Glutathione levels and related enzyme activities in vitamin B-6-deficient rats fed a high methionine and low cystine diet

We examined the change in glutathione metabolism in vitamin B-6-deficient rats. Vitamin B-6-deficient rats were fed a vitamin B-6-deficient diet containing 0.56% methionine and 0.075% cystine for 8 wk. Controls were fed an identical diet supplemented with 10 mg pyridoxine hydrochloride/kg diet. Glutathione concentrations in each organ examined were similar in control and vitamin B-6-deficient rats, and the values were comparably lower after intraperitoneal injection of diethylmaleate. However, buthionine sulfoximine caused a significantly greater decrease in glutathione levels in the liver and lungs of vitamin B-6-deficient rats relative to controls. Glutathione peroxidase activity in the liver of vitamin B-6-deficient rats was higher than in control animals; however, glutathione transferase activity in tissues other than liver of vitamin B-6-deficient rats was higher than in the controls. The activities of gamma-glutamyl-transferase in the liver and spleen of vitamin B-6-deficient rats were significantly lower than control values. The holoenzyme activities of cystathionine beta-synthase and cystathionine gamma-lyase in the liver of vitamin B-6-deficient rats were markedly reduced. These findings indicate that although the activities of enzymes that synthesize cysteine from methionine were decreased by vitamin B-6 deficiency, the level of synthesis and supply of cysteine in vitamin B-6-deficient rats were sufficient to maintain the same glutathione level as in controls, and that glutathione utilization in the liver was accelerated by vitamin B-6 deficiency.     

Evaluation of vitamin B-6 status and function of rats fed excess pyridoxine

We compared the vitamin B-6 status of 12-wk-old rats (n = 12) fed excess (1400 mg/kg diet) or the recommended level (7 mg/kg diet, control) of pyridoxine (PN) hydrochloride to test if excess vitamin B-6 would cause tissue depletion of pyridoxal phosphate (PLP), the active coenzyme form of vitamin B-6. Plasma PLP, tryptophan-load test results, food intake, and tissue and body weights were not different at wk 6. Red blood cell endogenous alanine aminotransferase activity and PLP concentration were elevated (P less than 0.01) in rats fed 1400 mg PN.HCl/kg diet. In contrast, PLP concentration in muscle was significantly lower (P = 0.01) in rats fed excess vitamin B-6 (9.7 +/- 0.8 nmol/g, mean +/- SEM) than in controls (14.9 +/- 1.4). PLP concentration in other tissues, including plasma, was not affected. In rats fed excess vitamin B-6, pyridoxal was increased in all tissues examined (P less than 0.05), and total vitamin B-6 was increased in plasma, red blood cells and kidneys (P less than 0.05). Total glycogen phosphorylase (a + b) activity in the gastrocnemius was not affected, but phosphorylase a activity was increased in rats fed excess vitamin B-6 (P = 0.025). Concentrations of dopamine and metabolites in the caudate nucleus of the basal ganglia were not affected. A transient, but significant, elevation in acoustic startle response, a central nervous system reflex, was observed in rats fed excess vitamin B-6. The depletion in muscle PLP could not hae been predicted by either plasma or red blood cell PLP concentration, although the latter did reflect vitamin B-6 intake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coenzyme A metabolism in vitamin B-12-deficient rats

Vitamin B-12 (cobalamin) deficiency results in decreased L-methylmalonyl-coenzyme A (CoA) mutase activity. The consequence of this defect on the cellular CoA pool was studied in rats with functional vitamin B-12 deficiency induced by administration of the cobalamin analogue hydroxy-cobalamin [c-lactam] or by dietary vitamin B-12 deficiency. Both types of vitamin B-12 deficiency were associated with methylmalonic acidemia (100-300-fold increases in plasma methylmalonic acid concentration compared with controls), but overall fuel homeostasis was intact. Liver from rats treated with hydroxy-cobalamin [c-lactam] contained a threefold greater concentration of total CoA (free CoA plus all acyl-CoA) compared with saline-treated rats. Fractionation of the CoA pool revealed higher levels of CoA, propionyl-CoA, methyl-malonyl-CoA, acid-insoluble CoA, as well as total CoA in the rats treated with hydroxy-cobalamin [c-lactam] compared with controls. Similar increases in liver CoA content were seen in dietary vitamin B-12 deficiency in both the fed and fasted states. To examine the hypothesis that sequestration of hepatic CoA as propionyl-CoA and methylmalonyl-CoA could increase CoA biosynthesis, the effect of propionate on CoA biosynthesis was studied in hepatocytes isolated from control rats. Propionate (1 mM) increased the formation of 14C-CoA from [14C]pantothenate (10 microM) by 27% in the hepatocyte system. When butyrate (1 mM) was provided as substrate, propionate (10 mM) increased [14C]CoA formation by 63%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic changes in golden hamsters fed vitamin B-12-deficient diets.

