A high performance gel filtration chromatography method for gamma-glutamyltransferase fraction analysis

The clinical relevance of serum gamma-glutamyltransferase (GGT) activity, in areas other than hepatic function, has recently been increased by several epidemiological associations. Still, GGT remains a nonspecific test because of the influence of various pathophysiological factors. We devised a procedure based on gel filtration chromatography, followed by postcolumn injection of fluorescent GGT substrate (gamma-glutamyl-7-amido-4-methylcoumarin), permitting the quantification of GGT fractions in serum or plasma. Plasma GGT molecular weight distribution was analyzed in healthy volunteers (20 males; mean+/-SD age 38+/-10 years; 20 females; age 44+/-13; total GGT 21+/-11 for males vs 13+/-7 for females; P<0.01). The method is highly sensitive (determination limit: 0.5 U GGT/L), with a linear dynamic range between 0.5 and 150 U/L for each fraction. Four GGT fractions of different molecular weight were detected in all subjects of both genders: b-GGT, m-GGT, s-GGT (likely lipoprotein-bound, molecular masses >2000, 940, and 140kDa, respectively), and a free fraction (f-GGT, 70kDa). f-GGT and s-GGT were the main fractions in subjects with lower and higher total GGT activity, respectively. Higher total GGT activity in males is related mainly to f-GGT (P<0.01). GGT fraction analysis may increase the sensitivity and specificity of the GGT activity test.