Plasma Plasma Pyridoxal 5'-phosphate Concentrations Research Articles

Pyridoxal-5′-phosphate and pyridoxal biokinetics in aging Wistar rats

Biokinetic parameters of plasma pyridoxal-5′-phosphate (PLP) and pyridoxal (PL) disposition were studied in male Wistar rats aged 8 and 27 months kept from weaning on a purified diet containing 250 g casein and 6 mg pyridoxine·HCl per kg. Baseline plasma PLP concentration was lower in the older animals (514 ± 56 nmol/L for young and 317 ± 124 nmol/L for old animals), whereas baseline plasma PL concentration did not differ between age group (average 235 nmol/L for both young and old animals). We hypothesized lower baseline plasma PLP in the older animals was caused by an increased PLP elimination rate, a decreased PLP synthesis rate, or a combination of these processes. Observations from earlier in vitro experiments suggest age-related changes occur in vitamin B-6 metabolizing enzyme activities. In the in vivo experiments described here no age-related difference in plasma PLP elimination rate nor in plasma PLP synthesis rate was observed to explain the observed decrease in plasma PLP concentration with age.

Aging and vitamin B-6 depletion: effects on plasma pyridoxal-5’-phosphate and erythrocyte aspartate aminotransferase activity coefficients in rats

Plasma pyridoxal-5'-phosphate (PLP) and erythrocyte aspartate aminotransferase activity coefficients were longitudinally determined in rats. Blood was obtained at weeks 0, 1, 2, 4, 6, 9, and 11 from rats initially aged 3 wk, 3 mo, 12 mo, and 23 mo (weeks 0, 1, 4, 6, and 9 only). The diet groups were ad libitum control (ALC), deficient (DEF), and pair-fed control (PF). Plasma PLP concentrations of controls were highest at 3 mo, intermediate at 3 wk and 12 mo, and lowest at 23 mo. When an additional group of 22-mo-old rats was fed a high-vitamin B-6 diet for 4 wk, their low baseline plasma PLP concentrations did not increase significantly. Plasma PLP decreased significantly within 1 wk and activity coefficients increased significantly by week 4 in all DEF rats. Depletion was most rapid and severe in the youngest DEF rats and least in 12-mo-old DEF rats. Mechanisms for the low plasma PLP control values and resistance to depletion in aged rats remain to be determined.

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.

Changes in Pyridoxal Phosphate and Pyridoxamine Phosphate in Blood, Liver and Brain in the Pregnant Mouse

A decrease in plasma pyridoxal-5'-phosphate (PLP) occurs during pregnancy in humans and experimental animals for reasons that are not known. To determine if mice also develop decreased plasma PLP concentrations during pregnancy, and if plasma PLP levels in pregnancy reflect tissue levels of PLP and pyridoxamine-5'-phosphate (PMP), we measured PLP concentrations in plasma, erythrocytes and whole blood, and liver and brain PLP and PMP in control and pregnant mice. Mice were fed a nonpurified diet containing 8.13 mg pyridoxine-HCl/kg. The PLP analyses were performed in our newly developed apotryptophanase method in which the substrate S-benzyl-L-cysteine is hydrolyzed to benzyl mercaptan, reacted with Ellman's reagent and measured spectrophotometrically. During pregnancy, plasma PLP levels decreased 50% below control levels, but erythrocyte and whole blood PLP levels increased 2.9- and 1.6-fold, respectively. Liver PLP and PMP decreased 25%, in parallel with plasma PLP, but brain PLP and PMP concentrations were unchanged during pregnancy. These results suggest that metabolism or utilization of vitamin B-6 is altered in pregnancy, and that plasma PLP concentrations alone may not be a good indicator of nutritional status in pregnancy.

Effect of oral vitamin B6 supplementation on in vitro platelet aggregation

A randomized, double-blind study was conducted with 12 healthy adult males to determine the effects of oral pyridoxine supplementation on in vitro platelet aggregation. Following a 4-wk baseline period, half the subjects received 100 mg/day of pyridoxine·HCl while the remaining subjects received a placebo for 6 wk. In vitro platelet responses to ADP and collagen and the plasma pyridoxal 5′-phosphate (PLP) concentrations were measured at biweekly intervals. Plasma PLP concentrations increased significantly (p < 0.001) for those receiving the vitamin B6 compared to baseline values or compared to those receiving the placebo. However, there was no significant effect of increased levels of plasma PLP on collagen-stimulated platelet aggregation and only a slight effect on ADP-stimulated aggregation. Acute administration of 100 mg pyridoxine·HCl failed to alter the in vitro response of platelets to either ADP or collagen. Reevaluation of conclusions based solely on in vitro studies suggesting the use of pyridoxine as an effective in vivo antithrombotic agent may be warranted.

