Validated UPLC-MS/MS method for the analysis of vitamin B6 pyridoxal 5́-phosphate, pyridoxal, pyridoxine, pyridoxamine, and pyridoxic acid in human cerebrospinal fluid.

Vitamin B6 and its metabolites play a crucial role in the development and interaction of brain metabolism. Following diagnostic improvements additional inherited disorders in vitamin B6 metabolism have been identified, most of them leading to a severe epileptic disorder accompanied by progressive neurological deficits including intellectual disability and microcephaly. Since early treatment can improve the outcome, fast and reliable detection of metabolic biomarkers is important. Therefore, the analysis of vitamin B6 metabolites has become increasingly important, but is, however, still challenging and limited to a few specialized laboratories. Until today, vitamin B6 metabolites are measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) using trichloroacetic acid for protein precipitation. In this work, we present the development and validation of a new, accurate and reliable method for analysis and quantification of the vitamin B6 vitamers pyridoxal 5́-phosphate (PLP), pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM) and pyridoxic acid (PA) in human CSF samples using acetonitrile for protein precipitation. The method is based on ultra-performance liquid chromatography-tandem mass spectrometry using electrospray ionization (UPLC-ESI-MS/MS). The calibration was performed in surrogate matrix Ringer solution and metabolites were quantified by their corresponding isotopically labelled internal standards. A protein precipitation by acetonitrile was applied greatly improving chromatographic separation of the metabolites in a 4.7 min chromatographic run. The method was validated following the European Medical Agency (EMA) and Food and Drug Administration (FDA) guidelines for bioanalytical method validation. The metabolites were quantified from 5 to 200 nmol/L with a seven-point calibration curve and minimum coefficient of regression of 0.99. The validation was performed with quality control samples at four concentration levels with surrogate matrix ringer solution and pooled CSF material. Within- and inter-day accuracy and precision in Ringer solution were within 85.4 % (PLP) and 114.5 % (PM) and from 2.6 % (PA) to 16.5 % (PLP). Within- and inter-day accuracy and precision in pooled CSF material were within 90.5 % (PN) and 120.1 % (PL) and from 1.7 % (PA) to 19.0 % (PM). The method was tested by measuring of 158 CSF samples to determine reference ranges. The B6 vitamers PLP and PL were determined in all CSF samples above 5 nmol/L while PN, PM and PA showed concentrations below or near LOQ. Probable supplementation of PLP was detected in eight CSF samples, which revealed high concentrations of PM, PN, PL, or PA, whereas PLP was in the reference range or slightly elevated. The method is suitable for the application within a routine diagnostic laboratory.

Vitamin B6 in plasma and cerebrospinal fluid of children.

BACKGROUND: Over the past years, the essential role of vitamin B6 in brain development and functioning has been recognized and genetic metabolic disorders resulting in functional vitamin B6 deficiency have been identified. However, data on B6 vitamers in children are scarce. MATERIALS AND METHODS: B6 vitamer concentrations in simultaneously sampled plasma and cerebrospinal fluid (CSF) of 70 children with intellectual disability were determined by ultra performance liquid chromatography-tandem mass spectrometry. For ethical reasons, CSF samples could not be obtained from healthy children. The influence of sex, age, epilepsy and treatment with anti-epileptic drugs, were investigated. RESULTS: The B6 vitamer composition of plasma (pyridoxal phosphate (PLP) > pyridoxic acid > pyridoxal (PL)) differed from that of CSF (PL > PLP > pyridoxic acid > pyridoxamine). Strong correlations were found for B6 vitamers in and between plasma and CSF. Treatment with anti-epileptic drugs resulted in decreased concentrations of PL and PLP in CSF. CONCLUSION: We provide concentrations of all B6 vitamers in plasma and CSF of children with intellectual disability (±epilepsy), which can be used in the investigation of known and novel disorders associated with vitamin B6 metabolism as well as in monitoring of the biochemical effects of treatment with vitamin B6.

Vitamin B-6 vitamers in human plasma and cerebrospinal fluid.

