Vitamin C: Newer Insights into Its Biochemical Functions Harish Padh, Ph.D. Ever since the discovery of vitamin C (ascorbic acid), scientists have been intrigued as to how ascorbic acid deficiency can lead to the diverse symptoms exhibited in scurvy. Only in recent years has it been appreciated that ascorbic acid has important functions in many cellular reactions and processes in addition to its role in collagen synthesis. The few such reactions that are understood at the molecular level make it apparent that ascorbic acid does not directly participate in enzyme-catalyzed conversion of substrate to product. Instead, the vitamin regenerates prosthetic metal ions in these enzymes in their required reduced forms. This is in agreement with other antioxidant functions of vitamin C, e.g., scavenging of free radicals. Ascorbate and other antioxidant nutrients are presumed to play a pivotal role in minimizing the damage from oxidative products, including free radicals. This protective function is twofold: the already-oxidized groups in prosthetic centers of enzymes are reduced and the oxidants and free radicals are removed.
precise biochemical reactions have not been identified. The relationship between ascorbic acid nutriture and a variety of clinical conditions has been reviewed by Clemet~on.~ The present review will first discuss the clearly defined biochemical steps affected by ascorbic acid and then propose that the prime function of vitamin C is that of an antioxidant.
Vitamin C (ascorbic acid) is an essential nutrient for human beings and a few other species that lack L-gulono-y-lactone oxidase (EC 1.1.3.8), the last enzyme in the ascorbic acid biosynthesis from glucose that converts L-gulono-y-lactone to ascorbic acid.’ Ascorbic acid is synthesized in the liver of mammals capable of its synthesis or in the kidney in reptiles and amphibians.’ Readers are referred to reviews on the metabolism of ascorbate,lS2 its historical background3and cellular function^.^^^ The historic link between collagen synthesis and ascorbic acid has dominated thinking about the biochemical functions of ascorbic acid. In recent years, however, it has been shown that, besides its role in the collagen synthesis, ascorbate is involved in many biochemical steps. In addition, many more physiologic effects of ascorbic acid have been described, but Dr. Padh is Research Associate (Assistant Professor) at the Department of Biochemistry and Molecular Biology, The University of Chicago, 920 East 58th Street, Chicago, IL 60637.
c
Biochemical Functions of Ascorbic Acid A number of dioxygenases that contain prosthetic Fe2+and monooxygenases with prosthetic Cu+ are stimulated by ascorbic acid (see Table 1). Dioxygenases require aketoglutarate and O2 while monooxygenases require O2 as cosubstrates. In spite of the fact that ascorbic acid affects a variety of biochemical processes listed in Table 1, none of these effects is specific to ascorbic acid. Many other reducing agents can replace ascorbic acid, at least to some extent, or partial enzyme activity for a few catalytic cycles can be detected in the absence of ascorbic acid. However, ascorbic acid shows the greatest stimulation of
TABLE 1 Enzymes Stimulated by Ascorbic Acid Enzyme
Process
Collagen synthesis
Carnitine biosynthesis
Metabolism of pyrimidine and its nucleotide in fungi Cephalosporin synthesis Catabolism of tyrosine Norepinephrine biosynthesis Conversion of inactive precursors to active hormones
Proly l-4-hydroxylase Prol yl-3- hydroxy lase Lysyl hydroxylase 6-N-trimethyl-L-l ysine hydroxy lase y-butyrobetaine hydroxylase Thymine 7-hydroxylase Pyrimidine deoxyribonucleoside 2 '-hydroxylase Deacetoxycephalosporin C synthetase 4-hydroxyphenyl pyruvate hydroxylase Dopamine P-monooxygenase Peptidylglycine a-amidating monooxygenase
these activities. As is evident from the discussion of a few examples that follows, the function of ascorbic acid is to provide electrons to keep prosthetic metal ions in their reduced forms. This includes cuprous ions in monooxygenases and ferrous ions in dioxygenases.
Siosynthesis of Co//agen Collagen synthesis is an elaborate process of protein synthesis, posttranslational modifications, protein secretion, and extracellular matrix formation. Collagen is a unique animal protein: up to one-third of its amino acid residues are glycine with an abundance of proline or 4-hydroxyproline and a few residues of 3-hydroxyproline and hydroxylysine. Respective hydroxylases catalyze the hydroxylation of prolyl and lysyl residues in collagen. The well-characterized enzyme requires ferrous pro1y I 4-hydroxyla~e~-~
D.Dtlda
a - KO10
Mono/Diox y genase
huAvmrulrtrA
A.corb.1.
