Vitamin C: biologic functions and relation to cancer. September 10-12, 1990, Bethesda, Maryland. Abstracts.

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To cite this article: (1991) Vitamin C: Biologic functions and relation to cancer, Nutrition and Cancer, 15:3-4, 249-280, DOI: 10.1080/01635589109514133 To link to this article: http://dx.doi.org/10.1080/01635589109514133

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Vitamin C: Biologic Functions and Relation to Cancer Sponsored by National Cancer Institute and National Institute of Diabetes and Digestive and Kidney Diseases September 10-12, 1990, Bethesda, Maryland

Introduction

G. Block, D. E. Henson, and M. Levine Vitamin C has been the subject of numerous investigations, almost 800 citations in Medline since 1989 alone. The very extent and diversity of the research, especially when competing with many other interesting research areas for attention, has meant that important results may go unremarked. Even more serious, the pattern represented by a set of research results may not be visible. Furthermore, there has been considerable public interest in the possibility of a role for this vitamin in cancer. In order that this debate might take place in a rigorous and informed manner, we attempted to bring together not only the latest research on basic actions, such as free-radical scavenging or enzyme functions, but also some of the basic laboratory and animal studies relating to cancer. To this end, the National Cancer Institute and the National Institute of Diabetes and Digestive and Kidney Diseases jointly sponsored a symposium at the National Institutes of Health on September 10-12, 1990, entitled Vitamin C: Biologic Functions and Relation to Cancer. Topics from several disciplines are presented in the following abstracts, which describe a wide range of biological actions of ascorbate. The well-known antioxidant and free-radical scavenging activities are discussed in the first series of papers. Because free-radical damage and formation of lipid peroxides are suspected in carcinogenesis as well as cardiovascular disease, this may be important for disease prevention. Several papers discuss enzyme-related functions, including collagen synthesis and gene expression, norepinephrine and carnitine synthesis, and transmembrane electron transfer. Nonenzyme functions include stimulation of acetylcholine receptor expression and chondrocyte function. A role in the immune system is indicated by the very high concentration of ascorbic acid in neutrophils, and several papers address effects on immune function. Approximately half of the symposium addressed the role of ascorbate in cancer prevention or as an adjuvant in cancer therapy, primarily in animal models. In vitro studies included research on oncogenic transformation and effects on HIV virus. In in vivo studies in animals, several speakers presented effects of ascorbate on tumor incidence, growth, or host survival. Moreover, several researchers presented data that suggest a role for ascorbate in reducing the toxicity or improving the effectiveness of conventional therapies. Finally, a


review is presented of all human epidemiologic studies on the relationship between vitamin C and cancer prevention. Other reports describe the nature of the genetic error responsible for the inability of humans to synthesize ascorbate, and others propose the use of in situ kinetics to determine human vitamin requirements. Several other abstracts describe the effects of experimental vitamin C depletion in humans, effects on copper transport, and methodologic issues regarding assay techniques. Even a cursory review of the scientific literature on vitamin C reveals a surprising number of leads, hints, and important functions. Many investigators who have made important contributions could not be included in this symposium, for which we apologize. It is our hope that their work and the work presented in the following abstracts will be examined and pursued. What seems to be before us now is an opportunity—indeed, a need—to follow up these leads with further research. Address requests for reprints of these abstracts to Dr. Gladys Block, Div. of Cancer Prevention and Control, National Cancer Institute, EPN Rm. 313, 9000 Rockville Pike, Bethesda, MD 20892.

ANTIOXIDANT AND FREE RADICAL SCAVENGING FUNCTIONS Ascorbic Acid Protects Plasma Lipids Against Oxidative Damage Balz Frei and Bruce N. Ames Prooxidant states have been linked to certain types of cancer, and antioxidants are known to act as anticarcinogens. We investigated the relative effectiveness of selected physiological antioxidants by exposing human blood plasma to various types of oxidative stress (Frei et al., Proc Natl Acad Sci USA 85, 9748-9752, 1988; Frei et al., Proc Natl Acad Sci USA 86, 6377-6381, 1989; Frei et al., Antioxidants in Therapy and Preventive Medicine, New York: Plenum, pp 155-163, 1990). Oxidative damage was assessed by measuring lipid hydroperoxides using high-performance liquid chromatography coupled to postcolumn chemiluminescence detection (Frei, Anal Biochem 175, 120-130, 1988). This selective and sensitive assay can detect lipid hydroperoxides in plasma at concentrations as low as 10 nM. We found that in plasma exposed to a constant flux of aqueous peroxyl radicals generated by the chemical radical initiator, 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH), the endogenous antioxidants are consumed in the temporal sequence ascorbic acid = protein thiols = ubiquinol-10 > albumin-bound bilirubin > uric acid > a-tocopherol. Most importantly, no lipid peroxidation could be detected as long as detectable concentrations (>1 ixM) of ascorbic acid were present. Immediately after the complete consumption of ascorbic acid, lipid peroxidation was initiated. Lipid peroxidation occurred despite the presence of physiological or near-physiological concentrations of protein thiols, albuminbound bilirubin, uric acid, and a-tocopherol. In plasma devoid of ascorbic acid, lipid peroxidation was initiated immediately on exposure of the plasma to AAPH, whereas supplementation of plasma in vitro with increasing amounts of ascorbic acid led to an increase of the lag phase preceding detectable lipid peroxidation. This lag phase increased sublinearly with increasing ascorbic acid concentrations, reflecting a slower rate of increase in radical-trapping ability of ascorbic acid as its concentration increases. However, ascorbic acid acted always protectively, rather than as a prooxidant, providing strictly increased benefit with increased concentration.

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Sequences of antioxidant consumption and lipid peroxidation very similar to those in plasma exposed to aqueous peroxyl radicals were observed in plasma exposed to other types of oxidative stress, such as activated neutrophils, the gas phase of cigarette smoke, and superoxide radicals and hydrogen peroxide generated by the xanthine/xanthine oxidase system. Under all these types of oxidative stress, ascorbic acid formed the first line of antioxidant defense in plasma and was the only endogenous antioxidant capable of completely protecting the lipids against oxidative damage. We have recently confirmed these results by using isolated human low-density lipoprotein (LDL) exposed to oxidative stress in the absence and presence of added ascorbic acid. In the absence of ascorbic acid, lipid peroxidation was initiated immediately upon exposure of LDL to AAPH or activated neutrophils, despite the presence of the LDL-associated, lipid-soluble antioxidants ubiquinol-10, a-tocopherol, lycopene, and /3-carotene. In the presence of added ascorbic acid, there was a lag phase preceding detectable lipid peroxidation in LDL, during which the added ascorbic acid was oxidized completely. In summary, our data demonstrate that ascorbic acid is the only endogenous antioxidant in plasma that can completely protect the lipids from detectable peroxidative damage induced by aqueous peroxyl radicals, the oxidants generated by activated neutrophils, by the xanthine/xanthine oxidase system, or present in the gas phase of cigarette smoke. Ascorbic acid appears to trap the oxidants in the aqueous phase with a rate constant large enough to intercept virtually all these oxidants before they can diffuse into the plasma lipids and initiate lipid peroxidation. Under all types of oxidative stress that we used in our experiments, ascorbic acid proved to be a much more effective antioxidant than were bilirubin, uric acid, protein sulfhydryl groups, ubiquinol-10, a-tocopherol, lycopene, and /3-carotene, all of which can only lower the rate of lipid peroxidation but not prevent its initiation. Only ascorbic acid can do so. Requests for further information should be addressed to Dr. Balz Frei, Dept. of Nutrition, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115.

Action of Ascorbic Acid as a Scavenger of Active and Stable Oxygen Radicals Etsuo Niki Free radicals attack lipids, proteins, enzymes, and DNA to eventually cause a variety of pathological events and cancer. Ascorbic acid is accepted to act as one of the primary defenses by scavenging oxygen radicals. We have been studying the action of various antioxidants including ascorbic acid in the oxidations of biologic molecules, membranes, and tissues both in vitro and in vivo. When aqueous radicals were generated in the whole blood, ascorbic acid scavenged them faster than did any other antioxidants and protected lipids and proteins more effectively than did bilirubin, uric acid, or tocopherol (vitamin E). When the radicals were formed in the aqueous suspensions of red blood cells or low-density lipoprotein (LDL), ascorbic acid scavenged radicals before they reached the membrane and LDL and suppressed their oxidative damage. However, hydrophilic ascorbic acid was not able to scavenge lipophilic radicals located within the interior of membranes and LDL. Tocopherols acted as the primary defense against lipophilic radicals. Interestingly, ascorbic acid reacted with the tocopheroxyl radical, which was formed when tocopherol scavenged oxygen radicals at the surface of the membrane, to regenerate tocopherol. Thus, ascorbic acid contributed in sparing and maintaining the concentration of tocopherol and acted as a synergist in preventing the oxidations of red blood cells and LDL. It may also be important in inhibiting the prooxidant action of tocopherol in food. Ascorbic acid also reduced the nitroxide radical rapidly, faster than uric acid, cysteine, and glutathione. The rate of reduction decreased as

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the nitroxide radical went deeper into the interior of the membranes and LDL. The prooxidant action of ascorbic acid observed in some model experiments in vitro is probably not important in vivo. In conclusion, our work and others show clearly that ascorbic acid acts as a primary hydrophilic defense and protects aerobic organisms from free radicalmediated oxidative damage. Requests for further information should be addressed to Dr. Etsuo Niki, Dept. of Reaction Chemistry, University of Tokyo, Hongo, Tokyo 113, Japan.

Ascorbic Acid and Oxidative Inactivation of Proteins


E.R. Stadtman Oxygen free radicals or other "active oxygen species" are likely implicated in the etiology or manifestations of a number of pathological processes. Ascorbate, in concert with vitamin E, occupies a unique role in the protection of cells against oxygen-mediated toxicity. Paradoxically, ascorbate can also under certain conditions promote the generation of oxygen free radicals and hence contribute to oxygen toxicity. Nearly 36 years ago, Udenfriend and colleagues (J Biol Chem 208, 731-739, 1954) demonstrated that a mixture comprised of ascorbate, a chelating agent, Fe(III) and O2, can catalyze the hydroxylation of aromatic compounds in a manner similar to that achieved by mixed-function oxidases. In the meantime, the ascorbate mixed-function oxidation system has been widely used to catalyze the oxidative modification of biologic molecules, especially nucleic acids, proteins, and lipids. Mechanistically, ascorbate serves as an electron donor for the reduction of Fe(III) or Cu(II) to Fe(II) and Cu(I) and also for the reduction of O2 to H2O2. In the case of nucleic acids and proteins, the H2O2 and Fe(II) or Cu(I) react at metal binding sites on the macromolecules to produce active oxygen species [«OH, (FeO)2+] that attack functional groups proximal to the metal binding site. For proteins, oxidation leads to the modification of the side chains of amino acid residues at the metal binding site. Among other as-yet-unidentified modifications, histidyl residues are converted to asparaginyl or aspartyl residues; prolyl residues are converted to glutamylsemialdehyde and glutamyl or pyroglutamyl residues; and arginyl residues are converted to glutamylsemialdehyde residues. Requests for further information should be addressed to Dr. Earl Stadtman, Chief, Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Building 3, Room 222, National Institutes of Heatlh, 9000 Rockville Pike, Bethesda, MD 20892.

