Marker Of Chronic Alcohol Abuse Research Articles

Use of Serum Carbohydrate‐Deficient Transferrin Values to Exclude Alcoholic Hepatitis from Non‐Alcoholic Steatohepatitis: A Pilot Study

Since no clinical or biochemical parameters allow an accurate diagnosis of non-alcoholic steatohepatitis (NASH), the diagnosis by exclusion of alcoholic hepatitis is necessary. However, it is difficult to get the accurate amount of alcohol consumed from the patients, especially from females. To differentiate between NASH and alcoholic hepatitis, we investigated whether serum biochemical markers of chronic alcohol abuse are useful or not. Sera were obtained from 13 patients with NASH and 26 patients with alcoholic hepatitis. Diagnoses in these patients were confirmed histologically by needle biopsy of the liver. All patients with alcoholic hepatitis consumed more than 80 g of ethanol/day for more than 10 years. As markers of chronic alcohol abuse, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), hyaluronate, mean corpuscular volume of red blood cells (MCV) and carbohydrate-deficient transferrin (CDT) were measured. Among alcohol markers, serum values of AST, AST/ALT ratio, GGT, CDT and MCV in patients with alcoholic hepatitis were significantly higher than those in patients with NASH, respectively. However, serum values of these markers, except for CDT, were overlapped in many cases of NASH and alcoholic hepatitis. Serum CDT values of all patients with NASH were lower than the cutoff value, 2.66%, and those of all patients with alcoholic hepatitis were higher than the cutoff value. The results of the present study suggest that serum CDT level could be used to differentiate between NASH and alcoholic hepatitis.

The diagnostic accuracy of carbohydrate-deficient transferrin, sialic acid and commonly used markers of alcohol abuse during abstinence

Background The normalization of alcohol abuse markers during the abstinence depends on the time since the last drinking. The aim of this study was to evaluate the diagnostic accuracy (area under the curve—AUC, sensitivity and specificity) of CDT, sialic acid and others biochemical and hematological markers of chronic alcohol abuse during abstinence. Methods We studied 75 patients admitted to the treatment of alcohol dependence. The blood samples were collected upon admission to the hospital. CDT was estimated using an immunoturbidimetric assay after anion-exchange chromatography and sialic acid by enzymatic colorimetric method. Results Mean values of all markers were significantly higher. All tests, except SA, negatively correlated with time of abstinence but not with age, duration of dependence and amounts of weekly alcohol consumption. The area under the curve (AUC) for all tested markers decreased progressively during the abstinence. The highest AUC was obtained for CDT (0.98) and the lowest for ALT (0.78) when alcohol was consumed in the last week. AUC for sialic acid was lower than of CDT but higher than of ALT. Conclusions We conclude that the diagnostic accuracy for tested laboratory markers depends on the self-reported time of abstinence being the highest for CDT in the first week of abstinence. The accuracy of sialic acid was observed between GGT and ALT.

A fatal clomipramine intoxication case of a chronic alcoholic patient: Application of postmortem hair analysis method of clomipramine and ethyl glucuronide using LC/APCI/MS

Toxicological investigations of postmortem specimens of a 26-year-old man were performed with the use of LC/APCI/MS. They revealed in the blood of the deceased clomipramine (9.49 μg/g) and its main metabolite norclomipramine (1.10 μg/g) at concentrations explaining the fatal outcome. The presence of these xenobiotics in a 12-cm-long strand of hair (clomipramine, 7.60 ng/mg in I segment; 4.19 ng/mg in II segment; 1.86 ng/mg in III segment; norclomipramine, 5.71 ng/mg in I segment; 9.71 ng/mg in II segment; 4.13 ng/mg in III segment) confirmed the fact obtained from the medical history that the deceased had been receiving clomipramine as an antidepressant for 1 year prior to his death. The analysis demonstrated ethanol in autopsy blood (2.5 mg/ml) and urine (3.2 mg/ml); ethyl glucuronide as a marker of chronic alcohol abuse was detected in the deceased's hair (0.44 ng/mg in I segment; 0.07 ng/mg in II segment; n.d. in III segment). These findings may suggest the contribution of alcohol in the mechanism of drug–ethanol interaction, which in consequence might have affected the biotransformation of clomipramine in the final period of his life and evoked the ultimate toxic effect.

Alanine aminopeptidase (AAP) activity in urine: A new marker of chronic alcohol abuse?

Alanine aminopeptidase (AAP) activity in urine: A new marker of chronic alcohol abuse?

