Absorption and metabolism of pyridoxamine in mice. II. Transformation of pyridoxamine to pyridoxal in intestinal tissues.

The absorption of pyridoxamine from the intestine of the mouse was studied in whole animals. [3H]Pyridoxamine was orally administered and the distribution of isotope between the six recognized forms of vitamin B6 was determined in portal blood after the administration. When small doses (1.4 or 14 nmol) were administered, labeled pyridoxamine could hardly be found in the portal blood, although labeled pyridoxal and pyridoxal phosphate were found in the same blood. However, when a large amount (46 or 140 nmol) was given, a significant amount of labeled pyridoxamine was found with labeled pyridoxal and pyridoxal phosphate in the portal blood. These results suggest that a physiological dose of pyridoxamine is rapidly transformed to pyridoxal in the intestinal tissues and then released in the form of pyridoxal into the portal blood.     

Transport and metabolism of pyridoxine and pyridoxal in mice

[3H]Pyridoxine or [3H]pyridoxal in physiological amounts was orally administered to mice and the distribution of isotope between the six recognized forms of vitamin B6 and pyridoxic acid was determined at different times after the administration in the intestine, liver, blood, and brain. After 7 min about 50% of the radioactivity in pyridoxine and pyridoxal had been absorbed by the intestine and transported to the blood and other organs. When [3H]pyridoxine was administered, labeled pyridoxal, pyridoxal- and pyridoxine-phosphate were found in the intestine and liver, and labeled pyridoxine could not be detected in the peripheral blood but substantial amounts of labeled pyridoxal and pyridoxal-phosphate were found in the blood. The time course of the blood [3H]pyridoxal levels following the administration of [3H]pyridoxine was similar to that following the administration of [3H]pyridoxal. These results suggest that the intestine and/or liver play a major role in converting dietary pyridoxine to circulating pyridoxal which is taken up and phosphorylated by other organs. Moreover, most of the blood [3H]pyridoxal was shown to be located in the plasma. This localization may facilitate utilization by the organs.     

Transport and metabolism of pyridoxine in the intestine of the mouse

The absorption of pyridoxine from the intestine of the mouse was studied in whole animals. [3H]Pyridoxine was orally administered and the distribution of isotope between the six recognized forms of vitamin B6 was determined in portal and peripheral blood after the administration. When small doses (1.4 or 14 nmol) were administered, labeled pyridoxine could hardly be found in the portal blood as well as in the peripheral blood, although labeled pyridoxal and pyridoxal phosphate were found both in the portal and peripheral blood. However, when a large amount (46 or 140 nmol) was given, a significant amount of labeled pyridoxine was found with labeled pyridoxal and pyridoxal phosphate both in the portal and peripheral blood. These results suggest that a physiological dose of pyridoxine is transformed to pyridoxal in the intestinal tissues and then is released in the form of pyridoxal into the portal blood.     

Transport and metabolism of pyridoxamine and pyridoxamine phosphate in the small intestine of the rat.

The vascularly perfused small intestine of the rat was utilized to study the absorption and metabolism of pyridoxamine (PM) and pyridoxamine-5'-phosphate (PMP), independent of other tissues including erythrocytes. [3H]PM or [3H]PMP was administered intralumenally with or without the addition of unlabeled PM, or PMP or inorganic phosphate. The percentage absorption of PM was 17.1 to 19.7% in 10 minutes and was unaffected by dosages from 0.02 to 200 mumole. The isotope from [3H]PMP at a physiological level (0.02 mumole) was absorbed at the same rate as that from [3H]PM, and the distribution of the 3H remaining in the lumen and in the intestinal tissue and perfusate indicated that hydrolytic removal of the phosphate was occurring extensively in the gut. The spectrum of labeled compounds isolated from the lumen, the perfusate and the mucosa clearly indicated that PMP, unhydrolyzed, can be absorbed slowly and converted to a number of vitamin B-6 forms in the intestinal mucosa. However, the results support the view that under normal physiological conditions the majority of dietary PMP is hydrolyzed to PM which seems to be absorbed by passive diffusion and transported to other organs and tissues via the blood stream.     

