Chapter 15. Ascorbic Acid

500 Ascorbic Acid and Gene Expression.... AscH2 reduced ascorbic acid $Asc[] ascorbate radical $ DHASC dehydroascorbic acid.. M ore biologi cal ap proaches involv e the us e of anovel en

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

INTRODUCTION AND HISTORY

Ascor bic acid (vitami n C) plays a role as a redox cofactor and catalyst in a broad array ofbioche mical react ions an d pro cesses Vi tamin C is designa ted as ascorbi c acid because of itsability to cure and preven t scurvy [1–6] Ascor bic acid co mes from the Scandinavi an terms,skjoer berg or skorbjug g, and from the Engl ish, scarf y or scorby Fro m a histo rical perspec-tive , it is instructi ve to visit the original treatis e on scurvy by Ja mes Lind, Treatise on theScu rvy , publis hed in 175 3 [7], althoug h it was over 100 years late r that a con nection be tweenscurvy and diet was establis hed and an additio nal 100 years before the first biological andchemi cal de scriptions of ascorb ic acid began to ap pear [1]

Sc urvy has had a direct influe nce on all of our lives Scurvy was endemic in many areasthrough out sevent eenth to ninete enth centuri es The milita ry diets of the sevent eenth andeight eenth ce nturies adhered to protocol s that pr omoted scurvy, that is, lack of frui ts andvegeta bles For exampl e, Britain’s general lack of success in earliest explora tions of the New

Wo rld comp ared with the Spa nish and French is an exampl e of how scurvy infl uenced thehist orical face of ‘‘New World’ ’ developm ent Over 2 milli on sailo rs are report ed to ha ve died

of scurvy dur ing the era, often call ed the ‘‘Age of Sail’ ’ [8] In deed, it was not until 1804, thatthe British Navy a dopted the use of lime juice as a part of rations, which resulted in thenickna me ‘‘limeys’’ for British sail ors [8–10] In the United States, thousan ds of settler s diedfrom scurvy, pa rticular ly in rou te to the west [11] Durin g the Civil War, poor nutri tion,resul ting in scurvy (and also pe llagra), also took its toll and debatabl y influen ced the outcome

of a number of key battl es [8–12]

An impor tant bre akthrough in the underst anding of scurvy was the observation thatguinea pigs wer e suscept ible to scurvy This observat ion, report ed in 1907 by Hols t an dFro hlich, was one of the first exampl es of use of an anima l model to study a nutri tionaldiseas e [13] Eventual ly, it was demon strated that primates were also suscept ible to scurvy [1].Next , Zilva an d his associ ates isol ated antis corbutic activit y from a crude fraction of lemon[4,9 ,10] Zilva showe d that the acti vity was destro yed by ox idation and pro tected by reducingagents Importan t to the evo lving nome nclature for vitamins , it was suggested that the newantis corbuti c factor be de signated ‘‘factor or vitamin C’’ since ‘‘A’’ an d ‘‘B’’ had beenprevious ly designa ted as potenti al healt h and grow th fact ors [9]

Thr ougho ut the 1930s, work pr ogressed rapidl y with validati on an d identi fication

of vita min C in a number of foods Early papers by Szent- Gyorgy i, Haw orth, King,and cowor kers docu ment in part this effort as wel l as chemi cal identifi cation and eluci dation

of ascorbi c acid’s struc ture [10] In 1937, both Szen t-Gyorgyi and Hawo rth recei ved NobelPrizes in medic ine and chemi stry, respectivel y, for work relat ed to vita min C

CHEMISTRY AND FOOD SOURCES

NOMENCLATURE AND STRUCTURE

The IUPA C–IUB Commis sion on Biochemi cal Nomencl ature changed vitamin C (2-oxo -Ltheo-he xon o-4-lact one-2, 3-enedio l) to asco rbic acid or L-ascor bic acid in 1965 The c hemicalstruc tures of ascorbi c acid are given in Figure 15.1 The molec ule has a ne ar planar five-member ring Ascor bic acid has two chiral cen ters, which con tain four stere oisomers.Dehyd roascor bic acid, the oxidiz ed form of ascorbi c acid retai ns vita min C activit y and canexist as a hydrate d hemiket al Cry stalline de hydro- L-ascor bic ac id can exist as a dimer [14–16 ]

-PHYSICAL ANDCHEMICALPROPERTIES

Phys ical and chemi cal featu res of ascorb ic acid are summ arized in Table 15.1 Data arealso available regarding X-ray crystallographic, [1H] and [13C]NMR spectroscopic, IR- and

UV-spectroscopic, and mass spectroscopic characteristics [12,17] The most important ical property of ascorbic acid is the reversible oxidation to semidehydro-L-ascorbic acid andoxidation further to dehydro-L-ascorbic acid [12,14,16] This property is the basis for itsknown physiological activities In addition, the proton on oxygen-3 is acidic (pK1¼ 4:17),which contributes to the acidic nature of ascorbic acid.