Alanine Aminopeptidase Activity in Urine: A New Marker of Chronic Alcohol Abuse?

The purpose of this study was to evaluate the activity of urinary alanine aminopeptidase (AAP), the enzyme released from the brush border membranes of renal proximal tubules, as a new biological marker of chronic alcohol abuse. The AAP activity was assayed and compared between a group of 76 alcoholics undergoing detoxification and a group of 37 alcoholics abstaining from alcohol for at least 6 weeks. In all patients, the enzyme activity was measured both in untreated urine (uAAP) and after removal of endogenous AAP inhibitors by molecular filtration on Sephadex (eAAP). There was a correlation between the uAAP and eAAP activities in both groups of patients (r = 0.61 and r = 0.81 in abstinent alcoholics and in alcoholics undergoing detoxification, respectively), and both the uAAP activity and the eAAP activity were significantly and markedly higher in alcoholics being detoxified than in their abstinent counterparts. As revealed by receiver operating characteristic analysis, the discriminative power of the eAAP activity assay was higher than that of the uAAP activity assay. The area under the corresponding receiver operating characteristic curves was 0.84 +/- 0.04 and 0.78 +/- 0.05 (mean +/- SE), respectively. The results of this study demonstrate that the assays of urinary AAP activity, which relate to the nephrotoxic effects of alcohol abuse, could be a valuable complement to the other presently used markers of chronic alcohol abuse that are generally based on ethanol hepatotoxicity. Compared with the uAAP activity test, the eAAP activity test is of clear diagnostic advantage.

Alcohol Abuse and Carbohydrate-deficient Transferrin Analysis: Are Screening and Confirmatory Analysis Required?

Carbohydrate-deficient transferrin (CDT; asialo- plus monosialo- plus disialo-Fe2-transferrin) is currently the most specific laboratory marker of chronic alcohol abuse (1)(2). Because of the high prevalence of chronic alcohol abuse in many countries, CDT plays an important role in the areas of employment, traffic safety, and forensic medicine. The potentially strong social impact of an increased CDT value justifies the need for maximally reliable preanalysis, analysis, and interpretation of CDT. There is a highly developed quality-assurance system in toxicologic and forensic drug analysis, including standards and calibration materials, internal quality-control materials, external quality-control programs, and a system of screening and confirmatory analyses (3)(4)(5). Although CDT quality-control materials and surveys have become available in recent years and have greatly contributed to the improvement of CDT analysis, an international CDT standard is still lacking. Furthermore, a system for CDT analysis comprising screening and confirmatory analysis, analogous to forensic analysis of cannabinoids, heroin, and cocaine, has not been established. This is surprising because the dependency potential (addictiveness) of ethanol is reported to be comparable to that of cocaine and heroin (6)(7) and much higher than that of cannabis (6)(7). Many state laws regulate medico-legal diagnosis of “acute alcohol abuse”, demanding ethanol analysis by two independent analysis methods: an enzymatic NADH method and headspace gas chromatography (8). In contrast to this, the forensic laboratory diagnosis of “chronic alcohol abuse” by measurement of CDT is essentially not regulated and is usually based on only one (immunologic) analytical method. Here we summarize some arguments in favor of confirmatory methods for CDT. CDT shows an overall diagnostic specificity that is distinctly higher when compared with other markers of chronic alcohol abuse, e.g., γ-glutamyl transferase and mean corpuscular volume of erythrocytes (1)(2). In their systematic …

Characterization of human transferrin glycoforms by capillary electrophoresis and electrospray ionization mass spectrometry

Carbohydrate Deficient Glycoprotein Syndrome (CDGS) is an inherited metabolic disease affecting all parts of the body. The biochemical diagnosis of this syndrome is based on the presence of a special marker in blood, Carbohydrate Deficient Transferrin (CDT), which is also a marker of chronic alcohol abuse. CDT is characterized by abnormal glycoforms of serum transferrin (Tf). In the present study, electrophoretic separation of human serum transferrin glycoforms was carried out using a bare fused-silica capillary and the glycoforms present in commercial Tf were baseline separated. The limit of detection (LOD) of human Tf was around the nmol concentration range. The LOD of the trisialo- and disialo-Tf, expressed as percentages of the tetrasialo-Tf peak area, were 0.5% for trisialo-Tf and 0.4% for disialo-Tf, and these values were appropriate for CDGS diagnosis. Moreover, Tf glycoforms were characterized using mass spectrometry (MS). The method was applied to the analysis of normal and pathological serum samples, after dilution. The results obtained suggest a way of making a rapid and simple CDGS diagnosis.

