Diabetologia 9 Springer-Verlag 1984

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Diabetlgia (1984) 27:392-396 Diabetlgia 9 Springer-Verlag 1984 12SI-glucagn-degrading activity in acid-saline extracts f rat salivary gland M. Tminaga, K. Yamatani, S. Marubashi, H. Kaneda, H.

1 Diabetlgia (1984) 27: Diabetlgia 9 Springer-Verlag SI-glucagn-degrading activity in acid-saline extracts f rat salivary gland M. Tminaga, K. Yamatani, S. Marubashi, H. Kaneda, H. Manaka, T. Kamimura, T. Katagiri and H. Sasaki Third Department f Internal Medicine, Schl f Medicine, Yamagata University, Yamagata, Japan Summary. The antibdy-binding ability f the glucagn-like substance in rat submaxillary gland acid saline extract was examined by affinity chrmatgraphy, and the bilgical activity studied using the islated liver perfusin methd. We fund that the glucagn-like substances in acid saline extract culd nt be bund t anti-glucagn antibdy and that the gel-filtratin peak n ultrgel AcA 54 culd increase neither glucse nr cyclic AMP utput frm islated perfused rat liver. Furthermre, the radiactivity peak f 125I-glucagn n Bi Gel P-6 clumn chrmatgraphy mved frm its riginal psitin and eluted in later fractins after incubatin with an acid saline extract f the submaxillary gland. In cnsequence, there was 125I-glucagn degrading activity in the submaxillary gland, but n glucagn-related peptide. Therefre, it is suggested that the glucagn-like substance, which has been reprted in acid saline extract f the rat salivary gland, may be an artifact due t tracer degrading activity. Key wrds: Glucagn, salivary gland, acid saline extract, 125Iglucagn-degrading activity, affinity chrmatgraphy The existence f glucagn-like substances in the rat salivary gland has been reprted in recent years [1-7]. It has been pstulated that salivary gland may be ne f the candidates fr extrapancreatic surces f circulating glucagn, which ccurs even in the pancreatectmized r eviscerated rat [1-3, 7]. Hwever, the questin whether r nt the glucagnlike substance in acid saline extract f the submaxillary gland is identical t pancreatic glucagn and/r its precursr has been cntrversial because glucagn-like substances in the salivary gland have been fund nly in high mlecular frms [2-5], and because f the difficulty in demnstrating it successfully by the immunhistchemical methd. Recently, Hattn et al. reprted that high cncentratins f glucagn-like immunreactants measured by radiimmunassay in brain acid saline extract were nly artifacts due t 125I-glucagn-degrading activity [8]. They suggested that the glucagn-like immunreactivity in acid saline extract f the submaxillary gland likewise culd be an artifact. This investigatin was carried ut t examine the antibdy-binding ability f the glucagn-like substance in acid saline extract by affinity chrmatgraphy, its bilgical activity using the islated liver perfusin methd and 125I-glucagn-degrading activity by simple incubatin methds. During the preparatin f this paper, findings similar t these have been reprted by Tahara et al. [9], Materials and methds Animals Wistar rats (weight g) were sacrificed by cutting the cartid artery and their submaxillary glands were remved and stred at - 20 ~ until extracted. Extractin prcedures Several methds f extractin were used. (1) Kenny's methd [10]: the glands were hmgenized in acid alchl by a Plytrn hmgenizer (Kinematica, Luzern, Switzerland) and centrifuged at 2,000 g. The supernatant was treated with 4.5 vlumes f an alchl-ether mixture (1.7 : 2.8) and the precipitate cllected. (2) Acid-ethanl methd: the glands were extracted as in methd (1) but the supematant frm the centrifugatin was evaprated t dryness. (3) Biling methd: the materials were hmgenized in distilled water, biled fr 10 min, and centrifuged at 2,000 g fr 30 rain. The supernatant was used as the extract. (4) 0.2N acetic acid-biling methd: after hmgenizatin in 0.2N acetic acid, the same prcedure was used as fr methd 3. (5) 2N acetic acid methd: the material was hmgenized in 2N acetic acid, centrifuged at 2,000 g fr 30 min and the supernatant fluid was cllected as extract. (6) Acid saline methd: after hmgenizatin in saline (0.154ml/1) acidified with HCI t ph2.8, the sample was centrifuged at 10,000 g fr 30 min and the supernatant was used as the extract [2-7].