Various metabolic changes were observed in male hamsters fed vitamin B-12-deficient diets with or without supplements of cobalt, methionine, and a previously untested cobalt-free pseudovitamin B-12. The effects observed after 31 weeks of consuming the vitamin B-12-deficient diets included a marked increase in the urinary excretion of both methylmalonic acid and formiminoglutamic acid, slight increases in red blood cell mean corpuscular volume, and higher tissue levels of glutathione and activities of glutathione reductase and glucose-6-phosphate dehydrogenase. Vitamin B-12 in the diet prevented these changes, as did inorganic cobalt. The cobalt-free pseudovitamin B-12 showed no vitamin B-12 activity, neither did it have any potent antagonistic effect. Methionine supplementation reversed some of the metabolic changes. Addition of inorganic cobalt to the diet resulted in a significant increase in tissue stores of vitamin B-12.     

Dietary lactose improves endochondral growth and bone development and mineralization in rats fed a vitamin D-deficient diet

Lactose promotes the intestinal absorption of calcium independent of the vitamin D endocrine system. The purpose of this study was to determine the effect of lactose supplementation on endochondral bone growth, bone development and mineralization in weanling rats fed a vitamin D-deficient diet. Rat pups were weaned from vitamin D-deficient dams and fed a vitamin D-deficient diet containing sucrose as the primary carbohydrate source or a similar diet but containing 20% lactose. After 4 wk, body weights, serum calcium levels and endochondral bone elongation rates in the lactose-fed animals were higher than in rats fed the sucrose diet. In addition, bone weights, bone calcium content, percent bone ash of bone dry weight, percent metaphyseal osseous tissues and bone osteoid content in the lactose-fed rats were different from those in the rats fed the sucrose diet. In all cases the changes in osseous tissues that were observed in the animals fed the lactose-supplemented diet were toward normal values as observed in age-matched animals fed a vitamin D-replete diet. The improvements in bone growth and development due to lactose supplementation occurred independent of the vitamin D endocrine system and are likely the result of improved calcium absorption in the intestine.     

Maternal aerobic exercise and vitamin B-6 status

Effects of an aerobic walking program during gestational weeks 22-30 on vitamin B-6 status and birth outcome were studied in 28 healthy pregnant women, aged 21-36 y, receiving vitamin-mineral supplements. Mean daily vitamin B-6 intake, excluding a 10-mg supplement, was 1.81 mg. Subjects in the walking (n = 18) and nonwalking (n = 10) groups had similar microbiologically assessed plasma total vitamin B-6 levels and radioenzymatically assessed plasma pyridoxal phosphate concentrations. One walker at 22 wk and at 30 wk and a second walker at 30 wk had plasma total vitamin B-6 concentrations in the low-normal range; the same was true for plasma pyridoxal phosphate levels except that the 30-wk value for the walker who was low at 22 wk was missing. Birth-outcome measurements were similar for both groups. Participation in the walking program slightly improved aerobic fitness without affecting vitamin B-6 status or birth outcome in pregnant women taking vitamin-mineral supplements.     

Assessment of vitamin B-6 status in young women consuming a controlled diet containing four levels of vitamin B-6 provides an estimated average requirement and recommended dietary allowance

The Recommended Dietary Allowance (RDA) of vitamin B-6 for young women was recently reduced from 1.6 to 1.3 mg/d based on an adequate plasma pyridoxal phosphate (PLP) concentration of 20 nmol/L. To assess vitamin B-6 requirements and suggest recommendations for intake, seven healthy young women consumed a controlled diet providing 1.2 g protein/kg body weight for a 7-d adjustment period (1.0 mg vitamin B-6/d) and three successive 14-d experimental periods (1.5, 2.1 and 2.7 mg/d, respectively). Direct and indirect vitamin B-6 status indicators were measured in plasma, erythrocytes and urine. Indicators most strongly correlated with vitamin B-6 intake [i.e., plasma and erythrocyte PLP, urinary 4-pyridoxic acid (4-PA) and total vitamin B-6] were regressed on vitamin B-6 intake and the dietary vitamin B-6 to protein ratio. Inverse prediction using adequate and baseline values estimated vitamin B-6 requirement. Adequate values were determined for plasma PLP and urinary 4-PA from baseline values of 60 previous subjects, using the statistical method suggested by Sauberlich. The current study suggests a vitamin B-6 Estimated Average Requirement (EAR) for young women of 1.1 mg/d or 0.016 mg/g protein, and a RDA of 1.5 mg/d or 0.020 mg/g protein. When results from this study are combined with data from four other recent studies, the combined data predict an EAR of 1.2 mg/d or 0.015 mg/g protein, and a RDA of 1.7 mg/d or 0.018 mg/g protein. This study suggests that the current vitamin B-6 RDA may not be adequate.     

