VITAMINS John H. Block Pharmaceutical Science, Oregon State University, Corvallis, OR 1. INTRODUCTION Vitamins are enjoying renewed popularity with the lay public that in some ways mimics that seen in the early part of the twentieth century when they were first being discovered. Paralleling the interest by the general public is increased focus on the biochemical role of vitamins at the molecular level. Some vitamins can be considered prototypes for pharmacological agents used to treat diseases that do not appear directly related to the patient's vitamin status. Although all animals require vitamins, the discussion in this chapter will focus on humans and human biochemical pathways. Vitamins are complex biochemically and functionally. There are many ways to classify vitamins. This chapter will examine two approaches: the traditional solubility model and a functional classification. Unfortunately, the loose regulation of nutritional products in the United States has led to misleading promotion of these items to the lay public such that individuals are confused as to what can be caused by a vitamin. General criteria for deciding if a substance truly is a vitamin will be presented. Another problem is to decide if a patient actually has a vitamin deficiency. As will be noted in the next section, this can be a complex question. Finally, deciding how much of a particular vitamin should be consumed has moved from the familiar Recommended Dietary Allowance (RDA) to four different ways to evaluate vitamin requirements and consumption. Unfortunately, today's vitamin product labels use older Food and Drug Administration (FDA) criteria for adult daily requirements. 1.1. What is Vitamin? In general, a vitamin must be a naturally occurring organic molecule that is a normal constituent of the diet. It should be essential and only required in minute amounts. Finally, it is required to maintain normal cellular biochemistry and tissue integrity. These criteria need to be examined in more detail. 1.1.1. Vitamins are Naturally Occurring This criterion frequently is misinterpreted. While the vitamin is a natural product, there is no difference in efficacy between ingesting the all natural or a synthetic product as long as the natural and synthetic products are chemically identical. This includes stereochemistry. l-ascorbic acid is twice as active as the racemic d,l-ascorbic acid. A similar statement can be made for d-pantothenic acid and S-biotin relative to their racemic mixtures. The situation for a-tocopherol (vitamin E) with three asymmetric centers is more complex and will be discussed in more detail later in this chapter. 1.1.2. Vitamins are Essential Because They Are Not Produced by Human Biochemical Pathways With two exceptions, this requirement is obvious. If a compound is required for a biochemical process and our pathways cannot produce it, it must be obtained from exogenous sources. The evolutionary history of the human species is such that we sought out sources in our environment that contained these essential substances, and these substances became our food. One of the substances commonly treated as a vitamin is niacin, which is synthesized from the essential amino acid tryptophan. In humans, the ratio currently is considered to requiring approximately 60 mg of tryptophan to produce lmg niacin [1]. This has led to niacin requirements being expressed as niacin equivalents (NE) based on the amount of tryptophan in the diet. It must be kept in mind that tryptophan is essential and is the precursor to the neurotransmitter serotonin plus being a part of protein structure. Therefore, niacin can be thought of as tryptophan sparing. The 60:1 ratio has been questioned based on examining the diets of patients diagnosed with pellagra, a disease caused by a niacin deficiency. The diets of these individuals contained adequate amounts of tryptophan [2,3]. The other exception is vitamin D3 or cholecalciferol. Assuming adequate sunlight, 637 Burger's Medicinal Chemistry, Drug Discovery, and Development, Seventh Edition, edited by Donald J. Abraham and David P. Rotella Copyright © 2010 John Wiley & Sons, Inc. 638 VITAMINS cholecalciferol is the photochemical product from ultraviolet irradiation of 7-dehydrocho-lesterol found in our skin. A significant proportion of the world's population produces all of the cholecalciferol it needs from this photochemical reaction. It is true that a photochemical reaction is not a biochemical pathway, but exposure to adequate sunlight does fulfill the requirement for cholecalciferol. 1.1.3. Vitamins Are Organic Chemicals Trace elements are not properly called vitamins. Therefore, iron, zinc, magnesium, manganese, chromium, selenium, and the other trace elements that, obviously, are obtained from food sources are essential, but they are not vitamins. Conversely and by tradition, many of the essential organic substances in human diets are not vitamins. These would include essential fatty acids, essential amino acids, antioxidants, and fiber. 1.1.4. Vitamins Are Normal Constituents of the Human Diet With the exception of cholecalciferol, all of the other compounds that we treat as vitamins are normal constituents of our diets with most found in more than one food group. The situation with cholecalciferol is such that, except for the small part of the world's population that obtains its protein from marine species, neither cholecalciferol nor ergocalciferol from irradiation of the plant sterol ergosterol is a normal component of foods. Its presence in dairy products is caused by food processors added to it during pasteurization—homogenization of milk. 1.1.5. Vitamins Are Required in Minute Amounts This is arbitrary but ranges from 2.0|ig(lCT6 gm) for cyanocobalamin to 90 mg for ascorbic acid. Other essential nutrients including the essential amino acids and fatty acids generally are required in larger amounts. For example, the National Research Council's recommended daily intake of amino acids range from a low of 245 mg for tryptophan to 980 mg for phenylalanine and tyrosine (combined) and leucine [4]. Note that the value for tryptophan will only provide about 4 mg of niacin assuming that all 245 mg of tryptophan is converted to niacin. 1.1.6. Vitamins Are Required to Maintain Normal Biochemical Functions of the Tissues Most vitamins function either as a hormone/ chemical messenger (cholecalciferol) structural component in some metabolic process (pantothenic acid), or as a coenzyme (phyto-nadione, thiamine, riboflavin, niacin, pyridox-ine, biotin, folic acid, cyanocobalamin). At least one vitamin has more than one biochemical role. Vitamin A as an aldehyde (retinal) is a structural component of the visual pigment rhodopsin and in its acid form (retinoic acid) a regulator of cell differentiation. In addition to a general antioxidant role, the precise biochemical functions of ascorbic acid and a-tocopherol are still not well defined. 1.2. Causes of Vitamin Deficiencies There are a variety of reasons why a person might be experiencing a vitamin deficiency ranging from economics to genetics to diseased intestinal tract to a variety of medical conditions to lifestyle to drug interactions. Table 1 contains a classification scheme of possible causes of vitamin deficiencies. These include inadequate ingestion of food, inadequate absorption of nutrients from the gastrointestinal tract, inadequate utilization of the nutrient once absorbed systemically, and increased nutritional requirements usually due to changes in lifestyle. 2. DIETARY REFERENCE INTAKES AND DAILY VALUES It has been a challenge to determine and present vitamin dosing to health professionals and the general public. Until 1989, the common dosage for a vitamin was expressed as the RDA and Estimated Safe and Adequate. The latter was used for those vitamins for which there was inadequate information. One could think of it as a best guess estimate. Even earlier, there were minimum daily requirements (MDR). Beginning in the 1990s, panels were assembled to develop a new reference system, Dietary Reference Intakes (DRIs), for nutrients that include vitamins, minerals, fiber, essential amino acids, essential fatty acids, and other nutrients. The DRIs are published by the Food and Nutrition Board of the DIETARY REFERENCE INTAKES AND DAILY VALUES 639 Table 1. Causes of Nutrient Deficiencies Cause Mechanism/Reason Remarks Inadequate ingestion usually from a poor diet Inadequate absorption Inadequate utilization Economic deprivation Self-imposed reducing diets Disease Diseased intestinal tract Mineral oil laxatives that may dissolve the oil-soluble vitamins Ion exchange resins (colestipol, cholestyramine, colestid) that complex with the bile salts and can interfere with the absorption of the oil-soluble vitamins Aluminum antacids can complex with some of the vitamins and, when used chronically, most definitely can cause hypophosphatemia Chronic alcohol intake Cystic fibrosis that can cause fat malabsorption (steatorrhea) due to inadequate production of pancreatic lipases Genetic diseases Drug—vitamin interactions Inability to purchase adequate amounts and a variety of food A 1200 calorie diet professionally selected from the four major food groups (dairy, fruits and vegetables, grains, and meat) containing neither fried food nor added sugar has been considered the least amount of food not requiring a vitamin supplement This is usually due to loss of appetite from such conditions as cancer chemotherapy, depression, and eating disorders Examples include chronic inflammatory conditions such as Crohn's disease and parasites This could include retinol, cholecalciferol/ ergocalciferol, a-tocopherol, and vitamin K from food A vitamin supplement can be taken 1 h before or 2 h after taking the resin Aluminum antacids are no longer commonly used Ethyl alcohol interferes with the uptake of some vitamins (folic acid, thiamine) or, because of liver disease, proper processing and/or storage of vitamins This could include retinol, cholecalciferol/ ergocalciferol, a-tocopherol, vitamin K from food, all of which are more readily absorbed when they can be part of normal mixed micelle process that occurs with lipid digestion and absorption Examples include variants of maple syrup urine disease (MSUD) that will respond to thiamine (vitamin Bx) supplements and homocystinuria that will respond to pyri-doxine (vitamin B6). The mutation lies with apoenzyme such that the equilibrium between the apoenzyme and the coenzyme lies to the left. To push the reaction to the right requires additional coenzyme. These can interfere with vitamin processing in the intestinal tract, tie up the vitamin preventing it from being used, or possibly promote elimination of the vitamin. Examples include isoniazid—pyridoxine, phe-nobarbital-cholecalciferol, methotrexate-folic acid, and phenytoin—folic acid (continued) 640 VITAMINS Table (Continued) Cause Mechanism/Reason Remarks Increased requirements above the recommended daily allowances (RDA) Reference dietary indices are based on an average population performing average duties Individuals performing more strenuous activities (e.g., competitive athletics, construction in the arctic) requiring additional intake of calories also will require more nutrients including vitamins. Patients recovering from a debilitating illness, severe burn injuries, and cancer also have increased nutritional needs. Nutritional assessments are becoming a more common part of medical treatment National Academy of Sciences, National Research Council (see Bibliography). 2.1. How Do the DRIs Differ from Earlier RDAs? There will be one set of reference values for both Canada and the United States, and there will be clear documentation on how reference values are selected. A goal is the promotion of nutrient function and biologic—physical well-being. Evidence concerning the prevention of disease and developmental disorders will be examined in addition to the tradition of how much nutrient is needed to prevent a deficiency symptom. Data supporting food components that, up to this time, have not been considered essential nutrient will be examined. Finally, there will be recommendations for future research. 2.2. Uses of Dietary Reference Intakes The DRIs consist of four components. Each type of reference value is calculated from daily intakes averaged over time (usually one or more weeks). The surveys include, but not limited to, random selection of healthy individuals and asking them to either report what they have eaten or maintain food diaries, monitoring overall food production and consumption, and correlating a defined population's health status with the group's food intake. The four components are estimated average requirement (EAR), RDA, adequate intake (AI) and tolerable upper intake level (UL). Table 2 describes each DRI in more detail. 2.3. Daily Values The FDA has a set of daily values (DV) that are used on the labels of multivitamin products and the nutrition labels required on many food products [5]. Many times the label will indicate the percentage of the daily value (%DV) of the nutrient. In general, the daily values do not have the age and gender differentiation seen with the DRIs. Table 3 shows a comparison of the FDA's daily values with the National Academy of Sciences adult RDA/AI values. 2.4. Determination of a Vitamin Dose This is not easy. Ideally, a dose—response curve could be constructed that would provide defined endpoints such as an ED50 or ED90 and to avoid serious toxicities, the LD50- The problem is defining a biological or pharmacological endpoint. Not all vitamin deficiencies have defined syndromes. Little is known of the pharmacokinetics of individual vitamins. In contrast with most drugs, the body stores vitamins, sometimes several months' supply. This should not be surprising when one examines human history. Until fairly recent times, humans regularly experienced food plenty and food deprivation (famine) as crops failed and animals moved away from settlements. Causes ranged from weather to war. To survive, the evolutionary development of humans included ways to store and reuse vitamins. Assuming a good diet, humans have a supply of 6—9 months of vitamin A. Humans efficiently recycle cobalamin by enterohepatic circulation. The intestinal flora produces a precursor to vitamin K. DIETARY REFERENCE INTAKES AND DAILY VALUES 641 Table 2. Methods for Estimating Vitamin Requirements Vitamin Determination Methodology Remarks Retinol (vitamin A) Calciferol (vitamins D2 and D3) a-Tocopherol (vitamin E) Vitamin K Thiamine (vitamin Bx) Riboflavin (vitamin B2) Niacin/niacinamide Vitamin B6 family Dark adaptation test Pupillary response test Plasma retinol concentration Relative dose response Serum 25(OH)D2 or D3 Lipid peroxidation markers Plasma a-tocopherol concentration Prothrombin time Factor VII Plasma or serum phylloquinone concentration Urinary y-carboxyglutamyl residues Erythrocyte transketolase activity Urinary thiamine excretion Erythrocyte thiamine concentration Erythrocyte glutathione reductase activity Erythrocyte flavin Urinary riboflavin excretion Urinary excretion Plasma concentration Erythrocyte pyridine nucleotides Plasma pyridoxal phosphate Erythrocyte and total blood pyridoxal phosphate Urinary pyridoxic acid Considered reasonably sensitive Data do not exit relating pupillary threshold sensitivity to retinol intake Insensitive to liver stores An initial plasma concentration is determined followed by a second concentration in a defined time after administration of a small dose of retinol. The ratio is proportional to the liver stores of retinol. This is considered a more accurate indicator of vitamins D status compared to serum vitamins D or l,25(OH)2D. They are not specific to vitamin E It does not seem to correlate with daily intake, but there is a linear relationship seen in tocopherol-depleted subjects It is used to assess patients on Coumadin anticoagulant therapy, but does not appear to be a reliable measure of vitamin K status in otherwise healthy subjects Even though factor VII only has a 6 h half-life, it does not appear to be reliable This