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ELSEVIER Topical Vitamin C in Aging ROY M. COLVEN, MD SHELDON R. PINNELL, MD itamin C (ascorbic acid) is essential for life in V humans. Its deficiency causes scurvy. Indeed, the name “ascorbic” is derived from ascorbutic (scorbutus = scurvy). Scurvy was described in writings by the ancient Egyptians, Greeks, and Romans and dur- ing the Crusades in the 13th century. In the 18th cen- tury, Lind, a British naval surgeon, found that citrus fauits rapidly cured the disease common to travelers, , _ ecially seamen.' Today scurvy still exists, not only in Third World countries but also in industrialized na- tions, largely as a result of urban poverty. The groups found to have modern-day scurvy in this country are alcoholics and the institutionalized elderly.” Since the discovery in the 1930s that vitamin C is the antiscorbutic factor, much research has been carried out elucidating vitamin C’s metabolic role and its uses in pharmacologic doses. Pertinent to dermatology are its roles in collagen synthesis and as an antioxidant. Most ‘of the supplementation research has considered in- gested or parenteral vitamin C. A relatively small body of literature has focused on topical application for pho- toprotection and treatment of photoaging. This review will highlight these data. By way of background, we shalll also discuss the biochemistry and the physiologic and pharmacologic effects of vitamin C. Structure and Biochemistry of Ascorbic Acid Chemically, ascorbic acid (AA) is an alpha-ketolactone with the structure shown in Fig. 1. At physiologic pH, AA exists as the monovalent hydroxyl anion. This and its polar moieties restrict ascorbate to aqueous compart- ments. By a stepwise donation of two electrons, ascor- bate is oxidized to dehydro-L-ascorbic acid (DHAA). The intermediate compound after donation of one elec- tron is the ascorbate free radical. Fortunately, as this transient compound is more stable than other free radi- cals, ascorbate is an effective free radical scavenger. DHAA can be reduced to ascorbate; this reaction can be catalyzed by DHAA reductase, or it decays to 2,3- diketogulonic acid, a step in which the lactone ring ir- reversibly opens. From the Division of Dermatology, Duke University Medical Center, Durham, Narth Carolina ‘Address correspondence to Dr. Roy M. Colven, Chief Resident of Der- ‘matology, Division of Dermatology. Duke University Medical Center, Box 3822, Durham, NC 27710. © 1996 by Elsevwer Science Inc 655 Avenue of the Americas, New York, NY 10010 Absorption and Excretion of AA Primates, including humans, and guinea pigs, w other mammals, do not synthesize ascorbate from g cose owing to a deficiency of t-gulono-y-lactone dase, the final enzyme in ascorbate synthesis. Vitamij must then be ingested for survival. Plants, espe citrus fruits and dark-green leafy vegetables, are richest sources. Intestinal absorption is 80-90% effia with normal dietary intake? Ascorbate is actively transported with sodium against an electroch gradient into the intestinal (small bowel) epithelial d Once it is inside, a concentration gradient is created both brush border absorption and intracellular red tion of DHAA to ascorbate. Facilitated diffusion ascorbate through the basolateral membrane is sodi independent and follows a concentration and el chemical gradient. Renal handling of ascorbate is by a similar mech nism, accounting for near-complete resorption of gif merular-filtered compound. Only in excess states high levels of ascorbate and, significantly, one oft metabolites, oxalate, found in the urine. The latteq4 pertinent, as it accounts for one of the few potenti clinical toxicities of systemic vitamin C supplement tion, oxalic acid renal stones. Intestinal absorptive ef ciency likewise declines with repleted or oversatural states. i i \ i 1 | i i i Biochemistry of AA Role in Redox Reactions in General AA’s stepwise oxidation and subsequent regenerati make it an effective reducing agent (electron dot biochemically. Besides its role as a free radical sca ger, AA functions in biologic systems as an enzym cofactor, providing electrons for ferric and cupric 1 talloions. Therefore, ascorbate’s biochemical functiq can be broken down broadly into its participation| enzymatic systems and its antioxidant role. Cofactor functions of AA Ascorbate serves as a cofactor in a number of enz; systems.‘ In general, these enzymes are monooxyg ases or dioxygenases utilizing reduced copper and i metalloions, respectively. For some of the enzyme tems, the need for ascorbate is not absolute. Other ducing agents can serve to keep the metalloion cof 228 COLVEN AND PINNELL guraon etry ne 0 —_ DHAA events of 0 Detvdro-1-ascorbic acid Figure 1. Chemical structure of ascorbic acid. tors in their reduced forms. The enzymes’ activities are ‘most stimulated, however, in the presence of ascorbate. In the case of dioxygenases utilizing Fe" and ascorbate as cofactors, alpha-ketoglutarate and O, are consumed, and succinate and CO, are produced. Often the reaction product entails hydroxylation of the peptide substrate, The general reaction is shown below: Peptide Ascorbate /Fe™* Product eckeloghutsrate (substrate-OH) + . succinate + CO, Hydroxylation of procollagen is one such enzyme system in which ascorbate participates and is of par- ticular importance in dermatology. Collagen is the most abundant animal protein and is uniquely synthesized. AA appears to influence production of collagen by posttranslational and transcriptional mechanisms. One of the best-known functions of AA is as a cofactor iri the hydroxylation of lysine and proline residues of procol- lagen as it is being translated within ribosomes. Three dioxygenase enzymes catalyze these reactions: prolyl hydroxylase, prolyl 4-hydroxylase, and lysyl hydrox- ylase. Formation of 4-hydroxyproline contributes to he- lical stability of the collagen molecule, while hydroxyl- -—ation of lysine allows intermolecular cross-links to form, stabilizing the collagen fiber. The exact function of 3-hydroxyproline is unclear. In all three reactions, as with the several dioxygenase reactions in which ascor- bate participates, ascorbate is consumed nonstoichio- metrically. One explanation for this is that AA is not involved in the substrate/product cycle catalytically utilizing reduced iron.’ Only when a nonhydroxylat- able sequence is encountered does the enzyme-bound Fe** get “wasted” to Fe’, then requiring reduction by AA. In recent years, ascorbate's role in influencing quan- titative collagen synthesis in addition to stimulating qualitative changes to the collagen molecule has been proposed. One way ascorbate may stimulate collagen synthesis is by directly and specifically activating col- lagen gene regulation, both by increasing transcription rate and by stabilizing procollagen mRNA.*” Another controversial mechanism is initiation of lipid peroxida- tion. Here, AA acts in a prooxidant capacity to effect membrane lipid free radical formation (peroxides). This increase in lipid peroxidation leads to an increase in Clinics in Dermatology * 1996;14: malondialdehyde, a product of lipid peroxi¢ which somehow stimulates collagen gene expi This mechanism has recently come into questio ever, as it may occur as an artifact of in vitro § Other examples of enzymatic processes utilizi are carnitine biosynthesis (two ascorbate-associalt droxylases), norepinephrine biosynthesis (AA with copper as a cofactor for a monooxygenasd} vation of a number of peptide hormones by jf amidation (monooxygenase), and tyrosine cata (another Fe”*-hydroxylase).* Antioxidant Function of AA OxIDATIVE STRESS Much of the recent research i tamin C has focused on its role as a free radica | ¢ enger. The term “free radical” as applied to 4 organisms translates to oxygen free radicals. If g cludes excited state oxygen species, such as si gen ('O,), which is not a free radical per se, o more broadly apply the term “reactive oxygen 57 (ROS). Endogenous or physiologic production of RG gins by the partial reduction of O, to the supe| radical, O;-. Single electrons are generated fro riety of physiologic sources, including mitocha electron transport chains, soluble oxidase enzyme! NADPH, as in activated neutrophils), various quit and cytochrome P-450.'° Approximately 1% of duced results in production of O;."" Superoxid mutates to form hydrogen peroxide, H,0,. Hyd} peroxide and O;, in the presence of catalytic ants of iron or copper metalloions, can generate tt droxyl radical (HO"). As free radical species are| ucts of these reactions, which can in turn prop more reactions, a single event can initiate a free chain reaction. Exogenous sources of ROS initiation includ light, ozone, cigarette smoke, and dietary quinondy quinoid drugs." Singlet oxygen is produced by al tion of incident light of particular wavelengths citable endogenous molecules. This energy is transferred to an adjacent triplet (unexcited) 03 molecule, raising O, to the singlet state. This ROS Hf fore exerts its effects primarily in the skin and the | “Oxidative stress” is a term given to the sum to} fects of ROS from these physiologic and exog sources.!? Oxidative damage occurs by a number of 1 nisms.'> DNA takes thousands of oxidative hits day, many of them mutagenic. Structural or enz; proteins can be distorted or inactivated directly b dative damage. This has been causally related to ract formation. Lipid peroxidation entails formatit peroxide radicals along the polyunsaturated fatt} | side chains of lipid. A single initiating event can | many cycles of peroxidation (another free radical Clinics in Dermatology + 1996;14:227-234 reaction), leading in turn to breakdown products that are themselves DNA or protein damaging. Lipid per- oxidation has been linked to atherosclerosis through oxidative damage to low-density lipoprotein (LDL)."* ANTIOXIDANT PROTECTION. Fortunately, elaborate sys- tems to deal with oxidative stress have evolved to main- tain cellular integrity."' Biologic antioxidants can be cat- egorized as enzymatic (superoxide dismutase, catalase, and glutathione peroxidase and reductase) and nonen- zymatic (ascorbate, tocopherol, beta-carotene, reduced coenzyme Q, or ubiquinol, and reduced glutathione). These antioxidant systems act as primary intracellular defenses against reactive oxygen species before their interaction with macromolecules. The enzymatic anti- Adants serve mainly intracellularly, being absent or }- T