Chapter 15. Ascorbic Acid

15 Ascorbic Acid Carol S Johnston, Francene M Steinberg, and Robert B Rucker CONTENTS Introduction and History 490 Chemistry and Food Sources 490 Nomenclature and Structure 490 Physical and Chemical Properties 490 Isolation 492 Chemical and Biological Synthesis 492 Analysis 493 Sources of Ascorbic Acid 494 Biochemical Functions 494 Plants 494 Animals and Animal Models 494 Selected Enzymes and Biochemical Processes 496 Ascorbic Acid and Glutathione Interrelationships 496 Norepinephrine and Adrenal Hormone Synthesis 498 Hormone Activation (a-Amidations) 499 Ascorbic Acid as an Antioxidant 499 Carnitine Biosynthesis 500 Extracellular Matrix and Ascorbic Acid 500 Ascorbic Acid and Gene Expression 502 Ascorbic Acid Metabolism and Regulation 503 Selected Clinical Features Important to Ascorbic Acid Status 504 Defining Ascorbic Acid Status 504 Clinical Features 504 Immune Function 504 Progression of Selected Chronic Diseases 506 Requirements, Allowances, and Upper Limits 508 Rebound Scurvy 508 Oxalic Acid and Uric Acid 508 Iron-Related Disorders 509 Vitamin B12 509 Summary 509 References 510 ß 2006 by Taylor & Francis Group, LLC INTRODUCTION AND HISTORY Ascorbic acid (vitamin C) plays a role as a redox cofactor and catalyst in a broad array of biochemical reactions and processes Vitamin C is designated as ascorbic acid because of its ability to cure and prevent scurvy [1–6] Ascorbic acid comes from the Scandinavian terms, skjoerberg or skorbjugg, and from the English, scarfy or scorby From a historical perspective, it is instructive to visit the original treatise on scurvy by James Lind, Treatise on the Scurvy, published in 1753 [7], although it was over 100 years later that a connection between scurvy and diet was established and an additional 100 years before the first biological and chemical descriptions of ascorbic acid began to appear [1] Scurvy has had a direct influence on all of our lives Scurvy was endemic in many areas throughout seventeenth to nineteenth centuries The military diets of the seventeenth and eighteenth centuries adhered to protocols that promoted scurvy, that is, lack of fruits and vegetables For example, Britain’s general lack of success in earliest explorations of the New World compared with the Spanish and French is an example of how scurvy influenced the historical face of ‘‘New World’’ development Over million sailors are reported to have died of scurvy during the era, often called the ‘‘Age of Sail’’ [8] Indeed, it was not until 1804, that the British Navy adopted the use of lime juice as a part of rations, which resulted in the nickname ‘‘limeys’’ for British sailors [8–10] In the United States, thousands of settlers died from scurvy, particularly in route to the west [11] During the Civil War, poor nutrition, resulting in scurvy (and also pellagra), also took its toll and debatably influenced the outcome of a number of key battles [8–12] An important breakthrough in the understanding of scurvy was the observation that guinea pigs were susceptible to scurvy This observation, reported in 1907 by Holst and Frohlich, was one of the first examples of use of an animal model to study a nutritional disease [13] Eventually, it was demonstrated that primates were also susceptible to scurvy [1] Next, Zilva and his associates isolated antiscorbutic activity from a crude fraction of lemon [4,9,10] Zilva showed that the activity was destroyed by oxidation and protected by reducing agents Important to the evolving nomenclature for vitamins, it was suggested that the new antiscorbutic factor be designated ‘‘factor or vitamin C’’ since ‘‘A’’ and ‘‘B’’ had been previously designated as potential health and growth factors [9] Throughout the 1930s, work progressed rapidly with validation and identification of vitamin C in a number of foods Early papers by Szent-Gyorgyi, Haworth, King, and coworkers document in part this effort as well as chemical identification and elucidation of ascorbic acid’s structure [10] In 1937, both Szent-Gyorgyi and Haworth received Nobel Prizes in medicine and chemistry, respectively, for work related to vitamin C CHEMISTRY AND FOOD SOURCES NOMENCLATURE AND STRUCTURE The IUPAC–IUB Commission on Biochemical Nomenclature changed vitamin C (2-oxo-Ltheo-hexono-4-lactone-2,3-enediol) to ascorbic acid or L-ascorbic acid in 1965 The chemical structures of ascorbic acid are given in Figure 15.1 The molecule has a near planar fivemember ring Ascorbic acid has two chiral centers, which contain four stereoisomers Dehydroascorbic acid, the oxidized form of ascorbic acid retains vitamin C activity and can exist as a hydrated hemiketal Crystalline