chem-Degradation reactions of L-ascorbic acid in aqueous solutions depend on a number offactors such as pH, temperature, the presence of oxygen, or metals In general, ascorbic acid is

O O DHAsc

2,3-Diketo- L -gulonic acid

CO2Oxalic acid

+

L -Threonic acid L -Xylonic acid + L -Lyxonic acid

HO O

OH

O

O −

O • Asc[ • −]

CO2

L -Xylose + CO2

FIGURE 15.1 Ascorbic acid and various oxidation products Ascorbic acid can exist in several differentforms The two predominant forms and some of their associated oxidation products are shown Insolution, ascorbic acid probably exists as the hydrated semiketal AscH2 (reduced ascorbic acid) $Asc[] (ascorbate radical) $ DHASC (dehydroascorbic acid) Under basic conditions, cleavage occursrapidly at carbon-1 or carbon-2

not very stable in aqueous media at room temperature Above pH 7.0, alkali-catalyzeddegradation 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 ormetaphosphoric acid Ascorbic acid is reasonably stable in blood or enteral or intravenoussolutions when stored at or below 208C [20–22].

As noted, in addition to redox and acid–base properties, ascorbic acid can exist as a freeradical [14,16,18,19,23] The ascorbate radical is an important intermediate in reactionsinvolving oxidants and ascorbic acid’s antioxidant activity The physiologically dominateascorbic 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,

104108

s1 When ascorbate radicals are generated by oxyanions, the rate constants are onthe order of 104107

s1, when generated by halide radicals, 106108

s1, and when generated

by tocopherol and flavonoids radicals, 106108 s1 [14,15] Once formed, the ascorbateradical decays slowly, usually by disproportionation [15,16] Changing ionic strength or pHcan influence the rate of dismutation of ascorbic acid (i.e., either increase or decrease).Certain oxyanions, for example, phosphate, accelerate dismutation [16] The acceleration isattributed to the ability of various protonated forms of phosphate to donate a protonefficiently to the ascorbate radical, particularly dimer forms of ascorbate

In biological systems, the unusual stability of the ascorbate radical dictates that accessoryenzymatic systems be made available to reduce the potential transient accumulation of theascorbate radical Excess ascorbate radicals may initiate free-radical cascade reactions ornonspecific oxidations In plants, NADH:monodehydroascorbate reductase (EC 1.6.5.4) hasevolved to maintain ascorbic acid in its reduced form NADH:monodehydroascorbate reduc-tase plays a major role in stress-related responses in plants In animal tissues, glutathionedehydroascorbate reductase (EC 1.8.5.1) serves this purpose Such enzymes keep vitamin Coperating at maximum efficiency, so that other enzyme systems may take advantage of theunivalent redox-cycling capacity of ascorbate [12] As an example, without an interactionbetween 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 itwith 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–containingethylenediamine tetraacetic acid (0.5%–2%), oxalic acid, dilute trichloroacetic acid, diluteperchloric acid, or 2,3-dimercaptopropanol are often used as solvents or solutions for tissueextraction [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 BIOLOGICALSYNTHESIS

The approach used for ascorbic acid synthesis often depends on the eventual use of the finalproduct [27] For example, strategies for radiochemical labeling of ascorbic acid involvecoupling 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 ascorbicacid or an analog to a suitable radiolabeled derivative Although procedural details arebeyond the scope of this chapter, most approaches in making or modifying ascorbic acidinvolve first derivatizing ascorbic acid [28,29] In this regard, selective derivatization ofascorbic acid can be difficult because of delocation of the negative charge of ascorbate inits anionic form For example, by protecting the C-2 and C-3 hydroxyl groups, alkylation or

acylation can take place at the more sterically access ible primary hydroxyl group on C-6.Reaction s at the C- 5 pos ition occur only after deriva tizations of the C-2, C-3, and C-6 arecomplet ed [30] The formati on of acetates or ketals of ascorbic acid is useful for protect ion ofthe molecule while reaction s at the other carbons are carried out [30] The chemi cal pathw ayfor indust rial syn thesis of ascorb ic acid from glucose is given in Fig ure 15.2 This process wasfirst developed in the 1930s and is sti ll in use M ore biologi cal ap proaches involv e the us e of anovel en zyme, for examp le, L-sorbos one dehyd rogenase , which directly conve rts polyal coho ls,such as L-sorbos one to L-ascor bic acid a nd 2-keto- L-guloni c acid [31, 32].

