The influence of dietary protein on ascorbic acid metabolism in rats

1. d-Glucuronolactone reductase, l-gulonolactone oxidase, uronolactonase, dehydroascorbatase, l-gulonate dehydrogenase and l-gulonate decarboxylase have been measured in the tissues of rats fed on diets containing variable amounts of protein. Rats fed on a protein-free or a 2% casein diet for 15 days showed a marked decline in the activities of d-glucuronolactone reductase, l-gulonolactone oxidase, uronolactonase and dehydroascorbatase in the liver, and no change in l-gulonate dehydrogenase and l-gulonate decarboxylase activities in the kidney when compared with rats fed on diets containing 9%, 18% or 25% casein. Giving diets containing 60% or 88% casein to rats did not appreciably alter the activities of uronolactonase, dehydroascorbatase, l-gulonate dehydrogenase and l-gulonate decarboxylase, but inhibited considerably the activities of d-glucuronolactone reductase and l-gulonolactone oxidase in the liver, resulting in decreased synthesis of ascorbic acid. 2. Rats fed on a 25% casein diet showed maximal weight gain, higher tissue reserve of ascorbic acid and higher urinary excretion of both ascorbic acid and glucuronic acid when compared with rats fed on diets containing lower or higher amounts of protein.     

Occurrence in yeast of L-galactonolactone oxidase which is similar to a key enzyme for ascorbic acid biosynthesis in animals, L-gulonolactone oxidase.

l-Galactonolactone oxidase is believed to catalyze the last step of l-ascorbic acid biosynthesis in yeast. A highly purified preparation of this enzyme from baker's yeast was obtained by a seven-step procedure. The molecular weight of the purified enzyme was estimated to be 290,000 by gel filtration, while the dissociated enzyme possessed a molecular weight of 56,000, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzes the reaction, l-galactono-γ-lactone + O₂ → l-ascorbic acid + H₂O₂. l-Gulono- and d-altrono-γ-lactone also serve as substrates. The enzyme was found to contain a flavin which is covalently bound to the enzyme protein. By comparing the properties of this enzyme with those of isofunctional enzymes of higher plants and animals, it became evident that the yeast enzyme is more like the l-gulonolactone oxidase (EC 1.1.3.8) of animals than the l-galactonolactone dehydrogenase (EC 1.3.2.3) of higher plants. Since phylogenetically lower animals are reported to lack l-gulonolactone oxidase, the finding of a similar enzyme in yeast is of great interest.     

Treatment of a metabolic disease, scurvy, by administration of the missing enzyme

Reaction of immunoprecipitated L-gulonolactone oxidase with glutaraldehyde allows multiple administrations of large amounts of this enzyme extracted from either chicken or rats to guinea pigs. L-Gulonolactone oxidase converts L-gulonolactone to ascorbic acid, and its absence from guinea pigs and primates results in their requirement for this vitamin. By administration of this enzyme guinea pigs are able to survive on an ascorbic-acid-deficient regimen.     

Is L-gulonolactone-oxidase the only enzyme missing in animals subject to scurvy?

Evidence has recently appeared implicating an unusual microsomal D-glucuronolactone reductase, which requires carbonyl reagents for activity, in the biosynthesis of ascorbic acid. It was also shown that this microsomal enzyme activity was missing in guinea pigs and primates suggesting that L-gulonolactone oxidase deficiency was not the only defect in animals subject to scurvy. However, we have shown that highly purified L-glulonolactone oxidase catalyzes the conversion of the oxime and semicarbazone of D-glucuronolactone to the corresponding ascorbic acid derivative. There is, therefore, no need to propose a second pathway to ascorbic acid, nor is there evidence for more than the one enzyme defect in scurvy-prone animals.     

Ascorbic acid-independent synthesis of collagen in mice

The mouse has become the most important model organism for the study of human physiology and disease. However, until the recent generation of mice lacking the enzyme gulanolactone oxidase (Gulo), the final enzyme in the ascorbic acid biosynthesis pathway, examination of the role of ascorbic acid in various biochemical processes using this model organism has not been possible. In the mouse, similar to most mammals but unlike humans who carry a mutant copy of this gene, Gulo produces ascorbic acid from glucose. We report here that, although ascorbic acid is essential for survival, its absence does not lead to measurable changes in proline hydroxylation. Vitamin C deficiency had no significant effect on the hydroxylation of proline and collagen production during tumor growth or in angiogenesis associated with tumor or mammary gland growth. This suggests that factors other than ascorbic acid can support proline hydroxylation and collagen synthesis in vivo. Furthermore, the failure of Gulo-/- mice to thrive on a vitamin C-deficient diet therefore suggests that ascorbic acid plays a critical role in survival other than the maintenance of the vasculature.     

