Sphingosine 1-phosphate lyase inhibition by 2-acetyl-4-(tetrahydroxybutyl)imidazole (THI) under conditions of vitamin B6 deficiency.

Caramel food colorant 2-acetyl-4-(tetrahydroxybutyl)imidazole (THI) causes lymphopenia in animals through sphingosine 1-phosphate lyase (SPL) inhibition. However, this mechanism of action is partly still controversial because THI did not inhibit SPL in vitro either in cell-free or in cell-based systems. It is thought that the in vitro experimental conditions which have been used so far were not suitable for the evaluation of SPL inhibition, especially in case of cell-based experiments. We speculated that the key factor might be the coenzyme pyridoxal 5'-phosphate (PLP), an active form of vitamin B6 (VB6), because media used in cell-based assays usually contain an excess amount of VB6 which leads to the activation of SPL. By the use of VB6-deficient culture medium, we could regulate apo- (without PLP) and holo- (with PLP) SPL enzyme in cultured cells, resulting in the successful detection of SPL inhibition by THI. Although the observed inhibitory effect was not as strong as that of 4-deoxypyridoxine (a VB6 analog SPL inhibitor), these findings may be useful for further understanding the mechanism of action of THI.

Novel spectrophotometric method for the quantitation of urinary xanthurenic acid and its application in identifying individuals with hyperhomocysteinemia associated with Vitamin B6 deficiency.

A novel spectrophotometric method for the quantification of urinary xanthurenic acid (XA) is described. The direct acid ferric reduction (DAFR) procedure was used to quantify XA after it was purified by a solid-phase extraction column. The linearity of proposed method extends from 2.5 to 100.0 mg/L. The method is precise, yielding day-to-day CVs for two pooled controls of 3.5% and 4.6%, respectively. Correlation studies with an established HPLC method and a fluorometric procedure showed correlation coefficients of 0.98 and 0.98, respectively. Interference from various urinary metabolites was insignificant. In a small-scale screening of elderly conducted at Penghu county in Taiwan (n = 80), we were able to identify a group of twenty individuals having hyperhomocysteinemia (>15 µ mole/L). Three of them were found to be positive for XA as analyzed by the proposed method, which correlated excellently with the results of the activation coefficient method for RBC's AST/B6 functional test. These data confirm the usefulness of the proposed method for identifying urinary XA as an indicator of vitamin B6 deficiency-associated hyperhomocysteinemic condition.

Perinatal hypophosphatasia presenting as neonatal epileptic encephalopathy with abnormal neurotransmitter metabolism secondary to reduced co-factor pyridoxal-5'-phosphate availability.

We describe two neonates presenting with perinatal hypophosphatasia and severe epileptic encephalopathy resulting in death. Both had increased levels of urinary vanillactate, indicating functional deficiency of aromatic amino acid decarboxylase, a pyridoxal-5-phosphate (PLP)-dependent enzyme required for dopamine and serotonin biosynthesis. Clinical findings and results of subsequent metabolic investigations were consistent with secondary pyridoxine-deficient encephalopathy. These patients highlight the importance of tissue non-specific alkaline phosphatase in the neuronal PLP-dependent metabolism of neurotransmitters. In addition, the disturbance of PLP metabolism appears to underlie the predominant neurological presentation in our patients. We recommend the measurement of serum alkaline phosphatase (ALP) during the assessment of perinatal seizures.

Hepatic alanine-glyoxylate aminotransferase activity and oxalate metabolism in vitamin B6 deficient rats.

