Radioimmunoassay for Luteinizing Hormone-Releasing Hormone (LHRH): Its Application to the Measurement of LHRH in Ovine and Human Plasma123

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Abstract
A radioimmunoassay for LHRH, MODIFIED FROM THe assay of Nett et al. (J Clin Endocrinol Metab 36: 880, 1973) and characterized in more detail, was applied to the measurement of endogenous plasma LHRH in the peripheral circulation of ewes and women around the time of gonadotropin release. Sensitivity was 1.2 pg/tube. Within- and between-assay coefficients of variation were 10% and 25%, respectively, over the range 10-100 pg/tube. Minimal cross reaction (less than 0.1%) was observed with LHRH analogues tested, except those which had undergone single amino acid alterations in the 4 or 8 positions (62-93%) and in the 1 position (1-3%). Inhibition curves parallel to synthetic LHRH were obtained with these immunoreactive analogs, and with crude hypothalamic and pituitary extracts from 16 cycling ewes and 3 pooled hypothalamic extracts from male rats. LHRH contents of ovine hypothalami and pituitaries ranged from 1.9-10.2 (4.5 plus or minus 2.1, mean plus or minus SD) and 0-27.5 (4.2 plus or minus 7.3, mean plus or minus SD) ng LHRH, respectively, with no obvious correlation between the contents of either tissue. LHRH contents of pooled hypothalami from normal, castrated-hypophysectomized male rats were 4.5, 2.9 and 1. 3 ng/hypothalamus. Recovery of synthetic LHRH added to plasma was quantitative, provided the storage time at 4 and 20 C was minimal. Synthetic LHRH administered to ewes by intravenous infusion or subcutaneous injection was readily detectable in peripheral plasma. The metabolic clearance rates of synthetic LHRH' in 3 sheep were 14, 11 and 14-1/day/kg, respectively. Endogenous immunoreactive material in single, unextracted plasma samples from sheep in various physiological states ranged from 0-400 pg/ml. However, the endogenous plasma immunoreactivity in these samples bone no resemblance to synthetic LHRH added to ovine plasma in that it was stable to gentle heat, and was undetectable after methanol extraction. No endogenous LHRH could be detected in methanol extracts of peripheral plasma obtained from estrous ewes or oophorectomized ewes injected iv with estradiol-17beta (40mug) and bled at 15 min intervals prior to gonadotropin release. Single daily plasma samples obtained from three normal women over their entire menstrual cycles contained no significant levels of endogenous LHRH.

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