of hydrophobic signal molecules (intracellular receptors) • Regulation of enzyme degradation Rates of degradation of specific enzymes are selectively regulated by enzymes that catalyze the rate limiting-steps or enzymes that represent important metabolic control points. These are short-lived proteins. The susceptibility of an enzyme to be proteolytically degraded depends upon its conformation which may be altered by the presence/absence of substrates, coenzymes and metal ions. Long-lived proteins, under physiological conditions are degraded at constant rates, mostly non-selectively. Starvation increases selectively the degradation rates of enzymes that can be missed and are not needed for the survival of the cell. Control of enzyme activity This is a more rapid type of control than the control of enzyme synthesis. There are many ways by which enzyme activities can be controlled: • Activation of proenzymes by partial proteolysis of the proenzyme Active enzymes are formed from proenzymes molecules by irreversible splitting of certain part(s) in their polypeptide chain. This is frequent among proteinases – it protects against unwanted breakdown of proteins Examples: -Extracellular “big” proteinases of the GIT (pepsin, chymotrypsin, trypsin, etc) -Extracellular proteinases in the blood clotting cascade (coagulation factors IX, X, XI and thrombin) -Intracellular proteinases – activation of caspases that initiate apoptosis • Allosteric control and cooperative effects of enzymes that consist of several identical subunits Regulatory enzymes are frequently oligomers that consist of several identical subunits (protomers). Their saturation curves usually deviate from hyperbolic (Michaelis) shape, they are sigmoid. Cooperative effect – binding of substrate to active site can affect affinity of active sites for substrates in other subunits. It is positively cooperative when it facilitates substrate binding to other subunits and so activating the enzyme. Allosteric effectors – molecules that are allosteric to the substrate and can bind reversibly to specific sites other than the enzymes active sites (to the allosteric sites) Biochemistry II Pouria Farsani 2013 305 the induced change in conformation results in either higher activity of the enzyme or in inhibition. • Control arising from interactions with regulatory proteins – such as in activation of enzymes by releasing of inhibitory subunits or another regulatory protein Examples: -Protein kinase A - forms inactive tetramers C2R2. If two regulatory subunits R bind four molecules cAMP, two catalytically active subunits C are released. The decrease in cAMP concentration supports interactions between C and R subunits, the inactive tetramer is restored. -Phosphoprotein phosphatase 1 – has a regulatory subunit, which keeps up active complex of glycogen with the catalytic subunit. If the regulatory unit is phosphorylated by protein kinase A, it releases the catalytic subunit (exhibiting low activity) that is then fully inactivated by binding with a similarly phosphorylated protein inhibitor. If this is phosphorylated at another site by insulin-dependent protein kinase, the phosphatase activity of the complex of glycogen and the catalytic subunit will increase. -Proteinases – often occur in the inactive forms, bound reversibly to the more or less specific protein (proteinase inhibitors). Plasma proteinase thrombin is inactivated by binding to antithrombin, intracellular Ser- or Cys- proteinases are inhibited by various types of serpins and cystatins. Biochemistry II Pouria Farsani 2013 306 • Control by reversible covalent modification of enzymes or of regulatory proteins: the most important example of this is reversible phosphorylation catalyzed by protein kinases and controlled by extracellular signals -Reversible covalent modification of proteins: *phosphorylation – protein kinases (dephosphorylation is carried out by phosphoprotein phostphatases) *acetylation *ADP-ribosylation – transfer of ADP-ribosyl from NAD+ , nicotinamide is relased *myristoylation, farnesylation (prenylation) *Gamma-carboxylation of glytamyl residues side chains (coagulation factors etc.) is irreversible, bit it is more important in formation of binding centers for Ca2+ ions, essential for the biological activity of the protein -Reversible phosphorylation of proteins – an intracellular reaction with ATP as the donor of phosphate. The reaction is catalyzed my protein kinases. For more details about protein kinases see q. 76. Control of transport across membranes Examples: -Insulin – stimulates glycolysis as it also promotes the uptake of glucose by muscle and adipose tissue. Insulin binding results in the rapid increase in the number of GLUT4 transporters in the plasma membrane of muscle cells and adipose cells -Fatty acid synthesis and degradation are reciprocally regulated so that both are not simultaneously active. Malonyl-CoA (present in cytosol when there is an abundant of nutrients to the cell) inhibits carnitine acyltranferase, thus preventing access of fatty acid acyl-CoAs. On the contrary, fatty acyl-CoAs (present in cytosol at a high level in fasting) inhibit the mitochondrial tricarboxylate transporter, thus preventing acetyl-CoA carboxylase by outflow of citrate from mitochondrial matrix Biochemistry II Pouria Farsani 2013 307 Extracellular signals The signals molecules can also be classified as: -Endocrine – carried by the blood, may act in the whole body -Paracrine – act within short distance of the site of their production -Autocrine – act on the cells that produce them The size and polarity of a signal molecule is decisive of how a cell will process and respond to information from the environment: -Proteins and small polar signal molecules – (amino acids, peptides, biogenic amines, eicosanoids) do not penetrate across the plasma membranes. They bind onto specific membrane receptor (integral membrane proteins) Binding => conformational change of intracellular domain => increase of concentration of second messenger or direct activation of a protein kinase -Non-polar signal molecules – (steroids, iodothyronines, retinoates) diffuse through the plasma membrane and bind to specific intracellular receptors. Hormone-receptors complexes then enter the nuclei, binds to a region of DNA (hormone response element, HRE) and activate (or repress) gene transcription. Biochemistry II Pouria Farsani 2013 308 70. General features of hormone synthesis, secretion, transport, and inactivation in relation to signal intensity received by the target cell -Types of signal molecules in neurohormonal regulations: • Hormones – secreted by endocrine glands, by dispersed glandular cells (eicosanoids by many other cellular types) • Neurohormones – secreted by neurons into the blood circulation • Neurotransmitters – secreted by neurons at nerve endings • Cytokines – secreted by immunocompetent cells • Growth factors – secreted by various types of cells Signal molecules can also be classified as: -Endocrine – carried by the blood, may act in the whole body -Paracrine – act within short distance of the site of their production -Autocrine – act on the cells that produce them Biochemistry II Pouria Farsani 2013 309 -Hormone synthesis • Protein and peptide hormone synthesis -Preprohormone synthesis occurs in the ER and is directed by a specific mRNA -Signal peptides are cleaved from the preprohormone, producing a prohormone, which is transported to the GA -Additional peptide sequences are cleaved in the GA to form the hormone, which is packaged in secretory granules for later release • Steroid hormone synthesis -Steroid hormones are derivatives of cholesterol. For more detailed information about their synthesis see q. 81-83 • Amine hormone synthesis -Amine hormones (thyroid hormones, epinephrine, norepinephrine) are derivatives of tyrosine. For more detailed information about their synthesis, see q. 78 and 80 Biochemistry II Pouria Farsani 2013 310 -Regulation of hormone secretion – on and off turning of secretion in order to maintain homeostasis • Negative feedback -Is the most commonly applied principle for regulating hormone secretion -Is self-limiting -A hormone has biogenic actions that, directly or indirectly, inhibit further secretion of the hormone -For example, insulin is secreted by the pancreatic beta cells in response to an elevated increase in blood glucose. In turn, insulin causes an increase in glucose uptake into cells that results in decreased blood glucose concentration. The decrease in blood glucose concentration then decreases further secretion of insulin • Positive feedback -Is rare -Is explosive and self-reinforcing -A hormone has biologic actions that, directly or indirectly, cause more secretion of the hormone -For example, the surge of luteinizing hormone (LH) that occurs just before ovulation is a result of positive feedback of estrogen on the anterior pituitary. LH then acts on the ovaries and causes more secretion of estrogen • Regulation of receptors – hormones determine the sensitivity of the target tissue by regulating the number of receptors 1. Down-regulation of receptors -A hormone decreases the number or affinity of receptors for itself or for another hormone -For example, in the uterus, progesterone down-regulates its own receptor and the receptor for estrogen 2.Up-regulation of receptors -A hormone increases the number or affinity of receptors for itself or for another hormone -For example, in the ovary, estrogen up-regulates its own receptor and the receptor for LH Biochemistry II Pouria Farsani 2013 311 71. Membrane receptors cooperating with G-proteins (types of receptors and Gproteins, corresponding intracellular messengers) Their response is a few minutes – neurotransmitter and hormones. We describe the common structural features of membrane receptors which act through G-proteins: -They are all seven alpha-helical segments (integral proteins) which are connected by intraand extracellular hydrophilic and more divergent loops -There is a binding site for the agonist which can be the accessory binding site for antagonists as well – extracellular domain -Intracellular domain has the binding site for the specific G-protein type G-proteins and types The heterotrimeric G-protein can move freely along the inner surface of the plasma membrane. They consist of three subunits, alpha, beta and gamma. The alpha subunit is the largest hydrophilic subunit which can bind to GTP or GDP => specific for particular mechanism of second messenger production. The gamma and beta subunits are hydrophobic => non-specific Biochemistry II Pouria Farsani 2013 312 1. Inactive state: GDP is bound to alpha-subunit which is associated with beta- and gamma-subunits 2. Binding of ligand => conformation of receptor changes => initiation of conformation change of G-protein 3. Decreased affinity of alpha-subunit to GDP => detachment of GDP => replacement by GTP 4. Concurrently: detachment of G-protein from receptor => interrupted interaction between alpha-subunit with beta-gamma complex 5. Diffusion of alpha-GTP and beta-gamma complex through membrane => binding to target proteins (effectors) 