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The AKR1A1 protein is a member of the aldo-keto reductase superfamily that is responsible for the conversion of D-glucuronate to L-gulonate in the ascorbic acid (vitamin C) synthesis pathway. In a pCAG-eGFP transgenic mouse line that was produced by pronuclear microinjection, the integration of the transgene resulted in a 30-kb genomic DNA deletion, including the Akr1A1 gene, and thus caused the knockout (KO) of the Akr1A1 gene and targeting of the eGFP gene. The Akr1A1 KO mice (Akr1A1eGFP/eGFP) exhibited insufficient serum ascorbic acid levels, abnormal bone development and osteoporosis. Using micro-CT analysis, the results showed that the microarchitecture of the 12-week-old Akr1A1eGFP/eGFP mouse femur was shorter in length and exhibited less cortical bone thickness, enlargement of the bone marrow cavity and a complete loss of the trabecular bone in the distal femur. The femoral head and neck of the proximal femur also showed a severe loss of bone mass. Based on the decreased levels of serum osteocalcin and osteoblast activity in the Akr1A1eGFP/eGFP mice, the osteoporosis might be caused by impaired bone formation. In addition, administration of ascorbic acid to the Akr1A1eGFP/eGFP mice significantly prevented the condition of osteoporotic femurs and increased bone formation. Therefore, through ascorbic acid administration, the Akr1A1 KO mice exhibited controllable osteoporosis and may serve as a novel model for osteoporotic research.

ENA-actimineral resource A (ENA-A) is an alkaline mineral water and has a few biological activities such as antioxidant activity. The aim of this study was to examine the effects of ENA-A on lifespan in mice using senescence marker protein-30 knockout mice. The present study had groups of 18-week-old mice (n = 24), 26-week-old mice (n = 12), and 46-week-old mice (n = 20). Each differently aged mice group was divided into three subgroups: a control group, a 5 % ENA-A-treated group, and a 10 % ENA-A-treated group. Mice in the 18-week-old group were treated with vitamin C drinking water 1.5 g/L. However, the mice in the 26-week-old and 46-week-old groups were not treated with vitamin C. The experiments were done for 18 weeks. All vitamin C-treated mice were alive at week 18 (100% survival rate). In the non-vitamin C group, the 10% ENA-A-treated mice were alive at week 18. The control and 5% ENA-A-treated mice died by week 15. As expected, vitamin C was not detected in the non-vitamin C-treated group. However, vitamin C levels were increased in an ENA-A dose-dependent manner in the vitamin C-treated group. In the TUNEL assay, a number of positive hepatocytes significantly decreased in an ENA-A dose-dependent manner. Periodic acid Schiff positive hepatocytes were significantly increased in an ENA-A dose-dependent manner. In addition, the expression level of CuZnSOD was increased by the ENA-A treatment. These data suggest that the intake of ENA-A has a critical role in the anti-aging mechanism and could be applied toward the lifespans of humans.

Aim: l-ascorbic acid (vitamin C) insufficiency is considered one of the major risk factors for the development of liver disease. However, its specific effects and related mechanisms in vivo are largely unknown. The objective of this study was to investigate the in vivo protective role of vitamin C and its related mechanisms in liver injury with Gulo(-/-) mice that cannot synthesize vitamin C like humans due to the lack of l-gulonolactone-γ-oxidase (Gulo), an essential enzyme for vitamin C synthesis., Results: When liver injury was induced in Gulo(-/-) mice by injection of concanavalin A (Con A), there was greater extensive liver damage accompanied by an increased number of apoptotic hepatocytes in vitamin C-insufficient Gulo(-/-) mice. Additionally, the plasma and hepatic levels of the proinflammatory cytokines, such as TNF-α and IFN-γ, were much higher in the vitamin C-insufficient Gulo(-/-) mice than in the control mice. Moreover, increased numbers of liver-infiltrating T-cells in the vitamin C-insufficient Gulo(-/-) mice were related to the increased hepatic levels of IFN-inducible factor (IP-10). Although the vitamin C-insufficient Gulo(-/-) mice had higher amounts of interleukin-22 (IL-22), a hepatoprotective cytokine, a defect in IL-22Rα expression and its downstream STAT3 activation in hepatocytes were found., Innovation: We first demonstrate the novel in vivo action mechanisms of vitamin C on the prevention of disease development in the liver, through the regulation of excessive immune activation and maintenance of the IL-22Rα signaling pathways., Conclusion: These results suggest that severe liver damage induced by inflammation could be prevented by sufficient supplementation with vitamin C.

Organisms using oxygen for aerobic respiration require antioxidants to balance the production of reactive oxygen species during metabolic processes. Various species--including humans and other primates--suffer mutations in the GULO gene encoding L-gulono-γ-lactone oxidase; GULO is the rate-limiting enzyme in the biosynthesis of ascorbate, an important cellular antioxidant. Animals lacking the ability to synthesize vitamin C develop scurvy without dietary supplementation. The Gulo-/- knockout (KO) mouse requires oral supplemental vitamin C; without this supplementation the animal dies with a scorbutic condition within several weeks. Vitamin C is known to be most abundant in the brain, where it is believed to play important roles in neuroprotection, neurotransmission and neuromodulation. We therefore hypothesized that ascorbate deficiency in Gulo-/- KO mice might lead to an abnormal behavioral phenotype. We established the amount of ascorbate in the drinking water (220 ppm) necessary for generating a chronic low-ascorbate status in the brain, yet clinically the mice appeared healthy throughout 100 days postpartum at which time all behavioral-phenotyping tests were completed. Compared with Gulo+/+ wild-type littermates, ascorbate-deficient Gulo-/- mice were found to be less active in moving in their environment; when in water, these mice swam more slowly in some tests, consistent with a mild motor deficit. We found no evidence of cognitive, anxiety or sensorimotor-gating problems. Despite being less active, Gulo-/- mice exhibited exaggerated hyperactivity to the dopaminergic agonist methamphetamine. The subnormal movement, combined with hypersensitivity to a dopamine agonist, point to developmental ascorbate deficiency causing long-term striatal dysfunction., (© 2012 The Authors. Genes, Brain and Behavior © 2012 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.)

