Choice of Extraction Procedure for Estimation of Anterior Pituitary Hormone Content.

Transcription

1 Endocrinol. Japon. 1987, 34(5), Choice of Extraction Procedure for Estimation of Anterior Pituitary Hormone Content. JUNKO ISHIKAWA*, YUSUKE FUSE* AND KATSUMI WAKABAYASHI** Common Research Institute, National Defense Medical College* 3-4 Namiki, Tokorozawa 359, Japan Institute of Endocrinology, Gunma University** , Showamachi, Maebashi 371, Japan Abstract 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 immuno- 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. Undoubtedly, solubilization of the whole hormone in a gland is essential in estimating the hormonal content and in biosynthetic Received April 1, 1987 studies. Though there have been many reports on the hormone content, biosynthesis, and molecular nature of the anterior pituitary hormones, it remained obscure whether or not the extraction of hormones in those studies was complete. The hormones contained in the anterior pituitary gland differ considerably from each other in their physico-chemical nature, and an extraction procedure which is quite efficient in solubilizing one hormone is not necessarily

2 756 ISHIKAWA et al. Endocrinol. Japon. October 1987 useful for others. In the present study, we examined the efficiency of various extraction procedures including media, additives, freezing-thawing treatment, and sonication for solubilization of prolactin (PRL), growth hormone (GH), lutropin (LH), follitropin (FSH), and thyrotropin (TSH) to find the best medium and treatment for the simultaneous measurement of pituitary hormone content. Animals Materials and Methods Adult male rats of Wistar strain were purchased from Imai Experimental Animal Farm, Kodama, Saitama Pref., at 8 weeks of age, and maintained with free access to food and water under controlled temperature (23+1 Ž) and illumination(light on from 5: 00 to 19: 00). They were killed by decapitation and the anterior pituitaries were removed. Extraction media Sixty percent ethanol solutions at various ph were prepared by mixing 1 volume of 0.05 M sodium-phosphate buffer of the target ph and 4 volumes of 75% EtOH just prior to use. Phosphate buffeted saline solutions(pbs) at various ph were prepared by mixing equal volumes of 0.28 M NaCl and 0.02 M phosphate buffer of the target ph, and the ph of the PBS was finally adjusted before use. PBS containing urea, ph 7, were prepared by mixing equal volumes of PBS and urea solution at double concentrations. The medium which had been employed by Hartree (1966), for the extraction of anterior pituitary glycoprotein-fraction was prepared by mixing equal volumes of 80% EtOH and 12% ammonium acetate ph 5.1. Treatment of anterior pituitary glands Anterior pituitary was homogenized in a motor-driven teflon-glass homogenizer with 10 slow strokes under cooling with ice-water. In some experiments, the homogenate was quickly frozen in CryoCool, CC-80f (Neslab, Portsmouth, N. H.) at -60 Ž and thawed. Sonication was carried out by repeating 10 second treatment at 20 watt, 23 KHz with 10 second intervals under ice-cooling employing a Microson ultrasonic cell disruptor (Heat Systems Ultrasonics Inc., Framingdale, N.Y.). Centrifugation was performed in a high-speed refrigerated centrifuge (Hitachi1 8PR-s) at 12,000 rpm for 20 mins. In a series of experiments, single anterior pituitary was homogenized with 1 ml medium, then centrifuged with or without freezing-thawing treatment (F-T), 100ƒÊl supernatant fluid was taken as the first sample, and the rest of the supernatant fluid, together with precipitates, was sonicated and centrifuged. Another 100ƒÊl of the supernatant fluid was taken as the second sample. Usually the sample was immediately added to 900ƒÊl of PBS ph 7.5 containing 1% bovine serum albumin, 0.01% triton X-100, and 0.05% NaN3 (1% BSA-PBS), and stored frozen until assay. In an experiment, the sample was stood in a refrigerator for 1 or 2 days, then diluted to see the stability of the hormones. In a second series of experiments, 10 anterior pituitaries were homogenized with 10 ml PBS ph 7, and 6 aliquots of 1 ml homogenate were prepared. One was immediately frozen and thawed, then centrifuged, and the supernatant fluid was sampled (F-T). The other 5 aliquots were centrifuged, and their supernatant fluids were pooled and 100ƒÊl was taken as a sample (HG). To the precipitates, PBS ph 7, containing 0 to 8 M urea was added in a volume of 1 ml, then they were sonicated for 3 ~10 sec, and centrifuged. A sample of 100ƒÊl was taken from each supernatant fluid, then sonication was repeated another 3 times (each 2 ~10 sec), with centrifugation and sampling of 100ƒÊl supernatant fluid. These samples were immediately diluted with 900ƒÊl of 1% BSA-PBS and assayed. Measurement of hormones Prolactin (PRL), growth hormone (GH), lutropin (LH), follitropin (FSH), and thyrotropin (TSH) were measured by double antibody radioimmunoassays employing NIDDK immuno-reagents according to the methods indicated in the instruction paper with minor modifications. The dilutions of the samples were 1: 200 for FSH, 1: 2,000 for LH and TSH, and 1: 10,000 for PRL and GH. The assay results were expressed in terms of miu of PRL and GH, ƒêg of NIH- LH S1, ƒêg of NIDDK rat FSH RP-2, and ƒêg of NIDDK rat TSH RP-2. The mean assay value for each group was shown with the SEM. The significance of the ultrasonic treatment was estimated by paired t-test.

