LC-MS/MS method for the differential diagnosis of treatable early onset inherited metabolic epilepsies.

Rapid diagnosis and early specific treatment of metabolic epilepsies due to inborn errors of metabolism (IEMs) is crucial to avoid irreversible sequalae. Nowadays, besides the profile analysis of amino- and organic acids, a range of additional targeted assays is used for the selective screening of those diseases. This strategy can lead to long turn-around times, repeated sampling and diagnostic delays. To replace those individual targeted assays, we developed a new liquid chromatography mass spectrometry method (LC-MS/MS) for the differential diagnosis of inherited metabolic epilepsies that are potentially treatable. The method was developed to simultaneously quantify 12 metabolites (sulfocysteine, guanidinoacetate, creatine, pipecolic acid, Δ1 -piperideine-6-carboxylate (P6C), proline, Δ1 -pyrroline-5-carboxylate (P5C), and the B6 -vitamers) enabling the diagnosis of nine different treatable IEMs presenting primarily with early-onset epilepsy. Plasma and urine samples were mixed with internal standards, precipitated and the supernatants were analyzed by LC-MS/MS. In comparison with previous assays, no derivatization of the metabolites is necessary for analysis. This LC-MS method was validated for quantitative results for all metabolites except P6C and P5C for which semiquantitative results were obtained due to the absence of commercially available standards. Coefficients of variation for all analytes were below 15% and recovery rates range between 80% and 120%. Analysis of patient samples with known IEMs demonstrated the diagnostic value of the method. The presented assay covers a selected panel of biochemical markers, improves the efficiency in the laboratory, and potentially leads to faster diagnoses and earlier treatment avoiding irreversible damage in patients affected with IEMs.

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

BACKGROUND: Recent 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. METHODS: We 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 (n = 50); pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency (n = 6); antiquitin (ATQ) deficiency (n = 21); tissue non-specific alkaline phosphatase (TNSALP) deficiency (n = 2) and epileptic encephalopathy (EE) of unknown etiology tested negative for ATQ and PNPO deficiency (n = 64). RESULTS: High 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 (p < 0.0001), 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. CONCLUSIONS: PM 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.