‹#› 1 Biochemistry of Selected Elements © Department of Biochemistry (Jiří Dostál) 2010 ‹#› 2 Distribution of main elements (mass %) Nature Human body Oxygen Silicium Aluminium Iron Calcium 50.0 26.0 7.5 4.7 3.4 Oxygen Carbon Hydrogen Nitrogen Calcium 63.0 20.0 10.0 3.3 1.5 ‹#› 3 Hydrogen in the human body •Elemental gaseous hydrogen (H2) formed in the large intestine by the action of microflora (not important in biochemistry) •Proton (H+) in body fluids determines the pH, constant pH values are kept by the three systems of buffers – see the table, the next slide • Hydrogen ‹#› 4 Buffer systems in blood (pH = 7.40) Buffer system Buffer base Buffer acid Abundance pKA Ratio [base] : [acid] Ideal buffer Ideal base Ideal acid 100 % 7.4 1 : 1 Hydrogen carbonate HCO3- H2CO3 + CO2 50 % 6.1 20 : 1 Protein* Protein-His Protein-His-H+ 45 % 6.0 – 8.0 NA Hydrogen phosphate HPO42- H2PO4- 5 % 6.8 4 : 1 * In plasma mainly albumin, in erytrocytes hemoglobin ‹#› 5 •Covalently bound H in the molecules of nutrients •in metabolic dehydrogenations are 2H transferred to cofactors NAD+, FAD. Oxidation of NADH and FADH2 in respiratory chain releases energy which is used to synthesize ATP (see lecture Bioenergetics) •Hydride anion (H-) •formed transiently during dehydrogenation reactions by the action of NAD+ (2 H ® H- + H+) •for more detailed explanation see the lecture Heterocycles ‹#› 6 Distinguish between proton and hydrogen A donor of H+ = acid A donor of H = reductant An acceptor of H+ = base An acceptor of H = oxidant ! ‹#› 7 Oxygen in nature •the most abundant element (water, CO2, minerals) •dioxygen (O2) in atmosphere, ozone (O3) in ozonosphere •air composition (vol. %): N2 78 O2 21 Noble gases 0.9 CO2 0.03 Oxygen Reserves of O2 in the adult body are very limited •free O2 in lungs (max. 1 litre) •bound to Hb (blood), Mb (muscles) (1.5 litre) •physically dissolved in blood (0.2 litre) ‹#› 8 Biochemical role of dioxygen •transported from lungs as oxy-Hb to cells, where it diffuses to mitochondria •terminal acceptor of electrons in respiratory chain •four-electron reduction of dioxygen gives water: O2 + 4 e- + 4 H+ ® 2 H2O ‹#› 9 Dioxygen is also a hydroxylation agent •phenylalanine ® tyrosine •tyrosine ® ® adrenaline •cholesterol ® ® calcitriol •cholesterol ® ® bile acids •hydroxylation of xenobiotics R-H + O2 + NADPH + H+ ® R-OH + H2O + NADP+ ‹#› 10 Reactive oxygen species (ROS) formed in the human body •Superoxide anion-radical •Hydroxyl radical •Singlet oxygen •Hydrogen peroxide Positive effects - bactericidal (respiratory burst) Negative effects – damage of biomolecules (membranes, enzymes, receptors, DNA) ‹#› 11 Superoxide anion-radical •O2- •One-electrone reduction of dioxygen • • O2 + e- ® •O2- [redox pair] ‹#› 12 Superoxide formation •Respiratory burst (in neutrophils) 2 O2 + NADPH ¾® 2 •O2- + NADP+ + H+ •Spontaneous oxidation of hemoproteins heme-Fe2+ + O2 ¾® heme-Fe3+ + •O2- ‹#› 13 Elimination of superoxide •Superoxide dismutase •Catalyzes the dismutation of superoxide 2 •O2- + 2 H+ ¾® O2 + H2O2 •Oxidation numbers of oxygen • -½ ¾® 0 -I Dismutation is a special type of redox reaction in which an element is simultaneously