Serum Urate as an Antioxidant for Ascorbic Acid Alex Sevanian, Kelvin Davies, and Paul Hochstein Ascorbic acid has received increasing attention as an important biologic antioxidant and, in serum, is proposed to be a primary antioxidant, protecting both serum lipids and proteins. This antioxidant function is rationalized on the basis of its action as a biologic reductant, particularly for the reduction of radical species and hypervalent metal catalysts. In humans, ascorbic acid levels must be maintained by ingestion of the vitamin because during human evolution there was a loss of ascorbate synthetase. In the past decade, it became recognized that the loss of ascorbate synthetase activity was accompanied by a loss in uricase along with an active kidney reabsorbtion mechanism for urate and that this double mutation could serve as a partial substitution for endogenous antioxidant activity. In a series of studies, we observed that urate not only behaved as a powerful antioxidant in several free radical

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systems but also served to stabilize ascorbic acid and hence could conserve this vitamin in humans. This dual function could enhance the antioxidant potency of serum and reduce vitamin C demand. Although urate functions as a free radical scavenger and as a reductant, much of its vitamin C protective action appears to be due to inhibition of iron-catalyzed oxidation reactions. In this respect, the iron-catalyzed oxidation of ascorbate (typical of iron-catalyzed free radical generating reactions) is inhibited by urate. This inhibition is seen in various buffers and in human serum and appears to be due to iron chelation, although reaction with H 2 O 2 (or derived «OH) affords a minor contribution. Indeed, the protection of ascorbate by urate is not accompanied by appreciable losses of urate, unlike other free radical scavenging reactions where urate is consumed. Chelation of iron by equimolar concentrations of EDTA (a much better chelator than urate) eliminates the protective action of urate. Likewise, depletion of serum urate results in a very rapid subsequent oxidation of ascorbate and a lower antioxidant capability of serum to a host of free radical reactions. Thus, the interaction of urate and ascorbate represents another relationship between two important physiological antioxidants. Requests for further information should be addressed to Dr. Alex Sevanian, Toxicology and Pathology, Institute for Toxicology, University of Southern California, 1985 Zonal Ave., Los Angeles, CA 90033.

ASCORBIC ACID REGULATION OF ENZYME FUNCTION Ascorbate Requirement for Hydroxylation and Secretion of Procollagen: Relationship to Inhibition of Collagen Synthesis in Scurvy Beverly Peterkofsky Scurvy, the disease caused by vitamin C deficiency, is associated with defective connective tissue, particularly in wound healing. Our early studies showed that ascorbate was required for the posttranslational hydroxylation of proline residues in procollagen to form hydroxyproline. In the absence of ascorbate, cultured cells, in most cases, produce unhydroxylated procollagen that is secreted at a slower rate than is the fully hydroxylated protein produced in its presence, although the rate of procollagen synthesis is unaffected. This results from the critical role of hydroxyproline in stabilizing the triple helical structure of collagen. These observations appeared to provide the basis for explaining the defects in connective tissue that occur during scurvy. A much different perspective, however, has emerged from recent studies in our laboratory on the effects of ascorbate deficiency on extracellular matrix production in connective tissues of young, rapidly growing guinea pigs. There was relatively little effect of ascorbate deficiency on the extent of proline hydroxylation in collagen of bone and cartilage. Nevertheless, there was a specific decrease in collagen synthesis, and in addition, there was inhibition of cartilage proteoglycan synthesis, for which there is no known requirement for ascorbate. Decreased synthesis of both matrix components was correlated with the extent of weight loss that is characteristic of the third and fourth week of ascorbate deficiency. Furthermore, guinea pigs that were fasted but received ascorbate so that proline hydroxylation was normal showed a similar correlation between the extent of weight loss and the decreases in the rates of collagen and proteoglycan biosynthesis. These observations led us to suggest that scurvy and fasting are equivalent with regard to regulation of the synthesis of extracellular matrix components and that regulation occurs independently of the role of ascorbate in proline hydroxylation. The in vivo defects in collagen and proteoglycan

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synthesis could be transmitted to cultured chick embryo chondrocytes and human fibroblasts by culturing the cells in media containing sera from fasted or scorbutic guinea pigs plus ascorbate. Decreased synthesis was caused by the presence of an inhibitor in these sera rather than by the lack of a stimulatory factor. Inhibition could be reversed by the addition of insulin-like growth factor (IGF)-I to cultures. Our current experiments suggest that the inhibitor consists of two IGF binding proteins with unsaturated binding sites that can be detected with [125I]IGF-I. These binding proteins inhibit binding of free IGF-I to its cellular receptor, which could result in inhibition of IGF-I-dependent functions such as collagen, proteoglycan, or DNA synthesis. We propose that induction of these circulating binding proteins during vitamin C deficiency also may be responsible for in vivo inhibition of collagen and proteoglycan synthesis. Requests for further information should be addressed to Dr. Beverly Peterkofsky, Laboratory of Biochemistry, Natural Cancer Institute, Bldg 37, Rm. 4C18, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. Ascorbic Acid Stimulation of Collagen Biosynthesis, Independent of Hydroxylation

Karl Houglum, David Brenner, and Mario Chojkier Ascorbic acid stimulates collagen gene expression in cultured fibroblasts (Lyons and Schwartz, Nucleic Acid Res 12, 2569-2579, 1984), but the mechanisms responsible for this effect are poorly understood. In the presence of the transitional metal iron, ascorbic acid could induce lipid peroxidation with the formation of reactive aldehydes. Because another aldehyde, acetaldehyde, the first metabolite of ethanol, also stimulates collagen transcription in cultured fibroblasts (Brenner and Chojkier, J Biol Chem 262, 17690-17696, 1987), we investigated whether ascorbic acid induces lipid peroxidation in cultured cells and if this is the mechanism by which ascorbic acid stimulates collagen gene expression. Ascorbic acid (0.2 mM) induced lipid peroxidation in cultured human fibroblasts judging by the production of thiobarbituric acid reactive substances and carbonyl groups and by the presence of malondialdehyde-protein and 4-hydroxynonenal-protein adducts. Ascorbic acid stimulated (two- to threefold) the net production of collagen relative to total proteins, the levels of procollagen a^IJmRNA, and the transcription of this gene. Inhibition of the ascorbic acid-induced lipid peroxidation in cultured human fibroblasts with a-tocopherol (50 pM) or methylene blue (10 jiM) prevented the stimulation of collagen gene expression. The addition of malondialdehyde (200 /tM), a product of lipid peroxidation, to cultured human fibroblasts also increased two- to threefold the collagen production and procollagen a,(I)mRNA levels. Thus, ascorbic acid induces lipid peroxidation and reactive aldehydes. This step may be necessary for stimulation of collagen gene expression by ascorbic acid in cultured human fibroblasts. Requests for further information should be addressed to Dr. Mario Chojkier, Dept. of Medicine (V-111D), University of California at San Diego, 3350 La Jolla Village Dr., San Diego, CA 92161. Multicompartmental Secretion of Ascorbic Acid and Its Dual Role in Dopamine |3-Hydroxylation

Emanuel J. Diliberto, Jr. and O. Humberto Viveros New information suggests that ascorbic acid participates in more complex neural and endocrine functions than earlier proposals where its function was limited to a cofactor for

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dopamine 0-hydroxylase (DBH). The final step in the biosynthesis of norepinephrine is catalyzed by DBH, an enzyme localized exclusively within catecholamine storage vesicles. Previous work in our laboratory has shown that two ascorbic acid molecules donate single electrons to this monooxygenase per /3-hydroxylation cycle with the formation of two molecules of semidehydroascorbate. These observations prompted the characterization and localization of the enzyme activity of semidehydroascorbate reductase on the outer membrane of mitochondria, suggesting that the cofactor could be reutilized. Clues to the mechanism by which the ascorbic acid redox couple within the vesicle is linked to the redox couple in the cytosol were provided by several groups (Njus et al., J Biol Chent 258, 27-30, 1983; Harnadek et al., Biochemistry 24, 2640-2644, 1985; Srivastava et al., JBiol Chem 259, 8072-8075, 1984) demonstrating the participation of a specific protein, cytochrome b 561 , on the vesicle membrane. In this model, the cytochrome is reduced by cytosolic ascorbic acid (with the formation of cytosolic semidehydroascorbate) and is oxidized by vesicular semidehydroascorbate generated from ascorbic acid by the DBH reaction and thus shuttles electrons across the vesicular membrane. On the cytosolic side, the semidehydroascorbate is reconverted to ascorbic acid by the mitochondrial semidehydroascorbate reductase. To study whether these components were indeed part of a regeneration system for the cofactor, we used two model systems: intact chromaffin cells in culture and isolated chromaffin vesicles. In the latter preparation, we demonstrated that an external electron donor such as ascorbic acid or glucoascorbic acid prevented the decrease of intravesicular ascorbic acid that otherwise occurs during /3-hydroxylation without the transfer of reductant molecules into the vesicles. The capacity of this regeneration system was demonstrated in situ in cultured chromaffin cells. The intracellular ascorbic acid is maintained in the reduced form under experimental conditions leading to large changes in intravesicular levels of ascorbic acid and hydroxylation rates. Thus, in this system, ascorbic acid functions not only as a cofactor for DBH but also as a component of a system for its own regeneration. Recent evidence also indicates that ascorbic acid may function as a chemical messenger or neuromodulator. In response to various secretagogues, chromaffin cells release ascorbic acid from both intra- and extravesicular compartments. Vesicular ascorbic acid is related by exocytosis along with all other soluble vesicular components. The largest proportion of secreted ascorbic acid appears to originate from the cytosol and is probably released by a transporter mechanism that can be activated independent of exocytosis. Activation of the adrenal medulla by stress depletes ascorbic acid from both the adrenal cortex and medulla, suggesting that adrenomedullary ascorbic acid may have different neural and endocrine roles in addition to serving as a cofactor for catecholamine and peptide biosynthesis. Requests for further information should be addressed to Dr. Emanuel Diliberto, Jr., Neuroscience Section, The Wellcome Research Laboratories, Burroughs Wellcome, 3030 Cornwallis Rd., Research Triangle Park, NC 27709.

Cytochrome b 561 , Ascorbic Acid, and Transmembrane Electron Transfer

Patrick J. Fleming and Ute M. Kent Cytochrome b 561 was first recognized as a component of catecholamine storage granules almost 30 years ago. Dopamine /3-hydroxylase, an enzyme known to prefer ascorbic acid as a reductant, was subsequently shown to be associated with these granules, causing several investigators to speculate that cytochrome b 561 was involved in electron transfer with the hydroxylase/ascorbate system. Elegant spectroscopic studies by other investigators showed that the cytochrome appeared to mediate equilibrium of ascorbate/semidehydroascorbate inside the granule with this redox pair in the cytoplasm. Thus, the cytochrome acted as a

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transmembrane electron shuttle between the cytoplasm and granule matrix. We showed that purified cytochrome in reconstituted systems did catalyze transmembrane electron transfer. We also demonstrated in reconstituted systems that the cytochrome did not donate electrons directly to dopamine /3-hydroxylase, that the intragranular ascorbate is the immediate donor to the hydroxylase, and that the role of cytochrome is to regenerate ascorbate from semidehydroascorbate. Cytochrome b 561 is found in many neuroendocrine tissues. Colocalization with peptidylglycine a-amidating monooxygenase and reconstitution experiments with this enzyme provide good evidence that cytochrome is also responsible for providing regenerated ascorbate for this enzyme. The structure of cytochrome b56i is slowly emerging. The primary sequence suggests six membrane-spanning regions, and topology studies have located the carboxy terminus on the cytoplasmic side of the granule membrane. Redox reactivity and comparison with other cytochrome sequences both suggest a hem binding pocket is located on the cytoplasmic side of the membrane. Preliminary evidence supports a structure with a single hem liganded to two histidines. This very hydrophobic transmembrane protein is also fatty acylated. Our current picture of cytochrome b 561 suggests that electron transfer between ascorbate and the cytochrome may involve the polypeptide itself as well as the hem prosthetic group. Elucidation of the structure and mechanism of redox activity of cytochrome b 561 may demonstrate paradigms for other ascorbate utilizing enzymes as well as provide insights into long-range biologic electron transfer. Requests for further information should be addressed to Dr. Patrick J. Fleming, Dept. of Biochemistry and Molecular Biology, Rm. 353, Basic Sciences Building, Georgetown University School of Medicine, Washington, DC 20007.