Enzymo-Fa"
88 NUTRfTlON REVlEWSlVOL 49,NO 3lMARCH 1991
+
DiDiDiDi-
Fe2+ Fe2 Fe2+ Fe2+
DiDiDi-
Fe2+ Fe2+ Fez+
Di-
Fez+
Di-
Fe2+
MonoMono-
Copper Copper
+
ion, a-ketoglutarate, oxygen, a proper hydroxyltable substrate peptide, and ascorbate for maximum activity (Figure 1). The P-subunit of the prolyl 4-hydroxylase displays two additional activities: protein disulphide isomerase and thyroid-hormone binding, the significance of which remains unclear. After extensive work in many laboratories, it has become evident that prolyl 4-hydroxylase requires Fe2+ ion. As shown in Figure 1, ascorbate stimulates the enzyme by reducing enzyme-bound Fe3+that forms during occasional futile decarboxylation.1° These observations imply that ascorbic acid is not required for the hydroxylation reaction per se but is required to keep the enzyme-bound iron in the ferrous state. Two hydroxylases involved in carnit ine biosynthesis exhibit similar cofactor requirements and may have similar mechanisms.
ta +
Enzyme-Fa"
Metal Ion
DHA
co,
Figure 1. Overall reaction catalyzed by prolyl4hydroxylase (EC 1.14. 11.2). Suggested role of ascorbate in the reaction is depicted at the bottom.%ee text for details. DHA = dehydroascorbate.
oopnrr p
Dopmlne + Asc + 0,
--
ECl14l71
1
3
Noreplnophrlne + rmlDHA
by dopamine P-monooxygenase in the formation of norepinephrine. A S ~= ascorbic acid; semiDHA = semidehydroascorbate.
precursor molecules that, after a series of modifications, are converted to their active forms. One such final step of activation is a-amidation, which is required for the biologic potency of the peptides. Examples of a-amidated peptides include melanotropins, calcitonin, releasing factors for growth hormone, corticotropin and thyrot ropin, pro-ACTH, vasopressin, oxytocin, cholecystokinin, and gastrin.l4-l6 Peptidy lglycine a-amidating monooxygenase (EC.1.14.17.3), the enzyme that carries out a-amidation, is found in secretory granules in many neuroendocrine tissues, including brain, pituitary, thyroid, and submaxillary gland^.^^^^^ The precursor neuropeptide with glycine at the C-terminal is amidated at the C-terminal by release of glyoxylate and the neuropeptide (Figure 3). A reductant and O2are required for the reaction in vitro and ascorbate is the best reductant. It is not known how a-amidation of neuropeptides is affected in vitamin C deficiency.
In a variety of cell types, ascorbate increases transcription, translation, and stability of mRNA for procollagen. Ascorbate also stimulates secretion of procollagen for the formation of extracellular matrix. This suggests that each step in collagen synthesis, hydroxylation, and secretion is efficiently regulated by the rest of the process. lsl*
Biosynthesis of Norepinephrine Synthesis of norepinephrine and a-amidation of neurohormones are two of the major functions of ascorbate, explaining in part its higher concentrations in brain and endocrine tissues. Dopamine p-hydroxylase (EC 1.14.17.1), present in catecholamine storage granules in nervous tissues as well as in chromaffin cells of adrenal medulla, catalyzes the final and probably rate-limiting step in the synthesis of norepinephrine (Figure 2). Dopamine p-hydroxylase is a tetramer containing two Cu+ ions per monomer that consumes ascorbate stoichiometrically with O2 during its reaction cycle. Recent findings suggest that at a steady state the predominant enzyme form is an enzyme-product complex and that ascorbate reduces copper in this complex.13 Only the reduced enzyme seems to be catalytically competent, with bound cuprous ions as the only reservoir of its reducing equivalents. It is not known how norepinephrine synthesis is affected during vitamin C deficiency.
