Screening for pyridoxine dependent epilepsy (PDE) by Tandem Mass Spectrometry (HPLC-MS/MS)

Introduction: Pyridoxine dependent epilepsy (PDE; MIM:266100) is a rare, auto-somal-recessive inborn error of metabolism. More than 120 cases have been reported up to now. Neonatal seizures are caused by a secondary vitamin B6 deficiency due to a lack of 2-aminoadipic acid semialdehyde dehydrogenase activity (2-AASA, allysine). Lysine degradation is disturbed. Besides seizures mental retardation, in some cases microcephaly and disturbed brain development may occur.

Method: Using HPLC and tandem mass spectrometry we established a method for quantitative determination of pipecolic acid, 2-AASA and lysine in urine, plasma and cerebrospinal fluid. Components were eluted with a mixture of water (70%) and methanol (30%) each containing 0.05% trifluoracetic acid. After butylation the eluate was analysed by an Applied Biosystems API 3000 tandem mass spectrometer. Turbo ion spray is used for ionization, MS/MS spectra were taken by multiple reaction monitoring (MRM) of characteristic ions.

Results: Limit of quantification is 1µmol/L for 2-AASA and pipecolic acid. Patients with seizures who respond to vitamin B6 initially show elevated 2-AASA levels in all body fluids. This proofs pyridoxine dependent seizures. According to these results pipecolic acid was also found in all body fluids (but not in all patients). Therefore excretion of 2-AASA seems to be a reliable marker for diagnosis and therapy monitoring of pyridoxine dependent seizures. Two cases are shown.

Discussion: The presented method offers a non-invasive, quick and reliable tool for diagnosis and therapy monitoring of PDE. Sample preparation is easy. Results will be available on the same day, when the sample arrives in the laboratory.