2 M. Tminaga et al.: Glucagn degradin in rat salivary gland 393 Radiimmunassay Radiimmunassay was perfrmed by the methd described previusly [11]. The values measured by radiimmunassay with C-terminal specific antibdy 30K (Texas University, Dallas, Texas, USA) [12] r OAL123 (Otsuka Assay Labratry, Tkushima, Japan) [13] are referred t as immunreactive glucagn (IRG), and thse measured with central prtin reactive antibdy K4023 (Nva, Cpenhagen, Denmark) [14] r OAL196 (Otsuka Assay Labratry, Tkushima, Japan) [15] are referred t as glucagn-like immunreactivity (GLI) in this text. The term glucagn-like substances is used as a general term fr IRG and GLI thrughut the text. Gel-filtratin The acid saline extract (0.5 ml nml/1 glucagn as measured by the 30K antibdy) was chrmatgraphed n an ultrgel AcA 54 clumn ( cm; LKB, Brmma, Sweden) in 10 mml/1 ammnium bicarbnate buffer (ph8.6). The flw rate was apprximately 6.0 ml/h and fractins f 1 ml were cllected. Liver perfusin The bilgical activities f acid saline extracts were examined by liver perfusin after the methd f Sugan et al. [16]. Crude acid saline extracts (- 0.9 nml/1 glucagn by the 30K antibdy) and the gel-filtratin peak (-= 0.3 nml/1 glucagn by the 30K antibdy) were perfused thrugh rat liver fr 10 rain. Glucse and cyclic AMP in the effluents were measured by the glucse xidase methd and radiimmunassay [17], respectively. Trypsin digestin The dried gel-filtratin peak (1.5 mg prtein equivalent) was disslved in 0.1 real/1 Tris-HC1 buffer (0.1 ml, ph 7.5) cntaining CaC12 (10retl/l) and trypsin (Sigma, St. Luis, Missuri, USA) at 100 ~tg/1. The slutin was incubated at 37 ~ fr a h. After biling fr 2 rain, aliqutes f enzyme-digested extracts were measured by radiimmunassay and rechrmatgraphed n ultrgel AcA 54 as described abve. Affinity chrmatgraphy Rabbit anti-glucagn serum YG8, prduced by urselves, was used in this experiment. Prcine glucagn (Nva, Cpenhagen, Denmark) was cnjugated with bvine serum albumin (Sigma) by the glutaraldehyde methd [22]. After immunizatin, anti-glucagn serum f lw titre was btained (final dilutin used in radiimmunassay 1 : 8,000). The partially purified GLI f the prcine dudenum given by Prfessr V. Mutt (Karlinska Institute, Stckhlm, Sweden) was measured by radiimmunassay using K4023, 30K and this antibdy, YG8. The 30K immunreactivity measured <2.3% f the K4023 immunreactivity in Prfessr Mutt's GLI, but the YG8 immunreactivity crrespnded t 36% that f antibdy K4023. Cnsequently, the anti-glucagn serum YG8 was crss-reactive, but nt cmpletely s, with GLI. The antiserum YG8 was purified t its g-glbulin fractin by ammnium sulphate precipitatin. The purified antiserum disslved in 0.01 real/1 Tris-HCl buffer (ph 8.4) was incubated with CN-Br-activated Sepharse 4B (Pharrnacia, Uppsala, Sweden) at rm temperature fr 2 h and washed twice with I0 vlumes f the same buffer. The cupled sepharse was put int a clumn (1.0 x 5.0 cm) and equilibrated with 0.01 ml/1 Tris-HCl buffer (ph 8.4). Crude acid saline extract was chrmatgraphed n this affinity clumn. After incubatin fr 24 h, 10 vlumes f 0.01 real/1 Tris-HC1 buffer (ph 8.4) were eluted at a flw rate f 10 ml/h, and then five vlumes f 1N acetic acid were eluted. Apprximately, I ml fractins were cllected. The ph was adjusted t with NH4OH and IRG was measured by radiimmunassay with antibdy 30K. Kenny's extracts f the rat hypthalamus and 125I-glucagn were subjected t the same prcedure as the cntrl experiments. 125I-ghtcagn degrading activity Prcine crystallized glucagn (Nva, Cpenhagen, Denmark) was labelled with the cartier-free slutin, 12SI-Na (Amersham, Internatinal, Bucks, UK), using the chlramine T methd [18], and the labelled 1251-glucagn was purified n a QAE Sephadex A-25 (Pharmacia) clumn ( cm), accrding t the methd f Jrgensen and Larsn [19]. lz5i-glucagn (-1.5 x 106 cpm, and apprximately 45 ng) was incubated with acid saline extract (10 mg prtein) f the submaxillary gland in 1.0 ml f 0.1 ml/l Tris-HC1 buffer (ph 7.5) cntaining CaC12 (10 mml/1) at 37 ~ fr 2 h and subjected t gel-filtratin n Bi-Gel P-6 (1.0 x ll0cm, Bi-Rad, Richmnd, Califrnia, USA). Fractins (-I ml) were cllected, and the radiactivities f these fractins were measured. As a cntrl experiment, 12SI-glucagn was incubated withut acid saline extact and subjected t gel-filtratin. Results IRG and GLI cncentratins in extract f the submaxillary gland Table 1 shws IRG cncentratins measured with the 30K antibdy and GLI cncentratins measured with antibdy K4023 in rat submaxillary gland and in extracts f rat pancreas using the varius extractin methds. In the extract f the submaxillary gland using Kenny's methd [10], the IRG and GLI cncentratins were and nml/g wet weight, respectively. These values were nearly equal t thse f the plasma cncentratin. Hwever, in the acid-saline extract f the submaxillary gland, IRG and GLI cncentratins were 1894 and 2171 nml/g wet weight, respectively. These cncentratins were similar t thse in the pancreas extract btained by Kenny's methd. IRG and GLI cncentratins in acid saline extract f the submaxillary gland measured with OAL123 and OAL196 were similar t thse measured with 30K and K4023, respectively. It was nted that the cncentratin f IRG in the acid saline extract was nearly equal t the GLI cncentra- Table 1. Cncentratins f glucagn-like substances in rat submaxillary gland and pancreas cmpared by varius extractin methds Immunreactive glucagn (nml/g wet weight) Submaxillary gland Kenny's methd [10] Acid ethanl (direct methd) Biling methd N acetic acid 1.04 biling methd 2 N acetic acid methd 41.5 HCl-saline methd 1894 HCl-saline methd 1806 a Pancreas Kenny's methd [10] 1471 HCl-saline methd 25.2 a measured with antibdy OAL123; b measured with antibdy OAL196 Glucagn-like immunreactivity (nml/g wet weight) b

3 394 "2 c :lk g B 3 2,, 12 E E c 9 ~g 6 O.- ~ = ~ g g g g I Fractin number Fig.l. Gel-filtratin prfile f acid saline extract f submaxillary gland n ultrgel AcA 54. M immunreacfive glucagn; ( glucagn-like immunreactivity. Markers: 1: blue dextran, 2: bvine serum albumin, 3: 12SI-human grwth hrmne, 4: 125I-glucagn, 5: 125I-Na Time (rain) Fig. 2. Effect f acid saline extract f submaxillary gland n glucse utput frm islated perfused liver. The crude acid saline extract f submaxillary gland (H) (n = 1) (-= 0.9 nml/1 glucagn measured by 30K antibdy), the gel-filtratin peak (13 13) (n=l) ( nml/l t glucagn measured by 30K antibdy) as well as prcine glucagn 0.01 nml/1 (O----O) (n=6) and 0.1 nml/1 (O-----Q) (n = 4) were perfused int islated liver, and the glucse levels in the effluents were measured by the glucse xidase methd. Data are shwn as mean values M. Tminaga et al.: Glucagn degradin in rat salivary gland Liver perfusin The glucse utput elicited by prcine glucagn 0.01 nml/1 (n = 6) and 0.1 nml/1 (n = 4) increased in a dse-dependent manner in this liver perfusin experiment (Fig.2). The crude acid saline extract frm the submaxillary gland (- 0.9 nml/1 glucagn with the 30K antibdy, n = 1) gave a glucse utput frm the liver nly ne hundredth that f the immunequivalent dse f glucagn. The pattern f glucse utput raised by the crude acid saline extract was different frm that raised by prcine glucagn. Furthermre, when the gelfiltratin peak (-0.3 nml/1 glucagn measured by the 30K antibdy, n = 1) was perfused, there was n effect n glucse utput. Prcine glucagn increased nt nly glucse but als cyclic AMP utput. Cyclic AMP utput was raised by prcine glucagn (0.01 and 0.1 nml/1) t and 8.11_+a.96pml/g f liver per min (mean_+sd, n = 6, n-- 4), respectively. Hwever, when the crude acid saline extract and the gel-filtratin peak were perfused, cyclic AMP utputs were less than the detectable level (1.27 pml/g liver per min). Trypsin digestin Althugh IRG cncentratin decreased frm 8.29 t 0.45 nml/1 (5.3%) after trypsin digestin, gel-filtratin prfiles did nt alter and trypsin digestin did nt elicit any new peak in the regin f the lwer mlecular weight fractin (Figs. 1 and 3). Affinity chrmatgraphy Mst IRG fractins in the acid saline extract f the submaxillary gland were eluted during washing prcedures (Fig. 4). There was n IRG peak after lwering the ph. This phenmenn was in cntrast t the affinity chrmatgraphy patterns f Kenny's extract f the rat hypthalamus and t25i-glucagn in which the main IRG and radiactivity peaks were eluted after lwering the ph qp qp V g v c 0.9 tin. IRG and GLI cncentratins in the submaxillary gland using varius ther extractin methds were variable and ranged between thse fund in Kenny's extracts and the acid saline extract. Gel-filtratin n ultrgel AcA 54 The gel-filtratin prfile f the acid saline extract n ultrgel AcA 54 demnstrated a single peak eluting at the psitin f 125I-HGH (human grwth hrmne), which reacted identically with 30K and K4023 (Fig. 1). The mlecular weight f the peak was ~20,000. There was n peak at the 125I-glucagn psitin. ~ 0.6 -~ gf i Fractin number Fig.3. Gel-filtratin prfile f trypsin-treated acid saline extract f submaxillary gland n ultrgel AcA 54. H immunreactive glucagn; ~ glucagn-like immunreactivity. Markers as fr Figure 1

.... M. Tminaga et al.: Glucagn degradin in rat salivary gland 395 12i -+ ;... A 12 0.

4 .... M. Tminaga et al.: Glucagn degradin in rat salivary gland i -+ ;... A c:m~4 ~ 6 E~ E 4 E=~ 0 2 i ~2 lo ~ = ~ 40 ~~ I 8 --~ 6 E 4 cl xlo _; I + t _~ ~" = Fractin number Fig.4A-C Affinity chrmatgraphy n sepharse 4B cupled with a rabbit anti-glucagn antiserum YG8 was used t examine the antibdy-binding ability f acid saline extract f submaxillary gland (A), Kenny's extract f rat hypthalamus (B) and 12SI-glucagn (C). The ph (O----0) f each fractin (1 ml) was adjusted t with NH4OH and immunreactive glucagn (IRG) cncentratin (H) was measured by radiimmunassay with 30 K antibdy. In the case f 12SI-glucagn, the radiactivity f each fractin was measured by a gamma-cunter xlo ~ 50 E e~ ~ 30 >, > ~ 2 n,-,,, L, 8,0,, Fractin number Fig. 5. Gel-filtratin prfile f 125I-glucagn incubated with (H) and withut (O----0) acid saline extract f submaxillary gland n Bi-Gel P I-glucagn-degrading activity When 125I-glucagn was incubated with acid saline extract f the submaxillary gland, the peak f radiactivity n Bi-Gel P-6 mved frm its riginal psitin t appear in later fractins (Fig. 5). Discussin In recent years, it has been reprted that there are cnsiderable amunts f glucagn-like substances in salivary gland extract [1-7]. Amng these investigatins there is a cnsensus that the cntent f glucagn-like substances is greater in the extracts frm the submaxillary gland than in thse frm ther salivary glands. Fllwing these bservatins, we examined the rat submaxillary gland. It is als recgnized that there are apparently high cncentratins f glucagn-like immunreactivity in acid saline extracts cmpared t thse in acid ethanl extracts. In agreement, these studies cnfirm the presence f high cncentratins f IRG and GLI in acid saline extracts f the submaxillary glands. The acid saline extractin methd was develped riginally fr the extractin f higher mlecular weight cmpnents which are nt extractable by the traditinal acid-ethanl methd f Kenny [3]. This prbably frms the basis f the cntrversy cncerning the questin whether glucagn-like substances in acid saline extract are identical t glucagn-related peptides including pancreatic glucagn and/r its precursrs. Hattn et al. [8] raised the pint that acid saline extract cntain tracer degrading activity, and suggested the apparently measurable glucagn-like material culd be an artifact. Perez-Castill and Blazques [6, 7] have shwn already that glucagn degradatin was 67% with the acid saline extractin prcedure, and had examined acid ethanl extracts. The findings reprted here that glucagn-like substances in acid saline extract f the rat submaxillary gland culd nt be bund t anti-glucagn antibdy by affinity chrmatgraphy and that there was t25i-glucagn-degrading activity in acid saline extract supprts Hattn's suggestin. The glucagn-like substance fund in the acid saline extract f the submaxillary gland is indeed an artifact due t tracer degrading activity. The gel-filtratin prfile f acid saline extract f the submaxillary gland revealed a single high mlecular weight cmpnent nly. Althugh Lawrence et al. [2, 3] reprted the mlecular weight as 90,000 and Bhathena et al. [4] and Smith et