Background and Purpose: Vascular tolerance to nitroglycerin (GTN) may be caused by impaired GTN bioactivation due to inactivation of mitochondrial aldehyde dehydrogenase (ALDH2). As relaxation to GTN is reduced but still sensitive to ALDH2 inhibitors in ascorbate deficiency, we compared the contribution of ALDH2 inactivation to GTN hyposensitivity in ascorbate deficiency and classical in vivo nitrate tolerance., Experimental Approach: Guinea pigs were fed standard or ascorbate-free diet for 2 weeks. Reversibility was tested by feeding ascorbate-deficient animals standard diet for 1 week. Nitrate tolerance was induced by subcutaneous injection of 50 mg x kg(-1) GTN 4 times daily for 3 days. Ascorbate levels were determined in plasma, blood vessels, heart and liver. GTN-induced relaxation was measured as isometric tension of aortic rings; vascular GTN biotransformation was assayed as formation of 1,2- and 1,3-glyceryl dinitrate (GDN)., Key Results: Two weeks of ascorbate deprivation had no effect on relaxation to nitric oxide but reduced the potency of GTN approximately 10-fold in a fully reversible manner. GTN-induced relaxation was similarly reduced in nitrate tolerance but not further attenuated by ALDH inhibitors. Nitrate tolerance reduced ascorbate plasma levels without affecting ascorbate in blood vessels, liver and heart. GTN denitration was significantly diminished in nitrate-tolerant and ascorbate-deficient rings. However, while the approximately 10-fold preferential 1,2-GDN formation, indicative for active ALDH2, had been retained in ascorbate deficiency, selectivity was largely lost in nitrate tolerance., Conclusions and Implications: These results indicate that nitrate tolerance is associated with ALDH2 inactivation, whereas ascorbate deficiency possibly results in down-regulation of ALDH2 expression.

Oxidative stress is implicated in the cognitive deterioration associated with normal aging as well as neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. We investigated the effect of ascorbic acid (vitamin C) on oxidative stress, cognition, and motor abilities in mice null for gulono-gamma-lactone oxidase (Gulo). Gulo-/- mice are unable to synthesize ascorbic acid and depend on dietary ascorbic acid for survival. Gulo-/- mice were given supplements that provided them either with ascorbic acid levels equal to- or slightly higher than wild-type mice (Gulo-sufficient), or lower than physiological levels (Gulo-low) that were just enough to prevent scurvy. Ascorbic acid is a major anti-oxidant in mice and any reduction in ascorbic acid level is therefore likely to result in increased oxidative stress. Ascorbic acid levels in the brain and liver were higher in Gulo-sufficient mice than in Gulo-low mice. F(4)-neuroprostanes were elevated in cortex and cerebellum in Gulo-low mice and in the cortex of Gulo-sufficient mice. All Gulo-/- mice were cognitively normal but had a strength and agility deficit that was worse in Gulo-low mice. This suggests that low levels of ascorbic acid and elevated oxidative stress as measured by F(4)-neuroprostanes alone are insufficient to impair memory in the knockouts but may be responsible for the exacerbated motor deficits in Gulo-low mice, and ascorbic acid may have a vital role in maintaining motor abilities.

The absence of L-ascorbic acid (L-AA, or AA) synthesis in scurvy-prone organisms, including humans, other primates, guinea pigs, and flying mammals, was traced to the lack of L-gulonolactone oxidase (GULO) activity. GULO is a microsomal enzyme that catalyzes the terminal step in the biosynthesis of L-AA. Clinical cases of scurvy were described in a family of Danish pigs. This trait is controlled by a single autosomal recessive allele designated od (osteogenic disorder). Here we demonstrate that the absence of GULO activity and the associated vitamin C deficiency in od/od pigs is due to the occurrence of a 4.2-kbp deletion in the GULO gene. This deletion includes 77 bp of exon VIII, 398 bp of intron 7 and 3.7 kbp of intron 8, which leads to a frame shift. The mutant protein is truncated to 356 amino acids, but only the first 236 amino acids are identical to the wild-type GULO protein. In addition, the od allele seems to be less expressed in deficient and heterozygous pigs compared with the normal allele in heterozygous and wild-type animals as determined by ribonuclease protection assay. We also developed a DNA-based test for the diagnosis of the deficient allele. However, we failed to identify the mutated allele in other pig populations.

l-Gulono-gamma-lactone oxidase (GULO) is a critical enzyme present in most mammalian species that is required for the terminal step in vitamin C biosynthesis. Primates are absolutely dependent on exogenously supplied dietary vitamin C due to inactivation of the Gulo gene by mutation over 40 million years ago. In this study, we report the cloning and expression of the murine l-gulono-gamma-lactone oxidase cDNA and gene. The cDNA (2.3 kb) encodes an open reading frame of 440 amino acids that shows high homology to the rat l-gulono-gamma-lactone oxidase (>94%). The Gulo gene is 22 kb long and contains 12 exons. The 11 introns range in size from 479 to 5641 bp. Northern blot analysis revealed high expression of Gulo transcript in the liver. To investigate whether metabolic loss of vitamin C biosynthesis in human cells can be corrected by heterologous expression of GULO, we constructed a first-generation adenoviral vector expressing the murine GULO cDNA under the transcriptional control of the murine cytomegalovirus (MCMV) early promoter. Low rescue efficiency of Gulo-expressing adenoviral constructs and reduced viral growth in HEK293 cells were observed, suggesting that overexpression of Gulo may be inhibitory to cell growth. Placement of a removable stuffer fragment flanked by lox sites between the MCMV promoter and the Gulo gene resulted in efficient vector rescue and normal viral replication in parental HEK293 cells and high-level expression of Gulo in HEK293 cells expressing Cre recombinase. Cells infected with Gulo-expressing vectors overexpressed an FAD-containing protein that corresponded in size to that predicted for recombinant GULO protein and expressed a functional enzyme as measured by the conversion of l-gulono-gamma-lactone to ascorbic acid in cell-free extracts. The cloning of the murine Gulo cDNA and the construction of Gulo-expressing adenoviral vectors are vital steps toward determining the role of vitamin C in basic metabolism and in disease.

