Carbohydrate-Deficient Transferrin Measured by Capillary Zone Electrophoresis and by Turbidimetric Immunoassay for Identification of Young Heavy Drinkers

Abstract

Carbohydrate-deficient transferrin (CDT) measured by capillary zone electrophoresis (CZE), particularly asialotransferrin (Tf), is purported to better differentiate between excessive and moderate drinkers than does CDT measured by turbidimetric immunoassay (TIA) (1,2 ). The use of biological markers such as CDT is of particular interest for identifying young heavy drinkers because other clinical signs of heavy drinking are generally absent and heavy drinking is a leading cause of morbidity and mortality in this age group (3, 4 ). Several authors have shown interest in the ability of CDT to identify nondependent heavy drinkers (5, 6 ); we therefore describe here the performance of CZE measurements of asialo-and disialo-Tf and TIA analysis of CDT in a large community sample of 19-year-old men, of whom 21% were heavy drinkers.From a sample of 1018 men attending a mandatory 1-day army recruitment process for all Swiss males at age 19 years, 1004 (98.6%) agreed to complete a research questionnaire. Of these, 581 young men (57.9%) consented to give blood for the measurement of asialo-Tf (CZE), disialo-Tf (CZE), and CDT (TIA). The Ethics Committee of the Lausanne University Medical School approved the study protocol. Volunteers were compensated for participation in the study.Volunters gave written informed consent and then completed an instrument entitled "Health and Lifestyle Questionnaire", which included questions assessing the typical quantity and frequency of alcohol consumption during the 12 months preceding the survey and the frequency of drunkenness over the last 30 days. One drink was defined as a 250-mL can or bottle of beer, a 120-mL glass of wine, or a 40-mL shot of liquor straight or in a mixed drink, and corresponded to ϳ12 g of pure ethanol.A study investigator was present during administration of the questionnaire to verify that participants answered all items. Serum samples were obtained by centrifugation of peripheral blood collected in 10-mL tubes. Samples were stored at Ϫ20°C before analysis.Total CDT was measured by anion-exchange chromatography and TIA with the Axis-Shield CDT (TIA) reagent set (7 ). To separate and measure Tf isoforms, we used a previously described and validated CZE method (8, 9 ) with the Ceofix CDT reagent set (Analis) on a Hewlett Packard (HP) 3D-CE instrument. The CZE conditions are described in Table 1 of the Data Supplement that accompanies the online version of this Technical Brief at http:// www.clinchem.org/content/vol51/issue6/. CZE electropherograms showing the serum Tf profiles for a heavy drinker before and after addition of anti-Tf polyclonal antibody to the serum are shown in Fig. 1 of the online Data Supplement, and CZE electropherograms showing the Tf profiles of a teetotaler and of 2 heavy drinkers are shown in Fig. 2 of the online Data Supplement.Peaks representing the different Tf isoforms were quantified as the amounts of the asialo-, disialo-, trisialo-, tetrasialo-, pentasialo-, and hexasialo-Tf (CZE) as a percentage of the total Tf content, in terms of va...

Monitoring of transferrin isoforms in biological samples by capillary electrophoresis

“…Although the CEofix kit was promoted for CE instruments of Beckman Coulter, it was also used on other CE instruments as was shown for the HP 3D‐CE system by Daeppen et al. . The kit comprised an initiator solution composed of a pH 4.8 malic acid/arginine buffer with the polycation and the pH 8.5 Tris/boric acid running buffer containing the polyanion.…”

“…10-004740 of Analis, Namur, Belgium) which provided a nice separation of most Tf isoforms (asialo-Tf to hexasialo-Tf) and thus the possibility to determine CDT in human serum to differentiate between chronic alcohol abusers and teetotalers [26,28,29, [80][81][82][83]. Although the CEofix kit was promoted for CE instruments of Beckman Coulter, it was also used on other CE instruments as was shown for the HP 3D-CE system by Daeppen et al [84]. The kit comprised an initiator solution composed of a pH 4.8 malic acid/arginine buffer with the polycation and the pH 8.5 Tris/boric acid running buffer containing the polyanion.…”

