Occurrence and tissue distribution of both NADH- and NADPH-linked aquacobalamin reductases in some vertebrates

To elucidate the synthesis of cobalamin coenzymes in view of comparative biochemistry, tissue distribution of activity of aquacobalamin reductase [EC 1.6.99.8] catalyzing the reduction of hydroxocobalamin to cob(II)alamin was studied in some vertebrates. Activity of NADH- or NADPH-linked aquacobalamin reductase (or both) was found in some tissues of monkey, rat, cow, hog, chicken, fish, and frog. The liver homogenates of these vertebrates contained both NADH- and NADPH-linked enzymes. Monkey and rat livers accounted for more than 80% of total activities of both NADH- and NADPH-linked enzymes. Ratios of activities of the liver NADH- and NADPH-linked enzymes were above 1.0 in some land animals (monkey, rat, cow, and hog) but below 1.0 in water animals (sea and freshwater fishes). These different ratios are presumably due to the difference in the metabolisms of cobalamin and/or in the other cellular components between land and water animals.     

Vitamin B-12 deficiency induces anomalies of base substitution and methylation in the DNA of rat colonic epithelium

Derangements of one-carbon metabolism can directly affect the integrity of the genome by producing inappropriate uracil insertion into DNA and by altering patterns of DNA methylation. Vitamin B-12, a one-carbon nutrient, serves as a cofactor in the synthesis of precursors of biological methylation and in nucleotide synthesis. We therefore examined whether vitamin B-12 deficiency can induce these molecular anomalies in the colonic mucosa of rats. Weanling male Sprague-Dawley rats (n = 30) were divided into 2 groups and fed either a vitamin B-12-deficient diet or a similar diet containing adequate amounts of the vitamin. Rats from each group were killed at 6 and 10 wk. Uracil misincorporation into DNA was measured by GC/MS and genomic DNA methylation was measured by LC/MS. Plasma vitamin B-12 concentrations in deficient rats were below detectable limits at 6 and 10 wk; in control rats, concentrations were 0.46 +/- 0.07 and 0.42 +/- 0.10 nmol/L at those times. Although the colon total folate concentration did not differ between the groups, the proportion that was methylfolate was marginally greater in the deficient rats at 10 wk (P = 0.05) compared with control, consistent with the "methylfolate trap" that develops during vitamin B-12 deficiency. After 10 wk, the colonic DNA of the deficient rats displayed a 35% decrease in genomic methylation and a 105% increase in uracil incorporation (P < 0.05). This vitamin B-12-deficient diet, which was of insufficient severity to cause anemia or illness, created aberrations in both base substitution and methylation of colonic DNA, which might increase susceptibility to carcinogenesis.     

Influence of zinc deficiency on retinal reductase and oxidase activities in rat liver and testes.

The influence of zinc deficiency on vitamin A metabolism in rats was investigated by assessing two specific enzymes involved in its metabolism viz retinal reductase and retinal oxidase in the liver as well as in the testes. The activity of retinal reductase in the liver was not altered in zinc deficiency. Retinal oxidase activity on the other hand, was increased approximately 1.5 fold over the pair-fed controls. In contrast, both retinal reductase and oxidase in the testes were decreased during zinc deficiency. The effects of zinic deficiency on vitamin A metabolism in the liver could be partly attributed to the secondary effect of reduced food intake and growth. However, the effects seen in the testes on the metabolic enzymes of vitamin A appear to be due to zinc deficiency per se. The liver concentration of vitamin A (microgram/g) as well as total vitamin A (microgram/liver) were higher in the zinc-deficient rats when compared to the zinc-sufficient rats although not significantly different from the pair-fed controls. In aggreement with previous reports, the plasma vitamin A in zinc-deficient rats were found to be markedly lowered compared to zinc sufficient ad libitum controls.     