does respond to changes in dietary intake within 24 h This is considered promising This is considered the most accurate This method is also considered good It is not as widely used It is the most common assay Some question the sensitivity of this test because the difference between adequacy and inadequacy is small Care must be exercised with the size of the dose and method of administration N^methylnicotinamide and N^-methyl-2-pyridone-5-carboxamide are the two metabolites that are considered sensitive measures of niacin status N1-methyl-2-pyridone-5-carboxamide plasma levels provide an indication of low niacin intake by dropping below detection limits Erythrocyte NAD levels may replace measuringmetabolites foundinthe urine It changes slowly in response to changes in vitamin intake There may be racial differences because of lower kinase activity It tends to reflect recent vitamin intake rather than general vitamin status (continued) 642 VITAMINS Table (Continued) Vitamin Determination Methodology Remarks Pantothenic acid Biotin Folic acid Vitamin Bi2 (Cobalamin) Ascorbic acid (vitamin C) Various erythrocyte transaminases Tryptophan catabolites Plasma homocysteine Urinary excretion Blood levels Urinary excretion Erythrocyte folate Plasma homocysteine Serum folate Urinary folate Hematological status Hematological response Serum or plasma Vitamin Bi Methylmalonic acid Homocysteine Holotranscobalamin II Antioxidant functions Cellular DNA damage Urinary markers Aspartate and alanine transaminases have been studied most extensively. There is a lack of consensus regarding their usefulness as indicators of vitamin B6 status While useful, some of the reactions are affected changes in steroid hormone status See discussion in the vitamin B6 section It is strongly dependent on intake There is poor correlation with urinary excretion values There is a correlation with induced deficiencies caused by eating raw egg white Only the developing erythrocyte takes up folate. Therefore, this test is an indicator of long-term status and not a measure of immediate changes in folate status There is no question that elevated homocysteine decreases with folate administration. There is not enough information to support its use for determining DRIs While a measure of dietary folate intake, it is not considered a reliable measure of folate status This test may underestimate folate needs Characteristic megaloblastic anemia develops late in folic acid deficiency These are the typical hemoglobin, hematocrit, and erythrocyte counts. Partial responses are of limited values A problem is that serum values may be maintained at the expense of tissue stores Studies need to be done to see if it will correlate with vitamin Bi2 status This can be elevated in folate and vitamin B6 deficiencies This protein is responsible for receptor-mediated uptake of Bi2 into cells. Further work needs to be done before it is adapted for routine clinical use A variety of markers have been evaluated including LDL, VLDL, malondial-dehyde, hydroxynonenal, and reduced glutathione These have not proven useful for estimating ascorbate requirements Oxidized DNA is nonspecific DIETARY REFERENCE INTAKES AND DAILY VALUES 643 Table 3. Comparison of Adult Daily Values (DV) with Adult Recommended Dietary Allowances (RDA) or Adequate Intakes (AI) Vitamin Daily Value RDA or AI* Retinol (A) 1500 ug 700-900 ug Ascorbic Acid (C) 60 mg 25-90 mg Cholecalciferol (D3) 400 IU *200-600 IU a-Tocopherol (E) 30 mg 7-15 mg Phylloquinone (K4) 80 ng *55-120 ug Thiamin (Bi) 1.5 mg 0.9-1.2 mg Riboflavin (B2) 1.7 mg 0.9-1.3 mg Niacin 20 mg 12-16 mg Pyridoxine (B6) 2.0 mg 1-1.7 mg Folate 400 ug 200-400 ug Cyanocobalamin (Bi2) 2.0 ng 1.8-2.4 ug Biotin 30 ng *20-30 ug Pantothenic Acid 10 mg *20 mg 'Adequate Intake for these vitamins. 2.5. Methods to Determine a Valid Dose of a Vitamin Each of the methods outlined in the following sections have significant problems in determining a dose—response curve for a vitamin. These include 2.5.1. Extrapolate from Animal Studies This method will be dependent on the species. The vitamin requirements differ among the common laboratory animals. The classic example is ascorbic acid, which is not a vitamin in most animals. a-Tocopherol, biotin, and pantothenic acid cause definite deficiency syndromes not seen in humans. 2.5.2. Metabolic Balance Studies in Humans This usually involves a specified length of time on a defined diet in which the urine and feces are monitored. The problem is that corrections have to be made for the vitamins that are stored. The pharmacokinetics of most vitamins has not been carefully determined. 2.5.3. Compare Nutrient Intake in Areas with and Without the Deficiency Disease First, not all vitamin deficiencies lead to a defined deficiency state. Second, rarely does one find an area deficient in only one nutrient. Most common deficiencies are due to inadequate diet, which means several nutrient deficiencies will result. 2.5.4. Saturation of Biochemical Function A reliable biochemical indicator is required. For niacin, which NAD+ or NADP+ containing enzyme should be selected? Which transaminase will be the indicator for pyridoxine? Which function should be selected for vitamin A: vision in the rods or cell differentiation? 2.5.5. Serum Levels This probably is the most reliable, but it does require very sensitive assay methods for those vitamins required in microgram (10~6) amounts. It also requires knowledge as to how the vitamin is transported: free or bound to a plasma protein or a specific transport protein. There are specific transport proteins for vitamin A and vitamin D3. The tocopherols will be found in the lipoproteins (VLDL, LDL, etc.). 2.6. Do Vitamins Really Help Beyond Treating Their Deficiency Diseases? Vitamins are essential nutrients, and their deficiencies cause diseases, some with definitive symptoms. It is also know that excess doses of some vitamins produce identifiable adverse reactions. The latter is the reason some vitamins have tolerable upper intake levels. But what about ingesting vitamins at doses between the RDA/AI and the UL? This is not an easy question to answer. Studies are expensive, usually are retrospective, and require years to complete. Many times the results are equivocal [6—9]. Nevertheless, there is evidence that doses of vitamins in excess of their RDA/AI, but less than their UL (for those vitamins with an UL), may, at a minimum, not be effective and possibly exacerbate certain diseases [10,11]. 2.7. Antioxidant Supplement Controversy Two vitamins, oil-soluble tocopherol family (vitamin E) and water-soluble ascorbic acid (vitamin C) plus the /3-carotene, are considered biological antioxidants along with specific vitamin functions or, in the case of /3-carotene, precursor to the vitamin A family. In addition to these three, there are other antioxidants in the foods we eat including lycopene. There have been several studies trying to answer the question if antioxidants can prevent or even treat cardiovascular dis- 644 VITAMINS ease and malignancies. In general, the results have been equivocal [9]. The one exception is /3-carotene that may exacerbate lung cancer in smokers [12,13]. 3. VITAMINS The order of vitamins in this chapter will follow the classical classification-based solubility: fat soluble and water soluble. This classification originated before there was any understanding of the structural chemistry of the vitamins. There were fat-soluble A and water-soluble B. Thiamine was the first vitamin (Bi) whose structure was elucidated. It is an amine leading to the term "vital amine" and finally vitamin (without the "e"). The letter designations, for the most part, are being relegated to history. Many of the vitamins are actually groups of structures and, therefore, using a single letter can be misleading. Also, the structures occurring naturally in food and commercial preparations may actually be provitamins that have to be converted to the biochemically active form. Unless specified differently, the information in the following summaries will be found in the "Dietary Reference Intakes" published by the Institute of Medicine listed in the general bibliography at the end of this chapter. 3.1. Retinol (Vitamin A) Family Early work on this vitamin was confusing because similar outcomes were seen with ingestion of "yellow vegetables" and colorless fish liver oils. It was finally shown that carotene (the yellow pigment) extracts from vegetables were converted to colorless retinal. 3.1.1. Chemistry The commercial form of vitamin A is all-trans retinol, usually formulated as the acetate or palmitate ester. The active forms are the two oxidation products (Fig. 1): retinal that is a structural component of the visual pigment, rhodopsin, and the two retinoic acids (all-trans and 9-cis) that are required for cell differentiation. There are specific nuclear receptors for retinoic acid, RAR and RXR. While the vitamin is marketed in the all trans form, both retinal and retinoic acid will be present in cis forms. There are also commercial forms related to the retinoic acid structure that have cis stereochemistry. Although promoted commercially, carotenes are sold for their antioxidant activity rather than a source of the vitamin A group. Nevertheless, the carotenes are the source of the vitamin in yellow vegetables. There are many carotenes of which three are shown in Fig. 2. Only (3-carotene is symmetrical (note the dashed line) and theoretically will produce two equivalents of retinal following the enzyme catalyzed oxidative cleavage. In reality, more recent studies indicate that vitamin A activity is six times the vitamin A activity derived from (3-carotene. The problem is one of capacity in the intestinal mucosa cell to cleave the carotenes. Furthermore, there appears to be regulation of the cleavage of (3-carotene. As the stores in the liver reach capacity, there is less conversion of (3-carotene being oxidized to retinal. This is one of the reasons that (3-carotene nutritional supplements enter the body intact. Furthermore, the bioavailability of (3-carotene is significantly lower than that of retinol [14]. 3.1.2. Uptake and Metabolism (Fig. 3) Both vitamin A esters, whether from animal tissues or a vitamin supplement, and (3-carotene must be incorporated into mixed micelles along with other lipid material. The vitamin A esters are hydrolyzed by intestinal esterases. Both retinol and (3-carotene are then absorbed into the mucosa cell where (3-carotene is oxidatively cleaved to retinal and then reduced to retinol. The retinol, independent of the source, follows the same steps as seen with 2-monoglyceride from triglyceride digestion. The retinol is re-esterified, usually with palmitic acid, and incorporated into the chylomicrons along with the other dietary lipids. The chylomicrons first enter the lymph and then move to the circulatory system. The triglycerides are removed from the chylomicrons and deposited in the adipose and skeletal muscle cells leaving chylomicron remnants that are transported to the liver where they are stored [15]. Transportation from the liver to tissues where required is done on specific retinol binding proteins. Humans consuming a balanced diet store several months of retinol esters in their livers. It must be pointed out that the retinol binding pro- 646 VITAMINS H3C^_ ..\\CH; HaC" d "CH3 a-Carotene y-Carotene Figure 2. Carotenes. teins have functions beyond that of transporting retinol esters. Retinol binding protein 4 (RBP4) is produced by adipocytes, and in obese subjects there is increased amounts of this protein. RBP4 may cause insulin resistance in skeletal muscle and increased gluconeogen-esis in the liver. The combination of these two responses may contribute to the hyperglycemia seen in type 2 diabetes [16,17]. 3.1.3. Biochemical Functions and Deficiency Three retinoids appear to have most of the biochemical functions attributed to vitamin A. The retinoic acids are required for cell differentiation and are the ligands for two families of nuclear receptors: £rcms-retinoic acid for RARaßY and 9-cis-retinoic acid for RXRa ßr These receptors are part of a family of super receptors that include the steroid hormones and cholecalciferol. Vitamin A deficiency can lead to a variety of symptoms depending on the age of the deficient person. The most serious syndrome is keratomalacia that results in desiccation, ulceration, and xerophthalmia of the cornea and conjunctiva. It is one of the leading causes of blindness in infants and children. Retinoic acid is required for the development of goblet mucous cells. A deficiency results in proliferation of basal cells with increased keratinization of the epithelial structures. Mucous is one of the essential physical barriers that prevents pathogens from entering the body. Therefore, a vitamin A deficiency increases the risk of infection. The aldehyde form, retinal, is an essential component of the visual pigment found in the rods of the eye. A very brief outline of the oxidation ß-Carotene-►Retinal mucosa cell Retinol esters (food or vitamin supplements) Retinal and retinoic acid transported on retinol binding protein (1) esterase (2) oxidation Liver storage Mucosa cell reductase reesterify Retinol-►Retinol palmitate mucosa cell Intestinal esterases Chylomicron^-remnant TGs 1' Chylomicron Figure 3. Uptake of dietary retinol and carotene. VITAMINS 647 Rhodopsin (visual pigment) (Light) 11 -c/s-Retinal Changes in the conformation of the rhodopsin complex and hydrolysis of the enamine Opsin + ŕrans-Retinal Nerve impulse to the brain -►Sight M frans-Retinol (transported to the eye from the liver on a retinol binding protein) t ) r Liver stores of retinol esters Figure 4. Outline of rhodopsin cycle. rhodopsin cycle is shown in Fig. 4. Retinol is transported from the liver to the eye where it is converted to 11-cis-retinal. In the rod, the aldehyde forms an enamine with a lysine on opsin forming rhodopsin. In the presence of light, trans-retinal forms with cleavage of the enamine sending a nerve impulse to the brain along the optic nerve. In the dark, 11-cis-re-tinol re-forms followed by oxidation to 11-cis-retinal and the cycle repeats. A deficiency causes night blindness, which is considered an early symptom of vitamin A deficiency. Night blindness refers to decreased ability to see in very dim light because there is an adequate amount of retinal in the eye to fully "stock" the rods with functional rhodopsin. There is some evidence that as retinol levels in the liver decrease, the equilibrium favors the movement of retinol from the eye back to the liver. 3.1.4. Dosage Forms Vitamin A is unstable. It easily dehydrates (Fig. 5) forming a resonance-stabilized carbonium ion. Therefore, the two most common forms for both oral and parenteral administration are the acetate or palmitate esters. With its extensive conjugation, vitamin A is light sensitive and subject to oxidation. Therefore, vitamin formulators must protect the product from light and air. 3.1.5. Hypervitaminosis A In high doses, vitamin A can be toxic. Acute poisoning is rare and dependent on the dosage form. Nausea and vomiting are the most common symptoms. Most rapidly absorbed are the "clear" emulsions (usually formulated with a Tween or other surfactant). Next in order are the standard emulsions, usually produced from fish liver oils. The most slowly absorbed are the dry tablet formulation or an oil solution in a 648 VITAMINS H,C HoO CH2OH &f^CH3 CH2OR •CH3 R = acetate or palmitate Figure 5. Vitamin A's commercial forms. capsule. Chronic hypervitaminosis is more common and is more commonly seen when people consume fish liver oil concentrates. The symptoms are nondescript and include fatigue, malaise, lethargy, abdominal discomfort, bone/joint pain, severe and throbbing headache, insomnia, restlessness, dry and scaly skin, loss of body hair, brittle nails, constipation, and irregular menses, symptoms that might make a users conclude that they are vitamin A deficient. Depending on the health of person's liver, there is risk of developing cirrhosis. Multiple vitamins containing vitamin A contain a warning that excessive intake of the vitamin may increase the risk of osteoporosis in postmenopausal women [18]. There is a daily tolerable upper intake level (UL) of 3000 |ig for this vitamin. The UL to RDA ratio is narrow, 3—5, relative to most vitamins. 