Determination of CDT, a marker of chronic alcohol abuse, for driving license issuing: immunoassay versus capillary electrophoresis

The present work is aimed at a validation of the carbohydrate-deficient transferrin (CDT) determination in real cases by comparison between the a commercial immunometric method and a method based on capillary electrophoresis. Overall, 650 serum samples from subjects applying to re-obtain their driving license, previously withdrawn for “drunk driving”, were investigated. A highly significant correlation (P<0.001) was found between the results from immunoassay and capillary electrophoresis. However, particularly in the samples with CDT values around the cut-off or moderately elevated, a wide dispersion of the correlation data was found. This finding stresses the need to confirm by alternative techniques all the results from CDT immunoassays. For this purpose, capillary electrophoresis, because of its inherent characteristics of high selectivity, easy operation, high productivity and low operative costs looks well-suited for becoming the method of choice.

Caveats in Carbohydrate-deficient Transferrin Determination

Carbohydrate-deficient transferrin (CDT) refers to a group of minor isoforms of transferrin that, according to most authors, includes asialo-, monosialo-, and disialo-Fe2-transferrin (1). CDT is a widely used marker of chronic alcohol abuse in Europe and, after its recent approval by the Food and Drug Administration, a candidate to become a popular diagnostic tool in the US (2). Arndt (1) recently concluded that “CDT is the most specific marker of chronic alcohol abuse to date”. Its specificity, however, is affected by the choice of analytical method, which must selectively determine only the CDT components, without interference from the other transferrin glycoforms (trisialo-, tetrasialo-, pentasialo-, and hexasialo-Fe2-transferrin), which are present in large excess. Interference from trisialo-Fe2-transferrin has been reported for most commercial CDT tests, which are based on anion-exchange microchromatographic separations followed by immunoassays. Unfortunately, much of the literature does not report any correlation between trisialo-transferrin and alcohol intake (3). Moreover, Dibbelt (4) recently demonstrated by HPLC that increased relative concentrations of disialo- and asialo-transferrin associated with alcohol abuse are not correlated with increased trisialo-transferrin concentrations and consequently stated that “trisialo-transferrin is obviously of no diagnostic value, I strongly recommend not including … [↵][1]bAuthor for correspondence. Fax 32-9-240-49-85; e-mail joris.delanghe{at}rug.ac.be. [1]: #xref-corresp-2-1

The Activity of β-Hexosaminidase (uHex) and y-Glutamyi-transferase (uGGT) in Urine as Non-Invasive Markers of Chronic Alcohol Abuse: II. Opiate-Dependent Subjects Receiving Methadone Substitution

Summary: The usefulness of β-hexosaminidase (uHex) and γ glutamyltransferase (uGGT) activity in urine as markers of chronic alcohol abuse was examined in male methadone programme participants. The group consisted of 24 non-abusing and 21 alcohol-abusing men. It has been demonstrated that the uHex test is one of the best alcohol abuse markers for opiate-dependent individuals (Receiver Operating Characteristic (ROC) area under the curve (AUC) 0.82, sensitivity 0.70, specificity 0.82). The uGGT test can be used as a marker of alcohol abuse in opiate-dependent men, however, its parameters are worse than those of uHex (ROC AUC 0.73, sensitivity 0.71, specificity 0.77). Among serum markers, only the relative amount of carbohydrate-deficient transferrin (s%CDT) has parameters sufficient for diagnostic use (ROC AUC 0.74, sensitivity 0.60, specificity 0.86). Factors other than alcohol can render the commonly used sGGT and sHex tests useless (differences between means for abusing and abstinent patients were insignificant, AUC ROC were lower than 0.7-0.65 and 0.69, respectively).Their good parameters, low costs, ease of use and non-invasive character are all features which make uHex and uGGT highly useful tools in the detection of chronic alcohol abuse in opiate-dependent patients participating in a methadone substitution programme.