Vitamin E deficiency and prostaglandin synthesis in rat testes

A group of 8 weanling rats was fed a vitamin E(VE) deficient diet (D) and a control group was fed a VE sufficient diet (C) for 19 weeks. Levels of docosapentaenoic acid (22:5w6) in testis parenchyma lipids were significantly lower (p<0.05) in group D compared with group C. This decrease in 22:5w6, however, was not accompanied by the documented decrease in total testis weight of the VE deficient group. These results suggest that the 22:5w6 level in testis parenchyma lipids may be a sensitive indicator of VE deficiency in rats. There was no difference in the arachidonic acid (AA) level of testis parenchyma lipids between two groups. The tunica was the major site of prostaglandin (PG) synthesis in the testicular tissue and prostacyclin was the major PG synthesized from exogenous AA in the tunica. Both PGE₂ and 6-keto-PGF_1α derived from endogenous AA in tunica homogenate affer 30 min incubation appear to be greater in the control group than in the deficient group, although the difference was not statistically significant. These results imply that increased lipid peroxidation in VE deficiency can result in greater inactivation of PGH synthetase and thus can reduce the synthesis of PGs during the incubation.     

Vitamin A deficiency increases hepcidin expression and oxidative stress in rat

ObjectiveThe interaction between vitamin A and iron status has been widely reported; however, the exact mechanism involved in this interaction has not been well characterized. The present study investigated the mechanism involved in tissue iron accumulation and changes in the oxidative status in vitamin A–deficient rats.MethodsRats were treated with a control diet, a vitamin A–deficient diet, or a vitamin A/iron-deficient diet for 57 d. The animals were sacrificed; the blood, liver, and spleen were collected for biochemical analysis. Analysis of variance or Mann-Whitney tests were used to compare groups and Pearson's or Spearman's tests to investigate the bivariate correlation.ResultsVitamin A deficiency increased liver hepcidin mRNA and iron spleen concentrations; however, iron deficiency in vitamin A–deficient rats deeply inhibits liver hepcidin mRNA expression and significantly increases divalent metal transporter-1 mRNA levels. Liver ferroportin and hereditary hemochromatosis gene mRNA levels did not change in either treatment. In the vitamin A–deficient groups, liver carbonyl protein increased, whereas catalase and glutathione S-transferase activities decreased, suggesting that vitamin A protects the liver against protein oxidation. A significant positive correlation was found between lipid oxidative damage and iron concentration in the liver and spleen (r = 0.611, P = 0.007; r = 0.558, P = 0.025, respectively).ConclusionThese results suggest that vitamin A maintains iron homeostasis by the modulation of liver hepcidin expression. The increase of lipid peroxidation in vitamin A deficiency seems to be iron dependent, whereas protein oxidation is not.     

Dietary Sources of Vitamin B-12 and Their Association with Vitamin B-12 Status Markers in Healthy Older Adults in the B-PROOF Study

Low vitamin B-12 concentrations are frequently observed among older adults. Malabsorption is hypothesized to be an important cause of vitamin B-12 inadequacy, but serum vitamin B-12 may also be differently affected by vitamin B-12 intake depending on food source. We examined associations between dietary sources of vitamin B-12 (meat, fish and shellfish, eggs, dairy) and serum vitamin B-12, using cross-sectional data of 600 Dutch community-dwelling adults (≥65 years). Dietary intake was assessed with a validated food frequency questionnaire. Vitamin B-12 concentrations were measured in serum. Associations were studied over tertiles of vitamin B-12 intake using P for trend, by calculating prevalence ratios (PRs), and splines. Whereas men had significantly higher vitamin B-12 intakes than women (median (25th–75th percentile): 4.18 (3.29–5.38) versus 3.47 (2.64–4.40) μg/day), serum vitamin B-12 did not differ between the two sexes (mean ± standard deviation (SD): 275 ± 104 pmol/L versus 290 ± 113 pmol/L). Higher intakes of dairy, meat, and fish and shellfish were significantly associated with higher serum vitamin B-12 concentrations, where meat and dairy—predominantly milk were the most potent sources. Egg intake did not significantly contribute to higher serum vitamin B-12 concentrations. Thus, dairy and meat were the most important contributors to serum vitamin B-12, followed by fish and shellfish.     