3.1.6. Hypercarotenosis This occurs from massive doses of carotene, which exceed the capacity of the mucosa cells to cleave the molecule to retinal derivatives. The excess carotene becomes deposited in the body tissues. Except for the yellow or bronze-orange skin, there seem to be no other symptoms. The skin coloration will slowly disappear when carotene intake stops and the absorbed carotene is slowly eliminated from the body. A commercial form of carotene is indicated for the photosensitivity seen in erythropoietic porphyria. Carotene capsules also are sold with claim that a person can have a tanned appearance without the need of UV radiation. Patients who drink large amounts of carrot juice sometimes show signs of hypercarotenosis. 3.1.7. Dietary Reference Intakes Vitamin A's DRIs are expressed at retinol activity equivalents (RAE) 1 (Xg RAE—1 (ig retinol, 12 (ig ß-carotene, and 24 (ig a-carotene AI (infants 1-12 months)400-500 (Xg/day EAR Children (1—8 years) Boys (9—18 years) Girls (9—18 years) Men (19-70 + years) Women (19-70 + ) Lactating RDA Children (1—8 years) Boys (9—18 years) Girls (9—18 years) Men Women Pregnant Lactating UL 210-275 (Xg/day 445-630 (Xg/day 420-485 (xg/day 625(xg/day 500(xg/day 880-900 (Xg/day 300-400 (Xg/day 600-900 (Xg/day 600-700 (Xg/day 900 (Xg/day 700(xg/day 750-770 (xg/day 1200-1300 (Xg/day (Applies only to preformed vitamin A in foods, fortified foods, and supplements. It does not apply to vitamin A derived from carotenoids) 3000 (Xg/day for all adults including pregnant women. There is some concern of teratogenic effects based on the experience of the retinoids used in therapy 3.1.8. Pharmacologically Active Retinoids Because vitamin A deficiency shows in the VITAMINS 649 keratinization of epithelial tissue, vitamin A was, at one time, recommended for skin conditions including acne. There is no clinical evidence that vitamin A is effective for this type of skin condition. Now that it is realized that the active form is retinoic acid, the focus has been on developing pharmacologically active compounds based on this structure. These are divided into three groups: treatment of (1) acne, (2) the autoimmune disease, psoriasis, and (3) malignancies. Retinoid and Retinoid-Like Drugs Used in the Treatment of Acne (Fig. 6) The first product introduced was tretinoin, which is topical all-irons-retinoic acid. Its effectiveness probably may not be related to any direct retinoid activity, but by its producing a complex response related to increasing the turnover of follicular epithelial cells. The result is decreased cohesiveness of follicular epithelial cells. Tretinoin also is used as an antiwrinkle cream. There is disagreement on the mechanism and there may be more than one. Some improvement may be due to the irritation the drug causes. There is an increase in epidermal cell turnover shedding older cells and thickening the skin. Also, the drug may combine with epidermal retinoic acid receptors decreasing keratin production. Keratin can contribute to skin wrinkling [19]. Isotretinoin, 13-cis-retinoic acid, is very effective in treating severe forms of acne. It also is very teratogenic. There are elaborate procedures involving the prescribing physician, dispensing pharmacist before a female patient can receive the drug. There is also some concern that the sperm of men using the drug might be affected [20]. Although used topically, the nonretinoid, adapalene, does bind to the retinoic acid nuclear receptor and does affect cell differentiation, keratinization, and inflammatory responses. Drug Based on the Retinoid Structure Used to Treat Psoriasis (Fig. 7) Etrinate is the ethyl ester of acitretin and is active after hydrolysis to the acidic drug. The terminal half-life after 6 months of etrinate therapy is 120 days. In contrast, the terminal half-life of acitretin is only 33—96 h. Both drugs are teratogenic and require elaborate warnings before being pre- scribed and dispensed. The third drug, tazar-otene gel, is administered topically and is indicated for both acne and psoriasis. While there appears to be minimal absorption if used over limited skin area, there is some absorption with retention by the body for up to 3 months. It can cause fetal damage and cannot be used by pregnant women or women who may become pregnant. Retinoids Used in the Treatment of Malignancies [21] (Fig. 8) Retinoic acid has been evaluated as a possible treatment for malignancies. This is based on the fact that it is required for proper cell differentiation, and products based on the retinoid structure are teratogenic. All-irons-retinoic acid is used in combination with other chemotherapeutic agents for the treatment of acute promyelocytic leukemia [22]. It does not kill the malignant cell. It seems to facilitate its differentiation into a nonprolifer-ating myelocyte. Alitretinoin, 9-cis-retinoic acid, indicated for Kaposi's sarcoma is administered topically. It binds to all six retinoic acid receptors. Bexarotene is most selective for the three RXRapY receptors. It also binds several other nuclear receptors including the RAR, vitamin D, thyroid, and peroxisome prolif-erator activator receptors, which probably explains its numerous adverse reactions. The drug is indicated for cutaneous T-cell lymphoma and is available as capsules and a topical gel. 3.2. Vitamin D Family Rickets was first reported in the mid-17th century. It could be lethal, but bone deformation was more common. Rickets, in the United States, continued to be a problem until the 1930s when vitamin D fortified milk became common. Older adults show the bow-legged characteristic of childhood rickets. There were many attempts at giving calcium and/or phosphorous supplements. Finally, it was realized that rickets was not found in sunny climates, and populations whose main source of protein was fish did not have rickets. In 1924, Professor Henry Steenbock, University of Wisconsin, demonstrated that irradiation of foods, including milk, produced food that was antirachitic [23,24]. 650 VITAMINS CH, Tretinoin (retin-A CH3 Retinoic acid CH3 O CH, ^CH3 Isotretinoin 13-c/s-Retinoic acid CH, OH H3CO OCH2CH3 Tazarotene gel Figure 6. Retinoid and retinoid-like drugs indicated for acne. When there is adequate sunlight, no dietary source of the vitamin is required. Indeed, an argument can be made that the vitamin D is not a normal component of the diet. In the United States, it is added to milk, other dairy products, and dairy substitutes. Fish is about the only natural food source. Cholecalciferol (vitamin D3) is produced in the body from endogenously synthesized 7-dehydrocholecal-ciferol (Fig. 9). Consistent with a hormone VITAMINS 651 Tazarotene gel Figure 7. Drugs based on the retinoid structure indicated for psoriasis. model, excess amounts of cholecalciferol can result in excess calcium uptake from the intestinal track leading to calcification of soft tissues. 3.2.1. Chemistry There are two forms of vitamin D, and originally both were considered biologically equivalent. Irradiation of the major plant sterol, ergosterol, produces ergocal-ciferol, also known as vitamin D2 (Fig. 10). However, kinetic studies show significant differences between 25-hydroxy D3 and 25-hydroxy D2- Whereas both showed similar initial rises in serum concentration for the first 3 days, 25-hydroxy D3 continued to increase over 14 days, but 25-hydroxy D2 returned to baseline. The area under the curve (AUC) was significantly greater for 25-hydroxy D3 [25,26]. Being photochemical reactions, in contrast to enzyme catalyzed biochemical reactions, the formation of cholecalciferol is not clean. Exposure of human skin to sunlight of 295—300 nm converts 7-dehydrocholesterol to previtamin D3. The isomerization to cholecal- ciferol is heat catalyzed. Continuous exposure to ultraviolet radiation from the sun results in the reversible formation of lumisterol and tachysterol [27,28]. Once the B ring of the two steroids has been cleaved, the products should no longer be referred to as steroids. 3.2.2. Vitamin Uptake and Metabolism This is very complicated and is dependent upon the source. From a biochemical viewpoint, it is a hormone because in the presence of adequate sunlight, cholecalciferol is produced from 7-dehydrocholesterol in the skin. In this context vitamin D, when administered in supplements, could be considered replacement therapy. When photochemically synthesized in the skin, it is transported to the liver by a specific binding protein formed in the skin where the cytochrome enzyme system (CYP P450) hy-droxylates it to 25-OH-cholecalciferol (25(OH) D3; Fig. 11). This pulls the reversible reactions shown in Fig. 9 toward the desired cholecalciferol. Dependency on the binding protein for transport from the skin also provides a control to prevent overproduction of the hormone and possible hypercalcemia. 652 VITAMINS Alitretinoin 9-c/s-Retinoic acid ct oh Bexarotene Figure 8. Retinoids indicated for malignancies. When taken orally, it follows the same route as other dietary lipids. It is part of the mixed micelles and ends up on the chylomicrons formed in the intestinal mucosa cells. It is then transported to the liver where, like the endogenously produced cholecalciferol from sunlight, it is hydroxylated to 25(OH)D3. The 25-hydroxylated product is then transported to the kidney where it is hydroxylated a second time by 25(OH)D-la-hydroxylase (kidney CYP P450) forming the active 1,25-dihy-droxycholecalciferol (l,25(OH)2D3). The latter is transported to the intestinal tract where it attaches to a nuclear receptor that signals the mucosa cell to synthesize a calcium transport protein. The final l,25(OH)2D3 product can be considered a kidney hormone that regulates calcium intake. Finally, l,25(OH)2D3 is oxidized to inactive calcitroic acid that is excreted through the kidney. The 24,24(OH)2D3 metabolite may be part of the degradation process for 25(OH)D3 that is not transported to the kidney, but it also can elevate serum calcium levels [16]. Patients with kidney failure can experience vitamin D-resistant rickets. Because they cannot carry out the final hydro-xylation step, l,25(OH)2D3 (calcitriol) is prescribed for their hormone replacement therapy. 3.2.3. Biochemical Function Vitamin D function is complex and, with the exception of calcium transport from the intestinal tract, is poorly understood. Specific vitamin D receptors (VDR) are found in 30 different tissues including bone, intestine, prostate, hematopoietic cells, and skin [29]. Like the retinoic acid receptors, the vitamin D receptor is a nuclear receptor belonging to the steroid hormone superfamily of nuclear receptors that includes receptors for estrogen, glucocorticoids, thyroid hormones, and retinoic acid. There are at least 50 genes that respond to hydroxylated calciferols regulating calcium release and uptake and cell division. VITAMINS 653 Tachysterol Cholecalciferol (Vitamin D3) Figure 9. Photochemical formation of vitamin D3 (cholecalciferol). Calcium Regulation There are at least three, possibly four, hormones that regulate calcium metabolism, parathyroid (PTH), calcitonin, l,25(OH)2, and gut-produced serotonin. Bone is the principal calcium reservoir, and it is a dynamic tissue with calcium being released and deposited. New calcium comes from our diet, and calcium is excreted through the kidneys. In response to low serum calcium levels, PTH stimulates the hydroxylation of 25(OH) D3, leading to formation of calcium transport protein and osteoclast cells required to release calcium from bone. PTH also inhibits calcium excretion by the kidney. In contrast, calcitonin (produced in the thyroid gland) acts when serum calcium levels are high. It promotes the deposition of calcium into bone and excretion of calcium by the kidney. Using knockout mice, evidence has been published that serotonin produced in the gut may be the "master" Vitamin D2 (ergocalciferol) Figure 10. Photochemical formation of vitamin D2 (ergocalciferol). Vitamin D3 Calcitroic acid Figure 11. Vitamin D metabolism. VITAMINS 655 regulator of calcium flux to and from bone. This serotonin must act in the periphery because it does not pass the blood—brain barrier. In the knockout mice, the more the serotonin is released, the greater the calcium loss from bone. The process is reversed with low production of serotonin [30]. Rickets in infants and children and osteomalacia in adults are caused by a deficiency of vitamin D. Osteoporosis is complex involving many variables including vitamin D status and lack of calcium in the diet. Today most vitamin D deficiencies are caused by a lack of sunlight or restricted diet. The lack of sunlight may be caused by living in northern latitudes. The latter also can be affected by the amount of skin pigmentation [31—33]. A strict vegetarian diet lacking vitamin D fortified dairy products or fatty fish, particularly in children, may also result in rachitic lesions in the bone [34,35]. Mechanistically, rickets and osteomalacia are similar in that both are being characterized by bone softening. Normal bone growth and maintenance require that the osteoblast cells lay calcium hydroxyapatite onto a cartilage matrix. A deficiency of vitamin D results in a lack of mixed calcium salt available to the osteoblast cells. In infants and children, the cartilage continues to grow. Cartilage, being soft, cannot support the child's weight, leading to the typical bowlegs seen in a rachitic child. An adult also will have bone deformations, particularly in the pelvic area, because the bones cannot support the upper torso. Osteoporosis is a different disease. It can be thought of as osteoclast cells removing calcium more quickly such that osteoblast cells can lay calcium down. The result is porous, brittle bones that break easily. Incidence of hip fractures correlates with 25(0H)D3 status [36]. A patient's vitamin D status can be monitored by measuring the patient's 25(0H) D3. At one time calcitonin sometimes was prescribed to decrease the release of calcium from bone by osteoclast cells. Calcium with vitamin D is currently recommended to replace calcium being released from bone and excreted through the kidney. Vitamin D Analogs Used in Chronic Renal Failure Because l,25(OH)2D3 is produced in the kidney, renal failure leads to a deficiency of this hormone and vitamin D-resistant rickets. The solution was prescribing synthetic 1,25 (OH)2D3 (generic name calcitriol). Nevertheless, these patients can experience hyperparathyroidism caused by the parathyroid gland attempting to compensate for the lack of 1,25 (OH)2D3. The result is increased osteoclast activity leading to a loss of calcium from the patient's bones, leading to hypercalcemia and either osteomalacia or osteoporosis. To overcome these complications, there are two synthetic ergocalciferol analogs (Fig. 12) indicated for secondary hyperparathyroidism associated with chronic renal failure. Note that both compounds contain the hydroxy group at position 1, the position where the kidney carries out its hydroxylation to produce the 1,3,25-trihydroxy product. Doxercalciferol is 1,3-dihydroxy-ergocalciferol and, in the liver, is hydroxylated to active l,25(OH)2D2 (the ergocalciferol analog of calcitriol). It is not clear why doxercalciferol is more selective than calcitriol. In contrast, paricalcitol is the 19-normethyl analog of l,3,25(OH)3D2 produced from ergocalciferol and requires no further hydroxylation reactions. Other Activities Attributed to Vitamin D in Its Active Forms The status of vitamin D today is where vitamins C and E were years ago. Because of the wide distribution of the vitamin D receptors, vitamin D's role is cell biochemistry and maintenance of health is being widely investigated. A search in Medline will produce papers in highly regarded refereed journals showing that vitamin D deficiency is associated with cardiovascular