Pyridoxine-deficient Rats Research Articles

Cerebral monoamine neurotransmitters in progeny of pyridoxine-deficient rats: H. Choi and S. A. Kang, Department of Food and Nutrition, Seoul National University, Seoul 151, Korea

Prothymosin α has been purified from human thymus and its amino acid sequence determined, except for a 15 amino acid segment including 10 glutamyl residues near the middle of the molecule. Like prothymosin α from rat thymus [A. A. Haritos, R. Blacher, S. Stein, J. Caldarella, and B. L. Horecker (1985) Proc. Natl. Acad. Sci. USA82, 343–346], human prothymosin contains the thymosin α1 sequence at its NH2-terminus. It contains a total of 109–110 residues compared to 111–112 for rat prothymosin α, with deletions corresponding to positions Gln39 and Lys108 of the rat polypeptide. Human prothymosin α also differs from rat prothymosin α at positions corresponding to residues 87, 92, and 102 of the latter, with substitutions of alanine for proline, alanine for valine, and aspartic acid for glutamic acid, respectively. Human prothymosin is significantly less active than rat prothymosin in protecting mice against infection with Candida albicans and in stimulating release in vivo of migration inhibitory factor. Thus, the differences in amino acid sequences, present mainly the COOH-terminal half of the polypeptides, may determine species specificity in boilogical properties.

Effect of dietary pyridoxine deficiency on intestinal functions in rats

Dietary pyridoxine deficiency in rats significantly reduced (20–43%) the intestinal uptake of D-glucose, glycine, L-alanine and L-leucine. An appreciable reduction in the activities of sucrase, lactase, alkaline phosphatase and leucine amino peptidase was also observed, but maltase activity was unaffected under these conditions. A con-siderable decrease (27–66%) in membrane proteins, sialic acid, phospholipids and free cholesterol fractions in vitamin deficiency was observed. Estercholesterol and triglyceride contents were not affected. Acetate-1- 14C incorporation into various lipid fractions indicated that observed decrease in membrane lipids in B6 deficiency was a consequence of reduced synthesis. It is concluded that pyridoxine deprivation produces marked changes in chemical architecture of brush borders, which probably explains the observed aberrations in epithelial cell function in this nutritional deficiency.

3-Hydroxykynurenine, 3-hydroxyanthranilic acid, and o-aminophenol inhibit leucine-stimulated insulin release from rat pancreatic islets.

Individual islets were isolated from rat pancreas to study the effects of tryptophan and its metabolites on leucine-stimulated release of insulin. 3-Hydroxykynurenine, 3-hydroxyanthranilic acid, and o-aminophenol were inhibitors at concentrations below 10 mM whereas tryptophan, kynurenine, kynurenic acid, xanthurenic acid, and anthranilic acid were ineffective inhibitors at concentrations up to 10 mM. A structure-activity analysis of these metabolites demonstrated that vicinal aromatic hydroxy and amino groups with their concomitant electron donating properties are required for inhibition of insulin release. Inhibition of islet insulin release by the three kynurenine metabolites may be involved in the depressed insulin levels found in vitamin B6-deficient rats by other workers.

Effect of pyridoxine deficiency in young rats on high-affinity serotonin and dopamine receptors.

The high-affinity bindings of [3H]-5-hydroxytryptamine to serotonin S-1 receptors, [3H]-ketanserin to serotonin S-2 receptors in the cerebral cortex, [3H]-fluphenazine to dopamine D-1 receptors, and [3H]-spiroperidol to dopamine D-2 receptors in the corpus striatum were studied in pyridoxine-deficient rats and compared to pyridoxine-supplemented controls. There was a significant increase in the maximal binding (Bmax) of serotonin S-1 and S-2 receptors with a significant decrease in their binding affinities (Kd). However, there were no significant changes either in the maximal binding or binding affinity of striatal dopamine D-1 and D-2 receptors. Receptor sensitivity seems to correlate negatively with the corresponding neurotransmitter concentrations in the pyridoxine-deficient rats.

Effect of pyridoxine administration on the induction of cytosolic aspartate aminotransferase in the liver of rats treated with hydrocortisone.