Expression of Ascorbic Acid Oxidase in Zucchini Squash (Cucurbita pepo L.)

The expression of ascorbic acid oxidase was studied in zucchini squash (Cucurbita pepo L.), one of the most abundant natural sources of the enzyme. In the developing fruit, specific activity of ascorbic acid oxidase was highest between 4 and 6 days after anthesis. Protein and mRNA levels followed the same trend as enzyme activity. Highest growth rate of the fruit occurred before 6 days after anthesis. Within a given fruit, ascorbic acid oxidase activity and mRNA level were highest in the epidermis, and lowest in the central placental region. In leaf tissue, ascorbic acid oxidase activity was higher in young leaves, and very low in old leaves. Within a given leaf, enzyme activity was highest in the fast-growing region (approximately the lower third of the blade), and lowest in the slow-growing region (near leaf apex). High expression of ascorbic acid oxidase at a stage when rapid growth is occurring (in both fruits and leaves), and localization of the enzyme in the fruit epidermis, where cells are under greatest tension during rapid growth in girth, suggest that ascorbic acid oxidase might be involved in reorganization of the cell wall to allow for expansion. Based on the known chemistry of dehydroascorbic acid, the end product of the ascorbic acid oxidase-catalyzed reaction, we have proposed several hypotheses to explain how dehydroascorbic acid might cause cell wall “loosening.”     

Vitamin C. Biosynthesis, recycling and degradation in mammals

Vitamin C, a reducing agent and antioxidant, is a cofactor in reactions catalyzed by Cu(+)-dependent monooxygenases and Fe(2+)-dependent dioxygenases. It is synthesized, in vertebrates having this capacity, from d-glucuronate. The latter is formed through direct hydrolysis of uridine diphosphate (UDP)-glucuronate by enzyme(s) bound to the endoplasmic reticulum membrane, sharing many properties with, and most likely identical to, UDP-glucuronosyltransferases. Non-glucuronidable xenobiotics (aminopyrine, metyrapone, chloretone and others) stimulate the enzymatic hydrolysis of UDP-glucuronate, accounting for their effect to increase vitamin C formation in vivo. Glucuronate is converted to l-gulonate by aldehyde reductase, an enzyme of the aldo-keto reductase superfamily. l-Gulonate is converted to l-gulonolactone by a lactonase identified as SMP30 or regucalcin, whose absence in mice leads to vitamin C deficiency. The last step in the pathway of vitamin C synthesis is the oxidation of l-gulonolactone to l-ascorbic acid by l-gulonolactone oxidase, an enzyme associated with the endoplasmic reticulum membrane and deficient in man, guinea pig and other species due to mutations in its gene. Another fate of glucuronate is its conversion to d-xylulose in a five-step pathway, the pentose pathway, involving identified oxidoreductases and an unknown decarboxylase. Semidehydroascorbate, a major oxidation product of vitamin C, is reconverted to ascorbate in the cytosol by cytochrome b(5) reductase and thioredoxin reductase in reactions involving NADH and NADPH, respectively. Transmembrane electron transfer systems using ascorbate or NADH as electron donors serve to reduce semidehydroascorbate present in neuroendocrine secretory vesicles and in the extracellular medium. Dehydroascorbate, the fully oxidized form of vitamin C, is reduced spontaneously by glutathione, as well as enzymatically in reactions using glutathione or NADPH. The degradation of vitamin C in mammals is initiated by the hydrolysis of dehydroascorbate to 2,3-diketo-l-gulonate, which is spontaneously degraded to oxalate, CO(2) and l-erythrulose. This is at variance with bacteria such as Escherichia coli, which have enzymatic degradation pathways for ascorbate and probably also dehydroascorbate.     