disease, autoimmune diseases, increased bone fractures, metabolic syndrome, and diabetes. With the exception of bone strength, there is little evidence that intervention with vitamin D supplements will treat these diseases with the possible exception that a person definitely is vitamin D deficient. The role of l,25(OH)2 vitamin D in regulating cell division is under active investigation. Populations living in areas with higher exposure to sunlight have lower incidence of prostate, breast, and colon cancers [37]. On the other hand, there is some evidence that excessive vitamin D might exacerbate prostate cancer [38]. It is beyond the scope of this chapter to provide an in-depth, referenced review. An extensive review of vitamin D and 656 VITAMINS H0V Doxercalciferol 1,3-dihydroxy-ergocalciferol Paricalcitol 19-nor-1,3,25-trihydroxy-ergocalciferol Figure 12. Ergocalciferol analogs indicated for chronic renal failure. cancer is available from the International Agency for Research on Cancer of the World Health Organization [39]. There are concerns that taking megadoses of vitamin D causes hypercalcemia leading to possible calcium deposits in the soft tissues and possibly blood vessel walls. In contrast with vitamins C and E, vitamin D can be considered a hormone with the cautions associated with dosing any hormone therapy. Therefore, the challenge is to develop compounds that are selective for receptors involved with controlling cell division but will not affect calcium transport leading to hypercalcemia. The initial analog on the market is calcipotriene (Fig. 13), which is indicated for psoriasis, a nonmalignant proliferation of cells. Its use is limited to topical application. When administered internally, hypercalcemia can result. Attempts have been made at pulse dosing of l,25(OH)2D3 to maximize inhibition of cell division with minimal calcemic activity [40]. Most of the calciferol analogs are based on modifications of the 17-alkyl side chain. Modifications of the A ring include 19-nor methylene and 3-nor hydroxy analogs with and without the 1-hydroxy moiety. The la-hydroxy-24-ethyl cholecalciferol analog (Fig. 14) was less calcemic in mice and inhib- HO Calcipotriene Figure 13. Calciferol analog indicated for psoriasis. ited the development of preneoplastic lesions in mammary glands [41]. Unsatura-tion at position 16 (Fig. 15) provides modest antiproliferative effects on prostate cancer cells with little hypercalcemia [42]. A series of 20-cyclopropyl-cholecalciferol analogs VITAMINS 657 HO^ v ^OH 1 -(OH)-24-ethyl-cholecalciferol Figure 14. la-Hydroxyvitamin D3. (Figs 16 and 17) showed good activity against human prostate, breast, and myeloid leukemia cell lines [43]. The most potent in this series were the 19-nor methylene analogs independent of whether the side chain had ethylene or acetylene unsaturation. Modification of the side chain with a 25-keto or oxime (Ri = O or NOH, respectively) with or without an additional hydrogen or methyl (R2 = H or CH3, respectively) produced analogs as antiproliferative in vitro as l,25(OH)2D3 (Fig. 18). The oximes were less calcemic [44]. 1,25-(OH)2-16-ene Figure 15. 16-Ene-25-yne D3 analogs. 658 VITAMINS F3C 1,25-(OH)2-26,27-F6-19-nor 3-Desoxy-1,25-(OH)2-26,27-F6 Figure 16. 23-i?-Ene-20-cyclopropyl D3 analogs. 3.2.4. Dosage Forms The commercial products are produced by irradiation of 7-dehy-drocholesterol or ergosterol under controlled conditions. The final yield is about 50%. Although more stable than vitamin A, vitamin D is sensitive to oxygen and tend to isomerize into inactive isomers in the presence of trace metals, which can cause problems in formulating a vitamin mineral supplement. It is stabilized with antioxidants and protective coatings. 3.2.6. Dietary Reference Intakes There is no RDA for this vitamin. It is difficult to derive a RDA for a population because a significant percentage will receive adequate amounts of the vitamin from sunlight because of the area of the country where they live. On the other hand, the diverse population of the United States means the people of color may need more of the vitamin from fortified milk relative to those whose ancestors came from Northern Europe. 3.2.5. Hypervitaminosis D Like vitamin A, the calciferols are one of the few vitamins that have tolerable upper intake levels (UL) with the UL to RDA ratio of 5—10, which is similar to vitamin A. Hypervitaminosis D causes increased absorption of calcium and phosphorous (P follows Ca), leading to calcification of the tissues, vomiting, kidney damage, and so on. It can be the most serious of the hypervi-taminoses. The reports that vitamin D inhibit proliferation of tissue and may protect against certain cancers could cause the public to overdose with this vitamin. Nevertheless, some medical specialties are recommending that adults take in 600-1000IU (15-25 ug) per day divided between morning and evening. Because of vitamin D's low concentration in breast milk, pediatric vitamin D is being recommended. AI Infants (0—12 months) Children (1—8 years) Boys (9—18 years) Girls (9—18 years) Men (19-50 years) Women (19—50 years) Men (51-70 years) Women (51—70 years) Men (70 + years) Women (70 + years) Pregnancy Lactation UL Infants Children (1—18 years) Adults (over 19 years) Pregnancy Lactation 5 (ig (200IU)/day 5 (ig (200IU)/day 5 (ig (200IU)/day 5 (ig (200IU)/day 5 (ig (200IU)/day 5 (ig (200IU)/day 10(ig(400IU)/day 10(ig(400IU)/day 15(xg(600IU)/day 15(xg(600IU)/day 5 (ig (200IU)/day 5 (ig (200IU)/day 25 (ig (1000IU)/day 50(xg(2000IU)/day 50(xg(2000IU)/day 50(xg(2000IU)/day 50(xg(2000IU)/day 1,25-(OH)2 ho^ -—- -▼oh 1,25-(OH)2-25,27-F6-19-nor l,25-(OH)2-25,27-F6 l,25-(OH)2-19-nor hov "—' 3-Desoxy-1,25-(OH)2 1a-F-25-(OH) Figure 17. 23-Yne-20-cyclopropyl D3 analogs. 660 VITAMINS HO Figure 18. Keto and oxime vitamin D3 analogs. 3.2.7. Drug Interactions Phenobarbital and possibly other anticonvulsants used in epilepsy induce liver hydroxylation leading to subsequent formation of the inactive end products. As long as the epileptic child receives a normal amount of fortified milk, there is no problem with this interaction. 3.3. Vitamin E Family (Tocopherols and Tocotrienols) This vitamin group was discovered in rat feeding experiments resulting in these animals unable to produce offspring. These same rats seemed normal in all other respects including physical growth [45]. The condition could be corrected by addition of lettuce, whole wheat, and cereal grains, with the best source being wheat germ followed by vegetable oils. Think of the tocopherols and tocotrienols (Table 4) as nature's antioxidants. Most of the defined vitamin activity in terms of rat fertility assays is found in a-tocopherol. "Tocopherol" means childbearing alcohol. 3.3.1. Chemistry The vitamin E family consists of tocopherols and tocotrienols. There has been considerable debate as to whether the RRR isomer is the only biologically active form of the vitamin. The vitamin of commerce is a-tocopherol either as a racemic synthetic mix- ture or the natural RRR-a-tocopherol. All of the tocopherols found in food have the ^-configuration at position 2. The synthetic form of the vitamin is a mixture of all eight stereoisomers (now referred to as rac-a-tocopherol rather than d,l-a-tocopherol). Because there is both general and stereospecific antioxidant activity, the RDA tables state that the activity ratio of RRR-a-tocopherol to rac-a-tocopherol is 1:1.36 Table 5 [46]. All isomers are antioxidants and probably do provide general antioxidant protection internally or in vivo. On the other hand, evidence points to the RRR isomer as being specific for a variety of reductase systems, involving selenium (Se) and glutathione (GSH/GSSG). The "relative biological activity" in Table 4 is based on a rat resorption—gestation assay that probably has no parallel in terms of vitamin E status in human health. 3.3.2. Uptake and Metabolism Like the vitamin A esters, tocopherols, both esterified and nonesterified, require bile salts to become part of the mixed micelles containing the dietary lipids. They are absorbed into the mucosal cell by passive diffusion. The tocopherols follow the dietary lipids onto the chylomicrons. Because the latter first enter the lymphatic system before the circulatory system, the Table 4. Tocopherols and Tocotrienols Relative Biological Activities1 hcl R2 6 ch3 . 1 2 ^0 Š h3c R h3c////(j R^h ch3 1 /H ^^Kh3 Tocopherols Ri R2 Relative Biological Activity0 a-Tocopherol ß-Tocopherol y-Tocopherol ô-Tocopherol CH3 CH3 H H CH3 H CH3 H 1 0.5 0.1 0.03 Ri hcl 6 ch3 ch3 ch3 <^ ch3 . 1 2 ^0 i h3c R ^^^^ ^^^ch3 Tocotrienols Ri R2 Relative Biological Activity0 a-Tocotrienol CH3 CH3 0.3 ß-Tocotrienol CH3 H 0.05 y-Tocotrienol H CH3 Inactive ô-Tocotrienol H H Inactive 1 Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium and Carotinoids, Food and Nutrition Board, Institute of Medicine, National Academy Press, p. 193, 2000. °Rat fetal reabsorption assay. Table 5. Factors for Converting International Units of Vitamin E to cx-Tocopherol (mg) to Meet Recommended Intake1 USP Molar a-Tocopherol Conversion Conversion Conversion Factors0 Factors Factors IU/mg \ mg/IU umol/IU mg/IU Synthetic Vitamin E and Esters a d,Z-a-Tocopherol Acetate 1.00 1.00 2.12 0.45 d,Z-a-Tocopherol Succinate 0.89 1.12 2.12 0.45 d, Z-a-Tocopherol 1.10 0.91 2.12 0.45 Natural Vitamin E and Esters6 d-a-Tocopherol Acetate 1.36 0.74 1.56 0.67 d-a-Tocopherol Succinate 1.21 0.83 1.56 0.67 d-a-Tocopherol 1.49 0.67 1.56 0.67 1 Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium and Carotenoids, Food and Nutrition Board, Institute of Medicine, National Academy Press, p 192, 2000. ° Synthetic vitamin E supplements labeled as d,/-a-tocopherol can consist of all eight possible isomers (RRR-, RSR-, RRS-, RSS-, SSS-, SRS,-, SRR-, and SSR-). 6 d-a-Tocopherol refers to the RRR-isomer, the only one found naturally in foods, and the other 2 R stereoisomers (RRS-, RSR-, and RSS). c The United States Pharmacopeia (USP) has defined one IU as 1 mg of all racemic a-tocopherol acetate based on a 1940s rat fetal resorption assay. 662 VITAMINS tocopherols do not go directly to the liver. Instead, the chylomicrons are distributed throughout the body, and some of the tocopherols enter the adipose tissue with the fatty acids that also were being transported on the chylomicrons. The chylomicron remnants finally reach the liver. The remaining tocopherols leave the liver on the very low-density lipoproteins (VLDL) that become the low-density lipoproteins (LDL). In other words, tocopherols will be found wherever there is a significant amount of lipid material including the high-density lipoproteins (HDL). There is no specific organ where the tocopherols are stored. Consistent with antioxidant model, the RDA is based on the polyunsaturated fatty acid (PUFA) consumption. The implication is that if increased PUFA intake is recommended, the RDA for tocopherol should be increased. There is increasing evidence that the RRR-stereoisomer is preferentially transferred in the liver onto the lipid transport proteins, but it is not absolute. This could explain why RRR-a-tocopherol is more active in vivo, but the other isomers are not inactive. Complicating the study of vitamin E in humans is the fact that the most common isomer in the human diet is y-tocopherol, and it has anti-inflammatory properties. Excess a-tocopherol from vitamin supplements and fortified food can displace y-tocopherol [47]. This raises the question if the conflicting report on the efficacy of vitamin E (as a-tocopherol) is related to its effects on y-tocopherol and mix of stereoisomers. 3.3.3. Biochemical Function The best way to describe tocopherol's role is that of a lipid -soluble antioxidant. It protects unsaturated lipids from oxygen-induced peroxide formation. There is evidence for both free-radical one-electron chemistry (Fig. 19) and two-electron quinone—hydroquinone (Fig. 20) [48]. The oxidized—reduced glutathione system may be part of the system that regenerates reduced a-tocopherol. At one time it was thought that the preference for the 2R stereoisomers indicated that the vitamin was part of a biochemical oxidation—reduction system, possibly as a coenzyme. So far that role for a-tocopherol has not been confirmed. The current evidence points to the hepatic tocopherol transfer protein's pre- ference for the i?.Rfl-stereoisomer as the explanation for the partial steric preference. It is well known that oxygen-generated free radicals are tightly controlled by a variety of antioxidants, some generated in vivo and others dietary. A part of the current model for several diseases, including cardiovascular, is based on inflammatory process of which oxidative process is a part. This has led to extensive investigations on the effectiveness of a-tocopherol and ascorbic acid (vitamin C) in preventing and/or treating these diseases. Initially, the results looked promising. More recent studies show a lack of effectiveness and possibly exacerbating the patient's condition [49-53]. 3.3.4. Deficiencies The current model for the cause of vitamin E deficiencies points to malabsorption of lipids. Thus, there may be malabsorption of other lipid-soluble vitamins. Little is know regarding the pharmacokinetics of the tocopherols. Part of the reason for this may be caused by there not being a specific storage organ for the vitamin. Correlating human medical conditions with the biochemical role of the tocopherols is difficult because of the lack of correlations between deficiency diseases seen in animals relative to what is seen in humans. Deficiency diseases seen in animals include reversible reproductive failure in female rats; irreversible degeneration of rat testicular tissue leading to mail sterility; nutritional muscular dystrophies in monkeys, rabbits, guinea pigs, lambs, calves, turkeys, and chicks; and an anemia in monkeys. Vitamin E does not treat human muscular dystrophy, nor various causes preventing a couple from conceiving a child or inability of pregnant women to go to full term. What is seen in humans is partially reversible set of neurological problems and hemolytic anemia in premature infants. Because of poor placental transfer, newborn have little of the vitamin. Human milk contains 2—5mg/L. Cow's milk contains less. 3.3.5. Hypervitaminosis E This is a relatively safe vitamin. Toxicities have been reported involving chronic administration of300—1200 mg per day. The symptoms can be very serious and Chromanoxyl radical Chromanol methide radical Chromanone-8oc radical Figure 19. Resonance stabilized tocopherol radicals. 664 VITAMINS Reduced tocopherol (hydroquinone) Oxidized tocopherol (quinone) Figure 20. Tocopherol oxidation—reduction. include thrombophlebitis, pulmonary embolism, hypertension, breast development in men andchildren, severe fatigue, andnonmalignant breast tumors. Nevertheless, the UL to RDA ratio is about 66 to 1 for adults, making it a very safe vitamin. 