A direct immunoradiometric assay for human plasma prorenin: concentrations in cycling women and in women taking oral contraceptives

Autor: A. Nanninga, T Delfs, F A Leidenberger, A K Mukhopadhyay, Matthias Schumacher
Rok vydání: 1992
Předmět:
Zdroj: The Journal of Clinical Endocrinology & Metabolism. 75:617-623
ISSN: 1945-7197
0021-972X
DOI: 10.1210/jcem.75.2.1639960
Popis: Two synthetic penta deca peptides corresponding to the N-terminal portion (amino acid sequence 1-15) and the C-terminal portion (sequence 32-46) of the pro moiety of the human prorenin (PR) molecule were coupled to BSA and used as antigens to generate antibodies against PR. In a RIA system using the 125I-labeled peptide as tracer, it could be shown that antibodies against peptide 32-46 bound the peptide and the native PR in the follicular fluid (FF) to a similar degree, whereas antibodies generated against peptide 1-15 did not specifically recognize native PR. The specificity of the PR-(32-46) antibodies for PR was demonstrated by comparative measurements of PR by RIA and by an indirect procedure (involving trypsin treatment of PR) in different individual FF samples, plasma samples, and fractions of FF obtained by gel filtration or immunoaffinity chromatography. Normal plasma PR levels could not be measured by RIA, since they were below the detection limit of the assay (0.5 micrograms PR/L approximately 10 pmol/L). For measurement of the low PR levels in plasma, a sensitive direct immunoradiometric assay with a detection limit of 0.1 fmol PR/tube was developed. It was based on the combined action of a commercially available solid phase renin antibody and the affinity-purified and 125I-labeled PR-(32-46) antibody. The measurement of 35 individual plasma samples with different PR concentrations showed an excellent correlation (r = 0.99) between the new direct and the conventional indirect assays. The direct assay of PR concentrations in plasma of healthy women during the length of a menstrual cycle resulted in a biphasic pattern of PR concentrations, with peak levels (approximately 3-fold increase) at the time of the LH surge. The intake of monophasic and triphasic contraceptives caused a suppression of normal PR concentrations (96.9 +/- 34 ng/L; early follicular phase; n = 12) by 39% and 25%, respectively, which was also observed in the pill-free phase of the artificial cycle.This paper studies the effects of different oral contraceptives on plasma prorenin (PR) levels in women as determined by a direct immunoradiometric assay (IRMA). 3 healthy and normally cycling women, aged 22, 30, and 37, volunteered for venipuncture every second day of one complete menstrual cycle. 18 healthy women taking oral contraceptives were studied for the effects of oral contraception on plasma PR concentrations. Results of this study showed that in a renin-angiotensin (RIA) system, using the I-labeled peptide, antibodies against peptide 32-46 bound the peptide and the native PR in the follicular fluid (FF) to a similar degree, while antibodies generated against peptide 1-15 did not recognize native PR. The absolute PR levels measured by the direct assay were approximately 20% lower than in the indirect assay, although both assay procedures indicate the same molecular measurement. The measurement of the different PR concentrations of the 35 individual plasma samples indicates an excellent correlation (r = 0.99) between the new direct and the conventional indirect assays. Sequential changes in the plasma PR levels of healthy women demonstrate that PR in the circulation was mainly due to ovarian origin. Lastly, PR concentration was significantly lower at any time during the artificial cycle than in the early follicular phase of spontaneous cycles. In conclusion, this PR IRMA is believed to be superior to the conventional indirect procedures.
Databáze: OpenAIRE
Externí odkaz: