Monique Albersen

Quantification of vitamin B6 vitamers in human cerebrospinal fluid by ultra performance liquid chromatography–tandem mass spectrometry

Since vitamin B6 is essential for normal functioning of the central nervous system, there is growing need for sensitive analysis of B6 vitamers in cerebrospinal fluid (CSF). This manuscript describes the development and validation of a rapid, sensitive and accurate method for quantification of the vitamin B6 vitamers pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxic acid (PA), pyridoxal 5-phosphate (PLP), pyridoxamine 5-phosphate (PMP) and pyridoxine 5-phosphate (PNP) in human CSF. The method is based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with a simple sample preparation procedure of protein precipitation using 50 g L(-1) trichloroacetic acid containing stable isotope labeled internal standards: PL-D(3) for PL and PM, PN-(13)C(4) for PN, PA-D(2) for PA and PLP-D(3) for the phosphorylated vitamers. B6 vitamers were separated (Acquity HSS-T3 UPLC column) with a buffer containing acetic acid, heptafluorobutyric acid and acetonitrile. Positive electrospray ionization was used to monitor transitions m/z 168.1→150.1 (PL), 169.1→134.1 (PM), 170.1→134.1 (PN), 184.1→148.1 (PA), 248.1→150.1 (PLP), 249.1→232.1 (PMP) and 250.1→134.1 (PNP). The method was validated at three concentration levels for each B6 vitamer in CSF. Recoveries of the internal standards were between 93% and 96%. Intra- and inter-assay variations were below 20%. Accuracy tests showed deviations from 3% (PN) to 39% (PMP). Limits of quantification were in the range of 0.03-5.37 nM. Poor results were obtained for quantification of PNP. The method was applied to CSF samples of 20 subjects and two patients on pyridoxine supplementation. Using minimal CSF volumes this method is suitable for implementation in a routine diagnostic setting.

Lysine-Restricted Diet as Adjunct Therapy for Pyridoxine-Dependent Epilepsy: The PDE Consortium Consensus Recommendations

BACKGROUNDnSeventy-five percent of patients with pyridoxine-dependent epilepsy (PDE) due to Antiquitin (ATQ) deficiency suffer from developmental delay and/or intellectual disability (IQ < 70) despite seizure control. An observational study showed that adjunct treatment with a lysine-restricted diet is safe, results in partial normalization of lysine intermediates in body fluids, and may have beneficial effects on seizure control and psychomotor development.nnnMETHODSnIn analogy to the NICE guideline process, the international PDE Consortium, an open platform uniting scientists and clinicians working in the field of this metabolic epilepsy, during four workshops (2010-2013) developed a recommendation for a lysine-restricted diet in PDE, with the aim of standardizing its implementation and monitoring of patients. Additionally, a proposal for a further observational study is suggested.nnnRESULTSn(1) All patients with confirmed ATQ deficiency are eligible for adjunct treatment with lysine-restricted diet, unless treatment with pyridoxine alone has resulted in complete symptom resolution, including normal behavior and development. (2) Lysine restriction should be started as early as possible; the optimal duration remains undetermined. (3) The diet should be implemented and the patient be monitored according to these recommendations in order to assure best possible quality of care and safety.nnnDISCUSSIONnThe implementation of this recommendation will provide a unique and a much needed opportunity to gather data with which to refine the recommendation as well as improve our understanding of outcomes of individuals affected by this rare disease. We therefore propose an international observational study that would utilize freely accessible, online data sharing technologies to generate more evidence.

Increased bioavailable vitamin B6 in field-grown transgenic cassava for dietary sufficiency.

Increased bioavailable vitamin B 6 in field-grown transgenic cassava for dietary sufficiency

Vitamin B6 Vitamer Concentrations in Cerebrospinal Fluid Differ Between Preterm and Term Newborn Infants

BACKGROUND AND OBJECTIVE: Vitamin B6 plays a pivotal role in brain development and functioning. Differences in vitamin B6 homeostasis between preterm and term newborn infants have been reported. The authors sought to investigate whether B6 vitamers in cerebrospinal fluid (CSF) of preterm and term newborn infants are different. METHODS: B6 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. B6 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 B6 homeostasis in brain differs between preterm and term newborns. These results should be taken into account for diagnosis and treatment of epilepsy and vitamin B6 deficiency in newborn infants.

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

BACKGROUNDnVitamin 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.nnnOBJECTIVEnTo 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).nnnDESIGNnConcentrations 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnWe 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.