BACKGROUND: Vitamin B-6 comprises a group of 6 interrelated vitamers and is essential for numerous physiologic processes, including brain functioning. Genetic disorders disrupting vitamin B-6 metabolism have severe clinical consequences. OBJECTIVE: To adequately diagnose known and novel disorders in vitamin B-6 metabolism, a reference set is required containing information on all vitamin B-6 vitamers in plasma and cerebrospinal fluid (CSF). DESIGN: Concentrations of vitamin B-6 vitamers in the plasma and CSF of 533 adult subjects were measured by ultra high-performance liquid chromatography-tandem mass spectrometry. RESULTS: The relative vitamin B-6 vitamer composition of plasma [pyridoxal phosphate (PLP) > pyridoxic acid (PA) > pyridoxal] differed from that of CSF (pyridoxal > PLP > PA > pyridoxamine). Sex influenced vitamin B-6 vitamer concentrations in plasma and CSF and should therefore be taken into account when interpreting vitamin B-6 vitamer concentrations. The strict ratios and strong correlations between vitamin B-6 vitamers point to a tight regulation of vitamin B-6 vitamer concentrations in blood and CSF. Given the unique design of this study, with simultaneously withdrawn blood and CSF from a large number of subjects, reliable CSF:plasma ratios and correlations of vitamin B-6 vitamers could be established. CONCLUSIONS: We provide an extensive reference set of vitamin B-6 vitamer concentrations in plasma and CSF. In addition to providing insight on the regulation of individual vitamers and their intercompartmental distribution, we anticipate that these data will prove to be a valuable reference set for the diagnosis and treatment of conditions associated with altered vitamin B-6 metabolism.

Typical and atypical phenotypes of PNPO deficiency with elevated CSF and plasma pyridoxamine on treatment.

Pyridox(am)ine phosphate oxidase (PNPO) deficiency causes severe early infantile epileptic encephalopathy and has been characterized as responding to pyridoxal-5'-phosphate but not to pyridoxine. Two males with PNPO deficiency and novel PNPO mutations are reported and their clinical, metabolic, and video-electroencephalographic (EEG) findings described. The first child showed electro-clinical responses to pyridoxine and deterioration when pyridoxine was withheld. At last review, he has well-controlled epilepsy with pyridoxal-5'-phosphate monotherapy and an autism spectrum disorder. The second child had a perinatal middle cerebral artery infarct and a myoclonic encephalopathy. He failed to respond to pyridoxine but responded well to pyridoxal-5'-phosphate. At the age of 21 months he has global developmental delay and hemiparesis but is seizure-free with pyridoxal-5'-phosphate monotherapy. Plasma and cerebrospinal fluid pyridoxamine levels were increased in both children during treatment with pyridoxine or pyridoxal-5'-phosphate. These observations indicate that differential responses to pyridoxine and pyridoxal-5'-phosphate treatment cannot be relied upon to diagnose PNPO deficiency.

Vitamin B6 vitamer concentrations in cerebrospinal fluid differ between preterm and term newborn infants.

BACKGROUND AND OBJECTIVE: Vitamin B(6) plays a pivotal role in brain development and functioning. Differences in vitamin B(6) homeostasis between preterm and term newborn infants have been reported. The authors sought to investigate whether B(6) vitamers in cerebrospinal fluid (CSF) of preterm and term newborn infants are different. METHODS: B(6) vitamer concentrations were determined in 69 CSF samples of 36 newborn infants (26 born preterm and 10 born term) by ultra performance liquid chromatography-tandem mass spectrometry. CSF samples, taken from a subcutaneous intraventricular reservoir, were bedside frozen and protected from light. RESULTS: Concentrations of pyridoxal (PL), pyridoxal phosphate (PLP), pyridoxic acid (PA), and pyridoxamine (PM) in preterm newborns (postmenstrual age 30-37 weeks) were at least twice as high as in older newborns (postmenstrual age ≥ 42 weeks). Pyridoxine and pyridoxamine phosphate concentrations were below limits of quantification in all newborns. In CSF of 2 very preterm newborns (postmenstrual age <30 weeks), significant amounts of pyridoxine were present besides high concentrations of PL, PA, and PM, whereas PLP concentrations were relatively low. B(6) vitamers in CSF were positively correlated, especially PA, PLP, and PL. CONCLUSIONS: In CSF of newborn infants, PL, PLP, PA, and PM are present, and concentrations are strongly dependent on postmenstrual age. Our results indicate that vitamin B(6) homeostasis in brain differs between preterm and term newborns. These results should be taken into account for diagnosis and treatment of epilepsy and vitamin B(6) deficiency in newborn infants.