Ascorbic Acid and Carnitine Biosynthesis

Charles J. Rebouche L-Carnitine is required for entry of long-chain fatty acids into mitochondria where they are utilized for cellular energy production. It also facilitates removal of excess short-chain acyl moieties from mitochondria. L-Carnitine is synthesized ultimately from lysine and methionine. In mammals, three methyl groups from methionine are transferred, via 5-adenosylmethionine, to the e-amino group of lysine, which must be in peptide linkage. e-N-Trimethyllysine is released from peptide linkage by proteolytic digestion. This amino acid is converted to L-carnitine via a series of four enzymatic steps: e-iV-trimethyllysine — /?-hydroxy-e-Af-trimethyllysine — y-iV-trimethylaminobutyraldehyde — y-butyrobetaine -• L-carnitine. Steps one and four in this sequence are catalyzed by a-ketoglutarate-dependent dioxygenases, which require ascorbate as cofactor for optimal activity in vitro. e-NTrimethyllysine hydroxylase activity is found in cardiac and skeletal muscle, brain, liver, and kidney. 7-Butyrobetaine hydroxylase distribution varies by species, but it is present in liver and absent in cardiac and skeletal muscle of all species studied (in humans, activity of this enzyme is abundant in kidney as well as liver). The role of ascorbate in these reactions is thought to involve maintenance of iron (an obligatory cofactor in these reactions) in the reduced (ferrous) state. In some studies of scorbutic guinea pigs, carnitine concentrations in one or more of several different tissues, including liver and cardiac and skeletal muscle, were decreased compared with normal animals. However, in other studies, carnitine concentrations in one or more of these tissues, as well as kidney, were found to be normal. The discrepancies may be related to various aspects of experimental design and dietary treatments. In one study, e-Ar-trimethyllysine administered

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via the inferior vena cava of the anesthetized guinea pig was taken up primarily by the kidney. Its conversion to 7-butyrobetaine was markedly reduced in scorbutic animals compared with ascorbate-sufficient, pair-fed, and control (ad libitum-fed) animals. e-NTrimethyllysine-hydroxylating activity was restored in scorbutic guinea pig kidney by injection of ascorbate with the substrate. In another study, no differences were found in the ability of perfused livers from scorbutic animals and control animals to convert e-iV-trimethyllsyine to 7-butyrobetaine. On the other hand, investigators from two laboratories independently showed that livers from scorbutic guinea pigs have impaired capacity to convert 7-butyrobetaine to L-carnitine, but again, demonstration of this effect of ascorbic acid deficiency depended on proper selection of experimental conditions. Restoration of 7-butyrobetaine-hydroxylating activity in liver of scorbutic animals was effected by administration of ascorbic acid immediately prior to the test dose in vivo or in the perfusion medium in vitro. The role of 7-butyrobetaine hydroxylase activity in reduced carnitine biosynthesis in ascorbic acid-deficient guinea pigs is unclear, because in rats and presumably in guinea pigs (guinea pig liver contains about five times more of this enzyme activity than does rat liver), the capacity for 7-butyrobetaine hydroxylation exceeds by at least 100 times the normal rate of carnitine biosynthesis. Thus, the preponderance of evidence indicates that ascorbic acid deficiency impairs carnitine biosynthesis, but the level at which inhibition of the pathway occurs in vivo has not been clearly established. Requests for further information should be addressed to Dr. Charles J. Rebouche, Dept. of Pediatrics, University of Iowa, Iowa City, IA 52242.

Peptidylglycine a-Amidating Monooxygenase, an Ascorbate-Dependent Enzyme Essential for the a-Amidation of Neuroendocrine Peptides

Betty A. Eipper and Richard E. Mains Many bioactive peptides have an a-amidated amino acid at their carboxyl terminus; the presence of an a-amidated amino acid is essential for the biologic activity of many neuroendocrine peptides such as gastrin, bombesin, corticotropin-releasing hormone, thyrotropin-releasing hormone, substance P, and neuropeptide Y. In their precursor proteins, these a-amidated peptides are flanked by endoproteolytic cleavage sites, with a glycine residue always situated to the carboxyl-terminal side of the amino acid that is a-amidated in the product peptide. Endo- and exoproteolytic cleavage events expose a carboxyl-terminal glycine resident, and an a-amidating enzyme converts the peptidylglycine intermediate into an a-amidating product. Because the a-amidating enzyme depends on copper, molecular oxygen, and ascorbate, it has been termed peptidylglycine a-amidating monooxygenase (PAM) (EC 1.14.17.3). cDNAs encoding PAM have been cloned from several tissues and species. Although the amidating enzymes purified from bovine neurointermediate pituitary are soluble 40- to 50-kD proteins, the cDNA for bovine PAM encodes a 108-kD protein with an aminoterminal signal sequence and a hydrophobic putative transmembrane domain located near its carboxyl terminus. Tissue-specific alternative splicing and endoproteolytic processing of these membrane precursors generates an array of proteins with enzymatic activity. Membraneassociated forms of PAM predominate in the heart atrium, whereas soluble forms predominate in the pituitary. When rat intermediate pituitary cells are maintained in primary culture in serum free medium lacking added ascorbate, they rapidly lose their ability to produce a-amidated peptides. On addition of physiological levels of ascorbate to the culture medium, cellular

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ability to produce a-amidated peptides Coining peptide and a-melanocyte-stimulating hormone) is restored. AtT-20 corticotropic tumor cells also produce a-amidated joining peptide from pro-ACTH/endorphin. In contrast to the primary cell cultures, AtT-20 cells depleted of ascorbate continue to produce substantial amounts of a-amidated joining peptide. The AtT-20 cells can utilize ascorbate as well as several other cofactors to support peptide a-amidation. In test tube assays, catecholamines replace ascorbate in supporting the a-amidation reaction. A lack of either copper or ascorbate can limit the ability of tissues to produce a-amidated peptides. In addition, in at least some tissues, the amount of PAM is rate limiting. On transfection of a cDNA encoding bovine PAM into AtT-20 cells, the ability of the cells to produce a-amidated joining peptide in the absence of ascorbate is increased. On reduction of endogenous PAM levels by expression of an antisense PAM RNA, the ability of the cells to produce a-amidated joining peptide in the presence or absence of ascorbate is reduced. Some of the poorly understood effects of ascorbate may involve its role in the production of a-amidated peptides with autocrine and paracrine roles. The PAM precursor protein consists of two separable enzymatic activities catalyzing the two steps of the a-amidation reaction. The amino-terminal third of the PAM precursor is a peptidylglycine a-hydroxylating monooxygenase, converting peptidylglycine into peptidyl-ahydroxyglycine. At alkaline pH, the peptidyl-a-hydroxyglycine is spontaneously converted into a peptidyl-a-amide. At the acidic pH generally encountered in secretory granules, conversion of peptidyl-a-hydroxyglycine into a-amidated product is catalyzed by an enzyme (peptidyl-a-hydroxyglycine a-amidating lyase) derived from the middle third of the PAM precursor. Requests for further information should be addressed to Dr. Betty Eipper, Dept. of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205.

NONENZYMATIC MECHANISMS OF ASCORBIC ACID ACTION Ascorbic Acid and Acetylcholine Receptor Expression

M.M. Salpeter, E. Liu, J.A.M. Wootton, and R.R. Minor Ascorbic acid has a stimulatory effect on acetylcholine receptor (AChR) synthesis. When 170 /iM ascorbic acid is added to cultures of the L5 cloned muscle cell or to primary rat muscle cultures, there is approximately a threefold increase in the AChR mRNA, selectively for the a-subunit (Horovitz et al., J Cell Biol 108, 1817-1822, 1989; Horovitz et al., J Cell Biol 108, 1823-1832, 1989). In the L5 cells, but not in the primary rat muscle cell cultures, the increase in the AChR a-subunit mRNA is correlated to an increase in surface AChR levels (Neugebauer et al., Brain Res 346, 58-69, 1985; Knaack et al., J Cell Biol 102, 795-802, 1986). However, both the increase in a-AChR mRNA and that of the surface AChR is delayed about 20-24 hours even after a single 5-hour dose of ascorbic acid. Because of this delay and the fact that the AChR response can be triggered by a short exposure to ascorbic acid, we suggested that an intermediate step may be involved and asked whether the other well-known effect of ascorbic acid, that on increasing collagen secretion, could be involved. Using L5 cells, we found that ascorbic acid caused a two- to fivefold increase in the secretion of collagen type I (al(I) and a2(I) chains) and type III ([a(IH)]3 homotrimer) and that the increase began by 3 hours and peaked by 12 hours after a single dose of ascorbic

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acid. Nevertheless, digestion of the secreted collagens with bacterial collagenase did not prevent the stimulation of surface AChR levels by ascorbate. Thus, the ascorbic acidtriggered effect on elevating AChR levels appears to be independent of its effect on collagen secretion. Requests for further information should be addressed to Dr. Miriam Salpeter, WII 3 Seeley Madd Hall, Dept. of Neurobiology, Cornell University, Ithaca, NY 14853.

Ascorbic Acid and Iron Metabolism


Kim E. Hoffman, Kathi Yanelli, and Kenneth R. Bridges Although iron is essential to cell metabolism, the element can promote free radical damage to membranes and lipids. Therefore, excess intracellular iron is stored as an inert crystal within the shell of the hollow ferritin molecule until it is required for metabolic use. Ascorbic acid retards ferritin degradation, thereby modifying the bioavailability of iron. When the growth medium of the human erythroleukemia cell, K562, is supplemented with 100 /iM ascorbic acid, the concentration of the vitamin in the cells as measured by high-performance liquid chromatography analysis increases from a negligible level to 1 nmol/107 cells. Ascorbic acid increases the stability of the iron cores of ferritin in cells prelabeled with 59Fe. Biosynthetic labeling of ferritin with [35S]methionine demonstrates that this enhanced stability of the ferritin iron core is derived from a slowing of the rate of degradation of the protein shells. A similar stabilization of ferritin is produced by generalized inhibitors of protein synthesis such as cloroquine and ammonium chloride. Unlike these agents, however, ascorbate does not have a general inhibitory effect on protein synthesis, as shown by the lack of effect of the vitamin on the turnover either of hemoglobin or lactate dehydrogenase in K562 cells. Ferritin normally is degraded in cytoplasmic lysosomes. Subcellular fractionation of 59 Fe-labeled cells using a Percoll density gradient technique shows that the shift of ferritin label from the cytosolic component to the lysosomal component is significantly delayed by ascorbic acid. A Sepharose CL-6B column can separate ferritin components of the cell into monomeric cytoplasmic ferritin, ferritin aggregates in the cytoplasm, and ferritin aggregates that have been taken into lysosomes by autophagy as a prelude to degradation. Cells pulse labeled with 59Fe show an initial distribution of iron into the cytoplasmic ferritin monomer and aggregate fractions with no label in the lysosomes. Over 24 hours, the label shifts from ferritin monomers to aggregates and from aggregates to lysosomes. Ascorbate does not affect the conversion of cytoplasmic ferritin monomers to aggregates. The vitamin greatly retards the autophagic uptake of ferritin aggregates into lysosomes, however, and ferritin synthesis is unaffected. The result is that the ferritin content of cells treated with ascorbate increases 4-fold over 24 hours. Because the iron stored within the ferritin shell is in equilibrium with iron in the cytoplasm, the amount of free iron in the cytoplasm increases secondary to the augmented ferritin content of the cells. Free iron can then promote the formation of injurious compounds such as the hydroxyl radical that produce oxidant damage to membranes and lipids within cells. The consumption of excessive amounts of ascorbate by patients with iron overload has produced severe tissue toxicity in some cases, manifested most profoundly by the development of heart failure and death in some instances. Enhanced iron availability in the cells of these individuals may produce free radical damage leading to substantial tissue injury. Requests for further information should be addressed to Dr. Kenneth R. Bridges, Hematology Div., Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115.