A NALYSIS

Ascor bic acid has strong UV absorpt ion, which is the basis of spectr ophot ometric methodsfor the measur ement of ascorbic acid (see Table 15.1) Treatm ent of mate rial to be analyze dwith ascorbi c acid ox idase is often us ed as a blank to correct for interfer ing substa nces inbiologi cal sampl es A num ber of high-pe rforman ce liquid chro matograp hic method s havenow been developed for isolatio n of ascorbi c acid [33–43 ]

Electr ochemic al detect ion is also used for measur ing ascorbi c acid an d deriva tives ineluat es [36] Electro chemical detection allows for the sim ultaneo us measur ement of ascorbi cand dehydro ascorbic acid, isom ers, an d de rivative s Chromatogr aph ic approache s includeion exchange, gas, revers ed phase, an d ion-pairing HPLC chromat ographic protocol s

In direct assays of ascorbi c acid in crude mixt ures, the 2,2 0 -dipyr idyl calorimet ric method

is often used, which is ba sed on the redu ction of Fe(III ) to Fe(II) by ascorbi c acid [39] Fe(II)react s with 2,2 0 -dipyr idyl to form a complex that can be qua ntified calorimet rically

In addition to 2,2 0 -dipyr idyl, ferozine an d Fol in phe nol reagent ha ve also been used Further,

methods based on fluorometr ic and chemi luminescen ce de tection provide highly sensi tiveapp roaches for the determinat ion of asco rbic acid [38,40].

Enzy matic methods using ascorbat e ox idase have the a dvantage of selec tively measur ingthe biologi cal acti vity of ascorbi c acid [44] Convent ional and isoto pe ratio mass spectro metrytechni ques ha ve also been used to analyze ascorb ic acid Isotope rati o mass spectr ometry isparti cularly useful and sensitiv e, when 13 C ascorb ic acid is avail able for use as a refer ence orstandar d in the analys is of complex matrices [40,43]

As a final point, in addition to the prob lems associ ated with accurat ely measur ing ascorbi cacid, the presence of ascorbic acid may also interfer e wi th many urine and blood chemi cal test s.Exa mples include the analys is of glucose , uric acid, creatinine, bili rubin, glycohem oglobin,hemog lobin A, cholest erol, triglyce rides , leuko cytes, and inorgan ic phos phate [45,46], because

as a reducta nt, ascorbic acid can ca use nonspecif ic co lor form ation

S OURCES OF A SCORBIC A CID

Ascor bic acid occurs in signi ficant amounts in vegeta bles, fruits, and anima l orga ns such aslive r, kidney, an d brain Potatoes and cabbage are also among the impor tant so urces ofvita min C Typical values are given in Tabl e 15.2

BIOCHEMICAL FUNCTIONS

P LANTS

Ascor bic acid is de tected in yeast and prokaryot es, except cyan obacter ia [12] Ascor bic acid issynthes ized in plants from D-gluc ose and other sugars Ascor bic acid function s in man ymono - and dioxygena ses to maintain meta ls in a reduced state For exampl e, mono- anddioxygen ases usuall y contai n copper or iron a s redox cofacto rs, respect ivel y As an a dditionalcharact eristic , dioxygen ases requir e a-ketog lutar ate and O2 as cosubst rates in react ionswher eas mon ooxygenases require only O2 The pathway for ascorbi c acid synthes is in plan tsand an imals is shown in Figure 15.3

A NIMALS AND ANIMAL MODELS

In the kidney of fish, reptiles, an d birds, and the live r of mamm als, the key enzyme inthe 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 functionalactivity disappeared in the guinea pig, some fruit-eating bats, and most primates, includingman [47]

Regarding specific steps in the pathway in animals,L-gulonolactone is generated by thedirect oxidation of glucose [48,49] In this regard, it may be asked whether the amounts ofascorbic acid synthesized per day in animals with gulonolactone oxidase correspond to theamounts needed in the diets of the guinea pig or primate Grollman and Lehninger [48] usedliver homogenates and gulonic acid as substrates for ascorbic acid synthesis They found thatthe amounts varied from ~0.01 g ofL-ascorbic acid synthesized per day per kilogram bodyweight 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 inhumans 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 oxidativepathway In a 70 kg person, this value ranges from 5 to 15 g=day Only ~1% of the gulonateflux is in the direction of ascorbate synthesis [48,51,52] Therefore, given that 5–15 g ofglucose and galactose are shunted through the glucuronate and gulonate pathway in humans,

this would amount to ~50–150 mg of ascorbate per day, if humans were capable of makingascorbate This is the same order of magnitude, as the reported need for ascorbic acid inhumans [51,53–55].

Interestingly, examination of two animal genetic models (1) the gulonolactone dase null mouse [56] and (2) the osteogenic disorder Shionogi (ODS) rat [57], in which amissense mutation ofL-gulono-g-lactone oxidase causes scurvy-prone disorders, leads to thesame conclusion The L-ascorbic acid requirement for normal growth and metabolism forthese two animal models is in the order of 300–400 mgL-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 optimalgrowth Expressed per unit of food energy intake, this amounts to 80–160 mg L-ascorbicacid=1000 kcal (4187 kJ), that is, 150–300 mg=day in ‘‘human terms.’’ Moreover, human milkcontains 50 mgL-ascorbic acid=L or 150–250 mg=kg of milk solids The point is that a strongcase may be made that for vitamins, ascorbate as a specific example, requirements in

oxi-TABLE 15.2

Vitamin C in Selected Food

homeo thermic (warm- blooded) anima ls are similar or are of the same magni tude whenexpress ed relative to units of en ergy intake