Antioxidant Role of Ascorbic Acid Free radicals, including hydroxy, hypochorite, peroxy, alkoxy, superoxide, hydrogen peroxide, and singlet oxygen are generated by autoxidation, radiation, or from activities of some oxidases, dehydrogenases, and peroxidases. Additional sources of free radicals are tobacco smoke, hyperoxic air, solvents, pesticides, and certain pollutants, including ozone. Free radicals can be extremely damaging to biologic system~.~' Ascorbic acid, a-tocopherol, and p-carotene are excellent antioxidants and free-
Activation of Honnones Many of the peptide hormones and hormone-releasing factors are synthesized as
""\u M A 01 nml-DtlA
NEUROPEPTIDE-plycln.
+ 0,
Figure 3. a-Amidation of neuropeptides by ascorbate requiring a-amidating monooxygenase. Asc = ascorbic acid; DHA = dehydroascorbate; semiDHA = semidehydroascorbate.
+
NEUROPEPIIDE
Peptidy1. glyone a-amdaltng monooryOInase
c
n,o +
OLIOXILATE
NUTRITION RWIIWSIVOL 49, NO 3IMARCH 1991 67
radical-scavenging nutrients protecting cells from damage by oxidant^.^^-^^ It is important to recall that evolutionary emergence of the ascorbate biosynthetic ability in amphibians suggests that a greater need for ascorbate may have been linked with a step from an aquatic to a terrestrial mode of life, where they faced higher oxygen tension and a hot climate.’ Ascorbic acid can react with and scavenge many types of free radicals, including singlet oxygen, superoxide, and hydroxy radicals. In addition, ascorbate can regenerate the reduced form of a-tocopherol, perhaps accounting for observed sparing effects of these vitamins. A cell’s major defense against free radicals and other oxidative damage includes the antioxidant vitamins like ascorbate and atocopherol, enzymes like catalase and superoxide dismutase, and compounds like glutathione. However, in blood and other extracellular fluids, vitamins C and E are the major antioxidants. Ascorbate can also inactivate and neutralize his tar nine.'^^ Synergistic actions and sparing effects between these vitamins and with trace elements like selenium suggest that the optimum requirement of one nutrient will depend on intake of the rest of the nutrients.
Other Effects of Ascorbic Acid Ascorbic acid is shown to have a number of physiologic effects, mechanisms of
TABLE 2 Detoxification of histamine’.) Phagocytic functions of leukocytesz3 Metabolism of drugs’.u Formation of nitrosamine= Tubulin functionz7 Expression of acetylcholine receptoP Leukotriene biosynthesism Lipid metabolismz Tetrahydrofolatereduction” Immunity” CanceP Diabetic complication^^*^^-^ Cataract formation3s Periodontal d i ~ e a s e ~ . ~ Rheumatoid arthritisM Inf&ons1.3.20.26.37.3a
which are not understood. In addition, the metabolism of ascorbate is altered in some disease condition^.^.^ Beneficial effects of ascorbate supplementation in some disease conditions are reported. It may be added that some of these effects are controversial and may not have been firmly established. A few of these effects are listed in Table 2, with the hope that they may be used as leads to identify yet undiscovered biochemical functions of ascorbic acid. A wealth of information about these and other aspects of ascorbic acid is available.‘-4.21.22
Summary and Conclusion Ascorbic acid seems to affect many enzyme activities and physiologic processes. The available data suggest that perhaps the most significant role of ascorbate is as a reductant that, along with other reducing agents, minimizes damage by oxidative processes. This role includes keeping iron and copper ions in some enzymes in their required reduced form and neutralizing harmful oxidants and free radicals. Several observations support the premise that the prime function of ascorbate is as an antioxidant. 1) From the known molecular mechanisms in two enzymatic reactions discussed in detail earlier, it is apparent that ascorbate reduces metal ions in these enzymes. 2) The lack of specificity: other reducing agents, albeit with less efficiency, can replace ascorbate in enzyme reactions. 3) The lack of any significantly strong binding site or binding affinity between ascorbate and the enzymes explains why other reducing agents can replace ascorbate in these reactions. 4) The antioxidant property of ascorbate, including its ability to scavenge free radicals, has already been established. 5) Compared to the aquatic life amphibians faced, the transition to a terrestrial existence meant excessive oxidative stress in a hot, dry climate, with much higher oxygen tension. This coincided with the evolutionary emergence of amphibians’ ability to synthesize ascorbate. 6) Plants do not spthesize collagen or norepinephrine. Yet plants in general,