Modification of vitamin A metabolism in rats fed a copper-deficient diet

The liver is the main storage site of vitamin A and copper. Inverse relationships between copper and vitamin A liver concentrations have been suggested. We have investigated the consequences of a copper-deficient diet on liver and blood vitamin A storage in Wistar rats. Animals were fed either a copper-deficient diet for 45 days from weaning, or an identical diet containing adequate amounts of copper. Concentrations of vitamin A were determined by isocratic high performance liquid chromatography using UV detection. We have observed in the liver of the rats fed a copper-deficient diet a significantly higher mean level of retinyl esters (148 +/- 37 micrograms/g of liver) and retinol (3.3 +/- 1.4 micrograms/g of liver) compared to the mean concentration of the retinyl esters (53 +/- 8.5 micrograms/g of liver) (p less than 0.01) and retinol (1.4 +/- 0.5 micrograms/g of liver) (p less than 0.01) in controls. Opposite results were observed in the serum of the group fed a copper-deficient diet as these rats had a significantly lower level of retinol (22 +/- 4 micrograms/100 ml) compared to the mean concentration in the controls (64 +/- 20 micrograms/100 ml) (p less than 0.01). These findings suggest that a copper-deficient diet may cause defective transport of vitamin A from liver to blood. This experimental model may be useful to further investigate unusual liver vitamin A and copper concentrations observed in children during various hepatobiliary diseases.     

Metformin improves liver antioxidant potential in rats fed a high-fructose diet

Increased lipid peroxidation plays a role in the pathology associated with fructose feeding. The present study reports the effects of metformin on the liver lipid peroxidation and antioxidant defence system of rats fed a high-fructose diet. The experimental animals were divided into two batches of 12 animals each. The control batch received a control diet containing 60% starch; the second batch was given a high-fructose diet containing 60% fructose as the sole source of carbohydrate. At the end of second week these were each subdivided into two groups. One was given metformin (50 mg/kg body weight/day in water) by intragastric intubation and the other group was left untreated. The rats were continued on the same dietary regimen for the next two weeks. After the experimental period of four weeks, liver lipid peroxidation and antioxidant status were quantified. Enhanced thiobarbituric acid-reactive substance reactivity and lipid hydroperoxides were observed in high-fructose-fed rats. However, the activities of enzymic antioxidants were lower in this group. Administration of metformin attenuated the rise in lipid peroxidation and improved the antioxidant potential in high-fructose-fed rats. Metformin did not have any effect on the antioxidant status of control rats. Attenuation of lipid peroxidation by metformin could be related to its insulin sensitising action.     

Improved vitamin B-6 status is positively related to lymphocyte proliferation in young women consuming a controlled diet

To examine the effect of increased intake levels of vitamin B-6 (B-6) on lymphocyte proliferation and interleukin 2 (IL-2) concentration, young women (n = 7) consumed a constant diet containing 1 mg (5.91 micro mol) B-6/d for a 7-d adjustment period, followed by three 14-d experimental periods during which the daily B-6 intake was 1.5, 2.1 and 2.7 mg (8.86, 12.41 and 15.95 micro mol)/d, respectively. Weekly fasting blood and daily 24-h urine samples were collected. Lymphocyte proliferation and IL-2 production were measured in response to phytohemagglutinin. Vitamin B-6 status improved with increased B-6 intake as measured by plasma pyridoxal 5'-phosphate (PLP) and urinary 4-pyridoxic acid. When subjects consumed 2.1 mg B-6/d for 7 d, lymphocyte proliferation increased by 35% (P < or = 0.05) compared with the mean value after consumption of 1.5 mg B-6/d for 14 d. There was no further enhancement after an additional week of 2.1 and 2.7 mg B-6/d for 2 wk. Lymphocyte proliferation was correlated (P < or = 0.01) with vitamin B-6 intake (r = 0.757), plasma PLP (r = 0.456) and erythrocyte aminotransferase activities (r = -0.361). Plasma IL-2 concentration and in vitro production did not change throughout the study, although five of seven subjects showed increases with intakes of 2.1 and 2.7 mg B-6/d, respectively, compared with the 1.5 mg/d intake. Concentrations of PLP in peripheral blood mononuclear cells were correlated (r = 0.357, P < or = 0.01) with plasma PLP, but not with proliferation. These results show that improving vitamin B-6 status by consuming a B-6 intake higher than the current Recommended Dietary Allowance enhances lymphocyte proliferation.     