dehydro-L-ascorbic acid can exist as a dimer [14–16] PHYSICAL AND CHEMICAL PROPERTIES Physical and chemical features of ascorbic acid are summarized in Table 15.1 Data are also available regarding X-ray crystallographic, [1 H] and [13 C]NMR spectroscopic, IR- and ß 2006 by Taylor & Francis Group, LLC OH OH O O HO OH O O −e−−2H+ HO −e− +e−−2H+ HO OH O O HO +e− O• AscH2 Chemical forms of ascorbic acid and major metabolites and degradation products O O O− Asc[• −] DHAsc 2,3-Diketo- L-gulonic acid CO2 L-Xylose + CO2 Oxalic acid + L-Threonic acid CO2 L-Xylonic acid + L-Lyxonic acid FIGURE 15.1 Ascorbic acid and various oxidation products Ascorbic acid can exist in several different forms The two predominant forms and some of their associated oxidation products are shown In solution, ascorbic acid probably exists as the hydrated semiketal AscH2 (reduced ascorbic acid) $ Asc[À] (ascorbate radical) $ DHASC (dehydroascorbic acid) Under basic conditions, cleavage occurs rapidly at carbon-1 or carbon-2 UV-spectroscopic, and mass spectroscopic characteristics [12,17] The most important chemical property of ascorbic acid is the reversible oxidation to semidehydro-L-ascorbic acid and oxidation further to dehydro-L-ascorbic acid [12,14,16] This property is the basis for its known physiological activities In addition, the proton on oxygen-3 is acidic (pK1 ¼ 4:17), which contributes to the acidic nature of ascorbic acid Degradation reactions of L-ascorbic acid in aqueous solutions depend on a number of factors such as pH, temperature, the presence of oxygen, or metals In general, ascorbic acid is TABLE 15.1 Selected Physical Properties of Ascorbic Acid Empirical formula Molar mass Crystalline form Melting point Optical rotation pH, at mg=mL at 50 mg=mL pK1 pK2 Redox potential (dehydroascorbic acid=ascorbate) (ascorbate –, Hþ =ascorbateÀ ) Solubility, g=mL Water Ethanol, abs Ether, chloroform, benzene Absorption spectra at pH at pH 6.4 ß 2006 by Taylor & Francis Group, LLC C6 H8 o6 176.13 Monoclinic, mix of platelets and needles 1908C–1928C [a]25=D þ 20.58 to 21.518 (cm ¼ in water) ~3 ~2 4.17 11.57 À174 mV þ282 mV 0.33 0.02 Insoluble Emax (1%, 10 mm) 695 at 245 nm Emax (1%, 10 mm) 940 at 265 nm not very stable in aqueous media at room temperature Above pH 7.0, alkali-catalyzed degradation results in over 50 compounds, mainly mono-, di-, and tricarboxylic acids [15,18,19] The vitamin can be stabilized in biological samples with trichloroacetic acid or metaphosphoric acid Ascorbic acid is reasonably stable in blood or enteral or intravenous solutions when stored at or below 208C [20–22] As noted, in addition to redox and acid–base properties, ascorbic acid can exist as a free radical [14,16,18,19,23] The ascorbate radical is an important intermediate in reactions involving oxidants and ascorbic acid’s antioxidant activity The physiologically dominate ascorbic acid monoanions and dianions have pKs of 4.1 (pK1 ) and 11.79 (pK2 ), respectively Rate constants for the generation of ascorbate radicals vary considerably, for example, 104À108 sÀ1 When ascorbate radicals are generated by oxyanions, the rate constants are on the order of 104À107 sÀ1, when generated by halide radicals, 106À108 sÀ1, and when generated by tocopherol and flavonoids radicals, 106À108 sÀ1 [14,15] Once formed, the ascorbate radical decays slowly, usually by disproportionation [15,16] Changing ionic strength or pH can influence the rate of dismutation of ascorbic acid (i.e., either increase or decrease) Certain oxyanions, for example, phosphate, accelerate dismutation [16] The acceleration is attributed to the ability of various protonated forms of phosphate to donate a proton efficiently to the ascorbate radical, particularly dimer forms of ascorbate In biological systems, the unusual stability of the ascorbate radical dictates that accessory enzymatic systems be made available to reduce the potential transient accumulation of the ascorbate radical Excess ascorbate radicals may initiate free-radical cascade reactions or nonspecific oxidations In plants, NADH:monodehydroascorbate reductase (EC 1.6.5.4) has evolved to maintain ascorbic acid in its reduced form NADH:monodehydroascorbate reductase plays a major role in stress-related responses in plants In animal tissues, glutathione dehydroascorbate reductase (EC 1.8.5.1) serves this purpose Such enzymes keep vitamin C operating at maximum efficiency, so that other enzyme systems may take advantage of the univalent redox-cycling capacity of ascorbate [12] As an example, without an interaction between dopamine hydroxylase (EC 1.14.17.1) and cytochrome b5 reductase (EC 1.10.2.1), increasing the concentration of