The metabolism of vitamin B6 in relation to genetic disease

Over the past years, interest in vitamin B6 has increased, since its essential role in the brain has been recognized and specific inborn errors of metabolism resulting in functional vitamin B6 deficiency have been identified. Patients suffering from vitamin B6 deficiency present with epilepsy and, frequently, developmental delay. Disturbances of vitamin B6 metabolism may be missed by biochemical profiling of the secondary effects of functional vitamin B6 deficiency. Direct analysis of the different B6 vitamers may overcome this diagnostic limitation and will increase our insight in normal human vitamin B6 metabolism and transport. We developed and validated a UPLC-MS/MS (ultra performance liquid chromatography-tandem mass spectrometry) method for simultaneous quantification of the different B6 vitamers in CSF (cerebrospinal fluid). With our method, decreased B6 vitamer concentrations can be detected and the biochemical effects of vitamin B6 supplementation can be monitored. We identified several pre-analytical factors that must be taken into account when studying vitamin B6 in body fluids. In addition, we observed that B6 vitamer concentrations in CSF of newborn infants are substantially higher than in CSF of children aged 1-18 years. Reference values for B6 vitamers in CSF must therefore be established for newborn infants and children separately. Since simultaneously studying B6 vitamer concentrations in plasma and CSF will deepen our understanding of normal human B6 vitamers and their interrelationships, we adapted our UPLC-MS/MS method for the analysis of CSF to enable the analysis of plasma. We show that the B6 vitamer composition of plasma differs from that of CSF, that concentrations of certain B6 vitamers in plasma and CSF are influenced by sex and age and that epileptic children with anti-epileptic drug treatment are at risk of vitamin B6 deficiency. To investigate the genetic regulation of vitamin B6, we conducted a GWAS (genome-wide association study) of B6 vitamers in plasma and CSF and studied the association with common genetic variants. We identified several genome-wide significant SNPs (single nucleotide polymorphisms) at loci containing transporter and neurotransmitter receptor genes. The mechanism by which these genes influence vitamin B6 remains to be elucidated. The organs that are important in the conversion of precursor B6 vitamers into the biologically active cofactor PLP (pyridoxal phosphate), have not been irrefutably identified. Although it has been generally accepted to be the liver, there is a discrepancy in literature regarding the main location of vitamin B6 metabolism, because the intestine has also been implicated. We therefore investigated the role of the intestine in human vitamin B6 metabolism using an in vitro model for intestinal enterocytes. Our results shed new light on human vitamin B6 metabolism, as we demonstrate a substantial role for the intestine. Although we show concentrations of the different B6 vitamers in plasma and CSF, neither the normal B6 vitamer distribution within brain cells nor the intracellular consequences of functional vitamin B6 deficiency and vitamin B6 supplementation are known, while vitamin B6-related neurotoxicity has been reported in literature. We therefore investigated vitamin B6 metabolism in an in vitro mouse model for neuronal cells. We show that neuronal cells take up, convert and excrete the different B6 vitamers. In CSF of children supplemented with vitamin B6, we observed supraphysiological concentrations of the different B6 vitamers, whereas concentrations of PLP remained almost within normal limits.