The effects of pyridoxine and hydrocortisone administrations on the rate of synthesis of cytosolic aspartate aminotransferase in adrenalectomized pyridoxine-deficient rat livers were examined. Induction of cytosolic aspartate aminotransferase by hydrocortisone was observed 6 to 10 h after the injection. Treatment with pyridoxine daily for 6 days but not for the 2 days, resulted in suppression of the enzyme synthesis. Similar suppression of enzyme synthesis was observed in rats without hydrocortisone treatment. The activity of tryptophan oxygenase, which is known to be induced by glucocorticoid and does not contain pyridoxal phosphates, was higher in the livers of pyridoxine-deficient rats than in that of controls. The possible effects of pyridoxal phosphate on the action of glucocorticoid are discussed based on the results.

Accumulation of Linoleic and γ-Linolenic Acids in Tissue Lipids of Pyridoxine-Deficient Rats

Young male Sprague-Dawley rats were fed diets containing added pyridoxine · HCl at 22 mg/kg (control), 0 mg/kg or 88 mg/kg for 6 weeks. In comparison with control or pyridoxine-supplemented (+PN) rats, growth of the pyridoxine-deficient (-PN) rats was significantly less after 2 weeks. After 6 weeks, liver weight was higher but thymus and epididymal fat weights, in relation to body weight, were significantly lower in -PN compared to control rats. In -PN rats, phospholipid levels of linoleic and γ-linolenic acids were increased, but arachidonic acid was decreased compared to controls in plasma, liver, thymus and skin. In liver triglycerides from -PN rats, all essential fatty acids (n3 and n6) were increased compared to both control and +PN rats. The n3 essential fatty acids were significantly increased in plasma, liver, and thymus phospholipids in the +PN compared to control rats. These results support previous reports of an effect of pyridoxine on essential fatty acid metabolism and suggest that both linoleic desaturation and γ-linolenic acid elongation may be impaired in -PN rats. In addition, the accumulation of essential fatty acids in the liver triglycerides of -PN rats suggests that essential fatty acid turnover between triglyceride and phospholipid may be influenced by pyridoxine.

Effect of pyridoxine deficiency on the exploratory behavior of rats

Malnutrition early in life impairs the development of brain as well as behavior. In the present study the behavioral effects of preweaning pyridoxine deficiency in rats, as reflected by their exploratoryscore, have been investigated. The results clearly indicated that the body weight of pyridoxine-deficient rats was significantl less when compared to the control rats. Besides this, the sign of maturation of sensory perceptual mechanisms like the first day of eye opening and signs of maturation of neuromotor coordination like the day of supported standing are delayed in vitamin-deficient rats. The open-field activity reflected by the exploratory score was observed to be significantly less in vitamin-deficient rats. In short, considerable effects of prenatal pyridoxine deficiency were observed in the neonatl pups as stunting of growth and in the delayed onset of neuromotor coordination as well as a low level of open-field activity. The importance of maternal pyridoxine supplementation during pregnancy has been emphasized.

Effect of pyridoxine deficiency on intestinal absorption of calcium and oxalate: Chemical composition of brush border membranes in rats

[U- 14C]oxalic acid and 45Ca uptake was measured in control and vitamin B 6-deficient rats. Calcium and oxalate uptake rates were significantly increased from the intestine of vitamin B 6-deficient rats as compared to pair-fed controls. Oxalate uptake in pair-fed control rats follows a passive diffusion. In pyridoxine-deficient rats, the oxalate uptake increases nonlinearly as the oxalate concentration in the incubation medium increased, indicating a two-component system—a saturable sodium-independent uptake and a linear nonsaturable passive-diffusion component. The brush border membrane composition reveals that membrane sialic acid, cholesterol, and protein contents were markedly reduced. These aberrations in the chemical composition of brush border membrane may be responsible for the enhanced oxalic acid uptake in vitamin B 6-deficient rats.

Enhancement of high affinity γ-aminobutyric acid receptor binding in cerebellum of pyridoxine-deficient rat

The high-affinity of [ 3H]γ-aminobutyric acid (GABA) to GABA A receptors and [ 3H]baclofen to GABA B receptors were studied in the cerebellum of pyridoxine-deficient rats and compared to pyridoxine-supplemented controls. There was a significant increase in the maximal binding (B max) of both GABA A and GABA B receptors with no significant difference in their binding affinities ( K d ). The changes observed suggest a supersensitivity of GABA A and GABA B receptors which seems to correlate negatively with the concentration of GABA in the cerebellum of pyridoxine-deficient rats.

Studies on the origin of triglyceride in fatty liver caused by pyridoxine-deficiency in rats

The effects of pyridoxine-deficiency on the incorporation of oral [1-14C]-linoleate into serum, dextran sulfate and CaCl 2 precipitable lipoprotein (B-lipoprotein) and liver lipid, and on the profiles of serum lipoprotein and apolipoprotein were studied. The incorporation of label into liver lipid in the deficient group was higher than that in the pair-fed controls and tended to be higher than that in the controls fed ad lib. The counts in lipid of B-lipoprotein as a percentage of total liver lipid was significantly lower in pyridoxine-deficient rats than in the two controls. The transport of lipid from the liver to the blood was studied by examining the compositions of lipids and apolipoproteins of various density classes of serum lipoproteins in pyridoxine-deficient, and pair-fed control rats. The concentrations of protein in chylomicrons and very low density lipoproteins (VLDL) were lower than those of pair-fed controls. These findings suggested the possibilities that the secretion of VLDL from liver into blood was impaired or catabolism of VLDL was increased in pyridoxine-deficient rats. The apolipo-protein profiles of VLDL and high density lipoprotein (HDL) as measured by 0.1% SDS, 10% polyacrylamide disc electrophoresis showed that pyridoxine-deficient rats had lower contents of apo-C relative to apo-E in VLDL or to apo-A-I in HDL than those of the pair-fed controls.

The use of cyst(e)ine in the removal of proteinbound homocysteine

Competition between homocyst(e)ine and cyst(e)ine for binding sites on plasma proteins was examined both in vivo and in vitro. Plasma-free cyst(e)ine concentrations were elevated in rats fed diets adequate or deficient in vitamin B6 containing 2.4% l-cystine; however, plasma protein-bound cysteine was not increased. Feeding high cystine diets did not slow the accumulation or decrease the concentration of plasma protein-bound or free homocyst(e)ine in vitamin B6-deficient rats. An in vitro experiment demonstrated that plasma protein-bound cysteine increased when plasma was incubated with increasing concentrations of cysteine, and decreased with increasing homocysteine concentrations. Plasma protein-bound homocysteine concentration was increased by increasing the concentration of homocysteine in the incubation medium; however, increasing the cysteine concentration failed to decrease bound homocysteine. The affinity of homocysteine for binding sites on plasma proteins appeared to be high because cysteine did not displace the bound homocysteine. Therefore, feeding a high cystine diet is unlikely to cause a decrease in bound homocysteine in homocystinuric patients due to competition for binding sites, but may still be beneficial because plasma cystine concentrations are below normal in individuals with homocystinuria.

Cysteine-dependent inactivation of hepatic ornithine decarboxylase.

When rat liver homogenate or its postmitochondrial supernatant was incubated with L-cysteine, but not D-cysteine, ornithine decarboxylase (ODC) lost more than half of its catalytic activity within 30 min and, at a slower rate, its immunoreactivity. The inactivation correlated with production of H2S during the incubation. These changes did not occur in liver homogenates from vitamin B6-deficient rats. A heat-stable inactivating factor was found in both dialysed cytosol and washed microsomes obtained from the postmitochondrial supernatant incubated with cysteine. The microsomal inactivating factor was solubilized into Tris/HCl buffer, pH 7.4, containing dithiothreitol. Its absorption spectrum in the visible region resembled that of Fe2+ X dithiothreitol in Tris/HCl buffer. On the other hand FeSO4 inactivated partially purified ODC in a similar manner to the present inactivating factor. During the incubation of postmitochondrial supernatant with cysteine, there was a marked increase in the contents of Fe2+ loosely bound to cytosolic and microsomal macromolecules. Furthermore, the content of such reactive iron in the inactivating factor preparations was enough to account for their inactivating activity. These data suggested that H2S produced from cysteine by some vitamin B6-dependent enzyme(s) converted cytosolic and microsomal iron into a reactive loosely bound form that inactivated ODC.

Effect of administration of triiodothyronine on aspartate aminotransferase in pyridoxine-deficient rats.