“…CZE‐based assays for CDT are primarily used to monitor CDT in clinical and forensic laboratories as an aid to investigate whether a person is consuming high amounts of alcohol over an extended period. Furthermore, CZE was employed to assess the performance of asialo‐Tf in detecting alcohol abuse , to estimate the apparent half‐lives of asialo‐Tf and disialo‐Tf by Tf isoform monitoring of a patient with a relapse drinking episode , to establish that disialo‐Tf is an accurate marker for alcoholic cause of acute pancreatitis , to find that primary biliary cirrhosis is not a clinical condition for increased CDT , to suggest that a distinctly incomplete separation between disialo‐Tf and trisialo‐Tf could be related to alcohol‐induced liver cirrhosis , to conclude that disialo‐Tf could be increased during sepsis , to find a high prevalence of trisialo‐Tf in patients with anorexia nervosa , to determine CDT in blood microsamples of newborns in the context of the search for glycosylation defects , to correctly measure CDT in sera of argininosuccinate lyase deficiency patients , to identify young heavy drinkers during the army recruitment process of 19‐year‐old Swiss males , to confirm the high prevalence of chronic alcohol abusers among drunken drivers and the usefulness of CDT as a predictor of the risk of driving under the influence of alcohol , to assess the use of CDT as a marker for driver's license renewal or regranting , to find a high incidence of chronic alcohol abusers in blood specimens collected from drivers that contained medicinal and abused drugs , to assess whether trisialo‐Tf can be used as an additional biomarker to improve detection of chronic excessive alcohol intake , to report the first case of disialo‐trisialo‐Tf bridging associated with alcoholic liver cirrhosis in Asia , to assess CDT in the Han Chinese population , to characterize Tf isoform patterns in pancreatic cancer and in liver diseases , and to determine CDT by use of finger‐prick dried blood spots which provided electropherograms similar to those obtained from serum .…”

Monitoring of alcohol markers by capillary electrophoresis

“…dilution of the serum with an Fe (III) containing reagent, CZE‐based CDT assays do not require any sample preparation. Routine methods with their cut‐off values are listed in Table , whereas comparative CDT data determined with CZE and other assays are summarized in Table . CDT routine analyses by CZE are performed on (i) the well distributed automated multicapillary platforms Capillarys 1, Capillarys 2, and MINICAP (that feature eight, eight, and two capillaries, respectively) of Sebia (Evry, France), and (ii) single capillary P/ACE instruments of Beckman Coulter (models 2200, 5500, MDQ, PA‐800; Fullerton, CA, USA) using the CEofix CDT reagents of Analis (Suarlée, Belgium) or homemade reagents (Verona assay).…”

“…CZE‐based assays for CDT are primarily used to monitor CDT in clinical and forensic laboratories as an aid to investigate whether a person is consuming moderate‐to‐high amounts of alcohol over an extended period. Furthermore, CZE was employed to assess the performance of asialo‐Tf in detecting alcohol abuse , to estimate the apparent half‐lives of asialo‐Tf and disialo‐Tf via Tf isoform monitoring of a patient with a relapse drinking episode , to establish that disialo‐Tf is an accurate marker for alcoholic cause of acute pancreatitis , to find that primary biliary cirrhosis is not a clinical condition for increased CDT , to suggest that a distinctly incomplete separation between disialo‐Tf and trisialo‐Tf could be related to alcohol‐induced liver cirrhosis , to conclude that disialo‐Tf could be increased during sepsis , to find a high prevalence of trisialo‐Tf in patients with anorexia nervosa , to determine CDT in blood microsamples of newborns in the context of the search for glycosylation defects , to correctly measure CDT in sera of argininosuccinate lyase deficiency patients , to identify young heavy drinkers during the army recruitment process of 19‐year‐old Swiss males , to confirm the high prevalence of chronic alcohol abusers among drunken drivers and the usefulness of CDT as a predictor of the risk of driving under the influence of alcohol , to assess the use of CDT as marker for driver's license renewal or regranting , and to find a high incidence of chronic alcohol abusers in blood specimens collected from drivers that contained medicinal and abused drugs .…”