and green leaves in particular, are rich in ascorbate. We may recall that chlorophyllcontaining cells carry out potent oxidative reactions known in biology. To keep the right balance of oxidative and reducing forces in tissues is an important service that ascorbate and other antioxidants seem to provide. This also explains to an extent the sparing effects of ascorbic acid, vitamin E, and other antioxidants observed in vivo and in vitro. Present understanding of the biochemical functions of ascorbate does not yet permit a correlation with the pathologic symptoms found in scurvy, an indication of a fundamental gap in the knowledge about its in vivo functions. Therefore, further studies of the antioxidant activity of ascorbate are warranted, especially as it relates to ascorbate deficiency symptoms. In that connection it will be important to understand interactions among various antioxidant nutrients, including ascorbate. 1. Chaterjee IB. Ascorbic acid metabolism. World Rev Nutr Diet 1978;30:69-87 2. Seib PA, Tolbert BM. eds. Ascorbic acid: chemistry. metabolism and uses. Adv Chem Series 200, Washington, DC: American Chemical Society, 1982 3. Clemetson CAB. Vitamin C. Vols. 1-3. Boca Raton, FL: CRC Press, 1988 4. Padh H. Cellular functions of ascorbic acid. Biochem Cell Biol 1990;68:1166-73 5. Englard S, Seifter S.The biochemical functions of ascorbic acid. Ann Rev Nutr 1986;6:365-406 6. Helaakoski T. Vuori K. Myllyla R. Kivirikko KI, Pihlajaniemi T. Molecular cloning of the a-subunit of human prolyl4-hydroxylase:the complete cDNA derived amino acid sequence and evidence for alternative splicing of RNA transcripts. Proc Natl Acad Sci USA 1989;86:4392-6 7. Parkkonen T, Kivirikko KI, Pihlajaniemi T. Molecular cloning of a multifunctional chicken protein acting as the prolyl4-hydroxylase p-subunit. protein disulphide isomerase and a cellular thyroid hormone binding protein. Comparison of cDNAdeduced amino acid sequences with those in other species. Biochem J 1988;256:1005-11 8. Kivirikko KI, Myllyla R, Pihlajaniemi T. Protein hydroxylation: prolyl 4-hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit. FASEB J 1989;3:1609-17 9. Pajunen L, Myllyla R, Helaakoski T. et al. Assign-
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23. c
24.
ment of the gene coding for both the p-subunit of prolyl 4-hydroxylase and the enzyme disulfide isomerase to human chromosome region. Cytogenet Cell Genet 1988;47:37-41 Kivirikko KI, Myllyla R. Post-translational processing of procollagens. Ann NY Acad Sci 1985;460:187-201 Schwarz RI, Kleinman P, Owens N. Ascorbate can act as an inducer of the collagen pathway because most steps are tightly coupled. Ann NY Acad Sci 1987;498:172-85 Kalcheim C, Leviel V. Stimulation of collagen production in vitro by ascorbic acid released from explants of migrating avian neural crest. Cell Differ 1988;22:107-14 Brenner MC. Murray CJ, Klinman JP. Rapid freeze- and chemical-quench studies of dopamine 8-monooxygenase: comparison of presteady-state and steady-state parameters. Biochemistry 1989;28:4656-64 Bradbury AF. Finnie MDA, Smyth DG. Mechanism of C-terminal amide formation by pituitary enzymes. Nature 1982;298:686-8 Glembotski CC. The role of ascorbic acid in the biosynthesis of neuroendocrine peptides a-MSH and TRH. Ann NY Acad Sci 1987;498:54-62 Murthy ASN, Keutmann HT, Eipper BA. Further characterization of peptidylglycine a-amidating monooxygenase from bovine neurointermediate pituitary. Mol Endocrinol 1987;1:290-9 Halliwell B, Gutteridge JMC. Free-radicals in biology and medicine. Oxford, England: Clarenton, 1985 Machlin L. Bendich A. Free radical tissue damage: protective role of antioxidant nutrients. FASEB J 1987;1:441-5 Anderson A, Theron AJ. Physiological potential of ascorbate, p-carotene and a-tocopherol individually and in combination in the prevention of tissue damage, carcinogenesis and immune dysfunction mediated by phagocyte-derivedreactive oxidants. World Rev Nutr Diet 1990;62:27-58 Lunec J, Blake DR. The determination of dehydroascorbic acid and ascorbic acid in the serum and synovial fluid of patients with rheumatic arthritis. Free Radical Res Commun 1985;1:31-9 Lewin S. Vitamin C: its molecular biology and medical potential. New York: Academic Press, 1976 Burns JJ, Rivers JM, Machlin W. eds. Third Conference on Vitamin C. Ann NY Acad Sci 1987; 498:l-538 Shilotri PG. Phagocytosis and leukocyte enzymes in ascorbic acid deficient guinea pigs. J Nutr 1977;107:1513-6 Zannoni VG, Susick RL, Smart RC. Ascorbic acid
25.
