Direct Assay of δ-Aminolevulinic Acid Dehydratase in Heme Biosynthesis for the Detection of Porphyrias by Tandem Mass Spectrometry
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- 作者：John R. Choiniere ; C. Ronald Scott ; Michael H. Gelb ; Frantiek Tureek
- 刊名：Analytical Chemistry
- 出版年：2010
- 出版时间：August 1, 2010
- 年：2010
- 卷：82
- 期：15
- 页码：6730-6736
- 全文大小：236K
- 年卷期：v.82,no.15(August 1, 2010)
- ISSN：1520-6882
文摘
We report a new assay of human δ-aminolevulinic acid dehydratase (ALAD), an enzyme converting δ-aminolevulinic acid (ALA) into porphobilinogen. The assay is developed for use in the clinical diagnosis of δ-aminolevulinic acid dehydratase-deficient porphyria, a rare enzymatic deficiency of the heme biosynthetic pathway. The assay involves the incubation of erythrocyte lysate with the natural substrate, ALA, followed by quantitative in situ conversion of porphobilinogen to its butyramide, and liquid−liquid extraction into a mass spectrometer-friendly solvent. Quantitation of the butyrylated porphobilinogen is done by electrospray ionization tandem mass spectrometry, using a deuterium labeled internal standard. The assay stays well within the range wherein ALAD activity is linear with time. The Km of ALAD for ALA was measured as 333 μM, and the Vmax was 19.3 μM/h. Average enzyme activity among a random sample of 36 anonymous individuals was 277 μmol/L erythrocyte lysate/hour with a standard deviation of 90 μmol/L erythrocyte lysate/hour. The tandem mass spectrometric assay should easily detect the enzyme deficiency, which causes a reduction of activity by 95−99%. The assay shows good reproducibility and low background, requires a simple workup, and uses a commercially available substrate.