S ELECTED E NZYMES AND B IOCHEMICAL PROCESSES

Ascor bic acid de privation and scurvy includ e a range of signs and sympt oms that involv edefect s in specific enzymat ic steps a nd process es (cf Tabl e 15.3) Othe r e xamples are sum-mari zed in Select ed Clinic al Fea tures Import ant to Ascor bic Aci d Status

Asco rbic Acid and Glutat hione Interrelati onships

Cells deal with excess ive oxidan ts by a numb er of mechani sms The most impor tant is theutilizat ion of L- g-glut amyl- L-cysteine- glycin e (GSH ) a s a reducta nt [59–67 ] GSH is synthe-sized by a two-step react ion involv ing g -L-glut amyl cyste ine synthet ase and GSH syntheta se

Wh en GSH synthes is is blocked, for exampl e, by use of inhibi tors, such as L-buthi ( SR )-sulfox imine, ne wborn anima ls die within a few da ys due to oxidat ive stre ss, which canresul t in pr oximal renal tubular da mage, liver damage, and disrupt ion of lamella bodi es inlung [65] The cellular damage involves mostly mito chondrial changes A role for ascorbi cacid is dep icted in Fi gure 15.4

onine-M eister and his associ ates reported that admini stration of ascorbic acid amel iorates mo st

of the signs of chemica lly ind uced GSH defic iency [65] The effect is very pro nounced innewbor n rats , whi ch do not efficie ntly synthes ize ascorbi c acid in contras t to adult rats, andguinea pigs When L-buthi onine-( SR )-su lfoximi ne is admini stered , in additi on to the loss inGSH, there is a marked increa se in dehydroasco rbic acid This has led to the hypothesi s thatGSH is ve ry impor tant to dehyd roascor bic acid redu ction and, as a conseq uence, ascorbi cacid recycling M oreover, in studies using guinea pigs, treatment wi th GSH ester significan tlydelays the onset of scurvy The sparing effect is probab ly due to the need for both ascorbi c

CH HO

CH2OH CH HO

H OH

H OH

H OH

H O O

O OH

H O

OH

O

O O

FIGURE 15.3 Cellular pathways for the synthesis of ascorbic acid The direct oxidative pathway forglucose 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 makeL-ascorbic acid (pathway tothe left), galactose and mannose, in addition toD-glucose can contribute to ascorbic acid production

acid and GSH in counteracting the deleterious effects of reactive oxidant species Keys tounderstanding the steps in the ascorbate–GSH relationships are the enzymes thioredoxin andthioredoxin reductase [68,69] The mammalian thioredoxin reductases are found within afamily of selenium-containing pyridine nucleotide–disulfide oxidoreductases They are catalyzed

by the NADPH-dependent reduction of thioredoxin, as well as of other endogenous and

TABLE 15.3

Functions of Ascorbic Acid Associated with Specific Enzymes

Associated Mechanism and Features

maturation (collagen biosynthesis) Prolyl-4-hydroxylase

Lysyl hydroxylase

Carnitine biosynthesis 6-N-Trimethyl- L -lysine hydroxylase Dioxygenase; Fe 2þ

g-Butyrobetaine hydroxylase Pyridine metabolism Pyrimidine deoxyribonucleoside Dioxygenase; Fe 2þ

Hydroxylase (fungi) Cephalosporin synthesis Deacetoxycephalosporin C synthetase Dioxygenase; Fe 2þ

Tyrosine metabolism Tyrosine-4-hydroxyphenylpyruvate hydrolase Dioxygenase; Fe 2þ

Norepinephrine biosynthesis Dopamine-b-monooxygenase or hydrolase Monooxygenase; Cu1þPeptidylglycine a-amidation in

the activation of hormones

Peptidylglycine a-amidating monooxygenase Monooxygenase; Cu1þ

Ribulose

Diffusible oxidants

Reduced oxidants

Diffusible oxidants

DHA GSH

NADP 6-Phosphogluconate

GRX GRD

6PGD Pentose

shunt

FIGURE 15.4 Interaction between ascorbic acid and glutathione Excess oxidants can be reduceddirectly and indirectly by ascorbic acid and glutathione by complex processes that are depicted con-ceptually 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 involvingL-glutamylcysteine synthetase and GSH synthetase In addition to reducing equivalents derived from the pentoseshunt or hexose monophosphate shunt pathway via NADPH (catalyzed by 6-phosphogluconatedehydrogenase [6-PGD] and transferred by glutathione reductase [GD]), reduced ascorbic acid cantransfer reducing equivalents to oxidized glutathione (GSSG) catalyzed by thioredoxin (TRX) andperhaps to some species of diffusible oxidants