By inactivating the gene for L-gulono-gamma-lactone oxidase, a key enzyme in ascorbic acid synthesis, we have generated mice that, like humans, depend on dietary vitamin C. Regular chow, containing about 110 mg/kg of vitamin C, is unable to support the growth of the mutant mice, which require L-ascorbic acid supplemented in their drinking water (330 mg/liter). Upon withdrawal of supplementation, plasma and tissue ascorbic acid levels decreased to 10-15% of normal within 2 weeks, and after 5 weeks the mutants became anemic, began to lose weight, and die. Plasma total antioxidative capacities were approximately 37% normal in homozygotes after feeding the unsupplemented diet for 3-5 weeks. As plasma ascorbic acid decreased, small, but significant, increases in total cholesterol and decreases in high density lipoprotein cholesterol were observed. The most striking effects of the marginal dietary vitamin C were alterations in the wall of aorta, evidenced by the disruption of elastic laminae, smooth muscle cell proliferation, and focal endothelial desquamation of the luminal surface. Thus, marginal vitamin C deficiency affects the vascular integrity of mice unable to synthesize ascorbic acid, with potentially profound effects on the pathogenesis of vascular diseases. Breeding the vitamin C-dependent mice with mice carrying defined genetic mutations will provide numerous opportunities for systematic studies of the role of antioxidants in health and disease.

Vitamin C deficient pigs, when fed a diet lacking L-ascorbic acid (AscA), manifest deformity of the legs, multiple fractures, osteoporosis, growth retardation and haemorrhagic tendencies. This trait was shown by others to be controlled by a single autosomal recessive allele designated as od (osteogenic disorder). The inability of AscA biosynthesis in primates and guinea pigs that exhibit similar symptoms, when they are not supplemented with AscA in the food, was traced to the lack of L-gulono-gamma-lactone oxidase, which catalyzes the terminal step in the biosynthesis of AscA. The non-functional GULOP was mapped to human chromosome 8p21 that corresponds to an evolutionarily conserved segment on either porcine chromosome 4 (SSC4) or 14 (SSC14). We investigated linkage between OD and SSC4- and 14-specific microsatellite loci in order to map the OD locus. Twenty-seven informative meioses in families from one sire and three dams revealed linkage of od with microsatellites SW857 and S0089, located in the subcentromeric region of SSC14. We isolated part of the GULO gene of the pig by screening a porcine genomic library using a pig GULO cDNA as a probe, and mapped it to SSC14q14 by fluorescence in situ hybridization (FISH). Thus, the porcine GULO gene is both a good physiological and positional candidate gene for vitamin C deficiency in pigs.

The contents of ascorbic acid (AsA) and erythrobic acid (ErA) in the tissues of guinea pigs intraperitoneally injected with AsA and/or ErA were determined to learn the difference in their retention in the tissues. After 10 d of AsA depletion, the guinea pigs were intraperitoneally injected with 5 mg of AsA, or 5 mg of ErA, or 5 mg of each. At day 5 of repletion, the guinea pigs were killed and liver, adrenal glands, spleen, and kidneys were removed. AsA and ErA in these tissues were measured by using HPLC. The contents of AsA in the tissues of only the AsA-injected guinea pigs were similar to those of the AsA- + ErA-injected guinea pigs. The contents of ErA in the tissues of the ErA-injected guinea pigs were higher than those of the AsA- + ErA-injected guinea pigs, but apparently lower than the contents of AsA in the AsA-injected guinea pigs. ErA was scarcely retained in the tissues of guinea pigs.

The effects of ascorbic acid (AA) or vitamin C in atherosclerosis has attracted considerable attention; however results of clinical studies are conflicting. Several studies indicate an increase in plasma triglyceride (TG) and cholesterol (CH) levels in guinea pigs (GP) that have been fed a diet containing a minimal amount of AA. Previous studies carried out in GP fed a diet devoid of AA showed a significant decrease in cytochrome P-450 level compared to GP fed high and adequate amounts; however, the level of cytochrome P-450 in the two groups were not significantly different. The enzymes that synthesize TG and CH are located in endoplasmic reticulum which is also the site for cytochrome P-450 synthesis. It is of interest to determine whether there is an association between TG and CH synthesis and cytochrome P-450 induction. Adult male Hartley GP weighing 350-400 g were fed a diet containing 2.5% (Group I), 0.1% (Group II) and 0% (Group III) AA. The food consumption and weight gain were not significantly different in different groups. After feeding the diet for four weeks, half of the animals in each group were starved. Blood was withdrawn and TG and CH were determined in the serum. TG and CH were markedly elevated in both starved and nonstarved Group III GP; however, these levels were not altered in Group 1 and Group II GP. Plasma AA showed significant differences in all three nonstarved and starved groups. Plasma alpha-lipoprotein was decreased and beta-lipoprotein was increased in Group III GP. Hepatic CH and TG were also significantly elevated in Group III GP, and Groups I and II showed no changes. TG and CH showed a negative correlation with cytochrome P-450, whereas CH and TG showed a positive correlation. We conclude that AA deficiency causes extensive hyperlipidemia, feeding high level of AA does not alter the lipid metabolism and induction ofcytochrome P-450 is inversely related to TG and CH synthesis.