6. Interaction with effector => stimulation of GTPase activity of alpha-GTP subunit => hydrolysis of bond between GTP and alpha-subunit 7. Detachment of alpha-GDP complex from effector => GDP remains bound and the subunit combines with the beta-gamma complex There are more than 20 different types of alpha subunits according to which G-proteins are classified. Activated beta-gamma complex is however able to mediate signal transduction as well! Biochemistry II Pouria Farsani 2013 313 Types of receptors and corresponding intracellular messengers -Adenylate cyclase The two schemes above are the same. Biochemistry II Pouria Farsani 2013 314 -Adenylate cyclase is the effector molecule that catalyzes the reaction of ATP => cAMP +PPi -cAMP is hydrolyzed by phosphodiesterase to AMP -The balanced action of adenylate cyclase and phosphodiesterase maintains the concentration of cAMP in the inactive state -Activation of adenylate cyclase is carried out by the alphas subunit of the G-protein -Inhibition of adenylate cyclase is carried out by the alphai subunits -Phosphodiesterase is also affected by some hormones, such as insulin, which decreases cAMP by activating phosphodiesterase cAMP action in the cells -Phosphorylation of proteins by activation protein kinase A (PKA) *Cytoplasm: metabolic enzymes (rapid response) *Nucleus: phosphorylation of gene specific transcription factor CREB (cAMP response element-binding protein) (slow response). CREB binds to a cAMP-response element in a nonphosphorylated state and acts as a weak transcription activator. Amplification of transcription is carried out by CBP (CREB-binding protein) See scheme above Protein kinase A (PKA) is a heterotetrameric molecule consisting of two regulation (R) subunits and two catalytic (C) subunits which are bound together in an inactive state. cAMP => binds to regulation subunits => induces detachment => catalytic subunits become active which catalyzes transfer of phosphate from ATP to serine or threonine side chain hydroxyl in target proteins. The effects of kinases can be directed to phosphorylation of specific proteins. Therefore there are specific proteins binding to kinases. For PKA it is the AKAPs (A kinase anchoring proteins) which acts as a support structure: it locates the position of PKA close to a specific Biochemistry II Pouria Farsani 2013 315 substrate which is to be phosphorylated. Similar proteins also affect the specific action of phosphatases and phosphodiesterases. Explaining Pathology: some bacterial enzymes modify the effect of G-proteins. Cholera is a result of entertoxin produced by Vibrio cholera. The toxin inhibits GTPase activity of the alphas subunit of Gs protein. This results in the “freezing” of the alphas in an active state thus cAMP is continually produced => activation of channels for Clin the bowel cell membrane => secretion chloride anions and water into intestinal lumen. The Bordetella pertussis bacteria produce a toxin which targets the inhibitory G-protein resulting in Gi protein inactivation => overproduction of cAMP. cAMP or cGMP can also bind to ion channels and affect their permeability – occurs mainly in activation of olfactory and visual perception. -Phosphatidyl inositol system This scheme illustrate, in further detail, one of the events, of the two schemes shown below. Biochemistry II Pouria Farsani 2013 316 The two schemes above show the same process. Biochemistry II Pouria Farsani 2013 317 These receptors bind to the q-isoform of the alpha-subunit. Ligand binding => activation of phospholipase Cbeta => hydrolysis of membrane-bound phosphatidylinositol bisphosphate (PIP2) => formation of two second messengers: diacylglycerol (DAG), and 1,4,5-inositol trisphosphate (IP3). The binding site for IP3 is located at the SR and ER. Binding => release of Ca2+ => Ca2+ forms a complex calmodulin (CM) => activation of calciumcalmodulin dependent kinases => phosphorylation of proteins. Calcium-dependent colmodulin kinase II influences CREB in the nucleus as well. DAG remains in the membrane => activates protein kinase C => extension of response by activation of target proteins. -Control of metabolism by changes of cytoplasmic concentration of Ca2+ *basal conc. in cytoplasm ~ 1.10-7 mol/l *conc. increase to ~1.106=> rapid and maximal activation various Ca2+ - regulated functions *increase of Ca2+ by: influx of Ca2+ across plasmatic membrane (e.g. smooth muscle contration) or by release from intracellular stores (ER, mitochondria). E.g. IP3 gated Ca2+ channels in ER or ryanodine receptors in ER/SR of cardiac or skeletal muscle. -Calmodulin as regulatory protein *when cytosolic Ca2+ increase => activation of various calcium-binding regulatory proteins of which calmodulin is the most important. It is a ubiquitously expressed protein in almost all cells. Calmodulin binds 4 Ca2+ => conformational change => facilitation of its interaction with downstream signaling proteins (kinases, phosphatases etc.) Ca2+ -calmodulin dependent kinases can be very specific or they can have a broad substrate specifity. Biochemistry II Pouria Farsani 2013 318 72. Receptors with tyrosine kinase activity. The most important signal pathways (RAS and MAP kinase pathway) This family of receptors includes receptors for: -Insulin -IGF-1 (insulin growth factor 1) -EGF (epidermal growth factor) -PDGF (platelet-derived growth factor) The growth factors stimulate mitosis, cell differentiation, cell migration and apoptosis. The intracellular tyrosine kinase domain is the common feature of the receptors. -Common features: *signal molecule binds to receptor => conformational change of the receptor => activation of tyrosine kinase activity of the receptor. *the receptor itself is the first protein substrate (autophosphorylation of tyrosine molecules in the receptor). Eventually other proteins are phosphorylated. *when tyrosines and other substrates are phosphorylated, they act as a recognition or anchoring site for other proteins, adaptor molecules. *adaptor proteins bind to phosphotyrosine residues by SH2 domains (Src homology 2 domain) and are phosphorylated. *the adaptor proteins react with other molecules => signal is transmitted through a cascade of other reaction, mainly phosphorylation/de-phosphorylation, exchange of quinine nucleotides, changes in conformation etc. Insulin receptor See also q.85 Biochemistry II Pouria Farsani 2013 319 -Dimeric structure *alpha-subunit: for binding of insulin (extracellular) *beta-subunit: tyrosine kinase activity (integral membrane protein) *both subunits are joined by a disulfide bond. A disulfide bond joins both monomers as well Insulin binds to receptor => tyrosine kinase activity => autophosphorylation of beta-subunits => phosphorylation of IRS 1-4 proteins (insulin receptor substrates 1-4 / adaptor proteins) at several sites => activation of binding sites in IRS for various proteins that transduce several insulin effects => activation of proteins (most proteins bind to binding sites in the IRS via specialized SH2 domains) -Grb2 (growth factor receptor-bound protein) binds to one of the sites. It is attached to the membrane by a phospholipid anchor. It activates the membrane-bound G-protein Ras Dimerizing receptor for EGF (epidermal growth factor) containing an intrinsic tyrosine kinase activity Binding of ligand => dimerization of receptor => activates tyrosine kinase in cytoplasmic domain => autophosphorylation of the receptor => adaptor proteins Grb2 (SH2 domains) binds to phosphorylated sites. G-protein Ras is activated by the action of SOS protein => activation of MAP-kinase cascade (Ras/Map-cascade) Biochemistry II Pouria Farsani 2013 320 Ras Ras is a monomer which exists in an inactive GDP and active GTP form. It binds GTP and has GTPase activity at the same time. It is structurally analogous to the alpha-subunit of the heterotrimeric G-protein). Activation of Grb2 => increase in number of active Ras-GTP molecules => activation of protein kinases => induction of Ras and MAP (mitogen activated protein) signal cascades => phosphorylation of proteins in the cytoplasm and nucleus (regulation of transcription). Inactivation of Ras is carried out by hydrolysis of GTP which is initiated by other regulating proteins. -Superfamily of Ras proteins *5 families: Ras, Rho, Arf, Rab, Ran *anchored to lipid membrane by lipid anchors (myristoyl, farnesyl) *monomeric G-protein which plays an important role in the regulation of morphogenesis, cell motility, cytokines etc. *mutations in Ras gene => pathologic proliferation and antiapoptosis => tumor (30% of all human tumors involve ells expressing mutated Ras oncogenes – Ras genes are named proto- oncogenes) Map-kinase signaling pathway Biochemistry II Pouria Farsani 2013 321 IP-3 kinase IP-3 kinase binds another phosphotyrosine site of the IRS => activated => phosphorylates inositol phospholipids in the membrane at position 3. e.g. conversion of phosphatidylinositol-4,5-bisphosphate to phosphatidyl-inositol-3,4,5- trisphosphate. Activated phospholipids act as second messengers => activation of other proteins such as: proteins kinase B and C that mediate many other insulin effects in a cell. Exposition of GLUT transporters into the membrane in the muscles and adipocytes is one of the effects Phospholipase Cgamma binds to another IRS site and becomes activated as well Biochemistry II Pouria Farsani 2013 322 Biochemistry II Pouria Farsani 2013 323 73. Receptors cooperating with non receptor kinases (JAK/STAT signal pathway) JAK-STAT receptors (Janus Kinase – Signal Transducer and Activator of Transcription) These receptors do not have kinase activity (intrinsic enzyme activity) – they are associated with tyrosinekinase JAK. The receptors dimerize (homo- or heterodimers) after binding of ligand. The activated receptor then binds to and activates JAK => JAKs phosphorylate the tyrosine residue of the receptor => formation of phosphotyrosine binding sites for proteins with SH2 domains (STAT). The STAT proteins associate with the receptor and are phosphorylated by JAK => dimerization of STAT phosphates => translocation to nucleus => biding to specific DNA elements => regulates transcription. This signaling pathway is used by receptors of cytokines (interleukins, interferon etc.) in order to regulate proliferation of cells involved in immune response. -Parts of the receptor: *extracellular domain *intermembrane segment *intracellular domain Biochemistry II Pouria Farsani 2013 324 74. Intracellular hormones receptors, their activation and consequences These receptors are present in the cytoplasm or nucleus. Lipophilic (non-polar) signal molecules (steroid hormones, thyroid hormones, retinoids and calcitriol) are transported in blood with the aid of transport proteins. They cross the cytoplasmic membrane when reaching the target cell. In the cytoplasm/nucleus they are transported bound to proteins. The hormone then binds to the intracellular receptor forming a hormone-receptor complex. The ligand binding domain (LBD) is located at the carboxyl-terminal of receptor. LBD selectively binds to a hormone and thus mediates its effect. LBD can also bind to heat-shock proteins (chaperons) The hormone-receptor complex => translocation to the nucleus => binding to a hormone response element (HRE) in a DNA regulation sequence => thus hormone-receptor complex = transcription factor. All of the intracellular receptors have a central DNA-binding domain for protein to bind to HRE. In order to bind with DNA special proteins domains are used such as zinc fingers. Cortisol as an example Cortisol => CBG (corticosteroid-binding protein) in ECF => penetrates cell membrane => enters the cell. Inactive receptor for glucocorticoids (forms a complex with hsp 90 dimer and other proteins) + cortisol => release of hsp 90 and other proteins => active receptor-ligand complex Biochemistry II Pouria Farsani 2013 325 (monomer) => formation of dimer => nucleus (via nuclear pores) => active complex binds onto DNA at GRE (glucocorticoid response element). Simultaneous attachment of the coactivator and specific hormone response element-binding proteins (GREB-proteins) => formation of a complex that acquires the ability to act as an enhancer which supports initiation of transcription on the promoter by means of mediator proteins => initiation of transcription by cortisol! Biochemistry II Pouria Farsani 2013 326 75. Intracellular Ca2+ distribution – calcium channels, carriers, Ca2+ -dependent proteins (e.g. calmodulin) and enxymes, relations to cell functions Intracellular Ca2+ distribution (see also schemes for Ca2+ distribution in q. 79) The basal intracellular (in cytoplasm) concentration of calcium is ~ 1.10-7 mol/l. There is almost no calcium found in the cytoplasm as we can see. Instead, calcium is kept in the ER and sarcoplasmic reticulum, vesicles, mitochondria and nuclei of the cell. The concentration of extracellular calcium is much higher. The reason for this is that Ca2+ is continuously pumped from the cytoplasm into the intracellular stores mentioned above, or it is pumped out from the cell. The processes by which calcium is pumped within our out from the cell are mainly active transport processes through Ca2+ /2 Na+ anitporters. When the calcium concentration in the cell is increased to ~1.10-6 mol/l it rapidly and maximally activates the various calcium regulated cell functions. How is this increased intracellular concentration achieved? By the following ways: • Influx of Ca2+ across the plasmatic membrane (e.g. during smooth muscle contraction) – depolarization of the cell membrane • Release of Ca2+ from intracellular stores (intracellular messengers) – such as: *IP3 gated Ca2+ channels in ER *Ryanodine receptors in ER/SR of cardiac or skeletal muscle • Stretching or heating of the cell membrane Calcium channels (see also schemes in q. 79) When we speak about calcium channels, we speak about voltage-dependent calcium channels and ligand-dependent calcium channels. -Voltage-dependent calcium channels – are found in excitable cells (muscle, nerve tissue). They have a permeability to Ca2+ ions and slightly to Na+ – calcium/sodium channels. These channels are closed during physiological conditions, but activated (opened) at depolarized membrane potential => Ca2+ can enter the cell => muscular contraction, excitation of neurons, increased gene expression, release of hormones or release of neurotransmitters. Biochemistry II Pouria Farsani 2013 327 -Ligand-gated – receptor operated calcium channels (in vasoconstriction). There are a few types: *IP3 receptors in ER/SR => release of calcium from ER/SR *Ryanodine receptor in ER/SR => calcium-induced calcium release in myocytes *Two-pore channel *Cation channels of sperm *Store-operated channels – indirectly gated by ER/SR depletion of calcium. Found in plasma membrane -Calcium channels in myocardium – voltage-operated channels are found here. There are two types: L-type and T-type: • L-type The major rout of entry of calcium into myocytes is via these slow calcium channels (long-duration). They are opened during depolarization by a spreading action potential. They are regulated by protein kinase A (stimulation) and cGMP-dependent protein kinases (inhibition). Calcium blockers (e.g. verapamil) act upon these channels in treatment for hypertension. • T-type (temporary opening) These channels are found in the sinus node. Their function is to control the spontaneous depolarization, of the sinus node, and thus the rate of heartbeat. An increased concentration of calcium in myocytes induced by the influx of extracellular calcium results in the opening of the Ca2+ channels (ryanodine receptor) in the sarcoplasmic reticulum. This process is known as Ca2+ -induced Ca2+ release (CICR). About 10% of the calcium which is involved in contraction comes from ECF; the other 90% come from the ER. Muscle relaxation between contractions requires a rapid reduce of sarcoplasmic Ca2+ . This is carried out by the Na+ /Ca2+ exchanger in the sarcoplasmic membrane and Ca2+ -ATPase (SERCA) in the membrane of SR. The Na+ /Ca2+ exchanger is the major route of transport of Ca2+ ions out of the cell. This facilitates relaxation. However, in some circumstances it also contributes to the increase of sarcoplasmic level of Ca2+ ions – the exchange proceeds in the opposite direction, Ca2+ uptake into SR is mediated by Ca2+ -ATPase. The level of calcium and phospholamban regulated the activity of Ca2+ -ATPase. Biochemistry II Pouria Farsani 2013 328 It is assumed that both the Na+ /Ca2+ exchanger and by Ca2+ -ATPase are activated by phosphorylation (calmodulin-dependent protein kinase II) The concentration of Na+ is what the Na+ /Ca2+ exchanger is dependent on. High Na+ in ICF => transport proceeds in the opposite direction => calcium enters the cell. This is why all factors which increase Na+ in ICF => Ca2+ ICF ↑ => contraction intensity ↑ => positive ionotropic effect. Digitalis is a known substance regarding this. Verapamil has a negative ionotropic effect on the other hand. Calcium dependent proteins –calmodulin Other examples of calcium proteins are annexins, troponin C. Calmodulin is a regulatory protein. When Ca2+ in cytoplasm increases it activates various calcium-binding regulatory proteins (family of small, Ca2+ dependent protein) the most important of which is calmodulin. It is a ubiquitously expressed protein in almost all cells. Binding of Ca2+ (4 biding sites) => conformation of calmodulin => facilitation of interaction with downstream signaling proteins such as kinases, phosphatases etc. Some of the Ca-calmodulin-dependent kinases are very specific; the others have broad substrate specificity. The Ca-calmodulin complex also activates, among the number of different enzymes, calmodulin-dependent protein kinase II and myosin light chain kinase which is involved in smooth muscle contraction. We can explain the action of calmodulin in more detail, which corresponds to the scheme above. 1. Hormone => receptor in cell membrane 2. Activation of G-protein => mobilization of intracellular Ca2+ stores => opening of Ca2+ channels in the cell membrane => increase in intracellular Ca2+ 3. Ca2+ binds to calmodulin => physiological actions. -The activated G-protein (alpha-unit + GTP) activates the effector = phospholipase C => catalyzes hydrolysis of PIP2 => DG + IP3. DG then activates proteinkinase C which phosphorylates… -IP3 opens Ca2+ channels in the ER => release into cytoplasm => activation with calmodulin => Ca-calmodulin (Ca-CM) complex activates the Ca-CM dependent kinases. The phosphorylated intracellular proteins = biological response to the signal molecule. Biochemistry II Pouria Farsani 2013 329 Calcium carriers/proteins Ca2+ -dependent enzymes Glycogenolysis is one of many processes which have enzymes involved in the process that are dependent on calcium. Depolarization (for muscle contraction) => ICF calcium ↑ => activation of muscle phosphorylase kinase => activation of glycogen phosphorylase => glycogenolysis Relaxation of muscle => calcium ICF ↓ => inactivation of phosphorylase kinase => stop of glycogenolysis. Other enzymes (among many others) are: -Ca-calmodulin-dependent kinases -Protein kinase C -NO synthase Relations to cell functions (see also q. 71) A rise in intracellular calcium concentration is a signal (2nd messenger!) for many important cell functions including: -Contraction of smooth and cardiac muscle (cardiotonics => ↑ Ca2+ ICF) -Exocytosis of neurotransmitters in presynaptic endings -Endocrine and exocrine hormone secretion – secretion of signal molecules (neurotransmitters, peptide hormones, catecholamines) -Excitation of certain sensory cells -Closure of gap junctions in various cells -Opening of other types of ion channels -Migration of leukocytes and tumor cells -Thrombocyte activation -Sperm mobilization -Hemocoagulation -Cell adhesion – tight junction (interactions with cadherins) -Neuromuscular excitation – hypocalcaemia increase the excitability of muscles and nerves (Ca2+ decreases membrane permeability for Na+ ) *decrease of Ca2+ by 50% => hypocalcaemic tetany, laryngospasmus *alkalosis => ↓ Ca2+ => ↑ nerve excitability => epileptic attack *acidosis (diabetic, uremic) => Ca2+ ↑ => ↓ nerve excitability => comatose condition Biochemistry II Pouria Farsani 2013 330 76. Protein kinases (main classes) and phosphoprotein phostphatases, regulation of their activity (This question is also related to q.69) Protein kinases Protein kinases (PK) catalyze phosphorylation of proteins which will either activate or inactivate them. When phosphorylating, a phosphate ester originates by the transfer of γphosphate from ATP. PK are the largest family of homologues enzymes known – there are more than 550 human types of protein kinases. The proteins are phosphorylated either on serine or threonine residues (alcoholic groups), or on residues of tyrosine (phenolic hydroxyl), at specific position within the polypeptide chain. PK are activated specifically (e.g. cAMP, cGMP, Ca2+ -calmodulin complex etc. see table). The signal that activates protein kinases is amplified (activation of one enzyme molecule results in phosphorylation of numerous of protein molecules). Main classes Biochemistry II Pouria Farsani 2013 331 Protein kinase A as an example: forms inactive tetramers C2R2. If two regulatory subunits R bind four molecules cAMP, two catalytically active subunits C are released. The decrease in cAMP concentration supports interactions between C and R subunits, the inactive tetramer is restored Phosphoprotein phosphatases Phosphoprotein phosphatases catalyzes dephosphorylation (hydrolysis of the ester bond) of phosphoproteins. Phosphoprotein phosphatase 1 has a regulatory subunit, which keeps up active complex of glycogen with the catalytic subunit. If the regulatory unit is phosphorylated by PK A, it releases the catalytic subunit (exhibiting low activity) that is then fully inactivated by binding with a similarly phosphorylated protein inhibitor. If it is phosphorylated at another site by insulin-dependent PK, the phosphatase activity of the complex of glycogen and the catalytic subunit will increase Biochemistry II Pouria Farsani 2013 332 77. The role of hypothalamic and pituitary hormones – a brief survey, functions Brief survey (see all the following