Vitamin B-12 deficiency and hyperhomocysteinemia are partly ameliorated by cobalt and nickel supplementation in pigs

Vitamin B-12 deficiency and hyperhomocysteinemia alter the metabolism of trace elements. This study tested the hypothesis that there is a reverse relationship in which diets high in iron, copper, nickel and cobalt would influence vitamin B-12 deficiency outcomes including hyperhomocysteinemia. Piglets (German Landrace x Pietrain) were assigned to six groups of 8 and fed one of the following diets for 166 d: a vitamin B-12-adequate and folate-fortified diet (30 microg/kg vitamin B-12 and 0.5 mg/kg folate) with normal trace element concentrations or one of five vitamin B-12-free, folate nonsupplemented diets (0.36 mg/kg), with either normal trace element concentrations or high concentrations of iron (300 mg/kg), copper (30 mg/kg), cobalt (1 mg/kg) or nickel (6 mg/kg). Feed intake and weight gain did not differ significantly among the groups. Vitamin B-12-deficient pigs developed diminished serum and liver concentrations of vitamin B-12 and folate, an accumulation of iron in the liver and hyperhomocysteinemia. The magnitude of changes differed among vitamin B-12-deficient groups. Vitamin B-12-deficient pigs fed 6 mg/kg nickel had distinctly higher vitamin B-12 concentrations in liver and serum and 45% lower serum concentration of homocysteine than the corresponding deficiency group fed 1 mg/kg nickel; iron concentration in liver was completely normalized. Vitamin B-12-deficient pigs fed 1 mg/kg cobalt had 47% lower homocysteine concentrations in serum than the vitamin B-12-deficient group fed 0.13 mg/kg cobalt, but the vitamin B-12 status was unaffected. Supplementation of iron and copper did not affect these variables. The dietary manipulations had no detrimental effects on variables symptomatic of oxidative stress. The findings indicate a collaborative relationship between vitamin B-12 metabolism and the trace elements nickel and cobalt.     

Vitamin B12 deficiency results in the abnormal regulation of serine dehydratase and tyrosine aminotransferase activities correlated with impairment of the adenylyl cyclase system in rat liver

The aim of the present study was to elucidate the mechanism of the vitamin B(12) deficiency-induced changes of the serine dehydratase (SDH) and tyrosine aminotransferase (TAT) activities in the rat liver. When rats were maintained on a vitamin B(12)-deficient diet, the activities of these two enzymes in the liver were significantly reduced compared with those in the B12-sufficient control rats (SDH 2.8 (sd 0.56) v. 17.5 (sd 6.22) nmol/mg protein per min (n 5); P < 0.05) (TAT 25.2 (sd 5.22) v. 41.3 (sd 8.11) nmol/mg protein per min (n 5); P < 0.05). In the B(12)-deficient rats, the level of SDH induction in response to the administration of glucagon and dexamethasone was significantly lower than in the B(12)-sufficient controls. Dexamethasone induced a significant increase in TAT activity in the primary culture of the hepatocytes prepared from the deficient rats, as well as in the cells from the control rats. However, a further increase in TAT activity was not observed in the hepatocytes from the deficient rats, in contrast to the cells from the controls, when glucagon was added simultaneously with dexamethasone. The glucagon-stimulated production of cAMP was significantly reduced in the hepatocytes from the deficient rats relative to the cells from the control rats. Furthermore, the glucagon-stimulated adenylyl cyclase activity in the liver was significantly lower in the deficient rats than in the controls. These results suggest that vitamin B(12) deficiency results in decreases in SDH and TAT activities correlated with the impairment of the glucagon signal transduction through the activation of the adenylyl cyclase system in the liver.     