A direct assay for pyridoxal 5'-phosphate using pig heart apo-aspartate transaminase.

The development of a simple enzymatic method for the determination of blood and plasma pyridoxal 5'-phosphate (PLP) using pig heart apo-aspartate transaminase (apo-AST) is described. The technique requires three steps: sample extraction using perchloric acid, a binding step in which PLP in the sample extract is attached to the apo-AST and the enzymatic assay of the reconstituted holo-AST. PLP extracts were analysed with and without a known concentration of added PLP to correct for variation in recovery between different specimens. Procedures are outlined for manual and automatic analysis of the PLP extracts. Using the KONE Clinical Analyser after the extraction step, it is possible to measure enzyme activity in 100 specimens (i.e. 400 tubes) in a 5 h period. Results are shown from 185 healthy women aged 20-45 years, in which plasma PLP concentrations ranged from 5 to 165 nmol/L, and 142 men and 56 women in government service aged 17-64 years, whose plasma PLP ranged from 8 to 169 nmol/L. Values less than 20 nmol/L are believed to indicate vitamin B6 deficiency and the method is able to measure 5 nmol/L.

Pyridoxal 5′-phosphate deficiency in uremic undialyzed, hemodialyzed, and non-uremic kidney transplant patients

In this study, we have investigated plasma pyridoxal 5'-phosphate (PLP) concentrations in undialyzed and dialyzed uremic patients and in kidney transplant subjects, using an enzymatic technique with thermal deproteinization to liberate PLP from plasma proteins. The specificity of the reaction indicates no interference with pyridoxal and only 3% interference with pyridoxamine phosphate. In 17 hemodialyzed patients, a deficiency of about 50% of plasma PLP concentration is found as compared to 25 healthy subjects (22.2 +/- 2.47 vs. 48.8 +/- 3.00 nmol . l-1), as mean +/- SEM). In seven undialyzed uremic patients with end-stage renal failure, the plasma PLP concentration is also decreased (29.3 +/- 1.74 nmol . l-1). The absence of PLP in plasma ultrafiltrates demonstrates that no loss of PLP occurs due to hemodialysis. The daily oral supplementation with 250-750 mg pyridoxal induces a supraphysiological increase in plasma PLP concentration in hemodialyzed as well as in undialyzed patients. In 116 non-uremic kidney transplant subjects, the mean plasma PLP concentration was 33.8 +/- 3.50 nmol . l-1). In 65% of these patients, a marked deficit (below 20 nmol . l-1) was observed. In conclusion, uremic patients have a deficient vitamin B6 state. Its correction with pyridoxal to restore physiological plasma PLP concentration necessitates oral supplementation with lower doses that those widely used at present. In kidney transplant patients a similar plasma PLP deficiency is observed in the absence of chronic renal failure.

Vitamin B6 metabolism in chronic alcohol abuse. Pyridoxal phosphate levels in plasma and the effects of acetaldehyde on pyridoxal phosphate synthesis and degradation in human erythrocytes.

The plasma pyridoxal-5'-phosphate (PLP) level of alcoholic subjects has been compared with that of non-alcoholic individuals in order to ascertain the incidence of abnormal vitamin B(6) metabolism in chronic alcohol abuse. 66 alcoholic subjects were selected on the basis that they did not exhibit abnormal liver function tests and hematologic findings. 35 of them had plasma PLP concentrations less than 5 ng/ml, the lowest value encountered in 94 control subjects, indicating a high incidence of deranged PLP metabolism in alcoholic patients even when hepatic and hematologic abnormalities are absent. The biochemical basis for the altered PLP metabolism in chronic alcohol abuse was examined. Low plasma PLP levels in alcoholics were not accompanied by decreased pyridoxal kinase and pyridoxine phosphate oxidase activities in erythrocytes. Further studies with erythrocytes demonstrated that the cellular content of PLP is determined not only by the activities of these PLP-synthesizing enzymes but also by the activity of a phosphate-sensitive, membrane-associated, neutral phosphatase, which hydrolyzes phosphorylated B(6) compounds.Acetaldehyde, but not ethanol, impaired the net formation of PLP from pyridoxal, pyridoxine, and pyridoxine phosphate by erythrocytes. However, when the B(6)-phosphate phosphatase activity was inhibited by 80 mM phosphate, this effect of acetaldehyde was abolished. By the use of broken cell preparations, it was possible to demonstrate directly that the action of acetaldehyde is mediated by the phosphatase, resulting in an acceleration of the degradation of the phosphorylated B(6) compounds in erythrocytes.