The value of plasma vitamin B6 profiles in early onset epileptic encephalopathies

BackgroundRecent decades have unravelled the molecular background of a number of inborn errors of metabolism (IEM) causing vitamin B6-dependent epilepsy. As these defects interfere with vitamin B6 metabolism by different mechanisms, the plasma vitamin B6 profile can give important clues for further molecular work-up. This has so far been investigated in only a small number of patients.MethodsWe evaluated the vitamin B6 vitamers pyridoxal 5’-phosphate (PLP), pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN) and the catabolite pyridoxic acid (PA) in the so far largest patient cohort: reference (nu2009=u200950); pyridox(am)ine 5’-phosphate oxidase (PNPO) deficiency (nu2009=u20096); antiquitin (ATQ) deficiency (nu2009=u200921); tissue non-specific alkaline phosphatase (TNSALP) deficiency (nu2009=u20092) and epileptic encephalopathy (EE) of unknown etiology tested negative for ATQ and PNPO deficiency (nu2009=u200964).ResultsHigh plasma PM concentration was found in all patients with PNPO deficiency irrespective of vitamin B6 supplementation. Their PM concentration and the PM/PA ratio was significantly higher (pu2009<u20090.0001), compared to any other patients analysed. One patient with TNSALP deficiency and sampling prior to PN supplementation had markedly elevated plasma PLP concentration. On PN supplementation, patients with TNSALP deficiency, ATQ deficiency and patients of the EE cohort had similar plasma vitamin B6 profiles that merely reflect the intake of supra-physiological doses of vitamin B6. The interval of sampling to the last PN intake strongly affected the plasma concentrations of PN, PL and PA.ConclusionsPM concentrations and the PM/PA ratio clearly separated PNPO-deficient patients from the other cohorts. The plasma PM/PA ratio thus represents a robust biomarker for the selective screening of PNPO deficiency.

Improved cognition, mild anxiety-like behavior and decreased motor performance in pyridoxal phosphatase-deficient mice

Pyridoxal 5-phosphate (PLP) is an essential cofactor in the catalysis of ~140 different enzymatic reactions. A pharmacological elevation of cellular PLP concentrations is of interest in neuropsychiatric diseases, but whole-body consequences of higher intracellular PLP levels are unknown. To address this question, we have generated mice allowing a conditional ablation of the PLP phosphatase PDXP. Ubiquitous PDXP deletion increased PLP levels in brain, skeletal muscle and red blood cells up to 3-fold compared to control mice, demonstrating that PDXP acts as a major regulator of cellular PLP concentrations in vivo. Neurotransmitter analysis revealed that the concentrations of dopamine, serotonin, epinephrine and glutamate were unchanged in the brains of PDXP knockout mice. However, the levels of γ-aminobutyric acid (GABA) increased by ~20%, demonstrating that elevated PLP levels can drive additional GABA production. Behavioral phenotyping of PDXP knockout mice revealed improved spatial learning and memory, and a mild anxiety-like behavior. Consistent with elevated GABA levels in the brain, PDXP loss in neural cells decreased performance in motor tests, whereas PDXP-deficiency in skeletal muscle increased grip strength. Our findings suggest that PDXP is involved in the fine-tuning of GABA biosynthesis. Pharmacological inhibition of PDXP might correct the excitatory/inhibitory imbalance in some neuropsychiatric diseases.

In response to “Prenatal screening of sialic acid storage disease and confirmation in cultured fibroblasts by LC-MS/MS” by van den Bosch et al.

With great interest we have read the recent paper by Van den Bosch et al. (2011) who reported reference values of free sialic acid (FSA) in amniotic fluid supernatant (AFS). Reference values in AFS were lower (0–8.2xa0μmol/L) at 15–25xa0weeks of gestation compared to 26–38xa0weeks of gestation (3.2–12.0xa0μmol/L), indicating that FSA in AFS tends to increase with advancing gestation. n nRecently, we developed a method for quantification of sialic acid (SA) in cerebrospinal fluid (CSF) (van der Ham et al. 2010) since brain specific disorders of SA metabolism are recognized nowadays. When establishing reference values, we did not take into account the impact of gestational age (GA). The study of Van den Bosch et al. (2011) suggested SA metabolism to be dependent on GA. We therefore performed a study to evaluate CSF SA values in newborn infants (both preterm and full term born infants). This knowledge is essential for the interpretation of SA concentrations in CSF in these populations. n nThe study included 27 CSF samples from preterm and 42 CSF samples from full term newborn infants. CSF was obtained for different clinical reasons. Prematurity was defined as a post menstrual age of less than 37xa0weeks at the time of CSF withdrawal. Post menstrual ages of preterm and full term infants were between 28 and 37xa0weeks and between 37 and 54xa0weeks, respectively. SA in CSF was quantified by HPLC-MS/MS (van der Ham et al. 2010). n nFSA concentrations were 72.2u2009±u200921.3xa0μmol/L and 49.3u2009±u200920.6xa0μmol/L in preterm and full term newborn infants, respectively (pu2009<u20090.0005). There was a significant negative correlation between FSA and post menstrual age (ru2009= −0.664) with a 3.0xa0μmol/L decrease in FSA for each week of advancing post menstrual age (pu2009<u20090.0005). No difference was observed between conjugated sialic acid from preterm and full term newborn infants (80.0u2009±u200919.1xa0μmol/L versus 73.6u2009±u200928.0xa0μmol/L (pu2009=u20090.303)). n nIn conclusion, with increasing gestational age, there was a considerable decrease in the amount of FSA in CSF. These results demonstrate that proper interpretation of metabolic investigations in preterm newborns depends on group-specific reference ranges, as concentrations of metabolites may differ significantly between full term and preterm born infants.