Vitamin B-6 deficiency prolongs the time course of evoked dopamine release from rat striatum

Vitamin B-6-deficient animals exhibit motor abnormalities. To investigate the possible physiologic alterations in the dopaminergic nervous system in vitamin B-6 deficiency, dopamine release in the striatum of vitamin B-6-deficient rats was determined using in vivo electrochemistry. Male Sprague-Dawley rats, 3 wk old, weighing 50-60 g, were randomly assigned to a control (7 mg pyridoxine HCl/kg diet), vitamin B-6-deficient (0 mg pyridoxine HCl/kg diet), or pair-fed (7 mg pyridoxine HCl/kg diet) group. After 8 wk of dietary treatment, plasma concentrations of pyridoxal 5'-phosphate as well as the striatal pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate were significantly lower in the vitamin B-6-deficient group than in the control and pair-fed groups. The dopamine concentrations of the striatum and the magnitude of the dopamine release after local application of KCl did not differ among the groups. However, the time required for KCl-evoked dopamine release to reach its peak level was significantly longer for the vitamin B-6-deficient rats than for controls. In addition, the decay time from the peak to one-half of the KCl-evoked dopamine release was also significantly prolonged in vitamin B-6-deficient rats compared with the control group. The results indicate that the cellular content of dopamine does not reflect the functional state of dopaminergic neurons in vitamin B-6 deficiency. The time course for release of dopamine and decay of the released dopamine is prolonged by vitamin B-6 deficiency, which might contribute to the motor abnormalities of the deficient rats.     

Vitamin B-6 metabolism and its relation to ornithine decarboxylase activity in regenerating rat liver

The metabolism of vitamin B-6 regenerating rat liver and liver from sham-operated control animals fed either a pyridoxine-sufficient or pyridoxine-deficient was investigated. The pyridoxal phosphate levels in plasma, regenerating liver and control liver were determined as were the activities of three enzymes involved in the metabolism of the vitamin, namely, pyridoxine kinase, pyridoxine phosphate oxidase, and pyridoxine phosphate phosphatase. In addition, total and holo-ornithine decarboxylase activities in the livers were measured. The results indicate that the metabolism of vitamin B-6 regenerating rat liver is different from that observed in Morris hepatomas (Thanassi et al. (1981) J. Biol. Chem. 256, 3370-3375). Vitamin B-6 metabolism in Morris hepatomas is concluded to be characteristic of the tumors rather than a property common to rapidly proliferating hepatic tissue. Regenerating liver ornithine decarboxylase holoenzyme activity in pyridoxine deprived rats was maintained at the same level as that in regenerating liver of pyridoxine-sufficient animals. The mechanism behind this maintenance of holo-enzyme activity appears to involve a pronounced increase in the amount of apoornithine decarboxylase. The time-dependent peak of ornithine decarboxylase activity following partial hepatectomy was shifted from four hours to twelve hours by vitamin B-6 deficiency.     

The effect of severe zinc deficiency on activity of intestinal disaccharidases and 3-hydroxy-3-methylglutaryl coenzyme A reductase in the rat

Specific activities of five enzymes were measured in intestinal mucosa of zinc-deficient rats and compared to activities in appropriate zinc-sufficient controls. Three disaccharidases were found to be significantly reduced in zinc deficiency. Alkaline phosphatase, a zinc metalloenzyme, also showed reduced activity. Activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme of cholesterol synthesis, was elevated. It is possible that impaired carbohydrate digestion (reflected in disaccharidase activity) and even defective lipid absorption (reflected in reductase activity) may contribute to the poor nutrition and diarrhea of zinc deficiency.     

Vitamin B-12 metabolism in HIV-infected patients in the age of highly active antiretroviral therapy: role of homocysteine in assessing vitamin B-12 status