ascorbate will scavenge the dopamine radical and replace it with an ascorbate radical Similarly, dopamine can reduce the radical intensity of ascorbate [24,25] Enzymatically coupled reactions reduce the potential of radical accumulation ISOLATION Ascorbic acid is stable in many organic and inorganic acids m-Phosphoric acid–containing ethylenediamine tetraacetic acid (0.5%–2%), oxalic acid, dilute trichloroacetic acid, dilute perchloric acid, or 2,3-dimercaptopropanol are often used as solvents or solutions for tissue extraction [20,26] Extraction of ascorbic acid should be carried out under subdued light and an inert atmosphere to avoid the potential for degradation [26] CHEMICAL AND BIOLOGICAL SYNTHESIS The approach used for ascorbic acid synthesis often depends on the eventual use of the final product [27] For example, strategies for radiochemical labeling of ascorbic acid involve coupling either a C-1 fragment to a C-5 fragment or a C-2 fragment to a C-4 fragment Alternatively, the approach may involve the conversion of the six-carbon form of ascorbic acid or an analog to a suitable radiolabeled derivative Although procedural details are beyond the scope of this chapter, most approaches in making or modifying ascorbic acid involve first derivatizing ascorbic acid [28,29] In this regard, selective derivatization of ascorbic acid can be difficult because of delocation of the negative charge of ascorbate in its anionic form For example, by protecting the C-2 and C-3 hydroxyl groups, alkylation or ß 2006 by Taylor & Francis Group, LLC Reichstein–Grussner synthesis Glucose Hydrogenation Fermentation D-Sorbitol Fermentation L-Sorbose L-Sorbose Acetone addition Diacetone-L-Sorbose Ox/Hydrolysis Fermentation 2-Keto-L-gulonic acid Esterification Methyl-2-keto-L-gulonic acid Lactonization Ascorbic acid L-Sorbosone Enzymatic FIGURE 15.2 Basic steps in the commercial synthesis of ascorbic acid from D-glucose via the Reichstein–Grussner synthesis pathway or fermentation starting with D-glucose or L-sorbitol If ample quantities of sorbosone are produced, ascorbic acid can be generated by the action of sorbosone dehydrogenase acylation can take place at the more sterically accessible primary hydroxyl group on C-6 Reactions at the C-5 position occur only after derivatizations of the C-2, C-3, and C-6 are completed [30] The formation of acetates or ketals of ascorbic acid is useful for protection of the molecule while reactions at the other carbons are carried out [30] The chemical pathway for industrial synthesis of ascorbic acid from glucose is given in Figure 15.2 This process was first developed in the 1930s and is still in use More biological approaches involve the use of a novel enzyme, for example, L-sorbosone dehydrogenase, which directly converts polyalcohols, such as L-sorbosone to L-ascorbic acid and 2-keto-L-gulonic acid [31,32] ANALYSIS Ascorbic acid has strong UV absorption, which is the basis of spectrophotometric methods for the measurement of ascorbic acid (see Table 15.1) Treatment of material to be analyzed with ascorbic acid oxidase is often used as a blank to correct for interfering substances in biological samples A number of high-performance liquid chromatographic methods have now been developed for isolation of ascorbic acid [33–43] Electrochemical detection is also used for measuring ascorbic acid and derivatives in eluates [36] Electrochemical detection allows for the simultaneous measurement of ascorbic and dehydroascorbic acid, isomers, and derivatives Chromatographic approaches include ion exchange, gas, reversed phase, and ion-pairing HPLC chromatographic protocols In direct assays of ascorbic acid in crude mixtures, the 2,20 -dipyridyl calorimetric method is often used, which is based on the reduction of Fe(III) to Fe(II) by ascorbic acid [39] Fe(II) reacts with 2,20 -dipyridyl to form a complex that can be quantified calorimetrically In addition to 2,20 -dipyridyl, ferozine and Folin phenol reagent have also been used Further, ß 2006 by Taylor & Francis Group, LLC methods based on fluorometric and chemiluminescence detection provide highly sensitive approaches for the determination of ascorbic acid [38,40] Enzymatic methods using ascorbate oxidase have the advantage of selectively measuring the biological activity of ascorbic acid [44] Conventional and isotope ratio mass spectrometry techniques have also been used to analyze ascorbic acid Isotope ratio mass spectrometry is particularly useful and sensitive, when 13 C ascorbic acid is available for use as a reference or standard in the analysis of complex matrices [40,43] As a final point, in addition to the problems associated with