Departments of 1 Clinical Chemistry and 2 Psychiatry, EP Central Hospital, Seinäjoki and 3 University of Tampere, Tampere, Finland
* Author to whom correspondence should be addressed at: EP Central Hospital, Laboratory, FIN-60220 Seinäjoki, Finland. Tel.: +358 6 415 4719; Fax: +358 6 415 4924; Email: onni.niemela{at}epshp.fi
(Received 4 August 2003; first review notified 18 September 2003; in revised form 20 October 2003; accepted 29 October 2003)
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ABSTRACT |
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INTRODUCTION |
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Various methods with different reactivities to different transferrin isoforms are currently available. The diagnostic characteristics of such methods in the clinical assays for detecting alcohol misuse have, however, not been fully established (Scouller et al., 2000). The new %CDT assay, which excludes the trisialo isoform (the isoform with three sialic acid side chains) from the measurement, has recently been suggested to yield improved specificity as compared to the conventional CDTect method, which has previously been the most widely used CDT assay (Anton et al., 2001
).
We designed this study to compare the characteristics of the different CDT methods in the detection and follow-up of alcoholics, who were admitted for detoxification. For comparison, the conventional alcohol markers (GT, AST, MCV) were also measured. The data suggest distinct differences between the different CDT assays in monitoring alcohol drinking and abstinence.
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MATERIALS AND METHODS |
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All serum samples were stored at 70°C until analysis. All participants in the study gave their informed consent and the study was carried out according to the provisions of the Declaration of Helsinki.
CDT analyses
The concentration of CDT was measured in serum samples by two different methods. First, a turbidimetric immunoassay after ion-exchange chromatography (%CDT; Axis-Shield, Oslo, Norway), which detects transferrin variants with 02 sialic acid residues, was used. In this assay, CDT concentration is expressed as a percentage of the total amount of serum transferrin. The measurements were carried out on Behring Nephelometer II (Dade Behring; Behring Diagnostics, Marburg, Germany) according to the instructions of the manufacturer's. Amounts exceeding 2.60% were considered high (Anton et al., 2001). In addition, CDT was measured by a competitive radioimmunoassay after micro-column separation (CDTect). In this assay, serum transferrin isoforms with 01 sialic acid residues (
- and monosialotransferrins) and minor amounts of isotransferrin with two sialic acid residues (disialotransferrin) are detected. The reference range is 020 U/l for men and 026 U/l for women (Anton et al., 2001
; Helander, 2002
).
Other methods
Serum -glutamyl tansferase (GT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, bilirubin and mean corpuscular volume (MCV) of erythrocytes were measured by standard clinical chemical methods in an accredited (SFS-EN 45001, ISO/IEC Guide 25) laboratory of EP Central Hospital, Seinäjoki, Finland. The reference ranges for these parameters were as follows. GT, men <80 U/l, women <50 U/l; AST, men <50 U/l, women <35 U/l; ALT, men <50 U/l, women <35 U/l; albumin 3650 g/l; bilirubin 220 µmol/l; MCV 7696 fl.
Calculations and statistical methods
The data are expressed as means ± SD. The comparisons between the alcoholic and non-alcoholic groups were carried out using Student's t-test or the MannWhitney test for parameters with skewed distributions of values. Comparisons of the analytical characteristics between the different markers were based on ROC-assays using Analyse-it software (Analyse-it Software, Leeds, UK) Calculations of marker normalization rates were based on observed values and related to the starting value and to the time point of the last drink. Correlations were calculated using the Pearson productmoment correlation coefficients. A P-value less than 0.05 was considered statistically significant.
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RESULTS |
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Elevated marker values in the alcoholics at the time of admission were observed with the following frequencies: %CDT 69%, CDTect 61%, GT 61%, AST 56% and MCV 47%. Except for MCV, the differences in the analytical characteristics between the above marker pairs were not statistically significant. Serum bilirubin and albumin did not differ significantly between the alcoholic and control populations, suggesting a lack of significant liver dysfunction in the present sample of alcoholic patients (Table 1). The amount of alcohol consumed, as estimated by a time-line follow-back method from different time intervals prior to sampling, correlated most significantly with the %CDT and MCV results (Table 2).
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DISCUSSION |
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Most of the current knowledge on CDT measurements has been based on the use of the conventional CDTect method. The more recently introduced %CDT measurements have offered the benefit of expressing the data conveniently as percentages of total transferrin with similar cut-offs for both sexes. However, the first versions of the %CDT assays suffered from poor analytical sensitivities (Viitala et al., 1998). More recent comparisons between the new %CDT and CDTect methods have shown essentially similar sensitivities (Anton et al., 2001
). Accordingly, the new %CDT method in the present relatively small material appears to yield even a slightly higher (although not statistically significant) sensitivity (69%) than the conventional CDTect method (61%) or the other conventional markers of ethanol consumption (Fig. 1) when analysed at the time of admission. It may be emphasized that, in assays for alcohol misuse, which has an extremely high prevalence (typically 1020% in many Western countries), even a small improvement in the analytical performance should lead to a more correct identification of a large number of hazardous drinkers, which, in turn, is of crucial importance in the clinical assessment of such patients. However, as the current material is relatively small future studies for testing marker function in larger materials appear warranted.
Interestingly, current data also show that the correlation between the marker results and the actual amount of ethanol consumed is stronger with the %CDT results than with the CDTect results, indicating that the measurements (which exclude the trisialofraction but include the entire disialofraction into the measurement) may reflect ethanol-induced changes in transferrin protein in a more specific manner. This may also be due to the fact that the CDTect method may respond to unspecific changes in transferrin protein metabolism, iron balance, or hormonal status (Niemelä et al., 1995; Sorvajärvi et al., 1996
; De Feo et al., 1999
; DiMartini et al., 2001
; Whitfield et al., 2001
). Such phenomena may also explain the previously reported poor sensitivities for CDTect assays in female patients (Anton and Moak, 1994
; Löf et al., 1994
; Reif et al., 2001
; Anton et al., 2002
). Preliminary analyses of serum sialic acid contents from these patients have indicated no significant relationship between the assay-dependent characteristics and sialic acid metabolism (data not shown).
In this study we included both abstainers and social drinkers in our control material. No significant differences in CDT values emerged between these subgroups, suggesting a lack of correlation between the consumption of low doses of alcohol and CDT values. In the alcoholics, the presence of severe liver disease has previously been found to be a significant confounding variable in the interpretation of CDT results (Anttila et al., 2003). In this study the possible effect of alcohol-related liver disease on the findings could not be assessed by the method-of-choice, because liver biopsies were not done. It should be noted, however, that based on clinical examination and laboratory findings indicating that serum bilirubin and albumin levels were not significantly different between the alcoholics and controls, the former group should have been devoid of any significant liver dysfunction.
The present follow-up data show a consistent decrease of the %CDT marker values during abstinence, which is also in line with the view that the %CDT results show a close association between the marker data and drinking habits. The discrepancies between GT, CDTect, AST and abstinence perhaps could be due to induction of liver pathology, which may be reflected in marker values also during abstinence. In particular, the CDTect method may be sensitive to changes occurring during liver pathology even in its early phase (Tsutsumi et al., 1994; Niemelä et al., 1995
). The present findings indicating that CDT methods may also have kinetic differences in their rates of normalization should also be considered in further method comparisons and in monitoring abstinence in alcoholic patients.
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ACKNOWLEDGEMENTS |
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