BACKGROUND: Before the advent of highly active antiretroviral therapy (HAART), 20% and 10% of HIV-infected patients had low vitamin B-12 and red blood cell folate (RBCF) concentrations, respectively. However, few patients had real vitamin B-12 deficiency. OBJECTIVE: We evaluated the prevalence of low vitamin B-12 and RBCF concentrations in HIV-infected patients receiving HAART and the usefulness of serum homocysteine (sHcy) for differentiating patients with deficiency from those with harmlessly low vitamin B-12. DESIGN: The prevalence of low vitamin B-12 and RBCF was evaluated in 126 HIV-infected patients receiving HAART. Moreover, sHcy concentrations were evaluated in 40 HIV-infected patients with low vitamin B-12 and in 37 HIV-infected patients with low RBCF and were compared with those in 128 HIV-infected patients with normal vitamin B-12 and RBCF. sHcy was used to monitor treatment with vitamin B-12 and folic acid in 28 patients (24 with low vitamin B-12 and RBCF and 4 with hyperhomocysteinemia but normal vitamin B-12 and RBCF). RESULTS: The prevalence of low vitamin B-12 was significantly lower in patients receiving HAART than in previously studied patients who did not receive HAART (8.7% compared with 27%). Nine of the 40 patients (22.5%) with low vitamin B-12 (< or = 200 pmol/L) had hyperhomocysteinemia (> 17.5 micromol homocysteine/L). Nineteen (51.4%) of the 37 patients with low RBCF (< or = 580 nmol/L, percentile 10) had hyperhomocysteinemia. Among the 9 patients with an RBCF concentration < or = 450 nmol/L (percentile 2.5), all had hyperhomocysteinemia. The treatment with vitamin B-12 and folic acid normalized sHcy concentrations. CONCLUSIONS: The prevalence of low vitamin B-12 decreased after the introduction of HAART. The study of sHcy is useful for detecting HIV-infected patients with low vitamin B-12 and real deficiency.     

Effects of maternal vitamin B-6 deficiency on specific regions of developing rat brain: the extrapyramidal motor system

Progeny of rats deficient in vitamin B-6 exhibit gross neurological symptoms during a critical period of brain development, which may be related to abnormalities of the extrapyramidal motor system. In this study concentrations of the neurotransmitters, gamma-aminobutyric acid (GABA) and dopamine (DA) and certain amino acids were determined in substantia nigra (SN) and caudate/putamen (C/P) in progeny of dams fed diets containing either deficient (D), 0.6 or control (C), 7.0 mg pyridoxine x HCl (PN x HCl) per kilogram diet throughout growth, gestation and lactation. These measurements in C/P were correlated with the number of synapses. Analysis of brains of pups at 15 days of age, which gross neurological symptoms were evident in group D, showed that concentrations of GABA were decreased in C/P, whereas aspartate (Asp), glutamate (Glu) and DA were not affected compared to C values. One hour postinjection of PN x HCl, D pups showed decreased Glu in C/P and increased Asp in SN and at 3 hours postinjection, GABA was increased in SN. Since some synapses in C/P were postulated to be from GABAergic striatal interneurons, fewer of these synapses in conjunction with decreased GABA in C/P may result in ineffective inhibition of the circuitry of C/P in vitamin B-6 deficiency.     

Effects of vitamin B-6 deficiency on selenium metabolism in the rat

Rats were fed for 23 d diets adequate or deficient in vitamin B-6 and containing selenium as either sodium selenite, selenocysteine (SeCys) or selenomethionine (SeMet). They were then injected with 75Se of the same chemical form and killed 2 d later. Tissue deposition of stable and radiotracer selenium and the activity of glutathione peroxidase (GSHPx) were used to assess selenium utilization. Erythrocyte levels of selenium and GSHPx were lower in vitamin B-6--deficient animals for all forms of selenium; however, 75Se deposition in erythrocytes was not affected by vitamin B-6 status. The activities of cystathionine lyase, aspartate aminotransferase and selenocysteine lyase were lower in livers of vitamin B-6--deficient rats than in vitamin B-6--supplemented rats. The proportion of liver and kidney 75Se soluble in 5% trichloroacetic acid and 0.1 M 2-mercaptoethanol was consistently lower in vitamin B-6--deficient animals, but cation-exchange chromatography of tissue extracts did not identify a specific low-molecular-weight species. Tissue retention of 75Se provided as SeMet was increased in vitamin B-6--deficient animals, but the proportion of 75Se retained in muscle and liver as SeCys was significantly reduced. These findings suggest that the conversion of SeMet to a form available for GSHPx synthesis is reduced by vitamin B-6 deficiency.