HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY IMPROVES DIAGNOSTIC EFFICIENCY OF CARBOHYDRATE-DEFICIENT TRANSFERRIN

Alcohol & Alcoholism Vol. 32, No. 1, pp. 71-77, 1997 fflGH-PERFORMANCE LIQUID CHROMATOGRAPHY IMPROVES DIAGNOSTIC EFFICIENCY OF CARBOHYDRATE-DEFICIENT TRANSFERRIN EGON WERLE*, GISELA E. SEITZ1, BRIGITTE KOHL, WALTER FIEHN and HELMUT K. SEITZ1 Central Laboratory, Medical Clinic and Policlinic, University of Heidelberg, Bergheimerstr. 58, 69115 Heidelberg and 'Laboratory of Alcohol Research, Liver Disease and Nutrition and Dept. of Medicine, Salem Medical Center, Zeppelinstr. 11-33, 69121 Heidelberg, Germany (Received 29 March 1996; accepted in revised form 21 August 1996) Abstract — Carbohydrate-deficient transferrin (CDT) is considered a useful biochemical marker of regular high alcohol intake. CDT was measured in the sera of 51 alcohol abusers, 20 patients with nonalcoholic liver disease and 30 healthy controls with an alcohol intake of <30g/day. The mean CDT levels of these three groups respectively were determined with high-performance liquid chromatography (HPLC; 4.6 ± 5.2%; 0.7 ± 0.2%; 0.7 ± 0.2%) and with a radioimmunoassay after microcolumn anionexchange chromatography (MAEC/RIA; 34.2 ± 26.9 U/l; 16.9 ± 3.8 U/l; 18.0 ± 5.7 U/l). CDT levels in patients with severe alcohol abuse (161.6 ± 96.4g/day) were significantly higher than in the two other groups under investigation (P < 0.0001). In heavily drinking subjects, the mean daily alcohol intake correlated with aspartate aminotransferase levels (ASAT) but not with the CDT levels determined either with HPLC or MAEC/RIA. With both methods, the CDT levels were slightly higher in patients with an ASAT concentration >30 U/l, which may indicate an advanced liver damage (P < 0.05). Analysis of receiver-operating characteristic (ROC) plots demonstrated that the diagnostic accuracy of the HPLC method, which determines the relative amount of CDT, was significantly higher than the established MAEC/RIA method, which measures the absolute amount of CDT (area under the ROC curve: 0.95 ± 0.02 vs 0.73 ± 0.05; P < 0.0001). At a specificity of >95%, the sensitivity of CDT determined with HPLC and MAEC/RIA was 80 and 47%, respectively. In addition, HPLC may be a useful and reliable method for the determination of this important biochemical marker, since the HPLC chromatogram is a visible document of the successful isotransferrin separation and measurement. INTRODUCTION Hazardous alcohol consumption may be diagnosed with a variety of laboratory tests; however, there is no satisfying marker of alcohol abuse with high sensitivity and specificity (Conigrave et al, 1995; Helander et al, 1996). Carbohydrate-deficient transferrin (CDT) has been reported to be a highly specific marker of alcohol abuse (Stibler, 1991). CDT may anse from an acetaldehyde-mediated inhibition of glycosyl transfer (Malagolini et al, 1989; Guasch et al, 1992). Microcolumn anionexchange chromatography followed by a radioimmunoassay of transferrin (MAEC/RIA) as well as isoelectric focusing (IEF), immunoblotting, and densitometry are commonly used for the absolute or relative quantification of CDT, respectively (Anton and Bean, 1994). An alternative method is high-performance liquid chromatography (HPLC; Jeppsson et al, 1993). We compared two methods for the evaluation of CDT as a marker of chronic alcohol abuse in a German population with or without alcoholic liver disease, MATERIALS AND METHODS Subjects Sera from 101 patients were analysed (Table 1). Fifty-one inpatient alcoholics (32 men, 19 women, mean age 48 years, range 18-71), admitted for detoxification, were repeatedly questioned about their drinking habits by a psychologist (G.S.). These self-reports revealed that they had a daily alcohol intake of >50 g, and in most cases (42 out of 51) >100g. The mean (±SD) alcohol intake •Author to whom correspondence should be addressed. 