Effect of exercise on the metabolism of vitamin B6 and some PLP-dependent enzymes in young rats fed a restricted vitamin B6 diet

The effect of exercise on vitamin B6 metabolism and PLP-dependent enzymes was studied in rats fed a diet with or without vitamin B6. Metabolism of some amino acids (citrulline, arginine, ornithine and threonine) inhibited in the B6-deficient rats was normalized during exercise. Exercise was also effective in storing vitamin B6 in the body by lowering excretion of vitamin B6, when intake of vitamin B6 was restricted. Aspartatae aminotransferase activity was higher in the red portion of the gastrocnemius muscle than that of the white one, whereas glycogen phosphorylase activity was vice versa and furthermore glycogen content in the white portion was very low in the vitamin B6-deficient rat. From the data obtained, it has been suggested that the red and white portions of the gastrocnemius muscle seemed to be more important in metabolizing amino acids and hydrolyze glycogen, respectively.     

Vitamin B-12 supplementation improves arterial function in vegetarians with subnormal vitamin B-12 status

Objective

Vegetarians are more vascular-healthy but those with subnormal vitamin B-12 status have impaired arterial endothelial function and increased intima-media thickness. We aimed to study the impact of vitamin B-12 supplementation on these markers, in the vegetarians.

Design

Double-blind, placebo controlled, randomised crossover study.

Setting

Community dwelling vegetarians.

Participants

Fifty healthy vegetarians (vegetarian diet for at least 6 years) were recruited. Intervention: Vitamin B-12 (500??g/day) or identical placebo were given for 12 weeks with 10 weeks of placebo-washout before crossover (n=43), and then open label vitamin B-12 for additional 24 weeks (n=41).

Measurement

How-mediated dilation of brachial artery (FMD) and intima-media thickness (IMT) of carotid artery were measured by ultrasound.

Results

The mean age of the subjects was 45+9 years and 22 (44%) were male. Thirty-five subjects (70%) had serum B-12 levels <150pmol/l. Vitamin B-12 supplementation significantly increased serum vitamin B-12 levels (p<0.0001) and lowered plasma homocysteine (p<0.05). After vitamin B-12 supplementation but not placebo, significant improvement of brachial FMD (6.3±1.8% to 6.9±1.9%; p<0.0001) and in carotid IMT (0.69±0.09mm to 0.67±0.09 mm, p<0.05) were found, with further improvement in FMD (to 7.4±1.7%; p<0.0001) and IMT (to 0.65±0.09mm; p<0.001) after 24 weeks open label vitamin B-12. There were no significant changes in blood pressures or lipid profiles. On multivariate analysis, changes in B-12 (??=0.25; p=0.02) but not homocysteine were related to changes in FMD, (R=0.32; F value=3.19; p=0.028).

Conclusions

Vitamin B-12 supplementation improved arterial function in vegetarians with subnormal vitamin B-12 levels, proposing a novel strategy for atherosclerosis prevention.     

Grape skin improves antioxidant capacity in rats fed a high fat diet

This study was conducted to investigate the effect of dietary grape skin on lipid peroxidation and antioxidant defense system in rats fed high fat diet. The Sprague-Dawley rats were fed either control (5% fat) diet or high fat (25% fat) diet which was based on AIN-93 diet for 2 weeks, and then they were grouped as control group (C), control + 5% grape skin group (CS), high-fat group (HF), high fat + 5% grape skin group (HFS) with 10 rats each and fed corresponding diets for 4 weeks. The hepatic thiobarbituric acid reacting substances (TBARS) were increased in high fat group as compared with control group, but reduced by grape skin. The serum total antioxidant status, and activities of hepatic catalase and superoxide dismutase, xanthine oxidase and glucose-6-phosphatase were increased by supplementation of grape skin. Glutathione peroxidase activity was significantly higher in CS group than in C group. Grape skin feeding tended to increase the concentration of total glutathione, especially in control group. The ratio of reduced glutathione to oxidized glutathione was lower in high fat groups than in control groups. The ratio was increased by dietary supplementation of grape skin in control group. These results suggest that dietary supplementation of grape skin would be effective on protection of oxidative damage by lipid peroxidation through improvement of antioxidant defense system in rats fed high fat diet as well as rats with low fat diet.     

