Quantitative determination of natriuretic peptides in human biological samples with a bioassay using cultured cells.
Up to now, members of the natriuretic peptide family have usually been determined by radioimmunoassays using antibodies more or less specific for the distinct peptides so far identified. However, natriuretic peptides differing significantly in their amino acid sequence from the one against which the antibody has been raised cannot be determined by this means, and still unknown natriuretic peptides cannot be detected. We therefore developed a new bioassay system sensitive to all members of the natriuretic peptide family by taking advantage of the biological activity of these hormones, the activation of the guanylate cyclase/cyclic GMP system. In this assay, cultured cells are incubated with the natriuretic peptides, and the amount of cyclic GMP produced by the cells is determined by radioimmunoassay. From the relative stimulation of the cellular cyclic GMP production, the concentration of natriuretic peptides in the sample is determined after calibration with synthetic atrial natriuretic peptide. For a qualitative identification of the various peptides, the bioassay is combined with a reversed-phase HPLC step. Using cultured bovine aortic endothelial or bovine kidney epithelial cells for the bioassay, we achieved detection limits of 1 fmol or 50 fmol, respectively, for human atrial as well as brain natriuretic peptide. Intra-assay coefficients of variation of 4.3% (aortic endothelial cells, at 0.65 nmol/l peptide) and 5.8% (kidney epithelial cells, at 6.5 nmol/l peptide) were obtained. The total content of natriuretic peptides as well as the amounts of the individual natriuretic peptides following HPLC separation were determined in extracts of human atria obtained at aortocoronary bypass operations.(ABSTRACT TRUNCATED AT 250 WORDS)
Abt. für Klinische Chemie, Robert-Bosch-Krankenhaus Stuttgart, Bundesrepublik Deutschland.
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Kohse
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Feifel
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Wisser
D001011:Aorta; D009320:Atrial Natriuretic Factor / Q000032:analysis* / Q000494:pharmacology; D001681:Biological Assay; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D006152:Cyclic GMP / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006162:Guanylate Cyclase / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry*; D020097:Natriuretic Peptide, Brain; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000494:pharmacology; D011863:Radioimmunoassay
D009419:Nerve Tissue Proteins; D020097:Natriuretic Peptide, Brain; D009320:Atrial Natriuretic Factor; D006162:Guanylate Cyclase; D006152:Cyclic GMP