PURPOSE: Urinary oxalate has an important role in the formation of calcium oxalate stone and approximately 50% to 60% of urinary oxalate is derived from the endogenous metabolism of glyoxylate. Glyoxylate is enzymatically converted to glycine by alanine-glyoxylate aminotransferase in the liver and vitamin B6 has a key role as a coenzyme. Therefore, we evaluated hepatic alanine-glyoxylate aminotransferase activity and oxalate metabolism in vitamin B6 deficient rats. MATERIALS AND METHODS: A total of 12 male Wistar rats were fed a control or a vitamin B6 deficient diet. After 4 weeks creatinine, oxalate, glycolate, glycine and citrate in the urine, and alanine-glyoxylate aminotransferase activity and its mRNA level in the liver were measured. RESULTS: Urinary oxalate-to-creatinine and glycolate-to-creatinine ratios were significantly higher in vitamin B6 deficient rats than in control rats but urinary glycine-to-creatinine and citrate-to-creatinine ratios were significantly lower. Hepatic alanine-glyoxylate aminotransferase activity and its mRNA level were significantly lower in vitamin B6 deficient rats than in control rats. CONCLUSIONS: Vitamin B6 deficiency not only decreased alanine-glyoxylate aminotransferase activity, but also down-regulated alanine-glyoxylate aminotransferase gene expression by hepatocytes and led to hyperoxaluria and hyperglycolic aciduria secondary to impaired metabolism of oxalate precursors. Hyperoxaluria with hypocitruria may also contribute to calcium oxalate stone formation in vitamin B6 deficiency.

Vitamin B-6 deficiency in rats reduces hepatic serine hydroxymethyltransferase and cystathionine beta-synthase activities and rates of in vivo protein turnover, homocysteine remethylation and transsulfuration.

Vitamin B-6 deficiency causes mild elevation in plasma homocysteine, but the mechanism has not been clearly established. Serine is a substrate in one-carbon metabolism and in the transsulfuration pathway of homocysteine catabolism, and pyridoxal phosphate (PLP) plays a key role as coenzyme for serine hydroxymethyltransferase (SHMT) and enzymes of transsulfuration. In this study we used [(2)H(3)]serine as a primary tracer to examine the remethylation pathway in adequately nourished and vitamin B-6-deficient rats [7 and 0.1 mg pyridoxine (PN)/kg diet]. [(2)H(3)]Leucine and [1-(13)C]methionine were also used to examine turnover of protein and methionine pools, respectively. All tracers were injected intraperitoneally as a bolus dose, and then rats were killed (n = 4/time point) after 30, 60 and 120 min. Rats fed the low-PN diet had significantly lower growth and plasma and liver PLP concentrations, reduced liver SHMT activity, greater plasma and liver total homocysteine concentration, and reduced liver S-adenosylmethionine concentration. Hepatic and whole body protein turnover were reduced in vitamin B-6-deficient rats as evidenced by greater isotopic enrichment of [(2)H(3)]leucine. Hepatic [(2)H(2)]methionine production from [(2)H(3)]serine via cytosolic SHMT and the remethylation pathway was reduced by 80.6% in vitamin B-6 deficiency. The deficiency did not significantly reduce hepatic cystathionine-beta-synthase activity, and in vivo hepatic transsulfuration flux shown by production of [(2)H(3)]cysteine from the [(2)H(3)]serine increased over twofold. In contrast, plasma appearance of [(2)H(3)]cysteine was decreased by 89% in vitamin B-6 deficiency. The rate of hepatic homocysteine production shown by the ratio of [1-(13)C]homocysteine/[1-(13)C]methionine areas under enrichment vs. time curves was not affected by vitamin B-6 deficiency. Overall, these results indicate that vitamin B-6 deficiency substantially affects one-carbon metabolism by impairing both methyl group production for homocysteine remethylation and flux through whole-body transsulfuration.

Vitamin B-6 deficiency and level of dietary protein affect hepatic tyrosine aminotransferase activity in cats.