reduced and oxidized so as to form two different products. ‹#› 14 Hydroxyl radical •OH •The most reactive species, very harmful •The reactions of •OH with various biomolecules produce secondary radicals • •O2- + H2O2 ® O2 + OH- + •OH Catalyzed by reduced metal ions (Fe2+, Cu+) (Fenton reaction) hydroxide hydroxyl ‹#› 15 H2O + ½ O2 Hydrogen peroxide H2O2 is formed as a side product in the deamination of some amino acids imino acid catalase oxo acid ‹#› 16 Elimination of H2O2 •Catalase: H2O2 ¾® ½ O2 + H2O •Glutathione peroxidase (selenocysteine) 2 G-SH + H-O-O-H ¾® G-S-S-G + 2 H2O • ‹#› 17 Compare: reduction of dioxygen Type of reduction Partial scheme (= redox pair) Four-electron O2 + 4 e- + 4 H+ ® 2 H2O One-electron O2 + e- ® ·O2- Two-electron O2 + 2 e- + 2 H+ ® H2O2 ! ‹#› 18 Antioxidant systems in the body •Enzymes superoxide dismutase, catalase, glutathione peroxidase •Low molecular antioxidants reducing substances with: phenolic / enolic -OH group, -SH group • containing extended system of conjugated double bonds • ‹#› 19 Lipophilic and hydrophilic antioxidants Antioxidant Sources Tocopherol Carotenoids Ubiquinol Plant oils, nuts, seeds, germs Fruits, vegetables (most effective is lycopene) Formed in the body from tyrosine L-ascorbate Flavonoids Dihydrolipoate Uric acid Glutathione Fruits, vegetables, potatoes Fruits, vegetables, tea, wine, cocoa Made in the body from cysteine Catabolite of purine bases Made in the body from cysteine ‹#› 20 Distribution of magnesium Human body Blood plasma (ECF) Bones 60 % Ionized 60 % Tissues (ICF) 39 % Associated with proteins 33 % ECF 1 % Chelated 7 % Magnesium ‹#› 21 Biological role of magnesium •typical intracellular cation •associated with negative charges of proteins and nucleotides •activator of many enzymes (kinases, ATPases) •antagonist of calcium ions - Mg2+ ions slow down neuromuscular transmission •mineral component of bones Food sources most animal and plant foods, higher content in: •green leafy vegetables (chlorophyl) •wholemeal cereals, legumes, nuts, sesame seeds, halvah •mineral waters (Magnesia) ‹#› 22 Distinguish: Two types of mineral waters Feature Magnesia (CZ) Šaratica (CZ) Main cation Mg2+ Mg2+ Main anion HCO3- SO42- Usage source of Mg osmotic laxative ‹#› 23 Calcium in nature is abundant •Minerals, sediments •CaCO3 (limestone, marble, chalk, karst ....) •UK: White Cliffs of Dover (chalk) •CZ: Moravian Karst (Macocha abyss) • Calcium ‹#› 24 Distribution of calcium In body In blood plasma (ECF) Bones 99 % Ionized 48 % Tissues (ICF) traces Associated with proteins 46 % ECF 1 % Chelated 6 % ‹#› 25 Calcium malate is an example of chelate does not dissociate !! ‹#› 26 Ca2+ ions in body fluids •Extracellular fluid •2.4-2.7 mmol/l •three forms •blood clotting •Intracellular fluid •cytosol (10-7 mol/l) •Mitochondria, ER - high deposits •Regulation functions •The second messenger •Muscle contraction • ‹#› 27 Calcium ion concentrations ECF ICF 10-3 M 10-7 M Difference by four orders ! ‹#› 28 Feature Parathyrin Calcitriol Calcitonin Produced in parathyroid glands skin – liver – kidneys thyroid gland Chemical type peptide secosteroid peptide Main effect release