3.3.6. Dosage Forms The tocopherols, being antioxidants, are very sensitive to oxygen. Sensitive to UV light is another problem. There are two provitamin esters that are used commercially (Fig. 21), the oil-soluble acetate and water-soluble hemisuc-cinate. The latter is commonly found in dry dosage forms requiring a free flowing powder. Oxidation to the quinone form is blocked by esterifying the free phenolic hydroquinone. R = acetate, hemisuccinate (sodium salt) Figure 21. a-Tocopherol dosage forms. VITAMINS 665 3.3.7. Dietary Reference Intakes (Based on o-a-Tocopherol) AI Infants (0—12 months) 0.6 mg/kg/day EAR Children (1—8 years) 5—6 mg/day Boys (9—18 years) 9-12 mg/day Girls (9—18 years) 9-12 mg/day Men (19-50 years) 12 mg/day Women (19—50 years) 12 mg/day Men (51-70 + years) 12 mg/day Women (51—70+ years) 12 mg/day Pregnancy 12 mg/day Lactation 16 mg/day RDA Children (1—8 years) 6—7 mg/day Boys (9—18 years) 11—15 mg/day Girls (9—19 years) 11—15 mg/day Men (19-50 years) 15 mg/day Women (19—50 years) 15 mg/day Men (51-70 + years) 15 mg/day Women (51—70+ years) 15 mg/day Pregnancy 15 mg/day Lactation 19 mg/day UL Infants Not established (do not give supple- ments; use only food and formula for sources) Children 200 mg/day (1-3 years) up to 600 mg/ day (9—13 years) Adolescents 800 mg/day Adults (19 + years) 1000 mg/day Pregnancy 800-1000 mg/day Lactation 800-1000 mg/day 3.4. Vitamin K Family Vitamin K was discovered by accident by Danish scientists who, using a special fat-free diet designed to determine whether chickens synthesize cholesterol, observed that the animals developed a hemorrhagic condition characterized by a prolonged clotting time. The condition could be cured by an organic factor found in fresh cabbage, ether extract of alfalfa, putrefied fish meal, cereals, or hog livers. It was named Vitamin K for coagulation vitamin. This may be the only vitamin that humans receive in significant amounts from their intestinal bacterial, and there is some question regarding this commonly held assumption. Because of this source, it has been very difficult to establish a recommended daily allowance. An estimated safe intake was established for this vitamin for the first time with the recent 1989. With the release of the Dietary Reference Intakes, there is an adequate intake, but no RDA. 3.4.1. Chemistry There are two series for this vitamin (Fig. 22). The vitamin Kx series is mostly obtained from green plants, whereas the K2 series is the product of bacteria. The active vitamin is in the K2 series. Menadione has sometimes been referred to as vitamin K3. The common commercial form is called phytonadione in the United States Pharmacopeia and phylloquinone in Chemical Abstracts. 3.4.2. Vitamin K Uptake and Metabolism Dietary vitamin Kx and the pharmaceutical form, phytonadione or vitamin Ki(20), must be converted to the K2 series known as menaqui-nones. The most common of these is menqui-none-4 or K2(20). This conversion to the K2 series occurs in the liver and possibly the intestinal flora. It involves removing the phy-tyl chain producing the intermediate menadione. Menadione sometimes is prescribed when there is impaired uptake of lipids from the intestine. There is little storage reserve in the liver, and a deficiency can result when dietary intake of vitamin K is restricted or absorption is impaired. Dietary vitamin K and supplements are processed similarly as with the other fat-soluble vitamins. Bile salts are required for emulsification and formation of mixed micelles. They travel to the liver on chylomicrons along with vitamins A and E. 3.4.3. Vitamin K Biochemistry and Deficiency Deficiencies of this vitamin lead to serious hemorrhaging. The vitamin is required for formation of proteins that complex calcium. This is done by functioning as a coenzyme in the y-carboxylation of glutamic acid (Fig. 23). Vitamin K2 is reduced to the hydroquinone. After several steps, a complex oxidation occurs resulting in the "vitamin K base" that is integral part of the carboxylation step. In a key step, the vitamin K oxide is reduced to the 666 VITAMINS Phytonadione (vitamin Ki; phylloquinone) Liver Removal of the side chain O Menadione Addition of the geranylgeranyl chain CH, CHp —CH = C- -CH2-|-H n Vitamin K2 (n = 4; menaquinone-4) Figure 22. Formation of vitamin K2. original vitamin K2. It is this final reduction coronary thrombosis. This interaction between that is inhibited by the Coumadin anticoagu- the Coumadin anticoagulants and the regen-lants widely used by patients susceptible to eration of vitamin K2 is the reason why pa-stroke, pulmonary embolism, phlebitis, and tients on Coumadin must monitor their vitamin y-Carboxyglutamate Figure 23. Outline of vitamin K carboxylation of glutamic acid. 668 VITAMINS K intake either by vitamin supplements or diet. This usually is done by regularly scheduled determinations of prothrombin time. The carboxylation reaction is required for production of several clotting proteins including prothrombin, protein C, protein S, and factors VII, IX, and X. It is also required for y-carboxylation of osteocalcin, an important calcium-binding protein found in the matrix of bone and required for proper deposition of calcium onto bone. This latter finding has led to several studies to determine if patients prescribed Coumadin anticoagulants are at increased risk for osteoporosis and fractures [54—57]. Originally, it was postulated that vitamin K might help prevent osteoporosis. If this were correct, it would appear that patients on anticoagulant therapy would be at increased risk. Although there is some evidence that vitamin Ki supplementation can increase bone mass density, there is little evidence that supplements reduce the incidence of fracture. Complicating the latter is an indication that vitamin Ki supplement's ability to increase bone mass density may be gender specific with benefits seen in women and not men [58]. Nevertheless, some calcium supplements have both vitamin D and K added to their formulation. 3.4.4. Causes of Vitamin K Deficiency Rarely is a vitamin K deficiency caused by insufficient diet. It more likely is due to a medical condition. At one time, multivitamin supplements rarely contained vitamin K. It is now routinely found in these products. Causes of a vitamin K deficiency include obstructive jaundice (now uncommon), loss of intestinal flora in preparation for intestinal surgery, and hemorrhagic disease of the newborn. Deficiencies caused by obstructive jaundice were caused by blockage of the bile duct, usually from cholelithiasis, preventing the release of bile salts into the intestinal tract for emulsifying the lipid contents. At one time, surgical removal of gallstones was delayed to see if the problem would correct itself. Menadione would be prescribed because it does not require micelle formation in that it is directly absorbed through the mucosa into the portal vein and flows to the liver. Alternatively, intramuscular injections of vitamin Ki were given. In nonemergency surgery of the intestinal tract, the patient would undergo 1—2 weeks of antibiotic therapy to reduce the level of intestinal bacteria. Usually the patient did not eat well because of the intestinal problem. The combination of reduced dietary vitamin K and vitamin from the intestinal bacteria could result in a vitamin K deficiency. Determination of prothrombin time is ordered by the surgeon to determine whether menadione or phytonadione is indicated. The third cause is hemorrhagic disease of the newborn. Infants are born with a sterile intestinal tract. Until the flora is established, the infants have to get along with the vitamin K they received from their mothers. In the past, an infant might die from hemorrhaging. Most states requires that each newborn receive an injection of vitamin K1(20). Menadione injection should not be given because it can cause a hemolytic anemia in the newborn. 3.4.5. Hypervitaminosis K While it is possible to overdose with this vitamin, the fact that it is only available over the counter in small doses in multivitamin preparations has resulted in little knowledge of any toxicities. Toxicities do not appear in animals administered large doses. It is known that excess intake of the vitamin does not promote clot formation. There is no tolerable upper intake level. 3.4.6. Dietary Reference Intakes AI Infants Children (1—8 years) Boys and girls (9—18 years) Men Women Pregnancy Lactation 2-2.5 (xg/day 30-55 p.g/day 60-75 (Xg/day 120(xg/day 90 (Xg/day 75-90 (Xg/day 75-90 (Xg/day 3.5. Thiamine (Vitamin Br) After 26 years of constant research, the vitamin preventative of the disease beri-beri has been isolated, its chemical constitution determined and the vitamin itself synthesized at a cost far lower than that of recovering it from bran.(Scientific American, February 1938; reprinted in 258, 12, Feb. 1988) VITAMINS 669 The vitamin B complex, of which thiamine is considered the first one, generally includes the group of water-soluble vitamins found in rice polishings, bean extracts, yeast, and liver. There are no chemical relationships in the B complex. The nomenclature is very confusing. The common name originally implied something about the chemical nature of the vitamin. Even the concept of water soluble is somewhat misleading as some of the vitamins in this group would be considered poorly soluble by most pharmaceutical standards. The one thing the B complex vitamins have in common is that nearly all of them function either as a coenzyme or a structural component of a coenzyme. 3.5.1. Chemistry (Fig. 24) Thiamine consists of a pyrimidine joined to a thiazole ring by a methylene bridge. The thiazole nitrogen is a quaternary with a permanent positive charge. H3C ~N Thiamine mononitrate NH3+ CI H,C Thiamine HCI (thiamine chloride HCI) ATP AMP J Thiamine kinase CH2 —CH2-OH CHp-CHo-0-P-O-P-Cr H3C 'N' Thiamine pyrophosphate Figure 24. Commercial forms of thiamine and formation of thiamine pyrophosphate. 670 VITAMINS There are two commercial salts. Thiamine hydrochloride is, in reality, thiamine chloride hydrochloride. It is a double salt consisting of an amine hydrochloride on the pyrimidine amine and a chloride on the thiazole quaternary nitrogen. Thiamine nitrate is correctly named in that the nitrate anion is found on the quaternary nitrogen, and the pyrimidine amine is not protonated. Once the vitamin enters the acidic stomach, it will exist as the chloride hydrochloride salt. Thiamine hydrochloride is very water soluble (1 gm/1 ml). It also is very hygroscopic, making it difficult to use in dry formulations. This salt is commonly used in liquid and injectable formulations. Thiamine nitrate is sufficiently water soluble (1 g/35 mL) that it can be used in liquid formulations since the RDAs are less than 2 mg per day. Because it is nonhygro-scopic, it is commonly used in dry dosage forms. 3.5.2. Uptake and Metabolism An active transport system in the jejunum provides efficient uptake of the vitamin into the intestinal mucosa cell. A thiamine kinase in the intestinal mucosa cell transfers a pyrophosphate from the ATP to the propyl alcohol at position 5 of the thiazole ring forming thiamine pyrophosphate (TPP) (Fig. 24). The latter product is the coenzyme form of the vitamin. There is some evidence that this phosphorylation is the rate-limiting step and controls the absorption of the vitamin. The coenzyme is transported to the tissues where needed. Thiamine pyrophosphate has two important coenzyme roles, both focusing mostly on carbohydrate metabolism (Figs 25 and 26). The active portion of the coenzyme is the thiazole ring. The first step in the oxidative decarboxylation of a-keto acids requires TPP. The two most common examples are pyruvate and a-ketoglutarate oxidative decarboxyated to acetyl CoA and succinyl CoA, respectively. The same reaction is found in the metabolism of the branched-chain amino acids valine, iso-leucine, and leucine and also methionine. In all cases, TPP is a coenzyme in a mitochondrial multienzyme complex consisting of TPP, lipoic acid, coenzyme A, FAD, and NAD. TPP is also the coenzyme in the transketo-lase reaction (Fig. 26) found in the pentose phosphate pathway that interconverts hexoses, pentoses, tetroses, and trioses. This reaction removes carbons 1 and 2 of a ketose and transfers them to an acceptor aldose. Examples include TPP transferring carbons 1 and 2 of xylulose-5-P to ribose-5-P producing glyceraldehyde-3-P (five carbons minus two carbons) and sedoheptulose-7-P (five carbons plus two carbons). This reaction is reversible. A second reversible reaction has TPP transferring carbons 1 and 2 of xylulose-5-P to erythrose-4-P producing fructose-6-P (four carbons plus two carbons) and glyceralde-hyde-3-P (five carbons minus two carbons). The dietary reference intakes for thiamine are basedon carbohydrate consumption. This is because most pyruvate substrate comes from aerobic glycolysis of glucose, much of the a-ketoglutarate substrate originates from carbohydrate sources, and the transketolase reactions are part of carbohydrate metabolism. 3.5.3. Thiamine Deficiencies Thiamine deficiencies were reported in the very early medical literature and were called beriberi, a Japanese term. Sailors in the Japanese navy experienced thiamine deficiencies when fed rice in which the polishings had been removed to prevent mold growth. This is somewhat analogous to removing the germ from wheat in order to prolong the shelf life of flour containing foods. There are two forms of beriberi, wet and dry. Wet beriberi is characterized by edema and enlarged heart. Dry beriberi is more neurological and can include muscle wasting. A beriberi patient can experience both. Assuming a reasonably balanced diet, most thiamine deficiencies today are caused by chronic alcoholism. This form of thiamine deficiency is called Wernicke—Korsakoff syndrome. It is common for emergency medical personal to add thiamine to the i.v. being administered to a comatose patient suspected of experiencing substance abuse. 3.5.4. Hypervitaminosis Thiamine The vitamin is considered very safe. There are no tolerable upper intake levels. Possibly the rate-limiting phosphorylation step in the intestinal mucosa reduces the risk of toxicity. The percentage of thiamine absorbed decreases as the dose increases. Figure 25. Thiamine pyrophosphate decarboxylatian of a-keto acids. VITAMINS 673 3.5.5. Dietary Reference Intake AI Infants 0.2-0.3 mg/day EAR Children (1—13 years) 0.4-0.7 mg/day Males (14-18 years) 1.0 mg/day Females (14—18 years) 0.9 mg/day Men (19-50 + years) 1.0 mg/day Women (19—50 + years) 0.9 mg/day Pregnancy 1.2 mg/day Lactation 1.2 mg/day RDA Children (1—13 years) 0.5-0.9 mg/day Males (14-18 years) 1.2 mg/day Females (14—18 years) 1.0 mg/day Men (19-50 + years) 1.2 mg/day Women (19—50 + years) 1.1 mg/day Pregnancy 1.4 mg/day Lactation 1.5 mg/day UL None reported 3.6. Riboflavin (Vitamin B2) Shortly after the discovery of thiamine from yeast concentrates, the presence of a second nutritional factor in such materials was suggested. This second factor was also reported to have a pellagra-preventative activity since it alleviated a deficiency-induced dermatitis in rats. It was called vitamin B2 in England and vitamin G in the United States. 3.6.1. Chemistry (Fig. 27) Riboflavin has a characteristic flavin ring system that gives it a unique spectroscopic and instability properties. There are two commercial forms. Riboflavin, itself, is poorly water soluble (1 g/ 10,000 mL) and is limited to oral dry dosage forms. Riboflavin phosphate, as the sodium salt, is very water soluble at 100 mg/mL. It is widely used in dry and liquid dosage forms. 3.6.2. Riboflavin Uptake and Chemistry (Fig. 27) Most dietary riboflavin is eaten as the FAD or FMN coenzymes. Intestinal pyrophosphatases and phosphatases produce free riboflavin that is actively transported through the proximal area of the small intestine into systemic circulation. Because of its poor water solubility, it is transported on albumin and immunoglobulin proteins. Conversion to the coenzyme forms occurs inside the cells that need these coenzymes. 3.6.3. Metabolic Role Riboflavin coenzymes are required for most oxidations of carbon-carbon bonds (Fig. 28). Examples include the oxidation of succinyl CoA to fumarate in the Krebs cycle and introduction of a,(3-unsatura-tion in (3-oxidation of fatty acids. Riboflavin is also required for the metabolism of other vitamins including the formation of niacin coenzymes from tryptophan (Fig. 29), reduction of 5,10-methylene tetrahydrofolate to 5-methyl tetrahydrofolate (Fig. 45), and interconver-sion of pyridoxine-pyridoxal phosphate-pyri-doxamine (Fig. 32). Because oxidation—reductions using FAD or FMN as the coenzyme is a two-step process, some flavin coenzyme systems contain more than one FAD or FMN. 3.6.4. Riboflavin Deficiency With riboflavin's central role in energy metabolism and conversion of folic acid and pyridoxine to their active forms, it is surprising that riboflavin deficiency does not produce a characteristic set of symptoms. One of the reasons maybe that it is rare to see a patient who is solely deficient in riboflavin. 