Intestinal absorption of pyridoxal 5'-phosphate at physiological levels in rats

The intestinal absorption of pyridoxal 5'-phosphate (PLP) at physiological levels (10(-7) -10(-6) M) was studied in comparison with that of pyridoxal (PL) in rat, using in vitro everted sac and an intestinal preparation that permitted continuous in situ collection of mesenteric venous blood. After PLP administration (10(-6) -10(-3) M) in situ, larger amounts of PLP were found in the mesenteric venous plasma than after PL administration at the same dose. The amount of PLP found in the mesenteric venous plasma was dependent on its dose at lower concentrations up to 10(-4) M but became independent at higher concentrations. After PL administration at various doses, the amount of PL found in the mesenteric venous blood increased linearly with the dose. When various concentrations of PLP were added to the mucosal side, under the in vitro condition with protection from alkaline phosphate hydrolysis, PLP was detected in the serosal side and the extent of PLP transport was dependent on the initial concentration of PLP in the mucosal side. When various concentrations of PL were added to the mucosal side, the extent of PL transport was independent of the initial concentration of PL in the mucosal side. In rat pretreated with actinomycin D, PLP transport in vitro was inhibited but not that of PL. N2-induced anoxia and pyridoxamine 5'-phosphate and anion transport inhibitor (4,4'-diisothiocyanostilben-2,2'-disulfonic acid disodium salt) showed no effect on PLP transport. These results suggest that PLP can be absorbed in the phosphorylated form and imply the presence of a saturable process for direct absorption of PLP itself and a diffusive process for PL absorption. In addition, the result of the in vivo neonatal experiment suggests that the neonatal intestine also can transport PLP in phosphorylated form.     

Pyridoxine-5'-beta-D-glucoside affects the metabolic utilization of pyridoxine in rats.

A major form of vitamin B-6 in plant-derived foods is pyridoxine-5'-beta-D-glucoside. Previous studies have shown that pyridoxine-5'-beta-D-glucoside is poorly available as a source of vitamin B-6 in rats and is partially utilized in humans. This research was conducted to determine whether unlabeled pyridoxine-5'-beta-D-glucoside affects the metabolic utilization of simultaneously administered isotopically labeled pyridoxine in rats. Three groups of rats (n = 6) were administered a single oral dose of 0, 36 or 72 nmol of unlabeled pyridoxine-5'-beta-D-glucoside along with 166.5 MBq (240 nmol) of [14C]pyridoxine. Twenty-four hours after administration of the dose the rats were killed, and the isotopic distribution of vitamin B-6 metabolites in liver and urine was determined. Urinary 14C and hepatic 14C-labeled pyridoxine phosphate and pyridoxal phosphate were directly related to pyridoxine-5'-beta-D-glucoside dose. Hepatic 14C, 14C-labeled pyridoxal, pyridoxine and pyridoxamine, and the concentration of urinary [14C]4-pyridoxic acid, relative total urinary 14C, were inversely proportional to the dose of pyridoxine-5'-beta-D-glucoside. These results provide evidence that pyridoxine-5'-beta-D-glucoside quantitatively alters the metabolism and in vivo retention of [14C]pyridoxine and that pyridoxine-5'-beta-D-glucoside may retard the utilization of nonglycosylated forms of vitamin B-6.     

Update on interconversions of vitamin B-6 with its coenzyme

Biosynthesis of pyridoxal 5'-phosphate (PLP) depends upon the relatively specific action of two consecutive enzymes, viz. pyridoxal (pyridoxine, pyridoxamine) kinase and pyridoxine (pyridoxamine) phosphate oxidase. Less specific phosphatases catalyze hydrolyses of the 5'-phosphates of the vitamers pyridoxal, pyridoxamine, and pyridoxine. From the recognition a generation ago of these processes by which the three forms of vitamin B-6 and their 5'-phosphates are interconverted, more recent studies have provided a fairly sophisticated understanding of the molecular characteristics of the enzymes involved. The evolutionary retention of homologous portions of pyridoxal kinase in humans as well as bacteria and the most recent finding of a highly conserved region of the pyridoxine (pyridoxamine) phosphate oxidase, also from both prokaryotic and eukaryotic organisms, emphasize the importance of these catalysts in the formation of a coenzyme that is essential for most organisms. Both kinase and oxidase involved in B-6 metabolism are potential targets for pharmacologic agents.     