of Ca from bones resorption of Ca from intestine deposition of Ca into bones Hormones regulating calcium level ‹#› 29 Calcium phosphate Ca3(PO4)2 •calcii phosphas •Insoluble in water •In body – apatites – binary phosphates (= two different anions) •Bones - hydroxylapatite Ca5OH(PO4)3 •Teeth - fluoroapatite Ca5F(PO4)3 • ‹#› 30 Biological apatites are presented by different formulas Name Stoichiometric formula (the least ratio of elements) Crystal unit (multiple) Hydroxylapatite Ca5(PO4)3OH Ca10(PO4)6(OH)2 Fluoroapatite Ca5(PO4)3F Ca10(PO4)6F2 Octacalcium phosphate Ca4H(PO4)3 Ca8H2(PO4)6 Tricalcium phosphate Ca3(PO4)2 Ca9(PO4)6 ‹#› 31 Solubility of calcium phosphates (see also Medical chemistry – Practicals, p. 30-31) Ca3(PO4)2 insoluble CaHPO4 insoluble Ca(H2PO4)2 soluble ‹#› 32 Solubility of calcium phosphates (see also Medical chemistry – Practicals, p. 30-31) pH > 12 pH 8-11 pH 4-6 (kidney stones) (urine) ‹#› 33 Calcium sulfate CaSO4 •calcii sulfas •Insoluble Caution! MgSO4 is soluble (laxative mineral waters) •hemihydrate (½ H2O) plaster hardening ‹#› 34 Calcium carbonate CaCO3 (calcii carbonas) •External aplication •insoluble •powders •tooth paste •Peroral aplication •basic properties •neutralizes gastric HCl antacid Tums (GB), Rennie (F) •Ca supplementation • CaCO3 + 2 R-COOH ® CO2 + (R-COO)2Ca + H2O • ‹#› 35 Calcium hydrogen carbonate Ca(HCO3)2 •calcii hydrogenocarbonas •Exists only in aqueous solution •Hardness of water •By boiling CO2 is removed to make hard scale • Ca(HCO3)2 ® CO2 + CaCO3 ¯ + H2O •Very common in mineral waters ‹#› 36 Hard scale formation Ca(HCO3)2 ® CO2 + CaCO3 ¯ + H2O Soap does not work: Ca2+ (aq) + 2 R-COONa ® (R-COO)2Ca ¯ + 2 Na+ (aq) R-COOH is a long-chain fatty acid (palmitic, stearic) Hard water makes problems ‹#› 37 Calcium in food •Animal food •milk •dairy products •bioavailability ~ 50 % • •Plant food •poppy seed •nuts, almonds •dates, legumes •bioavailability ~ 10 % Recommended intake: Infants 1200 mg Adults 1000 mg Older people 1500 mg ‹#› 38 Calcium content in some dairy products (mg/100 g) •Dried milk 1300 •Parmesan (I) 1200 •Emmental (CH) 1000 •Bryndza (SK) 600 •Processed cheese 400 - 500 •Yoghurts 100 - 160 •Milk 110 - 130 •Cottage cheese 100 - 140 •Butter 20 • ‹#› 39 Intestinal absorption of calcium •Increased by •vitamin D •proteins •milk fermented products (kefir) • •Decreased by •phosphates (Coca-Cola, processed cheese) •oxalates •fibre excess • ‹#› 40 Distribution in the body Sodium Potassium ECF 50 % ECF 2 % ICF 10 % ICF 98 % Bones 40 % Sodium + Potassium ‹#› 41 Mean concentrations of ions in blood plasma Cation mmol/l Anion mmol/l Cation Charge Anion Charge Na+ K+ Ca2+ Mg2+ 142 4 2.5 1.5 142 4 5 3 Cl- HCO3- Protein- HPO42- SO42- Org. A. 103 25 2 1 0.5 4 103 25 18 2 1 5 Total: 154 Total: 154 ‹#› 42 Biological roles of sodium and potassium •Na+ is main cation of ECF, contributes to plasma osmolality •Na+ is the most hydrated ion Þ sodium movement (retention, excretion) is followed by movement of water •K+ is main cation of ICF, associated with polyanions of proteins •the uneven distribution of ions is maintained by Na+,K+-ATPase Ion Ion diameter (nm) Free Hydrated Na+ 