schemes) Biochemistry II Pouria Farsani 2013 333 Hypothalamic hormones – abbreviations and functions Abbreviation Name Major actions Gn-RH Gonadotropin releasing hormone Stimulates secretion of LH and FSH PIH Prolactin inhibiting factor (dopamine) Inhibits secretion of prolactin TRH Thyrotropin-releasing hormone Stimulates secretion of TSH and prolactin SIH Somatotropin releaseinhibiting hormone (somatostatin) Inhibits secretion of growth hormone and TSH GH-RH (=SRH) Growth hormonereleasing hormone Stimulates secretion of growth hormone CRH Corticotropin-releasing hormone Stimulates secretion of ACTH Hormones of the anterior pituitary – abbreviations and function Abbreviation Name Major actions FSH Follicle-stimulating hormone Stimulates growth of ovarian follicles and estrogen secretion. Promotes sperm maturation LH Luteinizing hormone Stimulates ovulation, formation of corpus luteum, and synthesis of estrogen and progesterone (ovary). Stimulates synthesis and secretion of testosterone PRL Prolactin Stimulates breast milk production and breast development TSH Thyroid-stimulating hormone Stimulates synthesis and secretion of thyroid hormones GH Growth hormone (somatotropin) Stimulates protein synthesis and overall growth ACTH Adrenocorticotropic hormone Stimulates synthesis and secretion of adrenal cortical hormones - β-lipotropin ? humans MSH Melanocyte-stimulating hormone Stimulates melanin synthesis (? humans) Biochemistry II Pouria Farsani 2013 334 Hormones of posterior pituitary – abbreviations and functions Abbreviation Name Major actions - Oxytocin Milk ejection; uterine contraction ADH Antidiuretic hormone (vasopressin) Stimulates H2O reabsorption by renal collecting ducts A more detailed description of the functions of the hormones will follow below. However, the surveys above are of main importance for this oral question. Anterior pituitary -TSH, LH and FSH – these hormones belong to the same glycoprotein family. Each has an α- and a β-subunit. The α-subunits are identical. The β-subunits are more differentiated and are responsible for the unique biological activity of each hormone. -ACTH, melanocyte-stimulating hormone (MSH), β-lipoprotein, and β-endorphin *are derived from a single precursor, pro-opiomelnaocortin (POMC) *α-MSH and β-MSH are produced in the intermediary lobe, which is rudimentary in adult humans -Growth hormone *the most important hormone for normal growth to adult size *single-chain polypeptide that is homologous with prolactin and himan placental lactogen a) Regulation of growth hormone secretion *GH is relased ina pulsatile fashion *secretion is increased by sleep, stress, hormones related to puberty, starvation, exercise, and hypoglycemia *secretion is decreased by somatostatin, somatomedins, obesity, hyperglycemia, and pregnancy (1) Hypothalamic control – GHRH and somatostatin *GHRH stimulates the synthesis and secretion of GH *somatostatin inhibits secretion of GH by blocking the response of the anterior pituitary to GHRH (2) Negative feedback control by somatomedins *somatomedins are produced when GH acts on target tissues *somatomedins inhibit the secretion of GH by acting directly on the anterior pituitary and by stimulating the secretion of somatostatin from the hypothalamus (3) Negative feedback control by GHRH and GH *GHRH inhibits its own secretion from the hypothalamus *GH also inhibits its own secretion by stimulating the secretion of somatostatin from the hypothalamus b) Actions of GH *in the liver, GH generates the production of somatomedins [insulin-like growth factors (IGF)], which serve as the intermediaries of several physiologic actions *the IGF receptor had tyrosine kinase activity, similar to the insulin receptor (1) Directi actions of GH *glucose uptake into cells (diabetogenic) ↑ Biochemistry II Pouria Farsani 2013 335 *lipolysis ↑ *protein synthesis in muscle ↑ and lean body mass ↑ *production of IGF ↑ (2)Actions of GH via IGF *protein synthesis in chondrocytes ↑ and linear growth (pubertal growth spurt) ↑ *protein synthesis in muscle ↑ and lean body mass ↑ *protein synthesis in most organs ↑ and organ size ↑ -Prolactin *the major hormone responsible for lactogenesis *participates with estrogen, in breast development *it is structurally homologous to GH a) Regulation of prolactin secretion (1) Hypothalamic control by dopamine and TRH *prolactin secretion is tonically inhibited by dopamine [prolactin-inhibiting factor (PIF)] secreted by the hypothalamus. Thus, interruption of the hypothalamic-pituitary tract causes increased secretion of prolactin and sustained lactation *TRH increases prolactin secretion (2) Negative feedback control *Prolactin inhibits its own secretion by stimulating hypothalamic release of dopamine b) Actions of prolactin (1) stimulates milk production in the breast (casein, lactalbumin) (2) stimulates breast breast development (in a supportive role with estrogen) (3) inhibits ovulation by decreasing synthesis and release of Gn-RH (4) inhibits spermatogenesis (by decreasing Gn-RH) Factors that increase prolactin secretion Factors that inhibit prolactin secretion Estrogen Dopamine Breast-feeding Bromocriptine (dopamine agonist) Sleep Somatostatin TRH Prolactin (by neg. feedback) Dopamine antagonists Posterior pituitary ADH and oxytocin are homologous nonapeptides. They are synthesized in hypothalamic nuclei and are packaged in secretory granules with their respective neurophysins. They travel down the nerve axons for secretion by the posterior pituitary -ADH see q. 44 -Oxytocin *originates primarily from on the paraventricular nuclei of the hypothalamus *causes ejection of milk from breast when stimulated by suckling a) Regulation of oxytocin secretion (1) Suckling *major stimulus *afferent fibers carry impulses from the nipple to the spinal cord. Relays in the hypothalamus Biochemistry II Pouria Farsani 2013 336 tripper the release of oxytocin from the posterior pituitary *the sight or sound of the infant may stimulate the hypothalamic neurons to secrete oxytocin (2) Dilation of the cervix and orgasm *increase the secretion of oxytocin b) Actions of oxytocin (1) Contraction of myoepithelial cells in the breast *milk is forced from the mammary alveoli into the ducts and delivered to the infant (2) Contraction of the uterus *during pregnancy, oxytocin receptors in the uterus are up-regulated as parturition approaches, although the role of oxytocin in normal labor is uncertain *oxytocin can be used to induce labor and reduce postpartum bleeding Biochemistry II Pouria Farsani 2013 337 78. Synthesis of thyroid hormones (description, localization, secretion and its control) In the thyroid gland, follicle cells synthesize the two iodine-containing thyroid hormones, thyroxine (T4 tetraiodothyronine) and triiodothyronine, T3. Both of these hormones are bound to thyroglobulin and stored in the colloid of the follicles. Thyroglobulin is a dimeric glycoprotein. TSH stimulates its synthesis in the thyroid cells after which it is stored in vesicles and released into the colloid by exocytosis. Synthesis (follow scheme on next page!) The synthesis and release of T3/T4 is controlled by the thyrolibertin (= thyrotroponinreleaseing hormone, TRH)-thyrotroponin (TSH) axis. Iodine which is needed for the hormone synthesis is taken up from the bloodstream in the form of iodide (I). As it is taken into the thyroid cells by 2Na+ -Isymport carrier (NIS) the resulting intracellular concentration is 25 times higher than the concentration of blood plasma. The thyroid-stimulating hormone (TSH) increases the basolateral Iuptake by up to 250 times The I- /Clantiporter (pendrin) transports Iions continuously transported from the intracellular Ipool to the colloid (apical side), this is stimulated by TSH. In the colloid, thyroxine peroxidase (TPO) and H2O2, oxidize the Ito I+ => reacts with tyrosyl residues of the thyroglobulin => iodination of the phenol ring of the tyrosyl residue at position 3 and/or 5 => diiodotyrosine (DIT) and/or monoiodotyrosone (MIT) residues. These steps are stimulated by TSH as well and inhibited by thiouracil, thiocyanate, glutathione and other reducing substances. Furthermore, the structure of the thyroglobulin molecule, allows the iodinated tyrosyl residues to react with each other in the colloid. The phenol ring of one DIT (or MIT) molecule links or birdges with another DIT molecule, resulting in a thyroglobulin chain containing tetraiodothyronine residues and (less) triiodothyronine residues. These are the storage form of T4 and T3 Biochemistry II Pouria Farsani 2013 338 Secretion The secretion is also stimulated by TSH. Secretion occurs in the following steps Endocytosis of iodinated thyroglobulin from the colloid (reabsorption) Fusion of endosomes with primary lysosomes forming phagolysosomes Hydrolysis by proteases Release of T3 and T4 Secretion of T3 and T4 and DIT + MIT Release of Ifrom DIT + MIT by the action of deiodase Iis again available for synthesis In blood > 99 % of T3 and T4 is bound on thyroxine binding globulin. Control of T3 and T4 secretion -Hypothalamic-pituitary control – TRH and TSH The TSH secretion by the anterior pituitary is stimulated by TRH, a hypothalamic tripeptide, and inhibited by somatostatin (SIH). TSH increases both the synthesis and secretion of thyroid hormones by the follicular cells via cAMP mechanism. The T3 downregulates TRH receptors in the anterior pituitary and thereby inhibiting TSH secretion. Biochemistry II Pouria Farsani 2013 339 -Thyroid-stimulating immunoglobulins These are components of the IgG fraction of plasma proteins; they are antibodies to TSH receptors on the thyroid gland. As they bind to the TSH receptors, they stimulate the thyroid gland to secrete T3 and T4 – TSH does. Patients with Graves’disease have high circulating levels of these immunoglobulins leading to high concentrations of thyroid hormones and low concentrations of TSH (caused by feedback inhibition of thyroid hormones on the anterior pituitary) Except for thyroxine-binding globulin (TBG), T3 and T4 are also transported by other plasma proteins: -Thyroxin-binding prealbumin (TBPA) -Serum albumin Metabolism of thyroxin It is important to note that only about 20 % of the all circulating T3 originate from the thyroid; the other 80% are produced by the liver, kidneys and other target cells that cleave iodide from T4 – the conversion T4 to T3 which is catalyzed by 5`-deoiodase which removes iodine from the 5`position on the outer ring. T3 is therefore a more potent (acts more rapidly, has a shorter half-life) hormone while T4 is mainly ascribed a storage function in plasma. Biochemistry II Pouria Farsani 2013 340 Pathology -Goiter (struma) – diffuse or nodular enlargement of the thyroid gland. Diffuse goiter can occur due to an iodine deficiency, resulting T3/T4 deficits that ultimately lead to increased secretion of TSH. Chronic elevation of TSH leads to the proliferation of follicle cells, resulting in goiter development (hyperplastic goiter). This prompts an increase in T3/T4 synthesis, which sometimes normalizes the plasma concentration of T3/T4 (euthyroid goiter). This type of goiter often persists