| Objectives Alcohol is the most frequently abused drug throughout the world, leading not only to irreversible physical injuries, neuropsychiatric defects, social problems, and trauma, but also to substantial costs for society. ALD has a incidence of 80% among the alcoholics in the western countries. It is reported to be the second cause of cirrhosis besides the viral hepatitis. Conventional laboratory tests for GGT and MCV have low diagnostic sensitivity for detecting alcohol abuse before the stage of organic complications and are indicators of disease in a particular organ, with poor specificity for different etiological possibilities. Since Stibler's discovery that alcoholics demonstrate an abnormal transferrin electrophoretic pattern in 1976, CDT has been considered the most valuable marker for monitoring chronic alcohol abuse and diagnosis of alcoholism. Transferrin exhibits a microheterogeneityin its iron, amino acid, and carbohydrate content. Variations in iron content can be overcome by saturating samples with iron, and amino acid variations are seen only in rare genetic variants of transferrin. The commonly occurring isoforms of diferric transferrin depend on variations in carbohydrate content. Normal serum contains high concentrations of tetrasialotransferrin (pI 5.4) and low amounts disialo-, trisialo-, and pentasialotransferrin (pI 5.7, 5.6, and 5.2, respectively). In alcoholics, transferrin fractions with di- (pI 5.7), mono- (pI 5.8), and asialylated (pI 5.9) carbohydrate chains are increased. It has been estimated that a minimum alcohol consumption of 50-80 g/day for at least 1 week is needed to increase the serum CDT in 80% of subjects. The half-life of the marker is 14 days .Early methods for CDT used isoelectric focusing (IEF) with laser densitometric quantification after immunoblotting. Recently, commercial methods for easy, time-saving CDT detection have been developed that use anion-exchange chromatography followed by RIA (CDTectTMand %CDT-RIATM), enzyme immunoassay (CDTect-EIATM), or turbidimetric immunoassay (%CDT-TIATM and ChronAlcoI.D.TM). In 1989, capillary IEF was successfully applied to quantitative measurement of CDT. However, this method is not easily applied in a routine clinical environment.Although IEF served as gold standard for the validation of most CDT procedures, this technique was too complex and laborious for routine clinical purposes. Alternative procedures were introduced based on anion-exchange chromatography, chromatofocusing, HPLC and FPLC fast protein liquid chromatography. In this study ,we used anion-exchange chromatographic method for quantification of CDT on a Mono Q HR 5/5 column and try to chase a high diagnostic performance of alcoholism combining CDT and GGT.Methods In this study , samples from 26 alcoholics ,25 male(age 44.9 + 9.6) and 1 female (age 48) ,was collected from the 2nd Hospital of TianJin University. Reference serum samples were collected from 60 healthy subjects (36 males, age 41.1+9.9 years; 24 females, age 50.2+6.4 years) who showed no biochemical or clinical evidence of chronic alcohol abuse. Fresh serum or serum frozen at -22 C for < 6 months was saturated with iron by adding 25ul of NaHCO3(500mmol/L) and 18 ul FeCl3 (10mmol/L) per milliliter of serum .After mixing and storage at 4C overnight ,the lipoprotein were precipitated by adding 10 ul of dextran (100g/L) and 50 ul of CaCl2 (lmol/L) per milliliter of serum .This mixture was stored for 30-60 min at 4C and then centrifuged at 10 OOOg for 10 min . The supernate was diluted fivefold with water and filtered through a 0.22um filter unit,and 100 ul pretreated sample transferred to an HPLC autoinjector. The transferrin isoforms were separated on a Mono Q?HR 5/5 column (by Pharmacia ) by salt gradient elution and the process took 32 min, including regeneration of the column .The flow rate was maintained at 1ml/min ,and the monitor sensitivity range was 0.01 A full scale at the wavelength of 460 nm. GGT was determined by a clinical chemistry analyzer. A SPSS software was used to deal with the da... |
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