exogenous compounds The importance of thioredoxin to many aspects of cell functionappears 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 oflymphoid and myeloid cells, Puskas and associates [70–74] have shown that dehydroascor-bate, the oxidized form of vitamin C, also stimulates the antioxidant defenses in some cells bypreferentially importing dehydroascorbate over ascorbate While 200 -800 mM vitamin Ccaused apoptosis of Jurkat and H9 human T lymphocytes, pretreatment with 200 -1000 mMdehydroascorbate stimulates the activity of the pentose phosphate pathway enzymes glucose6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase, and transaldolase,and elevates intracellular glutathione levels A 3.3-fold elevation in glutathione was observedafter 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 theneed for a high concentration of ascorbic acid in brain tissue and the adrenal glands Ascorbicacid 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 asignificant decrease in tissue catecholamine levels in the adrenals The drop in ascorbic acid isaccompanied by decreased plasma levels of corticosterone and altered morphology of mito-chondrial membranes, that is, a clear validation of the importance of ascorbic acid on adrenalcortical 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 inchromaffin cells of the adrenal medulla This is the site of the final and rate-limiting step in thesynthesis of norepinephrine Dopamine-b-hydroxylase is a tetramer containing two Cu(I)ions per monomer, which consumes ascorbate stoichiometrically with O2 during its catalyticcycle At a steady state, the predominant enzyme form is an enzyme–product complex

+ Peptide COOH

N

O O

The pr imary functio n of ascorbate is to maintain copp er in a redu ced state in this complex Only the reduced en zyme seems to be catalyti cally comp etent, with bound cup rous ions as theonly reservoir of reducing equival ents Un der acute stress, ascorbi c acid levels in neural tissueare also rapidl y depleted [12, 77,78].

Hormon e Act ivation ( a -Amidatio ns)

Many hormones and hor mone-r eleasing fact ors are a ctivated by posttran slational stepsinvolv ing a-amidati ons [12] Exa mples of hor mone activati on include mela notropi ns, calci-tonin, relea sing facto rs for grow th hormone, corticotrophi n and thyrotrop in, pro-ACTH ,vasopres sin, ox ytocin , ch olecystokini n, and ga strin [79,80] Peptidyl glycine a -amidatin gmonoo xygenase (EC 1.14.17.3 ), the enzyme that carries out a-amidati on, is found in secre-tory granule s of neuroend ocrine cells in the brain, pitui tary, thyroi d, an d sub maxilla ry glands[81–83] For peptides that undergo amidation, a glycine must be at the C terminus Theprocess involv es C- terminal amida tion with the relea se of glyoxyl ate (Figur e 15.5) Similar todopamine-b-hydroxylase, in the ascorbate-dependent a-amidases, ascorbic acid serves as areductant to maintain copper in a reduced state at the active site of the enzyme

Ascorbic Acid as an Antioxidant

The Food and Nutrition Board’s panel on Dietary Antioxidant and Related Compounds ofthe NAS has defined an antioxidant as ‘‘any substance that, when present at low concentra-tions compared to those of an oxidizable substrate (e.g proteins, lipids, carbohydrates andnucleic acids), significantly delays or prevents oxidation of that substrate’’ [84] Ascorbic acidreadily scavenges reactive oxygen and nitrogen species, such as superoxide and hydroperoxylradicals, aqueous peroxyl radicals, singlet oxygen, ozone, peroxynitrite, nitrogen dioxide,nitroxide radicals, and hypochlorous acid Excesses of such products have been associatedwith lipid, DNA, and protein oxidation

Lipids

Although mostly inferential, numerous in vitro and in vivo studies have focused on the ability

of ascorbic acid to reverse lipid peroxidation [85,86] When the peroxyl radicals are generated

in plasma, vitamin C is consumed faster than other antioxidants, for example, uric acid,bilirubins, and vitamin E [86] Ascorbic acid is 103more reactive than a polyunsaturated fattyacid in reacting with peroxyl radicals In contrast, ascorbic acid is not as effective inscavenging hydroxyl or alkoxyl radicals [86]

In assays for lipid peroxidation, low-density lipoprotein (LDL) particles are often used asthe lipid source [85,87,88] LDL oxidation susceptibility is estimated by the lag time andpropagation rate of lipid peroxidation in LDL exposed to copper ions or catalyst to initiateoxidation Many studies in humans and animals support the observation that ascorbic acidgenerally retards peroxyl radical formation in LDL Studies have been carried out withsmokers, nonsmokers, and hypercholesterolemic subjects [89] In all cases, with the combin-ation of ascorbic acid and vitamin E, a significant reduction in oxidized LDL has beenreported [88] In studies using only vitamin C, however, the effect on LDL oxidation ismore varied

DNA

Oxidative damage to DNA is of particular importance in somatic cells, because of the risk

of mutations, which can lead to cancer or birth defects In addition to protection againstlipid peroxidative damage, ascorbic acid has also been shown to provide a degree ofprotection with respect to DNA oxidation In DNA, 8-oxoguanosine and its respective

nuc leoside 8-oxo -deoxygua nosine are the pro ducts [90–92] These mo dified nuc leic acid basesare found in all cell s, an d are excret ed in urine Estimati on of 8-oxo- deoxygu anosine deriva tivesand other modified bases has led to the view that cells must repair 104 to 1 0 5 ox id at iv e l es ions pe rcell per day [93] Fr aga et al [90,91] wer e amo ng the first to demonst rate a signific ant decreas e

in human sperm 8-oxo-deoxyguanosine levels following vitamin C supplementation However,since the nuc leus of many cells c ontains relative ly low concentra tions of ascorbi c acid, itremai ns unc lear as to the relative impor tance of ascorbi c acid to DNA protect ion