Peroxisomal fatty acyl-coenzyme A oxidation in chicken liver

The activities of antimycin A-insensitive palmitoyl-CoA oxidation and of palmitoyl-CoA oxidase in peroxisomes from chicken liver were similar to those of rat liver. Catalase and d-amino acid oxidase activities in peroxisomes from chicken liver were lower than those of rat liver and urate oxidase was not detected. Carnitine acetyltransferase and palmitoyltransferase levels in chicken liver were 18- and 2-fold higher, respectively, than those of rat liver. Peroxisomal palmitoyl-CoA oxidation of chicken liver was inhibited by cyanide, in contrast to that of rat liver, although it was insensitive to antimycin A. Subcellular distribution of this enzyme was similar to that of rat liver; i.e., it was located only in the peroxisomes. The fatty acyl-CoA oxidase had a higher affinity toward medium- to long-chain fatty acyl-CoAs (C₈ to C₁₆) than shorter-chain analogs. The fatty acyl-CoA dehydrogenase had a broad affinity toward fatty acyl-CoAs (C₄ to C₁₈). Carnitine acetyltransferase was distributed equally in both peroxisomes and mitochondria. Carnitine palmitoyltransferase was distributed in the proportion of 20 and 80% in peroxisomes and mitochondria, respectively.     

Regulation of ascorbic acid and of xylulose synthesis in rat-liver extracts. The effect of alloxan-diabetes on the glucuronic acid pathway.

1. The synthesis of l-ascorbic acid and of l-xylulose from d-glucuronolactone, d-glucuronate, l-gulonate and l-gulonolactone has been studied with liver extracts from normal and alloxan-diabetic rats. 2. In diabetic animals the synthesis of ascorbic acid is impaired, and more from glucuronolactone and glucuronate than from gulonate and gulonolactone, whereas the formation of xylulose from gulonate and gulonolactone is enhanced. These changes are reversed by insulin therapy. 3. The activity of the NAD-linked gulonate dehydrogenase is enhanced during diabetes.     

Catalytic activity of administered gulonolactone oxidase polyethylene glycol conjugates

Scurvy in guinea pigs provides a convenient model of inborn metabolic disease for the investigation of enzyme therapy protocols. Gulonolactone oxidase, the enzyme in ascorbic acid biosynthesis that is missing from the scurvy-prone species, was modified by attachment of polyethylene glycol. The catalytic properties of this enzyme were affected little by the modification. Intravenous injection of this modified form of the enzyme elicited ascorbic acid synthesis in a dose-dependent manner. The modified enzyme was stabilized to incubation at 37 degrees C but was not protected from inactivation by trypsin. The circulating half-life of enzyme activity was not prolonged by this modification. Further, attachment of polyethylene glycol did neither abolish the enzyme's ability to react with preformed antibodies nor eliminate its immunogenicity.     

Modification of analytical procedures for determining vitamin C enzyme (L-gulonolactone oxidase) activity in swine liver

Modifications of the analytical method to determine L-gulono-gamma-lactone oxidase (EC 1.1.8) enzyme activity were conducted in pig liver by evaluating the concentration of added substrate (L-gulono-gamma-lactone), glutathione, and various tissue sample-to-buffer ratios in the incubation mixture. Sampling different liver sites (lobes), the effect of different cooling temperatures of the liver immediately after collection, and the effect of tissue storage length on subsequent enzyme activity were evaluated. Our results demonstrated that 10 mM of substrate added to the reaction media maximized L-gulono-gamma-lactone oxidase enzyme activity, whereas increasing levels of glutathione did not greatly affect enzyme activity. High sample-to-buffer ratios resulted in higher L-gulono-gamma-lactone oxidase activities but sample analytical variations and background interferences were greater. A 1:4 tissue sample to buffer ratio (weight:weight) resulted in repeatable values, but the importance of maintaining the same ratio of the two components seems to be critical within an experiment. Expressing L-gulono-gamma-lactone oxidase enzyme activity on a liver protein rather than on a liver weight basis also resulted in more consistent results. No difference in liver L-gulono-gamma-lactone oxidase enzyme activities or ascorbic acid concentrations occurred between liver lobes. L-gulono-gamma-lactone oxidase enzyme activity from 0 to 90 day of storage was not affected when tissue samples were immediately frozen in liquid nitrogen, or placed on crushed ice. During a 90-day storage the oxidized form of ascorbic acid (dehydroascorbic acid) decreased (P < 0.01), the reduced (ascorbic acid) form increased (P < 0.01), while total ascorbic acid concentration remained constant.     