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The Role of Ascorbic Acid in Regulating Cartilage Maturation in the Epiphyseal Growth Plate

Irving M. Shapiro, Phoebe S. Leboy, Toshikazu Tokuoka, Sherrill L. Adams, Ellis E. Golub, and Maurizio Pacifici Bones grow in length because of the activities of cartilage cells in a strip of tissue called the epiphyseal growth plate. The cellular architecture of the growth cartilage facilitates analysis of spatial and temporal events that lead to cartilage calcification and bone formation. Earlier investigations clearly demonstrated that as the tissue matures, epiphyseal chondrocytes undergo a series of well-characterized events in which there are synchronous changes in both morphology and function. These events include elemental loading, matrix vesicle biogenesis, mineral deposition, and macromolecular synthesis. In addition, our studies of the growth cartilage have documented the importance of metabolic factors in regulating the mineralization of the epiphysis. It is possible to mimic selected events that occur in the growth cartilage by using cells in culture. We characterized many of these events by using biochemical, immunohistochemical, and molecular biology techniques. Our studies indicate that ascorbate regulates chondrocyte function at a number of levels. First, while it is recognized that ascorbate is required for the posttranslational modification of collagen, we showed that ascorbate is required for the expression of type X collagen. This collagen species is unique to cartilage and comprises a major component of the extracellular matrix of both cultured cells and the hypertrophic zone of the growth plate. Second, ascorbate stimulates alkaline phosphatase activity of cells in culture. Although the role of this protein in the mineralization process is not known, there is no doubt that phosphatase activity is required for mineral formation. Third, ascorbate regulates the energy status of the maturing chondrocyte. We have found that in the presence of ascorbate, there is a change in oxidative activity. Thus, while lactate formation is inhibited, there is a decrease in the adenylate energy charge and the redox ratio (NADH/NAD). Recent work indicates that a fourth layer of control may exist. We found that ascorbate induces an increase in oxygen radical metabolism in chondrocyte cultures. Following treatment with ascorbate, there is an increase in thiobarbituric acid-reactive substances (TBARS) synthesis by chondrocytes. Ascorbate supplemented with Fe(II) further increases TBARS production. Experiments performed to determine whether the ascorbate-induced radical activity modulates the synthesis of matrix proteins showed that ascorbate induced changes in the electrophoretic mobility of type II and X collagen. These changes could be partially reversed by the addition of Fe(II) to the culture medium. In summary, these studies indicate that in development and maturation of the cartilage, ascorbate has multiple effects on cellular activity. These effects include modulating chondrocyte energy metabolism, regulating the activity of genes concerned with the synthesis of the extracellular matrix, increasing the activity of alkaline phosphatase, and by serving as a prooxidant and thereby influencing the synthesis of extracellular proteins. Requests for further information should be addressed to Dr. Irving M. Shapiro, Dept. of Biochemistry, School of Dental Medicine, University of Pennsylvania, 4001 Spruce St., Philadelphia, PA 19104-6003. ASCORBIC ACID AND THE IMMUNE SYSTEM Transport and Accumulation of Ascorbic Acid Into Human Neutrophils

P. Washko, D. Rotrosen, and M. Levine Ascorbic acid is found in human neutrophils in high (i.e., millimolar) concentrations, but the precise biochemical role of the vitamin in neutrophils is not well understood. We 260



investigated the transport, accumulation, and distribution of ascorbic acid in isolated human neutrophils by using high-performance liquid chromatography with coulometric electrochemical detection. The effect of extracellular glucose on the uptake and accumulation of ascorbic acid by isolated neutrophils was also studied. Freshly isolated human neutrophils contained 1.0-1.4 mM ascorbic acid, which was localized 94% to cytosol, was not protein bound, and was present only as ascorbic acid and not as dehydroascorbic acid. Transport of ascorbic acid into neutrophils occurred via a high- and a low-affinity transporter. The high-affinity transporter had a Michaelis constant of 2-5 /tM by Lineweaver-Burk and Eadie-Hofstee analyses, and the low-affinity transporter had a Michaelis constant of 6-7 mM by similar analyses. Both transporters were saturable and temperature dependent. Concentration gradients as large as 50-fold were maintained by the two transporters. In normal human blood, the high-affinity transporter should be saturated, while the lowaffinity transporter should be in its linear phase of uptake. Transport of ascorbic acid by both transporters was optimum at an extracellular glucose concentration of 1 mM. Concentrations of extracellular glucose greater than 1 mM had an inhibitory effect on both transporters; however, this inhibition was found to be completely reversible. The inhibitory effect of high concentrations of extracellular glucose on ascorbic acid uptake by neutrophils may have important implications for diabetics whose blood glucose levels are not well controlled. Requests for further information should be addressed to Dr. Philip W. Washko, Laboratory of Cell Biology and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, Bldg. 8, Rm. 419, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892.

Reduced Bactericidal Activity in Neutrophils From Scorbutic Animals and the Effect of Ascorbate on These Target Bacteria In Vivo and In Vitro

Millicent C. Goldschmidt Neutrophils from normal guinea pigs have 16 times more ascorbic acid than do scorbutic counterparts. The deficiency does not alter phagocytosis, but it distorts leukocyte nuclear morphology and reduces chemotactic responses. The ability of the neutrophils to kill cells of Actinomyces viscosus, whether ingested, cell associated, or extracellular, is greatly decreased, dropping from 83% to 12%. The addition of ascorbic acid to the diet of the scorbutic animals reversed these effects. An acridine orange staining technique was used to study leukocyte health and bacterial viability. Actinomyces viscosus and other actinomycetes are oral pathogens that cause root caries and periodontal diseases. In immunocompromised or immunoincompetent hosts, serious systemic diseases can result from infections with these bacteria. Degranulation of leukocytes at the site of an inflammation or infection releases two compounds closely involved with iron metabolism: lactoferrin and ascorbic acid. Iron is an essential mineral for most microorganisms. Their ability to actively acquire iron is crucial to their survival and growth both in vitro and in vivo. It is well known that ascorbic acid complexes with iron. In a modified disc antimicrobial susceptibility assay, ascorbic acid was bactericidal aerobically to 11 actinomycetes and several Gram-negative pathogens in concentrations as low as 12 /tg/ml of medium. Iron salts reversed this bacterial inhibition. Several reducing agents, such as cysteine or dithiothreitol, had no effect. Other iron chelating agents such as 2'2'-bipyridine were inhibitory. Utilizing the fact that pharmacological concentrations of ascorbic acid were inhibitory to bacteria, the oral flora of four Rhesus monkeys was lowered. A marker strain of an Actinomyces viscosus was implanted. The monkeys were given the organism in their drinking water and I g/day of ascorbic acid. Representative plaque and blood samples were taken at Vol. 15, Nos. 3 & 4

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intervals for bacterial counts and agglutinating antibody titers to the actinomycete. The bacterial counts dropped by six logs, and antibody titers also decreased. When the additional vitamin was removed, counts and titers again rose. The teeth of 15 male marmosets {Callitrix jaccus) were cleaned. Baseline gingival and plaque indices as well as initial actinomycete concentrations were obtained. The animals received topical applications of water or 1% ascorbic acid twice daily for 15 weeks and were monitored for oral health, plaque accumulation, and actinomycete count for 29 weeks. The ascorbic acid-treated animals had the lowest gingival indices (the healthiest gingiva). In animals treated with water, plaque increased more rapidly, although it also accumulated in the group treated with ascorbic acid. Actinomycetes increased in plaque samples but were usually 50% lower in the ascorbic acid group than in the water-treated group. When both rinses were stopped, all indices increased rapidly. The essential roles of ascorbic acid in leukocyte function, integrity, and antibacterial effectiveness are thus elaborated. Because pharmacological doses of the vitamin are also bactericidal in vitro, it may be acting similarly in vivo to augment the leukocytes' ability to kill both ingested and extracellular microorganisms (various animal studies point out its importance in increasing oral health and decreasing the presence of target oral pathogens). These multiple functions of ascorbic acid need to be recognized, especially as our population ages. In the presence of subclinical deficiencies of the vitamin, oral and other diseases will increase in severity as a result. Requests for further information should be addressed to Dr. Millicent C. Goldschmidt, Dental Branch, The University of Texas Health Science Center at Houston, PO Box 20068, Houston, TX 77225. Complement Component Clq Activity and Ascorbic Acid Nutriture in Guinea Pigs

Betty E. Haskell and Carol S. Johnston Our studies examined the effect of ascorbic acid nutriture on the concentration of complement component Clq in guinea pig plasma. Clq is a protein with structural similarities to collagen in that it contains a helical region abundant in hydroxyproline residues. Because hydroxyproline requires ascorbate for its biosynthesis, it seemed reasonable to expect that ascorbate nutriture might affect Clq biosynthesis. If so, this could affect host defense. As the recognition unit of the classical complement pathway, Clq binds to pathogens and initiates a sequence of events leading to their lysis. It also acts as an opsonin, thus promoting the destruction of the pathogen by phagocytic cells. Previously, other investigators had shown that Clq (measured indirectly as protein-bound hydroxyproline) was significantly lower in serum of scorbutic guinea pigs than in that of pair-fed controls. Our studies have shown that Clq is 30-50% higher in guinea pigs fed tissue-saturating amounts of ascorbic acid than in those fed only enough ascorbate for adequate growth and prevention of scurvy. This was true whether we quantified Clq by chemical assay (protein-bound hydroxyproline in the euglobulin fraction of plasma), by immunodiffusion against anti-Clq, or by hemolytic assay (testing the ability of experimental serum to restore hemolytic activity to Clq-depleted serum). Working with young male Hartley guinea pigs weighing approximately 500 g, we depleted the animals of ascorbate for 3 weeks to bring ascorbate stores to a uniform minimal level. The animals were then assigned randomly to 4 treatment groups and repleted for 4 weeks with the following daily doses of ascorbic acid (mg/100 g body wt): Group 1, 0.5 (adequate for growth and prevention of scurvy); Group 2, 2.0 (enough for normal incisor development); Group 3, 10 (ample for growth and dental development); and Group 4, 50 (tissue saturating; elevates liver stores to nearly maximal levels).

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Weight gain, food intake, and liver weight did not differ significantly among the repleted groups. However, liver ascorbate (mg/100 g wet wt) differed significantly for each treatment group [p < 0.05, one way analysis of variance and least significant difference (LSD) test]. Values (means ± SE) were as follows: Group 1, 14.8 ± 0.7; Group 2, 21.0 ± 0.8; Group 3, 29.5 ± 1.3; and Group 4, 38.7 ± 2.3. Plasma Clq values (in micrograms per milliliter) were significantly higher in Group 4 animals than in Group 1 animals (p < 0.05, one way analysis of variance and LSD test) regardless of the analytic method used to measure Clq. Values (means ± SE) were as follows: for protein-bound hydroxyproline: Group 1, 54.3 ± 7.0 and Group 4, 83.7 ± 9.1; for immunodiffusion: Group 1, 163 ± 1 1 and Group 4, 245 ± 12; for hemolytic assay: Group 1, 97.5 ± 9.5 and Group 4, 129 ± 13.4. No consistent differences were observed among plasma Clq values for Groups 1, 2, and 3. Whether changes in plasma Clq are physiologically significant in relation to host defense remains to be determined. Requests for further information should be addressed to Dr. Betty E. Haskell, Dept. of Human Ecology, 117 Gearing Hall, University of Texas at Austin, Austin, TX 78712.