71 © 1997 Medical Council on Alcoholism 72 E. WERLE et al. Table 1. Characterization and laboratory results in healthy social drinkers, patients with non-alcoholic liver disease and heavily drinking persons Group Social drinkers (n = 30) Non-alcoholic liver disease (/. = 20) Alcoholic patients (n = 51) Sex (female/male) Age (years) yGT (IU/1) MCV (fl) ASAT (IU/1) CDT (U/l) CDT (%) (MAEC/RIA) (HPLQ 18/12 9/11 36 ± 5 58 ± 9 11.1 ± 2 170 ±98 92 + 5 94±4 12 + 3 152 ±82 18.0 + 5.7 16.9 ± 3.8 0.7 ± 0.2 0.7 + 0.2 19/32 48 + 5 200±63 98 ± 6 44±11 43.2 ± 26.9 4.6 + 5.2 The healthy social drinkers had a daily alcohol intake of <30g, whereas the heavily drinking alcoholic patients drank 162 ± 96 g of alcohol/day. Values are means + SD was 161.7 ± 96.4g/day and the mean duration of alcohol abuse was 16.9 ± 13.8 years. In 17 patients, the CDT levels were determined three times within 3 weeks of alcohol abstinence in order to determine the half-life of CDT. Twenty patients (11 men, nine women, mean age 58 years, range 24—90) with liver damage unrelated to alcohol abuse (alcohol intake <30g per day; acute and chronic aggressive virus hepatitis C and B, non-alcoholic cirrhosis of the liver, autoimmune hepatitis, liver metastasis, e.g. of a prostate cancer) participated in the study. Thirty healthy persons (12 men, mean age 37 ± 3 years, 18 women, mean age 36 ± 5 years) with an alcohol consumption of <30g/day served as controls. In healthy controls, the y-glutamyl-transferase (yGT) activity (measured at 25°C) was 9 ± 1 U/l and 14 ± 2 U/l in women and men, respectively. of FeCl3 (10 mmol/1 Fe) to 1 ml of serum. After an overnight storage at 6CC, 10 ul of dextran sulphate (100 g/1) and 50 ul of CaCl2 (1 mol/1) were added per ml of serum in order to precipitate lipoproteins. After 1 h at 6°C a centrifugation step (10 OOOg, 10 min) was done, the supernatant was diluted fivefold with water and injected into the HPLC column Mono Q HR5/5 (Pharmacia, Freiburg, Germany). Iron-saturated isoforms of transferrin were eluted with a salt gradient and monitored at a detector wavelength of 460 nm (HPLC System Gold, Beckman, Munich, Germany). The valley-to-valley method was used to integrate the chromatography profile. The a-, mono- and disialotransferrin peaks are referred to as CDT and the result is expressed as a percentage of total transferrin (%CDT). The detection limit is <0.5%. Analytical methods Aspartate aminotransferase (ASAT) and yGT activities were measured with the analyser Cheml and the mean corpuscular volume of erythrocytes (MCV) was determined with the H3 (Technicon, Bayer, Munich, Germany). Serum transferrin concentrations were determined by nephelometry (Behring Nephelometer Analyzer II, Behringwerke, Marburg, Germany). A commercially available test kit for the quantification of CDT with MAEC/RIA (MAEC/RIA CDT) was used according to the manufacturer's instructions (CDTect, Kabi Pharmacia Diagnostics, Uppsala, Sweden). Sera were analysed in duplicates and results are given in units per litre (U/l). We adapted an HPLC method for the determination of CDT (Jeppsson et aL, 1993). In brief, iron saturation of transferrin wa (...truncated)