Intravascularly administered crosslinked immunoprecipitates of gulonolactone oxidase are toxic and rapidly cleared from the circulation

Glutaraldehyde crosslinked, immunoprecipitated gulonolactone oxidase, injected intraperitoneally, has significant catalytic activity and is capable of providing long-term therapeutic benefit for the enzyme deficiency disease scurvy. The enzyme is tolerated even in repetitive doses. In the present study, however, we have found that when administered intra-arterially this modified enzyme is quite toxic even in single doses. Prior to administration the enzyme complex was filtered through a 5-microns filter. When administered intravascularly the enzyme is not nearly as active catalytically. In spite of this, activity can be detected in vivo as an elevation of plasma ascorbic acid and prolonged survival of guinea pigs fed without the vitamin. Following administration both activity and the enzyme complex are rapidly removed from the circulation. Liver and spleen are largely responsible for this uptake. Because of its toxicity intra-arterial injection of this form of the enzyme does not appear suitable for enzyme therapy.     

Heterologous immunoprecipitates also have potential for therapeutic use

Potential therapeutic usefulness of administered enzymes is limited by toxicity and allergenicity. To overcome these problems we are using scurvy to test various enzyme modifications that may be suitable for therapy. L-Gulonolactone oxidase, which catalyzes the final step in ascorbic acid biosynthesis, is immunoprecipitated with specific antisera from rabbits and then cross-linked with glutaraldehyde. The modified enzyme retains activity sufficient to elicit ascorbic acid synthesis in scorbutic guinea pigs. Intraperitoneal injection of this altered enzyme to animals supplemented with L-gulonolactone increases plasma concentrations of the vitamin. Importantly, multiple doses of the complex are tolerated. Therefore, it is possible to prolong survival time of animals fed an ascorbic acid-deficient diet by this enzyme replacement therapy. This procedure can also be applied to other enzymes that have potential therapeutic use. Serum cholinesterase and asparaginase both retain activity after this modification and are tolerated in single or in weekly repeated injections. Following three or four weekly injections, an anaphylactic reaction to serum but not to enzyme can be elicited if they are injected intravascularly. We conclude that the stability of the immobilized foreign enzyme is a critical factor in lessening the toxicity to multiple injections of these foreign proteins.     

Structural insights into sulfite oxidase deficiency

Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.     

Alpha-tocopherol downregulates gulonolactone oxidase activity in sturgeon

Gulonolactone oxidase (GLO) is the enzyme responsible for the last step of ascorbic acid biosynthesis. The aim of this study was to investigate the effect of dietary alpha-tocopherol and ascorbic acid on GLO activity in a lower vertebrate, the white sturgeon (Acipenser transmontanus). Both alpha-tocopherol and ascorbic acid modulated renal GLO activity. The increase of dietary levels of alpha-tocopherol and/or ascorbic acid significantly raised the liver concentrations of these two antioxidants and concomitantly lowered kidney's GLO activity. The results suggest that the enzyme of ascorbic acid synthetic pathway responded to the animal's antioxidant status and that its activity was downregulated by alpha-tocopherol. This is the first record of alpha-tocopherol being involved in the regulation of ascorbic acid synthesis. This new observation may provide a hypothesis for the evolutionary loss of GLO expression in teleost fishes.     

Reaction Properties of the Ascorbic Acid Oxidase from Myrothecium verrucaria

Ascorbic acid oxidase activity in Myrothecium verrucaria extracts resulted in O(2) uptake exceeding 0.5 mole per mole of ascorbic acid and in CO(2) evolution. Measurement of oxidized ascorbic acid at completion of the reaction demonstrated that an average of 10% of the oxidized product disappeared. A comparison of the gas exchange data with the amount of ascorbic acid not accounted for indicated that the reaction could not be explained by independent oxidase and oxygenase systems. Chromatographic examination of the reaction mixtures identified l-threonic acid. Experiments with ascorbic acid-1-(14)C showed that C-1 was partially decarboxylated during the oxidation. Test of the fungal extracts for enzymes that might explain the deviation from expected stoichiometry showed that phenolase, glutathione reductase, cytochrome oxidase, peroxidase and oxalic decarboxylase were not involved. Addition of azide in concentrations sufficient to block catalase increased excess O(2) consumption about 65%. No enzymes were found that could directly attack oxidized ascorbic acid. H(2)O(2) accumulated during oxidation in azide-blocked systems.The O(2) excess could be explained by assuming the enzyme had peroxidative capacity on a reductant other than ascorbic acid. An intermediate of ascorbic acid oxidation appeared to function as the substrate yielding CO(2) and l-threonic acid on degradation. The increase in excess O(2) utilized in azide-blocked systems and the H(2)O(2) accumulation also were explained by the proposed scheme.Another interpretation would involve production of free radicals during ascorbic acid oxidation. Evidence for this was the ability of extracts to oxidize DPNH in the presence of ascorbic acid. Oxygen radicals formed in such reactions were considered possible agents of degradation of ascorbic acid.     

Biosynthesis of ascorbic acid by extant actinopterygians

Polypterus senegalus , the longnose gar Lepisosteus osseus and the bowfin Amia calva had gulonolactone oxidase activity in the kidney and thus can synthesize ascorbic acid de novo . The enzyme activity was associated with the microsomal fraction. The common carp Cyprinus carpio and the goldfish Carassius auratus had no gulonolactone oxidase activity. Antibodies directed against white sturgeon gulonolactone oxidase showed cross-reactivity with lake sturgeon, bowfin and longnose gar kidney enzymes, but not with enzymes from Polypterus , sea lamprey, and tadpole kidney or pig liver. Given cross-reactivity, gulonolactone oxidase relatedness matched actinopterygian phylogeny, and suggested homology of the character throughout fishes. Modern teleosts may have lost the ability to synthesize ascorbic acid since the late Triassic as a result of a single reversal in the founding population. Wild bowfin and longnose gar exhibited high ascorbate concentrations in liver and spleen when compared with the teleosts rainbow trout Oncorhynchus mykiss and common carp fed vitamin C-supplemented diets.     

The ascorbic acid-dependent oxidation of reduced nicotinamide–adenine dinucleotide by ciliary and retinal microsomes: The effect of some pharmacologically active compounds on this reaction

1. The presence of an ascorbic acid-dependent NADH oxidation in ocular tissues has been established. Subcellular fractionation revealed that the enzyme is localized in the microsomes. The distribution of the enzyme in some ocular tissues has been determined; microsomes from the ciliary processes and the retina have comparable activities, which are much higher than those from the cornea or lens. 2. NADPH cannot replace NADH, and cysteine, reduced glutathione, ergothioneine and dehydroascorbic acid cannot be substituted for ascorbic acid in the reaction. The rate of NADH oxidation was greatly increased in the presence of cucumber ascorbate oxidase, and the enzyme appears to be NADH–monodehydroascorbate transhydrogenase. 3. Cytochrome b₅ is present in retinal microsomes. 4. The enzyme is inhibited by p-chloromercuribenzoate and iodoacetate, but not by cyanide, Amytal or malonate. 5. High concentrations of chloroquine cause a partial inhibition of the reaction, probably owing to interaction of this compound with the enzyme thiol groups. Low concentrations of Diamox, comparable with those attained in tissues during therapy with this drug, bring about partial inhibition of the reaction. Eserine, cortisone, hydrocortisone, 11-deoxycorticosterone and dexamethasone have no effect on the rate of oxidation. 6. The possible role of ascorbic acid and NADH–monodehydroascorbate transhydrogenase in the formation of aqueous humour and secretory mechanisms is discussed.     

A conserved cysteine in molybdenum oxotransferases

The amino acid sequences of peptides derived from rat hepatic sulfite oxidase have been determined by a combination of amino acid analysis and Edman degradation of the purified protein. The data obtained showed the rat liver enzyme contained 3 cysteine residues which was confirmed by thiol modification studies using 4,4'-dithiodipyridine of the native enzyme. Combining these data with that previously published for chicken liver sulfite oxidase (Neame, P. J., and Barber, M. J. (1989) J. Biol. Chem. 264, 20894-20901) indicates that 2 cysteines (Cys186 and Cys430, based upon the numbering for the chicken sequence) are conserved in both chicken and rat liver enzymes with all the cysteine residues being present in the molybdenum-containing domain. Further comparison of the sequences of the molybdenum domains of rat and chicken liver sulfite oxidase with the amino acid sequences published for the molybdenum domains of a variety of assimilatory nitrate reductases suggests that only a single cysteine residue (Cys186) is conserved in all these enzymes, indicating that it may play a role in the binding of Mo-pterin to the protein.     

Determination of ascorbic acid with immobilized green zucchini ascorbate oxidase

Ascorbate oxidase from zucchini squash was immobilized onto CH-Sepharose via carbodiimide. The properties of the immobilized enzyme were found to be similar to those of the free ascorbate oxidase. The immobilized enzyme was utilized in a flow-through system equipped with a polarographic detector which monitors the oxygen depletion due to the reaction ascorbic acid + 1/2 O2----dehydroascorbic acid + H2O. This method, the response of which is linear between 3 X 10(-7) and 5 X 10(-4) M ascorbate, was utilized to measure the ascorbic acid in biological samples such as human plasma and fruit juices at a rate of about 60 determinations every hour with a standard deviation lower than 5%.