0,19 0,52 K+ 0,27 0,46 Food sources Sodium – table salt, salty food, mineral waters Potassium – most foods, higher content in plant foods: potatoes, legumes, fresh and dried fruits, nuts ‹#› 43 Hormones regulating sodium Feature Aldosterone Atrial natriuretic peptide (ANP) Produced in adrenal cortex heart atrium Chemical type steroid peptide Main effects resorption of Na+ excretion of K+ in kidneys excretion of Na+ and water in kidneys ‹#› 44 Elemental carbon occurs in various modifications •diamond, graphite, soot, coke, charcoal, fullerens •graphene – Nobel Prize in Physics 2010 •adsorption coal (carbo adsorbens, carbo activatus) is non-polar intestinal adsorbent • Carbon ‹#› 45 Carbon monoxide CO •Colourless, odourless gas, molecule makes a dipole •Exogenous sources: incomplete combustion of carbon and organic compounds (cigarette smoke) •Toxicity: strong affinity to Fe2+ in hemoglobin to give • Carbonylhemoglobin (CO-Hb) – does not transport O2 •Endogenous source: heme catabolismus heme ® Fe2+ + CO + biliverdin (® bilirubin) • (-)CºO(+) ‹#› 46 Hem degradation provides CO and bilirubin oxidative splitting CO + biliverdin + 3 H2O bilirubin 3 O2 + 3 NADPH+H+ A B ‹#› 47 Carbonylhemoglobin (CO-Hb) in blood Subject / Situation CO-Hb (%)* Newborns Adults (rural areas) Adults (big cities) Smokers Traffic policemen Poisoning Death 0.4 1-2 4-5 10-12 12-15 20-50 55-60 Endogenous CO Exogenous CO * Percentage of total hemoglobin ‹#› 48 Carbon dioxide CO2 •carbonei dioxidum •colourless gas, easily gets liquified •linear molecule Þ zero dipol moment Þ non-polar compound Þ poorly soluble in water •acidic oxide = acid anhydride (H2O + CO2 ® H2CO3) O=C=O ‹#› 49 Endogenous production of CO2 •CO2 is produced in decarboxylation metabolic reactions •oxidative decarboxylation of pyruvate ® acetyl-CoA •two decarboxylations in CAC (isocitrate, 2-oxoglutarate) •Other sources: • decarboxylation of amino acids ® biogenous amines • non-enzymatic decarboxylation of acetoacetate ® acetone ‹#› 50 Carbonic acid in vitro •weak diprotic acid (pKA1 = 6.37; pKA2 = 10.33) •does exist only in aq. solution, easily decomposes to CO2 and water •CO2 predominates 800 ´ in sol. Þ therefore CO2 included into KA • H2O + CO2 D H2CO3 D HCO3- + H+ 800 : 1 : 0.03 KA eff = effective dissociation constant ‹#› 51 Carbonic acid in blood plasma •formation catalyzed by carbonic anhydrase •under physiological conditions: pKA1 = 6.10 •CO2 is continually eliminated from body by lungs •the overall concentration of carbonic acid: • [CO2 + H2CO3] = pCO2 × s = 0.23 pCO2 (kPa) H2O + CO2 D H2CO3 D HCO3- + H+ 1 : traces : 20 directly measurable quantity ‹#› 52 Compare: CO2 in water and blood Liquid pH [CO2] : [HCO3-] Carbonated watera Bloodb 3.50 – 5.00 7.36 – 7.44 800 : 0.03 1 : 20 a Closed system (PET bottle), 25 °C, pKA1 = 6.37 pH ~ pCO2 ~ the pressure of CO2 applied in carbonating process b Open system, 37 °C, pKA1 = 6.10 CO2 continually eliminated, pCO2 in lung alveoli ~ 5.3 kPa, acid component of hydrogen carbonate buffer ‹#› 53 Metabolic pathway of nitrogen Nitrogen Intake Food proteins (meat, eggs, dairy products) Metabolism digestion (stomach, intestine): protein ® AA anabolic reactions: AA ® tissue proteins / other products catabolism (transamination + deamination): AA ® NH3 ® urea Urine excretion urea (300 - 600 mmol/day) NH4+ (30 - 50 mmol/day) see lectures: Biochemical reactions Amino acids ‹#› 54 Compare Feature Nitrates Nitrites Formula NO3- NO2- Food sources water, vegetables sausages Limits for drinking water 50 mg/l 0.5 mg/l Endogenous source arginine ® NO ® NO3- - Acute toxicity no yes Toxic mechanism - Hb-Fe(II) ® Hb-Fe (III) R1R2NH ® R1R2N-N=O ‹#› 55 Phosphorus Biochemical features of phosphorus Intake Most foods, phospholipids (meat, eggs), phytates (cereals), phosphoproteins (milk - casein) Distribution in body ICF: mostly phosphoesters (metab. of glucose), macroergic compounds Nucleus: DNA Cell membranes: phospholipid bilayer ECF: hydrogen phosphate buffer, phospholipid monolayer of lipoproteins Bones, teeth: biological apatites Urine excretion H2PO4- (30 - 50 mmol/day) ‹#› 56 Compare Liquid pH Prevailing species Blood 7.4 HPO42- Urine 5.5 H2PO4- Coca-Cola 2.5 H3PO4 ! ‹#› 57 Metabolic pathway of sulfur Sulfur Intake food proteins (AA cysteine and methionine) Metabolism Anabolic: Cys, Met ® tissue proteins methionine ® SAM ® methylation reactions cysteine ® taurine ® conjugation reactions cysteine ® glutathione (GSH) ® antioxidant, conjugation reactions cysteine ® SO42- ® PAPS ® sulfation reactions Catabolic: Cys, Met ® HSO3- ® SO42- (excretion) bacterial decomposition (large intestine) ® thiols (R-SH) + sulfane (H2S) Urine excretion SO42- (8 - 35 mmol/day) see also lecture Amino acids ‹#› 58 Distinguish Sulfite anion SO32- (alkyl sulfite R-O-SO2H ester) Sulfide inorganic anion S2-, ZnS zinc sulfide Sulfide organic R-S-R dialkylsulfide R-S-S-R dialkyldisulfide Sulfate anion SO42- (alkyl sulfate R-O-SO3H ester) ‹#› 59 Iron in human body •Essential microelement •In body: about 4-5 g Fe •Most in blood hemoglobin (60-85 %) myoglobin (10 %; muscles), and ferritin (10 %; liver) •Iron in body is always associated with proteins (heme and non-heme) • Free Fe2+ ions are toxic Iron ‹#› 60 Hemoproteins Protein Redox state Function Hemoglobin Myoglobin Catalase Peroxidase Cytochromes Fe2+ Fe2+ Fe3+ Fe3+ Fe2+ D Fe3+ transport of O2 in blood O2 deposit in muscles H2O2 decomposition peroxide decomposition components of resp. chain ‹#› 61 Other iron-proteins Protein Redox state Function Transferrin Ferritin Hemosiderin Fe-S proteins Fe3+ Fe3+ Fe3+ Fe2+ D Fe3+ transport of Fe in plasma cell storage of Fe cell storage of Fe components of resp. chain ‹#› 62 Food sources of iron •Animal •pork blood •blood sausage •liver, liver pate •red meat •bioavailability ~ 20 % • •Plant •broccoli •green leafy vegetables •bioavailability ~ 10 % • ‹#› 63 Intestinal resorption of iron •Only as Fe2+ •Needed ascorbic acid (vitamin C), Fe3+ is reduced to Fe2+ •Daily intake 10-30 mg •Daily need 1 mg (males), 2 mg (females) •Resorption hindered by: tanins, polyphenols (tea), phytates, oxalic acid (spinach), excess of dietary fibre (vegetarians) ‹#› 64 The Latin names of iron salts •ferrosi ´ ferri Fe2+ Fe3+ ‹#› 65 Distinguish ferrosi sulfas FeSO4 ferri sulfas Fe2(SO4)3 ferrosi sulfis FeSO3 ferri