The cytochrome P-450 monooxygenase system plays a central role in the oxidation of a wide variety of structurally unrelated compounds. Its contribution is affected by nutritional and several other factors. Ascorbic acid (AA) deficiency decreases the content of cytochrome P-450 in liver microsomes of guinea pigs (GPs). Included in the group of cytochromes P-450 are the phenobarbital and 3-methylcholanthrene inducible moieties. In the present study the effect of AA status on another specific cytochrome P-450, CYP4A1, laurate omega-hydroxylase was investigated. Ascorbic acid may selectively increase or decrease certain forms of cytochromes. For four weeks adult male Hartley GPs were fed a diet containing 2.5 (Group I), 0.1 (Group II) and 0% (Group III) AA. The liver microsomes were isolated at this stage and cytochrome P-450 content was determined. Group III showed a significant decrease in cytochrome P-450 compared to groups I and II. They also showed a marked decrease in aminopyrine N-demethylase activity. The expression of CYP4A1 was evaluated using Western blot and anti-CYP4A1 antibody. Group III GPs showed a marked decrease in CYP4A1 expression. Groups I and II showed similar expression. This study demonstrates that CYP4A1, a specific cytochrome induced by hypolipidemic agents, is decreased by AA deficiency.

The goal of this study was to test the hypothesis that the ratio of liver to bone alkaline phosphatase in blood plasma reflects the ascorbate status in scurvy-prone teleost fish (rainbow trout [Oncorhynchus mykiss]). The studies focused on finding a method for distinguishing bone alkaline phosphatase present in blood plasma from other alkaline phosphatase isoforms. We tested temperature optima and thermostability of liver, kidney, gill cartilage, and intestinal alkaline phosphatases. We did not observe differences among liver, bone, and kidney enzymes with respect to temperature optima and thermostability. We partially purified alkaline phosphatase from juvenile rainbow trout vertebrae and liver using n-butanol solubilization and ammonium sulfate fractionation. We found a difference between bone alkaline phosphatase, which precipitated in 0%-20% ammonium sulfate saturation, and liver enzyme, which required 40%-50% ammonium sulfate saturation to precipitation. We conducted a series of urea inactivation studies on partially purified enzymes from liver and vertebrae. Urea differentially inhibited the enzymes with t 1/2 = 1.1 and 0.4 min, for bone and liver, respectively. Subsequently, we subjected blood plasma alkaline phosphatase to urea inhibition, and using regression analysis we calculated the ratio of liver to bone alkaline phosphatase. We found that thus obtained ratios of bone enzyme in blood plasma correlated with liver ascorbate concentration. Bone alkaline phosphatase declined in ascorbate deficiency 10-fold, whereas low ascorbate status resulted in a 3.5-fold decrease. In order to draw a general conclusion on the linearity of the response of blood plasma/bone alkaline phosphatase as an indicator of ascorbate deficiency in fish, further studies must include analysis of individual fish followed in the process of developing avitaminosis.

Ascorbic acid (AsA) deficiency causes a decrease in hepatic concentration of cytochrome P-450 and a decrease in hepatic activity of drug-metabolizing enzymes in rats unable to synthesize AsA (ODS rats). To study the mechanism of the decrease in hepatic concentration of cytochrome P-450 isozymes by AsA deficiency, we chose the xenobiotics-inducible cytochrome P-450 and performed the experiments indicated below. AsA-deficient rats were fed polychlorinated biphenyls (PCB) which markedly induce both CYP1A subfamily and several isozymes in CYP2B subfamily. First, we assayed the activities of two drug-metabolizing enzymes so that one could be functionally distinguished from another. AsA deficiency significantly reduced the hepatic activity of aminopyrine-N-demethylase in ODS rats with and without dietary PCB, but had no effect on benzo(a)pyrene hydroxylase activity. Secondly, quantitative immunoblot analyses demonstrated that the levels of CYP2B1/2B2 and CYP1A1 in the AsA-deficiency rats fed PCB were approximately 60 and 80% lower than those found in rats fed AsA-supplemented diet. The degree of reduction in CYP2B1/2B2 was greater than CYP1A1. Thirdly, AsA deficiency caused a decrease in hepatic abundance of CYP2B1/2B2 mRNA, whereas it had no effect on the levels of CYP1A1 and 1A2 mRNA. These results indicated that dietary AsA selectively affects the levels of CYP2B1/2B2 mRNA among cytochrome P-450 induced by PCB and plays important roles for optimum induction of drug-inducible cytochrome P-450. We concluded that AsA deficiency decreases specific froms of drug-inducible cytochrome P-450, especially CYP2B1/2B2 and that the reduction of CYP2B1/2B2 mRNA level in AsA-deficient rats caused a decrease in cytochrome P-450 concentration and hepatic activity of drug-metabolizing enzymes.

Because ascorbic acid is an important contributor to the oxidant defense system in body tissues, we studied the effects of a low dietary intake of ascorbic acid on various indicators of oxidant defense and oxidant damage. During a 13-wk study eight healthy men (25-43 y), residing in a live-in metabolic unit, were fed controlled diets containing different amounts of ascorbic acid for four consecutive periods: period 1 = 250 mg/d for 4 d; period 2 = 5 mg/d for 32 d; period 3 = 10 or 20 mg/d for 28 d and period 4 = 60 or 250 mg/d for 28 d. Measurements were made at several time intervals of the activities of glutathione peroxidase and superoxide dismutase in RBC, DNA strand breaks in mononuclear leucocytes, glutathione concentrations in plasma and RBC and NAD and NADP in RBC. After 60 d of low ascorbic acid intakes and associated with plasma ascorbic acid levels less than 6 mumol/L, the total glutathione concentration and the reduced glutathione:oxidized glutathione ratio were decreased in plasma. At the same time NAD and NADP levels in RBC were elevated. It seems that chronic marginal vitamin C deficiency states may be associated with selected biochemical changes in oxidant defense indices.

