Choice of extraction procedure for estimation of anterior pituitary hormone content

Searching for the best procedure for simultaneous estimation of the anterior pituitary hormones, extraction efficiencies of various media, additives such as urea and triton X-100, and physical treatments such as freezing-thawing (F-T) and sonication, were examined by measuring prolactin (PRL), growth hormone (GH), lutropin (LH), follitropin (FSH), and thyrotropin (TSH) in the extracts. Ethanolic media (60% EtOH) gave high yields of PRL at neutral to alkaline pH, but poor extraction of GH accompanied by a marked loss of its immunoreactivity during storage. Ethanolic media also gave a poor yield of LH even at high pH. Aqueous media like PBS at various pH, 0.1 M acetic acid and distilled water were considerably effective in the extraction of GH, LH, FSH and TSH if they were coupled with F-T and sonication. However, high yields of PRL could not be obtained with these aqueous media even with F-T and sonication. Hartree's 40% EtOH-6% ammonium acetate, pH 5.1, solubilized considerable amounts of glycoprotein hormones, but yielded almost no GH and only a small amount of PRL. The addition of triton X-100 to PBS (pH 7) at 0.1% resulted in the maximum extraction of glycoprotein hormones with homogenization and F-T, but further sonication was necessary for GH and PRL. When the anterior pituitaries were homogenized and frozen-thawed in PBS (pH 7) containing 1 M urea, yields of PRL, GH, LH, FSH, and TSH were maximum, and sonication did not cause any additional extraction, indicating that this procedure, i.e. homogenization and F-T in 1 M urea-PBS, would be the best for the simultaneous estimation of these anterior pituitary hormones.