Direct Assay of δ-Aminolevulinic Acid Dehydratase in Heme Biosynthesis for the Detection of Porphyrias by Tandem Mass Spectrometry

Abstract

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/hr. 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, low background, requires a simple workup, and uses a commercially available substrate.

Laboratory Diagnosis of Porphyria

“…HMB spontaneously cyclizes to uroporphyrinogen I and is detectable in spectrofluorometry as oxidized uroporphyrin I. Recently, tandem mass spectrometry was applied to the ALAD assay [91]. The incubation of red blood cells (RBC) lysate with ALA was followed by the quantitative in situ conversion of PBG into its butyramide and the extraction of the derivative product into a mass spectrometer-friendly liquid solvent.…”

Elimination of the Effect of Some Herbicides on the Growth of Zea mays and Accumulation in the Soil Using Urea

“…The reaction mixtures were allowed to react at 37 o for an hour, and then the reaction was stopped by the addition of 1.0 mL M mercuric chloride in 10% trichloacetic acid followed by centrifugation. The quality of propholbilinogen was determined spectrophotometrically at 555 nm by the reaction with an equal volume of modified Ehrlich's reagent according to the procedure of Choiniere (2010).…”

1-Butanol as a Solvent for Efficient Extraction of Polar Compounds from Aqueous Medium: Theoretical and Practical Aspects

“…Unfortunately, although quantitative conversion was apparent from other data, the isolated yield turned out to be disappointingly low. We then turned to 1-butanol, which had previously been used only sporadically as an extraction solvent in organic chemistry. − The solvent switch proved to be successful, leading to an almost quantitative isolated yield of cyclohexane-1,2-diol (94–98%) . This raises the question of how “universal” 1-butanol is as an extraction solvent for polar (and likewise nonpolar) compounds.…”