3 Vol.34, No.5 EXTRACTION FOR PITUITARY HORMONE CONTENT 757 Results Extraction with 60% EtOH and PBS at various ph As shown in Fig. 1, homogenization of anterior pituitary with 60% EtOH of neutral to alkaline ph resulted in excellent extraction of PRL, but resulted in a poor yield of GH and LH. In an acidic condition, 60% EtOH solubilized only small amounts of all the hormones. It was also indicated that sonication did not cause further solubilization with 60% EtOH, but rather reduced the amounts. This tendency was clear with GH. In contrast, PBS solubilized most of the hormones except PRL. Simple homogenization with PBS caused only very poor solubilization of PRL. In the case of PBS, sonication significantly improved the efficiency of extraction with all the hormones. But even with sonication, the extraction of PRL and GH at neutral to acidic ph was poor. Stability of GH in 60% EtOH Fig. 2 shows important information about the stability of GH after the extraction with 60% EtOH at various ph. The anterior pituitaries were homogenized with 60% EtOH of ph 3-9.5, and the supernatant fluids were allowed to stand in a refrigerator for 0-2 days. Extraction of GH was very poor with 60% EtOH except in a highly acidic condition, and while standing for 1-2 days the extracted GH markedly lost its immunoreactivity (note that Fig. 2 is expressed with a logarithmic scale). Only at ph 3, GH was extracted efficiently and remained rather stable. Though data are not shown here, other hormones such as PRL, LH, FSH, and TSH, were fairly stable in 60% EtOH of ph 3-9.5, losing no significant immunoreactivity until 48 hours except PRL at ph 9.5 where a slight decrease in immunoreactivity was observed. In PBS with ph 3-9, all the hormones stayed stable for 2 days. Effects of urea and sonication As described in Materials and Methods, precipitates of PBS (ph 7) homogenates were treated with various concentrations of urea and repeated sonication. Fig. 3 indicates that the effects of urea and sonication differ according to the hormone. In the case of PRL, simple homogenization and F-T with PBS yielded only 10% extraction of the hormone, and repeated sonication was only partially effective. The presence of urea in the medium, on the other hand, caused concentration-dependent solubilization of PRL. The effect of repeated sonication was clear only with 1 M urea. The effect of F-T was quite evident with GH, LH and FSH. Repeated sonication did not always increase the yield. In the PBS-urea media 3 ~10 sec sonication seems to be enough. For LH and FSH, additional sonication to F-T was not effective. Effects of F-T and sonication in the presence of urea, Hartree's medium, and distilled water F-T is a very popular treatment carried out just after the homogenization of tissue. It makes the precipitation of cell fragments easy and gives clear supernatant fluid. The pevious experiment indicated that the solubilization of hormones was promoted also by this treatment. Fig. 4 shows that in the presence of only 1 M urea, homogenization followed by F-T resulted in the maximum yield of all the hormones, and higher concentrations of urea did not cause further extraction over 1 M urea. Hartree's medium (40% EtOH-6% ammonium acetate, ph 5.1) extracted LH, FSH, and TSH with good yields though not the best, and small amounts of PRL and almost no GH. Homogenization and F-T with distilled water solubilized GH, LH, FSH, and TSH with excellent yields but only a little PRL. In order to confirm the efficiency of PBS-urea and F-T, especially for PRL, PBS