The state of thyroid function and the effect of triiodothyronine (T3) administration on aspartate aminotransferase isozymes in the livers of rats fed on a diet with or without pyridoxine were examined. Decreased thyroid function in pyridoxine-deficient rats was demonstrated using malic enzyme as a marker of thyroid function in the liver. Administration of T3 increased cytosolic aspartate aminotransferase in the liver of pyridoxine-deficient rats, but not of control rats.

Impaired collagen maturity in vitamins B 2 and B 6 deficiency—Probable molecular basis of skin lesions

Solubility of collagen was increased and the proportion of insoluble collagen was reduced in the skin of both riboflavin as well as pyridoxine-deficient rats. Collagen content of the skin, and aldehyde concentration of salt-soluble collagen were also lower in the deficient groups. The α:β subunit ratio of salt-soluble collagen was higher in riboflavin deficiency. In food-restricted weight-matched control groups, similar changes in collagen solubility, but of lesser magnitude were observed. Both food restriction and riboflavin deficiency decreased plasma PLP concentration. Increase in the solubility of collagen, decrease in the aldehyde content of soluble collagen and increase in the α:β subunit ratio of soluble collagen, suggest that the maturation of collagen may be affected in pyridoxine or riboflavin deficiency. These molecular events may be etiologically related to the pathogenesis of the skin lesions in vitamin B 2 or B 6 deficiency.

Metabolism of methylmalonic acid in rats. Is methylmalonyl-coenzyme a racemase deficiency symptomatic in man?

Vitamin B12-deficient and normal rats were loaded with methylmalonic (MMA) and ethylmalonic acids labeled with 13C in the carboxyl groups and with 2H in the alkyl groups. Significant fractions of the administered acids were excreted in both the B12-deficient and the normal animal, having undergone exchange of both their 13C-labeled carboxyl groups with endogenous 12C. The exchange of the alpha-1H of MMA in 2H2O at 25 degrees C and pH 7.5 was found by 1H-nuclear magnetic resonance to have a half-life of 28.3 min. These results show that a fraction of in vivo metabolism through the propionate-to-succinate pathway occurs via a shunt involving free MMA. The enzymes of this pathway are thought to utilize only coenzyme A (CoA) esters. To allow for the exchange of the second CoA-bound carboxyl group, we propose the deacylation of the once exchanged acid with spontaneous racemization (relative to the 13C-carboxyl group), followed by reacylation, thus exposing the labeled carboxyl to decarboxylation. The significance of this mechanism involving free MMA is that racemization of methylmalonyl (MM)-CoA may also occur without the intervention of MM-CoA racemase. A deficiency of this enzyme need not result in symptomatic methylmalonic aciduria.

Consequences of decreased brain serotonin in the pyridoxine-deficient young rat

1. 1. The concentrations of serotonin in various brain areas were significantly decreased in the pyridoxine-deficient young rat. 2. 2. There was no change in the concentration of dopamine. 3. 3. Both Bmax and Kd of [ 3H] serotonin binding to membrane preparations from cerebral cortex were increased in deficiency and were restored to normal upon pyridoxine supplementation. 4. 4. There was no change in [ 3H] spiroperidol binding to corpus striatal membrane preparations in pyridoxine-deficient rats.

Increase of inactive form of aspartate aminotransferase in pyridoxine-deficient rat liver.

Cytosolic aspartate aminotransferase from rat liver was separated into at least 3 subforms, focused at pH 6.2, 5.9, and 5.7, by isoelectric focusing. Increase of subforms with low pI values was observed in pyridoxine-deficient rat liver. These subforms with low pI values showed low catalytic activities relative to their antigenic activities. The Km values for substrate and optimal pH values of the two main subforms were not significantly different in pyridoxine-deficient and control rat livers. Some conformational change of enzyme in the cytosol of pyridoxine-deficient rat liver was suggested by circular dichroic and fluorescent spectra. The N and C terminals of the enzyme from both pyridoxine-deficient rats and controls were shown to be alanine and glutamine, respectively.

Effect of pyridoxine-deficiency on the syntheses of aspartate aminotransferase in rat liver and muscle in vivo.

