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半胱氨酸尿至关重要。该研究项目如果成功，将基于蛋氨酸裂解酶催化的反应，对生物体液中的L-同型半胱氨酸和L-蛋氨酸进行选择性定量分析。对L-蛋氨酸和L-同型半胱氨酸具有高度选择性的酶将从一株假单胞菌中分离出来并进行鉴定。通过将l -蛋氨酸或l -同型半胱氨酸的酶分解偶联到nadh氧化脱氢酶，将实现快速分光光度读出。将建立l -蛋氨酸和l -同型半胱氨酸的定量条件，并确定灵敏度的上下限。将评估生物流体成分可能造成的干扰。最后，蛋氨酸γ裂解酶将固定和测试，以建立快速，常规测量试纸的可行性。这项工作的商业应用是立即取代新生儿筛查中的Guthrie微生物测定法，后者无法检测到L-同型半胱氨酸。改善同型半胱氨酸尿的早期诊断将允许更及时和有效的治疗，从而降低成本并显著改善同型半胱氨酸尿的预后。存在着进一步发展快速试纸条的机会。拟议的商业应用：新生儿同型半胱氨酸尿筛查试验；用于测量尿或血中l -蛋氨酸和l -同型半胱氨酸浓度的家庭和医生办公室测试条。

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.