Total activity [pyridoxal 5'-phosphate (PLP) added in the assay] of hepatic tyrosine aminotransferase (TAT) measured in cats at 0300, 0900, 1500 and 2100h was 10.3 +/-1.1, 14.0 +/- 0.7, 9.8 +/- 1.3 and 11.0 +/- 0.7 nkat/g liver, indicating little diurnal variation. Activity after 18 h of food deprivation was 10.0 +/- 0.3 nkat/g liver, also not different from cats that were eating ad libitum. These findings support the idea that cats have only limited changes in the activity of hepatic TAT compared with rats. Total TAT activity was measured in cats fed high protein (550 g/kg) and low protein (180 g/kg) diets for 4 wk. Cats fed a high protein diet had activities significantly higher (about twice) than cats fed the low protein diet. Hepatic TAT activity of vitamin B-6-deficient cats (diet without pyridoxine for 9 wk) was compared with cats given the same diet with 8 mg pyridoxine/kg. Total hepatic TAT activity in deficient cats was significantly (P < 0.05) lower per gram soluble or total protein (but not per gram liver) than control cats; holoenzyme activity and percentage of active enzyme in deficient cats were also significantly lower by 75 and 64%, respectively. The apparent Km of TAT from cats for tyrosine (2.1 mmol/L) was similar to that for rats (1.9 mmol/L), but higher for PLP in cats (0.16 micromol/L) than rats (0.034 micromol/L). Part of the reason for the higher plasma tyrosine in vitamin B-6-deficient cats than rats is the higher Km of TAT for PLP in cats than rats.

Vitamin B6 deficiency accelerates metabolic turnover of cystathionase in rat liver.

Although most of cystathionase was found to exist as an inactive apoenzyme in the liver of vitamin B6-deficient rats, the concentrations of the immunoreactive enzyme protein were virtually the same for control and vitamin B6-deficient livers. Under vitamin B6 deficiency, however, the rate of synthesis of cystathionase, measured by incorporation of labeled amino acid into the immunoprecipitated enzyme, was increased severalfold due to an increased level of cystathionase mRNA. Western blot analysis of lysosomal proteins showed that the amount of cystathionase in the lysosomes from the liver of vitamin B6-deficient rats was also increased severalfold. This observation suggests that lysosomes specifically recognize the apocystathionase for sequestration in preference to the holoenzyme. The present study provides the molecular basis for dual roles of vitamin B6 in controlling the metabolic turnover of cystathionase; it regulates synthesis of the enzyme by modulating the expression of cystathionase gene, and it regulates degradation of the enzyme by different susceptibilities of apo- and holoenzymes to lysosomal proteolysis.

Pyridoxal 5'-phosphate modulates expression of cytosolic aspartate aminotransferase gene by inactivation of glucocorticoid receptor.

The level of mRNA for cytosolic aspartate aminotransferase (cAST) in the liver of vitamin B6-deficient rats was found to be 7-fold higher than that of the control rats. The administration of hydrocortisone to adrenalectomized vitamin B6-deficient rats induced expression of hepatic cAST mRNA and the induction was suppressed by the simultaneous administration of pyridoxine. Since the 5' regulatory region of the rat cAST gene contains several sequences showing homology to glucocorticoid-responsive elements, we synthesized an oligonucleotide probe of glucocorticoid-responsive element sequence and assayed the binding activity of liver nuclear extract to the oligonucleotide by gel mobility shift analysis. We found that the binding activity of nuclear extract prepared from the liver of vitamin B6-deficient rats was far greater than that of the control rats, indicating that the DNA-binding activity of glucocorticoid receptor was enhanced by vitamin B6 deficiency. We further found that preincubation of the nuclear extract from the vitamin-deficient liver with pyridoxal 5'-phosphate brought about a rapid and extensive decrease in the binding of the extract to the glucocorticoid-responsive element. Congeners of pyridoxal phosphate, such as pyridoxamine 5'-phosphate, pyridoxal, pyridoxamine and pyridoxine, did not show an inhibitory effect. These observations suggest that pyridoxal 5'-phosphate modulates cAST gene expression by inactivating the binding activity of glucocorticoid receptor to glucocorticoid-responsive elements.

Alteration in the phosphatidylcholine biosynthesis of rat liver microsomes caused by vitamin B6 deficiency.