Abnormal molecules of mitochondrial aspartate aminotransferase in the liver of vitamin B-6--deficient rats may be produced in the mitochondrial matrix

The distribution of mitochondrial aspartate aminotransferase (AspATm) in liver cells was studied in rats fed pyridoxine-deficient and control diets. Mitochondrial aminotransferase activity was found mainly in the matrix fraction, with smaller amounts in the outer membranes, intermembrane space and cytosol. The precursor of the enzyme was detected in the liver cytosol of both vitamin B-6--deficient and control rats, and its amount was similar in the two groups. When pyridoxal phosphate was added to the assay system, the ratio of enzyme activity to antigenic activity (E/A) of mitochondrial aspartate aminotransferase in the cytosol of both vitamin B-6--deficient and control rats was about 70% of that in the matrix of control rats. On the other hand, the E/A of the matrix enzyme in deficient rats was 53% of that of controls. From these results we concluded that pyridoxal phosphate is not necessary for translocation of mitochondrial aspartate aminotransferase into mitochondrial matrix and that abnormal molecules of the enzyme may be formed in the matrix of vitamin B-6--deficient rat liver.     

Control Of Enzyme Levels In Vitamin Deficiency

Pyridoxins deficiency in rats resulted in an increase in the activity of an enzyme which inactivates pyridoxal phosphate dependent apoenxymes by splitting the peptide chain. The activity of this enzyme increased in intestine and skeletal muscle, but not in liver or heart, and may function to relcase pyridoxal phosphale from muscle for more vital functions in other tissues.     

Hepatic control of food intake

Russek's (1981 a) review of the "hepatostatic" theory states that food absorbed from the intestine causes a change in liver metabolism that in turn affects food intake. The results of two of my experiments are in conflict with the theory. In one experiment, food absorbed in physiological amounts from the intestine of the rat failed to cause a decrease in subsequent food intake. In the other experiment, food absorbed from the intestine was diverted into the systemic blood through the use of a portacaval shunt. In spite of a decrease in the amount of absorbed food that would flow to the liver, there was no increase in food intake. These experiments fail to support the "hepatostatic theory".     

Relationship between maternal and neonatal vitamin B6 metabolism: perspectives from enzyme studies.

Enzymes involved in vitamin B6 metabolism, i.e., pyridoxal kinase, pyridoxamine (pyridoxine) 5'-phosphate oxidase, and pyridoxal 5'-phosphate phosphatase, were assayed in hemolysates prepared from cord, maternal, and control blood samples. Mean cord and control pyridoxamine (pyridoxine) 5'-phosphate oxidase activities were significantly higher than maternal activities (p less than 0.001 and p less than 0.05, respectively). A significant correlation (p less than 0.001) was observed between maternal and cord vitamin B6-metabolizing enzymes. Cord pyridoxal 5'-phosphate levels correlated significantly with maternal pyridoxal 5'-phosphate levels (p less than 0.001) and with cord pyridoxal kinase activity (p less than 0.05). Maternal pyridoxal 5'-phosphate levels appear to be the most important factor determining fetal vitamin B6 status.     

Activities of the hepatic enzymes of vitamin B6 metabolism for patients with cirrhosis

Patients with cirrhosis and other hepatic diseases frequently exhibit lower concentrations of plasma pyridoxal 5'-phosphate (PLP), which is derived primarily from liver. To determine the biochemical basis for this abnormality, the enzymes of vitamin B6 metabolism--pyridoxal kinase, pyridoxine (pyridoxamine) 5'-phosphate oxidase, PLP phosphatase(s), and pyridoxal oxidase(s)--were analyzed in liver. The activities of the two biosynthetic enzymes, pyridoxal kinase and pyridoxine (pyridoxamine) 5'-phosphate oxidase were similar for both. The phosphatase activities were significantly higher (mean +/- SD of 9.55 +/- 8.03 versus 3.97 +/- 2.36 nmol X min X mg protein, p less than 0.05) for cirrhotics. Pyridoxal oxidase activities appeared slightly lower for cirrhotics. There was considerable variation in many indices of liver function, which suggests that the defects contributing to altered vitamin B6 metabolism may be complex and individualistic. These analyses have shown that cirrhotics are capable of apparently normal PLP synthesis and that increased hepatic dephosphorylation may be responsible for low levels of plasma PLP.     

Assessment of vitamin B 6 status. Studies on pregnant women and oral contraceptive users.

