Determination of the components in erythrocytes using an automated analyzer.

Determination of the biochemical components in erythrocytes should provide unique pathophysiological information. We optimized a simple alcohol binding method for the selective removal of hemoglobins from hemolysates, and enabled simultaneous determination of several components in erythrocytes using commercially available assay kits in an automated analyzer. Venous blood was collected in a vacutainer containing lithium heparin. The washed cells were hemolyzed with distilled water, frozen, and then thawed. Nine volumes of the hemolysates were mixed with one volume of Tris-HCl buffer. One volume of n-butanol was then added to nine volumes of the buffered hemolysates. After vigorous mixing, the mixture of n-butanol and hemolysates was left to stand. The butanol-bound hemoglobins were precipitated by centrifugation, and the clear supernatant below the butanol layer was applied directly to an automated analyzer. Using sera, we determined the effects of the hemoglobin removal procedures on the chemical analytes. Sufficient recovery was noted in most analytes, except for several enzyme activities and lipids. Accordingly, we determined five components present in erythrocytes: creatine, potassium, magnesium, and aspartate aminotransferase as well as superoxide dismutase activities in healthy subjects. We suggest that our simple method is applicable to the simultaneous determination of erythrocyte components in routine laboratory tests.

Enhanced GLUT2 gene expression in an oleic acid-induced in vitro fatty liver model.

Previous reports have shown that the liver-type glucose transporter GLUT2 gene expression is upregulated in the liver of obese and diabetic animals and in human subjects. This phenomenon is correlated with an increased glucose output from the liver; however, the regulatory mechanism is not clear. To investigate the relationship between hepatic steatosis, frequently found in obese and diabetic patients, and GLUT2 gene expression, we developed an oleic acid-induced in vitro fatty liver model. Lipid-accumulated cells morphologically mimicking hepatic steatosis were successfully induced in the human HepG2 cell line by 24-h culture with oleic acid at 1 mM. The cells with steatosis showed increased levels of intracellular triglycerides and apolipoprotein B, which were reduced in the presence of bezafibrate at 100 &mgr;g/ml. GLUT2 mRNA expression estimated by semi-quantitative reverse transcription polymerase chain reaction was increased in the cells with steatosis. Bezafibrate inhibited GLUT2 mRNA expression in the cells with and without steatosis. These results suggest that hepatic steatosis causes the enhancement of hepatic GLUT2 mRNA expression, which may be associated with gluconeogenesis and insulin resistance.