Standardization of homocysteine on HPLC using DTT as reductant

Abstract

Determination of plasma total homocysteine by high-pressure liquid chromatography (HPLC) usually requires reduction of protein bound or free homocysteine-disulphides into thiols by a reducing agent and the liberated thiols are then derivatized by a fluorescent marker. In this study we have standardized the HPLC method for homocysteine measurement using dithiothreitol (DTT) as reductant. The results of plasma total homocysteine values obtained by HPLC were compared with IMx method. The difference between the two means was statistically insignificant [P=0.616847 (two tail)] Linear regression analysis showed strong correlation between the two methods (r=0.983). Using this method we have analyzed 132 controls and 130 Coronary Heart Disease (CHD) patients for plasma total homocysteine, wherein, the mean plasma total homocysteine levels were 10.51 ± 8.36 and 11.51 ± 10.06 μmol/L respectively. Our research study suggests that DTT method is a simple and inexpensive assay for homocysteine determination in human plasma for research application.