Abstract
Background: Hereditary tyrosinemia type I (HT) fulfills the criteria for inclusion in neonatal screening programs, but measurement of tyrosine lacks clinical specificity and quantitative assay of succinylacetone is laborious. We developed a semiquantitative assay based on inhibition of δ-aminolevulinate dehydratase (ALA-D) by succinylacetone.
Methods: Preincubation of 3-mm discs from dried-blood spots and reaction of the enzyme with δ-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 was measured at 550 nm in a plate reader.
Results: The detection limit for succinylacetone was 0.3 μmol/L; imprecision (CV) was <5.5% within-run and 10–16% between-run. Storage of blood spots at ambient temperature for several days led to a significant decrease of ALA-D activity. Enzyme activity was lost in filter cards at 45 °C, but remained stable at 2–37 °C. Enzyme activity was decreased in EDTA blood. The absorbance at 550 nm was 0.221 (± 0.073) in healthy neonates and 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 sensitive test for HT. This represents a basis for further examination of its general reliability as a confirmatory test if tyrosine is found to be increased.
405. Correction of the Murine Model of Hereditary Tyrosinemia Type I Using Messenger RNA as a Source of Transposase for Sleeping Beauty Mediated Integration of the FAH Gene