CANCER I: TUMOR CELL GROWTH AND MALIGNANT TRANSFORMATION IN VITRO

Mechanisms of Ascorbic Acid-Induced Inhibition of Chemical Transformation in C3H/10T1/2 Cells Luminita L. V. Ibric, Andrew R. Peterson, Alex Sevanian, and William F. Benedict

Ascorbate blocked morphological cell transformation when added as late as 23 days after methylcholanthrene exposure, and this effect, unlike that of vitamin A, was irreversible (Benedict et al., Cancer Res 40, 2796, 1980). Daily treatments with ascorbic acid, isoascorbic acid, or dehydroascorbic acid prevented expression of morphological transformation induced by methylcholanthrene in C3H/10T1/2 cells. To determine the mechanisms of this inhibition, we studied the influx/efflux, the time and dose dependency of the uptake, and the metabolism of ascorbic acid in vitro. We also analyzed the effects of acute or chronic treatments with ascorbic or dehydroascorbic acid on the redox potential (as NAD+/NADH), matrix protein production (such as collagen and glycoproteins), and the total lipid composition of C3H/10T1/2 cells. We found that 16.80 nM ascorbic acid was sufficient to saturate 106 cells, and the same concentration reduced the NADH/NAD + ratio by a factor of three. Daily treatments with 28 nM ascorbic acid for 21 days (a regimen that inhibits chemical oncogenic transformation) kept a steady-state concentration of intracellular ascorbic acid and maintained a reduction by half of the total NADH levels. Using high-performance liquid chromatography and spectrophotometry (Ibric et al., In Vitro 24, 669, 1988), we found that ascorbic acid taken up by cells was converted rapidly to dehydroascorbic acid and released back into the medium as ascorbic acid and with time, was accumulated inside cells as dehydroascorbic acid (which may explain the persistent depletion in NADH observed). Insoluble collagen production, after daily treatments with ascorbic, isoascorbic, or dehydroascorbic acid, was stimulated in a dose-dependent manner; however, only ascorbic and isoascorbic acid but not dehydroascorbic acid stimulation of the collagen synthesis might be correlated with the inhibition of transformation. On the other hand, all three reagents showed a strong association between the stimulation of glycoprotein synthesis

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and the inhibition of transformation. Daily ascorbic acid treatments of 10T1/2 cells also resulted in a gradual accumulation of lipophilic bodies in culture. Both ascorbic and dehydroascorbic acids reduced the total lipids over 21 days. Neutral Iipids were dramatically increased after seven days but were largely released into the media. Significant changes in phospholipids were observed in treated cells. The cholesterol/phospholipid ratios progressively declined for all groups, and an inverse correlation between unsaturation index and cholesterol/phospholipid ratios was apparent. Our data suggest an association between the regulation of the redox potential of the cells and the inhibition of transformation produced by ascorbic, isoascorbic, or dehydroascorbic acid. There may also be a relationship between the inhibition of oncogenic transformation and the changes in the lipid metabolism following chronic treatment with ascorbic or dehydroascorbic acid, but there was no consistent correlation between matrix collagen production and the inhibition of chemical transformation in C3H/10T1/2 cells. Requests for further information should be addressed to Dr. Luminita L.V. Ibric, PO Box 8302, La Crescenta, CA 91214.

Growth Modulation of Human Leukemic and Preleukemic Progenitor Cells by L-Ascorbic Acid

Chan H. Park and Bruce F. Kimler L-Ascorbic acid was shown to promote the in vitro growth of colonies of human and mouse myeloma progenitor/stem cells using a unique cell culture method employing daily feeding of new medium (Park et al., Science 174,720,1971). L-Ascorbic acid was also shown to modulate the in vitro growth of leukemic colony-forming cells (L-CFC) from bone marrow of patients with acute myeloid leukemia (AML) (Park et al., Cancer Res 37, 4595-4601, 1977; Park et al., Cancer Res 40, 1062-1065, 1980; Park et al., Exp Hematol 8, 853-859, 1980). In nearly 500 patients studied, L-ascorbic acid enhanced the growth of L-CFC in 35% of patients and suppressed the growth of L-CFC in 15% of patients (Park et al., Cancer Res 45, 3969-3973, 1985). These two effects were consistently demonstrated in multiple repeat studies of the same patients. In 50 hematologically normal individuals, only 10% showed even a mild suppression of normal CFC (N-CFC), and none showed evidence of enhancement. Dose-response studies indicate that the effective concentration range of added L-ascorbic acid is 0.03-0.3 mM (0.5-5 mg%), with concentrations below 0.01 mM having no apparent effect. This L-ascorbic acid effect persists over an extended period (up to 10 wks) of culture. The modulating effect is specific to L-ascorbic acid because other redox compounds (cysteine) or antioxidants (tocopherol) are without effect. Glutathione is ineffective by itself but does potentiate the L-ascorbic acid effect. Ascorbic oxidase can reverse the L-ascorbic acid-modulating effect, probably because of destruction of residual L-ascorbic acid effect (Park et al., J Nutr Growth Cancer 4, 141-146, 1987). The biologic nature of the L-ascorbic acid effect has been clearly established in a study that showed D-ascorbic acid, the optical isomer of L-ascorbic acid, to be far less effective than L-ascorbic acid. From the cell kinetic standpoint, the L-ascorbic acid effect is cytostatic rather than cytocidal. Leukemic cells are held in a resting stage at unfavorable L-ascorbic acid concentrations, yet can be induced to proliferate on changing to permissive L-ascorbic acid conditions (Park et al., J Nutr Growth Cancer 3, 131-134, 1986; Park et al., Blood 55, 595-601, 1980). The induced proliferation of leukemic cells by L-ascorbic acid appears to be associated with self-renewal rather than with differentiation (Park et al., Exp Hematol 16, 509, 1988). Chronic myelogenous leukemia, as with normal bone marrow, is not affected by L-ascorbic acid. However, preleukemia, that is, myelodysplastic syndrome (MDS), is as

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sensitive as AML to L-ascorbic acid. Further, these L-ascorbic acid effects have prognostic value in MDS: patients who are sensitive to L-ascorbic acid display shorter survival than do patients who are insensitive to L-ascorbic acid; this establishes a clinical implication for this L-ascorbic acid effect. MDS appears to be the ideal disease for clinical trials involving in vivo L-ascorbic acid manipulation to control the disease process. Such a trial is currently being planned. Requests for further information should be addressed to Dr. Chan H. Park, Div. of Oncology/Hematology, Health Sciences Center, Texas Tech University, 3601 4th St., Lubbock, TX 79430.

Ascorbate Stabilizes the Differentiated State and Reduces the Ability of Rous Sarcoma Virus to Replicate and to Uniformly Transform Cell Cultures Downloaded by [New York University] at 17:33 11 February 2015

Richard I. Schwarz

In primary avian tendon cells, Rous sarcoma virus can coexist or completely take over the cell. Infection, at high multiplicity or under conditions that promote high virus production (no ascorbate and high serum levels), results in the oncogenic transformation of almost all the cells in the culture. This is indicated in part by a radical change in morphology, the ability to grow a high cell density, and a dramatic drop in the production of their primary differentiated product, procollagen, from about 50% to about 3% of total protein synthesis. In contrast, infection at low multiplicity (< 1:1), infection with a replication defective virus, or the presence of ascorbate restricts the ability of the virus to transform the culture. Thus, there appears to be a balance between the normal and transformed states of the cell that can be shifted depending on the cellular environment and the level of infection. Ascorbate stabilizes the normal state by reducing virus production and promoting the synthesis of differentiated proteins. Requests for further information should be addressed to Dr. Richard I. Schwarz, Cell and Molecular Biology Div., Lawrence Berkeley Laboratory, Bldg. 83, University of California at Berkeley, 1 Cyclotron Rd., Berkeley, CA 94720.

Effects of Ascorbate on HIV Replication in T-Lymphocyte Cell Lines

S. Harakeh and R.J. Jariwalla

We investigated the effects of ascorbic acid (vitamin C) and its salts and metabolites against human immunodeficiency virus (HIV) in vitro. The cytoxicity of these compounds was assayed by measuring cell viability, host metabolic activity, and rate of protein synthesis in both chronically and acutely HIV-infected cells. Based on these tests, a range of nontoxic concentrations was established for each compound tested: ascorbic acid, sodium ascorbate, and oxidized-C. In chronically infected cells exposed to the highest nontoxic dose for four days at 37°C, reverse transcriptase (RT) activity was reduced by > 99% in the presence of ascorbic acid and about 94% with sodium ascorbate. Under the same conditions, oxidized-C caused an approximately 77% reduction in extracellular RT levels. Furthermore, levels of p24 HIV core antigen in the culture supernatant were reduced approximately 90% by ascorbic acid. In acutely infected cells, ascorbic acid reduced syncytia formation (by approx 93%). Exposure of cell free virus to ascorbate for one day at 37°C had no effect on RT or syncytia formation. Prolonged exposure of virus in the presence of ascorbate for four days

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at 37°C resulted in a drop by a factor of 3-14 in RT activity, compared with a reduction by a factor of 25-172 in extracellular RT released from chronically infected cells. The long-term effects of ascorbate on RT levels were investigated by growing chronically infected cells for 30 days in the presence and absence of ascorbate. Removal of compound at different time intervals resulted in the restoration of virus replication, indicating that the continuous presence of ascorbate is necessary for HIV suppression. Our results demonstrate that ascorbate mediates an anti-HIV effect by diminishing viral protein production in infected cells and RT stability in extracellular virions. Requests for further information should be addressed to Dr. Raxit J. Jariwalla, Head, Viral Carcinogenesis and Immunodeficiency Program, Linus Pauling Institute of Science and Medicine, 440 Page Mill Rd., Palo Alto, CA 94306.


CANCER II: IN VIVO CARCINOGENESIS AND TUMOR GROWTH

Reduced Incidence and Tumor Burden in Spontaneous Mouse Mammary Tumors and UV-Induced Tumors With Increasing Ascorbic Acid

Linus Pauling A few years ago my associates and I, at the suggestion of officers of the National Cancer Institute, carried out two studies on the effects of intake of L-ascorbic acid on the incidence of malignant tumors in mice. The first study (Dunham et al., ProcNatl Acad Sci USA 19,7352-7356, 1982) was of large malignant skin tumors (squamous cell carcinoma) and other lesions in hairless mice (groups of 38-45) intermittently exposed to ultraviolet light over a period of 15 weeks, beginning when the mice were about 10 weeks old. The several groups were given a standard diet with 0%, 0.3%, 5%, and 10% added L-ascorbic acid throughout the study. No lesions developed in unirradiated control groups. The lesions were counted every 14 days for four months, beginning four weeks before the end of the period of irradiation. The observed incidence of lesions of several sizes during successive time periods was analyzed by the statistical method recommended by a committee of the International Agency for Research on Cancer (Lyon, France). A pronounced effect of vitamin C in decreasing the incidence and delaying the onset of the malignant lesions was observed with high statistical significance. By 20 weeks, approximately five times as many mice had developed serious lesions in the zero-ascorbate group as in the high-ascorbate group. The second study (Pauling et al., Proc Natl Acad Sci USA 82, 5185-5189, 1985) was on the incidence of spontaneous mammary tumors in RIII mice in relation to different amounts of L-ascorbic acid (between 0.076% and 8.3%) contained in the food. It involved 10 groups of RIII mice (7 ascorbic acid and 3 control groups) with 50 mice in each group. With an increase in the amount of ascorbic acid, there is a highly significant decrease in the first-order rate constant for appearance of the first spontaneous mammary tumor after the lag time to detection by palpation. There is also an increase in the lag time. Median age at appearance of first tumor was 82.5 weeks in ad libitum controls and 124.9 weeks in the highest-dose ascorbate group. The proportion of mice with tumors was also substantially reduced. The mean body weight and mean food intake were not significantly different for the seven ascorbic acid groups. Striking differences were observed between the 0.76% ascorbic acid (the lowest dose above zero) and the control groups (which synthesize the vitamin): smaller food intake, decreased lag time, and increased rate constant at occurrence of the first mammary tumor. A similar

