1 Departments of Clinical Chemistry and
2 Psychiatry, EP Central Hospital, Seinäjoki,
3 University of Tampere, Finland and
4 Addiction Research Foundation, Toronto, Ontario, Canada
(Received 3 March 2003; first review notified 7 April 2003; in revised form 23 April 2003; accepted 1 May 2003)
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ABSTRACT |
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INTRODUCTION |
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Recently, the relative importance of the various CDT isoforms in the clinical assays for detecting alcohol misuse has received increasing attention (Scouller et al., 2000; Whitfield, 2002
). Although measurements with improved specificity towards such isoforms have been introduced, only a few studies are available on the comparisons between the different methods in clinical materials (Helander, 1999
; Anton et al., 2001
). The new assay approaches, which exclude the trisialo isoform (the isoform with three sialic acid side-chains) from the measurement, have recently shown high diagnostic accuracies in the detection of alcohol misuse (Legros et al., 2002
). Previous studies have also indicated that the diagnostic characteristics of the CDT assays may be markedly different from each other. Studies with the conventional CDTect method have found high diagnostic sensitivities, although also a high rate of false-positive results among women and in patients with high serum transferrin levels (Viitala et al., 1998
). Concerns over the impact of liver status have also been raised (Tsutsumi et al., 1994
; Niemelä et al., 1995
; DiMartini et al., 2001
).
We designed the present study to compare the conventional CDTect method and %CDT assays for their diagnostic value to identify problem drinking in alcoholic patients with or without liver disease. Separate versions, which have been made available for the %CDT assay, allow the comparisons between the assays with (%CDTTIA) or without (%CDT) reactivity towards the trisialofraction. The data suggests that the %CDT assay, which is devoid of such reactivity, offers several advantages over the previous CDT methods.
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SUBJECTS AND METHODS |
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CDT analyses
The concentrations of CDT were first measured by a turbidimetric immunoassay (%CDT) after ion exchange chromatography (Axis-Shield, Oslo, Norway). This assay measures the transferrin variants with 02 sialic acid residues and excludes the trisialofraction containing 3 sialic acids. The concentration of CDT is expressed as a percentage of the total amount of serum transferrin. The measurements were carried out on a Behring Nephelometer II (Dade Behring, Behring Diagnostics, Marburg, Germany). Values exceeding 2.6% were considered to be high.
For comparisons, CDT was also measured with a competitive radioimmunoassay after microcolumn separation (CDTect). This assay excludes the trisialofraction and part of the disialofraction. Thus, the isoforms with 01 sialic acid residues (a- and monosialotransferrins) and minor amounts of isotransferrin with 2 sialic acid residues (disialotransferrin) are detected (Arndt et al., 1998; Helander et al., 2001a
). The assay results are expressed as absolute CDT concentrations. The reference range in this assay is 020 U/l for men and 026 U/l for women. Measurements of the %CDTTIA, which also measures the trisialylated fraction of serum transferrin were carried out on a Kone Optima Clinical Chemistry Analyzer (Kone Instruments, Espoo, Finland), as described previously (Viitala et al., 1998
). The cut-off value for this assay is 6% of the total serum transferrin.
The cut-off values for all of the above assays were based on the manufacturers recommendations. For initial method evaluation, pooled sera from control and alcoholic patients with low and high CDT concentrations were first analysed to determine the analytical characteristics of the different methods. The within-run precisions (n = 10) for the %CDT were 5.2% for the low concentration and 4.4% for the high CDT content. The corresponding values for the CDTect method were 6.2 and 10%, respectively. The day-to-day CVs (n = 13) for the %CDT were 10% for the low and 5.6% for the high concentration, which were lower than those of the CDTect method.
Other laboratory procedures
Serum gamma glutamyl transpeptidase (GT), serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), albumin, transferrin, 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. For the parameters used as alcohol markers in this study, the following cut-off values were used: GT, 80 U/l (men), 50 U/l (women); AST 50 U/l (men), 35 U/l (women); MCV 96 fl.
Statistical methods
The data are expressed as mean ± SD. The statistical analyses were carried out using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). The comparisons between the alcoholic and non-alcoholic groups were carried out using Students t-test or by analysis of variance (ANOVA), with the Bonferronis method for multiple comparisons. Correlations were calculated using Pearsons product-moment correlation coefficients or Spearmans rank correlations, as required. A P-value of less than 0.05 was considered statistically significant.
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RESULTS |
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DISCUSSION |
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The present data indicate that the new %CDT assay may offer several benefits for assessing excessive alcohol consumption. First, there is an improved sensitivity when compared to the previous %CDT assays. This finding appears to stem from the fact that the new assay is devoid of reactivity towards the trisialylated fraction of transferrin, which is consistent with the view that the generation of this isoform in vivo is not significantly influenced by ethanol consumption. This finding is also in line with other recent observations on transferrin desialylation as a result of alcohol misuse (Arndt et al., 2002; Legros et al., 2002
, 2003
). Second, the data show a higher accuracy of the new %CDT method when compared to the conventional CDTect method. This observation appears to be primarily due to the higher specificity among women and in patients with liver disease, which may be explained by previous findings indicating that the CDTect method may be sensitive to unspecific changes in serum transferrin metabolism (Sorvajärvi et al., 1996
; Viitala et al., 1998
; De Feo et al., 1999
; DiMartini et al., 2001
; Whitfield et al., 2001
; Arndt and Kropf, 2002
; Whitfield, 2002
). Third, the analytical precision profiles of the %CDT method are also superior to those of CDTect, suggesting that this method should yield improved reproducibility and thus be more suitable for routine laboratory use.
The lack of sensitivity for CDT as a marker of alcohol misuse among women has been previously recognized in several reports (Anton and Moak, 1994; Löf et al., 1994
; Reif et al., 2001
). With the new %CDT method, despite this improvement, the diagnostic sensitivity in women continues to be lower than that in men. Our data further suggest that patients with mild to moderate alcoholic liver pathology, as judged from morphological indices, tend to show higher CDTect values than do alcoholics without liver pathology. These findings may be explained in the light of previous observations indicating that transferrin synthesis rate in (human) alcoholics may be accelerated in patients with early-stage liver disease but diminished in cirrhotic patients (Potter et al., 1985
). In severe liver disease, CDT levels when measured with the CDTect method, are expected to decrease upon decreased transferrin protein synthesis capacity. The %CDT method in turn appears to be less sensitive to the effects of liver disease per se. The strong correlation between the %CDT results and the amount of ethanol consumed indicates that this method may reflect ethanol-induced changes in the process of desialylation more specifically than CDTect.
The majority of previous studies that have compared CDT and other markers of ethanol consumption (GT, MCV, AST) have concluded that the sensitivity of CDT exceeds that of the conventional markers (see Scouller et al., 2000). Although current data suggest high sensitivities for GT measurements, it should be noted that GT is known to lack specificity in assays of hospitalized materials where several conditions may lead to elevated values, including non-alcoholic liver diseases, diabetes, obesity or use of various drugs (Salaspuro, 1999
). Despite the fact that CDT, as previously measured with the CDTect method, has also been reported to lack specificity, this may occur more rarely, including primarily conditions which affect body iron stores and thus alter serum transferrin levels (Viitala et al., 1998
; De Feo et al., 1999
; Arndt and Kropf, 2002
; Whitfield, 2002
). Because such effects are not expected to occur with the %CDT method, it should also further improve the clinical value of CDT measurements in alcohol screening programmes in hospitalized materials.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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