3.6.5. Hypervitaminosis Riboflavin The combination of regulated active transport and conversion to the coenzyme forms prevents hypervitaminosis problems with this vitamin. Toxicities from the water-soluble riboflavin phosphate have not been reported. There are no tolerable upper intake levels. 3.6.6. Dietary Reference Intakes AI Infants 0.3-0.4 mg/day EAR Children (1—13 years) 0.4-0.8 mg/day Males (14-19 years) 1.1 mg/day Females (14—19 years) 0.9 mg/day Men (19-70 + years) 1.1 mg/day Women (19-70 + years) 0.9 mg/day Pregnancy 1.2 mg/day Lactation 1.3 mg/day RDA Children (1—13 years) 0.5-0.9 mg/day Males (14-19 years) 1.3 mg/day Females (14—19 years) 1.0 mg/day Men (19-70 + years) 1.3 mg/day Women (19-70 + years) 1.1 mg/day Pregnancy 1.4 mg/day Lactation 1.6 mg/day UL None reported OH OH OH CH2 —CH—CH — CH — CH2-OH Flavokinase ATP ADP H,C H,C OH OH OH CH, —CH— CH — CH — Riboflavin Riboflavin phosphate flavin mononucleotide (FMN) OH OH OH CH2 —CH—CH—CH—CH2 — O- FAD pyrophosphorylase Riboflavin adenine dinucleotide (FAD) Figure 27. Riboflavin dosage and coenzyme forms. VITAMINS 675 l-UC fad or fmn (oxidized) e- + H"1 R h Cr fadh. or fmnh- (semiquinone) e- + H4 l-UC l-UC fadh2 or fmnh2 (reduced) Figure 28. FAD/FADH2-FMN/FMNH2 oxidation-reduction. Tryptophan-2,3-dioxygenase Tryptophan -CH2-CH—C02- NH, -CHO Kynurenine formamidase Formylkynurenine Kynurenine Kynurenine hydroxylase (FAD dependent) vi ,co2- NH+ C02-Quinolinic acid nonenzymatic CHO "02C 3-Hydroxyanthranilic 'NH3+ acid oxidase Amino-carboxymuconic semialdehyde • NH + Kynureninase OH 3-Hydroxyanthranilic acid OH 3-Hyd roxykyn u ren i ne t NAD+ Figure 29. Biosynthesis of NAD + from tryptophan. VITAMINS 677 3.7. Niacin (Nicotinic Acid)—Niacinamide (Nicotinamide) The history of niacin revolves around trying to find a way to prevent andcure pellagra, the late-stage deficiency disease caused by a niacin deficiency. Pellagra has been a serious nutritional disorder in the United States, mostly in the southeast. Two thousand deaths from pellagra were reported in 1941. This is ironic because nicotinic acid, later known as niacin, was first reported in during the structure elucidation of the alkaloid nicotine in the nineteenth century. Like some of the other deficiency diseases, there was disagreement between those who thought pellagra was caused by poor sanitation versus those who concluded it was a nutritional disorder. Niacin deficiency, even today, is found in economically poor areas. Even when niacin was first isolated from foods, it was ignored because it did not cure beriberi. 3.7.1. Niacin/Niacinamide Chemistry, Uptake, and Metabolism Niacin is the simplest of the vitamins but has some of the most complex biochemistry of the vitamins. Structurally, it is pyridine-3-carboxylic acid. Strictly speaking, it is nonessential because the essential amino acid tryptophan is a source (Fig. 29). The bio-synthetic route does not produce "free" niacin by decarboxylation of quinolinic acid. In a complex reaction, quinolinic acid loses the 2-car-boxyl group and adds 5'-phosphoribose to form nicotinate mononucleotide (Fig. 30) [59,60]. Niacin and niacinamide in pharmaceutical dosage forms undergo a similar ribosylation reaction. Most vitamin products contain niacinamide because niacin can cause a distracting vasodilation leading to flushing. Niacin and niacinamide are rapidly absorbed from both stomach and intestine. As the dose increases, absorption decreases. It is not clear if there is a feedback mechanism operating. Conversion to the coenzyme forms occurs in the cells where NAD and NADP are needed. NAD primarily is the coenzyme required for oxidation—reduction of carbon—oxygen bonds and is required for oxidative catabolism (glycolysis, (3-oxidation, Krebs cycle). NADP is the coenzyme inbiosynthetic routes (fatty acid and cholesterol synthesis) and will be part of oxidation—reduction reactions involving both carbon—oxygen and carbon—carbon bonds. The active part of the coenzyme is the pyridine ring (Fig. 31). When the substrate is labeled with deuterium, it has been shown that NAD systems can be categorized by the deuterium ending up on the A or B face of the pyridine ring. Examples of NAD dehydrogenases where the hydride anion attaches to the A face are isocitrate dehydrogenase, ma-late dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase. Dehydrogenases involving the B face include a-ketoglutarate dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase. 3.7.2. Niacin Deficiency Niacin deficiency, manifested as pellagra, is characterized by the four Ds: dermatitis, diarrhea, depression, and death. The dermatitis is characterized by a pigmented rash that develops on skin exposed to heat. Changes to the gastrointestinal tract can lead to vomiting, constipation, or diarrhea. Depression is one of the neurological symptoms that can also include apathy, headache, fatigue, and memory loss. Deficiencies were common in populations whose main source of calorie was corn. Zein is the main protein found in corn and it is very low in both niacin and tryptophan. When corn is ground with lime water, the small amount of niacin and tryptophan becomes more bioavail-able. Populations who consume corn meal by this process have a smaller incidence of pellagra. At the same time, it must be remembered that pellagra is the extreme form of a niacin deficiency. The dietary reference intakes for this vitamin use clinical chemistry assays listed in Table 2 to determine DRIs rather than the first appearance of pellagra symptoms. 3.7.3. Hypervitaminosis Niacin Niacin is considered nontoxic, but there are tolerable upper intake levels based on its use as a vitamin. These refer only to niacin and niacinamide, but not tryptophan and niacin equivalents (see next section). Large doses of niacin and niacinamide do have adverse reactions. These are sometime seen with patients prescribed niacin in doses up to 2 g daily for hyperlipide-mia including both hypercholesterolemia and hypertriglyceridemia. For the former, there is 678 VITAMINS Tryptophan NH+ Quinolinic acid O o CT o "NH+ Nicotinic Acid (niacin) O NH+ Nicotinamide (niacinamide) PRPP PPi PRPP PPi \ Quinol + C02>\ Quinolinate PRPP transferase Nicotinate PRPP transferase PRPP PPi Nicotinamide PRPP transferase -Q-P-O- cr H H. 0 O-P-O- 1 ct -o- .H H, OH OH OH OH Nicotinate mononucleotide Nicotinamide mononucleotide 2 ATP + Gin + H20 ADP + PPi + Glu NHo 1. NAD+ pyrophosphorylase 2. NAD+ synthetase NAD+ pyrophosphorylase H2c- OR OH OH OH Nicotinamide adenine dinucleotide (NAD+); R = H Figure 30. Biosynthesis of NAD + from niacin and niacinamide. decreased LDL and increased HDL. There is striction seen with this disease, but it has the vasodilation from niacin, particularly in largely been replaced by more specific vasodi- the head area, caused by increased intercra- lators. Liver toxicities have been experienced nial blood flow. Niacin had been used for by patients prescribed sustained release nia- Raynaud's syndrome to treat the vasocon- cin products for hyperlipidemia. VITAMINS 679 O n+ r nad+ i_ T a face; nadh b face; nadh Figure 31. Stereochemistry of NAD + reduction. 3.7.4. Dietary Reference Intakes Niacin/niacinamide DRI units are expressed as milligram niacin equivalents (mg NE). These units assume that approximately 60 mg tryptophan produce 1 mg of niacin. For adult males, the RDA assumes a diet containing a mixture of tryptophan and niacin equivalent to 960 mg of tryptophan or 16 mg of niacin. AI Infants (0—5 months) Infants (6—11 months) EAR Children (1—13 years) Boys (14—18 years) Girls (14-18 years) Men (19-70 + years) Women (19-70 + years) Pregnancy Lactation RDA Children (1—13 years) Girls (14-19 years) Boys (14—19 years) Men (19-70 + years) Women (19-70 + years) Pregnancy Lactation UL Infants (0—12 months) Children (1—13 years) Adolescents (14—18 years) Pregnancy (14—18 years) Pregnancy (19 years older) Lactation (14—18 years) Lactation (19 years older) 2 mg/day of preformed niacin 4 mg NE/day 5- 9 mg NE/day 12 mg NE/day 11 mg NE/day 12 mg NE/day 11 mg NE/day 14 mg NE/day 13 mg NE/day 6- 12 mg NE/day 1.3 mg NE/day 16 mg NE/day 16 mg NE/day 14 mg NE/day 18 mg NE/day 17 mg NE/day Source of intake should be formula and food only 10-20 mg/day of niacin 30 mg/day of niacin 30 mg/day of niacin 35 mg/day of niacin 30 mg/day of niacin 35 mg/day of niacin 3.8. Vitamin B6 Family This group was discovered in the 1930s while conducting feeding experiments on rats. Its importance was realized tragically when the heat process for an infant formula reduced the bioavailability of the vitamin. Children developed convulsive disorders. Before realizing that the problem was caused by an induced pyridoxine deficiency, it was thought a contaminant had been introduced. 3.8.1. Uptake and Metabolism The vitamin B6 family consists of pyridoxine, pyridoxal, pyridoxamine, pyridoxine phosphate, pyridoxal phosphate (PLP), and pyridoxamine phosphate (Fig. 32). Pyridoxal phosphate is the coenzyme form. Pyridoxal phosphate and pyridoxamine phosphate are from animal tissues. Pyridoxine is from plant tissues. All phosphorylated forms are hydrolyzed in the intestinal tract by phosphatases before being absorbed passively. Conversion to the phosphorylated forms occurs in the liver. Notice that niacin (NAD) and riboflavin (FMN, FAD) are required for interconversion among the vitamin B6 family. The phosphorylated forms are transported to the cells where needed. The major excretory product is 4-pyridoxic acid. 3.8.2. Pyridoxal Phosphate Biochemistry Pyridoxal phosphate is required for amino metabolism and reactions involving amino acids. PLP is covalently bound to the apoenzyme through an enamine linkage between an e-amino group of lysine and the aldehyde moiety of PLP (Fig. 33). The most common of the PLP catalyzed reactions are transaminations coo- ,CH2OH H3C CH2OH 4-Pyridoxic acid (metabolite) ^FADH2 Oxidase v FAD CHO CH2NH3+ ,CH2OH NAD+ NADH + H+ H0- ,CH2OH H3C Oxidase Oxidase N Pyridoxine H3C N Pyridoxal FMNH2 FMN H c CH2OH Pyridoxamine Pi ■* Phosphatase ¥ATP Kinase k ADP Pi-*^ Phosphatase /ADP Kinase *V ATP Pi Phosphatase CH2OH CHO ,CH2OP FMN FMNH2 ,CH2OP H3C N Pyridoxine P Oxidase Oxidase H3C CH2OP Pyridoxal P (PLP) Figure 32. Interconversions among the vitamin B6 family. FMNH2 FMN ^ ^- Pyridoxamine P VITAMINS 681 r C*AAA j vw»N-C-H Enzyme < h | (CH2)4 H CH2OP Pyridoxal P Figure 33. Pyridoxal phosphate bound to the enzyme. (Fig. 34). One-half of all transamination reactions involves a-amino acid + a-ketoglutarate -^a-keto acid + glutamic acid a-ketoglutarate as the acceptor of the amine group forming glutamic acid. Alternatively, glutamic acid donates the amine group and an a-keto acid is the acceptor. Another PLP catalyzed reaction is decarboxylation of amino acids (Fig. 34). These are part of the biosynthesis of neurotransmitters including histamine, serotonin, dopamine, and y-aminobutyric acid. Although not well understood, PLP is also required for the phosphorylase catalyzed gly-cogenolysis producing glucose-l-phosphate. 3.8.3. Vitamin B6 Deficiency There is no distinct deficiency syndrome, but a deficiency can be serious. Seborrheic dermatitis, microcytic anemia, and eleptiform convulsions are observed. The anemia may be caused by decreased heme biosynthesis and the convulsions by imbalances in neurotransmitter biosynthesis. At one time there was a hypothesis that vitamin B6 deficiency might contribute to arteriosclerosis or possibly could be used with folic acid and vitamin B12 to reduce the risk of cardiovascular disease. This was based on an observation that elevated homocysteine correlated with increased incidence of arteriosclerosis, and homocysteine might contribute to the pathology of the vessel walls. Vitamins B6, B12, and folic acid are required for the metabolism of homocysteine (Fig. 35). The first two are cofactors in the "remethylation" back to methionine, and the latter for the initial step in the glycogenic catabolism of the amino acid. Note in Fig. 35 that bio tin and a coenzyme form of vitamin Bi2 are also required for the catabolic reactions. While vitamins B6, B12, and folic acid will reduce homocysteine, there is little evidence that reduced homocysteine levels parallel a reduced risk of cardiovascular disease [61,62]. A better indicator is C-reactive protein (CRP). There is further discussion of vitamin B6 supplementation for improving cognitive levels in the folic acid discussion. 3.8.4. Vitamin-Drug Interactions [63] The two most clinically significant interactions are pyridoxal phosphate with l-Dopa or isoniazid. Examine Fig. 34 and note that dopa decarboxylase requires PLP. This enzyme is found both centrally and peripherally. The latter includes the intestinal mucosa. The precursor to dopamine, l-Dopa is indicated for the treatment of Parkinson's disease. l-Dopa is prescribed because little dopamine crosses the blood—brain barrier relative to its precursor l-Dopa. A patient with Parkinson's disease prescribed l-Dopa and who takes a vitamin supplement with amounts of pyridoxine greater than the vitamin's RDA can experience an increase in Parkinsonian tremors. This is because l-Dopa will undergo decarboxylation in the intestinal mucosa and never reach the locations in the brain where it is converted to the needed dopamine. Isoniazid is widely prescribed for tuberculosis. It can chemically react with pyridoxal and pyridoxal phosphate, significantly reducing the availability of this coenzyme (Fig. 36) [64]. Pyridoxine supplements commonly are recommended to prevent iso-niazid-caused peripheral neuropathy, but they do not reduce the effectiveness of isoniazid. h Amine Figure 34. Pyridoxal phosphate catalyzed deaminations, transaminations, and decarboxylations. NH2 +H3N-CH — C-CT CH2 CH2 S CH3 Methione O C-SCoA CH2 — CH2 C-CT O Succinyl CoA Several steps +H3N-CH — C-CT ATP PPi + Pi Methionine adenosyl transferase CH2 I CH2 I S+— CH3 / -O- . H H . H Methyl transferases OH OH S-Adenosylmethionine (SAM) R-CH3 (carnitine, choline, methlated DNA, epinephrine) +H3N-CH—C- CH2 CH2 S+- H H h, H H Tetrahydrofolate OH OH S-Adenosylhomocysteine (SAH) Adenosine N5-Methyltetrahydrofolate O 0 = C-C- CH2 O -O" 1. Biotin 2. B12 coenzyme Cystathionine y-lyase CH3 a-Ketobutyrate -<-—\— / PLP >. NH4 SH NH3+ O CH2-CH—C- Cysteine H20 +H3N-CH — C- CH2 I CH2 I S NH3+ O +H3N-CH—C — CH2 I CH2 I S H Homocysteine H20 CH2-CH—C- Cystathionine Figure 35. Methionine metabolism. 684 VITAMINS NHp ,CH2OP + Isoniazid (INH) Isonicotinic acid hydrazide H3CT Pyridoxal P H20 J H CH2OP HO- / \ H3C Isoniazid pyridoxal adduct Figure 36. Pyridoxal phosphate—isoniazid interaction. 3.8.5. Hypervitaminosis Pyridoxine A certain mystique has built up around this vitamin resulting in individuals overdosing themselves with commercial vitamin supplements. Because of its role in glycogenolysis, some athletes will take large doses in an attempt to increase blood glucose levels. Serious neurological problems have been seen in doses of 2-6gm/day for 2-40 months [65-67]. Mega-dosing below 2 gm/day seems safe, but all of this information is based mostly on anecdotal reports. There is a tolerable upper intake level, but the UL to RDA ratio is a comfortable 50-60. 