Radioenzymic determination of homocysteine in plasma and urine

Abstract

Using a modification of the radioenzymic assay described previously (J Biol Chem 259: 2360-2364, 1984) we measured homocysteine in freshly prepared plasma and urine from volunteers. The concentration of free homocysteine--i.e., the amount measurable in plasma after deproteinization by strong acid--was 2.27 (SEM 0.11) mumol/L for 18 men and 1.95 (SEM 0.13) mumol/L for 16 women (p greater than 0.05, not significant). About 70% of the total homocysteine in human plasma was associated with plasma proteins, and was precipitated with strong acid. The concentration of protein-bound homocysteine in plasma was 6.51 (SEM 0.32) mumol/L for men and 7.29 (SEM 0.65) mumol/L for women, a significantly (p less than 0.01) different spread. Homocysteine was rapidly released from plasma proteins in the presence of a reducing agent, dithioerythritol. By gel filtration of plasma on a "high-performance" liquid-chromatographic column, albumin was shown to be the sole carrier of homocysteine in plasma. Because the fraction bound to protein as determined by this procedure equaled that obtained by precipitation of plasma proteins with acid, we conclude that homocysteine is bound to albumin in vivo. The concentration of homocysteine in urine ranged from 3.5 to 9.5 mumol/L, about 6 mumol of homocysteine being excreted per 24 h.