Rats fed with a vitamin B6-deficient 70% casein diet for 5 weeks were found to have decreased considerably in the content of phosphatidylcholine (PC) in liver microsomes, presumably because of the depressed PC biosynthesis from choline or phosphatidylethanolamine (PE). The activities of choline phosphokinase and choline phosphotransferase in liver decreased, apparently, as compared with the pair-fed control or control rats. The hepatic level of the PE methyltransferase co-substrate, S-adenosylmethionine (SAM), decreased about 1/3, but the level of the inhibitory metabolite, S-adenosylhomocysteine (SAH), was elevated due to the marked reduction in the activities of cystathionine beta-synthase and gamma-cystathionase. The resultant molar ratio of SAM/SAH decreased drastically such that the methylation of PE to PC was decreased in vivo, as confirmed by lowering the activity of PE methyltransferase in vitro in response to a decreased molar ratio of SAM/SAH. A similar effect on the PE methylation was also observed in the pair-fed control rats, but the PC biosynthesis from choline clearly compensated for the drop of PC biosynthesis from PE. Results of this study demonstrate that vitamin B6 deficiency modified methionine metabolism and decreased choline utilization, and thus indirectly affected the biosynthesis of PC in liver microsomes.

Connective tissue integrity is lost in vitamin B-6-deficient chicks.

The objective of the present investigation was to characterize further the connective tissue disorder produced by pyridoxine (vitamin B-6) deficiency, as previously evidenced by electron microscopy. Following the second post-natal week, fast growing male chicks were deprived of pyridoxine for a 1-mo period. Six weeks post-natally, blood concentrations in the experimental deficiency group had declined to deficiency levels as registered by low concentrations of pyridoxal phosphate (coenzyme form) in erythrocytes, but did not reach levels associated with neurological symptoms. Light microscopic study showed abnormalities in the extracellular matrix of the connective tissues. Collagen cross-links and the aldehyde contents were not significantly lower in cartilage and tendon collagens of vitamin B-6-deficient animals than in age-matched controls; also, their proteoglycan degrading protease and collagenase activities measured in articular cartilages were not greater. Thus, proteolysis was an unlikely alternative mechanism to account for the loss of connective tissue integrity. These results point to the need for further investigation into adhesive properties of collagen associated proteoglycans or other proteins in vitamin B-6-deficient connective tissue.

Overt vitamin B-6 deficiency affects rat pancreatic digestive enzyme and glutathione reductase activities.

Previous reports suggest that vitamin B-6 deficiency contributes to pancreatic insufficiency. However, the susceptibility of pancreatic function to marginal vitamin B-6 intake has not been defined. The present study examines digestive enzyme activity and steady-state mRNA levels, as well as antioxidant enzyme status from rats fed different vitamin B-6 diets. Groups (n = 12) of adult female Long-Evans rats were assigned to five dietary groups and fed their respective diets for 6 wk. Control and food-restricted rats were fed the control diet (7 mg pyridoxine/kg diet) freely, or food intake was restricted to the lowest intake of the experimental groups. The experimental groups were fed purified diets containing 0 (deficient), 0.25 or 1 (marginal) mg pyridoxine/kg diet. Plasma amylase and pancreatic amylase, trypsin and chymotrypsin activities were significantly lower in deficient rats compared with rats fed the control diet. Lower enzyme activities were accompanied by 83 and 55% lower amylase and trypsinogen mRNA levels compared with levels in rats fed the control diet. Other than low glutathione reductase in deficient rats, pancreatic antioxidant enzyme activity was similar in all dietary groups. These data suggest that the exocrine pancreas is impaired by vitamin B-6 deficiency, but marginal pyridoxine intake maintains function.

Effects of pyridoxine deficiency on the metabolism of N-nitrosodimethylamine in the rat.

The alteration in the metabolic activation of N-nitrosodimethylamine (NDMA) was investigated in the rat during dietary pyridoxine deficiency. The in vitro metabolism of NDMA by demethylase system was measured in both liver and kidney microsomes. The profile of the kidney enzyme appears similar to that of the liver indicating that at least two forms of isozymes with the low and the high Km's are present. Pyridoxine deficiency significantly increased the activity of NDMA-demethylase of both organs. The increase in the activity of NDMA-demethylase induced by dietary pyridoxine deficiency can be reversed by supplementation of pyridoxine (500 micrograms), i.p., daily for two consecutive days. The increase in the NADPH cytochrome c reductase activity was observed after 6 weeks on pyridoxine-deficient diet.

Vitamin B6 metabolism and binding to proteins in the blood of alcoholic and nonalcoholic men.

Blood obtained from nonalcoholic and alcoholic subjects was incubated with 100 nM [3H]pyridoxine to study its uptake and metabolism by erythrocytes and the binding of vitamin B6 metabolites to proteins in plasma and erythrocytes. Erythrocytes of the alcoholics accumulated tritium faster than those of the controls; however, they contained the same total amount of tritiated compounds by 15 min. After incubation for 30 min, the erythrocytes had converted most of the pyridoxine to pyridoxal phosphate and pyridoxal. Pyridoxal-P remained in the erythrocytes, and approximately 40% of the pyridoxal diffused into the plasma. [3H]Pyridoxal and [3H]pyridoxal-P levels in the erythrocytes and plasma of the alcoholics were similar to those in the controls. However, dialyzed hemolysates of the alcoholics had more [3H]pyridoxal and a lower percentage of [3H]pyridoxal-P than those of the controls. The total concentration of plasma pyridoxal-P was lower in the alcoholics than in the controls and did not change upon incubation of whole blood with pyridoxine or upon dialysis. The erythrocytes of the alcoholics and controls had similar concentrations of pyridoxal-P that increased 2.5-fold upon incubation of whole blood with pyridoxine for 30 min and returned to the initial concentrations upon dialysis. The amount of [3H]pyridoxal and [3H]pyridoxal-P bound to protein was assessed by treating hemolysate and plasma samples with borohydride before dialysis. More 3H was bound to protein in the erythrocytes than in the plasma. The amount of protein-bound 3H in the erythrocytes of the alcoholics was lower than that of the controls, whereas the amount of protein-bound 3H in plasma was similar in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitamin B6 deficiency causes activation of RNA polymerase and general enhancement of gene expression in rat liver.

The effect of vitamin B6 deficiency on the activity of RNA polymerase and expression of several mRNAs in rat liver was investigated. The activities of RNA polymerase I and II in the liver of vitamin B6-deficient rats were found to be higher than the control rats by 30%. The expression of several mRNAs, including mRNAs for beta-actin and glyceraldehyde-3-phosphate dehydrogenase, and the content of poly(A)+ RNA were also increased in vitamin deficiency. These observations suggest that vitamin B6 influences gene expression in the liver, at least in part, by modulating the activity of RNA polymerase.

Effect of pyridoxine deficiency on immunological phenomena.

Measurements of serine hydroxymethyltransferase (SHMT) in resting lymphocyte cultures showed that the level of activity of this enzyme was very low. Under the influence of mitogenic stimuli serine hydroxymethyl-transferase activity was induced 5-20-fold. Addition in the cultures of 4-deoxypyridoxine, a potent antagonist of vitamin B6 coenzymes, concurrently with the mitogen, inhibits the induction of serine hydroxymethyltransferase. Thus deoxypyridoxine exerts its effects not only at the level of the enzyme itself by antagonizing the coenzyme but also at the level of its biosynthesis. Synchronous addition of vitamin B6 with deoxypyridoxine effectively reverses the inhibitory effects. The T helper cells of the lymphocyte culture are very sensitive to deoxypyridoxine action in contrast to T suppressor cells. The effect of phytohaemagglutinin or concanavalin A on the production of interleukin-1b, interleukin-2 and interleukin-2 receptors is profoundly affected by deoxypyridoxine. These results give a deeper insight of the mechanisms by which pyridoxine deficiency causes significant reduction of humoral and cellular immune responses to antigenic stimuli. On the basis of the data of this report a detailed illustration of the events that follow the T cell activation is presented. From this investigation the enzyme serine hydroxymethyltransferase emerges as a key element in the processes of immune responses and cell proliferation. Based on this finding we advance the following two propositions for possible future medical application: (i) combination of deoxypyridoxine with immunosuppressive drugs in case of immunosuppressive therapy or organ transplantation. (ii) the enzyme presents an excellent target for chemotherapy and therefore development of special agents directed against its apoprotein may prove to be a very valuable medical tool.

