Elevated levels of homocysteine in urine and blood have been correlated with the development of atherosclerosis and mortality from people with existing cardiovascular disease. Even moderate homocysteinemia is now regarded as a riskfactor for cardiac and vascular diseases. In order to meet the need for a simple, high through-put assay for homocysteine that can utilize modern analyzers, we propose to develop a single-enzyme homocysteine assay. Toward this goal we have cloned a unique recombinant homocysteine alpha, gamma-lyase (rHCYase) from Trichomonas vaginalis which has been overexpressed in E.coli.The cloned rHCYase produces hydrogen sulfide from homocysteine and is not interfered with by cysteine. The amount of homocysteine in the sample is measured by the amount of hydrogen sulfide using N,N-diethylphenylene diamine (DEPDA) and related chromophores which have absorbance peaks between 670 nm and 680 nm and a sensitivity of 1 mu m homocysteine or less. The assay will be developed by determining optimal conditions for use of the enzyme and chromophore with respect to all aspects of performance including specificity, sensitivity, rapidity and simplicity. The total concentration of homocysteine present in biological samples, includes homocysteine molecules that are not present in free form, being instead covalently coupled to other molecules. Disulfide- bound homocysteine is released using dithiotreotal (DTT) which does not inhibit the color development in the assay. The methods developed here will be validated against standard HPLC methods of measuring homocysteine using banked plasma samples with a wide range of homocysteine levels. Diagnostic kits are proposed for use in determining the'amount of homocysteine in any biological sample. In Phase II, the optimized assay for both plasma and urine will be validated by clinical trial and commercial kits suitable for all major types of automated analyzers will be finalized.
Hyperhomocysteinemia and hyperhomocysteinuria are strong risk factors for cardiovascular disease. Current methods use cumbersome HPLC techniques not amenable for a widely-used diagnostic. The enzymatic diagnostic being developed in this application is ideal for current analyzers and could serve current market estimates of 50-l00 million tests per year.
| Tan, Y; Tang, L; Sun, X et al. (2000) Total-homocysteine enzymatic assay. Clin Chem 46:1686-8 |