The vitamin B6 status of 10 pregnant women (third trimester), 9 oral contraceptive agent users, and 12 notnpregnant women (controls) was investigated over a 10-week period by means of the erythrocyte glutamate-oxaloacetate transaminase (E-GOT) activation test and by measurements of whole blood pyridoxal phosphate levels. Blood pyridoxal phosphate levels in oral contraceptive agent users (7.6 plus or minus 1.1 ng/ml) were significantly lower than in the control group (9.6 plus or minus 1.7 mg/ml)indicating a relative vitamin B6 deficiency. Pyridoxal phosphate levels in pregnant women(5.1 plus or minus 1.3ng/ml) were even lower; in fact, no overlap was found in individual mean values between the pregnant and control groups. The E-GOT activation test did not indicate a vitamin deficiency in the pregnant women or in oral contraceptive agent users and the E-GOT activation factor did not correlate with blood pyridoxal phosphate levels unless results obtained after pyridoxine hydrochloride (20 mg/day) administration were included. The E-GOT activation test appears to be a poor indicator of vitamin B6 status, except in pronounced deficiency as it is less responsive to vitamin depletion than blood pyridoxal phosphate levels, and suffers from relatively large variations in individual control values. This may be a result of factors unrelated to vitamin B6 as blood pyridoxal phosphate levels remained fairly constant in the individuals investigated.     

Semi-automated system for analysis of vitamin B6 complex by ion-exchange column chromatography.

A simple system for semi-automatic analysis of pyridoxal, pyridoxine and pyridoxamine has been developed using diazide of 5-chloroaniline 2,4-disulfonyl chloride as a color-producing reagent in combination with desoxypyridoxine as an internal standard. It employs Aminex A-5 column, 10x0.6? cm, as an adsorbent. Adsorbates were eluted successively with 3 discrete phosphate buffers (0.4 N Na+). The effluent is mixed continuously with capillary streams of the diazide reagent and of sodium acetate. The mixture, maintained at 65 degrees C in a heating bath, then passes through a spiral of Teflon tubing with a residence time of 2 min. Characteristic orange colored products formed by a diazo coupling reaction are continuously monitored at 440 nm in a flow photometer. The individual peaks on the recorded chromatogram are manually integrated by a conventional HW method. The elution position and recovery of desoxypyridoxine permits correction for sensitivity changes or mechanical losses which might occur during a series of analyses. The analyzer system described allow quantitation from 2 to 25 mug of pyridoxal, pyridoxine and pyridoxamine in a single sample within 2 hr and with a precision of 100 +/- 4%. It is also found suitable as a routine procedure for the analysis of varied biological samples.     

Vitamin B-6 metabolism in the pregnant rat: effect of progesterone on the (re)distribution in maternal vitamin B-6 stores

Parameters of vitamin B-6 metabolism were studied in pregnant rats, nonpregnant control rats and progesterone-supplemented ovariectomized rats. Plasma pyridoxal 5'-phosphate (PLP) and pyridoxal concentrations in pregnant rats were 20 and 40% of those of nonpregnant rats, respectively. Excretion of 4-pyridoxic acid in the urine in pregnant rats was about 40% of that of nonpregnant rats. Liver PLP content was also lower during pregnancy, but liver pyridoxamine 5'-phosphate (PMP), kidney PLP and PMP and muscle PLP contents did not change significantly. Progesterone administration to ovariectomized rats resulted in slightly lower plasma, liver and kidney PLP levels than in intact untreated control rats. Liver and kidney pyridoxal kinase (PK) and pyridoxamine 5'-phosphate oxidase activities were similar in pregnant and nonpregnant rats. Progesterone treatment resulted in a significantly lower PK activity in the kidney of the treated rats than in untreated controls. It is concluded that the pregnancy-induced changes in vitamin B-6 metabolism were unlikely to be related directly to progesterone. However, progesterone may secondarily affect maternal vitamin B-6 stores during pregnancy, by temporary deposition and increased retention of vitamin B-6.     

Studies on the effect of dose size on the absorption of beta-carotene by the rat in vivo

This study was carried out to choose between two hypotheses with respect to the regulation of beta-carotene (BC) conversion to retinol in the whole animal: uptake of BC into intestinal mucosa is limited by saturation of an intestinal receptor; or the conversion to retinol is limited by saturation of the conversion enzyme(s). Groups of rats were given five different dose levels of labeled BC by stomach tube. Labeled and total BC and retinol were isolated from tissues and intestinal contents after 4 h. Results showed a positive linear relationship between BC in the intestinal wall and the dose administered, with no saturation level up to 1440 micrograms administered. Per cent formation of newly formed retinol from newly absorbed (i.e., labeled) BC was 20-26% of the three lower dose groups, 10% for the highest dose. No retinyl esters could be detected in the intestine. Most of the administered BC was in the intestinal contents, about 100-times more than in the intestinal wall and mucosa. Newly formed retinol in plasma was about 10-times that in liver. Small amounts of newly absorbed BC were found in liver, but no labeled retinyl esters. These results suggest that the absorption of BC is very inefficient; that it does not occur through an intestinal receptor; that the formation of retinol is regulated at the level of the conversion enzyme(s).     