26.
27.
28.
29.
30.
as it relates to the metabolism of drugs and environmental agents. Curr Concepts Nutr 1984;13: 21 -35 Horio F, Ozaki K. Kohmura M. Yoshida A, Makino S, Hayashi Y. Ascorbic acid requirement for the induction of microsomal drug-metabolizing enzymes in a rat mutant unable to synthesize ascorbic acid. J Nutr 1986;116:2278-89 Tannenbaum S. Wishnok JS. Inhibition of nitrosamine formation by ascorbic acid. Ann NY Acad Sci 1987;498:354-63 Nath J, Gallin JI. Effect of vitamin C on tubulin tyrosinolation in polymorphonuclear leukocytes. Ann NY Acad Sci 1987;498:216-28 Knaack D, Podleski TR. Salpeter MM. Ascorbic acid and acetylcholine receptor expression. Ann NY Acad Sci 1987;498:77-89 Schmidt KH, Steinhilber D, Moser U, Roth HJ. Lascorbic acid modulates 5-lipoxygenase activity in human polymorphonuclear leukocytes. Int Arch Allergy Appl lmmunol 1988;85:441-5 Stone KJ, Townsley BH. The effect of L-ascorbate on catecholamine biosynthesis. Biochem J 1973;131:611-3
70 NUTRlTlON REVIEWSIVOL 49, NO 3IMARCH 1991
31. Anderson R. The immunostimulatory, anti-inflammatory and anti-allergic properties of ascorbate. Adv Nutr Res 1984;6:19-45 32. Wittes RE. Vitamin C and cancer. N Engl J Med 1985;312:178-9 33. Pecoraro RE, Chen MS. Ascorbic acid metabolism in diabetes mellitus. Ann NY Acad Sci 1987;498:248-58
34. McLennan S, Yue DK, Fisher E, et al. Deficiency of ascorbic acid in experimental diabetes. Diabetes 1988;37:359-61 35. Gerster H. Antioxidant vitamins in cataract prevention. 2.Ernahrungswiss 1989;28:56-75 36. Aleo JJ. Diabetes and periodontal disease: possible role of vitamin C deficiency. J Periodontol 1981;52:251-4 37. Padh H. Aleo JJ. Activation of serum complement leads to inhibition of ascorbic acid transport. Proc SOCExp Biol Med 1987;185:153-7
38. Padh H, Aleo JJ. Ascorbic acid transport by 3T6 fibroblasts: regulation by and purification of human serum complement factor. J Biol Chem 1989 ;264 :6065- 9
precise biochemical reactions have not been identified. The relationship between ascorbic acid nutriture and a variety of clinical conditions has been reviewed by Clemet~on.~ The present review will first discuss the clearly defined biochemical steps affected by ascorbic acid and then propose that the prime function of vitamin C is that of an antioxidant.
Vitamin C (ascorbic acid) is an essential nutrient for human beings and a few other species that lack L-gulono-y-lactone oxidase (EC 1.1.3.8), the last enzyme in the ascorbic acid biosynthesis from glucose that converts L-gulono-y-lactone to ascorbic acid.’ Ascorbic acid is synthesized in the liver of mammals capable of its synthesis or in the kidney in reptiles and amphibians.’ Readers are referred to reviews on the metabolism of ascorbate,lS2 its historical background3and cellular function^.^^^ The historic link between collagen synthesis and ascorbic acid has dominated thinking about the biochemical functions of ascorbic acid. In recent years, however, it has been shown that, besides its role in the collagen synthesis, ascorbate is involved in many biochemical steps. In addition, many more physiologic effects of ascorbic acid have been described, but Dr. Padh is Research Associate (Assistant Professor) at the Department of Biochemistry and Molecular Biology, The University of Chicago, 920 East 58th Street, Chicago, IL 60637.