The rates of synthesis of aspartate aminotransferase isozymes in the liver and skeletal muscle in pyridoxine-deficient rats were examined. The rates of synthesis were compared in rats given pyridoxine-deficient diet ad libitum, rats given control diet ad libitum and rats pair-fed with those on the deficient diet. The rates of incorporation of 3H-L-leucine by both cytosolic and mitochondrial enzymes were highest in pair-fed controls. Incorporation of radioactivity into the cytosolic enzyme was higher in deficient rat liver than in that of controls fed ad libitum, but the rate of incorporation into the mitochondrial enzyme was similar in these two groups. In muscle the rates of incorporation of labeled leucine into both isozymes were similar in all groups when expressed relative to total protein synthesis. It was suggested that increase of glucocorticoid receptor might result in increased synthesis of cytosolic aspartate aminotransferase in pyridoxine-deficient rat liver.

Role of glucose on fatty liver formation in pyridoxine-deficient rats.

Studies were made on whether glucose starvation causes fatty liver in pyridoxine-deficient male Wistar rats. Pyridoxine deficiency resulted in significantly lower levels of liver glucose than in pair-fed controls but no significant change in the serum glucose concentration. In non-starving animals, serum immuno-reactive insulin (IRI) was significantly lower in pyridoxine-deficient rats than in pair- or ad libitum-fed controls. Liver glucokinase activity in pyridoxine-deficient rats was also significantly lower than in ad libitum-fed controls. The extent of insulin deficiency was evaluated by examining the effect of administration of insulin on pyridoxine-deficient rats. Administration of insulin had no effect on the activity of liver glucokinase in pyridoxine-deficient rats, but induced the enzyme in ad libitum-fed controls. In response to a decrease in the activity of liver glucokinase or hexokinase in the deficient group, glycolytic activity, estimated as lactate production from glucose in the liver supernatant spun at 100,000 X g, was reduced to half the control level in pyridoxine-deficient rats. The effects of glucose administration on the liver lipid content, serum insulin and serum glucose were investigated. The serum glucose concentration was not significantly different in pyridoxine-deficient and control rats at any time after the glucose load. The level of serum IRI after the load was similar in the two groups after 30 min but then gradually decreased in the deficient group. The liver lipid content of the deficient rats tended to decrease whereas that of the controls remained unchanged throughout the experiment. Thus glucose starvation in pyridoxine-deficient rats is one factor responsible for fatty liver formation. Possible mechanisms of this phenomenon are discussed.

Alterations of phospholipid and triglyceride metabolism in fatty liver caused by pyridoxine deficiency in rats.

When weanling rats were given 70% casein diet deficient in pyridoxine, they showed marked accumulation of liver lipid consisting mainly of triglyceride (TG) and cholesterol ester (CE). The serum concentrations of phospholipid (PL) and cholesterol (CH) in pyridoxine-deficient rats were significantly lower than those in pair-fed controls, but their serum levels of TG and free fatty acid (FFA) were not significantly different from those of controls. A significant inverse relation was shown between the concentration of liver TG and PL of pyridoxine-deficient rats. Measurement of incorporation of radioactivity from [1-14C]acetate into lipid in vivo showed that lipogenesis in the liver of pyridoxine-deficient rats was depressed, although the acetate pool was significantly higher than that in pair-fed controls. The incorporations of radioactivity from [1-14C]acetate into liver TG and PL in vivo were also lower in pyridoxine-deficient rats than in the pair-fed controls, but the ratio of the radioactivity in liver TG to that in liver PL was higher in pyridoxine-deficient rats than in pair-fed controls. This higher ratio was not due to increased mobilization of serum FFA from extra-hepatic tissues or decreased hydrolysis of liver TG, because labeling of serum FFA and the activity of liver lipase were not significantly different in deficient and control rats. The ratio of total radioactivity in serum TG to that in liver TG was lower in pyridoxine-deficient rats than in pair-fed controls in the latter period of the experiment. Studies were made on the components of PL contributing to liver TG accumulation in pyridoxine-deficient rats. The concentration of liver phosphatidylcholine (PC) was significantly lower in pyridoxine-deficient rats than in pair-fed controls. In pyridoxine-deficient liver, only PC showed an inverse correlation with the liver lipid concentration. These results suggest that a decreased concentration of liver PC and impaired secretion of TG from the liver into blood may be responsible for the inverse relationship of liver TG and PL concentrations in pyridoxine-deficient rats.