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effect was involved also in the first study. Consequently, two studies (Tsao and Young, Life Sci 45, 1553-1557, 1989; Tsao et al., JNutr 117, 291-297, 1987) on the effect of exogenous ascorbic acid intake on tissue ascorbate and biosynthesis of ascorbic acid in mice were made. These studies have shown that a small amount of exogenous ascorbic acid intake leads to a decrease in rate of biosynthesis of ascorbic acid and to a decreased level of ascorbic acid in various tissues in mice, probably from a feedback control mechanism that regulates ascorbic acid biosynthesis. This phenomenon, the intake of a small amount of ascorbic acid decreasing the amount of the vitamin in the plasma and tissues of the animal, occurs in mice and possibly in other animals that synthesize ascorbic acid but has no significance for guinea pigs and primates, which do not synthesize the vitamin. It does suggest the need for caution in the design and interpretation of ascorbate studies in species which synthesize ascorbic acid. Requests for further information should be addressed to Dr. Linus Pauling, Linus Pauling Institute of Science and Medicine, 440 Page Mill Rd., Palo Alto, CA 94306. Inhibition by Vitamin C of Incidence and Severity of Renal Tumors Induced by Estradiol or Diethylstilbestrol

Joachim G. Liehr

Chronic administration of estradiol or of the synthetic estrogen diethylstilbestrol (DES) to male Syrian hamsters for 6-8 months induces kidney tumors with 90-100% incidence. Dietary supplementation of vitamin C to such estrogen-treated hamsters decreases the incidence of estrogen-induced carcinogenesis by about 50%. The severity of the disease when measured as number of tumor foci was also decreased in hamsters treated with estradiol plus vitamin C. Additional experiments were carried out to examine the hypothesis that vitamin C reduces genotoxic estrogen metabolites to unreactive intermediates and thus inhibits tumor initiation. In vitro and in vivo, DES is oxidized metabolically to diethylstilbestrol4',4"-quinone (DES Q), which has been shown to bind covalently to DNA. A unique and distinct DNA adduct pattern was observed by 32P-postlabeling analysis in the kidney, liver, and uterus of hamsters treated with a single injection of DES. This set of DNA adducts closely matched patterns generated in vitro by reaction of DES Q with DNA or 2'-deoxyguanosine 3'-monophosphate. In the kidney and liver of hamsters treated with vitamin C and then with a single injection of DES, DES Q metabolite concentrations were decreased by 50% from levels in animals receiving only DES. Correspondingly, concentrations of DES-DNA adducts, which are formed by the reactive metabolite DES Q, were decreased by 70-90% in the liver and kidney of hamsters pretreated with vitamin C compared with animals receiving only a single injection of DES. It is concluded that vitamin C inhibits estrogen-induced carcinogenesis by reducing concentrations of estrogen quinone metabolites and their DNA adducts. Requests for further information should be addressed to Dr. Joachim G. Liehr, Dept. of Pharmacology, University of Texas Medical Branch, 11th and Market Sts., Galveston, TX 77550-2782. Effect of Combined Ascorbic Acid and B 12 on Survival of Mice With Impaired Ehrlich Carcinoma and L1210 Leukemia

Eymard Poydock, Hiroshi Takita, and Ralph Bernacki Previous results with a combination of dehydroascorbic acid (the oxidized form of vitamin C) and hydroxycobalamin (vitamin B12) demonstrated complete inhibition of mitoses in

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Sarcoma 37, Krebs 2, and Ehrlich carcinoma in vivo and in vitro without damage to normal cells (Poydock et al., Exp Cell Biol 47, 210-217, 1979). This study was designed to test the effect of these vitamins on the survival of mice bearing Ehrlich carcinoma and L1210 leukemia. Two identical experiments were conducted with a total of 80 mice. In each assay, 40 DBA/2 mice were implanted intraperitoneally with 0.1 ml (105 cells) taken from a stock mouse. After 24 hours, the mice were divided into 20 test and 20 control. Test mice were injected intraperitoneally with 0.2 ml (0.4 g/kg body wt) of the vitamin mixture for a total of 10 treatments. Results showed a significant increase in survival of treated mice when compared with the controls (p < 0.0001). All control mice were dead by Day 19. More than 50% of the treated mice were alive and without evidence of tumor after 60 days. In vitro findings revealed that the vitamins inhibited mitoses of L1210 leukemia cells, but division of the nonneoplastic L929 cells was not affected. Recent research involved testing a mixture of cobalt ascorbate and vitamin C to prove published evidence that when B ^ is in the presence of vitamin C, a series of reactions takes place during which the cobalt nucleus of B, 2 is released and attaches to one of the carbons of the vitamin C molecule, possibly forming cobalt ascorbate. Cobalt ascorbate was found to inhibit division of Ehrlich ascites tumor cells. Requests for further information should be addressed to Sr. M. Eymard Poydock, Director, Cancer Research Unit, Mercyhurst College, 501 E. 38th St., Erie, PA 16546.

Effect of Ascorbic Acid and Its Synthetic Lipophilic Derivative Ascorbyl Palmitate on Phorbol Ester-Induced Skin Tumor Promotion in Mice

R.C. Smart and C.L. Crawford Utilizing the mouse skin multistage model of carcinogenesis, we examined the effect of topical application of ascorbic acid and ascorbyl palmitate on 12-O-tetradecanoylphorbol-13 acetate (TPA)-induced tumor promotion in 7,12-dimethylbenz[a]anthracene-initiated CD-I mice. A single topical application of TPA resulted in a 45-50% decrease in epidermal ascorbic acid content. Large topical doses of ascorbic acid inhibited TPA-induced tumor promotion and ornithine decarboxylase activity but not epidermal DNA synthesis. Topical application of 6 and 28 jimol ascorbic acid simultaneously with 2 nmol TPA inhibited the average number of tumor-bearing mice by 39% and 75%, respectively, and decreased the numbers of tumor-bearing mice by 3% and 86%, respectively, after 21 weeks of promotion. In contrast, relatively small doses of ascorbyl palmitate had a marked inhibitory effect on TPA-induced ornithine decarboxylase activity, DNA synthesis, and tumor promotion. Topical application of 0.16,0.8, and 4.0 ftmol ascorbyl palmitate simultaneously with 5 nmol TPA markedly inhibited the average number of tumors per mouse by 22%, 50%, and 90%, respectively, and decreased the numbers of tumor-bearing mice by 5%, 18%, and 76%, respectively, after 20 weeks of promotion. To test the hypothesis that the attenuated antitumor activity of ascorbic acid was due to poor cutaneous absorption, we evaluated the effect of dietary ascorbic acid on tumor promotion and associated parameters. Supplementation of drinking water with ascorbic acid (27 mg/ml) for four weeks increased epidermal and hepatic ascorbic acid levels by 50% and 27%, respectively, compared with mice that did not have water supplemented with ascorbic acid. Dietary intake of ascorbic acid inhibited the induction of ornithine decarboxylase by TPA but did not inhibit TPA-induced tumor promotion or epidermal DNA synthesis, in this ascorbate-synthesizing species. Preliminary data suggest that the mice not receiving ascorbic acid developed increased epidermal ascorbic acid levels in response to the tumor-promoting regimen. Requests for further information should be addressed to Dr. Robert C. Smart, Dept. of

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Toxicology, Campus Box 7633, College of Agriculture and Life Sciences, North Carolina State University, Raleigh, NC 27695-7633. Inhibiting Effect of Ascorbic Acid on the Growth of Human Mammary Tumor Xenografts in Mice Constance S. Tsao, Wolcott B. Dunham, and Ping Y. Leung


The effect of ascorbic acid on the growth of human mammary tumor xenografts has been investigated with the six-day subrenal capsule assay method. The results indicated that ascorbic acid administered in the drinking water (1 or 5 g ascorbic acid/1) significantly inhibited the growth of tumor fragments implanted beneath the renal capsule of immunocompetent mice. Tumor growth was also inhibited when ascorbic acid was administered in the diet (50 g ascorbic acid/kg diet) together with cupric sulfate in the drinking water (18 or 90 mg/1) or by intraperitoneal injection of a mixture of ascorbic acid (150 mg/kg body wt/day) and cupric sulfate (3 mg/kg body wt/day). However, administration of ascorbic acid alone in the diet or by injection without the addition of cupric sulfate did not affect the growth of the human mammary tumor fragments within the six-day experimental period. The results support the hypothesis that certain oxidation or degradation products of ascorbic acid were active antineoplastic agents for the human mammary carcinoma studied. A stereoisomer of ascorbic acid, D-isoascorbic acid, which has 5% of the antiscorbutic potency, had similar inhibiting activity. This indicates that the antitumor activity of ascorbic acid was not due to the metabolism of ascorbic acid as a vitamin but was due to its chemical properties. Requests for further information should be addressed to Dr. Constance Tsao, Dept. of Physiochemistry, Linus Pauling Institute of Science and Medicine, 440 Page Mill Rd., Palo Alto, CA 94306.

CANCER III: ADJUVANT OR TOXICITY-REDUCING THERAPEUTIC APPLICATIONS

Interactions Between Ascorbic Acid, Radiation Therapy, and Misonidazole Paul Okunieff

Previous studies have shown that ascorbic acid reduces the toxic effects of radiation and in in vitro studies enhances the effects of certain nitroimidazole drugs. In vivo studies evaluating the direct effects of ascorbic acid alone or in combination with other drugs have been few and contradictory. In this study, we evaluated the effects of ascorbic acid given to animals with established tumors and given (in various doses on an every-other-day basis) to animals commencing with the day of tumor cell inoculation. Early generation isotransplants of a C3H/fSed murine fibrosarcoma were used (designated FSall). Very high drug doses were possible because the ascorbic acid was buffered to pH = 7.35; the maximum dose was 4.5 g/kg body wt. Even at the highest ascorbic acid dose, there was minimal modification in tumor growth rate and no animals were prevented from developing tumors. When ascorbic acid was given immediately preceding irradiation, there was a significant reduction in the radiation reaction incurred by the skin. Specifically, the radiation dose required to obtain a skin desquamation increased from 46.4 Gy to 55.7 Gy. A similar relative

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radioprotection of bone marrow was induced by ascorbic acid (the LD50 increased from 7.2 Gy to 8.5 Gy), suggesting that the radiation tolerance of normal tissues increases by approximately 25%. The mechanism of this improved radiation tolerance is unclear because similar changes in the LD50 can be caused by high-dose hydralazine, and some effect was observed with high-dose mannitol and pentobarbital. It may be that a toxic acidosis induced by high-dose ascorbic acid simply caused a generalized hypoxia. There was, however, some selectivity to the ascorbic acid effect because the radiation dose required to cure half the tumors treated was not increased, and the growth delay induced by subcurative doses of radiation was not modified. When combined with misonidazole, ascorbic acid partially protected the tumor from the radiosensitizing effect of misonidazole. This effect was greater for the normal tissues than for tumor and contradicts the in vitro observations made by others. Presumably, ascorbic acid enhances the metabolism and detoxification of misonidazole in vivo, and the expected interaction of dehydroascorbate with misonidazole is minimal. In summary, ascorbic acid, either directly or indirectly (due to toxicity-induced hypoxia), radioprotected both skin and bone marrow. It was not toxic to tumor and did not radioprotect tumor. In contrast to in vitro studies, it reduced the potency of misonidazole as a radiation sensitizer of tumor. The therapeutic gain of giving combined radiation and ascorbic acid is 1.25. Great caution, however, must be exercised before radiation dose escalation is applied clinically, particularly because there have been no studies on the combined effects of radiation and ascorbic acid on late-responding tissues. Requests for further information should be addressed to Dr. Paul Okunieff, Dept. of Radiation Medicine, Massachusetts General Hospital, Fruit St., Boston, MA 02114.

