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.

Pyridoxal in the Cerebrospinal Fluid May Be a Better Indicator of Vitamin B6-dependent Epilepsy Than Pyridoxal 5'-Phosphate.

BACKGROUND: We aimed to demonstrate the biochemical characteristics of vitamin B6-dependent epilepsy, with a particular focus on pyridoxal 5'-phosphate and pyridoxal in the cerebrospinal fluid. METHODS: Using our laboratory database, we identified patients with vitamin B6-dependent epilepsy and extracted their data on the concentrations of pyridoxal 5'-phosphate, pyridoxal, pipecolic acid, α-aminoadipic semialdehyde, and monoamine neurotransmitters. We compared the biochemical characteristics of these patients with those of other epilepsy patients with low pyridoxal 5'-phosphate concentrations. RESULTS: We identified seven patients with pyridoxine-dependent epilepsy caused by an ALDH7A1 gene abnormality, two patients with pyridoxal 5'-phosphate homeostasis protein deficiency, and 28 patients with other epilepsies with low cerebrospinal fluid pyridoxal 5'-phosphate concentrations. Cerebrospinal fluid pyridoxal and pyridoxal 5'-phosphate concentrations were low in patients with vitamin B6-dependent epilepsy but cerebrospinal fluid pyridoxal concentrations were not reduced in most patients with other epilepsies with low cerebrospinal fluid pyridoxal 5'-phosphate concentrations. Increase in 3-O-methyldopa and 5-hydroxytryptophan was demonstrated in some patients with vitamin B6-dependent epilepsy, suggestive of pyridoxal 5'-phosphate deficiency in the brain. CONCLUSIONS: Low cerebrospinal fluid pyridoxal concentrations may be a better indicator of pyridoxal 5'-phosphate deficiency in the brain in vitamin B6-dependent epilepsy than low cerebrospinal fluid pyridoxal 5'-phosphate concentrations. This finding is especially helpful in individuals with suspected pyridoxal 5'-phosphate homeostasis protein deficiency, which does not have known biomarkers.

Vitamin B6 in acute encephalopathy with biphasic seizures and late reduced diffusion.

BACKGROUND: The initial presentation of acute encephalopathy with biphasic seizures and late reduced diffusion (AESD) is indistinguishable from that of complex febrile seizures (FS), which poses a great diagnostic challenge for clinicians. Excitotoxicity is speculated to be the pathogenesis of AESD. Vitamin B6 (VB6) is essential for the biosynthesis of gamma-aminobutyric acid, an inhibitory neurotransmitter. The aim of this study is to investigate our hypothesis that VB6 deficiency in the brain may play a role in AESD. METHODS: We obtained cerebrospinal fluid (CSF) samples from pediatric patients with AESD after early seizures and those with FS. We measured pyridoxal 5'-phosphate (PLP) and pyridoxal (PL) concentrations in the CSF samples using high-performance liquid chromatography with fluorescence detection. RESULTS: The subjects were 5 patients with AESD and 17 patients with FS. Age did not differ significantly between AESD and FS. In AESD, CSF PLP concentration was marginally lower (p = 0.0999) and the PLP-to-PL ratio was significantly (p = 0.0417) reduced compared to those in FS. CONCLUSIONS: Although it is impossible to conclude that low PLP concentration and PLP-to-PL ratio are causative of AESD, this may be a risk factor for developing AESD. When combined with other markers, this finding may be useful in distinguishing AESD from FS upon initial presentation.

Pyridoxal 5'-phosphate, pyridoxal, and 4-pyridoxic acid in the paired serum and cerebrospinal fluid of children.

BACKGROUND: We quantified pyridoxal 5'-phosphate (PLP), pyridoxal (PL), and 4-pyridoxic acid (PA) in paired serum and cerebrospinal fluid (CSF) samples from children and investigated the effect of age on the concentrations and CSF-to-serum ratios of these vitamers. METHODS: Serum and CSF samples prospectively collected from 49 pediatric patients were analyzed. PLP, PL, and PA were measured using high-performance liquid chromatography with fluorescence detection, using pre-column derivatization by semicarbazide. Effects of age on these vitamers, the PLP-to-PL ratio, CSF-to-serum PLP ratio, and CSF-to-serum PL ratio were evaluated using correlation analysis. RESULTS: The PLP, PL, and PA concentrations in the serum and CSF were higher at younger ages, except for CSF PA concentrations that were mostly below the limit of detection (<1.2nmol/l). The PLP-to-PL ratios in the serum and CSF correlated positively with age. The CSF-to-serum PLP ratio and CSF-to-serum PL ratio were independent of age. CONCLUSIONS: Age-related changes in PLP, PL, and PA in serum and in CSF from pediatric patients and CSF-to-serum ratios of PLP and PL demonstrated in this study will provide valuable information for evaluating PLP supply to the central nervous system from the peripheral blood.

Measurement of pyridoxal 5'-phosphate, pyridoxal, and 4-pyridoxic acid in the cerebrospinal fluid of children.

BACKGROUND: We quantified pyridoxal 5'-phosphate (PLP), pyridoxal (PL), and 4-pyridoxic acid (PA) in the cerebrospinal fluid (CSF) of children and to investigate the effect of age, sex, epilepsy, and anti-epileptic drug (AED) therapy on these vitamers. METHODS: CSF samples prospectively collected from 116 pediatric patients were analyzed. PLP, PL, and PA were measured using high-performance liquid chromatography with fluorescence detection, using pre-column derivatization by semicarbazide. Effects of age, sex, epilepsy, and AEDs on these vitamers and the PLP/PL ratio were evaluated using multiple linear regression models. RESULTS: The PLP, PL, and PA concentrations were correlated negatively with age and the PLP/PL ratio was correlated positively with age. Multiple regression analysis revealed that the presence of epilepsy was associated with lower PLP concentrations and PLP/PL ratios but sex and AED therapy had no influence on these values. The observed ranges of these vitamers in epileptic and non-epileptic patients were demonstrated. CONCLUSIONS: We showed the age dependence of PLP and PL in CSF from pediatric patients. Epileptic patients had lower PLP concentrations and PLP/PL ratios than non-epileptic patients, but it is unknown whether this is the cause, or a result, of epilepsy.

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.

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.