Vitamin B6 Acquisition and Metabolism in Schistosoma mansoni.

Schistosomes are parasitic platyhelminths that currently infect >200 million people globally. The adult worms can live within the vasculature of their hosts for many years where they acquire all nutrients necessary for their survival and growth. In this work we focus on how Schistosoma mansoni parasites acquire and metabolize vitamin B6, whose active form is pyridoxal phosphate (PLP). We show here that live intravascular stage parasites (schistosomula and adult males and females) can cleave exogenous PLP to liberate pyridoxal. Of the three characterized nucleotide-metabolizing ectoenzymes expressed at the schistosome surface (SmAP, SmNPP5, and SmATPDase1), only SmAP hydrolyzes PLP. Heat-inactivated recombinant SmAP can no longer cleave PLP. Further, parasites whose SmAP gene has been suppressed by RNAi are significantly impaired in their ability to cleave PLP compared to controls. When schistosomes are incubated in murine plasma, they alter its metabolomic profile-the levels of both pyridoxal and phosphate increase over time, a finding consistent with the action of host-exposed SmAP acting on PLP. We hypothesize that SmAP-mediated dephosphorylation of PLP generates a pool of pyridoxal around the worms that can be conveniently taken in by the parasites to participate in essential, vitamin B6-driven metabolism. In addition, since host PLP-dependent enzymes play active roles in inflammatory processes, parasite-mediated cleavage of this metabolite may serve to limit parasite-damaging inflammation. In this work we also identified schistosome homologs of enzymes that are involved in intracellular vitamin B6 metabolism. These are pyridoxal kinase (SmPK) as well as pyridoxal phosphate phosphatase (SmPLP-Ph) and pyridox(am)ine 5'-phosphate oxidase (SmPNPO) and cDNAs encoding these three enzymes were cloned and sequenced. The three genes encoding these enzymes all display high relative expression in schistosomula and adult worms suggestive of robust vitamin B6 metabolism in the intravascular life stages.

The genetic basis of human erythrocyte pyridoxal kinase activity variation.

Thirty years ago we reported that erythrocyte pyridoxal kinase activity of African-Americans was strikingly lower than that of persons with European ancestry in a tissue-specific manner. At the time, it was impossible to elucidate the mechanism by which evolution had selectively lowered the enzyme activity in one cell type but not in others. We have now identified a promoter mutation with potential erythroid-specific properties that could be the basis of a novel mechanism of controlling cell-specific decreased activity of an essential enzyme.

Abnormally high plasma levels of vitamin B6 in children with autism not taking supplements compared to controls not taking supplements.

BACKGROUND: There have been many studies of the effect of high-dose supplementation of vitamin B6 on children and adults with autism, with all but one reporting benefits. OBJECTIVE: The aim of this study was to investigate the biochemical basis for vitamin B6 therapy by measuring the level of total vitamin B6 in the plasma of unsupplemented children with autism spectrum disorder compared to unsupplemented control subjects. PARTICIPANTS: Children with autism spectrum disorders (n = 35, age 3-9 years) and unrelated typical children (n = 11, age 6-9 years), all from Arizona, were studied. (This includes the data from 24 children with autism from our previous study.) METHODOLOGY: A microbiologic assay was used to measure the level of total vitamin B6 (including phosphorylated and unphosphorylated forms), in a blinded fashion. RESULTS: Children with autism had a 75% higher level of total vitamin B6 than the controls (medians of 56 versus 32 ng/mL, respectively, p = 0.00002). Most of the autistic children (77%) had levels that were more than 2 standard deviations above the median value of the controls. The autistic girls (n = 5) also had elevated levels (mean of 54.6 ng/mL, median of 60 ng/mL). DISCUSSION: These results are consistent with previous studies that found that: (1) pyridoxal kinase had a very low activity in children with autism and (2) pyridoxal 5 phosphate (PLP) levels are unusually low in children with autism. Thus, it appears that the low conversion of pyridoxal and pyridoxine to PLP results in low levels of PLP, which is the active cofactor for 113 known enzymatic reactions, including the formation of many key neurotransmitters. CONCLUSIONS: Total vitamin B6 is abnormally high in autism, consistent with previous reports of an impaired pyridoxal kinase for the conversion of pyridoxine and pyridoxal to PLP. This may explain the many published studies of benefits of high-dose vitamin B6 supplementation in some children and adults with autism.