even after iodide deficiency is rectified. -Hypothyroidism – occurs when TSH-driven thyroid enlargement is no longer able to compensate for the T3/T4 deficiency (hypothyroid goiter). This type of goiter can also occur due to a congenital disturbance of T3/T4 synthesis or thyroid inflammation. -Hyperthyroidism – occurs when a thyroid tumor (hot node) or diffuse struma (e.g. in Graves’disease) result in the overproduction of T3/T4, independent of TSH. In the latter case, an autoantibody against the TSH receptor binds to the TSH receptor. Its effect mimics those of TSH, stimulation of T3/T4 secretion. Biochemistry II Pouria Farsani 2013 341 79. Calcium and (inorganic) phosphate metabolism – intake, distribution in the body, mineral deposits and soluble forms, the role of PTH, calcitriol, calcitonin. Plasmatic concentration of calcium, factors affecting it Calcium Intake of calcium can be divided into animal food and plant food: -Animal food *milk and dairy products *eggshells (90 % CaCO3) – mix with lemon juice and drink – the ultimate calcium intake! *bioavailability ~ 50% -Plant food *broccoli, poppy seed *nuts, almonds, dates, legumes *bioavailability ~10% We can clearly see that animal sources of calcium are a better source for calcium intake due to the higher bioavailability Some factors affect the intestinal absorption of calcium: -Increase *vitamin D *proteins *fermented milk products (yoghurt, kefir) -Decrease *phosphates (Coca-Cola, processed cheeses) *oxalates and magnesium (leafy vegetables) *excess of dietary fibers The recommended daily intake: -Infants – 1.2 g -Adults – 1.0 g -Pregnant, lactating women – 1.4 g -Elderly – 1.5 g Biochemistry II Pouria Farsani 2013 342 Biochemistry II Pouria Farsani 2013 343 Looking at the first scheme we can see that the serum levels of calcium are dependent on the interplay between intestinal absorption, renal excretion and bone remodeling (resorption and formation) – each of these components is hormonally regulated (will be explained below). In order to maintain the calcium balance, the net intestinal absorption must be balances by urinary excretion! Furthermore we can see a positive and negative Ca2+ balance where in: -Positive: *seen in growing children *intestinal calcium absorption > urinary excretion, and the excess is deposited in the growing bones -Negative: *seen in pregnant or lactating women *intestinal absorption < urinary excretion, and the deficit comes from the maternal bones Looking at the latter scheme, we can see the three main distributions of calcium in the body, where the majority is the free ionized calcium. The ionized calcium is the biologically active which participates in regulations of numerous of cell functions (see details in q. 75). The scheme above illustrates the different kinds of calcium chelates (10%) found in the body. Biochemistry II Pouria Farsani 2013 344 As seen in the scheme above, protein levels in blood will affect calcium levels. Regarding the calcium that is bound to proteins, an important mark can be made regarding the pH of the blood. At physiological pH almost all proteins are in a pH higher than the isoelectric point, hence their negative charge and ability to bind to Ca2+ . That is why pH ↑ => more bindings sites for Ca2+ => decrease of calcium level in alkalosis. The opposite reaction takes place in acidosis. Note that the x- and y-axes are upside down for calcium Biochemistry II Pouria Farsani 2013 345 Looking at the scheme above, we can see the three main ways of calcium intestinal absorption -Facilitated diffusion -Active transport -Paracellular transport Biochemistry II Pouria Farsani 2013 346 Looking at the latter scheme, we can see that calcium transport across membranes takes place with: -Calcium channels -Ca2+ -ATPase -Na+ /Ca2+ exchanger (NCX) Phosphate The phosphate metabolism is closely related to calcium metabolism but is less tightly controlled. The daily intake is about 1.4 g where about 0.9 g is absorbed and usually excreted by the kidneys. The concentration of phosphate in serum ranges from 0.7-1.6 mmol/l. Looking at the scheme above, we can see that 30-80 mmol/d of phosphate is ingested. 10-40 mmol/d is excreted whilst 15-55 mmol/d is absorbed. Furthermore we can see that the greatest deposit of phosphate is found in bones the turnover of which (resorption and formation) is about 8-15mmol/d. The excretion fraction (EF) of phosphate which is less than 25% is either increased or decreased depending on different factors – growth hormone (GH) decreases it (increasing factors, see purple box). Biochemistry II Pouria Farsani 2013 347 The role of PTH, calcitriol and calcitonin The individual effects of each hormone will be presented in the following schemes, the last two of which illustrates their cooperative and combined effects. -Parathormone (PTH, parathyrin) – is a peptide hormone (84 AA, t1/2 ~ 3-5 min) secreted from the chief cells of the parathyroid gland. The Ca2+ sensors in the gland register changes in serum Ca2+ levels. Biochemistry II Pouria Farsani 2013 348 -Calcitriol – is secreted from the cells of the proximal tubule of the nephron in kidneys. It is a secosteroid, 1,25-dihydroxyvitamin D t1/2 ~4-6h. For more details see q. 58 -Calcitonin – is secreted from the parafollicular cells (C cells) of the thyroid gland. It is a peptide hormone 32AA t1/2 ~ 10 min (salmon ~ 60 min, used in the treatment for osteoprorosis). There are Ca2+ in the thyroid gland as well. Biochemistry II Pouria Farsani 2013 349 Biochemistry II Pouria Farsani 2013 350 Plasmatic concentration of calcium and factors affecting it The total calcium concentration in plasma is 2.25-2.75 mmol/l. The amount which is biologically active, free, is 1.12-1.37 mmol/l What regulates the calcium metabolism and its concentration can be summarized into three regulating factors: 1. Homeostasis of Ca2+ in plasma – regulated by PTH and calcitonin -It is mainly the Ca2+ exchange between bones and ECF, the excretion of Ca2+ into urine in a lesser extent. -Rapid exchangeability 2. Homeostasis of Ca2+ in cytoplasm -Regulated by membrane ATPases, NCX 3. Calcium homeostasis in body – regulated by calcitriol -Control of the balance between intake from the intestine and output into urine -It is a hormonal, slower, regulation Biochemistry II Pouria Farsani 2013 351 Mineral deposits and soluble forms Calcium phosphates are sparingly soluble. When the product of Ca2+ concentration times the phosphate concentration (solubility product) exceeds a certain threshold, calcium phosphate starts to precipitate in solutions, and the deposition of calcium phosphate salts occurs. The salts are mainly deposited in bone, but can also precipitate in other organs. Bone is composed of both organic (proteins) and inorganic material. Hydroxyapatite is the inorganic material along with sodium, magnesium, carbonate and fluoride. Hydroxyapatite confers on bone the strength and resilience required by its physiological roles. Biochemistry II Pouria Farsani 2013 352 80. Synthesis and inactivation of catecholamines, degradation products Synthesis and storage • Tyrosine (tyrosine hydroxylase) => DOPA (DOPA-decarboxylase) => dopamine in cytosol Dopamine is then transported into vesicles (ATP dependent transport) => vesicles to nerve terminals. • The beta-hydroxylation by dopamine-beta-hydroxylase in the vesicles produces noradrenaline. • Noradrenaline can be transformed into epinephrine by the action of phenylethanolamine-N-methyltransferase DOPA is therefore the parent substance for dopamine, noradrenaline and epinephrine. The stepwise formation of each of these is shown below. Catecholamines are produced in the adrenal medulla: -The chromafinn cells of the adrenal medulla are modified sympathetic neurons – they do not have axonal fibers -They release their chemical transmitter directly into the circulation – adrenalin being the main one (95%) -Catecholamines are stored in the chromafinn granules together with cotransmitters (neuropeptides), ATP and chromogranine -Secretion is stimulated by cholinergic neurons in the adrenal medulla – nicotinic receptors Biochemistry II Pouria Farsani 2013 353 Inactivation and degradation products The degradation is carried out by MAO (Monoamine oxidase) and COMT (catechol-O – methyltransferase) -MAO – found in neural tissue, the gut and the liver. In the neurons it functions as a “safety value” to oxidatively deaminate and inactivate any excess neurotransmitter that may leak out of synaptic vesicles when the neuron is at rest. -COMT – carries out O-methylation (links with phase 2 of xenobiotics) In degradation: firstly, the aldehyde products of MAO reaction are oxidized to corresponding acids. Secondly, the metabolic products of these reactions are excreted into urine as: -Vanilmandelic acid -Metanephrine -Normetanephrine The steps of degradation can be seen in the scheme below Biochemistry II Pouria Farsani 2013 354 81. Glucocorticoids – structure, biosynthesis, function, regulation of secretion Questions 81-83 regard steroid hormones. Therefore the general biosynthesis of steroid hormones will be discussed in this question before actually discussing glucocorticoids. We divide the steroid hormones and steroidogenic organs accordingly: • Corticosteroids – Adrenal cortex -Glucocorticoids – zona fasciculata and zona reticularis -Mineralocorticoids – zona glomerulosa • Sex hormones – Adrenal cortex & Gonads -Androgens – Leydig cells of testes, (ovaries in a lesser extent) -Estrogens – ovarian follicles and corpus luteum -Progestogens – corpus luteum The precursor of steroid hormones in cholesterol (cholest‐5‐en‐3beta‐ol). Cholesterol is mainly synthesized in the liver, arising from acyl-CoA. It is then transported to the endocrine glands by lipoproteins. The adrenal cortex is also capable is synthesizing cholesterol de novo, the placenta is not however. The steroid hormones are synthesized as needed since only a small amount are stored in the steroidogenic organs. The following pathway shows the formation of steroid hormones from cholesterol as the precursor => Biochemistry II Pouria Farsani 2013 355 Pregnenolone Initially, steroidogenesis is followed by these steps: • Free cholesterol availability -Mostly of extracellular origin -Intracellular CE-pools -Activation of esterases • Transport into mitochondria -Mediated by steroidogenic acute regulatory protein (StAR) -Activated by PK-A (LH, ACTH) • Synthesis of pregenolone Side chain cleavage of cholesterol is carried out by desmolase => pregnenolone -Cytochrome P450 side chain cleavage enzyme (P450scc) -The rate limiting enzyme of steroidogenesis -Activation of P450-subunit of desmolase leads to the phosphorylation of desmolase Biochemistry II Pouria Farsani 2013 356 Progesterone Progesterone is not only a potent hormone itself but it can act as the precursor of all other steroid hormones. It is produced and secreted by corpus luteum. Its primary effects are: -Secretory phase of uterus and mammary glands -Implantation and maturation of fertilized ovum Glucocorticoids The initial hydroxylation at C17 results in the synthesis of glucocorticoids mainly in zona fasciculata and reticularis. Biochemistry II Pouria Farsani 2013 357 The glucocorticoids are cortisol and cortisone. -Cortisol has a primary effect on the intermediary metabolism: *gluconeogenesis (liver) *protein degradation (skeletal muscles) *fatty acid mobilization (adipose tissue) *anti-inflammatory effect (stabilize lymphokine synthesis and histamine release) *increase of myocardial contractility and vasoconstriction due to enhancement of catecholamine effects *increase of epinephrine and angiotensinogen *maintain a normal glomerular filtration rate by delaying the excretion of water *weakening of protective functions of the gastric mucosa hence high doses or stress => ↑ risk of gastric ulcers *Suppression of the immune response Most of the plasma cortisol is bound to transcortin or cortisol-binding globulin (CBG). Cortisol is released in response to conformational changes of CBG due to inflammation etc. Biochemistry II Pouria Farsani 2013 358 Regulation of secretion The synthesis and secretion is regulated by CRH and ACTH. The following scheme illustrates the feedback regulation of cortisol synthesis There is also a circadian rhythm of CRH secretion allowing observation of changes of ACTH and cortisol levels throughout the day – secreted in 2-3 hour episodes. Biochemistry II Pouria Farsani 2013 359 82. Mineralocorticoids – structure, biosynthesis, function, regulation of secretion, the renein-angiotensin system Biosynthesis Synthesis of the mineralocorticoids takes place in zona glomerulosa of the adrenal cortex. Hydroxylation at C21 which is carried out in this zone makes the steroid insensitive to the effects of 17-hydroxylase. As a result, only mineralocorticoids like corticosterone and aldosterone can be synthesized. Only the initial hydroxylation provides glucocorticoids (see q. 81). Biochemistry II Pouria Farsani 2013 360 Structure Function, regulation of secretion, the renin-angiotensin system Besides the very short information provided in the schemes above, see q. 44 and 58 for more detailed information. Biochemistry II Pouria Farsani 2013 361 83. Sex hormones (strructure, biosynthesis, function, regulation of secretion, inactivation) Sex hormones (androgens 19 C) can be synthesized, both by gonads and the adrenal cortex. In general we need to explain that precursors for steroid hormone synthesis are present in all steroid hormone glands. The type of hormone produced and the site of hormone synthesis depends on: 1) The type of receptors available for the superordinate control hormones (ACTH, FSH, LH, etc.) 2) The dominant enzyme responsible for changing the structure of the steroid molecule in the hormone-producing cells of the gland in question Male androgens Steroid hormones with 19C. These hormones include the potent hormones like testosterone and dihydrotestosterone (DHT). The less potent type would be DHEA. About 95% of testosterone in males is synthesized by the testes (Leydig cells). The other 5% is Biochemistry II Pouria Farsani 2013 362 synthesized by the adrenal cortex. In females, the ovaries and adrenal cortex stand for the testosterone production. The difference is that in the testes and ovaries the reaction for production is promoted by LH (formation of cAMP via the G protein-coupled LH receptor. cAMP increases the formation of cholesterol and the formation to pregnenolone), rather than ACTH which promotes the reaction in the adrenal cortex. Furthermore, the testes do not have 11- and 21-hydroxylases, which is why gluco- or mineralocorticoids are not synthesized in gonads. 17-alpha-hyroxylase is present in both testes and adrenal cortex however. Testosterone is bound to albumin (98%) and sex hormone-binding globulin in plasma. The testes produce, besides DHT, estradiol (E2) as well. However, larger quantities of DHT (via 5-alpha-reductase) and estradiol are synthesized from testosterone (via aromatase) by their respective target cells. Estradiol influences many functions in male, such as epiphyseal cartilage and ejaculate formation as well as pituitary and hypothalamic activity. Function of testosterone -Sexual development and differentiation: *need of testosterone in the steps of somatic sex development and sex differentiation -Secondary male characteristics -Testicular function – sperm maturation and semen production, along with FSH. -Sperm protein production in Sertoli cells -Inhibitory feedback on LH secretion -Protein-anabolic growth-promoting effect Function of dihydrotestosterone -More potent (3x) than testosterone -Prenatal differentiation of external genitalia -Anabolic effect -Virilization Biochemistry II Pouria Farsani 2013 363 Regulation of secretion Luetinizing hormone (LH) is the main hormone which regulates the secretion stimulating the testosterone release from the Leydig cells. The secretion rate is 4-9mg/d in normal adult males. Small amounts are secreted in females as well – the ovaries being the major source and secondly the adrenal gland. A negative feedback by testosterone and estradiol inhibit the LH secretion. Also, Gn-RH is inhibited since it is the responsible hormone for the pulsatile secretion of LH. Furthermore, Gn-RH also induces the release of FSH which stimulates the secretion of inhibin and induces the expression androgen-binding protein (ABP) in Sertoli cells. ABP is needed in order for testosterone to induce spermatogenesis. FSH also induces formation of LH receptors in Leydig cells. Testosterone, DHT, estradiol and inhibin inhibit the secretion of FSH via negative feedback. Summary of secretion regulation: • Hypothalamic control – GnRH –from arcuate nuclei => stimuli for LH and FSH secretion • Anterior pituitary – FSH and LH – FSH => Sretoli cells => spermatogenesis LH => Leydig cells => testosterone synthesis • Negative feedback control – testosterone, DHT, estradiol, inhibin => testosterone => X Gn-RH => X LH Inhibin => X FSH Biochemistry II Pouria Farsani 2013 364 Female androgens The female androgens are estrogens (18C) and progesterone (21C). -Estrogens – they are primarily synthesized from the 17-ketosteroid androstendione (production of Estrone E1). Testosterone is also a precursor which is synthesized in the Theca cells, to the diffuse to the nearby granulosa cells which contain aromatase and convert testosterone to 17-beta-esrtradiol (Estradiol E2). Furthermore, the adrenal cortex and Leydig cells (in men) are also sites of estrogen synthesis. Estradiol E2 is the most potent estrogen – the potency of Estrone E1 is relatively low in comparison. Actions of estrone E1 -In extraovarian tissues, principally skeletal muscle, adipose, liver -Major source of estrogens in postmenopausal women Biochemistry II Pouria Farsani 2013 365 Actions of Estradiol E2 -Control of menstrual cycle -Female secondary sex characteristics -Inhibits bone resorption (increase in osteoblast activity) -Promotes renal salt and water retention -12x more potent than E1 -Peripheral aromatization of TST => ~80% more in men -During pregnancy adrenal androgens => 50% -Stimulates prolactin secretion (but then blocks its action on the breasts) -Lowers uterine threshold to contractile stimuli during pregnancy Progesterone Progesterone is the most potent progestational (pregnancy-sustaining) hormone. It is synthesized from cholesterol via pregnenolone in corpus luteum, ovarian follicles and placenta in females. It is also synthesized in the adrenal cortex in both men and women. Actions of progesterone -Negative feedback effects on FSH and LH secretion during luteal phase -Maintains secretory activity of the uterus during the luteal phase -Maintains pregnancy -Raises the uterine threshold to contractile stimuli during pregnancy -Participates in the development of the breasts The uterus is the chief target organ of progesterone, progesterone induces endometrial thickening, stimulates growth of the myometrium, restructures the endometrial glands, alters blood supply and changes the glycogen content. Regulation of secretion • Hypothalamic control – GnRH As in the male, pulsatile GnRH stimulates the anterior pituitary to secrete FSH • Anterior pituitary – FSH and LH Biochemistry II Pouria Farsani 2013 366 These two hormones stimulate the following in the ovaries: -Steroidogenesis -Follicular development beyond the antral stage -Ovulation -Luteinization • Negative and positive feedback – estrogen and progesterone -Follicular phase – estrogen has a negative feedback effect on the anterior pituitary -Midcycle phase – estrogen has a positive feedback effect on the anterior pituitary -Luteal phase – estrogen has a negative feedback effect on the anterior pituitary. Progesterone has a negative feedback effect on the hypothalamus Aromatase Found in: -Ovaries and other tissues such as adipose, liver, skin -Its increase activity contributes to: *liver cirrhosis *hyperthyroidism *aging *obesity -Inhibitors of aromatase are used as therapeutic agents such as in estrogen-dependent tumours (breast cancer) Inactivation of Androgens Estrogens and progesterone are removed from the bloodstream during their first passage through the liver. In general degradation of steroid hormones takes place in the liver. Their OH groups are usually linked to sulfate or glucuronic acid molecules. The coupling products are ultimately excreted into the bile or urine. The main urinary metabolite of estrogens is estriol while that of progesterone and 17alphahydroxyprogesterone is pregnanediol (measured to exclude pregnancy) A small amount of testosterone is converted to estradiol, but most of the testosterone is converted to 17-ketosteroids, principally androsterone and its isomer etiocholanolone and excreted into urine. Biochemistry II Pouria Farsani 2013 367 84. Insulin (synthesis, regulation of secretion, fate, insulin receptor and results of its activation). Oral glucose tolerance test Insulin: -Formed in the pancreatic beta-cells -51 AA, 2 chains with 3 disulfide bridges -3 min t1/2 -Inactivation takes place in the liver (kidneys) -Inactivation is carried out by GSH-insulin-transdehydrogenase and insulinase Synthesis The synthesis takes place in the rough ER, Preproinsulin synthesis takes place in the rough ER Transfer of product into the ER trough the hydrophobic signal Removal of 23 AA sequence Production of proinsulin in the ER cisternae – its structure includes three disulfide bridges Transfer of proinsulin into the GA cisternae Conversion to insulin by hydrolytic enzymes Formation of insulin which consists of two unequally long polypeptide chains A (21 AA) + B (30 AA), and C-peptide Storage of both insulin and C-peptide in secretory granules Release of both insulin and C-peptide into the blood by exocytosis (C-peptide determination is used in clinical biochemistry in order to indicate endogenous insulin production) Regulation of secretion Insulin is continuously produced and secreted into the blood in healthy people, regardless of food intake – basal secretion. The basal secretion represents ~50% of all the insulin which is secreted within 24 hours. The remaining 50% is food stimulated (prandial) secretion. This secretion is thus regulated by the glucose level in blood, partially. The other regulating mechanisms are signals from CNS Biochemistry II Pouria Farsani 2013 368 (by somatostatin) and GIT (e.g. by cholecystokinin and GLP-1). Certain amino acids also participates in the stimulation of secretion, in a lesser extent however than that of glucose. -Secretion ↑ *blood glucose ↑ *Signals from CNS (by somatostatin) *Signals from GIT (cholecystokinin and GLP-1) *Certain AA -Secretion ↓ *Adrenaline ↑ *Glucagon ↑ (Glc ↓ => glucagon from pancreatic alpha cells ↑ => binds to receptors in pancreatic beta- and delta cells => somatostatin from delta cells ↑ => binding to beta-cells => inhibition of insulin release => insulin ↓) Glucose sensors detect elevated glucose levels => release of insulin. Entrance of glucose into beta-cells via GLUT 2 Phosphorylation of glucose by glucokinase Glucose ↑ => Glycolysis and CAC ↑ => ATP ↑ => inhibition of ATP-sensitive K+ channels => membrane depolarization => influx of Ca2+ through channels which are dependent on the membrane potential Ca2+ ↑ => stimulation of exocytosis of insulin (blocked by adrenaline and noradrenaline) It is the pancreatic glucokinase which mediated a connection between elevated blood glucose and insulin release this it is referred to as a glucose sensor. Sulfonylurea is another glucose sensor, an oral anti-diabetic-drug which influences K+ channel activity (same effect as ATP). Biochemistry II Pouria Farsani 2013 369 Fate Insulin is released with a periodicity of 11-15 min – frequency and amplitude of these pulses increases upon secretion stimuli. The insulin is released into the portal blood, hence it must pass through the liver before it can proceed to the systematic circulation – approx. half of the insulin is metabolized during this first passage through the liver. Fasting (20-100pmol/l) => 350-580 pmol/l (after a typical meal). Most of the insulin which is released is taken up by the liver again or by other tissues that have insulin receptors. In the liver: -GSH-insulin-transhydrogenase disrupts the disulfide bonds: 2GSH = insulin-disulfide => GS-S-G + insulin-dithol -Insulinase (insulysin, insulin-specific protease, insulin-glucagon protease) – a cytosolic protease which degrades insulin, glucagon and other polypeptides. It has no action on proteins. Insulin in the brain There are insulin receptors in numerous areas of the CNS. In the brain insulin participates in the regulation of energy balance of the body, and also plays a role in insulin interaction with other factors, such as leptin and serotonin. In hypothalamic neurons insulin action => increased proopiomelanocortion expression, melanocortion formation and a decrease in agouti-related peptide (AgRP) level => anorexigenic effect. Insulin thus contributes to the decrease in food intake together with leptin. Oral glucose tolerance test (oGTT) It is one of the diagnostic methods for diabetes mellitus. If fasting glucose in blood serum or plasma is within 6-8 mmol/l or if the venous or capillary blood is within 5-7mmol/l, the effectiveness of glucose metabolism should be verified by this test. It is based on the monitoring of changes in glycaemia after a per-oral administration of a standard dose of glucose by mouth. This test is not performed at higher glucose concentrations, especially if the characteristic symptoms of diabetes are present. -Standard procedure in adults – no restriction of saccharides three days before the test. Night fasting (10-14 hours) Blood sample is taken Patient is asked to drink 75 g of glucose in approx. 300 ml of tea Blood sample is taken 1 or 2 hours after drinking the tea Patient is not allowed to smoke, eat or drink or do any physical activity Biochemistry II Pouria Farsani 2013 370 -Evaluation (mmol/l) Glc tolerance 0 h (fasting) 1h post-glucose load 2h post-glucose load Normal <5.6 <11 <7.8 Impaired 5.6-6.9 >11 7.8-11 Diabetes mellitus >7 >11 >11 In case the values of glycaemia do not fit into one of the evaluation types, the level of glycaemia at is taken at fasting and 2 hours after load. Results which cannot be evaluated according to oGTT is a reason to repeat the test several days (up to several weeks) later. Examinants older than 50 years have upper reference levels shifted by 0.55 mmol/l for each decade. Some diseases such as hepatic, may disrupt the oGTT. Biochemistry II Pouria Farsani 2013 371 85. Insulin receptor and effects of its activation (See also q. 72) -The insulin receptor belongs to the family of membrane receptors with tyrosine kinase activity -The insulin receptor is located in membranes as a dimer. Each monomer consists of the extracellular subunit alpha unit and the subunit beta, which is an integral membrane protein. -Alpha and beta subunits are joined by a disulfide bond. A disulfide bond also joins both monomers. -Binding sites for insulin are found in the alpha subunits -Beta subunits contain domains with their own tyrosine kinase activity – these subunits mutually phosphorylate each other after insulin binding. Activated phosphorylated receptor binds to proteins called IRS (insulin receptor substrate, also called adaptor proteins) Phosphorylation of IRS at several sites (approx. 20 tyrosine residues) Activation of binding sites in the IRS for various proteins that transduce several insulin effects Binding of proteins to biding sites in the IRS => activation of these proteins (most of these proteins bind to these sites via specialized SH2 domains, src homology) Binding of protein Grb2 (growth factor receptor-bound protein) to one of the sites and attached to the membrane by a phospholipid anchor Biochemistry II Pouria Farsani 2013 372 Grb2A activates membrane-bound G-protein Ras. Since Ras is a monomer existing in an inactive GDP form and an active GTP form. Activation => increase of active RasGTP molecules that activate proteinkinases and induce Ras and MAP (mitogen activated protein) signal cascades => phosphorylation of proteins in the cytoplasm and nucleus where transcription factors are regulated PI-3 kinase binds at another phosphotyrosine site at the IRS and is thereby activated Phosphorylates inositol phospholipids in the membrane at position 3 (for example, conversion of phosphatidylinositol-4,5-bisphosphate to phosphatidyl-inositol-3,4,5- trisphosphate) Activated phospholipids acts as second messenger => activation of other proteins including protein kinases B and C (mediate many other insulin effects in a cell) Expulsion of GLUT4 transporters into the membrane in muscles and adipocytes (one of the effects) Phospholipase Cgamma binds to another IRS site and becomes activated as well Biochemistry II Pouria Farsani 2013 373 86. The metabolism of xenobiotics – stage I of their biotransformation (various types of transformations, examples, mixed-function monooxygenases – function of cyt P450) The three principal entries of xenbiotics into the body are the intestine, lungs and skin. The xenobiotics need to cross en epithelium barrier (phospholipid bilayer) between blood (ECF) and tissues (ICF). Penetration is dependent on physical and chemical properties where hydrophobicity facilitates transport through the cell membrane. Entry takes place through simple diffusion, facilitated diffusion, active transport or endocytosis. As xenobitotics enter the body, they need to undergo biotransformation which occurs mostly in the liver in two phases – the first of which will be discussed below. The kidneys and intestines also participate in the metabolism – the ultimate aim is to eliminate the xenobiotics from the body which requires them to be metabolized into water-soluble products. Without biotransformation, very hydrophobic substances would persist in adipose tissue for unlimited time. The first phase of biotransformation Hydroxylation reactions take place predominantly – the substance is oxidized by addition of one or more hydroxyl groups. The product may still be biologically active or its activity may be changed in this phase, for instance, the medicinal effect of a drug may be changed. The table below summarizes the reactions taking place during the first phase: Biochemistry II Pouria Farsani 2013 374 Cyt P450 and its function Oxidative reactions of the first phase are often monooxygenase reactions that use cytP450 (P450) as an electron carrier. Besides participating in the oxidative reactions of xenobiotics, P450 is also found in the steroid glands where it is involved in the synthesis of steroids. P450 is a superfamily of heme enzymes with many isoforms. It can catalyze different reaction types, hydroxylation being the main one. Its advantage is that it has a wide substrate specificity. Furthermore it can be induced and inhibited. P450 is found in most tissues (bound in the ER membrane or the inner mitochondrial membrane) except for muscles and erythrocytes. The highest amount is found in the liver (ER). Genetic polymorphism of P450 leads to atypical biotransformation. The microsomal cytP450 contains three cofactors and two enzymes: • NADPH + H+ , FAD, heme • NADPH:CYP reductase (separates 2H => 2e+ 2H+ ) • Cytochrome P450 (hydroxylase) (Mitochondrial cytP450 contains adrenodoxin reductase + adrenodoxin Fe-S) -P450 functions by forming a hydroxyl group -It is called monooxygenase as one O atom from O2 is incorporated into substrate between C and H (R-H => R-OH) -The second O atom + 2H from NADPH + H+ give water General reaction is hydroxylation reaction is: Biochemistry II Pouria Farsani 2013 375 The hydroxylation reactions by cytP450 occur both in endogenous and exogenous substrates where in: -Mitochondria: *steroid hormones -Endoplasmic reticulum: *squalene *cholesterol *bile acids *calciol *FA desaturation *prostaglandins *xenobiotics Biochemistry II Pouria Farsani 2013 376 There are many isoforms of human cytP450 where the various isoforms prefer different substrates. They also have different inducers and inhibitors: -The main P450 in drug biotransformation is isoform CYP3A4 -Induction – the metabolic capacity of CYP is enhanced: -Administration of inducer + drug, both metabolized by the same CYP isoform => faster metabolism of drug => drug is less effective -Examples of inducers: *binds to DNA => enhances transcription of mRNA => stimulation of CYP synthesis *decreases the degradation of mRNA and/or CYP *influences the post-transcription modifications of mRNA *may cause hypertrophy of ER -Administration of inhibitor + drug => increased drug level => overdosing => side effects -Examples of inhibitors – speak about inhibitors of CYP3A4: *macrolide antibiotics (erythromycin) *grapefruit juice *antifungal ketoconazole -Examples of inducers of CYP3A4: *rifampicin *herbal preparations from St John’s wort (Hypericum performatum) Biochemistry II Pouria Farsani 2013 377 -Genetic polymorphism of CYP450 – affects the individual’s ability to metabolize drugs. It is mostly found in the CYP2D6 isoform. Based on the phenotype, the population may be divided into two groups, the slow and fast metabolizers. In the case of 2D6 we distinguish between four groups – slow, medium, fast and very fast metabolizers. Where the slow metabolizers may be exposed to overdosing and fast metabolizers need higher doses in order to achieve therapeutic efficiency. Below there are two examples of biotransformations: Biochemistry II Pouria Farsani 2013 378 87. The metabolism of xenobiotics – stage II (conjugation). (Reaction types, reactant activation, products – examples) -The second phase of metabolism of xenobiotics involves conjugation with hydrophilic molecules – catalyzed by transferases. It is the xenobiotic from the 1st phase which reacts with endogenous conjugation reagent. -The synthesis is an endergonic reaction where one of the reactants must be activated. -The conjugate is more polar, less active/no more biologically active and easily excreted by urine and/or bile (stool) (GASSAM) -Glucuronidation – the most common conjugation: *O-glycosides – ether type (Ar-O-glucouronate, R-O-glucouronate) and ester type (Ar-COO- glucouronate) *N-, S-glycosides *substrates: aromatic amines, amphetamines, (acetyl) salicylic acid, flavonoids … *endogenous substrates: bilirubin, steroids Biochemistry II Pouria Farsani 2013 379 -Sulfation – carried out by PAPS (phosphoadenosyl phosphosulfate) Biochemistry II Pouria Farsani 2013 380 -Methylation – involved in the inactivation of catecholamines: Biochemistry II Pouria Farsani 2013 381 -Sulfide formation – glutathione conjugation Biochemistry II Pouria Farsani 2013 382 -Amide formation – conjugation with amino acids – glycine and taurine *Xenobiotics with –COOH groups *Amide bond formation *An endogenous example is conjugation of bile acids Biochemistry II Pouria Farsani 2013 383 88. Alcohols and phenols as xenobiotics and their transformation (ethanol and ethylene glycol, salicylates and acetaminophen) Ethanol Ethanol which is a small molecule, both soluble in water and lipids, is absorbed mainly by the small intestine by passive diffusion. A small portion (0-5%) is directly metabolized by mucosal cells of upper GIT (tongue, mouth and stomach) – the reaming part is transferred into the blood. The liver performs about 90% of the ethanol metabolism; the rest is metabolized by the kidneys and lungs. However, about 5% is excreted unchanged in the urine, by sweat or by exhalation. The following factors influence the level of ethanol in blood: • Quantity of ingested substance • Body weight • Absorption rate • Rate of detoxification About 7-10g/h of ethanol is eliminated; the level in blood decreases at a constant rate of about 0.1-0.2 ‰ per hour. The formula below allows calculation of blood alcohol level assuming immediate and complete absorption of ethanol, its instantaneous distribution in the body without concomitant elimination: f can also be 0.45 for obese women and 0.90 for muscular men. Biochemistry II Pouria Farsani 2013 384 The main reaction of ethanol metabolism is oxidation by alcohol dehydrogenase (AD) – taking place in the cytoplasm. Acetaldehyde which is the product is further metabolized in the liver (~90 %) to acetate by acetaldehyde dehydrogenase (AcD). Acetaldehyde is toxic if not broken down; its accumulation (due to large amounts of ingested alcohol) is what causes the hangover with its symptoms. Acetate which is not toxic can be activated in the liver (partly in the muscles and other tissues as well) to acyl-CoA where it can enter the CAC or FA synthesis. -Alcohol dehydrogenase – metalloenzyme (Zn) which has many isoforms. It is found in liver, kidneys, lungs, intestine and other tissues. Some of the isoforms are less active in females. The different isoforms have different specificity for different alcohols – ethanol is nonspecifically metabolized by most of the isoenzymes. (mistake? Class V should be class IV?) Biochemistry II Pouria Farsani 2013 385 -Class I – greatest specificity for ethanol -ADH1B and ADH1C – polymorphism expressed by point mutations. This explains familiar differences in the rate of alcohol elimination by different individuals and populations. ADH1B*2 allele has a reduced affinity for alcoholism since the enzyme produced by this gene has a high affinity for alcohol thus alcohol is metabolized very quickly in these individuals => quick accumulation of acetaldehyde which cannot be oxidized quickly enough by AcD => nausea => aversion to alcoholic beverages. This allele is more common in East Asian populations and less common among Europeans. -Aldehyde dehydrogenase – Found in the liver, two main isoforms, ALDH1 (cytosol) and ALDH2 (mitochondria, performs more than 80% of acetaldehyde oxidation, due to its higher affinity). -Commonly among Chinese, Japanese and Koreans (not among Europeans and Africans), polymorphism is found in the case ALDH2 as well => almost complete loss of enzyme activity => accumulation of acetaldehyde after alcohol ingestion => high sensitivity to alcohol. In the case of homozygotes, there is absolute intolerance to alcohol, thus alcohol addiction is never found in these individuals. -Antabuse (disulfiram) aims to inhibit ALDH2 –treatment for alcoholism. Alternative pathways for alcohol metabolism -Microsomal oxidizing system, CYP2E1 (MEOS) – conversion of ethanol by oxidation to acetaldehyde. This pathway takes place in the ER. CYP2E1 uses NADPH as an additional electron donor and O2 as an electron acceptor: CH3-CH2-OH + O2 + NADPH+H+ → CH3-CH=O + 2 H2O + NADP+ MEOS is activated when intake of alcohol is high (> 0.5 ‰) since the Michaelis constant of CYP2E1 is much higher than that of class I AD. Therefore it only significantly contributes to the metabolism of alcohol when intake is high => increased production of acetaldehyde -Peroxisomes – oxidation of ethanol by H2O2, catalase CH3-CH2-OH + H2O2 → CH3-CH=O + 2 H2O Biochemistry II Pouria Farsani 2013 386 The CYP2E1 isoform is inducible by ethanol alone, as well as some other substrates. Chronic alcohol intake => increasing of levels of hepatic CYP2E1 by 5-10 times. Other P450 are also increased with chronic alcohol intake. The ER also proliferates resulting in the increase of microsomal enzymes – even of those that are not involved in alcohol metabolism. CYP2E1 induction => acceleration of alcohol degradation => increased tolerance. As ethanol acts as an inhibitor of CYP2B2 which oxidases phenobarbital, intake of ethanol with phenobarbital will result in phenobarbital not being degraded => intoxication. Gain of energy from alcohol metabolism Depending on the pathway by which ethanol is metabolized: -Oxidation by cytosolic AD and mitochondrial AcD => 2 equivalents of NADH which can provide ATP in the respiratory chain. Additional ATP is provided when acyl-CoA from acetate is oxidized. Two ATP are consumed in the conversion of acetate to acyl-CoA (ATP => AMP) -No ATP is gained in the MEOS pathways since NADPH is consumed – lower total energy. -The nutritional value of alcohol is 29kJ/g. A daily intake of 100-120 g can cover up to half of the basal energy requirements by its metabolism. Adverse effects of alcohol Adverse effects affects the liver metabolism mainly. -Adverse effects due to increased NADH/NAD+ ratio – at lower doses of alcohol consumption, the oxidation rate is regulated by the rate of intake and the rate at which NADH is re-oxidized in the respiratory chain. AD is not reversely regulated by NADH, ATP, ADP or AMP hence metabolism of ethanol is preferred to the metabolism of other nutrients. When NADH is not metabolized quickly enough it accumulates, resulting in the increase in NADH/NAD+ ratio: -TAG increase Inhibition of FA oxidation Accumulation of FA in the liver FA reacts with glycerol to form TAG Increased NADH/NAD+ stimulates glycerol synthesis from glycolytic intermediates => increased availability of glycerol FA are incorporated into VLDL => accumulation in the liver => entering the blood TAG ↑ => storage in the liver => liver steatosis Biochemistry II Pouria Farsani 2013 387 -KB increase Beta-oxidation of FA is limited as acyl-CoA utilization in the CAC is blocked by high NADH/NAD+ High NADH/NAD+ Ketoneogenesis is increased Tissues do not utilize ketone bodies since they preferentially utilize acetate which is formed by alcohol metabolism Ketone bodies increases in blood -Lactate increase High NADH/NAD+ => shift of equilibrium of lactate dehydrogenase reaction Accumulation of lactate Lactoacidosis Reduced excretion of uric acid by the kidneys (why alcohol intake should be limited by people suffering from gout) -Hyperglycemia High NADH/NAD+ causes hypoglycemia when alcohol is drunken on an empty stomach – lack of substrates for gluconeogenesis Oxaloacetate and pyruvate occurs as reduced forms – malate and lactate Intake of alcohol with a filled stomach may result in the sensation of hyperglycemia – the high NADH/NAD+ ratio inhibits glycolysis -Adverse effects due to accumulation of acetaldehyde Decreased protein synthesis in the liver Decreased tubulin synthesis => impairment of protein secretion into blood Accumulation of protein in the liver cells Increased flow of water into the cells Portal hypertension and disturbances of the liver structure Initiation of tissue damage by oxygen radicals by formation of acetaldehyde adducts Consumption of glutathione by direct reaction with acetaldehyde Reduction of capacity to eliminate hydrogen peroxide and lipid peroxidation products Mitochondria can also be damaged by acetaldehyde => disturbances in the coupling of respiratory chain and oxidative phosphorylation -Chronic alcoholism – can cause neurological symptoms due to the lack of thiamine (B1) and pyridoxine (B6). The deficiency is partially due to poor diet in alcoholics, partially due to pathological changes in GIT and partially due to liver damage. Biochemistry II Pouria Farsani 2013 388 Vitamin deficiency has its most serious outcome as Wernicke-Korsakoff syndrome – due to thiamine deficiency which is a result of reduced intake and reduced storage in the liver. The syndrome results in: -Psychological disorders -Ataxia (impaired motor coordination and unsteady gait) -Uncoordinated eye movements Symptoms can be reduced by the administration of thiamine. Furthermore, alcoholics tend to have a lower bone density – they have an increased tendency to osteoporosis. It can be explained as a defect in C-25 hydroxylation of calciol in the liver and increased conversion of calciol by CYP450 to inactive products. The scheme below summarizes the adverse metabolic effects of alcohol: Biochemistry II Pouria Farsani 2013 389 Tests for detection of ethanol intake -Liver enzymes: GGT, AST, ALT ....↑, AST/ALT > 2, CHS .... ↓ -Carbohydrate-deficient transferrin (CDT) – detected in blood ~ 4 weeks after substantial alcohol intake = marker of abstinence -Fatty acids ethyl esters (FAEE) – appears in blood 12-18 hours after drinking and can be detected even 24 hours after alcohol in blood is no more increased. Traces are deposited in hair for months and may serve as a measure of alcohol intake. -Ethyl glucosiduronate (EtG) – blood ethanol ↓ EtG ↑. It is detected (in urine as well) after a few days, even up to 5 days. -Phosphatidyl ethanol (PEth) – present in the blood of individuals who have been drinking moderate ethanol doses daily, even 3 weeks after the last drink. Biochemistry II Pouria Farsani 2013 390 Ethylene glycol Salicylates Biochemistry II Pouria Farsani 2013 391 Acetaminophen