Comparison of HPLC and Small Column (CDTect) Methods for Disialotransferrin

Current methods for determination of carbohydrate-deficient transferrin (CDT) are based on separation of the CDT fraction by ion-exchange chromatography on minicolumns and quantification by immunoassay. Alternatively, the transferrin isoforms can be separated by HPLC anion-exchange chromatography and quantified by absorbance. This method has been reported to improve the validity of CDT as a marker of chronic alcohol abuse. HPLC on either MonoQ or ResourceQ anion-exchange columns was used to separate and quantify isoforms of transferrin with detection at 460 nm. The result was expressed as the percentage of the disialo form (pI 5.7) of total transferrin (DST). The commercial CDTect assay was used as a comparison method. Serum samples from nondrinkers (n = 57), moderate drinkers (n = 77), and heavy drinkers (n = 139) were analyzed. In ROC analysis for differentiation between moderate and heavy drinkers, the area under the curve (AUC) for the HPLC method was 0.87 (95% confidence interval, 0.81-0.93), whereas that for CDTect was 0.72 (95% confidence interval, 0.64-0.80). At 90% specificity, the sensitivity of DST was 63% (95% confidence interval, 53-73%) compared with 33% (22-44%) for CDT. The reference interval of the HPLC method was 0.68-1.7%. The HPLC anion-exchange method for quantification of CDT provides substantially better separation between moderate and heavy drinkers than the CDTect method.

Carbohydrate-deficient Transferrin as a Marker of Chronic Alcohol Abuse: A Critical Review of Preanalysis, Analysis, and Interpretation

Carbohydrate-deficient transferrin (CDT) is used for diagnosis of chronic alcohol abuse. Some 200-300 reports on CDT have been published in impact factor-listed journals. The aims of this review were to condense the current knowledge and to resolve remaining issues on CDT. The literature (1976-2000) was searched using MEDLINE and Knowledge Server with "alcohol and CDT" as the search items. The data were reviewed systematically, checked for redundancy, and organized in sequence based on the steps involved in CDT analysis. The review is divided into sections based on microheterogeneity of human serum transferrin (Tf), definition of CDT, structure of human serum CDT, pathomechanisms of ethanol-induced CDT increase, preanalysis, analysis, and medical interpretation (postanalysis). Test-specific cutoff values for serum CDT and causes of false positives and negatives for chronic alcohol abuse are discussed and summarized. Asialo- and disialo-Fe(2)-Tf, which lack one or two complete N-glycans, and monosialo-Fe(2)-Tf (structure remains unclear) are collectively referred to as CDT. Diminished mRNA concentration and glycoprotein glycosyltransferase activities involved in Tf N-glycan synthesis and increased sialidase activity most likely account for alcohol-induced increases in CDT. Knowledge about in vivo and in vitro effects on serum CDT is poor. Reliable CDT and non-CDT fractionation is needed for CDT measurement. Analysis methods with different analytical specificities and recoveries decreased the comparability of values and statistical parameters of the diagnostic efficiency of CDT. CDT is the most specific marker of chronic alcohol abuse to date. Efforts should concentrate on the pathomechanisms (in vivo), preanalysis, and standardization of CDT analysis.

The Activity of β-Hexosaminidase (uHex) and γ-Glutamyl-Transferase (uGGT) in Urine as Non-Invasive Markers of Chronic Alcohol Abuse: I. Alcohol-Dependent Subjects

It has been demonstrated that in alcohol-dependent men the uHex test is one of the most powerful discriminating tools, while uGGT has a discriminating power similar to that of sHex but worse than that of uHex, sGGT and s%CDT. Receiver Operating Characteristic (ROC) areas under the curves (AUC) for uHex, uGGT, s%CDT, sHex and sGGT were 0.92, 0.79, 0.88, 0.79 and 0.92, respectively. Due to their good parameters, low cost, ease of use and noninvasive character the uHex and uGGT tests are useful tools for the detection of chronic alcohol abuse.

Investigation by Isoelectric Focusing of the Initial Carbohydrate-deficient Transferrin (CDT) and non-CDT Transferrin Isoform Fractionation Step Involved in Determination of CDT by the ChronAlcoI.D. Assay

