Spectrophotometric microassay for delta-aminolevulinate dehydratase in dried-blood spots as confirmation for hereditary tyrosinemia type I

Background: Hereditary tyrosinemia type I (HT) fulfills the criteria for in clusion in neonatal screening programs, but measurement of tyrosine lacks c linical specificity and quantitative assay of succinylacetone is laborious. We developed a semiquantitative assay based on inhibition of S-aminolevuli nate dehydratase (ALA-D) by succinylacetone. Methods: Preincubation of 3-mm discs from dried-blood spots and reaction of the enzyme with delta -aminolevulinic acid as substrate were performed in microtiter plates. After separation of the supernatant and 10 min of color reaction with modified Ehrlich reagent, the formation of porphobilinogen wa s measured at 550 nm in a plate reader. Results: The detection limit for succinylacetone was 0.3 mu mol/L; imprecis ion (CV) was <5.5% within-run and 10-16% between-run. Storage of blood spot s at ambient temperature for several days led to a significant decrease of ALA-D activity. Enzyme activity was lost in filter cards at 45 degreesC, bu t remained stable at 2-37 degreesC. Enzyme activity was decreased in EDTA b lood. The absorbance at 550 nm was 0.221 (+/- 0.073) in healthy neonates an d 0.043-0.100 in 11 patients with HT. All neonates with increased tyrosine (above the 99.5th centile) in neonatal screening (97 of 47 000) had normal results by the new assay. Conclusions: The spectrophotometric microassay for ALA-D is a simple and se nsitive test for HT. This represents a basis for further examination of its general reliability as a confirmatory test if tyrosine is found to be incr eased. (C) 2001 American Association for Clinical Chemistry.