The effect of lipid peroxidation in vitro on the amounts of several forms of cytochrome P-450 in liver microsomes from guinea-pigs was investigated. Lipid peroxide formation in liver microsomes from ascorbic acid (VC)-deficient animals was much higher than that observed in control animals. The antibodies to rat P-450IA2 (P-448-H), P-450IIB1 (P-450b) and human P-450IIIA4 (P-450NF) recognized one or two forms of cytochrome P-450 in liver microsomes of guinea-pigs. Neither cytochrome P-450 cross-reactive with anti-P-450IIB1 antibodies nor cytochrome P-450 cross-reactive with antibodies to P-450IIIA4 was virtually affected by microsomal lipid peroxidation induced by NADPH in vitro. In contrast, the forms of cytochrome P-450 immunochemically related to P-450IA2 were decreased with the increased level of lipid peroxide formation. The form-specific degradation of cytochrome P-450 due to lipid peroxidation was in agreement with our previous observation that the amounts of cytochrome P-450 cross-reactive with antibodies to P-450IA2 but not with antibodies to P-450IIIA (P-450PB-1) were predominantly decreased in VC-deficient guinea-pigs compared to control animals in vitro.

Ascorbic acid (VC) deficiency resulted in a decrease in the activities of aminopyrine N-demethylase, aniline hydroxylase, and p-nitroanisole O-demethylase and in the content of cytochrome P-450, as spectrally determined, whereas it caused an increase in the activities of 6 beta-hydroxylases for testosterone and progesterone in liver microsomes of guinea pigs. Western blot analysis of liver microsomes with antibodies to rat P-448-H (P-4501A2), P-450j (P-450IIE), P-450 PB-1 (P-450IIIA), and P-450b (P-450IIB1) showed that VC deficiency decreased the amount of cytochrome P-450 immunochemically related to P-450IA2 and P-450IIE but did not change the amount of the form that was cross-reactive with antibodies to P-450IIB1 and tended to slightly increase (not statistically significantly) the amount of the form of the cytochrome immunochemically related to P-450IIIA. The larger decrease by VC deficiency in the amount of cytochrome P-450 that was cross-reactive to the rat P-450IA2 resulted in a lower capacity of liver microsomes to activate promutagens, such as 2-amino-3-methyl-imidazo(4,5-f)quinoline and aflatoxin B1. These results indicate that VC deficiency in guinea pigs differentially affects the content of individual forms of cytochrome P-450.

The effect of diet on induction of monooxygenases and distribution of label from 2-14C-lysine in fractions of liver homogenate, muscle homogenate and blood of male rats treated with phenobarbital (80 mg/kg, three days) was studied. 2-14C-lysine was injected intraperitoneally 24 h before the first injection of phenobarbital. It was demonstrated that monoxygenase induction, increase of relative liver weight and incorporation of label from 2-14C-lysine into fractions of liver homogenate in phenobarbital-treated rats were more pronounced as compared with the similarly treated rats that were fed a balanced diet. The possibility of mobilization of deficient essential components to liver from other organs and tissues for maintenance of monoxygenase induction is discussed.

The effect of dietary ascorbate on hepatic UDP glucuronyltransferase (UDPGT) appears to be selective in that only certain isozymes of UDPGT are jeopardized. In this study, ascorbic acid deficiency produced a 68% reduction in the specific activity of hepatic UDPGT towards p-nitrophenol. Earlier studies showed a reduction in UDPGT activity towards p-aminophenol in ascorbate-deficient guinea pigs, whereas bilirubin and acetaminophen glucuronidation were unaffected. Kinetic studies suggest that p-aminophenol and p-nitrophenol are metabolized by a single isozyme in that p-nitrophenol was found to be a competitive inhibitor of p-aminophenol glucuronidation. Both qualitative and quantitative studies on partially purified UDPGT from ascorbate-deficient and ascorbate-supplemented guinea pigs were carried out to investigate the biochemical role of the vitamin. Qualitative differences were observed in UDPGT from ascorbate-deficient animals and included an increased lability to: thermal inactivation; storage at 4 degrees; and purification with UDP-glucuronic acid agarose column chromatography. Furthermore, an analysis of the microsomal membrane showed a 14% increase in membrane fluidity in ascorbate deficiency. Ascorbic acid added in vitro could not reverse the increase in fluidity observed in ascorbate-deficient microsomal membranes; however, ascorbylpalmitate, a more lipophilic form of the vitamin, was effective. Palmitic acid had no effect on membrane fluidity in microsomes from either the ascorbate-supplemented or ascorbate-deficient animals. This increase in membrane fluidity could not be explained by differences in cholesterol, total phospholipid, or phosphatidylcholine content of hepatic microsomes. Furthermore, a quantitative reduction in UDPGT partially purified from ascorbate-deficient guinea pigs was indicated by a marked reduction in protein banding at 55,000 daltons when compared to UDPGT partially purified from ascorbate-supplemented animals.

The effects of L-ascorbic acid deficiency on guinea pig hepatic and brain lysosomal hydrolases were examined. In general, hepatic beta-N-acetylhexosaminidase, beta-D-glucoronidase, alpha-D-galactosidase, alpha-D-mannosidase, and acid phosphatase were elevated in scorbutic animals. This appears to be independent of the starved state. Brain beta-D-glucoronidase and acid phosphatase followed a similar pattern to that observed with the liver enzymes, but brain beta-N-acetylhexosaminidase was not affected by L-ascorbic acid decreased the activity of hepatic beta-N-acetylhexosaminiadase was unaffected by dietary treatments although the activity of beta-N-acetylhexosaminidase A tended to increase in the scorbutic animals. Subcellular fractions were obtained from the three groups of animals and the recoveries of protein, beta-N-acetylhexosaminidase, and glucose-6-phosphatase estimated.

Balanced diet during 60-day hypokinesia leads to inhibition of lipoprotein lypase (LPLA) and liver triglyceride lypase (L-TGLA) activity of the rat blood serum. The level of very low density lipoproteins (VLDLP) grows, and suppression of lecithin-cholesteryl-acyltransferase (LCAT) activity is accompanied by reduction of the share of cholesterol derivatives with polyunsaturated fatty acids. Combined effects of protein-vitamin insufficiency and hypokinesia result in parversion of the lipolysis processes, that manifests in prevalence of L-TGLA over LPLA. The levels of VLDLP increase, and growth of LCAT activity is acompanied by the growth of cholesteryl linoleate share and level. Hypokinesia combined with the studied experimental diets was found to lead to increase of the free fatty acid level and to decrease of the blood serum levels of phospholipids and triglycerides.