The introduction of a new set of reagents for the determination of carbohydrate-deficient transferrin (CDT) as a marker of chronic alcohol abuse requires an independent evaluation of the analytic specificity of the test. This information is needed for correct interpretation and classification of test results. Isoelectric focusing on the PhastSystem(TM) followed by immunofixation, silver staining, and densitometry was used to validate the initial transferrin isoform fractionation step on anion-exchange microcolumns involved in the ChronAlcoI.D. assay. The in vitro transferrin iron load was complete and stable. The CDT and non-CDT transferrin fractionation on anion-exchange microcolumns was reliable and reproducible (CV < or = 10%). Except for quantitatively unimportant traces of trisialo-Fe(2)-transferrin (<5% of total CDT), only asialo-, mono-, and disialo-Fe(2)-transferrin were detected in the microcolumn eluates (n = 170). There was a loss of proportionally similar amounts of asialo-Fe(2)-transferrin (during column rinsing) and disialo-Fe(2)-transferrin (on the anion exchanger). Thus, the peak height ratios for disialo- and asialo-Fe(2)-transferrin did not change from >1 (serum) to <1 (eluates) as described for the CDTect assays. The transferrin patterns in the ChronAlcoI.D. eluates were representative of those in serum. Transferrin D variants with isoelectric points close to that of trisialo-Fe(2)-transferrin C1 did not cause overdetermination of CDT by the ChronAlcoI.D. test. The initial CDT and non-CDT fractionation step involved in determination of CDT by the ChronAlcoI.D. assay is efficient for eliminating non-CDT transferrins from serum before quantification of CDT in the final turbidimetric immunoassay. We recommend IEF for validation of other (commercial) CDT analysis methods and of odd CDT results.

Capillary electrophoresis: a new analytical tool for forensic toxicologists.

In capillary electrophoresis, electrophoretic or electrokinetic separations are carried out in tiny capillaries at high voltages (10-30 kV), thus achieving high efficiency (N > 105), resolution power, and mass sensitivity (down to 10(-18)-10(-20) moles). The main characteristics of capillary electrophoresis are versatility of application (from inorganic ions to large DNA fragments), use of different separation modes with different selectivity, low demands on sample volume, negligible running costs, possibility of interfacing with different detection systems including mass spectrometry, and the ruggedness and simplicity of the instrumentation. Capillary electrophoresis applications in the forensic sciences are now rapidly growing, particularly in forensic toxicology. The present paper briefly describes the basic principles of capillary electrophoresis and presents a selected review of its main applications to the analysis of illicit/controlled drugs in biologic samples. An original analytical approach to the determination of carbohydrate deficient transferrin, a new marker of chronic alcohol abuse, based on capillary electrophoresis is also described. It is concluded that the peculiar separation mechanisms and the high complementarity of capillary electrophoresis to chromatography make it a new powerful tool of investigation in the hands of forensic toxicologists.

Review of factors susceptible of influencing post-mortem carbohydrate-deficient transferrin

Seric carbohydrate-deficient transferrin (CDT) is a biochemical marker of chronic alcohol abuse. Assessment of the influence of factors likely to modify CDT concentration is necessary to justify its use in the analysis of post-mortem blood samples. Hemolysis, site of collection and storage were tested. Hemolysed samples showed decreased CDT concentration. Statistical analysis of CDT concentration in cardiac blood versus femoral blood revealed no significant differences. Storage for fifteen days at +4°C or +20°C did not affect CDT concentration but repeated freezing and thawing resulted in decreased levels of CDT.

Carbohydrate-deficient transferrin, a sensitive marker of chronic alcohol abuse, is highly influenced by body iron

Carbohydrate-deficient transferrin (CDT), a microheterogeneous form of serum transferrin (Tf), has been proposed as the most reliable marker of chronic alcohol consumption, although unexplained false-positive and -negative results have been reported. We investigated whether body iron influenced CDT serum levels by studying alcohol abusers with or without iron overload and nonabusers with iron deficiency or iron overload caused by genetic hemochromatosis (GH). In alcohol abusers, CDT was significantly lower in the presence of iron overload than in the absence (24.6 +/- 16.5 U/L vs. 33.3 +/- 11.7 U/L; P <.01), with false-negative results almost exclusively in patients with iron overload. Similarly, in nonabusers with GH, CDT was lower than in normal controls (9.6 +/- 2. 2 U/L vs. 15.7 +/- 3.3 U/L; P <.0001), whereas, patients with iron deficiency anemia had significantly higher levels than controls (28. 1 +/- 5.8 U/L vs. 15.7 +/- 3.3 U/L; P <.0001). In nonabusers, iron supplementation therapy significantly decreased CDT levels in patients with iron deficiency anemia (33.7 +/- 6.6 U/L vs. 21.7 +/- 5.2 U/L; P =.0007), while iron-depletion treatment significantly increased CDT levels in patients with GH (9.7 +/- 2.0 U/L vs. 14.7 +/- 4.0 U/L; P =.001). Alcohol abusers had a significant relationship between liver iron concentration (LIC) and the reciprocal of CDT (r =.65; P <.0001), while in nonabusers, there was a significant correlation between Tf and CDT (r =.72; P <.0001). In conclusion, CDT serum levels are markedly affected by the patient's iron status, with iron overload reducing its sensitivity in alcohol abusers and iron deficiency its specificity in nonabusers. CDT can be considered a reliable marker of alcohol abuse only when iron stores are normal.