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5 Vol.34, No.5 EXTRACTION FOR PITUITARY HORMONE CONTENT 759 (ph 7) containing 0.5 M and 1 M urea were compared with 60% EtOH ph 9.5. As shown in Table 1.PBS-1 M urea with F-T had equal efficiency for PRL to 60% EtOH ph 9.5. Again sonication caused no additional solubilization. The adverse effect of sonication on GH with ethanolic medium was also confirmed. Table 2 shows the efficiency of 0.1 M acetic acid extraction. Acetic acid gave fairly good yields for GH, LH, FSH and TSH, but only a very poor yield for PRL. It seems that PRL gradually loses immunoreactivity in 60% EtOH at ph 9.5 and 0.1 M acetic acid. Effect of triton X-100 and sonication Table 3 shows the effect of trito X-100 added to PBS ph 7. As the control, the results of the extraction with 60% EtOH ph 9.5 were shown. Increased concentrations of triton caused more solubilization of PRL and GH, and sonication gave higher yields than F-T alone, while for LH, FSH and TSH, the effect of sonication was obvious only when the concentration of triton was low (0.01%). Discussion Many investigators measured the anterior pituitary hormone content under various physiological conditions and the influence of various drugs. Some also worked on de novo biosyn-

6 ISHIKAWA et al. Endocrinol. Japan, October 1987 Fig. 2. Extraction and stability of GH with 60% EtOH at various ph. An anterior pituitary was homogenized and centrifuged and the supernatant fliuid was stored for 0 to 2 days in a refrigerator, then diluted to 1: 10 with 1% BSA-PBS ph 7.5 and immunoreactive GH was assayed. Each bar shows the mean for 3 glands and SEM. thesis of hormones which included the incubation of pituitary glands with radioactive precursors followed by the extraction of hormones. However, little attention was paid to the efficiency of their methods in extracting all of the hormones concerned. A wide variety of media have been employed. Against PRL, for example, MacLeod and Abad(1968) used 0.06M phosphate buffer ph 7.2. Mena et al. (1982) used polyacrylamide sample gel at ph 7.2. Distilled water was used by Yanai and Nagasawa(1969), Yamamoto et al. (1970), and Nagy et al. (1983). Voogt et al. (1970) and Dickerman et al. (1972) used PBS ph 7 with sonication, while MacLoed and Robyn (1976) used PBS with triton. Carbonatebicarbonate buffer, 0.05 M, ph 10(Lewis et al., 1971, Sinha et al., 1976, Mena et al., 1982), tris-glycine buffer ph 8.6 (Nicoli et al., 1976), Hanks's balanced salt solution (Nicoli et al., 1969), and Earle's balanced salt solution ph 8.5 (Asawaroengchai and Nicoli, 1977) were also used. PRL has been known as a hydrophobic hormone. Ellis (1961) described, in his serial extraction procedure for anterior pituitary hormones, how extraction with water then with 0.25 M ammonium sulfate leaves most of the PRL in the insoluble residue and that PRL was solubilized with 70% EtOH at ph Later he used 60% EtOH ph 9.5 (1969). Zanini and Giannattasio (1972) compared the efficiency of the extraction media by using electrophoresis and electron microscopy, concluding that 2.5 M urea completely solubilized PRL and GH, while water and phosphate buffer solubilized only a part of these hormones. Haggi and Aoki (1981) also confirmed the

