We have found a hepatotrophic factor in plasma or sera of patients with fulminant hepatic failure and have purified human hepatocyte growth factor from plasma of these patients. In this study we developed an enzyme-linked immunosorbent assay with high specificity and sensitivity for human hepatocyte growth factor in human serum. This assay for serum human hepatocyte growth factor is a sandwich method consisting of three steps. The standard curve for human hepatocyte growth factor appeared to be linear in the range of 0.20 to 12.50 ng purified human hepatocyte growth factor/ml (2.35 to 147 pmol/L). The assay took about 4 hr. Serum human hepatocyte growth factor values in patients with fulminant hepatic failure measured by enzyme-linked immunosorbent assay showed a strong positive correlation with that by bioassay using rat hepatocytes in primary culture. The mean value of serum human hepatocyte growth factor for 30 normal subjects was 0.24 +/- 0.12 (S.D.) ng/ml; that for 23 patients with fulminant hepatic failure was 8.06 +/- 1.76 (S.E.M.) ng/ml- greater than 30 times greater than the mean value for normal subjects. Serum human hepatocyte growth factor levels in patients with acute hepatitis, chronic hepatitis and cirrhosis were found to be slightly higher than those in normal subjects, but only the increase in serum human hepatocyte growth factor of acute hepatitis patients was statistically significant. The enzyme-linked immunosorbent assay for serum human hepatocyte growth factor should prove useful for serum human hepatocyte growth factor level measurement in patients with various liver diseases.
Human hepatocyte growth factor has been purified from the plasma of patients with fulminant liver failure, but where this factor is produced in organs or cells of subjects with liver diseases is unknown. Therefore, we used a monoclonal antibody to human hepatocyte growth factor to stain cells in three normal and 29 diseased liver tissues by immunohistochemical techniques. By light microscopy, the immunostained cells seemed to be polymorphonuclear leukocytes because of their segmented nuclei. Some biliary epithelial cells also were stained. Electron microscopy confirmed that the immunostained cells with segmented nuclei were polymorphonuclear leukocytes and that the stained grains were on the membranes of rough endoplasmic reticulum, around specific or azurophilic granules and in the cell sap. Stained grains in the biliary epithelial cells were found sporadically on the inside and outside of the membranes of rough endoplasmic reticulum near the nuclei. Human hepatocyte growth factor is now known to be the same protein as scatter factor and tumor cytotoxic factor, both of which are produced by human fibroblasts in culture, but our results suggest that polymorphonuclear leukocytes in diseased livers are one cellular source of circulating human hepatocyte growth factor. The immunostaining properties of biliary epithelial cells in diseased livers also suggest that the cells produce and secrete human hepatocyte growth factor.
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