Effect of pyridoxine-deficiency on degradation of cytosolic aspartate aminotransferase in rat liver lysosomes.

Highly purified lysosomes were isolated from the livers of control and pyridoxine-deficient rats. The calculation of the lysosomal protein contents indicated that the livers of both groups of rats contain virtually the same amounts of the lysosomal proteins (12.0 and 13.0 mg lysosomal proteins/g liver proteins for the control and pyridoxine-deficient rats, respectively). The immunoblotting of the lysosomal proteins with anti-cytosolic aspartate aminotransferase (cAspAT) showed 46 kDa band, corresponding to the subunit molecular weight of cAspAT, as well as the bands representing degradative intermediates of cAspAT. The relative amounts of the immuno-reactive substances were estimated by scanning the immuno-stained bands and measuring the densitometric tracings. It was found that the lysosomes in the pyridoxine-deficient rat liver contain almost twice as much cAspAT and its degradative intermediates as those in the control rat liver. On the basis of these observations, it was concluded that the increased rate of degradation of cAspAT in the liver of the pyridoxine-deficient rats is brought about by the increased rate of sequestration of cAspAT into lysosomes.

Response of vitamin B-6 content of muscle to changes in vitamin B-6 intake in men.

Previous reports indicated that in growing rats the vitamin B-6 pool in muscle was relatively stable during deficiency but increased in response to increased vitamin B-6 intake. To determine whether human muscle would show a similar response 10 college-aged males received a low vitamin B-6 diet (1.76 mumol/d) for 6 wk followed by 6 wk on a self-selected diet supplemented with 0.98 mmol pyridoxine HCl/d. During depletion, excretion of pyridoxic acid rapidly adjusted to approximate the intake. Plasma pyridoxal phosphate concentrations at the end of the baseline, depletion, and supplementation periods were 81 +/- 51, 9 +/- 3, and 455 +/- 129 nmol/L, respectively, whereas muscle concentrations were 21 +/- 9, 20 +/- 4, and 25 +/- 7 nmol/g, respectively and total vitamin B-6 in muscle was 28 +/- 10, 27 +/- 4, and 35 +/- 10 nmol/g, respectively. These data provide further confirmation that the vitamin B-6 pools in skeletal muscle are resistant to depletion. They also demonstrate that in humans with constant body weight, vitamin B-6 supplementation is not associated with marked increases in vitamin B-6 in muscle.

High-performance liquid chromatographic assay of human lymphocyte kynureninase activity levels.

Human lymphocyte kynureninase activity was assessed in homogenized cells by determination of 3-hydroxyanthranilic acid production as a function of time after addition of the substrate, 3-hydroxykynurenine. The product, 3-hydroxyanthranilic acid, was determined by isocratic high-performance liquid chromatography and fluorescence detection. Mean (+/- S.D.) lymphocyte kynureninase activity in a group (n = 12) of vitamin B6-deficient men was 5.04 +/- 0.81 pmol 3-hydroxyanthranilic acid formed per mg protein per min, which was significantly (p = 0.005) lower than the 6.69 +/- 1.70 pmol 3-hydroxyanthranilic acid formed per mg protein per min in men with a normal vitamin B6 status. This indicates that lymphocyte kynureninase activity is depressed during a vitamin B6 deficiency.