c
Biochemical Functions of Ascorbic Acid A number of dioxygenases that contain prosthetic Fe2+and monooxygenases with prosthetic Cu+ are stimulated by ascorbic acid (see Table 1). Dioxygenases require aketoglutarate and O2 while monooxygenases require O2 as cosubstrates. In spite of the fact that ascorbic acid affects a variety of biochemical processes listed in Table 1, none of these effects is specific to ascorbic acid. Many other reducing agents can replace ascorbic acid, at least to some extent, or partial enzyme activity for a few catalytic cycles can be detected in the absence of ascorbic acid. However, ascorbic acid shows the greatest stimulation of
TABLE 1 Enzymes Stimulated by Ascorbic Acid Enzyme
Process
Collagen synthesis
Carnitine biosynthesis
Metabolism of pyrimidine and its nucleotide in fungi Cephalosporin synthesis Catabolism of tyrosine Norepinephrine biosynthesis Conversion of inactive precursors to active hormones
Proly l-4-hydroxylase Prol yl-3- hydroxy lase Lysyl hydroxylase 6-N-trimethyl-L-l ysine hydroxy lase y-butyrobetaine hydroxylase Thymine 7-hydroxylase Pyrimidine deoxyribonucleoside 2 '-hydroxylase Deacetoxycephalosporin C synthetase 4-hydroxyphenyl pyruvate hydroxylase Dopamine P-monooxygenase Peptidylglycine a-amidating monooxygenase
these activities. As is evident from the discussion of a few examples that follows, the function of ascorbic acid is to provide electrons to keep prosthetic metal ions in their reduced forms. This includes cuprous ions in monooxygenases and ferrous ions in dioxygenases.
Siosynthesis of Co//agen Collagen synthesis is an elaborate process of protein synthesis, posttranslational modifications, protein secretion, and extracellular matrix formation. Collagen is a unique animal protein: up to one-third of its amino acid residues are glycine with an abundance of proline or 4-hydroxyproline and a few residues of 3-hydroxyproline and hydroxylysine. Respective hydroxylases catalyze the hydroxylation of prolyl and lysyl residues in collagen. The well-characterized enzyme requires ferrous pro1y I 4-hydroxyla~e~-~
D.Dtlda
a - KO10
Mono/Diox y genase
huAvmrulrtrA
A.corb.1.
Enzymo-Fa"
88 NUTRfTlON REVlEWSlVOL 49,NO 3lMARCH 1991
+
DiDiDiDi-
Fe2+ Fe2 Fe2+ Fe2+
DiDiDi-
Fe2+ Fe2+ Fez+
Di-
Fez+
Di-
Fe2+
MonoMono-
Copper Copper
+
ion, a-ketoglutarate, oxygen, a proper hydroxyltable substrate peptide, and ascorbate for maximum activity (Figure 1). The P-subunit of the prolyl 4-hydroxylase displays two additional activities: protein disulphide isomerase and thyroid-hormone binding, the significance of which remains unclear. After extensive work in many laboratories, it has become evident that prolyl 4-hydroxylase requires Fe2+ ion. As shown in Figure 1, ascorbate stimulates the enzyme by reducing enzyme-bound Fe3+that forms during occasional futile decarboxylation.1° These observations imply that ascorbic acid is not required for the hydroxylation reaction per se but is required to keep the enzyme-bound iron in the ferrous state. Two hydroxylases involved in carnit ine biosynthesis exhibit similar cofactor requirements and may have similar mechanisms.
ta +
Enzyme-Fa"
Metal Ion
DHA
co,
Figure 1. Overall reaction catalyzed by prolyl4hydroxylase (EC 1.14. 11.2). Suggested role of ascorbate in the reaction is depicted at the bottom.%ee text for details. DHA = dehydroascorbate.
oopnrr p
Dopmlne + Asc + 0,
--
ECl14l71
1
3
Noreplnophrlne + rmlDHA
by dopamine P-monooxygenase in the formation of norepinephrine. A S ~= ascorbic acid; semiDHA = semidehydroascorbate.
precursor molecules that, after a series of modifications, are converted to their active forms. One such final step of activation is a-amidation, which is required for the biologic potency of the peptides. Examples of a-amidated peptides include melanotropins, calcitonin, releasing factors for growth hormone, corticotropin and thyrot ropin, pro-ACTH, vasopressin, oxytocin, cholecystokinin, and gastrin.l4-l6 Peptidy lglycine a-amidating monooxygenase (EC.1.14.17.3), the enzyme that carries out a-amidation, is found in secretory granules in many neuroendocrine tissues, including brain, pituitary, thyroid, and submaxillary gland^.^^^^^ The precursor neuropeptide with glycine at the C-terminal is amidated at the C-terminal by release of glyoxylate and the neuropeptide (Figure 3). A reductant and O2are required for the reaction in vitro and ascorbate is the best reductant. It is not known how a-amidation of neuropeptides is affected in vitamin C deficiency.