sulfis Fe2(SO3)3 ‹#› 66 Iron salts for peroral supplementation •Iron(II) sulfate (ferrosi sulfas) •Iron(II) fumarate (ferrosi fumaras) •Iron(II) gluconate (ferrosi gluconas) • ‹#› 67 Fluorine minerals: fluorite CaF2, fluoroapatite Ca5(PO4)3F Chlorine NaCl (rock salt), seawater (~ 3 %), salt lakes (up to 25 %): Dead Sea, Great Salt Lake Bromine seawater Iodine seawater, seaweed Halogens in nature Halogens ‹#› 68 Halogens in human body Fluorine bones, teeth (fluoroapatite) Chlorine Cl- is the main anion of blood plasma HCl in stomach (H+ from H2CO3, Cl- from plasma) Bromine traces in tissues and body fluids Iodine thyroid gland (thyroxine) ‹#› 69 Halogens in food Fluorine tap water, mineral waters, sea food, black tea (Camellia sinensis) Chlorine table salt NaCl Bromine sea food Iodine sea food, iodinated salt, Czech mineral water Vincentka ‹#› 70 Fluorine compounds •Sodium fluoride NaF (natrii fluoridum) in tablets – caries prevention in infants, tooth paste, oral rinse, dental floss •Sodium monofluorophosphate Na2PO3F (dinatrii fluorophosphas) tooth paste etc. • ‹#› 71 NaCl in medicine Osmolarity Osmolality Definition i c i cm Unit mmol/l mmol/kg H2O Temperature dependent yes no Saline solution NaCl (natrii chloridi solutio) is isotonic with blood plasma: 154 mmol/l (0.9 %) Osmolarity i c = 2 × 154 = 308 mmol/l Usage: infusions Osmolality of blood plasma: 280-295 mmol/kg H2O » 2 [Na+] + [glucose] + [urea] ‹#› 72 Compare concentrations (mmol/l) Blood plasma Saline sol. NaCl Na+ Cl- Na+ Cl- 133-150 97-108 154 154 Saline solution of NaCl has increased concentration of chloride ions compared to plasma!! ~ 100 ‹#› 73 Chloride balance •Usual daily intake: 5-12 g NaCl •Chloride loss: vomiting Þ increased HCO3- in plasma Þ alkalosis •Chloride excess: infusions of saline sol. Þ decreased HCO3- in plasma Þ acidosis • ‹#› 74 HCl in stomach •acidum hydrochloricum, strong acid •H+ comes from H2CO3 (parietal cells), Cl- from plasma •Concentration ~ 0.1 mol/l, pH 1-2 •Bactericidal effect •Protein denaturation – for better digestion •Pepsin activation (pepsinogen ® pepsin) • ‹#› 75 Iodine in human body •Essential microelement •Daily need: 150-300 μg •the best food source: sea products •For the production of iodinated thyronines - amino acids thyroxine and triiodothyronine • ‹#› 76 Conversion of tyrosine to thyroxine bound to thyreoglobulin tyrosine 3’,5’-diiododtyrosine thyroxine ‹#› 77 The iodination of table salt •35 mg I / kg salt •Sodium/potassium iodide (NaI, KI) - not very stable due to possible oxidation and sublimation: 2 NaI + ½ O2 + CO2 ® Na2CO3 + I2 •Sodium iodate NaIO3 – stable substance • ‹#› 78 Elementary iodine is a strong disinfectant •But has some disadvantages: •Possible allergen •Be very carefull in thyroid gland diseases!! •Only short-term application!! ‹#› 79 Solutions of elemental iodine English name Latin name Composition Tincture of iodine Lugol’s solution Povidone-iodinea tinctura iodi solutio iodi aquosa sol. povidoni iodinati I2 + ethanol I2 + KI + H2O I2 + povidoneb + H2O a Commercial names: Jodisol, Betadine, Jox b Synthetic polymer polyvinylpyrrolidone Elemental iodine is non-polar, insoluble in water.