METHOD FOR NEONATAL SCREENING FOR HOMOCYSTINURIA

检测L-同型半胱氨酸和L-蛋氨酸的能力对于准确筛查同型胱氨酸尿症至关重要。本研究项目，如果成功，将导致一个选择性的，定量测定L-同型半胱氨酸和L-蛋氨酸在生物体液中的蛋氨酸γ-裂解酶催化的反应的基础上。将从假单胞菌菌株中分离出对L-甲硫氨酸和L-高半胱氨酸具有高度选择性的酶并进行表征。通过将L-甲硫氨酸或L-高半胱氨酸的酶促分解与NADH氧化脱氢酶偶联，将实现快速分光光度读数。将开发L-蛋氨酸和L-同型半胱氨酸的定量条件，并确定灵敏度的上限和下限。将评估生物液体组分的可能干扰。最后，将固定蛋氨酸γ-裂解酶并进行测试，以确定用于快速常规测量的测试条的可行性。这项工作的商业应用是立即作为新生儿筛查中古特里微生物测定法的替代品，该测定法无法检测L-同型半胱氨酸。改善同型半胱氨酸尿症的早期诊断将允许更及时和有效的治疗，从而降低成本和显着改善同型半胱氨酸尿症的结果。存在开发快速测试条的进一步机会。拟议的商业应用：高胱氨酸尿症的新生儿筛查试验;家庭和医生办公室测试条，用于测量尿液或血液中的L-蛋氨酸和L-高半胱氨酸浓度。

The ability to detect both L-homocysteine and L-methionine is critical for accurate screening for homocystinuria. This research project, if successful, will result in a selective, quantitative assay for L-homocysteine and L- methionine in biological fluids based on the reaction catalyzed by methionine gamma-lyase. The enzyme which is highly selective for L- methionine and L-homocysteine will be isolated from a strain of Pseudomonas and characterized. By coupling the enzymatic decomposition of L-methionine or L-homocysteine to an NADH-oxidizing dehydrogenase, rapid spectrophotometric read-out will be achieved. Conditions for quantitation of L-methionine and L-homocysteine will be developed, and the upper and lower limits of sensitivity will be determined. Possible interference by components of biological fluids will be assessed. Finally, methionine gamma-lyase will be immobilized and tested to establish the feasibility of a test strip for rapid, routine measurements. The commercial application for this work is immediate as a replacement for the Guthrie microbiological assay in neonatal screening, which is unable to detect L- homocysteine. Improved early diagnosis of homocysteinuria will permit more timely and effective treatment, resulting in lower costs and dramatically improved outcomes for homocystinurics. Further opportunities exist for development of a rapid test strip. PROPOSED COMMERCIAL APPLICATIONS: Neonatal screening assay for homocystinuria; home and doctor's office test strip for measurement of L-methionine and L-homocysteine concentrations in urine or blood.