In a variety of cell types, ascorbate increases transcription, translation, and stability of mRNA for procollagen. Ascorbate also stimulates secretion of procollagen for the formation of extracellular matrix. This suggests that each step in collagen synthesis, hydroxylation, and secretion is efficiently regulated by the rest of the process. lsl*
Biosynthesis of Norepinephrine Synthesis of norepinephrine and a-amidation of neurohormones are two of the major functions of ascorbate, explaining in part its higher concentrations in brain and endocrine tissues. Dopamine p-hydroxylase (EC 1.14.17.1), present in catecholamine storage granules in nervous tissues as well as in chromaffin cells of adrenal medulla, catalyzes the final and probably rate-limiting step in the synthesis of norepinephrine (Figure 2). Dopamine p-hydroxylase is a tetramer containing two Cu+ ions per monomer that consumes ascorbate stoichiometrically with O2 during its reaction cycle. Recent findings suggest that at a steady state the predominant enzyme form is an enzyme-product complex and that ascorbate reduces copper in this complex.13 Only the reduced enzyme seems to be catalytically competent, with bound cuprous ions as the only reservoir of its reducing equivalents. It is not known how norepinephrine synthesis is affected during vitamin C deficiency.
Antioxidant Role of Ascorbic Acid Free radicals, including hydroxy, hypochorite, peroxy, alkoxy, superoxide, hydrogen peroxide, and singlet oxygen are generated by autoxidation, radiation, or from activities of some oxidases, dehydrogenases, and peroxidases. Additional sources of free radicals are tobacco smoke, hyperoxic air, solvents, pesticides, and certain pollutants, including ozone. Free radicals can be extremely damaging to biologic system~.~' Ascorbic acid, a-tocopherol, and p-carotene are excellent antioxidants and free-
Activation of Honnones Many of the peptide hormones and hormone-releasing factors are synthesized as
""\u M A 01 nml-DtlA
NEUROPEPTIDE-plycln.
+ 0,
Figure 3. a-Amidation of neuropeptides by ascorbate requiring a-amidating monooxygenase. Asc = ascorbic acid; DHA = dehydroascorbate; semiDHA = semidehydroascorbate.
+
NEUROPEPIIDE
Peptidy1. glyone a-amdaltng monooryOInase
c
n,o +
OLIOXILATE
NUTRITION RWIIWSIVOL 49, NO 3IMARCH 1991 67
radical-scavenging nutrients protecting cells from damage by oxidant^.^^-^^ It is important to recall that evolutionary emergence of the ascorbate biosynthetic ability in amphibians suggests that a greater need for ascorbate may have been linked with a step from an aquatic to a terrestrial mode of life, where they faced higher oxygen tension and a hot climate.’ Ascorbic acid can react with and scavenge many types of free radicals, including singlet oxygen, superoxide, and hydroxy radicals. In addition, ascorbate can regenerate the reduced form of a-tocopherol, perhaps accounting for observed sparing effects of these vitamins. A cell’s major defense against free radicals and other oxidative damage includes the antioxidant vitamins like ascorbate and atocopherol, enzymes like catalase and superoxide dismutase, and compounds like glutathione. However, in blood and other extracellular fluids, vitamins C and E are the major antioxidants. Ascorbate can also inactivate and neutralize his tar nine.'^^ Synergistic actions and sparing effects between these vitamins and with trace elements like selenium suggest that the optimum requirement of one nutrient will depend on intake of the rest of the nutrients.