accurately measuring ascorbic acid, the presence of ascorbic acid may also interfere with many urine and blood chemical tests Examples include the analysis of glucose, uric acid, creatinine, bilirubin, glycohemoglobin, hemoglobin A, cholesterol, triglycerides, leukocytes, and inorganic phosphate [45,46], because as a reductant, ascorbic acid can cause nonspecific color formation SOURCES OF ASCORBIC ACID Ascorbic acid occurs in significant amounts in vegetables, fruits, and animal organs such as liver, kidney, and brain Potatoes and cabbage are also among the important sources of vitamin C Typical values are given in Table 15.2 BIOCHEMICAL FUNCTIONS PLANTS Ascorbic acid is detected in yeast and prokaryotes, except cyanobacteria [12] Ascorbic acid is synthesized in plants from D-glucose and other sugars Ascorbic acid functions in many mono- and dioxygenases to maintain metals in a reduced state For example, mono- and dioxygenases usually contain copper or iron as redox cofactors, respectively As an additional characteristic, dioxygenases require a-ketoglutarate and O2 as cosubstrates in reactions whereas monooxygenases require only O2 The pathway for ascorbic acid synthesis in plants and animals is shown in Figure 15.3 ANIMALS AND ANIMAL MODELS In the kidney of fish, reptiles, and birds, and the liver of mammals, the key enzyme in the synthesis of ascorbic acid is L-gulonolactone oxidase (EC 1.1.3.8; cf Figure 15.3) During the course of evolution, the ability to express L-gulonolactone oxidase functional activity disappeared in the guinea pig, some fruit-eating bats, and most primates, including man [47] Regarding specific steps in the pathway in animals, L-gulonolactone is generated by the direct oxidation of glucose [48,49] In this regard, it may be asked whether the amounts of ascorbic acid synthesized per day in animals with gulonolactone oxidase correspond to the amounts needed in the diets of the guinea pig or primate Grollman and Lehninger [48] used liver homogenates and gulonic acid as substrates for ascorbic acid synthesis They found that the amounts varied from ~0.01 g of L-ascorbic acid synthesized per day per kilogram body weight for the pig to 0.2 g=kg body weight for the rat Linus Pauling in his monograph, Vitamin C and the Common Cold [50], used such data to infer that the ascorbic acid needs in humans were in grams per day range What is ignored is that ascorbic acid production can be no more than the amount of glucose or galactose shunted through the gulonate oxidative pathway In a 70 kg person, this value ranges from to 15 g=day Only ~1% of the gulonate flux is in the direction of ascorbate synthesis [48,51,52] Therefore, given that 5–15 g of glucose and galactose are shunted through the glucuronate and gulonate pathway in humans, ß 2006 by Taylor & Francis Group, LLC TABLE 15.2 Vitamin C in Selected Food Sources Edible Portion (mg=100 g) Sources Animal products Cows milk Human milk Beef Pork Veal Ham Liver, chicken Beef Kidney, chicken Heart, chicken Gizzard, chicken Crab muscle Lobster Scrimp muscle 0.5–2 3–6 1–2 1–2 1–1.5 20–25 15–20 10 6–8 5–7 1–4 2–4 Vegetables Asparagus Avocado Broccoli Beet Beans, various Brussels sprout Cabbage Carrot Cucumber Cauliflower Eggplant Chive Kale Onion Fruits Apple Banana Blackberry Cherry 3–30 8–16 8–10 15–30 Pea Potato Pumpkin Radish Spinach Currant, red Currant, black Grape Grapefruit Kiwi fruit Lemon Melons Mango Orange Pear Pineapple Plums Rose hips Strawberry Tomato 20–50 150–200 2–5 30–70 80–90 40–50 9–60 10–15 30–50 2–5 15–25 2–3 250–800 40–70 10–20l Spices and condiments Chicory Coriander (spice) Garlic Horseradish Lettuce, various Leek Parsley Papaya Pepper, various Edible Portion (mg=100 g) 15–30 10 80–90 6–8 10–15 100–120 30–70 5–10 6–8 50–70 15–20 40–50 70–100 10–15 8–12 4–30 15 25 35–40 33 90 16 45 10–30 200–300 39 150–200 this would amount to ~50–150 mg of ascorbate per day, if humans were capable of making ascorbate This is the same order of magnitude, as the reported need for ascorbic acid in humans [51,53–55] Interestingly, examination of two animal genetic models (1) the gulonolactone oxidase null mouse [56] and (2) the osteogenic disorder Shionogi (ODS) rat [57], in which a missense mutation of L-gulono-g-lactone oxidase causes scurvy-prone disorders, leads to the same conclusion The L-ascorbic acid requirement for normal growth and metabolism for these two animal models is in the order of 300–400 mg L-ascorbic acid=kg of diet [58], that is, about the same as that for the guinea pig, ~200 mg L-ascorbic acid=kg diet for optimal growth Expressed per unit of food energy intake, this amounts to 80–160 mg L-ascorbic acid=1000 kcal (4187 kJ), that is, 150–300 mg=day in ‘‘human terms.’’ Moreover, human milk contains 50 mg L-ascorbic acid=L or 150–250 mg=kg of milk solids The point is that a strong case may be made that for vitamins, ascorbate as a specific example, requirements in ß 2006 by Taylor & Francis Group, LLC D-Glucuronic D-Glucose L-Gulonic acid acid D-Glucose-6-P L-Gulono-g -lactone D-Frucose-6-P CH2OH O CH O HO D-Mannose-6-P L-Gulonolactone oxidase H OH D-Mannose-1-P O L-2-Oxogulono-g -lactone GDP-D-Mannose O CH2OH CH O HO GDP-L-Galactose L-Galactose CH2OH H H OH OH O CH O HO L-Galactose-1,4-lactone H H OH OH L-Ascorbic acid FIGURE 15.3 Cellular pathways for the synthesis of ascorbic acid The direct oxidative pathway for glucose is utilized in animals that make ascorbic acid Gulonolactone oxidase is compromised or absent in animals that cannot make ascorbic acid In plants and bacteria that make L-ascorbic acid (pathway to the left), galactose and mannose, in addition to D-glucose can contribute to ascorbic acid production homeothermic (warm-blooded) animals are similar or are of the same magnitude when expressed relative to units of energy intake SELECTED ENZYMES AND BIOCHEMICAL PROCESSES Ascorbic acid deprivation and scurvy include a range of signs and symptoms that involve defects in specific enzymatic steps and processes (cf Table 15.3) Other examples are summarized in Selected Clinical Features Important to Ascorbic Acid Status Ascorbic Acid and Glutathione Interrelationships Cells deal with excessive oxidants by a number of mechanisms The most important is the utilization of L-g-glutamyl-L-cysteine-glycine (GSH) as a reductant [59–67] GSH is synthesized by a two-step reaction involving g-L-glutamyl cysteine synthetase and GSH synthetase When GSH synthesis is blocked, for example, by use of inhibitors, such as L-buthionine(SR)-sulfoximine, newborn animals die within a few days due to oxidative stress, which can result in proximal renal tubular damage, liver damage, and disruption of lamella bodies in lung [65] The cellular damage involves mostly mitochondrial changes A role for ascorbic acid is depicted in Figure 15.4 Meister and his associates reported that administration of ascorbic acid ameliorates most of the signs of chemically induced GSH deficiency [65] The effect is very pronounced in newborn rats, which not efficiently synthesize ascorbic acid in contrast to adult rats, and guinea pigs When L-buthionine-(SR)-sulfoximine is administered, in addition to the loss in GSH, there is a marked increase in dehydroascorbic acid This has led to the hypothesis that GSH is very important to dehydroascorbic acid reduction and, as a consequence, ascorbic acid recycling Moreover, in studies using guinea pigs, treatment with GSH ester significantly delays the onset of scurvy The sparing effect is probably due to the need for both ascorbic ß 2006 by Taylor & Francis Group, LLC TABLE 15.3 Functions of Ascorbic Acid Associated with Specific Enzymes Function Associated Mechanism and Features Associated Enzymes Extracellular matrix maturation (collagen biosynthesis) Cq1 Complement synthesis Carnitine biosynthesis Pyridine metabolism Cephalosporin synthesis Tyrosine metabolism Norepinephrine biosynthesis Peptidylglycine a-amidation in the activation of hormones Prolyl-3-hydroxylase Prolyl-4-hydroxylase Lysyl hydroxylase Prolyl-4-hydroxylase 6-N-Trimethyl-L-lysine hydroxylase g-Butyrobetaine hydroxylase Pyrimidine deoxyribonucleoside Hydroxylase (fungi) Deacetoxycephalosporin C synthetase Tyrosine-4-hydroxyphenylpyruvate hydrolase Dopamine-b-monooxygenase or hydrolase Peptidylglycine a-amidating monooxygenase Dioxygenase; Fe2þ Dioxygenase; Fe2þ Dioxygenase; Fe2þ Dioxygenase; Fe2þ Dioxygenase; Fe2þ Dioxygenase; Fe2þ Monooxygenase; Cu1þ Monooxygenase; Cu1þ acid and GSH in counteracting the deleterious effects of reactive oxidant species Keys to understanding the steps in the ascorbate–GSH relationships are the enzymes thioredoxin and thioredoxin reductase [68,69] The mammalian thioredoxin reductases are found within a family of selenium-containing pyridine nucleotide–disulfide oxidoreductases They are catalyzed by the NADPH-dependent reduction of thioredoxin, as well as of other endogenous and Ribulose 5-phosphate Pentose shunt NADPH 6PGD 6-Phosphogluconate GSSG GRD NADP AA Diffusible oxidants GRX GSH Diffusible oxidants DHA Reduced oxidants FIGURE 15.4 Interaction between ascorbic acid and glutathione Excess oxidants can be reduced directly and indirectly by ascorbic acid and glutathione by complex processes that are depicted conceptually although in a very simplified fashion The most important reductant in the cell is glutathione (L-g-glutamyl-L-cysteine-glycine, GSH), which is synthesized by a two-step reaction involving L-glutamyl cysteine synthetase and GSH synthetase In addition to reducing equivalents derived from the pentose shunt or hexose monophosphate shunt pathway via NADPH (catalyzed by 6-phosphogluconate dehydrogenase [6-PGD] and transferred by glutathione reductase [GD]), reduced ascorbic acid can transfer reducing equivalents to oxidized glutathione (GSSG) catalyzed by thioredoxin (TRX) and perhaps to some species of diffusible oxidants ß 2006 by Taylor & Francis Group, LLC exogenous compounds The importance of thioredoxin to many aspects of cell function appears in part related to the recycling of ascorbate from its oxidized form [66,67] Although ascorbic acid also has pro-oxidant properties and may cause apoptosis of lymphoid and myeloid cells, Puskas and associates [70–74] have shown that dehydroascorbate, the oxidized form of vitamin C, also stimulates the antioxidant defenses in some cells by preferentially importing dehydroascorbate over ascorbate While 200 -800 mM vitamin C caused apoptosis of Jurkat and H9 human T lymphocytes, pretreatment with 200 -1000 mM dehydroascorbate stimulates the activity of the pentose phosphate pathway enzymes glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase, and transaldolase, and elevates intracellular glutathione levels A 3.3-fold elevation in glutathione was observed after 48 h stimulation with 800 mM dehydroascorbate [73] Norepinephrine and Adrenal Hormone Synthesis Synthesis of norepinephrine (Figure 15.5) depends on ascorbic acid and explains in part the need for a high concentration of ascorbic acid in brain tissue and the adrenal glands Ascorbic acid is a cofactor required both in catecholamine biosynthesis and in adrenal steroidogenesis In studies using animal models with a deletion in the ascorbic acid transporter SVCT2 gene, reduced tissue levels of ascorbic acid occur Animals die soon after birth and there is a significant decrease in tissue catecholamine levels in the adrenals The drop in ascorbic acid is accompanied by decreased plasma levels of corticosterone and altered morphology of mitochondrial membranes, that is, a clear validation of the importance of ascorbic acid on adrenal cortical function [75,76] At the enzymatic level, a primary effect is on dopamine-b-hydroxylase (EC 1.14.17.1), which is present in catecholamine storage granules in nervous tissues and in chromaffin cells of the adrenal medulla This is the site of the final and rate-limiting step in the synthesis of norepinephrine Dopamine-b-hydroxylase is a tetramer containing two Cu(I) ions per monomer, which consumes ascorbate stoichiometrically with O2 during its catalytic cycle At a steady state, the predominant enzyme form is an enzyme–product complex Dopamine hydroxylation HO CH2 CH2 NH2 CH HO CH2 NH2 OH Tyrosine Dopamine-β-hydroxylase Cu and ascorbic acid HO HO Dopamine Norepinephrine Peptidyl α-amidation O H Peptide N H H 2H+, 2e−, O2 O Peptide COOH O + NH H2 COOH Cu2+ Ascorbate + O2 O HO Peptide N H H COOH FIGURE 15.5 Steps in norepinephrine synthesis catalyzed by dopamine-b-hydroxylase (dopamine hydroxylation) and C-terminal amide formation via peptidyl a-amidations (peptidyl a-amidation) Mechanistically, these reactions occur in two steps The enzyme receives single eÀ sequentially from two ascorbic acid molecules resulting in ascorbate free radical as intermediate This intermediate is later reduced back to ascorbate by transmembrane electron transfers via cytochrome b561 ß 2006 by Taylor & Francis Group, LLC T-cell number or T-cell proliferation in assays in vitro [175]; and the decline in lymphocyte proliferation noted in aged populations is not restored by vitamin C in vitro [176] In another metabolic depletion–repletion study, vitamin C ingested daily in amounts ranging from to 251 mg for 92 days also did not affect mitogen-induced lymphocyte proliferation [177] Indeed, it may be that it is the restoration of function after a deficiency rather than response to supplementation that will prove to be the most important factor Moreover, the mechanism is complex and appears to involve mitogen-induced proliferative processes [169,178–180], as well as reduction in or in combination with a reduction in the rate of apoptosis in T cells maintained in culture Vitamin C may also influence other immune system parameters Vitamin C status in guinea pigs is directly related to serum concentrations of the complement component C1q, a protein that, in association with the other complement proteins, mediates nonspecific humoral immunity [181,182] Vitamin C dose-dependently inhibits