Human hepatocyte growth factor (hHGF) has been purified 209,000-fold with 18% yield from plasma of a patient with fulminant hepatic failure. The purification involves heat treatment of.plasma, ammonium sulfate precipitation, and chromatography on Affi-Gel Blue, heparin-Sepharose, and hydroxylapatite. Purified hHGF shows several bands with molecular weights between 76,000 and 92,000. Each band shows growthstimulating activity on cultured hepatocytes which is proportional to the intensity of the band. After reduction of the sample with 2-mercaptoethanol, SDS-PAGE yields two chains with molecular weights of 31,500-34,500 and 54,000-65,000. The effect of hHGF on DNA synthesis by hepatocytes is halfmaximal at 3.5 ng/ml. hHGF stimulates proliferation of cultured hepatocytes more effectively than human epidermal growth factor (hEGF) or insulin, and the effect of hHGF is additive or synergistic with the maximal effects of hEGF and insulin. These results suggest that hHGF is a new growth factor which is different from hEGF.
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.
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