The decrease of tyrosine hydroxylase activity in adrenal homogenate in scurvy was recovered after the administration of ascorbic acid. The causes of the increase in the enzyme activity after the administration of ascorbic acid have been studied. 1. No significant elevation in the enzyme activity was observed after the administration of reserpine to the scortutic guinea pig. 2. A dose of metal chelating agent, alpha, alpha'-dipyridyl, prevented the ascorbic acid-induced or reserpine-induced increase in enzyme activity in the scorbutic and the non-scorbutic guinea pigs, respectively. 3. Tyrosine hydroxylase activity was partially recovered by the administration of FeSO4 to the scorbutic guinea pig. From these results, it became clear that the induction of tyrosine hydroxylase which was not observed in scurvy was due to the deficiency of Fe2+. These results suggested that ascorbic acid affected the induction of this enzyme via Fe2+.

A chronic deficiency of ascorbic acid was induced in guinea pig. The level of catecholamines, copper and the activities of ceruplasmin, catecholamine oxidase, monoamineoxidase and acetylcholinesterase were checked in brain, liver and serum. Also the levels of ascorbic acid and glutathione were measured in the organs of ascorbic acid-deficient animals. The most important changes due to the ascorbic acid deficiency were observed in the brain were monoamineoxidase, catecholamineoxidase, acetylcholinesterase and the concentration of catecholamines were altered. The statement that brain is the organ most affected by the ascorbic acid deficiency is discussed.

Previous work supporting the vitamin A dependency of adrenal function in rats neglected to take into account a secondary effect of the deficiency, a decrease in hepatic ascorbic acid biosynthesis. Vitamin A-depleted rats maintained on a diet free of ascorbate had a decrease in the activity of adrenal 3 beta-hydroxysteroid dehydrogenase, and extensive adrenocortical degeneration. The use of an ascorbate supplement prevented the symptoms. The results suggest that previous evidence for direct involvement of vitamin A in steroidogenesis may have been due to the production of a secondary deficiency, a chronic scorbutic condition.

Studies were carried out to characterize the response of hepatic mixed function oxidase (MFO) activity to chronic ascorbic acid deficiency and excessive ascorbic acid intake in the guinea pig. When guinea pigs were fed excessive ascorbic acid, there was a small increase in hepatic cytochrome P-450 which was unaccompanied by any alteration in drug-metabolizing enzyme activity. Similarly, induction of MFO activity by phenobarbital was not modified by excessive ascorbic acid administration. Chronic ascorbic acid deficiency resulted in depressed metabolism of aniline, aminopyrine, ethoxycoumarin and benzphetamine, but not of ethylmorphine, in comparison with animals fed diets containing control and/or excessive amounts of ascorbic acid. In contrast to the metabolism of all drugs studied, the 7 alpha-hydroxylation of cholesterol was depressed by both inadequate and excessive vitamin C intake, demonstrating the unique sensitivity of cholesterol 7 alpha-hydroxylase to dietary ascorbate.

Activity of the antioxidant enzymes under conditions of experimental syndrome of peroxidation was studied in rabbits maintained at a diet deprived of antioxidants. Peroxidation of lipids was increased in the animals; at the same time, content of the vitamin-antioxidants was decreased in their tissues. Activity of the antioxidant enzymes was distinctly increased within the first steps of the experiment after which it was decreased. The data obtained suggest that deficiency of the vitamin-antioxidants may be transitory compensated by an increase in activity of the antioxidant enzymes; long-term deprivation of bioantioxidants led, however, to inhibition of biosynthesis of the antioxidant enzymes.

It was observed that both glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities were significantly increased in different tissues of both rats and guinea pigs, during a short period of altitude exposure, as well as in acclimatized animals. Interestingly, it was found that oral administration of glucose showed no such significant change at either sea level or in a hypoxic condition. Studies on the activity of GOT and GPT in different tissues of normal guinea pigs showed a little change in liver and brain during short periods of exposure. Kidney and heart showed a significant increase in GOT activity without any change in GPT activity. Increased activities were observed in all the tissues of the acclimatized animals. Of both unexposed and acclimatized animals, there was no change in the enzyme of unexposed and acclimatized animals except in heart following injection of thyroxine (16 micrograms X kg-1), but the enzyme activities were most pronounced in scorbutic animals.

Changes of the phosphofructokinase (PFK; KF.2.7.I.II) activity in the skeletal and heart muscle, in the liver and skin of guinea pigs with different ascorbic acid allowances of their organism were studied. In animals receiving physiological and high vitamin "C" doses no changes in the activity of the tissues under study were observed. In cases of acute C-hypovitaminosis the activity of the enzyme in the skin fell by 26 per cent, in scurvy it declined in the liver by 28 per cent and in the skin -- by 46 per cent. In these conditions the PFK activity in the skeletal and heart muscles remained invariable. In one group of the animals chronic latent C-hypovitaminosis was simulated it attended by a diminishing fermentative activity in the heart by 24 and in the skin -- by 20 per cent. No changed activity in the liver was revealed.