intracytoplasmic production of the proinflammatory cytokines IL-10 and TNF-a in whole blood cultures [183], possibly by inhibiting NFkB activation [184] Finally, several investigators have suggested that the antihistamine effect of vitamin C may indirectly enhance immune responsiveness, that is, ascorbic acid enhances mitogen-dependent lymphocyte blastogenesis by inhibiting histamine production in spleen cell cultures [185] Progression of Selected Chronic Diseases Atherosclerosis Epidemiologic studies have shown that death due to cardiovascular disease is inversely related to regular use of vitamin C supplements [186–190] Individuals participating in the NHANES II Mortality Study with tissue-saturating concentrations of serum ascorbic acid (!62 mmol=L) were 34% less likely to die from cardiovascular disease [186–190] Males with vitamin C deficiency (plasma vitamin C 45 mmol=L) was associated with a 30%–50% lower risk of stroke [196,201,202] However, the regular use of vitamin C supplements was not associated with reduced risk for stroke [203] suggesting that other factors in fruits and vegetables may be responsible for the beneficial effects of diets rich in vitamin C ß 2006 by Taylor & Francis Group, LLC Cancer In epidemiologic studies, all-cause cancer incidence and deaths appear inversely related to serum vitamin C concentrations in men but not women [185–187] Risk of fatal lung cancer was inversely related to serum vitamin C in both men and women [204] The regular use of vitamin C supplements is related to a reduced risk of gastric and intestinal cancers in large US cohorts [205]; yet, a short-term, intervention trial in a Chinese population at high risk for stomach cancer did not demonstrate a benefit of vitamin C supplementation (120 mg=day) on the incidence of gastric cancer [206–208] The chemopreventive properties of vitamin C may be linked to antioxidant effects that protect against oxidative DNA damage, or to protective effects against carcinogenic mechanisms that disrupt the cell cycle [209,210] Although the pro-oxidant effects of ascorbic acid in vitro suggest a possible role for vitamin C in mutagenesis, investigations utilizing physiologically relevant cell culture systems demonstrate that vitamin C is most often associated with decreased frequency of DNA mutations Cataracts Epidemiologic studies have also shown that the risk of cataract is significantly higher in individuals with moderate to low blood concentrations of vitamin C [211–213] After controlling for potentially confounding variables, including diabetes, smoking, sunlight exposure, and regular aspirin use, taking vitamin C supplements for ~10 years was associated with reduced risk for early (odds ratio, 0.23; 95% CI, 0.09–0.60) and moderate (odds ratio, 0.17; 95% CI, 0.03–0.87) age-related lens opacities in women In a separate study, high dietary vitamin C (>200 mg=day) was associated with a 30% reduction in cataract risk, and serum ascorbic acid concentrations >49 mmol=L were associated with a 60%–70% reduction in all types of cataract [211] Interestingly, a high-quality diet rich in fruits, vegetables, and whole grains is associated with a reduced prevalence of cataract in nonusers of vitamin C supplements but not in users of vitamin C supplements, suggesting an important role for vitamin C in cataract prevention [211–214] Pulmonary Function In large population-based studies, vitamin C is associated with forced expiratory volume (FEV1 ) and forced vital capacity, the common functional markers for pulmonary function [215,216] Vitamin C appears to decrease oxidant damage to the lung [210,217] and may modulate the development of chronic lung diseases and declines in lung function A prospective study of diet and lung function demonstrated that an additional 100 mg higher than average intake of vitamin C was related to a significantly smaller reduction in FEV1 after a year follow-up [218] Dietary vitamin C is inversely related to self-reported respiratory symptoms (morning cough, chronic cough, and wheezing) in adults, a relationship that tends to be related to smoking status [219,220] Hence, smokers, as well as populations with chronic exposure to air pollutants [217,219,221], benefit from optimizing intakes of vitamin C Bone Density Ascorbic acid increases collagen accumulation and alkaline phosphatase activity in osteogenic cells thereby affecting bone formation Abnormal bone development and fractures are noted in scurvy, yet the role of vitamin C in protecting against age-related bone loss is less clear Vitamin C from the diet is not consistently associated with bone mineral density in postmenopausal women [222,223], but dietary