Ascorbic Acid and Treatment of Experimental Transplanted Melanoma

Gary G. Meadows, Herbert F. Pierson, and Rokia M. Abdallah The direct antitumor activity of sodium ascorbate on primary and metastatic melanoma and the use of ascorbate as an adjuvant to diet and chemotherapy in vivo is reported. Sodium ascorbate (30 mg/ml) supplementation in the drinking water of female B6D2F1 mice inhibited subcutaneous B16 melanoma growth, enhanced levodopa methylester (LDME) chemotherapy, and singly or in combination increased survival of tumor-bearing mice. The effect of ascorbate was most prominent in mice given diets restricted in tyrosine and phenylalanine (restricted diet). Ascorbate supplementation did not modify food consumption in mice fed either a basal or restricted diet, but it partially protected against the LDME-induced decrease in food intake during treatment. Tyrosine and phenylalanine restriction and LDME treatment decreased tumor invasion of B16 melanoma into the pulmonary cavity. In both basal and restricted dietary groups receiving ascorbate, the primary tumor masses were smaller, more well defined, and less invasive. Secondary tumor masses, although few in number, appeared to be encapsulated. The combination of ascorbate and LDME reduced the size and distribution of secondary tumors, with the greatest effect in mice fed the restricted diet. Dehydroascorbate increased tumor growth and decreased survival of B16-bearing mice. Ascorbate supplementation did not alter the numbers of pulmonary tumors that developed after intravenous inoculation of the highly invasive B16-BL6 melanoma into mice fed the basal and restricted diet, but tumors were smaller in ascorbate-supplemented mice fed the restricted diet compared with untreated dietary control mice. Ascorbate augmented the growth inhibitory effect of LDME on lung colony tumors, an effect that was most

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pronounced in mice fed the restricted diet. Female B6D2F1 and both male and female C57BL/6 mice inoculated into the pinna of the ear with B16/BL6 melanoma developed spontaneous lung metastases when fed the basal diet, and the number of lung metastases were greatly inhibited when mice were fed the restricted diet. Ascorbate supplementation did not influence spontaneous metastasis in mice fed the basal diet but almost totally prevented tumor colony formation in mice fed the restricted diet. Sodium ascorbate supplementation doubled plasma ascorbate concentration in mice bearing B16 melanoma. Ascorbate concentration was twofold higher in tumors from mice fed the restricted diet compared with those fed the basal diet. Sodium ascorbate supplementation increased tumor ascorbate 3.7-fold in mice fed basal diet and 5.6-fold in mice fed the restricted diet compared with respective dietary control mice. LDME treatment and ascorbate supplementation alone and in combination increased tumor malondialdehyde levels. Although tumor peroxidation explains in part the activity of ascorbate against B16 melanoma, it does not fully explain the antitumor activity attributable to ascorbate. In summary, ascorbate alone has some inherent antitumor activity against B16 melanoma in vivo. However, its primary effect is as an adjuvant to enhance the antitumor activity of LDME and augment the antimetastatic activity of tyrosine and phenylalanine restriction. Requests for further information should be addressed to Dr. Gary G. Meadows, College of Pharmacy, Washington State University, Pullman, WA 99164-6510. Hypovitaminosis C in Patients Treated With High-Dose Interleukin 2 and Lymphokine-Activated Killer Cells

S.L. Marcus, D.P. Petrylak, J. P. Dutcher, E. Paietta, N. Ciobanu, J. Strauman, P.H. Wiernik, S.H. Hunter, O. Frank, and H. Baker Patients with metastatic malignant melanoma, hypernephroma, and colon carcinoma received a three-phase adoptive immunotherapy protocol. Phase 1 was 105 units (high-dose) interleukin-2 (IL-2) given intravenously every eight hours. Phase 2 was six and one-half days rest and leukopheresis. Phase 3 was four days of high-dose IL-2 plus three infusions of autologous lymphokine-activated killer (LAK) cells. Toxicities of treatment included fever, chills, tachycardia, hypotension, vomiting, diarrhea, and fluid retention. Ascorbic acid is known to be important in cell-mediated immunity, and various reports suggest that it is also depleted during physiologically stressful events. Therefore, we determined plasma ascorbic acid levels in patients before adoptive immunotherapy and before and after Phases 1, 2, and 3 of therapy. Patients entering the trial were not malnourished, and mean plasma ascorbic acid levels before therapy were normal (0.64 ± 0.25 mg/dl). Mean levels dropped by 80% after the first phase of treatment with high-dose IL-2 alone (to 0.13 ± 0.08 mg/dl). Mean plasma ascorbic acid remained at severely depleted scorbutic levels (between 0.08 and 0.13 mg/dl) throughout the remainder of the 15-day treatment. Ascorbic acid levels became undetectable ( 1 mM) in aqueous humor and lens, whereas the levels in nocturnal species such as rat, frog, and the nocturnal mouse are very low.

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The function of ascorbic acid in the different regions of the eye is not really known. It certainly must be important in the metabolic reactions described for other tissues such as in collagen biosynthesis. However, because the concentrations are high, particularly in the lens and aqueous humor, the major function proposed has been that of protection as an antioxidant. Ringvold {Ada Ophthalmol Scand 58, 69-82, 1980) proposed that ascorbic acid protects the eye from ultraviolet radiation. Other studies support a major function of ascorbic acid in protecting the eye from photooxidative damage. Ascorbic acid protects against photoperoxidation in the lens (Varma et al., Ophthalmic Res 14, 167-175, 1987), light-induced retinal damage in rats (Organisciak et al., Invest Ophthalmol Vis Sci 26, 1580-1588, 1985), and ultraviolet-induced eye lens protein damage in guinea pigs (Blondin et al., JFree Rad Biol Med 2, 275-281, 1986). The 35-fold difference in the ascorbic acid levels in aqueous humor of diurnal species of mice relative to that of a nocturnal species also suggests a role in protection against solar radiation (Koskela et al., Invest Ophthalmol Vis Sci 30, 2265-2267', 1989). It is well known that ascorbic acid can also participate in reactions as a prooxidant. A number of recent studies have implicated ascorbic acid in causing protein modifications of lens proteins that accumulate with aging. Ascorbic acid forms covalent adducts with the proteins (Bensch et al., ProcNatl Acad Sci USA 82, 7193-7196, 1985), causes formation of browning products as found in brunescent lenses (Lohmann et al., Exp Eye Res 43, 859-862, 1986), causes precipitation and crosslinking of lens proteins (Ortwerth et al., Exp Eye Res 47, 155-168, 1988), and causes a conversion of the crystallins to more acidic forms as found in aged lenses (Russell et al., FASEB J1, 32-35, 1987; Garland et al., Arch Biochem Biophys 251, 771-776, 1986). The oxidation of ascorbic acid also causes lens epithelial cytotoxicity in vitro (Wolff et al., Exp Eye Res 45, 777-789, 1987). Decreased ascorbate free radical reductase has been correlated with increased insolubilization of lens proteins (Bando et al., Exp Eye Res 50, 779-786, 1990). It is obvious that ascorbic acid must have an important role in protecting the eye against photooxidative and free radical damage. However, as the lens ages and ascorbic acid becomes oxidized or there is a change in the availability of metal to catalyze its oxidation, ascorbic acid may participate in reactions that have a deleterious effect on the lens. Requests for further information should be addressed to Dr. Donita Garland, National Eye Institute, Bldg. 6, Rm. 235, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892.

A Role for Ascorbic Acid in Copper Transport

E.D. Harris and S.S. Percival Scurvy-like symptoms brought on by deficiencies of L-ascorbic acid have also been seen in experimental copper deficiency. This forecasts a possible role for the vitamin in copper metabolism. Earlier, this laboratory showed that copper-deficient chicks injected with CuSO4 to activate lysyl oxidase, a copper-dependent enzyme, responded more strongly to the copper when ascorbate was injected 75 minutes after the copper. Ascorbate given before or with copper lessened the activation response. The enhancement was not seen when D-isoascorbate was used in place of L-ascorbate. It was concluded that ascorbate may have a postabsorption role in copper transport. The site of the vitamin's action was not identified, although a strong rise in ceruloplasmin, the serum copper protein, seemed to correlate with the ascorbate effect. Recently, we studied the transfer of 67Cu from 67Cu-labeled ceruloplasmin to human erythroleukemic K562 cells. This temperature-dependent and ceruloplasmin-dependent

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reaction results in 67Cu accumulating in an acid-resistant compartment of the cell. Transfer of 67Cu from ceruloplasmin to the cells occurred 4-10 times faster when L-ascorbic acid (100 nM) was in the incubation medium. D-Isoascorbic acid was as effective as L-ascorbic acid. Control studies showed that 67Cu-labeled ceruloplasmin did not dissociate spontaneously with up to 1 mM ascorbate. Approximately 20% of the 67Cu absorbed into the cytosol was precipitated by antibodies to Cu.Zn superoxide dismutase. Bathocuproine disulfonate, a chelator of Cu(I), inhibited uptake, suggesting the 67Cu was reduced on dissociating from ceruloplasmin. Present understanding is that L-ascorbic acid (or its epimer) chemically reduces copper atoms in ceruloplasmin, which facilitates breaking the bonds holding copper to the protein. Whether ascorbate reacts directly with ceruloplasmin or in conjunction with a membrane redox system is under investigation. One holds the possibility that physiological exchange of copper atoms in ceruloplasmin with tissue enzymes could depend on the availability of ascorbic acid. Requests for further information should be addressed to Dr. Edward D. Harris, Dept. of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128.

Accuracy and Precision of Ascorbic and Dehydroascorbic Acid Measurement in Human Blood and Plasma

Sam A. Margolis and Regina G. Ziegler

The original purpose of our studies was to develop a reference material that would be suitable for quality control in the measurement of ascorbic acid in human serum or plasma. Preparation of a stable reference pool has been achieved by adding dithiothreitol to ascorbic acid-supplemented plasma. Dithiothreitol can effectively be used for stablizing ascorbic acid standards for high-performance liquid chromatography (HPLC) assays, because ascorbic acid and dithiothreitol can be resolved. However, dithiothreitol interferes with colorimetric assays for vitamin C. We have characterized a HPLC procedure that resolves ascorbic acid from dithiothreitol, as well as from common electrochemically active plasma constituents. This method, when evaluated with the ascorbic acid reference material, is precise, accurate, and free from significant systematic bias. Our results, along with those from a collaborative study, indicate that ascorbic acid in plasma lyophilized with dithiothreitol is stable for at least 80 weeks when stored at -70°C. We used this method to measure the total vitamin C (both ascorbic acid and dehydroascorbic acid) in the plasma of children with juvenile rheumatoid arthritis and observed that the mean plasma vitamin C of 12 patients with pauciarticular disease was 7.0 ± 1 . 7 mg/1, of 14 patients with polyarticular disease was 4.8 ± 1.6 mg/1, and of 8 patients with systemic disease was 3.3 ± 1.6 mg/1. These differences were significant at p = 0.01. The stability of ascorbic acid in blood and plasma can be evaluated by measuring the formation of its initial oxidation product, dehydroascorbic acid, because all degradation of ascorbic acid goes through this pathway. We developed a method to make such measurements. The results of these experiments indicate that both ascorbic acid and dehydroascorbic acid can be accurately assessed and that both compounds are stable at -70°C for a minimum of 90 days when stored in 5% metaphosphoric acid. We measured the ascorbic acid and dehydroascorbic acid concentrations in plasma prepared from whole-blood samples collected from six volunteers and stored as whole blood at 4°C for 24 hours. The most striking observation was the absence of any clear change in the amounts of ascorbic acid and dehydroascorbic acid during the first 6 hours and only a small change in the next 18 hours. This suggests that ascorbic acid may initially be relatively stable in whole blood. This is not the case in plasma or serum where significant ascorbic acid conversion to dehydroascorbic

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acid is observed within the first hour at 4°C. These results suggest that blood collected in the field can be processed up to six hours after collection without modifying the plasma ascorbic acid levels. We are evaluating the effect of storage time, age, race, smoking, recent diet, vitamin C supplementation, and other parameters on serum ascorbic acid and dehydroascorbic acid levels in females selected as controls for a case-control study of cervical cancer. Preliminary results indicate that ascorbic acid is reduced among smokers, relative to nonsmokers; but dehydroascorbic acid is not noticeably altered. In 35 representative women, the serum ascorbic acid and dehydroascorbic acid concentrations were generally similar to those in freshly drawn samples, even though the serum had been stored from four to six years in metaphosphoric acid at — 70° C. This suggests that the ascorbic and dehydroascorbic acid values in the stored samples may represent physiologically meaningful values. Requests for further information should be addressed to Dr. Sam A. Margolis, National Institute of Standards and Technology, Quince Orchard Rd., Gaithersburg, MD 20899.