There is increasing evidence that the liver microsomal drug metabolizing system is affected by various vitamins such as ascorbic acid, riboflavin, and alpha-tocopherol. In regard to ascorbic acid deficiency there is a decrease in the quantity of hepatic microsomal electron transport components such as cytochrome P-450 and NADPH-cytochrome P-450 reductase, as well as decreases in a variety of drug enzyme reactions such as N-demethylation, O-demethylation, and steroid hydroxylation. In addition, young animals given high supplements of vitamin C have increased quantities of electron transport components and overall drug metabolism activities. Kinetic studies indicate no change in the apparent Km of N-demethylase, O-demethylase or hydroxylase for drug substrates in animals depleted or given high amounts of the vitamin. However, there are qualitative changes in both type I and II substrate-cytochrome P-450 binding. Ascorbic acid is not involved in microsomal lipid peroxidation or in any qualitative or quantitative change in phosphatidylcholine. Replenishing vitamin C-deficient animals with ascorbic acid required 3 to 7 days for the electron transport components and drug metabolism activities to return to normal levels. Induction with phenobarbital and 3-methylcholanthrene is not impaired in the deficient animal since drug metabolism activities are induced to the same extent as normal controls; however, the administration of delta-aminolevulinic acid, a precursor of heme synthesis, to deficient animals caused an increase in the quantity of cytochrome P-450. The effects of riboflavin deficiency on electron transport components and drug metabolism activities have been noted only in adult animals after prolonged periods of deficiency. Decreases in drug metabolism activities occur with both type I (aminopyrine and ethylmorphine) and type II (aniline) substrates. As was found with ascorbic acid deficiency, drug enzyme induction occurred to the same extent with phenobarbital in deficient and normal animals. In addition, it required from 10 to 15 days for the drug metabolism activities to return to normal levels when deficient animals were replenished with riboflavin. The effect of vitamin E on drug metabolism is specific in N-demethylase activities decrease while O-demethylase activities are not affected in the deficient state. This vitamin differs from ascorbic acid and riboflavin in that several laboratories have reported no quantitative decrease in cytochrome P-450, although there are some reports that it and delta-aminolevulinic acid dehydratase are lowered quantity of cytochrome in E-deficient animals. The effect of vitamin E, if any, on the P-450 is unresolved; an important question that requires further clarification. As with ascorbic acid there is no difference in the apparent Km of N-demethylase enzymes for varous substrates and the protective effect of vitamin E does not appear to be one of an antioxidant inhibiting microsomal lipid peroxidation.

Guinea pigs well supplied with vitamin C show a linear relation between serum alkaline phosphatase activity and body weight. Investigations in guinea pigs induced to low ascorbic-acid levels by two different procedures (complete and partial deprivation of vitamin C) demonstrated that the activity of the alkaline phosphatase is not generally decreased in the serum of ascorbic-acid-deficient guinea pigs. Decreases of the serum enzyme levels reported in guinea pigs completely deprived of vitamin C, i.e. in acute deficiency, may be due to stress factors.

It has been demonstrated that the deficiency of 3 essential amino acids (lysine, threonine and methionine) combined with polyhypovitaminosis A, C and E for 2 months leads to a decrease in the para-hydroxylase and N-demethylase activity in rat liver microsomes. The content of cytochromes P-450 and b5 was also reduced as compared to that in animals given balanced nutrition. However, in such a polynutrient deficiency, the activity of microsomal enzymes was induced by phenobarbital to a greater degree than in balanced nutrition. It is suggested that in polynutrient deficiency, the lacking essential factors mobilized from other organs and tissues are involved in the process of induction of microsomal enzymes.

1. The dependency of product formation on reaction time and on enzyme concentration was studied with certain purified enzymes and liver microsomal mixed-function oxidase system. 2. The product-formation-time relation was estimated with different enzyme and/or substrate concentrations for the reactions limited by diffusion. 3. The kinetic constants of the product-time relations in these diffusion systems have been verified experimentally to give a more specific and detailed characterization of an enzyme system under a variety of physiologic and reaction conditions. 4. The influence of inhibitors and activators in vitro, the effect of enzyme preparation technic, aging, starvation, pretreatment with somatotropic hormone, phenobarbital or 3-methylcholanthrene, and the effect of a tumor were studied. 5. The influence of vitamin C deficiency in guinea pig was also studied.

We investigated the requirement of ascorbic acid for the induction by polychlorinated biphenyls (PCB) of hepatic drug-metabolizing enzymes in ODS-od/od rat (OD rat) which is a rat mutant unable to synthesize ascorbic acid. ODS- +/+ rats (+/+ rat), which can synthesize ascorbic acid, were used as controls. In OD rats, the dietary requirement of ascorbic acid to maintain normal growth and prevent any signs of scurvy is about 300 mg of ascorbic acid per kilogram diet. In this study, dietary levels of ascorbic acid tested were 0, 50, 300, 1000 and 3000 mg ascorbic acid per kilogram diet with or without 200 mg of PCB per kilogram diet. Feeding PCB did not affect growth in rats of either genotype. When statistical analysis was done within groups fed diets without PCB, ascorbic acid deficiency caused significant decreases in body weight gain, hepatic activities of drug-metabolizing enzymes and level of hepatic cytochrome P-450. When OD rats were fed a diet without PCB, the supplementation of about 300 mg ascorbic per kilogram diet was sufficient to maintain normal activities of hepatic aminopyrine N-demethylase, aniline hydroxylase, cytochrome c reductase and reduction of cytochrome P-450 and a normal level of hepatic cytochrome P-450. However, when OD rats were fed a diet supplemented with 200 mg PCB per kilogram of diet, significantly higher activities of hepatic aminopyrine N-demethylase and aniline hydroxylase and significantly higher level of hepatic cytochrome P-450 were observed in OD rats fed a diet supplemented with 1000 mg or 3000 mg ascorbic acid per kilogram of diet than in rats fed a diet supplemented with 300 mg of ascorbic acid. It is concluded that the dietary requirement of ascorbic acid is increased severalfold by the administration of xenobiotics, such as PCB, for the maximum induction of hepatic drug metabolism.

Polynutrient deficiency in essential amino acids and vitamins A, C and E led to induction of liver tissue microsomal monooxygenase system in male rats of WAG strain within first 2 hrs and to inhibition of the induction within the subsequent hours after a single dose administration of a pesticide lindane (18 mg/kg) as compared with control animals maintained on a normal diet. The deficiency in essential nutrients caused also a delay in the monooxygenase induction in rats treated daily within 3 months with lindane at a dose of 0.9 mg per kg of body mass. No distinct nutritionally-dependent differences in the rate of the monooxygenase induction were observed after 6 months of the chronic treatment with the pesticide. Activity of monooxygenases in nutritionally-deprived and lindane-treated rats was decreased as compared with the similarly treated rats which were maintained on a normal diet. The induction of monooxygenases in experimental rats was accompanied by an increase in relative mass of liver tissue. The increase in liver tissue mass appears to occur as an adaptive response to the polynutrient deficiency and the effect of xenobiotics in order to maintain the enzymatic system at an adequate functional level.