Prote in

Exa mples of pro tein oxidat ion include the life long oxidat ion of long-l ived pro teins, such asthe cryst allin in the lens of the eye, the oxidat ion of a-prot einase inhibitor, and the ad vancedglycat ion end produ cts associ ated with diabetes Tyrosine , N-term inal amino acids, a ndcysteine are often targets of such reactions [94–96] Although data are limited, ascorbic acidsupplementation appears to have a protective effect and certain categories of protein oxidation.Protein carbonyl formation, a precursor for glycation products, is increased in scorbuticguinea pigs and reduced on ascorbic acid repletion [97] Vitamin C supplementation has alsobeen shown to reduce the formation of nitrotyrosine levels in patients with Helicobacter pylorigastritis [98]

In summ ary, ascorbate is an electron donor , which may accoun t for many of its knownfuncti ons How ever, much remains to be resol ved In spite of the ab ility of ascorbi c acid toinflu ence the produ ction of hyd roxyl a nd alkoxyl radica ls, wheth er this is the princi ple effect

or mech anism that occu rs in vivo remains unc ertain Ther e seems to be goo d evidence for theantiox idant protect ion of lipid s by vita min C in biologi cal fluids (both with and without ironcosupplementation, e.g., Ref [88]) with the data on protein oxidation and DNA oxidationsomewhat inconsistent

Carnitine Biosynthesis

Researchers have suggested that early features of scurvy (fatigue and weakness) may beattributed to carnitine deficiency [99–101] Ascorbate is a cofactor of two-enzyme hydroxyla-tion in the pathw ay of carn itine biosynt hesis (Figur e 15 6), g-butyr obetai ne hy droxyla se, ande-N-trimethyllysine hydroxylase [98–100] High doses of ascorbic acid in guinea pigs fed high-fat diets contribute to enhanced carnitine synthesis Ascorbate deficiency results in as much as

a 50% decrease in carnitine in heart and skeletal muscle compared with guinea pig fedascorbic acid [49,100,102]

Extracellular Matrix and Ascorbic Acid

Ascorbic acid is an essential cofactor in extracellular matrix (ECM) metabolism Vitamin Cdeficiency differentially affects the expression of collagen, laminin, various cell surfaceintegrins, as well as elastin [103] The effects of ascorbic acid may be observed at both thefunctional and regulatory gene levels Ascorbic acid is a cofactor for enzymes important tothe posttranslational modification of matrix proteins and perhaps the transcriptional regula-tion of specific proteins As examples, the role of ascorbic acid in prolyl and lysyl hydroxylasewill be highlighted

Collagen is the predominant structural protein in animals To date, 25 (possibly more)distinct types of collagen polypeptide chains have been identified Each of these formsassembles into distinct fibrillar or laminar structures [104] The fibrillar types are typicallyformed by triple helical arrangements of glycine- and proline-enriched polypeptide chains.For such chains to form stable structures, specific prolyl residues in each chain must behydroxylated The hydroxylation is catalyzed by prolyl hydroxylase

Moreover, lysyl groups in collagen are also hydroxylated Some of the resulting residues

of hydroxyl lysine becomes a part of the complex network of cross-links, which aid instabilizing the helical forms of collagen into stable fibers [79,80,105]

When collagen fibers are under-hydroxylated, intracellular cellular assembly of collagenfibers is compromised, with possible alterations in the distribution and types of cross-links.The result is excessive degradation and turnover of collagen Physiologic consequences rangefrom impaired wound healing to capillary fragility, the hallmarks of scurvy An importantperspective is that some collagens have very long half-lives and are developmentally regulated[105] Consequently, if scurvy occurs at critical times in development there may be veryprofound and long-lasting consequences affecting the deposition of bone, the modeling ofthe vascular, and pulmonary alveolar matrix [79,80,105–108]

Elastin is another protein that is hydroxylated, although it is the over-hydroxylation, incontrast to the under-hydroxylation, that causes decreased production of the ultimate prod-uct, an insoluble elastin fiber [105–108] When elastin producing cells are subjected to mediumcontaining high concentrations of ascorbic acid (e.g., near millimolar concentrations), there is

a decrease in expression of elastin The short-term response is abnormal assembly, whichcauses a shift in the partitioning of elastin into insoluble matrix to more ‘‘soluble’’ and easilydegraded forms of elastin The net result is less insoluble elastin [106,107]

Peptidyl lysine

3 SAM

Peptidyl trimethyllysine

Proteolysis Trimethyllysine

α-Ketoglutarate + O 2

Succinate + CO2

α-Ketoglutarate + O 2

Succinate + CO2N+

CH3

CH3

CH3OH

O HO

in a reaction requiring a-ketoglutarate, O2, and ascorbic acid Following the loss of glycine andoxidation to trimethylammoniobutyrate, a second hydroxylation involving an ascorbic acid-assistedstep results in carnitine

As noted, the effects of ascorbate on collagen and elastin are both posttranslational andpossibly transcriptional Collagen fibers begin to assemble in the endoplasmic reticulum (ER)and Golgi complexes Specific proline residues are hydroxylated by two hydroxylases, prolyl4-hydroxylase and prolyl 3-hydroxylase Vitamin C is necessary to maintain the enzymeprolyl hydroxylase in an active form by keeping iron, an essential cofactor for prolyl hydro-xylase, reduced Note that for the mono- and dioxygenases that utilize ascorbic acid as areductant, copper and iron are often the targets for reduction Reduced iron and copper arerequired for coordination with oxygen, the principal cosubstrate for the mono- and dioxy-genases [79,80].

Another cosubstrate is a-ketoglutarate Oxidative decomposition of a-ketoglutarateforms CO2 plus succinate and leads to the generation of Fe(IV)-oxo or activated oxygenspecies, which next interacts with and hydroxylates the primary substrate Ascorbate’s role is

to return the metal to its reduced state for another catalytic cycle In this regard, ascorbic acidpersists for 6–12 catalytic cycles (Figure 15.7)

Regarding ECM expression and ascorbate status, there is the need to resolve a number offactors In cell culture, high concentrations of ascorbate can act as a pro-oxidant Inversecorrelations exist between antioxidants (e.g., vitamin E added to cultures) and ascorbic acid infibroblast and smooth muscle cultures when collagen mRNA or collagen production is used

as the dependent variable [109] Ascorbic acid promotes collagen production, which may becountered by the addition of a-tocopherol to cultures The phenomenon appears most related

to the pro-oxidant effects of ascorbic acid and malonaldehyde formation as a product of lipidperoxidation Malonaldehyde has been shown to promote collagen expression, possibly byupregulation of c-jun nuclear kinase [110]

In summary, the response to ascorbic acid with respect to ECM is complex The role ofascorbate as a cosubstrate or cofactor for prolyl and lysyl hydroxylase (dioxygenases) is clearand mechanistically understood Many facets of ascorbate role in gene expression, however,remain unresolved, given that changes in redox potential, oxidation productions, and thestability of ECM can all impact changes in gene expression

Ascorbic Acid and Gene Expression

The influence of ascorbic acid on the mRNA transcription of specific genes remains unclear[12,99,111–113] As illustrated by the ECM and ascorbic acid interactions, it is clear that

COOH

CH2 + CH2+

+

CH2COOH

COOH H

HO

H2C

CHCOR' N

R

O2CHCOR'

(prolyl-in a reduced state

controlling for changes in oxidative potential is essential to truly determine the specific effects

of ascorbic acid on the expression of given genes For example, disruption of the ECM hasprofound effects on cell differentiation and gene expression Further, an interesting phenom-enon is the formation of the complex of ascorbate and RNA (at GC and AU base pairs) Thiscan apparently occur with little change in RNA secondary structure [114], which mayinfluence the stabilization or destabilization of given transcripts With regard to hydroxylatedproteins, particularly collagen, transcription may be influenced by the amounts of intracel-lular under-hydroxylated products or peptides [109,115–117] If there is a redox-controlledmechanism that involves ascorbic acid, additional detail is often needed to determine theinfluence of products of lipid peroxidation, which may also influence gene expression[109,116–118] The opening and closing of certain ion channels (e.g., the cystic fibrosistransmembrane chloride channel) is also sensitive to ascorbic acid, which may in turninfluence gene expression [119]

Regarding genes that are known to be influenced by ascorbic acid, the transcription of the

72 kDa type IV collagenase (matrix metalloproteinase-2) is downregulated by ascorbic acid incultured human amnion-derived cells [113] Tyrosine hydroxylase transcription [120] and themRNA encoding of various forms of cytochrome P450 in liver microsomes are enhanced byascorbic acid Other mRNAs whose transcription appears to be regulated by ascorbic acid arethe ubiquitins, the collagen-related integrins, and the fra-1 gene, which encodes a transcrip-tion factor of the Fos family and downregulates the activator protein-1 (AP-1) target gene[74,121] In plants, the maize Hrgp gene is induced by ascorbic acid [113]

ASCORBIC ACID METABOLISM AND REGULATION

Ascorbic acid is transported into all types of cells Simple diffusion accounts for some of thismovement The major transport, however, is carrier mediated [75] For ascorbate, there are

