Kinetic studies of vitamin B6 metabolism in humans

Abstract

The primal aim of this thesis was to establish whether kinetic aspects of vitamin B6 metabolism predispose to earlier observed racial differences found in plasma pyridoxal-5'-phosphate (PLP). The active forms of vitamin B6 namely plasma PLP and pyridoxal (PL) as well as the three enzymes expressed in the erythrocyte involved in B6 metabolism, PL kinase, PLP phosphatase and pyridoxamine -5'- phosphate (pyridoxine -5'- phosphate) [PMP(PNP) ] oxidase were measured by high performance liquid chromatography. Phase one supported earlier experimental evidence and lower plasma PLP concentrations were found in blacks in a group of200 male volunteers recruited from the South African National Defence Force (SANDF). The respective enzyme activities involved in vitamin B6 metabolism, from the same test subjects, suggested similar PLP production from PMP and PL as well as PLP dephosphorylation which result in the release of PL into the circulating fluid. Since applied exclusion criteria eliminated the majority of biochemical, physiological, genetical - and disease related factors that influence vit B6 status, dietary factors and individual preferences regarding food intake, were most likely to be responsible for the significantly lower circulating plasma PLP encountered in blacks. Phase two compared pharmacokinetic parameters between 7 black - and 9 white test subjects recruited from the South African Police Services after a single 10 mg oral supplement ofpyridoxine hydrochloride. Statistical analysis of the parameters elimination half-life, elimination rate constant, clearance, volume of distribution, mean residence time, maximum peak concentration and time to maximum peak concentration failed to demonstrate any significant differences between the two groups. These results suggest consistent appearance rate, distribution and metabolism for the metabolites PLP and PL in the study population. A tendency in slower appearance rate, for both the metabolites PLP and PL, were observed in blacks and needs to be investigated further. The end product of vitamin B6 metabolism, 4-pyridoxic acid, which was expressed in terms of 24 hour urine volume, again failed to illustrate any significant differences between blacks and whites. These results suggested similar excretion properties in my population study. Furthermore, the pharmacokinetic parameters calculated for plasma PLP and PL respectively, were found to display one-compartment - and two-compartment pharmacokinetic model characteristics. This mono- and bi exponential elimination characteristics displayed by PLP and PL respectively could be of value in future research efforts in terms of sampling time. The distribution half-life can be determined by the calculation of two-compartment macro-rate constants. Fasting blood-samples should be collected when true baseline values are needed in the case of PL. Following vit B6 supplementation, one should allow at least 5 times the distribution half-life (5-6 hr in the case of PL) before blood-sampling in order to achieve true pharmacological response. Phase three of this study was conducted to illustrate the metabolic interplay ofthe enzymes PL kinase and PMP (PNP) oxidase involved in PLP production. The kinetic parameters, Michaelis- Menten constant and maximum velocity rate, at varying substrate concentrations, for the enzymes PL kinase and PMP (PNP) oxidase, were compared in 14 white - and 14 black male test subjects recruited from the SANDF. Both the average Michaelis-Menten constant and maximum velocity rate were higher in whites, but these differences were not statistically significant. The high individual variability for both parameters calculated, can possibly be ruled out if a crystalline enzyme form is used and should be investigated further.