Inhibin-like activity in plasma from ovariectomized ewes and serum albumin: pitfalls for radioimmunoassay.
Progress to understand mechanisms that regulate inhibin secretion and action in farm animals has been handicapped by the shortage of simple, accurate assay methods to quantify inhibin in circulation. RIA would seem to provide the needed quantitative capability, but results of the following studies using inhibin RIA procedures reveal reasons to interpret inhibin immunological potency estimates with caution. Two sets of inhibin RIA reagents and various assay buffers were used. Initially, inhibin immunoactivity was estimated with an antiserum to a 32 amino acid peptide fragment from the alpha subunit of porcine inhibin [pI alpha(1-32)] and tracer to the peptide with tyrosine added in position 0 to permit radioiodination, pI alpha(Tyr1-32). Later, an antiserum to pI alpha(1-29Tyr30) peptide and pI alpha(1-29Tyr30) tracer was evaluated as were several combinations of assay buffer and assay conditions. Both sets of assay reagents provided quantitative recovery of pI alpha(1-32) peptide from plasma, parallel response between the peptide and either ovine or bovine plasma, as well as adequate sensitivity to measure inhibin immunoactivity in 25 microliters of plasma. However, plasma from long-term ovariectomized female sheep, swine or cattle appeared to contain nearly as much inhibin immunoactivity as intact animals. To explore the possibility that the adrenals may produce sufficient inhibin to account for unexplained high levels of inhibin immunoactivity in plasma from ovariectomized animals, ewes on days 12 and 13 of the estrous cycle were injected with either corn oil (CONT) or large doses of an adrenal steroid agonist, dexamethasone (DEX), to alter adrenal function. Likewise, ewes were either ovariectomized (OVX) on day 12 or injected on days 12 and 13 with estradiol-17 beta plus progesterone (E2 + P4) to alter ovarian function. The plasma concentration of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) increased following ovariectomy (P less than .001), and LH decreased following ovarian steroids (P less than .001). Treatment with DEX did not change plasma gonadotropin values (P greater than .1). When plasma was assayed using pI alpha(1-32) reagents and an assay buffer consisting of gelatin/phosphate/Tween-20 (GelT20), inhibin immunoactivity was not affected by any of the four treatments (P greater than .1), even including ovariectomy. Re-assay of these same samples with an RIA procedure that used gelT20 assay buffer and pI alpha(1-29Tyr30) reagents produced good agreement with the previous assay (partial correlation P less than .0001), but there was no statistical evidence that ovariectomy or treatment with ovarian or adrenal steroids changed the level of immunoassayable inhibin in plasma.(ABSTRACT TRUNCATED AT 400 WORDS)
Bolt
D J
DJ
Reproduction Laboratory, U.S. Department of Agriculture, Beltsville, Maryland 20705.
Caldwell
D W
DW
D000818:Animals; D004305:Dose-Response Relationship, Drug; D005260:Female; D005640:Follicle Stimulating Hormone / Q000097:blood; D007265:Inhibins / Q000097:blood*; D007986:Luteinizing Hormone / Q000097:blood; D010052:Ovariectomy / Q000662:veterinary*; D011485:Protein Binding; D011863:Radioimmunoassay; D012709:Serum Albumin / Q000378:metabolism*; D012756:Sheep / Q000097:blood*
D012709:Serum Albumin; D007265:Inhibins; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone