Waring, 1941]. Pig glands, however, can be five times as potent as ox

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1 A PURIFIED EXTRACT OF THE INTERMEDIATE LOBE OF THE PITUITARY. By F. W. LANDGREBE and G. M. MITCHELL. From the Department of Pharmacology, Welsh National School of Medicine, Cardiff. (Received for publication 18th November 1953.) THE pituitary gland of a domestic animal readily divides, on dissection, at the hypophyseal cleft into the anterior lobe (ALP) and the posterior lobe (PLP). The latter contains the pars intermedia and the pars nervosa, and these cannot easily be separated by dissection. So posterior lobe is preferable to whole gland material for the manufacture of potent intermediate lobe extracts. The pars intermedia secretes a hormone called "B" [Hogben and Slome, 1931] which expands the melanophores in the skin of amphibia and fish, and the assay of extracts is based on the degree of expansion obtained. Many methods have been suggested to prepare potent extracts of this activity from ox pituitaries [Zondek and Krohn, 1932; Stehle, 1936; Teague, 1939; Landgrebe and Waring, 1941]. Pig glands, however, can be five times as potent as ox glands in "B" [Landgrebe and Waring, 1949]. So we decided to use dried pig posterior lobe powder as our starting material. One difficulty has been the separation of the pressor and oxytocic activities of the pars nervosa from the " B ". Some workers have deliberately destroyed these contaminants by alkaline treatment of their original extract before purification. Such treatment, however, changes the character of the "B ", and the material is qualitatively different from that present in simple extracts [Landgrebe, Munday and Waring, 1950]. Our method necessitates no such treatment and allows recovery of pressor and oxytocic activity. A high yield of "B" is obtained in a final product many times more potent than any previously described and virtually free of oxytocic and pressor activities. In other experiments designed to test the separate identity of ACTH and "B ", we found that commercial extracts of ACTH (usually manufactured from whole gland) were also highly potent extracts of " B ". In view of this and the close chemical similarity of ACTH and "B ", we attempted purification of the latter using modifications of techniques already suggested for ACTH. The early stages of the method finally evolved are modifications of those used by Astwood, Raben, Payne and Grady [1951] in their oxycellulose adsorption method or preparation of ACTH. 11

2 12 Landgrebe and Mitchell MATERIALS. Melanophore dispersing activity is the first to deteriorate in badly prepared or stored dried pituitary material. A water content of only 4 per cent (estimated by drying to constant weight over phosphorus pentoxide in vacuo) is sufficient for this to occur, even at a temperature as low as C. In such material, deterioration can occur to 10 per cent of the original in a space of a few months [Landgrebe and Waring, 1949]. We find that the usual pig posterior lobe commercially available for extraction contains up to 7 per cent water and only from 1 to 3 " I.U." per mg. (Table I), whereas well collected and properly stored material may contain 10 " I.U." per mg. (The "International Unit " has been suggested by Landgrebe and Waring [1944] as that amount of activity present in 0 5 mg. of the International Standard ox posterior lobe pituitary powder extracted by the standard method. This conforms with the official definition of an International Unit of pressor and oxytocic activity.) Therefore the first problem is the acquisition of sufficient well-collected, well-dried and well-stored pig posterior lobe material. Oxycellulose (Eastman Kodak Batch C1205) containing per cent carboxyl units was used for adsorption of "B" and, judging from its activity in this respect, it is not as active as that used by Astwood. The amount of oxycellulose used was determined by measuring the unadsorbed "B " in the extract after 18 hours' contact. In our experiments about 10 per cent of "B" was unadsorbed. If other batches of oxycellulose are used, different quantities may be needed. All reagents used were of a purity suitable for analytical work. METHOD OF ASSAY. The method of measuring the "B" activity was that suggested by Landgrebe and Waring [1944] using Xenopu& UCeVi8. The degree of expansion of the melanophores is estimated microscopically and a numerical result (melanophore index) is allocated on an arbitrary scale (see fig. 1). They found intact animals suitable for testing pituitary extracts, but our results have also been checked on hypophysectomised Xenopus. Results accurate to 10 per cent are obtained easily and quickly by this method. METHOD OF PREPARATION. Ten g. of dried pig posterior lobe powder is damped with 25 ml. acetone, mixed into a smooth paste, and 100 ml. glacial acetic acid is added gradually with stirring. The mixture is heated on a sand bath to 500 C., stirring all the time (3-5 min.). The mixture is then centri-