Naþ cotransporters that actively transport ascorbate into cells [75] In contrast, corbate is taken into cells by the facilitative glucose transporters

dehydroas-Cell accumulation of ascorbic acid occurs as dehydroascorbate is converted to ascorbicacid This also serves to keep the intracellular concentration of dehydroascorbate low,favoring uptake into the cell along a concentration gradient As examples, the erythrocyteand neutrophil dehydroascorbate reduction system are capable of rapidly generating ascorbicacid [75] Neutrophils utilize ascorbate as a reductant to generate H2O2 for ‘‘killer activity.’’Inside the neutrophil, dehydroascorbate is converted to ascorbate by a dehydroascorbic acidreductase that utilizes glutathione as a reductant Activated neutrophils accumulate ascorbate

to levels that range from 2 mM to as much as 10 mM and can effectively recycle ascorbic acid.The increase in the intracellular ascorbic acid concentration occurs at a time when the cell needsmaximum antioxidant protection against the products of its own oxidative burst reactions

Of clinical significance, diabetes can influence ascorbate cellular transport High glucosecan compete for ascorbic acid, presumably by interfering with ascorbic acid uptake via theglucose transporter isoforms, GLUT1 and GLUT2, which transport the oxidized form ofvitamin C, dehydroascorbic acid [122–125] Dehydroascorbic acid also enters mitochondriavia the facilitative glucose transporter 1 (Glut1) and accumulates in the mitochondria asascorbic acid

Specific active transporters for ascorbic acid have also been identified and are found intissues that accumulate ascorbic acid [124–127] The activity of the transporters has beenobserved to vary inversely with intracellular ascorbate, with the net result of maintaining arelatively constant intracellular concentration Two different isoforms of specific sodium–vitamin C cotransporters (SVCT1=SLC23A1 and SVCT2=SLC23A2) have been cloned Thetissue distributions of the transporters differ, with SVCT1 occurring primarily in epithelial

tissues such as intestine, liver, and kidney Whereas SVCT2 is widely distributed and detected inchoroid plexus cells and neurons, cardiac muscle, placenta, and most other tissues [128–130].With regard to intestinal absorption, in guinea pigs and in humans, the ileum and jejunumare major sites of absorption The process is efficient Ascorbic acid is easily absorbed (cf.Requirements and Allowances) Similar to the cellular uptake of ascorbic acid by red cells andneutrophils, enterocyte uptake is Naþdependent [122].

Ascorbic acid circulates in plasma at micromolar concentrations, whereas it is found insome tissues, often at millimolar concentrations Ascorbic acid oxidation to CO2 and C-4 andC-5 derivatives and excretion are major paths by which ascorbic acid is lost from the body [131]

SELECTED CLINICAL FEATURES IMPORTANT TO ASCORBIC ACID STATUS

DEFININGASCORBICACIDSTATUS

Although leukocyte concentrations are generally considered to be the best indicator ofvitamin C status, the measurement of leukocyte vitamin C is technically complex [131,132].The varying amounts of ascorbic acid in differing leukocyte fractions and the lack ofstandardized reporting procedures [131–134] also complicate interpretation of data Hence,the measurement of plasma vitamin C concentration is currently the most widely applied testfor vitamin C status Plasma concentrations between 11 and 28 mmol=L represent marginalvitamin C status At this level, there is a moderate risk for developing clinical signs of vitamin

C deficiency due to inadequate tissue stores of vitamin C

Data from the Second National Health and Nutrition Examination Survey, 1976–1980(NHANES II) [135], indicated that the prevalence of vitamin C deficiency (plasma vitamin Cconcentrations <11 mmol=L) ranged from 0.1% in children (3–5 years of age) to 3% in females(25–44 years of age) and 7% in males (45–64 years of age) A decade later, the more sensitivemeasures utilized by NHANES III indicated that the prevalence of vitamin C deficiency was12% for adult females and 17% for adult males [136] Marginal vitamin C status (plasma vitamin

C concentrations from 11 to 28 mmol=L) was noted in 20%–23% of adults [136] Smokers aremore likely to have marginal vitamin C status compared with nonsmoking adults Several studiessuggest that smokers require >200 mg ascorbate daily to maintain plasma concentrations at alevel equivalent to nonsmokers consuming 60 mg vitamin daily (cf Ref [137] and referencescited therein) The current vitamin C recommendation for smokers is ~40% greater than thatfor nonsmokers, 110 and 125 mg daily for females and males, respectively [138]

In its most extreme form, scurvy is characterized by subcutaneous and intramuscularhemorrhages, leg edema, neuropathy, and cerebral hemorrhage, and, if untreated, the condi-tion is ultimately fatal Presently, even in affluent countries, scurvy should be consideredwhen signs and symptoms such as cutaneous and oral lesions are observed, particularly inalcoholics, the institutionalized elderly, or persons who live alone and consume restrictivediets containing little or no fruits and vegetables Patients may also complain of lassitude,weakness, and vague myalgias, and seek medical attention following the appearance of a skinrash or lower extremity edema Discussion of the functional roles of ascorbic acid in bodysystems and prevention of chronic disease is provided later

CLINICAL FEATURES

Immune Function

Since the publication of Vitamin C and the Common Cold by Pauling [50], the role of vitamin

C in immune function has been a topic of lively debate The high concentration of vitamin C

in leukocytes, and the rapid decline in plasma and leukocyte vitamin C concentrations during