New insight into vitamin B6 metabolism and related diseases

Vitamin B6 is present in the human body as six structurally related vitamers: pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM), and their respective 5’-phosphate esters pyridoxal 5’-phosphate (PLP), pyridoxine 5’-phosphate (PNP) and pyridoxamine 5’-phosphate (PMP). PLP is the metabolically active form of vitamin B6 and, although it is essential for survival of all organisms, only bacteria, yeast and plants synthesize it de novo. Humans rely on its uptake from the diet and on the vitamin B6 salvage pathway to recycle the different B6 vitamers to PLP.
In this thesis we present the current knowledge on vitamin B6, especially focusing on the roles of vitamin B6 in health and disease; the enzymes known to play a role in the human vitamin B6 metabolism; and the inborn errors of vitamin B6 metabolism: pyridoxine-dependent epilepsy, hyperprolinemia type II, pyridox(am)ine 5’-phosphate oxidase deficiency, hypophosphatasia and pyridoxal phosphate binding protein deficiency. Additionally, there are other, yet uncharacterized vitamin B6-responsive conditions.
Although the discovery of vitamin B6 dates back to the early 1930’s and pyridoxine-dependent epilepsy (PDE) is known since the 1950’s, the pathophysiology of vitamin B6 deficiency is still not completely understood. We showed that vitamin B6 insufficiency strongly impairs de novo serine synthesis and consequently leads to low glycine and 5-mTHF levels. These findings explain why some patients with PDE clinically respond to supplementation of folinic acid, a 5-mTHF precursor. Furthermore, we developed and optimized a sensitive and accurate stable isotope-based UPLC-MS/MS method that serves as a screening tool in the study of de novo serine biosynthesis in cultured cells.
This work also describes a new genetic vitamin B6-responsive condition: mitochondrial glutamate oxaloacetate transaminase (GOT2) deficiency. Our functional studies characterized the disease, explored putative treatment options and showed that GOT2 deficiency is a PLP-responsive disorder. In addition, our findings suggest that serine and pyruvate supplementation may be important therapeutical options to correct the biochemical abnormalities caused by GOT2 deficiency.
We also provide evidence for the presence of PL reductase activity in humans. Our study started when a strong accumulation of PN was detected in cerebrospinal fluid samples of two PLP-treated patients. To further investigate the existence of PL reductase activity, we treated four mammalian cell lines with PL and found that all cell lines reduce PL to PN in a time- and dose-dependent manner. Although further studies are needed to identify the responsible PL reductase protein(s), our findings clearly expand the number of enzymes with a role in vitamin B6 salvage pathway.
In conclusion, this study adds novel insight into mammalian metabolism of vitamin B6 by presenting compelling evidence for the existence of PL reductase activity in humans. In addition, we show that vitamin B6 is essential for de novo serine biosynthesis. We described and characterized a new vitamin B6-responsive IEM, GOT2 deficiency, and revealed that a (secondary) de novo serine biosynthesis defect may partially underlie the pathophysiology of this disease. To improve studies on primary and secondary de novo serine biosynthesis defects we developed a metabolic flux method.