Analysis of the Radioimmunoassay for Gonadotropin-Releasing Hormone (GnRH): Studies on the Effect of Radioiodinated GnRH

Abstract

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% B/Bt 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% B/Bt value of 20 +/- 0.9 pg (n=11). The sensitivity of both assays is approximately 2.0 pg. Gn RH concentrations in plasma and serum samples assayed with band I were consistently greater than those assayed with band IIa. Normal adult male plasmas assayed with band I measured 21 +/- 0.9 pg/ml, whereas band IIa values were 8 +/- 0.4 pg/ml. No difference between plasma and serum was detected, nor was there any difference among adult men, adult women, prepubertal children, hypogonadal patients, or hypopituitary patients with either assay. Plasma GnRH concentrations were also similar in jugular and vena cava samples from intact and castrated male rats. Because many of the samples were at or below the sensitivity of the band IIa assay, they were concentrated after extraction with either methanol or acid-ethanol. However, endogenous immunoreactive GnRH could not be concentrated by these extraction procedures. 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: 1) the characteristics of the radioiodinated hormone can influence the quantitation of GnRH; and 2) endogenous plasma concentrations of GnRH are much lower than previously reported.

Expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in control of GnRH secretion1

“…GnRH was iodinated by chloramine T technique as described [9] . Purification was achieved on a sephadex G25 column, using 0.1 mol/L acetic acid with 0.25% BSA as eluant.…”

Effect of Castration and Steroid Replacement on Immunoreactive Gonadotropin-Releasing Hormone in the Hypothalamus and Preoptic Area*

“…3) A major function of GnRH release is the stimulation of synthesis and release of LH and FSH, which are markedly elevated after castration (26). Additional evidence which could support the above-mentioned concept would be the presence of elevated levels of GnRH in peripheral serum after castration; however, reports of such levels are conflicting and the validity of RIAs of serum GnRH has been seriously questioned (27).…”

Radioimmunoassay of LH-RH. Standard Curves and Specificity

“…As important discrepancies in the blood concentrations of LH-RH remain, with values which are often under lOpg/ml or at the limit of sensitivity, the validity of the procedures of radioimmunological quantification has been questioned (4,6,9,17,26,27). In spite of the high sensitivity attained, the specificity, which could considerably affect the results, varied greatly with the variety of the procedure itself as well as with the nature of the conjugate used to elicit antibodies.…”