3 A Purified Extract of the Intermediate Lobe of the Pituitary 13 fuged and the supernatant collected. The residue is stirred thoroughly with a further 100 ml. of glacial acetic acid in the cold and centrifuged. The supernatants are combined and the volume measured. Half this volume of acetone and 1 ml. of saturated sodium chloride solution are added, and the mixture centrifuged. The volume of the supernatant fluid is measured and 2 volumes of ansesthetic ether are added. The material is placed in the deep freeze ( -150 C.) for 1 hour. It is then centrifuged and the precipitate washed and dried with acetone. The dried powder is dissolved in 75 ml. of N/IO acetic acid and centrifuged to remove an inert gummy precipitate. 300 mg. of powdered oxycellulose is ground with a few ml. of N hydrochloric acid, washed twice with distilled water and once with N/10 acetic acid. The oxycellulose is added to the acetic acid extract and stirred continuously for 18 hours. The oxycellulose is removed by centrifuging and washed twice with N/10 acetic acid and once with distilled water. The oxycellulose is extracted for 20 minutes with 10 ml. N/IO hydrochloric acid. After centrifuging, the oxycellulose is washed with a further 5 ml. of N/10 hydrochloric acid and centrifuged. The supernatants are combined and adjusted to ph 6-5 with N ammonium hydroxide and centrifuged to remove inert solid. Five volumes of acetone are added to the supernatant and the whole placed in the deep freeze for 1 hour. The precipitate is filtered by positive pressure through a Whatman No. 544 paper and dried by washing with acetone. About 25 mg. of material is obtained of a potency depending on the original material according to the following table: TABLE I. Material. Potency of original Potency of extract in.u./mg. "B" inli.u./mg. Oxytocin. '- -P-ressor. "B " Ox PLP. 1-6 < 0-2 < Pig PLP (1) 3-0 < 0-5 < Pig PLP (2) 9-0 < 0-25 < The yield is per cent of the original, and the product is virtually free of pressor and oxytocin ,ug. of the most potent " B " extract injected into the dorsal lymph sac of each test animal gives a response similar to that of 2-0,ug. of International Standard PLP (ox) (see figs. 2 and 3). Solutions in 0-25 per cent acetic acid (0.1 mg./ml.) are stable at room temperature for at least four months if heated in small ampoules in a boiling water bath for one minute and sealed while still hot. From the results shown (Table I) it is clear that good original material is necessary for a highly purified product. Such pig original material is five times as potent as similar ox material and yields an extract five times as active. So either the product obtained by this

4 14 a is. *4 Landgrebe and Mitchell i* As I FIG. 1.-The assessment of the melanophore index (M.I.). M.l. TIME'IN HOURS FIG. 2.-Response curve obtained in each case on the same group of test animals. Temperature 160 C. X X-2 4g. I.S. PLP. * 0-1 pg. I.S. PLP after sodium hydroxide treatment. 4" M I 2 i 4 S * a8 TIME IN HOURS FIG. 3.-Response curve obtained in each case on the same group of test animals. Temperature 16 C. X X p4g. "B". * pg. "B" after sodium hydroxide treatment.

5 A Purified Extract of the Intermediate Lobe of the Pituitary 15 method from pig material is more highly purified, or the melanophore expanding polypeptide obtainable from pig glands is different from that from ox pituitaries and is five times as potent. Further evidence for the latter view is obtained from the results of caustic soda treatment. Fig. 3 shows that after adjusting a dilute solution (10 "I.U."/ml.) to ph 13 with N sodium hydroxide and heating for two minutes on a boiling water bath [Landgrebe et al., 1950], the response curve of both the International Standard Posterior Lobe Pituitary (I.S. PLP) and the purified "B" is prolonged. But whereas standard extracts of ox material (both commercial and I.S. PLP) show an increased activity after treatment with NaOH and can be said to be " potentiated," extracts of pig material are, in fact, largely destroyed (Table II). A standard extract (1 mg./ml.) of the original pig material, too, is neither potentiated nor destroyed, but is "protected". TABLE II. Effect of caustic soda Material. Potency of extract treatment. in I.U./mg. Potentiation. Flattening index. Ox PLP I.S x Ox PLP Original Pig PLP Pig PLP Pig PLP x 0.2 (i.e per cent destruction) The term " protection" has been retained to describe this prolongation in order to avoid ambiguity, though it is not certain that the parent assumption is correct, namely, that such treatment results in an activity less easily destroyed by the body. Experiments on melanophores in isolated skin of dogfish show that treatment of extracts with NaOH results in both " potentiation " and " protection " [Landgrebe et al., 1950]. So if the "protection" shown by isolated skin melanophores is the same as that shown in the intact animal, it cannot be argued that it is the reduced rate of destruction by body tissue other than skin which gives the protection. The degree of protection has been defined as "the ratio of the increase in time necessary for the melanophores to move from melanophore index 3 0 to melanophore index 2-0 when injected with treated extract as compared with the time to make the same transition after injection with the same extract untreated in dose sufficient to attain the same peak melanophore index". We find this

6 16 A Purified Extract of the Intermediate Lobe of the Pituitary index for the same extracts varies with temperature and with the length of time the test animals have been on a white background. Using this index on the same animals at 160 C., the degree of protection of the present "B" extract is not significantly different from that obtained with the International Standard PLP (Table II). SUMMARY. 1. A method is described for the preparation of an extract of melanophore dispersing hormone 500 times as potent as International Standard posterior lobe powder. 2. Evidence is recorded suggesting that the polypeptide derived from pig glands is different from that obtained from ox material. ACKNOWLEDGMENT. Our best thanks are due to Dr. W. J. Tindall of Organon Laboratories Limited for a supply of suitable pig posterior lobe powder. REFERENCES. ASTWOOD, E. B., RABEN, M. S., PAYNE, R. W., and GRADY, A. B. (1951). J. Amer. Chem. Soc. 73, HOGBEN, L., and SLOME, D. (1931). Proc. roy. Soc. B, 108, 10. LANDGREBE, F. W., MUNDAY, K. A., and WARING, H. (1950). Aust. J. exp. Biol. med. Sci. 28, 619. LANDGREBE, F. W., and WARING, H. (1941). Quart. J. exp. Physiol. 31, 31. LANDGREBE, F. W., and WARING, H. (1944). Quart. J. exp. Physiol. 33, 1. LANDGREBE, F. W., and WARING, H. (1949). Aust. J. exp. Biol. med. Sci. 27, 331. STEHLE, R. L. (1936). J. Pharmacol. 57, 1. TEAG-UE, R. S. (1939). Endocrinology, 25, 953. ZONDEK, B., and KROHN, H. (1932). Klin. Wschr. 11, 405 and 1293.