Feeding dried purple laver (nori) to vitamin B12-deficient rats significantly improves vitamin B12 status.

To clarify the bioavailability of vitamin B12 in lyophylized purple laver (nori; Porphyra yezoensis), total vitamin B12 and vitamin B12 analogue contents in the laver were determined, and the effects of feeding the laver to vitamin B12-deficient rats were investigated. The amount of total vitamin B12 in the dried purple laver was estimated to be 54.5 and 58.6 (se 5.3 and 7.5 respectively) microg/100 g dry weight by Lactobacillus bioassay and chemiluminescent assay with hog intrinsic factor respectively. The purple laver contained five types of biologically active vitamin B12 compounds (cyano-, hydroxo-, sulfito-, adenosyl- and methylcobalamin), in which the vitamin B12 coezymes (adenosyl- and methylcobalamin) comprised about 60 % of the total vitamin B12. When 9-week-old vitamin B12-deficient rats, which excreted substantial amounts of methylmalonic acid (71.7(se 20.2) micromol/d) in urine, were fed the diet supplemented with dried purple laver (10 microg/kg diet) for 20 d, urinary methylmalonic acid excretion (as an index of vitamin B12 deficiency) became undetectable and hepatic vitamin B12 (especially adenosylcobalamin) levels were significantly increased. These results indicate that vitamin B12 in dried purple laver is bioavailable to rats.     

Pyridoxal-5'-phosphate and pyridoxal biokinetics in male Wistar rats fed graded levels of vitamin B-6

Biokinetic parameters of plasma pyridoxal-5'-phosphate (PLP) and pyridoxal (PL) disposition were studied in male Wistar rats (age 8 mo) fed a purified diet containing less than 0.5, approximately 3 or approximately 6 mg pyridoxine.HCl/kg diet from weaning, with animals fed the 6 mg/kg diet serving as the control group. Basal plasma PLP concentration was lower in both the less than 0.5 and 3 mg/kg diet groups than in control animals (98 +/- 12, 314 +/- 40 and 514 +/- 56 nmol/L, respectively). Basal plasma PL concentration was lower in the less than 0.5 mg/kg diet group only [60 nmol/L (measured in pooled samples), 190 +/- 73 and 235 +/- 63 nmol/L for less than 0.5, 3 and 6 mg/kg diet groups, respectively]. In both the less than 0.5 and 3 mg/kg diet groups, PLP clearance was lower than in control rats (0.158 +/- 0.025, 0.131 +/- 0.040 and 0.240 +/- 0.051 L.h-1.kg body weight-1, respectively). In the less than 0.5 mg/kg diet group, PLP synthesis was more efficient than in control animals (34.7 +/- 9.3, 12.1 +/- 2.5 and 16.7 +/- 11.4% for less than 0.5, 3 and 6 mg/kg diet groups, respectively). In both the less than 0.5 and 3 mg/kg diet groups, volume of distribution of PLP as well as of PL was larger than in controls. It is concluded that B-6 vitamer metabolism is influenced by vitamin B-6 status. The metabolic pathway involved (PLP synthesis and/or PLP degradation) was observed to depend on degree of vitamin B-6 deficiency.     

Testicular injury to rats fed on soybean protein-based vitamin B12-deficient diet can be reduced by methionine supplementation

We have previously reported that rats fed on a vitamin B12 (B12)-deficient diet containing 180 g soybean protein per kg diet showed marked histologic damage in their testes. In this paper, we report the effect of B12-deficiency on B12-dependent methionine synthase in the rats' testes and the effect of methionine supplementation of the diet on testicular damage. Rats were fed the soybean protein-based B12-deficient diet for 120 d. We confirmed that those rats were in serious B12-deficiency by measuring urinary methylmalonic acid excretion and B12 content in tissues. Methionine synthase activity in the testis of the B12-deficient rats was less than 2% of that in B12-supplemented (control) rats. To complement disrupted methionine biosynthesis, methionine was supplied in the diet. A supplement of 5 g D,L-methionine per kg diet to the B12-deficient diet did not affect urinary methylmalonic acid excretion of B12-deficient rats. The testicular histology of rats fed the methionine-supplemented B12-deficient diet was almost indistinguishable from that of control rats. Thus, we conclude that the lowered testicular methionine synthase activity is the primary cause of the histologic damage due to B12-deficiency and that methionine supplementation to the diet can reduce the damage. These findings would indicate the importance of the methionine synthase activity, especially for testicular function.