Carbohydrate-deficient transferrin, a sensitive marker of chronic alcohol abuse, is highly influenced by body iron.

Carbohydrate-deficient transferrin (CDT), a microheterogeneous form of serum transferrin (Tf), has been proposed as the most reliable marker of chronic alcohol consumption, although unexplained false-positive and -negative results have been reported. We investigated whether body iron influenced CDT serum levels by studying alcohol abusers with or without iron overload and nonabusers with iron deficiency or iron overload caused by genetic hemochromatosis (GH). In alcohol abusers, CDT was significantly lower in the presence of iron overload than in the absence (24.6 +/- 16.5 U/L vs. 33.3 +/- 11.7 U/L; P <.01), with false-negative results almost exclusively in patients with iron overload. Similarly, in nonabusers with GH, CDT was lower than in normal controls (9.6 +/- 2. 2 U/L vs. 15.7 +/- 3.3 U/L; P <.0001), whereas, patients with iron deficiency anemia had significantly higher levels than controls (28. 1 +/- 5.8 U/L vs. 15.7 +/- 3.3 U/L; P <.0001). In nonabusers, iron supplementation therapy significantly decreased CDT levels in patients with iron deficiency anemia (33.7 +/- 6.6 U/L vs. 21.7 +/- 5.2 U/L; P =.0007), while iron-depletion treatment significantly increased CDT levels in patients with GH (9.7 +/- 2.0 U/L vs. 14.7 +/- 4.0 U/L; P =.001). Alcohol abusers had a significant relationship between liver iron concentration (LIC) and the reciprocal of CDT (r =.65; P <.0001), while in nonabusers, there was a significant correlation between Tf and CDT (r =.72; P <.0001). In conclusion, CDT serum levels are markedly affected by the patient's iron status, with iron overload reducing its sensitivity in alcohol abusers and iron deficiency its specificity in nonabusers. CDT can be considered a reliable marker of alcohol abuse only when iron stores are normal.

Evaluation of the Cut-off for Serum Carbohydrate-deficient Transferrin as a Marker of Chronic Alcohol Abuse Determined by the ChronAIco I.D.™-Assay

Evaluation of the Cut-off for Serum Carbohydrate-deficient Transferrin as a Marker of Chronic Alcohol Abuse Determined by the ChronAIco I.D.™-Assay

Optimized determination of carbohydrate-deficient transferrin isoforms in serum by capillary zone electrophoresis.

Carbohydrate deficient transferrin (CDT) is one of the most reliable markers of chronic alcohol abuse. It consists of a group of minor isoforms of human transferrin (the main iron transport serum protein) deficient in sialic acid groups (asialo, monosialo and disialo) with a pI > 5.7, while the main isotransferrin (tetrasialo) has a pI of 5.4. The aim of the present work was to develop a capillary electrophoretic method to determine CDT in serum, suitable for routine use as a confirmatory technique of the current screening methods based on immunoassays. Serum samples (0.5 mL) were saturated with iron by incubation with 10 mM FeCl3 (9 microL) and 500 mM NaHCO3 (12 microL) for 30 min, then diluted 1/10 in water and injected by positive pressure (0.5 psi for 10 s). Separation was performed with a capillary zone electrophoretic method using bare fused-silica capillaries (20 microm ID, 37 cm in length) and a buffer composed of 100 mM sodium tetraborate adjusted with boric acid to pH 8.3. Applied voltage was 10 kV and temperature 25 degrees C. Detection was by UV absorption at 200 nm wavelength. Under the described conditions, asialo-, monosialo-, disialo-, trisialo- and tetrasialo-transferrin were separated in human serum. The limit of detection (signal-to-noise ratio of 2) was about 0.3% for disialo-transferrin, and 0.4% of trisialo-transferrin, expressed as percentages of the terasialo-transferrin peak area. Relative standard deviations (RSD) of absolute migration times were < 1%, while RSD of relative migration times (on the basis of tetrasialo-transferrin) were < 0.1%. Intra-day and day-to-day peak quantitation precision studies showed RDS ranging from 4 to 9% and from 13 to 24% for disialo- and trisialo-transferrin, respectively. The results from 30 control subjects, including social drinkers, and 13 alcoholics showed disialo- and trisialo-transferrin significantly increased in patients by a factor of about 4.5 (P < 0.0001).