An LC-MS/MS-Based Method for the Quantification of Pyridox(am)ine 5'-Phosphate Oxidase Activity in Dried Blood Spots from Patients with Epilepsy

Abstract

We report the development of a rapid, simple, and robust LC-MS/MS-based enzyme assay using dried blood spots (DBS) for the diagnosis of pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency (OMIM 610090). PNPO deficiency leads to potentially fatal early infantile epileptic encephalopathy, severe developmental delay, and other features of neurological dysfunction. However, upon prompt treatment with high doses of vitamin B 6 , affected patients can have a normal developmental outcome. Prognosis of these patients is therefore reliant upon a rapid diagnosis. PNPO activity was quantified by measuring pyridoxal 5'-phosphate (PLP) concentrations in a DBS before and after a 30 min incubation with pyridoxine 5'-phosphate (PNP). Samples from 18 PNPO deficient patients (1 day-25 years), 13 children with other seizure disorders receiving B 6 supplementation (1 month-16 years), and 37 child hospital controls (5 days-15 years) were analyzed. DBS from the PNPO-deficient samples showed enzyme activity levels lower than all samples from these two other groups as well as seven adult controls; no false positives or negatives were identified. The method was fully validated and is suitable for translation into the clinical diagnostic arena.Pediatr Neurol. 2016 Jun;59:6-12. (PMID: 26995068); JIMD Rep. 2016;25:71-75. (PMID: 26108646); Eur J Biochem. 2004 Jun;271(12):2452-61. (PMID: 15182361); Anal Chem. 2011 Feb 1;83(3):1152-6. (PMID: 21192662); Neurology. 2014 Apr 22;82(16):1392-4. (PMID: 24658927); Biochim Biophys Acta. 1980 Jun 13;613(2):401-9. (PMID: 7448195); Am J Hum Genet. 2016 Dec 1;99(6):1325-1337. (PMID: 27912044); Brain. 2014 May;137(Pt 5):1350-60. (PMID: 24645144); JIMD Rep. 2014;17:67-70. (PMID: 25256445); J Inherit Metab Dis. 2013 Jan;36(1):139-45. (PMID: 22576361); Anal Chem. 2014 Aug 5;86(15):7962-8. (PMID: 25019629); J Inherit Metab Dis. 2016 Sep;39(5):733-41. (PMID: 27342130); J Chromatogr. 1988 Oct 14;431(2):406-12. (PMID: 3243796); Biochim Biophys Acta. 1972 Sep 15;279(2):310-5. (PMID: 5082501); J Pharm Pharmacol. 2017 Apr;69(4):480-488. (PMID: 28220480); Mass Spectrom Rev. 2016 Jan-Feb;35(1):71-84. (PMID: 25952022); Methods Enzymol. 1986;122:110-6. (PMID: 3010031); Hum Mol Genet. 2012 Dec 15;21(24):5359-72. (PMID: 22949513); J Child Neurol. 2015 Aug;30(9):1218-25. (PMID: 25296925); J Inherit Metab Dis. 2006 Apr-Jun;29(2-3):317-26. (PMID: 16763894); J Nutr. 2004 Apr;134(4):842-6. (PMID: 15051835); Nature. 2016 Aug 17;536(7616):285-91. (PMID: 27535533); Clin Biochem. 2011 Dec;44(17-18):1445-50. (PMID: 21963384); Pediatrics. 1963 Sep;32:338-43. (PMID: 14063511); Am J Clin Nutr. 1993 May;57(5):666-72. (PMID: 8480684); Am J Hum Genet. 1976 Jan;28(1):9-17. (PMID: 2009); Nat Rev Neurol. 2015 Oct;11(10):567-84. (PMID: 26392380); Am J Clin Nutr. 2009 Nov;90(5):1151-9. (PMID: 19740970); J Biol Chem. 1983 Jan 25;258(2):840-5. (PMID: 6822512); Clin Chim Acta. 1986 Nov 15;160(3):307-11. (PMID: 3791637); J Biol Chem. 2003 Dec 12;278(50):50040-6. (PMID: 14522954); Hum Mol Genet. 2005 Apr 15;14(8):1077-86. (PMID: 15772097); Pediatr Neurol. 2015 Oct;53(4):277-86. (PMID: 26194033); Hum Nutr Clin Nutr. 1985 Mar;39(2):107-15. (PMID: 4019261); Enzyme Protein. 1996;49(5-6):291-304. (PMID: 9252787); Neurology. 2014 Apr 22;82(16):1425-33. (PMID: 24658933); Anal Biochem. 1980 Jan 15;101(2):435-41. (PMID: 7362038); J Child Neurol. 1996 Jan;11(1):35-40. (PMID: 8745383). Linking ISSN: 00032700. Subset: MEDLINE; Grant Information: United Kingdom Wellcome Trust Date of Electronic Publication: 2017 Aug 17. ; Original Imprints: Publication: Washington, American Chemical Society. <br />