Concerted Proton/Electron Transfer Between Ascorbic Acid and Cytochrome bs61

David Njus, Vishram Jalukar, Jian Zu, and Patrick M. Kelley Ascorbic acid is required as a reducing agent in a number of vital metabolic reactions, but its reducing power in biologic systems at physiological pH is paradoxical. At alkaline pH, there is a significant concentration of the ascorbate dianion (A=), a powerful electron donor that forms the relatively stable semidehydroascobate radical anion (A—) on oxidation. The concentration of the dianion at neutral and acidic pH, however, is insufficient to account for the rate of reduction supported by ascorbate in biologic systems. The predominant species at physiological pH, the ascorbate monoanion (AH~), is a poor electron donor because loss of an electron yields the energetically unfavorable neutral free radical (AH«). For that reason, ascorbate reduces cytochrome c relatively slowly at low pH. Moreover, this rate is highly pH dependent, increasing by a factor of about 10 with every pH unit. This is consistent with reduction of cytochrome c by the ascorbate dianion. Cytochrome b 561 , responsible for ascorbate regression in adrenal medullary chromaffin vesicles, has a lower midpoint reduction potential than does cytochrome c and should be reduced by ascorbate more slowly. We find, however, that it is reduced much more rapidly. Moreover, the rate of cytochrome b56i reduction is not nearly as pH dependent as would be expected for reduction by the ascorbate dianion. The rate of oxidation of b56i by semidehydroascorbate is also faster than expected when compared with its rate of oxidation by ferricyanide. The anomalously fast rate of electron transfer between cytochrome b 561 and ascorbate/ semidehydroascorbate may be rationalized in terms of a mechanism involving concerted proton/electron transfer. If the ascorbate monoanion donates a proton along with the electron, the reaction yields the semidehydroascorbate anion (A—); this allows the monoanion to act as a much stronger reducing agent. Because this permits reduction of cytochrome b 561 by the abundant ascorbate monoanion, this reaction may occur much more quickly than does reduction by the ascorbate dianion. Moreover, the rate will be much less pH dependent. Concerted proton/electron transfer will also facilitate the oxidation of cytochrome b 561 by semidehydroascorbate because semidehydroascorbate will go to the ascorbate monoanion directly, avoiding the energetically unfavorable ascorbate dianion. We propose that this is a general mechanism of ascorbic acid utilization in biologic systems. Enzymes using ascorbic acid as a reducing agent react with the ascorbate

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monoanion via a mechanism involving concerted proton/electron transfer. The term "concerted proton/electron transfer" is intended to emphasize the distinct roles of H + and e~ and not to convey any mechanistic connotations. In terms of mechanism, one could imagine that when ascorbate passes an electron to the heme, the proton is picked up by a protonatable group on the cytochrome. One could also imagine that H + is transferred not to the protein but to water. Ionic bonding between the ascorbate monoanion and a cationic group in the cytochrome may cause the pK for ascorbate in the reaction complex to be considerably lower than pK2 for free ascorbate. This could substantially increase the dissociation of the second proton from ascorbate in the reaction complex and therefore facilitate electron transfer. Requests for further information should be addressed to Dr. David L. Njus, Dept. of Biological Sciences, Wayne State University, Detroit, MI 48202.


Influence of Dietary Intakes of Erythorbic Acid on Plasma Vitamin C Analyses

H.E. Sauberlich, S.M. Wood, T. Tamura, and L.E. Freeberg

Erythorbic acid (D-isoascorbic acid) is an epimer of L-ascorbic acid and is commonly used as a food additive. Assessment of ascorbic acid nutritional status in the human is generally performed by measurements of the vitamin in plasma. Erythorbic acid lacks antiscorbutic activity. Because all commonly used assays for ascorbic acid measure both isomers equally, inclusion of erythorbic acid in the diet may cause misleadingly high values for vitamin C. We developed a high-performance liquid chromatography amperometric method for the simultaneous determination of ascorbic acid and erythorbic acid (Kutnink et al., / Liquid Chromatogr 8, 31-46, 1985) that was applicable to blood and food samples. In subsequent studies, we observed that supplements to young women of 600 mg/day of erythorbic acid resulted in marked accumulations of erythorbic acid in plasma, red cells, and leukocytes (Sauberlich et al., Am J Clin Nutr 50, 1039-1049, 1989). In a recent study with seven volunteer women, we demonstrated that the level of erythorbic acid present in food items commonly consumed was sufficient to produce an interference in plasma vitamin C analyses. Three meals were prepared (breakfast, lunch, and dinner) that included food items analyzed for their erythorbic acid content. The items selected for inclusion in the meals were ham, bacon, smoked turkey breast, smoked sausage, and Mountain Dew beverage. Each meal provided approximately 650 calories and 3-4 ounces of meat products. The meals were provided at 8 AM, 12 noon, and 6 PM. Blood samples (EDTA treated) were obtained at 10 AM, 2 PM, and the following morning fasting at 8 AM. Analyses revealed that the morning plasma contained an average of 9.14 mg/1 ascorbic acid and 1.29 mg/1 erythorbic acid and that the afternoon plasma contained 9.58 mg/1 ascorbic acid and 1.98 mg/1 erythorbic acid. The fasting morning plasma contained 9.77 mg/1 ascorbic acid and no erythorbic acid. By conventional analysis, the erythorbic acid would have represented 12-17% of the apparent vitamin C. For some subjects, erythorbic acid represented as high as 22% of the apparent vitamin C. As noted in this study and in additional pharmacokinetic studies, erythorbic acid is readily absorbed but rapidly cleared from the plasma. Hence, to avoid misleadingly high ascorbic acid values as a result of erythorbic acid ingestion, the analyses should be conducted on morning fasting blood samples. Requests for further information should be addressed to Dr. Howerde E. Sauberlich, Div. of Experimental Nutrition, Dept. of Nutrition Sciences, UAB Station, University of Alabama at Birmingham, Birmingham, AL 35294.

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Ascorbic Acid, Dehydroascorbic Acid, and Total Vitamin C in Foods: Implications for Diet Studies and Plasma Level Determinations


Joseph T. Vanderslice and Darla J. Higgs A recent survey of foods that constitute the major sources of vitamin C in the American diet yielded information on the total content of this vitamin as well as the amount of its two forms, ascorbic acid and dehydroascorbic acid, in these foods. The liquid chromatographic procedure used allowed for the separation and quantitation of the individual vitamers and included isoascorbic acid as the internal standard. Postcolumn chemistry involving reaction with orthophenylene diamine allowed for sensitive fluorometric detection with a minimum of interferences. Extraction of the vitamer was primarily with metaphosphoric acid with modifications when the samples contained fat or starch. Samples of individual foods showed a surprisingly large range of vitamin content even for foods collected from the same regions of the country and from the same source. The amount of dehydroascorbic acid in the different foods varied from approximately 10-20% of the total vitamin content. The internal standard, isoascorbic acid, added to the samples before extraction showed, for most foods, no evidence of oxidation to dehydroisoascorbic acid, suggesting that no oxidation of ascorbic acid occurred during extraction and analysis. The large range of values for the vitamin content in a given food suggests further that in human diet studies, when the major sources of vitamin C are from a few foods, daily analyses are required for the necessary precision. In a recent diet study, use of US Dept. of Agriculture Handbook 8 values for broccoli would have underestimated the amount of vitamin C being fed. In addition, daily analyses of batches over 21 days gave variances in average daily intake four times higher than would have been estimated from analysis of the variability within a single crate of broccoli. Recent studies on blood and plasma show that, in constrast to foods, substantial amounts of isoascorbic acid are being converted to dehydroisoascorbic acid during extraction and analysis. This indicates that ascorbic acid is also being converted to dehydroascorbic acid during processing, so reported values of dehydroascorbic acid in blood and plasma should be viewed with caution. Requests for further information should be addressed to Dr. Joseph T. Vanderslice, Nutrition Composition Laboratory, Bldg. 161, Rm. 202, BARC East, US Dept. of Agriculture, Beltsville, MD 20705.

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Abstracts of the Seventh Scientific Meeting of The TMJ Association, 7-9 September 2014, Bethesda, Maryland.

Ascorbic acid: biologic functions and relation to cancer.

Abstracts and lectures from the Annual Meeting Sponsored by the Laboratory of Tumor Cell Biology, National Cancer Institute. Bethesda, Maryland, September 1-8, 1991.

Nottingham international breast cancer meeting. 26th-28th September, 1990, Nottingham. Abstracts.

Biophysical Journal: program and abstracts, thirty-fourth annual meeting. February 18-22, 1990, Baltimore, Maryland.

Sixth International Symposium on Biomechanics and Medicine in Swimming. Liverpool, 7-11 September 1990. Abstracts.

September 14, 1990: the beginning.

Abstracts of poster and platform presentations: Fourth International Conference on Computerized and Quantitative EMG. Mainz, Germany, September 13-15, 1990.

Abstracts of poster and platform presentations: 37th annual meeting. American Association of Electrodiagnostic Medicine. Chicago, Illinois, September 7-8, 1990.

vaginal cytologic diagnoses: developed and approved at the National Cancer Institute Workshop in Bethesda, Maryland, December 12-13, 1988.

Proceedings of the NIH Consensus Development Conference on diagnosis and management of asymptomatic primary hyperparathyroidism. Bethesda, Maryland, October 29-31, 1990.

Demyelination--background and mechanisms. Kyoto, Japan, 9-11 September 1990.

Paediatric Research Society. Plymouth, 15 and 16 September 1978: Abstracts.

Abstracts Nederlandse Vereniging voor Thoraxchirurgie: 26 september 2003.

Vitamin C: newer insights into its biochemical functions.

Epidemiologic evidence regarding vitamin C and cancer.

Changing clonal patterns of Salmonella enteritidis in Maryland: evaluation of strains isolated between 1985 and 1990.

Heart disease, cancer, and stroke in Maryland.

[Abstracts of the 24th German Skin Cancer Congress and ADO Annual Meeting, 11-13 September, 2014, Frankfurt, Germany].

Symposium on the physics of electron transport. An international conference. Gaithersburg, Maryland, 2-3 April 1990.

Dietary modulation of cytokine production and biologic functions.

The cancer burden attributable to biologic agents.

Anti-cancer effects of vitamin C revisited.

Vitamin C therapy of advanced cancer.