7 Vol.34, No.5 EXTRACTION FOR PITUITARY HORMONE CONTENT 761 Table 1. Effects of urea and sonication on pituitary hormone extraction. Anterior pituitary was homogenized with 1 ml of medium, then frozen-thawed and centrifuged. A hundred Đl of the supernatant fluid was taken as the first sample. The rest of the supernatant, together with the precipitate, was sonicated for 3 ~10 seconds, and centrifuged, then 100 Đl of the supernatant was taken as the second sample. Mean for 3 pituitaries and SEM. Table 2. Effect of 0.1 M acetic acid on pituitary hormone extraction, and stability of the hormones in the media. Anterior pituitary was homogenized with 1 ml of medium, and centrifuged. Three 100 Đl aliquots were taken from the supernatant fluid. One was immediately diluted to 1:10 with 1% BSA-PBS, ph 7.5, and stored frozen until assay. Other aliquots were stored in a refrigerator for 1 and 2 days, then diluted and frozen until assay. Mean for 3 pituitaries and SEM. Table 3. Effects of triton X-100 and sonication on pituitary hormone extraction. Anterior pituitary was homogenized with 1 ml of medium, then frozen-thawed and centrifuged. A hundred Đl of the supernatant fluid was taken as the first sample. The rest of the supernatant fluid, together with precipitate, was sonicated for 3 ~10 seconds, and centrifuged, then 100 Đl of the supernatant fluid was taken as the second sample. Mean for 3 pituitaries and SEM.

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9 Vol. 34, No. 5 EXTRACTION FOR PITUITARY HORMONE CONTENT 763 effect of urea, and further reported that F-T, sonication, or 1% triton X-100, though enhancing the efficiency of phosphate buffer, did not cause complete solubilization of PRL. In our previous report (Ishikawa et al., 1985), we demonstrated that PRL, which was solubilized with 0.25 M ammonium sulfate, ph 5.5, had isoelectric points quite different from those of PRL which remained in the residue and was solubilized with 60% EtOH ph 9.5. The problem of extraction is not only with PRL but also with other hormones. In our present study, we systematically examined various media with additives, and physical treatments, measuring PRL, GH, LH, FSH, TSH, to elucidate the differences in the nature of these hormones and find out the best procedure for the simultaneous estimation of hormonal content. The present results, firstly, revealed contrasting profiles of PRL and GH. PRL could be solubilized with 60% EtOH of neutral to alkaline ph, but only a fraction of the GH was extracted (Fig. 1). Moreover, GH lost its immunoreactivity while standing in ethanolic media (Fig. 2). Sonication seemed to enhance this in activation by ethanol(fig. 2). The only exception was 60% EtOH at ph 3 with which considerable amounts of GH could be extracted, and which was

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11 Vol.34, No.5 EXTRACTION FOR PITUITARY HORMONE CONTENT 765 comparatively stable though the inactivation was still significant (p<0.05, paired t-test). Although PBS, at alkaline ph and with the help of sonication, solubilized GH, PRL could be only partially extracted even with sonication(fig. 1 and 3). The effect of urea was quite significant. If urea was not present at the time of homogenization, a higher concentration( 4 M) was necessary to solubilize PRL(Fig. 3). But when the gland was homogenized and frozen-thawed in the presence of urea, only 1 M was enough to solubilize maximum amounts of PRL(Fig. 4, Table 1), and in this case, sonication did not further solubilize PRL. The presence of 1 M urea in neutral PBS also caused maximum extraction of GH, LH, FSH, and TSH. Zanini and Giannattasio(1972) and Haggi and Aoki(1981) examined the residual pellets after extraction with 2.5 M urea, and confirmed that all the granules were empty. In our present experiment, homogenization with neutral PBS-1 M urea followed by F-T resulted in the maximum extraction of all the hormones, and no further increase was observed with higher concentrations of urea, indicating that whole amounts of hormones were solubilized by this procedure. The addition of triton X-100 to PBS, at a concentration of 0.1 %, also caused maximum solubilization of all the hormones, but in this case sonica-

