ISOELECTRIC-FOCUSING WESTERN BLOTTING - A NOVEL AND PRACTICAL METHOD FOR QUANTITATION OF CARBOHYDRATE-DEFICIENT TRANSFERRIN IN ALCOHOLICS

Carbohydrate-deficient transferrin (CDT) has been described as the single, most accurate marker of chronic alcohol consumption. Rapid, sensitive, and specific measurement of serum CDT levels can thus provide important clinical information concerning patient diagnosis and treatment. To date, however, methods used for assessing CDT concentrations [e.g., analytical isoelectric focusing combined with immunofixation and micro anion-exchange chromatography followed by radioimmunoassay (RIA)] have not been practical enough for widespread laboratory application. In the present study, we examined the use of a different technique, namely isoelectric focusing (IEF) combined with Western blotting (IEF/WB). Serum proteins (20-40-mu-g) were first focused according to isoelectric points (pI) on high-resolution agarose IEF gels (ampholyte pH range of 5-8) containing nonionic detergent. The focused proteins were transferred electrophoretically to nitrocellulose filters, and then stained immunochemically with antihuman transferrin IgG. IEF/WB completely resolved CDT (focusing at pI 5.7 and 5.9) from other serum transferring isoforms, as assessed with neuraminidase-generated CDT standards. Computerized densitometric scanning of the immunoblots allowed CDT levels to be quantitated directly rather than as a quotient. Serum CDT content determined by IEF/WB was highly correlated (r2 = 0.962; n = 17) with values determined previously by RIA. In a larger subject group, CDT levels (mg/liter) measured by IEF/WB were 139 +/- 54 in recently-drinking alcoholics (n = 58), 81 +/- 8 in abstaining alcoholics (n = 7), and 68 +/- 16 in healthy control subjects (n = 16). These IEF/WB values for serum CDT are highly similar to those reported using other quantitation methods. Importantly, CDT levels as measured by IEF/WB were not influenced by the severity of liver disease among the recently-drinking alcoholics nor did nondrinkers with liver disease exhibit elevated IEF/WB CDT values. Serum CDT/total transferrin ratios (the latter measured by ELISA) offered no advantage over serum CDT alone to distinguish active and heavy drinking. In conclusion, we have developed a sensitive, accurate, and most importantly, practical method for quantitation of serum CDT, a highly-reliable marker of chronic alcohol consumption. Routine implementation of this technique by clinical laboratories can rapidly provide the physician with a powerful diagnostic tool.