3.8.6. Dietary Reference Intake AI (any form of vitamin B6) Infants 0.1-0.3 mg/day EAR (any form of vitamin B6) Children (1—13 years) 0.4-0.8 mg/day Males (14-19 years) 1.1 mg/day Females (14—19 years) 1.0 mg/day Men (19-50 years) 1.1 mg/day Men (51 + years) 1.4 mg/day Women (19—50 years) 1.1 mg/day Women (51 + years) 1.3 mg/day Pregnancy 1.6 mg/day Lactation 1.7 mg/day VITAMINS 685 RDA (any form of vitamin B6) Children (1—13 years) 0.5-1.0 mg/day Males (14-19 years) 1.3 mg/day Females (14—19 years) 1.2 mg/day Men (19-50 years) 1.3 mg/day Men (51 + years) 1.7 mg/day Women (19—50 years) 1.3 mg/day Women (51 + years) 1.5 mg/day Pregnancy 1.9 mg/day Lactation 2.0 mg/day UL (as pyridoxine) Children (1—13 years) 30-60 mg/day Adolescents (14—18 years) 80 mg/day Adults (19 + years) 100 mg/day Pregnancy (14—18 years) 80 mg/day Pregnancy (19+ years) 100 mg/day Lactation (14—18 years) 80 mg/day Lactation (19 + years) 100 mg/day 3.9. Pantothenic Acid [68] Pantothenic acid is essential and is a normal component of our diet. There has been little research done on this vitamin, and therefore, it has adequate intakes and no RDAs. 3.9.1. Chemistry, Uptake, and Metabolic Role This vitamin, which can be considered a derivative of (3-alanine, is asymmetric (Fig. 37). The natural form has the d( + ) configuration. The l(—) stereoisomer is inactive. The reduced alcohol form, pantothenol, is considered as equally active as the parent acid. Many of the multiple vitamin products use a synthetic, racemic mixture. This means that double the amount of synthetic vitamin must be used to obtain equivalent active vitamin. Dietary pantothenic acid is consumed as coenzyme A and the intermediates from coenzyme A's biosynthesis (Fig. 38). These are hydrolyzed to free pantothenic acid. Absorption is by saturable, active transport. Calcium pantothenate is commonly used in dry dosage forms. It is moderately hygroscopic with a solubility of 1 g/2.8 mL and is unstable for autoclaving. Neither the parent pantothenic acid nor the sodium salt is commonly used in dosage forms. Pantothenol (panthenol) is reasonably stable and freely soluble and is used both in injectable and oral dosage forms. Although widely used in cosmetics including skin creams and shampoos, there is no evidence that this vitamin is effective as a vitamin topically. It apparently has good emollient properties, but these have nothing to do with its systemic role. Pantothenic acid is a structural component, but not the active site, of coenzyme A. The acyl thiol esters form on the mercaptan moiety that originates from a cysteine (Fig. 38). The biosynthesis of coenzyme A occurs in the tissues requiring it. Because coenzyme A is required for nearly all acyl transfers, biosynthesis takes place in nearly all cells. 3.9.2. Hypervitaminosis Pantothenic Acid There have been no reports of toxicity and no tolerable upper intake levels. Because its active transport is saturable, excessive uptake is doubtful. Also, this vitamin does not have the mystique that would prompt marketing "high potency" formulations. 3.9.3. Dietary Reference Intakes There are too few studies to provide sufficient information to estimate EAR or RDA. AI Infants Children (1—13 years) Everyone else Pregnancy Lactation EAR None reported RDA None reported UL None reported 1.7-1.8 mg/day 2—4 mg/day 5 mg/day 6 mg/day 7 mg/day 3.10. Biotin [69] Biotin (Fig. 39) is essential, a normal constituent of the diet, and required for four biotin-dependent carboxylation reactions. Eating raw egg white can induce a deficiency. 3.10.1. Uptake In foods, most biotin is cova-lently bound (Fig. 40) to the apoenzyme where it is the coenzyme for carboxylation reactions. Intestinal enzymes hydrolyze the amide link- 686 VITAMINS O OH CH. HO-C-CH2-CH2 —NH — C-CH — C-CH2OH CH, Pantothenic acid O OH CH. fNa "O-C-CHp —CHp —NH— C-C-C-CH2OH Hl CH, Sodium pantothenate Ca; 2+ O OH CH3 "O-C-CHp —CHp —NH — C-C-C-CH2OH Hl CH, Calcium pantothenate O OH CH3 HO-CH2 —CH2 —CH2 —NH — C-CH — C-CH2OH CH, Pantothenol Figure 37. Forms of pantothenic acid. age to produce free biotin. Biotin is actively transported through the intestinal flora into the portal vein and to the liver where it may be stored. It appears that adults may store several months of biotin. From the liver, it is transported to tissues where it is needed. 3.10.2. Chemistry Biotin consists of two five-membered rings cis fused to each other that can be drawn either as the keto (urea) or enolic form (Fig. 39). The enolic d-isomer is the active stereoisomer, but many times commercial multivitamin products contain the synthetic racemic d,l mixture. There is no activity with the l-stereoisomer. 3.10.3. Metabolic Role Biotin picks up carbon dioxide that has been activated by combining with an ATP donated phosphate producing the mixed anhydride of phosphoric and carbonic acids (Fig. 41). The biotin enolate receives the carbon dioxide producing the keto carbon dioxide releasing coenzyme. There are four biotin-dependent carboxyla-tion reactions (Fig. 42), three of which are in the mitochondria. They are (a) Pyruvate carboxylase This reaction, which converts pyruvate to oxaloace-tate, is in the mitochondria and has two functions. First, it is the initial reaction VITAMINS 687 "02C CH2 OH CH, "C "OH O H3C CH3 Pantothenic acid ATP ADP Pantothenate kinase OH .CH2 /Nv. ^CH /CH2 -Oacr "-CH, C Y vOP03"2 Q H3C CH3 4'-Phosphopantothenate Phosphopantothenoylcysteine synthetase OH .CH2 /CH2 ^-N\ /CH /CH2 HS VCH C ^CH2 C "^OPOa"2 C02" O O H3C CH3 4'-Phosphopantothenoylcy steine Phosphopantothenoylcysteine decarboxylase OH H H ? -.CH2 /CH2 ^-Nv. /CH /CH2 HS *"CH2 C VCH2 C VOP03-; I I hA, 4'-Phosphopantetheine 2 ATP ADP + PPi 1. Dephospho-CoA pyrophosphorylase 2. Dephospho-CoA kinase CH2 XH2 ___.NN HS VCH2 X VCH2 C' OH O O f II II ,CH -CH2-0-P-O-P-O- /\ o- 0- H3C CH3 .h h : H I [ H OPO3-2 OH Coenzyme A (CoASH) Figure 38. Formation of coenzyme A from pantothenic acid. NHo J in gluconeogenesis to overcome the 14kcal energy barrier to form phos-phoenolpyruvate. Second, this same reaction, sometimes referred to as an anapleurotic reaction, insures that there is adequate oxalocetate when there is large amount of acetyl CoA entering the Krebs cycle. 688 VITAMINS n ^ nh u JL s' g ***(ch2)4 ^oh h Enolic form Keto or ureido form Figure 39. Biotin. (b) Acetyl CoA carboxylase This reaction, found mostly in the cytosol, is the committed step in the synthesis of fatty acids. (c) Propionyl CoA carboxylase Propionyl CoA is the product from the catabolism of valine, isoleucine, methionine, and odd numbered fatty acids. The carbox-ylation reaction, found in the mitochondria, produces methylmalonyl CoA. The latter undergoes a cobalamin (vitamin B12) catalyzed rearrangement forming succinyl CoA, which is further metabolized in the Krebs cycle. (d) (3-Methylcrotonyl CoA Carboxylase This mitochondrial reaction permits the final steps in the catabolism of the branched-chain amino acid, leucine. The final products, acetoacetate and acetyl CoA, either are oxidative metabolized to carbon dioxide and water or enter other reactions in lipid metabolism. 3.10.4. Biotin Deficiency Relative to many of the vitamins, it is easy to induce a biotin deficiency by feeding volunteers raw egg white. Avidin, a basic protein found in egg white, forms salt linkages with acidic biotin and prevents its transport across the intestinal barrier. Cooked egg white is not a pro- n Z nh h q Lys residue q .JL. s- (CH2)4 ^n' h .(ch2)4____ .nh Figure 40. Coenzyme form of biotin. Figure 41. Biotin binding carbon dioxide. o o CH3-C — c-Pyruvate CH3-C-SCoA Acetyl CoA CH3-CH2-CSC0A Propionyl CoA CO 1 O Biotin ATP ADP + Pi Pyruvate carboxylase ATP ADP + Pi Acetyl CoA carboxylase C02 Biotin ATP ADP + Pi Propionyl CoA carboxylase -C-CH2-C-C-Cr Oxalocetate O O "O-C-CH2-C-SCoA Malonyl CoA O CH3 O -O-C-CH — C- Methyl malonyl CoA -SCoA Leu Several -SCoA steps CH C ß-Methylcrotonyl CoA carboxylase Biotin H3C "CH3 ATP + C02 ADP + Pi ß-Methylcrotonyl CoA C-SCoA CH O ,C. H3C CH2 'O- ß-Methylglutaconyl CoA Figure 42. Biotin catalyzed carboxylations. VITAMINS 691 blem. Because biotin is found in the yolk, eating whole raw egg will not induce a deficiency. Deficiencies were also caused in patients on total parenteral nutrition (TPN) because biotin was not included in the early formulations. Symptoms include dermatitis, loss of hair color, and central neurological effects. 3.10.5. Hypervitaminosis Biotin None has been reported in humans and there are no tolerable upper intake levels. 3.10.6. Dietary Reference Intakes These have been difficult to determine. There has been some speculation that humans might obtain part of their biotin requirements from the intestinal flora in the colon. The question that has not been adequately answered is whether there is significant absorption of bacterial produced biotin from the colon. AI Infants 5—6 pg/day Children (1-13 years) 8-20ug/day Adolescents (14—18 years) 25ug/day Adults 30 (ig/day Pregnancy 30ug/day Lactation 3 5 ug/day EAR None reported RDA None reported UL None reported 3.11. Folic Acid [70] With all vitamins being essential, it is difficult to state that one vitamin is more important than another. Nevertheless, folic acid, with its coenzyme role in purine biosynthesis, can be considered crucial for some of the cells most fundamental biochemistry, cell division. This vitamin is intimately tied to vitamin Bi2 (co-balamin), which has made estimating its DRIs difficult. Also, conditions that can cause a folic acid deficiency can also result in a vitamin B12 deficiency. 3.11.1. Chemistry The commercial form of the vitamin is folic acid (Fig. 43). It consists of a pteridine ring attached to a p-aminoben-zoic acid that is attached to the a-amine of glutamic acid. Two biosynthetic changes must occur before it is active. First, it must be reduced to tetrahydrofolate by dihydrofolate reductase in a two-step reduction (Fig. 44). Notice that niacin is required for this reduction. Second a polyglutamate chain must be attached to the y-carboxyl of the parent glutamic acid (Fig. 43). The remaining linkages of the polyglutamate chain are traditional a-amino-a-carboxyl peptides. The natural vitamin is made up of a family of polyglutamates all connected to the initial glutamic acid at the previously described y-carboxyl group. The length of this polyglutamate chain varies with the source of the vitamin, but lengths of three, five, and seven amino acids are seen. The most common polyglutamate found in food is 5-methyltetrahy-drofolate polyglutamate (Fig. 43). 3.11.2. Uptake The dietary polyglutamates are cleaved to the monoglutamate vitamin by a y-l-glutamylcarboxy peptidase commonly called conjugase. Folic acid is absorbed as the monoglutamate. Conjugase is found in the brush border of the intestine. Therefore, chronic inflammatory conditions in the intestine lead to low conjugase activity, which can result in significant decreased folic acid absorption. The absorbed vitamin must be converted to the coenzyme form. This requires addingback a five to seven member glutamate chain and commercial folic acid also must undergo the two-step reduction to tetrahydrofolic acid. There are two common abbreviations for the reduced form, FH4 and THF. These reactions apparently occur in a wide variety of tissues. The liver contains about 3—6 months supply of the vitamin, presumably in the polyglutamate form. 3.11.3. Metabolic Roles There are five forms of tetrahydrofolate polyglutamate, all but one of which are coenzymes (Fig. 45). The most highly oxidized is 10-formyl tetrahydrofolate polyglutamate produced by the addition of formic acid to tetrahydroformate polygluta- 692 VITAMINS NT ^CH2 10 OH Pteridine ring O J L CH ß ""CH;, i co2- . p-Aminobenzoate Folic acid (FA) Glutamate H?N OH CH3 5-Methyltetrahydrofolate polyglutamate HoN CH2 ß c- II O O C>AAA H I N-CH CH2 CH2 COO" n = 3, 5, 7 CH2 ^CO^f VCHo Methotrexate Figure 43. Folates and the antifolate, methotrexate. mate. It is the coenzyme for two reactions in purine biosynthesis: (1) the synthesis of for-mylglycine ribotide (FGAR) from glycine amide ribotide (GAR) and (2) the formation of aminoimidazole carboxamide ribotide (AICAR) to formamidoimidazole carboxamide ribotide (FAICAR) (Fig. 46). 5,10-Methenyl tetrahydroformate polyglutamate is the cyclic enamime formed from 10-formyl tetrahydroformate polyglutamate. It also is formed from the catabolism of histidine and can be considered an intermediate between the 10-formyl compound and, upon VITAMINS OH N CH2— NH _____/ \ COO" ■C-N-CH H (CH; 2^2 Folic acid (FA) COO" NADPH + H+ NADP H H Dihydrofolate reductase OH •H *N "CH2— NH coo- 2^2 Dihydrofolate (DHF; FH2) NADPH + H+ NADP Dihydrofolate reductase COO" H H Tetrahydrofolate (THF; FH4) Figure 44. Reduction of folic acid to tetrahydrofolate. COO" 694 VITAMINS Figure 45. Formation of tetrahydrofolate coenzymes polyglutamates. reduction, 5,10-methylene tetrahydroformate polyglutamate. The latter is the coenzyme required for the interconversion of serine and glycine and the methylation of deoxyuridylic acid forming deoxythymidylic acid (Figs 45 and 47). Most of the one-carbon units carried on position 5 or 10 or 5,10 bridge come from serine. The most reduced coenzyme is 5-methyl tetrahydrofolate polyglutamate. It is the source of the methyl group added to homocysteine regenerating methionine and tetrahydrofolate, the latter capable of accepting a one-carbon unit from formate or serine. This last reaction is where folic acid and vitamin B12 come together (Figs 45 and 49). The implications of this reaction and how folic acid can mask pernicious anemia is discussed in the section on vitamin B12 (cyanocobalamin). Note that the formation of 5-methyl-THF H?N 0 -o-P- o- -O-CH OH OH 5-Phosphoribosylglycinamide (GAR) N -Formyl-THF DHF -O-CH OH OH 5-Phosphoformylribosylglycinamide (FGAR) 5'-Phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) N10-Formyl THF DHF NADPH + H+ Dihydrofolate reductase NADP+ f THF Figure 46. 10-Formyl THF coenzyme roles. 5'-Phosphoribosyl-5-formamidoimidazole-4-carboxamide (AICAR) 696 VITAMINS normally is not reversible. Tetrahydrofolate can only be regenerated if there is adequate methyl cobalamin coenzyme. The fifth tetrahydrofolate compound is 5-formyl THF (folinic acid, citrovorum factor). This compound is not a coenzyme, but it can be converted to any of the active coenzyme forms. It is administered following treatment with the dihydrofolate reductase inhibitor, methotrexate (Fig. 43), as a form of rescue therapy. Because it already is in the reduced tetrahydrofolate form, it does not need dihydrofolate reductase to become an active coenzyme. 3.11.4. Folic Acid Deficiency It is obvious that folic acid is a very important vitamin for biosynthetic reactions, particularly those required for the biosynthesis of purines, methy-lation of deoxyuridylic acid, and regeneration of methionine from homocysteine. The main deficiency is a characteristic megaloblastic anemia due to a shortage of nucleotides required for the production of erythrocyte precursor cells. Another clinical sign of folic acid deficiency is neural tube defects including spina bifida and anencephaly. Neural tube defects are one of the main reasons that Federal regulations mandate supplementing cereal grain-based foods with folic acid along with thiamine, riboflavin, and niacin [71-73]. While prenatal multiple vitamins contain adequate amounts of folic acid, pregnant women may not start taking these products until the second or third month of the their pregnancies, and this may be too late. A third indication of inadequate folic acid is elevated blood homocysteine levels. As described in the vitamin B6 section, at one time there was a hypothesis that elevated homocysteine correlated with increased risk of cardiovascular disease. This hypothesis was based on an observation that individuals with increased blood vessel plaque build-up also show increased levels of homocysteine. The elevated homocysteine can be corrected, at least partially, with folate supplements. Figure 35 illustrates why the three vitamins, pyridoxine, folic acid, and vitamin B12, are indicated for elevated levels of homocysteine. Pyridoxal phosphate and cobalamin (B12) are required for the catabolism of homocysteine to succinyl CoA. Methyl cobalamin (methyl B12) is required for the conversion of homocysteine back to methionine. There are reports that plasma homocysteine concentrations are inversely related to cognitive function in older people. While supplements of folic acid, vitamin Bi2, and vitamin B6 did lower homocysteine, there were no significant differences between the vitamin and placebo groups in the scores on tests of cognition [74]. There are many causes of folic acid deficiencies. Inadequate nutrition during periods of increased requirements is one of the main causes of megaloblastic anemia of pregnancy and neural tube defects. Alcoholism is considered the leading cause of folic acid deficiency in the United States [75,76]. There are two ways that excessive alcohol consumption can interfere with folic acid activity: impairment of folic acid reduction to the active THF forms and interference with folic acid storage and release from the liver. A third cause of folic acid deficiency is chronic inflammation of the intestinal mucosa. Inflammation can reduce production of the required conjugase enzyme, which removes the polyglutamate chain, and/or an inflamed mucosa inhibits folate transport. Finally, anticonvulsants such as phenytoin somehow interfere with folic acid uptake or utilization. The dihydrofolate reductase inhibitor, methotrexate (Fig. 43), was developed as an anticancer drug whose inhibition of formation of folic acid coenzymes would block purine synthesis. In other words, it was designed to induce a folic acid deficiency. Notice in Figs 46 and 47 that formation of dTMP, FGAR, and AICAR also causes the oxidation of tetrahydrofolate to dihydrofolate. The latter must be reduced by dihydrofolate reductase to tetrahydrofolate before active coenzyme can form again. Thus, not only does methotrexate inhibit the initial formation of the tetrahydrofolate moiety from folic acid supplements, but it also blocks regeneration of the coenzyme form. 3.11.5. Hypervitaminosis Folic Acid With important exception of masking pernicious anemia characteristic of a vitamin Bi2 deficiency, excessive intake of folic acid has not been considered a problem. Transport across the VITAMINS 697 intestinal mucosa may be regulated by a feedback mechanism or the rate of hydrolysis of the polyglutamate chain or a combination of both. What is very important is that taking the vitamin in doses above 400 u.g (800 u.g in pregnant and lactating women) can mask the macrocytic anemia seen with pernicious anemia. The tolerable upper limit is based on trying to avoid this masking. Therefore, the UL to RDA ratio is low, 2—2.5, in adults. There have been many trials to determine if folic acid supplements can ameliorate certain medical condition. These have not been promising and possibly might exacerbate the patient's disease, particularly if it is a malignancy [77-81]. 