vitamin C, as well as fruit and vegetable consumption, is protective against bone loss in young and early menopausal women [222,223] Long-term vitamin C supplementation (400–750 mg=day for >10 year), however, is positively associated with bone mineral density in postmenopausal women [222–226] In a ß 2006 by Taylor & Francis Group, LLC population at risk for hip fracture (female smokers aged 40–76 years), low dietary vitamin C was associated with a threefold increased risk for hip fracture as compared with nonsmoking women; smokers consuming adequate dietary vitamin C (>67 mg daily) were not at an increased risk of hip fracture [226] Wound Healing and Connective Tissue Metabolism As discussed, the mechanisms that link ascorbic acid intake to connective regulation and deposition are related to its role as an enzymatic cofactor (and stabilizing factor) for prolyl and lysyl hydroxylases REQUIREMENTS, ALLOWANCES, AND UPPER LIMITS The 2000 RDA for vitamin C, 75 mg daily for adult females and 90 mg daily for adult males, represents a 25%–50% increase over the 1989 RDA, 60 mg [227] Plasma vitamin C concentrations range from 45 to 50 mmol=L in well-nourished individuals with a typical vitamin C intake of 90–100 mg dietary vitamin C daily [227] Plasma vitamin C concentrations in people who regularly consume vitamin C supplements are 30%–70% higher, ranging from 50 to 60 mmol=L to 75–80 mmol=L in individuals who regularly supplement 100 mg to 500–1000 mg vitamin C [227–229], respectively Plasma vitamin C concentrations in newborn infants are much higher, ~150 mmol=L [230–234] In humans, vitamin C bioavailability is nearly 100% for single oral doses 200 mg but falls to ~75% at an oral dose of 500 mg and to ~50% at an oral dose of 1250 mg [57] At the higher oral doses, 500 and 1250 mg, nearly 100% of the absorbed dose is excreted nonmetabolized in urine; thus, effective homeostatic mechanisms operate to control plasma vitamin C concentrations over wide ranges of intake REBOUND SCURVY There is some evidence that accelerated metabolism or disposal of ascorbic acid may occur after prolonged supplementation of high doses Presumably, when vitamin C supplementation ceases abruptly, the accelerated disposal of vitamin C creates a vitamin C-deficient state, that is, ‘‘rebound scurvy.’’ The phenomenon seems to have some support from animal studies [12] and historical records [10] However, there are concerns regarding rebound scurvy that come largely from work by Cochrane [233] Of 42 cases of infantile scurvy at Children’s Hospital in Halifax, Nova Scotia (from October 1959 to January 1961), only two could not be attributed to inadequate dietary vitamin C The possibility of rebound scurvy was considered, because the mothers of both of the infants reported taking vitamin C supplements during pregnancy (400 mg daily) However, the regression plots of plasma ascorbic acid depletion during withdraw from low-dose vitamin C (60 mg=day for weeks) and from high-dose vitamin C (600 mg=day for weeks) display similar slopes, indicating similar rates of vitamin C metabolism and disposal independent of initial vitamin C status In guinea pigs, high intakes of vitamin C upregulate enzymes important to ascorbic acid degradation [12] Whether this is truly the case in humans and in the appropriate setting lead to as suggested by the observations of Cochrane [233] needs further clarification and validation OXALIC ACID AND URIC ACID About 75% of kidney stones contain calcium oxalate; another 5%–10% is composed of uric acid High doses of vitamin C have been shown to increase urinary excretion of both oxalic acid and uric acid; and thus, theoretically promote the formation of kidney stones [235–238] ß 2006 by Taylor & Francis Group, LLC Case reports have described the development of hyperoxaluria with associated pathologies (tubular necrosis and hematuria) in previously healthy individuals consuming 5–8 g vitamin C daily [235–237] These individuals appeared to possess abnormally high capacities to absorb dietary ascorbic acid and to convert ascorbic acid to oxalate Yet clinical investigations indicate that calcium oxalate stone-formers and normal subjects respond similarly to megadoses of vitamin C [238,239] The crystallization of calcium oxalate in urine was increased ~60% in both stone-forming patients and healthy subjects consuming g vitamin C for days [239] A prospective cohort study of 45,600 men without a history of nephrolithiasis demonstrated a significant association between incident kidney stones and vitamin C ingestion after a 14 year follow-up [240,241] The multivariate risk ratio in men who consumed !1000 mg daily as compared with