Other Effects of Ascorbic Acid Ascorbic acid is shown to have a number of physiologic effects, mechanisms of
TABLE 2 Detoxification of histamine’.) Phagocytic functions of leukocytesz3 Metabolism of drugs’.u Formation of nitrosamine= Tubulin functionz7 Expression of acetylcholine receptoP Leukotriene biosynthesism Lipid metabolismz Tetrahydrofolatereduction” Immunity” CanceP Diabetic complication^^*^^-^ Cataract formation3s Periodontal d i ~ e a s e ~ . ~ Rheumatoid arthritisM Inf&ons1.3.20.26.37.3a
which are not understood. In addition, the metabolism of ascorbate is altered in some disease condition^.^.^ Beneficial effects of ascorbate supplementation in some disease conditions are reported. It may be added that some of these effects are controversial and may not have been firmly established. A few of these effects are listed in Table 2, with the hope that they may be used as leads to identify yet undiscovered biochemical functions of ascorbic acid. A wealth of information about these and other aspects of ascorbic acid is available.‘-4.21.22
Summary and Conclusion Ascorbic acid seems to affect many enzyme activities and physiologic processes. The available data suggest that perhaps the most significant role of ascorbate is as a reductant that, along with other reducing agents, minimizes damage by oxidative processes. This role includes keeping iron and copper ions in some enzymes in their required reduced form and neutralizing harmful oxidants and free radicals. Several observations support the premise that the prime function of ascorbate is as an antioxidant. 1) From the known molecular mechanisms in two enzymatic reactions discussed in detail earlier, it is apparent that ascorbate reduces metal ions in these enzymes. 2) The lack of specificity: other reducing agents, albeit with less efficiency, can replace ascorbate in enzyme reactions. 3) The lack of any significantly strong binding site or binding affinity between ascorbate and the enzymes explains why other reducing agents can replace ascorbate in these reactions. 4) The antioxidant property of ascorbate, including its ability to scavenge free radicals, has already been established. 5) Compared to the aquatic life amphibians faced, the transition to a terrestrial existence meant excessive oxidative stress in a hot, dry climate, with much higher oxygen tension. This coincided with the evolutionary emergence of amphibians’ ability to synthesize ascorbate. 6) Plants do not spthesize collagen or norepinephrine. Yet plants in general,
and green leaves in particular, are rich in ascorbate. We may recall that chlorophyllcontaining cells carry out potent oxidative reactions known in biology. To keep the right balance of oxidative and reducing forces in tissues is an important service that ascorbate and other antioxidants seem to provide. This also explains to an extent the sparing effects of ascorbic acid, vitamin E, and other antioxidants observed in vivo and in vitro. Present understanding of the biochemical functions of ascorbate does not yet permit a correlation with the pathologic symptoms found in scurvy, an indication of a fundamental gap in the knowledge about its in vivo functions. Therefore, further studies of the antioxidant activity of ascorbate are warranted, especially as it relates to ascorbate deficiency symptoms. In that connection it will be important to understand interactions among various antioxidant nutrients, including ascorbate. 1. Chaterjee IB. Ascorbic acid metabolism. World Rev Nutr Diet 1978;30:69-87 2. Seib PA, Tolbert BM. eds. Ascorbic acid: chemistry. metabolism and uses. Adv Chem Series 200, Washington, DC: American Chemical Society, 1982 3. Clemetson CAB. Vitamin C. Vols. 1-3. Boca Raton, FL: CRC Press, 1988 4. Padh H. Cellular functions of ascorbic acid. Biochem Cell Biol 1990;68:1166-73 5. Englard S, Seifter S.The biochemical functions of ascorbic acid. Ann Rev Nutr 1986;6:365-406 6. Helaakoski T. Vuori K. Myllyla R. Kivirikko KI, Pihlajaniemi T. Molecular cloning of the a-subunit of human prolyl4-hydroxylase:the complete cDNA derived amino acid sequence and evidence for alternative splicing of RNA transcripts. Proc Natl Acad Sci USA 1989;86:4392-6 7. Parkkonen T, Kivirikko KI, Pihlajaniemi T. Molecular cloning of a multifunctional chicken protein acting as the prolyl4-hydroxylase p-subunit. protein disulphide isomerase and a cellular thyroid hormone binding protein. Comparison of cDNAdeduced amino acid sequences with those in other species. Biochem J 1988;256:1005-11 8. Kivirikko KI, Myllyla R, Pihlajaniemi T. Protein hydroxylation: prolyl 4-hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit. FASEB J 1989;3:1609-17 9. Pajunen L, Myllyla R, Helaakoski T. et al. Assign-
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