Analysis of the radioimmunoassay for gonadotropin-releasing hormone (GnRH): studies on the effect of radioiodinated GnRH

When GnRH is radioiodinated by the chloramine-T method, two immunoreactive labeled species are formed at pH 6.5 with a chloramine-T:GnRH molar ratio of 11:1, whereas four bands (I, IIa, IIb, and III) are separated by polyacrylamide gel electrophoresis when the hormone is iodinated at pH 7.5 in a system containing a 97:1 molar ratio of chloramine-T:GnRH. Because they were more stable and were more immunoreactive than the other products, band I and band IIa from the latter system were used separately as tracers with Niswender antiserum R-42 in radioimmunoassays for GnRH. The standard curves of each tracer are distinct: when analyzed after log-logit transformation, the band I curve had a mean slope of -3.31 +- 0.2 (SE) and a 50 percent B/B/sub T/ level of 9 +- 0.8 pg (n = 8) of synthetic GnRH, whereas the band IIa standard curve had a slope of -2.30 +- 0.6 and a 50 percent B/B/sub T/ value of 20 +- 0.9 pg (n = 11). As measured in the band IIa assay, hypothalamic extracts from control adult male rats contained 3.1 +- 0.4 ng while hypothalami from castrated rats contained 1.4 +- 0,1 ng. Similar but slightly lower values were obtained with band I. In contrast, the GnRH content of pineal glands from intact and castrated male rats was similar (approximately 150 pg) when determined in either assay. These studies emphasize that: the characteristics of the radioiodinated hormone can influence the quantitation of GnRH; and endogenous plasma concentrations of GnRH are much lower than previously reported