Mechanisms of the inhibition of human erythrocyte pyridoxal kinase by drugs.

The aim of this study was to investigate the interaction between drugs chosen for their clinical neurotoxicity or chemical structure and vitamin B6 metabolism. After a preliminary screening of drugs to determine their potential inhibitory effect on erythrocyte nonpurified pyridoxal kinase (PLK) (EC 2.7.1.35), additional investigations, including kinetic studies and detection of chemical reactivity between the inhibiting drugs and pyridoxal (PL) or pyridoxal-5'-phosphate (PLP), using UV-visible spectrophotometry and mass analysis, were carried out to specify the mechanism of PLK inhibition. Depending on the results, the inhibiting drugs were divided into three groups. The first group included theophylline and progabide and inhibited PLK using either PL or pyridoxamine (PM) as substrate and thereby were true inhibitors. Moreover, they did not form covalent complexes with PL or PLP. The second group, which included cycloserine, dopamine, isoniazid, and thiamphenicol glycinate, inhibited PLK using PL, but not PM, as substrate. They were able to react with PL or PLP to form covalent complexes, and kinetic studies suggested that the observed PLK inhibition was due to these formed complexes. A third group, which consisted of levodopa, D-penicillamine, and muzolimine, inhibited PLK using PL, but not PM, as substrate. They formed, with PL or PLP, chemical derivatives that probably had no inhibitory effect on PLK. These results and the clinical consequences of such interactions are discussed and compared with results of previous studies.

Kinetic studies of the effects of K+, Na+ and Li+ on the catalytic activity of human erythrocyte pyridoxal kinase.

Kinetic studies were conducted to examine the effects of K+, Na+ and Li+ on human erythrocyte pyridoxal kinase (PK) activity. A dialyzed hemolysate served as the PK source. The substrates used were pyridoxal (PL) and ATP. Determination of the enzymatic activity was based on HPLC separation and fluorimetric detection of PL and pyridoxal 5'-phosphate as semicarbazone derivatives. In comparison to the poor activity of PK assayed without monovalent cation, all tested cations are activators. Among them, K+ is the most effective, improving both PK affinity for the substrates and maximal velocity. Na+ increases maximal velocity and PK affinity for ATP but decreases it for PL. Li+ is a poor activator which seems to modify the enzymatic mechanism from a random to an ordered sequential pattern with ATP bound before PL. Results suggest that K+ and Na+ bind to PK on the same site while Li+ binds on another site. This hypothesis and the mechanism of monovalent cation-PK interaction are compared to other well-known K(+)-activated enzymes.

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)

Theophylline increases pyridoxal kinase activity independently from vitamin B6 nutritional status.

Asthmatics treated with theophylline, a potent inhibitor of pyridoxal kinase and therefore a vitamin B6 antagonist, demonstrated a significant correlation (r = 0.71; p < 0.001) between drug plasma levels and erythrocyte pyridoxal kinase activities. A cross-over, placebo controlled study was completed on 15 healthy volunteers to investigate the mechanism by which theophylline induces pyridoxal kinase activity. The subjects were supplemented with vitamin B6 or placebo for two weeks before theophylline therapy was started. Vitamin B6 supplementation resulted in a four-fold increase in circulating pyridoxal 5'-phosphate levels, while placebo had no effect. When theophylline therapy was commenced, erythrocyte pyridoxal kinase activities increased significantly (p < 0.001) irrespective of whether vitamin B6 or placebo was supplemented. It is concluded that a depressed vitamin B6 status is not responsible for higher erythrocyte pyridoxal kinase activities encountered during theophylline therapy, but that the drug is directly responsible for elevated enzyme levels.

Increased activity of pyridoxal kinase in tongue in Down's syndrome.

The concentrations of B6 vitamins, and the activities of pyridoxal kinase, pyridoxamine phosphate oxidase and pyridoxal phosphate phosphatase were measured in tongue. Pyridoxal kinase activity was significantly greater (P less than 0.01) in Down's syndrome subjects compared with controls.

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.

Relationship between vitamin B-6 status and elevated pyridoxal kinase levels induced by theophylline therapy in humans.

Theophylline administration to seven healthy male volunteers resulted in a rapid and significant decline in both plasma and erythrocyte pyridoxal-5'-phosphate levels. Total erythrocyte pyridoxal kinase levels increased during 15 wk of theophylline treatment from a mean initial activity of 19.23 +/- 5.03 (mean +/- SD) to 62.64 +/- 11.59 nmol pyridoxal-5'-phosphate formed/(g hemoglobin.h). Although plasma pyridoxal levels remained normal, the threefold increase in total erythrocyte pyridoxal kinase activity levels did not normalize plasma and erythrocyte pyridoxal-5'-phosphate levels. Pyridoxal-5'-phosphate hydrolysis was not affected by theophylline therapy. Increased pyridoxal oxidation was confirmed by elevated urinary 4-pyridoxic acid excretion after 15 wk of theophylline treatment. Mean erythrocyte alanine aminotransferase activity declined by 70%, and aspartate aminotransferase activity declined by 50%, indicating that decreased availability of pyridoxal-5'-phosphate can have widespread metabolic consequences. We conclude that the effect of theophylline on vitamin B-6 metabolism is not transitory and cannot be overcome by elevated intracellular levels of pyridoxal kinase. However, pyridoxine supplementation (10 mg/d for 1 wk) normalized indices of vitamin B-6 status and reversed the downward trend in both alanine aminotransferase and aspartate aminotransferase activity levels.

Vitamin B-6 metabolic enzymes in blood and placenta of pregnant mice.

Plasma pyridoxal 5'-phosphate (PLP) concentrations decrease 50% in pregnant mice and erythrocyte PLP levels increase threefold over nonpregnant levels. These studies were designed to determine whether changes in the enzymes involved in synthesis and degradation of PLP in blood are altered during pregnancy. We measured net synthesis of PLP in erythrocytes and the activity of enzymes involved in the regulation of plasma and erythrocyte PLP concentration: erythrocyte pyridoxal kinase (PLK) and neutral phosphatase, and plasma and tissue alkaline phosphatase (ALP). Net synthesis of PLP and activities of erythrocyte PLK and neutral phosphatase in erythrocytes remained unchanged during pregnancy. We were unable to detect any dephosphorylation of PLP in erythrocytes of pregnant or nonpregnant mice. Mouse erythrocytes were devoid of ALP activity; neutral phosphatase was inactive with PLP and PLP was an uncompetitive inhibitor of the enzyme. Plasma ALP activity decreased 50% in the pregnant mice; therefore, it likely does not participate in the reduction of plasma PLP levels during pregnancy. Placenta had high levels of PLP-phosphatase activity (ALP) and, if it is active as an ectoenzyme in this tissue as it is in others, it may be the most important mediator of plasma PLP levels in pregnancy.

Concentration of vitamin B6 and activities of enzymes of B6 metabolism in the blood of alcoholic and nonalcoholic men.

The purpose of this study was to compare concentrations of vitamin B6 compounds and the activities of enzymes that synthesize or catabolize pyridoxal 5'-phosphate in the plasma and erythrocytes of nonalcoholic and alcoholic subjects. Blood was obtained from male nonalcoholics and chronic alcoholics with minimal liver damage and normal hematology. Plasma, erythrocyte, and urinary B6 compounds were analyzed by high performance liquid chromatography, and pyridoxal phosphate was also measured enzymatically. Erythrocyte pyridoxine kinase and pyridoxine phosphate oxidase and erythrocyte and plasma pyridoxine phosphate phosphatases were assayed. Plasma pyridoxal phosphate concentration was significantly lower in the alcoholics (31.3 +/- 3.6 nmol/liter) than in the nonalcoholics (58.7 +/- 7.5 nmol/liter). The concentrations of the other B6 compounds in plasma, erythrocytes, and urine were not different in the two groups. Plasma alkaline pyridoxine phosphate phosphatase activity was significantly higher in the alcoholics (4.05 +/- 0.36 nmol/(h.mg] than in the nonalcoholics (3.01 +/- 0.18 nmol/(h.mg]. The activities of erythrocyte kinase, oxidase, and phosphatases were not significantly different in the two groups. The relationship of plasma pyridoxal phosphate concentration to its metabolites and the activities of the enzymes involved in its metabolism was determined. Plasma pyridoxine phosphate phosphatase activity assayed at pH 9.0 or 7.4 correlated negatively with plasma pyridoxal phosphate concentration. The low pyridoxal phosphate concentration observed in the plasma of the alcoholic subjects may in part be related to increased plasma phosphatase activity.

Vitamin B6 status of women suffering from premenstrual syndrome.

The vitamin B6 status of women suffering from premenstrual symptoms (PMS, n = 19) and a group of matched controls (n = 19) has been investigated. The women volunteering in the study were selected on strictly defined criteria. Several biochemical parameters of the metabolism of vitamin B6 and tryptophan were studied in blood and urine samples during a full menstrual cycle. No significant differences in plasma pyridoxal and pyridoxal-5'-phosphate (PLP) concentrations, the holo and total EGOT activity, the erythrocyte pyridoxine kinase activity and the urinary 4-pyridoxic acid excretion between the two groups were observed. The excretion of the tryptophan metabolites xanthurenic acid (XA) and 8-methyl-xanthurenic acid (MXA), before as well as after an oral tryptophan load, tended to be higher for the PMS group. Each of the groups showed a significant cyclic variation during the menstrual cycle in the holo and total EGOT activities and in the excretion of XA and MXA before as well as after an oral tryptophan load. It is concluded that PMS is not related to a 'cyclic' vitamin B6 status. The slight differences observed for tryptophan metabolism between the PMS and the control group deserve further study.

Purification and characterization of pyridoxal kinase from human erythrocytes.

Pyridoxal kinase has been purified 50,000-fold from human erythrocytes. The purification procedure included dextran-induced aggregation of red blood cells, ammonium sulphate fractionation of the haemolysate, DEAE-cellulose chromatography, hydroxyapatite chromatography. Sephadex G-100 gel filtration and omega-aminooctyl agarose chromatography. The enzyme preparation migrated as a single protein and activity band on analytical gel electrophoresis. Determination of the Michaelis constants for pyridoxal, pyridoxine and pyridoxamine using a new assay gave comparable values of 33 microM, 16 microM and 6.2 microM respectively. Various amines were shown as competitive inhibitors of pyridoxal kinase with respect to ATP. The inhibition order was: N-dansyl-1,8-diaminooctane greater than 1,8-diaminooctane greater than 1,6-diaminohexane greater than 1,4-diaminobutane greater than gamma-aminobutyric acid, whereas octane, hexane and butane were not inhibitors. Results suggest that the amino groups on the above inhibitors are essential for competitive inhibition at saturating concentrations of pyridoxal. It was also observed that increasing the chain length of the hydrophobic backbone of these competitive inhibitors can facilitate its action.

Regulation of vitamin B6 metabolism in human red cells.

The effects of pyridoxine and pyridoxal 5-phosphate (PLP) administration on pyridoxine kinase (PnK) and asparate aminotransferase (EGOT), a PLP-dependent enzyme, were studied in human red cells separated into young and old populations by density centrifugation. After a delay of 48 hr, both pyridoxine and PLP increase EGOT activity in mature red cells by activating preformed GOT apoenzyme. In addition, in young erythroid cells, pyridoxine therapy induces synthesis of PnK, while both pyridoxine and PLP induce synthesis of GOT apoprotein. Thus, PLP stimulates EGOT induction without a change in PnK activity, suggesting that PLP enters erythroid precursor cells without prior dephosphorylation. However, with both pyridoxine and PLP, the full induction of enzyme activities reflect the gradual replacement of circulating red cells by newly formed cells with higher enzyme levels. Therefore, the use of EGOT as a measure of vitamin B6 nutritional status requires recognition of the complexities of intracellular enzyme regulation.

Vitamin B6 metabolism in idiopathic sideroblastic anaemia and related disorders.

