Direct assay of delta-aminolevulinic acid dehydratase in heme biosynthesis for the detection of porphyrias by tandem mass spectrometry.
Anal Chem
; 82(15): 6730-6, 2010 Aug 01.
Article
en En
| MEDLINE
| ID: mdl-20583792
ABSTRACT
We report a new assay of human delta-aminolevulinic acid dehydratase (ALAD), an enzyme converting delta-aminolevulinic acid (ALA) into porphobilinogen. The assay is developed for use in the clinical diagnosis of delta-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 K(m) of ALAD for ALA was measured as 333 microM, and the V(max) was 19.3 microM/h. Average enzyme activity among a random sample of 36 anonymous individuals was 277 micromol/L erythrocyte lysate/hour with a standard deviation of 90 micromol/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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Porfirias
/
Espectrometría de Masa por Ionización de Electrospray
/
Espectrometría de Masas en Tándem
/
Porfobilinógeno Sintasa
/
Hemo
Tipo de estudio:
Diagnostic_studies
Límite:
Humans
Idioma:
En
Revista:
Anal Chem
Año:
2010
Tipo del documento:
Article
País de afiliación:
Estados Unidos