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 B₆, 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 B₆ 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.

Figure 1

Vitamin B₆ metabolism. (a) Pyridoxine, (b) pyridoxal, (c) pyridoxamine, (d) pyridoxine 5′-phosphate, (e) pyridoxal 5′-phosphate, and (f) pyridoxamine 5′-phosphate. PK = pyridoxal kinase; PNPO = pyridox(am)ine 5′-phosphate oxidase.

Figure 2

Effect of humidity on the short-term stability of the PNPO enzyme in dried blood spots. Error bars indicate SEM. DBS were stored in sealed foil bags with desiccant (▲) or under ambient conditions (■) at room temperature (22 °C). Data points represent the mean percentage of activity as compared to that found after 1 day (n = 2).

Figure 3

Effect of temperature (4 °C [●], −20 °C [■], and −80 °C [▲]) on the long-term stability of the PNPO enzyme in dried blood spots (DBS). Data points represent the mean percentage of PNPO activity relative to measurement at time zero. Error bars indicate SEM. No significant difference was seen in activities of samples with/without desiccant; the data from DBS stored both with (n = 2) and without (n = 2) desiccant at each temperature have therefore been combined (n = 4).

Figure 4

DBS PNPO enzyme activity in PNPO-deficient patients and control samples. Box plots indicate range, interquartile range, and median. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test; ** = P < 0.01; **** = P < 0.0001.