Patients with idiopathic anaemias associated with abnormal sideroblasts were defined according to morphologic and ferrokinetic criteria and the haematologic and biochemical effects of vitamin B6 therapy were evaluated. While all patients presented similar clinical pictures, peripheral blood changes and bone marrow abnormalities, two distinct groups were identified by sideroblast morphology and ferrokinetics. Patients with more than 5% true ring sideroblasts in the marrow (IRSA) uniformly had marked ineffective erythropoiesis, while those with abnormal sideroblasts but few true ring forms were hypoproliferative. Measurements of red cell pyridoxine kinase (PnK) and intracellular pyridoxal 5-phosphate availability (PLP) as assessed by the activity of the PLP-dependent enzyme asparate aminotransferase (EGOT), revealed slightly decreased PnK levels in IRSA subjects but normal intracellular PLP activities in both groups. Furthermore, when treated with pyridoxine, all patients showed increases in both red cell PnK and EGOT activities which were similar to those seen in normal subjects. Treatment with PLP also effectively increased erythrocyte vitamin B6 activity. Even so, neither pyridoxine nor intramuscular PLP improved erythropoiesis as determined by serial haematocrits, reticulocyte counts and erythron iron turnover measurements.However, since both therapies increased red cell protoporphyrin levels and the excretion of urinary coproporphyrin in a number of subjects, the possibility that impaired haem synthesis in the sideroblastic anaemias is associated with abnormal vitamin B6 metabolism at the level of the mitochondrion must still be considered.

Vitamin B6 metabolism in anaemic and alcoholic man.

Physiological and pathological factors affecting intracellular red cell vitamin B6 metabolism in normal, anaemic and alcoholic man were studied using a new assay for pyridoxine kinase (PnK) together with saturated and total aspartate aminotransferase (EGOT) activities as indirect indices of intracellular pyridoxal 5-phosphate (PLP) availability. In studies of anaemic states, subjects with iron deficiency anaemia demonstrated elevated levels of both PnK and saturated EGOT, while seven out of 17 subjects with inflammatory anaemia had subnormal PnK but variable saturated EGOT activities. Despite a high incidence of complicating inflammatory disease, alcoholic subjects with or without ring sideroblastic anaemia had elevated levels of both PnK and saturated EGOT. As judged from the saturated EGOT and the ratio unsaturated EGOT/saturated EGOT, intracellular PLP availability was always appropriate to the higher levels of PnK activity.

Pyridoxine kinase in Plasmodium lophurae and duckling erythrocytes.

Pyridoxine kinase enzyme activity was greatly increased in duckling erythrocytes infected with Plasmodium lophurae. Pyridoxine kinase activity in parasites freed from erythrocytes was much greater than that of uninfected erythrocytes. The apparent Km for pyridoxine of the parasite enzyme was 6.6 times 10(-5) M whereas the host red cell enzyme Km was 1.9 times 10(-6) M. Deoxypyridoxine inhibited host and parasite pyridoxine kinase activity with an apparent Ki of 1.5 times 10(-6) and 8.6 times 10(-6) M, respectively. These results suggest that the vitamin B6 metabolism of the malaria parasites is distinct and separate from that of the host erythrocytes.

Vitamin B6 metabolism in human red cells. I. Variations in normal subjects.

Physiologic and pharmacologic factors affecting intracellular red cell vitamin B6 metabolism in normal human subjects were studied using a new assay for pyridoxine kinase (PnK) together with saturated and total aspartate aminotransferase (AST) activities as indirect indices of intracellular pyridoxal 5-phosphate (PLP) availability. The presence of reduced PnK activity in Blacks was confirmed but this could not be explained on the basis of increased enzyme inactivation during red cell aging in vivo. Racial differences were also noted in the metabolism of AST and, in Caucasians, net dissociation of PLP from the apoprotein was demonstrated to occur in vivo. Despite the wide variation in Pn5 activity, AST levels were maintained within relatively narrow limits. However, when pharmacologic doses of pyridoxine were administered, PnK and AST activities increased proportionately. These findings suggest that when the supply of B6 vitamers is not limiting, PnK may play a role in regulating red cell PLP levels.