12 ISHIKAWA et al. tion is indispensable in addition to F-T. The presence of triton in the media sometimes causes trouble due to foam formation. The effect of sonication became apparent only if the condition of the media was critical to the hormone aimed at, e. g. PBS for glycoprotein hormones, and PBS with a lower concentration of urea or detergent for PRL (Fig. 2, Tables 1 and 3). Our present research indicated that homogenization and F-T in 1 M urea-pbs, ph 7, would be the best procedure for the simultaneous measurement of anterior pituitary hormones. Acknowledgements The authors are much obliged to Dr. A. F. Par low of Pituitary Hormones and Antisera Center, Harbor-UCLA Medical Center, and the National Hormone and Pituitary Program, University of Maryland School of Medicine, for kindly supplying NIDDK immune-reagents. This study was supported by Grant-in Aid for Developmental Scientific Research No from The Ministry of Education, Science and Culture, Japan. References Endocrinol. Japan. October 1987 Asawaroengchai, H. and C. S. Nicoli (1977). Relationships among bioassay, radioimmunoassay and disc electrophoretic assay methods for measuring rat prolactin in pituitary tissue and incubation medium. J. Endocrinol. 73, Deckerman, S., J. Clark, E. Deckerman and J. Meltes (1972). Effects of haloperidol on serum and pituitary prolactin and on hypothalamic PIF in rats. Neuroendocrinology 9, Ellis, S. (1961). Studies on the serial extraction of pituitary proteins. Endocrinology 69, Eilis, S., R. E. Grindeland, J. M. Nuenke and P. X. Callahan (1969). Purification and properties of rat prolactin. Endocrinology 85, Haggi, E. and A. Aoki (1981). Prolactin content in rat pituitary gland. RIA of prolactin after different extraction procedures. Acta. Endocr. 97, Hartree, A. S. (1966). Separation and partial purification of the protein hormones from human pituitary glands. Biochem. J. 100, Ishikawa, J., K. Wakabayashi and M. Igarashi (1985). Charge heterogeneity of rat prolactin in relation to the estrous cycle, gonadectomy, and estrogen treatment. Endocrinol. Japan. 32, Lewis, U. J., R. N. P. Singh and B. K. Seavey (1971). Human prolaction: isolation and some properties. Biochem. Biophys. Res. Comm. 44, MacLeod, R. M. and A. Abad (1968). On the control of prolactin and growth hormone synthesis in rat pituitary glands. Endocrinology 83, MacLeod, R. M. and C. Robyn (1976). Mechanism of increased prolactin secretion bysurpiride. J. Endocrinol. 72, Mena, F., G. Martinez-Escalera, C. Clapp, D. Aguayo, C. Forray and C. E. Grosvenor (1982). A solubility shift occurs during depletion-transformation of prolactin within the lactating rat pituitary. Endocrinology 111, Nagy, I., G. Rappay, G. B. Makara, G. Horvath, E. Bacsy and R. M. MacLeod (1983). Is there a direct correlation between the activities of various lysosomal enzymes and prolactin secretion in the rat anterior pituitary? Endocrinology 112, Nicoli, C. S., J. A. Parsons, R. P. Fiorindo and C. W. Nichols, Jr. (1969). Estimation of prolactin and growth hormone levels by polyacrylamide disc electrophoresis. J. Endocrinol. 45, Nicoli, C. S., F. Mena, C. W. Nichols, Jr., S. H. Green, M. Tat and S. M. Russell (1976). Analysis of suckling-induced changes in adenohypophyseal prolactin in the lactating rat by three assay methods. Acta. Endocr. 83, Sinha, Y. N., C. B. Salocks, U. J. Lewis and W. P. Vanderlaan (1974). Influence of nursing on the release of prolactin and GH in mice with high and low incidence of mammary tumors. Endocrinology 95, Voogt, J. L., C. C. Chen and J. Meltes (1970). Serum and pituitary prolactin levels before,

13 Vol.34, No.5 EXTRACTION FOR PITUITARY HORMONE CONTENT 767 during, and after puberty in female rats. Am. J. Physiol. 218, Yamamoto, K., L. M. Taylor and F. E. Cole (1970). Synthesis and release of GH and prolactin from the rat anterior pituitary in vitro as functions of age and sex. Endocrinology 87, Yanai, R. and H. Nagasawa (1969). Quantitative analysis of prolactin by disc electrophoresis and its relation to biological activity. Proc. Soc. Exp. Biol. Med. 131, Zanini, A. and G. Giannattasio (1972). Polyacrylamide gel electrophoresis of rat anterior pituitary gland after different extraction procedures. J. Endocrinol. 53,