ИСТИНА

Background Pyridoxal kinase (PLK) and pyridoxine-5’-phosphate oxidase (PNPO) produce pyridoxal-5’-phosphate, the coenzyme derivative of vitamin B6, which is dephosphorylated by pyridoxal-5’-phosphate phosphatase (PLPP). Pyridoxal-5’-phosphate is particularly important for biosynthesis of major neurotransmitters. Hence, regulation of its metabolism in the brain is of medical significance. Activity of PLK is regulated by thiamine (vitamin B1) [1], its main cellular derivative, the coenzyme thiamine diphosphate [1], and a regulatory derivative thiamine triphosphate [2]. PLK transcription is also under circadian control [3], but how this affects the brain PLK protein level and function is unknown. On the other hand, the daytime of thiamine administration determines the thiamine-dependent regulation of the brain glutamate dehydrogenase [4]. For PNPO and PLPP, neither the circadian, nor the thiamine-dependent regulation have been studied. The aim of this work is to reveal how the thiamine administration affects the enzymes involved in metabolism of pyridoxal-5’-phosphate in the rat brain, regarding potential daytime dependence of the effects. Methods Male Wistar rats received thiamine in the morning or evening, as described [4]. PLK activity was determined in cerebral cortex homogenates clarified by centrifugation, by the established method [5]. Our PNPO assay in the homogenates was based on the Amplex UltraRed quantification of hydrogen peroxide produced in the PNPO reaction. Assay of recombinant human PNPO employed the pyridoxal-5’-phosphate absorbance at 388 nm. Protein abundances were estimated using quantification of peptides by LC-MS/MS. Changes in the proteins of interest were determined after normalization of their relative abundances to the summarized abundances of actin and tubulin in the same sample. Results Administration of thiamine in the morning causes an increase in PLK activity (p = 0.05), not observed upon the evening administration of thiamine. A diurnal difference in the brain PLK activity arises due to the daytime-dependent administration of thiamine (p = 0.02), which is absent in the control animals. All the studied animal groups do not significantly differ in the PLK protein level. Hence, activation by thiamine triphosphate, established earlier using recombinant human PLK in vitro [2], may be involved in the increase of the brain PLK activity after the morning thiamine administration. In contrast to PLK, activity of the recombinant human PNPO is not regulated by thiamine or its phosphates in vitro. In good accordance with this result, the PNPO activity assayed in the rat brain homogenates does not change after the thiamine administration. In addition to PLK activity, the PLPP protein level exhibits a diurnal difference after administration of thiamine (p=0.05), not observed in the control animals. Remarkably, the activity of PLK which produces pyridoxal-5’-phosphate, is higher after the morning thiamine administration, compared to the evening one, whereas the protein level of PLPP, which hydrolyzes pyridoxal-5’-phosphate, is higher after the evening thiamine administration, compared to the morning one. Conclusions Compared to the morning administration of thiamine, its evening administration decreases activity of PLK, increases protein level of PLPP, and does not change activity of PNPO.