Multiple forms of alkaline phosphatase in horse bone, liver, kidney, duodenum, caecum and serum separated by agarose gel electrophoresis with and without neuraminidase pretreatment.

Horse bone, liver, duodenum, caecum and kidney alkaline phosphatases were separated by a commercial agarose gel electrophoresis method with and without neuraminidase pretreatment, following the manufacturer's directions. Tissue extracts were obtained in saline solution and ALP extracted from cell membranes by the butanol method. Electrophoresis was performed using a TRIS/barbital/sodium barbital buffer with detergent, pH 8.6 to 9.0, at 250 V for 30 minutes. Bone, liver and kidney untreated extracts showed two ALP bands each, but with different relative migration (compared to albumin migration). When they were preincubated with neuraminidase, the two bone bands showed a marked decrease in their migration, followed by the kidney ALP bands and the most anodic band of liver Both intestinal untreated extracts showed three bands but with different mobilities. After preincubation with neuraminidase, the three bands of caecum mucosa decreased in their migration, and the most anodic duodenum band disappeared, overlapping the second one. When tissue extracts were incubated with wheat germ-lectin (WGL), 74.5% of bone extract ALP and 67.2% of caecum extract ALP precipitated, which demonstrated that the ALP band of both tissues have similar groups in the carbohydrate side chains. Horse serum showed two electrophoretic bands, which increased to three bands when treated with neuraminidase. ALP from hepatocytes was the dominant isoform, followed by a caecum band. Because the electrophoretic mobilities of some of the tissue bands studied were identical, the neuraminidase agarose electrophoretic method appeared to be a satisfactory alternative to separate them.

Bile acid fractionations by high-performance liquid chromatography in equine liver disease.

Serum bile acids were fractionated by high-performance liquid chromatography (HPLC) in 13 control and 8 cases of liver disease in horses. The severity and type of liver injury was determined by histopathological examination of biopsy and/or necropsy specimens. The total serum bile acids (tSBA) were determined in these horses by an enzymatic method (SBA-EA) and by summation of the bile acids (SBA-LC) as fractionated by the HPLC. The SBA-LC were generally higher than the SBA-EA in both the controls and liver disease and they did not parallel each other. The primary bile acids, total cholates and total chenodeoxycholates accounted for most of the tSBA increases in liver disease. There was a shift in profile from taurocholate to free (unconjugated) cholate in direct relation to the severity of the liver injury. Among the secondary bile acids, total deoxycholates and total taurodeoxycholates increased at random. The pattern of the SBA profile in relation to the severity of the liver disease suggested that hepatocellular excretion is the most sensitive step in the enterohepatic circulation of the bile acids.