Effects of vitamin B-6 deficiency and 4'- deoxypyridoxine on pyridoxal phosphate concentrations, pyridoxine kinase and other aspects of metabolism in the rat

Male rats about 100 days old were fed a B-6 deficient diet supplemented with 4'-deoxypyridoxine (1 g/kg diet) and/or pyridoxine hydrochloride (22 mg/kg diet) for 30 to 35 days. Addition of 4'-deoxypyridoxine to the B-6-deficient diet produced greater losses in body weight (P less than 0.05) and thymus weight (P less than 0.01) than in B-6-deficient pair-fed controls. 4'-Deoxypyridoxine combined with a B-6-deficient diet produced no decreases in the concentration of pyridoxal phosphate or pyridoxine kinase in the tissues examined when compared with B-6-deficient controls. Addition of deoxypyridoxine to a diet containing adequate B-6 tended to reduce that absolute weight of the adrenal glands and increased (P less than 0.05) plasma cholesterol compared with animals receiving only vitamin B-6. Compared with the B-6-deficient groups, pyridoxal phosphate concentrations in animals receiving normal B-6 were significantly (P less than 0.01) increased in the liver, muscle and adrenal glands but not in the thymus. In all groups the pyridoxine kinase activity was highest in the adrenal glands (3.6-6.3 pmole pyridoxine phosphate/minute/mg tissue) followed by the liver (1.3-3.7) and thymus (0.7-1.3). These high kinase values and the weight changes suggest an important role for vitamin B-6 in these organs. Recent evidence that pyridoxal phosphate may interact with glucocorticoid receptors raises the possibility that the role of vitamin B-6 in these and other organs may involve metabolic regulation by a mechanism independent of the well-established coenzyme function of this vitamin.     

Mode of binding of pyridoxal 5'-phosphate in rat liver ornithine aminotransferase

1. Pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate were effective for the association of apo-form II of ornithine aminotransferase [EC 2.6.1.13]; whereas other B6 derivatives, such as pyridoxal, pyridoxamine, pyridoxine and pyridoxine 5'-phosphate, had no effect on form II of this apoenzyme. 2. The pyridoxal 5'-phosphate contents of the native enzyme, and reconstituted forms I and II were determined by two different methods to be 1.5 moles, 2.5 moles and 3.3 moles of pyridoxal 5'-phosphate/mole of enzyme, respectively.     

Individual vitamin B6 contents in selected Japanese sushi toppings

The contents of six natural vitamin B(6) forms in popular Japanese sushi toppings were determined by a 4-pyridoxolactone-conversion HPLC method. The half-baked bonito exhibited the highest total vitamin B(6) content and the northern shrimp sashimi the lowest. Pyridoxal 5'-phosphate plus pyridoxal was predominant in nine samples, and pyridoxamine 5'-phosphate plus pyridoxamine in two other samples. Pyridoxine 5'-phosphate plus pyridoxine was minor. The raw meats (sashimi) of fatty seawater fishes contain a lot of pyridoxal 5'-phosphate and/or pyridoxamine 5'-phosphate. Five portions of sushi with 20 g of fatty seawater sashimi toppings would supply with vitamin B(6) recommended by the Japanese Recommended Daily Allowance.     

Individual vitamin B6 contents in selected Japanese sushi toppings

The contents of six natural vitamin B₆ forms in popular Japanese sushi toppings were determined by a 4-pyridoxolactone-conversion HPLC method. The half-baked bonito exhibited the highest total vitamin B₆ content and the northern shrimp sashimi the lowest. Pyridoxal 5′-phosphate plus pyridoxal was predominant in nine samples, and pyridoxamine 5′-phosphate plus pyridoxamine in two other samples. Pyridoxine 5′-phosphate plus pyridoxine was minor. The raw meats (sashimi) of fatty seawater fishes contain a lot of pyridoxal 5′-phosphate and/or pyridoxamine 5′-phosphate. Five portions of sushi with 20 g of fatty seawater sashimi toppings would supply with vitamin B₆ recommended by the Japanese Recommended Daily Allowance.