3.11.6. Dietary Reference Intakes AI Infants 65-80 (xg/day EAR Children (1—8 years) 120-160 (Xg/day Children (9—13 years) 250(xg/day Adolescents (14—18 years) 330 (Xg/day Adults (19-50 + years) 320(xg/day Pregnancy 520(xg/day Lactation 450 (Xg/day RDA Children (1—8 years) 150-200 (Xg/day Children (9—13 years) 300 (Xg/day Adolescents (14—18 years) 400 (Xg/day Adults (15-50 + years) 400 (Xg/day Pregnancy 600 (Xg/day Lactation 500 (Xg/day UL (from fortified foods or supplements) Children (1—3 years) 300 (Xg/day Children (4—8 years) 400 (Xg/day Children (9—13 years 600 (Xg/day Adolescents (14—18 years) 800 (Xg/day Adults (19 + years) 1,000 (Xg/day Pregnancy (14—18 years) 800 (Xg/day Pregnancy (19+ years) 1,000 (Xg/day Lactation (14—18 years) 800 (Xg/day Lactation (19 + years) 1,000 (Xg/day 3.12. Vitamin B12 (Cobalamin) [82] This chemically very complex vitamin is required for two reactions, the methylation of homocysteine to methionine and rearrangement of methylmalonyl Co A to succinyl Co A. A deficiency leads to pernicious anemia, at one time a disease whose prognosis was death. Because folic acid can mask the blood picture of a cobalamin deficiency, it has become important for physicians to order tests specific for the vitamin, particularly in older patients who begin to show symptoms. 3.12.1. Chemistry The cobalamin family consists of a corrin ring (Fig. 48). It is similar to that of the porphyrin ring system except that there is no methylene or methine bridge between pyrrole rings A and D, and it contains cobalt rather than iron. The commercial form sold in the United States is cyanocobalamin. The hydroxy dosage form has also been used. The two coenzyme forms are methylcobalamin (cytoplasm) and adenosylcobalamin (mitochondria). The commercial vitamin is produced from bacterial fermentation. The di-methylbenzimidazole ring comes from rings A and B of the reduced form of riboflavin (Fig. 28) [83]. 3.12.2. Uptake Uptake of the vitamin from food and vitamin products is complex. Indeed, most deficiencies are not from inadequate diet, but result from defects in the uptake process. Dietary cobalamin requires a fully functioning stomach and ileum of the small intestine [84]. Parietal cells in the stomach produce hydrochloric acid to free the vitamin from the food, R-factor that complex the dietary cobalamins and "intrinsic factor," a glycoprotein. The R-factor—cobalamin complex dissociates in the alkaline intestine, and the now free cobalamin combines with the intrinsic factor that also was produced in the stomach. In the presence of calcium supplied by the pancreas, specific receptors in the ileum in the mucosa take up the intrinsic factor—cobalamin complex. Without intrinsic factor, only about 1% of cobalamins are absorbed. Eventually the vitamin passes into systemic circulation and is transported by a series of plasma binding proteins, the transcobalamins. About 50% of the absorbed vitamin reaches the liver with the remaining transported to other tissues. Note that dietary cobalamins require both R-factor and intrinsic factor. Vitamin B12 supplements only require intrinsic factor. Humans are efficient recyclers of vitamin B12 because of enterohepatic circulation. The vitamin is secreted in the bile. Upon OH H Deoxyuridylic acid N5,N10-Methylene THF DHF OH H Deoxythymidylic acid NADPH + H+ NADP Dihydrofolate reductase THF Figure 47. Methylation of deoxyuridylic acid forming deoxythymidylic acid. VITAMINS 699 H2NOCH2CH2C H2NOCH2C CH2CONH2 CH2CH2CONH2 R = -CN, -OH, -CH3 OH OH Adenosyl Figure 48. Vitamin B12 and coenzyme forms. combining with intrinsic factor, the absorption process is repeated. This recirculation probably explains why dietary deficiencies are uncommon and why the inability to produce intrinsic factor results in vitamin B12 deficiency even though there may be adequate dietary intake. 3.12.3. Biochemical Role Cobalamin is a coenzyme in only two reactions, but they are basic to the health of the individual. Methyl cobalamin is required for the regeneration of methionine from homocysteine (Fig. 35). 5- Methyltetrahydrofolate polyglutamate is also required (Figs 45 and 49). The second reaction is the rearrangement of methylmalonyl CoA to succinyl CoA with adenosylcobalamin as the coenzyme (Fig. 50). Odd numbered fatty acids and the amino acids valine, isoleucine, and methionine produce propionyl CoA as part of their catabolism. Biotin catalyzed carboxyla-tion (Fig. 42) yields d-methylmalonyl CoA, which must be epimerized to the l-stereoi-somer. The latter undergoes a rearrangement to succinyl CoA, which enters the Krebs cycle for final degradation. There has been 700 VITAMINS considerable debate as to the mechanism of this rearrangement, but the general consensus is that there are free radical intermediates [85—87]. 3.12.4. Cobalmin Deficiency Pernicious anemia is the disease associated with vitamin Bi2 deficiency. It is usually caused by the inability to produce intrinsic factor. Deficiencies can also result from total gastrectomy and sometimes resection of the ileum [88]. Indeed, many times the vitamin must be administered by injection although oral megadoses, and up to 1000 u.g are usually tried initially. The blood picture, a megaloblastic anemia, is indistinguishable from that caused by folic acid deficiency. Indeed folic acid supplements can mask the blood picture. This is illustrated in Fig. 49. Removal of adenosylcobalamin eliminates the regeneration of tetrahydrofolate during the methylation of homocysteine to methionine. Folic acid supplements provide a fresh source of tetrahydrofolate coenzymes. DNA synthesis can continue and new erythrocytes form. Excess folic acid also may compete for the available vitamin, further exacerbating vitamin Bi2 deficiency. Pernicious anemia can be lethal if not treated because of nerve damage. There are two explanations for the cause of this damage, both involving an excess of methylmalonyl CoA. Methylmalonyl CoA is a competitive inhibitor of malonyl CoA during fatty acid synthesis. This may impede repair of the myelin sheath surrounding nerves. Alternatively, methylmalonyl CoA replaces malonyl CoA as a substrate in fatty acid synthesis producing fatty acids with methyl substituents. These are incorporated into the lipids components of the myelin sheath producing a nonfunctioning myelin sheath. 3.12.5. Hypervitaminosis B/2 The vitamin is considered nontoxic. There has been some concern that the presence of the CN~ anion in the commercial vitamin might cause problems with megadoses. However, 1000 u.g of cyanocobalamin contains only 0.02 mg of CN. There are no tolerable upper intake levels. dUMP Dietary folates 5,10-CH2-THF FADH2 / K Gly FAD DHF (dihydrofolate) DHF reductase 5-CH3-THF THF [CHa DHF reductase DHF DHF reductase Folic acid (standard monoglutamate dosage form) Homocysteine Met Figure 49. Methyl trap hypothesis. VITAMINS 701 3.12.6. Dietary Reference Intake Levels AI Infants 0.4-0.5(xg/day EAR Children (1-13 years) 0.7-1.5 (xg/day Adolescents (14-18 years) 2.0(xg/day Men and women (19—50+ years) 2(xg/day Pregnancy 2.2 (xg/day Lactation 2.4 (xg/ day RDA Children (1-13 years) 0.9-1.8 (Xg/day Adolescents (14-18 years) 2.4(xg/day Men and women (19—50+ years) 2.4(xg/day Pregnancy 2.6(xg/day Lactation 2.8 (xg/ day UL None reported 3.13. Vitamin C (Ascorbic Acid) [89] Deficiencies of this vitamin have been associated with the early sailors who lacked fresh fruit and vegetables. However, it was a problem on land and was seen in the Irish potato famine, the California gold miners, and territorial prisons. There long has been a mystique \ H3C- / -CH \ ■SCoA Biotin surrounding this vitamin with interest sharply increasing when Linus Pauling published his book "Vitamin C and the Common Cold" forcing the medical, nutritional, and biochemical professions to reexamine carefully the role of this essential nutrient in human health. A significant problem with studying this vitamin is the fact that ascorbic acid is not a vitamin in most animals. It was not until the discovery that guinea pigs also require ascorbate that animal experiments could be conducted. 3.13.1. Chemistry Ascorbic acid is derived from the aldonic acid form of l-gulose (Figs 51 and 52). There are two enolic proton donor groups with the one at position 3 being the most acidic with a pKa 4.1. Ascorbic acid is easily oxidized to the dehydro form without loss of vitamin activity, but the lactone ring now hydrolyzes easily producing inactive open chain product. The triketo representation has been the common way to represent dehydroas-corbic acid (DHA), but in the aqueous in vivo environment of the cell, it most likely is a bicyclic hemiacetal [90]. V / C-SCoA H T H3C-CH CO^ Propionyl CoA D-Methylmalonyl CoA \ CoAS- li r>_ / - fM / \ \ H Adenosyl cobalamin Methylmalonyl-CoA mutase Succinyl CoA Methyl malonyl-CoA epimerase ^ 1 \ / H3C-CH ■SCoA L-Methylmalonyl CoA Figure 50. Vitamin B12 catalyzed mutase reaction. L-2,3-Diketogluonate Figure 51. Ascorbic acid (vitamin C) chemistry. VITAMINS 703 Most animals, except for primates and guinea pigs, produce their own ascorbic acid from glucose (Fig. 52). The pathway follows the standard route to glucuronic acid. The aldose carbon is reduced to an alcohol and, following normal carbohydrate naming convention, the former carbon 6 of d-glucuronic acid becomes carbon 1 of l-gulonic acid. Cyclic l-gulonolactone forms and is oxidized to l-ascorbic acid. Humans and primates lack gulonolactone oxidase. Because humans do not produce ascorbic acid, we have developed an efficient method of recycling it. The glucose transporter on human erythrocytes is altered by another protein, stomatin, such that dehydroascorbate, instead of glucose, is transported into the erythrocyte where it is reduced to ascorbic acid that can return to cells where needed [91]. 3.13.2. Uptake Ascorbic acid is absorbed from the intestine by a sodium-dependent active transport system that is saturable. As the concentration of vitamin C increases in the intestinal tract, the absorption changes to passive diffusion. Once in systemic circulation, there are specific transporters based on cell types. 3.13.3. Metabolic Roles Ascorbic acid is an electron donor required for a variety of oxidative processes. It is readily regenerated by glutathione, NAD, and NADP and, therefore, has a long biological half-life. Currently, there are eight known human enzymes that require ascorbic acid. They are listed in Table 6. The precise metabolic roles have not been completely elucidated, but it appears that in the metalloenzym.es, ascorbate reduces the active metal site. In addition to these specific enzymes, ascorbic acid seems to function as a free radical scavenger in the aqueous phase of plasma and cells. 3.13.4. Ascorbic Acid Deficiency Scurvy is the classical disease associated with ascorbate deficiency. It is a disease of the connective tissue and probably is caused by inadequate cross-linking because there is a lack of hydro-xylated proline and lysine. Many consider scurvy to be an advanced stage of ascorbate deficiency. Chronic deficiencies may also (1) increase risk for malignancies as evidenced by oxidized DNA markers and increased concentrations of reactive oxygen species, (2) decrease immune function as evidenced by less vitamin in neutrophils and lymphocytes, (3) cardiovascular disease caused by the inflammatory response on the blood vessel walls, and (4) cataract formation caused by decreased concentrations of ascorbate in the ocular tissues. Nevertheless, see the discussion in the vitamin E section questioning the effectiveness of large doses of antioxidants. 3.13.5. Hypervitaminosis C The vitamin is considered very safe. At one time, many of the over-the-counter products contained significant amounts of sodium ascorbate, which would be contraindicated in people on low sodium diets. Today's products are virtually Table 6. Metabolic Roles of Ascorbic Acid (Vitamin C) Enzyme Reaction Contribution Dopamine-P-hydroxylase Hydroxylate dopamine Synthesis of norephrine phenethyl chain Peptidyl-glycine Monooxygenase Amidate carboxyl end of peptide Biosynthesis of peptide hormones hormones 4-Hydroxyphenylpyruvatedioxygenase Hydroxylate phenylalanine Synthesis of tyrosine Proline hydroxylase Post-translational Hydroxylation Cross linking of collagen of proline Lysine hydroxylase Post-translational hydroxylation Cross linking of collagen of lysine Trimethyl Lysine Hydroxylase Hydroxylation of trimethyl lysine Carnitine synthesis 4-Butyrobetaine Hydroxylase Oxidation of 4-butyrobetaine Carnitine synthesis aldehyde Cytochrome P450 Isozymes Oxidation of steroids Corticosteroid biosynthsis 6 C02- 6 CHpOH /-° OH OH D-Glucose 6 CO; Several steps Glucuronate 1 reductase OH - HO CH2OH C: CH \ NADPH + H+ NADP+ [2H] ^-1 HO-CH HO-CH HC-OH / \ HO OH Gulonolactone oxidase L-Ascorbic acid Figure 52. Outline of ascorbic acid biosynthesis. HO-CH 1 CH2OH L-Gulonic acid H20 CH2OH Aldono-lactonase HO __-H JO. / ^CH \ CH — CH / \ HO OH i_-Gulonolactone REFERENCES 705 sodium free unless labeled otherwise. Nevertheless, there are intermittent reports of adverse reactions associated with high doses. Therefore, there are tolerable upper intake levels, but these are very high relative to the RDAs. The UL to RDA ratio averages about 20. 3.13.6. Dietary Reference Intakes AI Infants 40-50 mg/day EAR Children (1—8 years) 13-22 mg/day Boys (9—18 years) 39-63 mg/day Girls (9—18 years) 39-56 mg/day Men (19-70 + years) 75 mg/day Women (19-70 + ) 60 mg/day Pregnancy 66-70 mg/day Lactation 96-100 mg/day RDA Children (1—8 years) 15—25 mg/day Boys (9—18 years) 45-75 mg/day Girls (9—18 years) 45—65 mg/day Men (19-70 + years) 90 mg/day Women (19-70 + ) 75 mg/day Pregnancy 80-85 mg/day Lactation 115-120 mg/day UL Infants Not established; use formula and food only Children (1—8 years) 400-650 mg/day Boys and girls (9—13 years) 1200 mg/day Adolescents (14—18 years) 1800 mg/day Adults (19 + years) 2000 mg Pregnancy 1800-2000 mg/day Lactation 1800-2000 mg/day BIBLIOGRAPHY Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D and Fluoride. Food and Nutrition Board, Institute of Medicine, National Academy Press, 1997. 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