Activity of the flavin-containing monooxygenase (FMO) was reduced significantly in ascorbic acid deficient guinea pigs. Reduction in oxidation of dimethylaniline (DMA) and of thiobenzamide was associated with a decrease in the activity of the FMO. In both ascorbate supplemented and deficient guinea pig hepatic 12,000 g supernatant fractions, SKF-525A and n-octylamine did not inhibit DMA N-oxidation. Phenobarbital pretreatment did not increase the rate of N-oxidation of DMA. In addition, hepatic supernatant fractions thermally treated at 50 degree were unable to N-oxidize DMA, but 80% of the cytochrome P-450 activity was retained. Also, N-oxidation of DMA was reduced by 53% at pH 7.0, while oxidation of cytochrome P-450 specific substrates was inhibited by only 19%. Kinetic studies of DMA N-oxidation indicate no significant change in the apparent Km in ascorbate supplemented or deficient animals. The in vitro addition of ascorbic acid had no effect on the activity of the FMO. The toxicological implications of the reduction in FMO activity in ascorbic acid deficiency are discussed.

Modulation of the flavin-containing monooxygenase (FMO) by varying the ascorbic acid and food intake was investigated. Hepatic activity of the FMO in ascorbic acid-deficient guinea pigs fed a restricted amount of diet which resulted in a 10-15% body weight loss, was 17% of that in animals fed restricted amounts of the adequate diet. FMO hepatic activity in ascorbic acid-supplemented guinea pigs on a food-restricted regimen was 176% of that found in animals fed the adequate diet ad libitum. This increase in activity was not related to stress. Alteration in the activity of this important drug-metabolizing enzyme system by a combination of ascorbic acid deficiency and reduced food intake could potentially alter the rate of metabolism of a great variety of pharmaceutical drugs and environmental chemicals.

Cholesterol 7alpha-hydroxylase activity was assayed in liver microsomes from guinea pigs supplemented with ascorbate and from guinea pigs in a state of ascorbate deficiency. A mass fragmentographic method was used by which the 7alpha-hydroxylation of endogenous cholesterol could be measured. The 7alpha-hydroxylation was markedly reduced in the ascorbate-deficient animals as compared to animals treated with ascorbate. Addition of ascorbate to the incubations did not increase this activity. 11- and 12-Hydroxylation of laurate as well as 25- and 26-hydroxylation of 5beta-cholestane-3alpha, 7alpha-diol were not significantly affected by the ascorbate status of animals. In the presence of excess NADPH-cytochrome P-450 reductase and a phospholipid, partially purified cytochrome P-450 from the microsomal fraction of liver of an ascorbate-deficient guinea pig had a much lower capacity to 7alpha-hydroxylate [4-14C]cholesterol than a corresponding system containing cytochrome P-450 from liver of an ascorbate-supplemented guinea pig. It is suggested that ascorbate affects the synthesis or breakdown of the 7alpha-hydroxylating system, in particular the cytochrome P-450 component.

Ascorbic acid deficiency causes qualitative and quantitative differences in the guinea pig hepatic flavin-containing monooxygenase (FMO). Kinetic studies with purified FMO indicated no significant change in the apparent Km of dimethylaniline or NADPH in ascorbate-supplemented or -deficient animals. Following purification of ascorbate-deficient guinea pig FMO by DEAE-cellulose and blue agarose chromatography, exogenous FAD was required for 15% of the FMO microsomal activity recovered. In contrast, only 5% of the total microsomal enzyme recovered from ascorbate-supplemented animals required exogenous FAD. Furthermore, there was an enhanced sensitivity to time-dependent nonlinearity with the purified ascorbate-deficient guinea pig FMO. The degree of time-dependent nonlinearity was related to the concentration of substrate. Also, purified ascorbate-supplemented guinea pig FMO was stable for 4 weeks at -20 degrees, whereas the ascorbate-deficient enzyme was inactivated. A decrease in the quantity of ascorbate-deficient guinea pig FMO compared to ascorbate-supplemented was indicated by a marked reduction in total FMO activity recovered from blue agarose chromatography and reduced protein staining intensity with SDS-PAGE at 56,000 daltons.

The effect of dietary ascorbic acid on hepatic microsomal UDP-glucuronyltransferase (UDPGT) activity towards p-aminophenol, bilirubin, and acetaminophen was investigated. Ascorbate deficiency produced a 33% reduction in the specific activity of UDPGT towards p-aminophenol, whereas there was no difference between microsomes from ascorbate-deficient and supplemented guinea pigs in the activity towards bilirubin and acetaminophen. This suggests that the effect of the vitamin is on a specific isozyme. This reduction was correlated with the reduced quantity of hepatic microsomal cytochrome P-450, which has been previously reported for ascorbate-deficient guinea pigs. No difference was found in the apparent affinity for the substrate, p-aminophenol, or the cofactor, UDP-glucuronic acid. Differences in microsomal UDPGT activity towards p-aminophenol occurred between the two groups with membrane-perturbing processes such as sonication and Triton X-100. Sonication and magnesium chloride were found to increase activity 329% in ascorbate-supplemented animals and 138% in the ascorbate-deficient group. The addition of ascorbate acid in vitro, or its analog d-isoascorbic acid, could protect against the detrimental effects of excess substrate by maintaining a linear enzymatic rate over a 30-min time period; there was no significant effect on the initial rate of hepatic microsomal UDPGT activity in the ascorbate-supplemented animals whereas there was a significant increase in the ascorbate-deficient group. Glutathione was as effective as ascorbic acid in protecting against the detrimental effects of excess substrate whereas cysteine and dimethyltetrapteridine were only partially effective. Ascorbyl-2-sulfate and alpha-tocopherol had no significant effect.(ABSTRACT TRUNCATED AT 250 WORDS)