血中proapo A-Iの酵素免疫測定法の開発および肝硬変患者における測定

Abstract

Human plasma apolipoprotein A-I (apo A-I), the major protein component of high density lipoprotein (HDL), is a single polypeptide that consists of 243 amino acids and has a molecular weight of about 28, 000. It is secreted from the cells of the intestine and liver as proapo A-I which contains a six amino acid prosegment: Arg-His-Phe-Trp-Gln-Gln.We describe a specific enzyme immunoassay (EIA) for quantification of plasma proapo A-I levels using monospecific antibodies raised against the synthetic peptide, copying a nine amino acid N-terminus of proapo A-I: Arg-His-Phe-Trp-Gln-Gln-Asp-Glu-Pro. The assay is a non-competitive sandwich EIA in which oligoclonal anti-proapo A-I antibodies are immobilized on the surface of polystyrene beads and other polyclonal anti-apo A-I antibodies are used as horseradish peroxidase conjugated antibodies. The working range of the assay was 10 to 500ng/ml of proapo A-I. Intraassay and interassay coefficients of variation were 5.8% and 7.0%, respectively. A delipidation step using diisopropylether-butanol was necessary to expose the antigen sites of proapo A-I in native lipoproteins.In this assay, we determined plasma proapo A-I levels and the proapo A-I ratio (proapo A-I/total apo A-I) of patients with liver cirrhosis and chronic hepatitis. The mean levels of proapo A-I in 6 patients with decompensatory liver cirrhosis, in 17 with compensatory liver cirrhosis and in 9 with chronic hepatitis were higher than in 21 normal subjects (8.8±2.5, 10.5±3.6, 9.3±2.0, 6.9±2.5mg/dl, respectively). Furthermore, the mean proapo A-I ratio of each liver disease was higher than that in normal subjects (10.0±3.5, 10.2±3.9, 6.6±0.8, 4.6±1.6%, respectively). Proapo A-I levels indicated a positive correlation to HDL2-C levels (r=0.736), though they showed no correlation to biochemical indicators of residual hepatic functions, apo A-I and HDL3-C levels. Proapo A-I ratios correlated positively with ICG-R15 and HDL2-C (r=0.544, r=0.508, respectively), and inversely with serum albumin, ChE, L-CAT and HDL3-C (r=-0.456, r=-0.402, r=-0.395, r=-0.609, respectively).We next measured proapo A-I converting enzyme activity in 10 normal subjects and 10 patients with liver cirrhosis. The activities were calculated on the basis of changes of serum proapo A-I levels during incubating for 6 hours at 37°C. Activities in patients with liver cirrhosis (6.2±3.0×10-2nmol ml/hr) were significantly lower than those in normal subjects (17.2±5.5×10-2nmol/ml/hr).These results indicated that the elevations of plasma proapo A-I levels and ratios in patients with liver cirrhosis are caused by a decline of proapo A-I converting enzyme, which may be produced in the liver. The strong relationships of proapo A-I levels and proapo A-I ratios to HDL2-, HDL3-C levels indicated that proapo A-I may play an important role in the maturation and catabolism of HDL.