al. [5] reprted it as 29,000, there is at least cncrdance that it is a single peak f high mlecular weight. Cnsidering the high mlecular weight and similarity f IRG and GLI, the putative peptide may be a large mlecular precursr f which the C-terminal peptide may be identical t the C-terminal residues f pancreatic glucagn. Hwever, glucagn precursrs studied in the gut and pancreas were reprted t cntain C-terminal extensin peptides which mask the reactivity with C-terminal specific antibdy [20, 21]. This cntradictin can be easily reslved if the glucagn-like substance measured by radiimmunassay is due t tracer degrading activity. If the degrading activities are the same in bth the IRG and GLI assay systems, the same values shuld be shwn n the standard curve. Therefre, there is n relatinship between the values btained and the specific reactivity f the antibdy used t the prtins f the peptide sequence. Indeed, as reprted here, the values f glucagn-like substances measured with fur different antibdies (30K, K4023, OAL123, OAL196) were almst identical.

5 396 M. Tminaga et al.: Glucagn degradin in rat salivary gland Hattn et al. [8] reprted that stepwise dilutin f acid saline extracts f the brain were parallel t the apparent immunreactivity, but this may be errneus. Affinity chrmatgraphy clearly identified the antibdybinding ability. The discrepancy in apparent cncentratins f glucagn-like substance using varius extractin methds may depend n the amunt f denaturatin caus'ed by the varius extractin prcedures; ethanl and biling may accelerate the denaturatin. Since trypsin digestin f acid saline extracts did nt elicit any new lwer mlecular weight cmpnents and the gel-chrmatgraphy prfile f acid saline extracts did nt change befre r after trypsin digestin, the apparent immunreactivity may be due t the tracer degrading activity, which culd be a prtein capable f degradatin by trypsin. Difficulty in immunhistchemical demnstratin f glucagn psitive cells in the salivary gland has been thught curius fr a lng time. Only ne uncnfirmed reprt has mentined the psitive immunflurescence f glucagn [4]. Hwever, as substances in the salivary gland are nw believed t degrade 125I-glucagn but nt glucagn related peptides, this accunts fr failure f immunhistchemical detectin. There is a reprt that the bilgical activity f the acid saline extract was examined by displacement f 12SI-glucagn frm rat liver plasma membrane [5]. Hwever, if 12SI-glucagn can be degraded by acid saline extract, the apparent radiactivity bund t the plasma membrane wuld be decreased and the reprt's cnclusin may be errneus. One reprt described glycgenlytic activity in the crude extract f rat salivary gland [4]. We fund als that the crude acid saline extracts increased glucse, but nt cyclic AMP utput. Hwever, the finding that the gel-filtratin peak f the acid saline extract did nt reveal any increase in glucse utput shuld negate this pssibility. It can be argued that the effect f crude extract n glucse utput may be due t ther cmpnents, such as adrenaline. On the ther hand, even if the abve cnsideratins shuld deny the presence f glucagn-like substances in acid saline extracts f the submaxillay gland(the pssibility f their presence in acid ethanl extracts may remain. Hwever, since, as shwn here and as mentined by ther authrs [2-5], the values in the extracts using Kenny's methds were extremely lw, the pssible prductin f glucagn-like substances is negligible. In cnclusin, it is unlikely that the salivary gland is a surce f circulating extrapancreatic glucagn. References 1. Dunbar JC, Silverman H, Kirman E, F~ PP (1977) Rle f the submaxillary gland and kidney in the hyperglucagnemia f eviscerated rats. In: F~ PP, Bajaj JS, F/l NL (eds) Glucagn: its rle in physilgy and clinical medicine. Springer, Berlin Heidelberg New Yrk, pp Lawrence AM, Tan S, Hjvat S, Kirsteins L (1976) Salivary gland hyperglycemic factr: an extrapancreatic surce f glucagn-like material. Science 195 : Lawrence AM, Tan S, Hjvat S, Kirsteins L, Mittn J (1976) Salivary gland glucagn in man and animals. Metablism 25 (Suppl 1): Bhathena SJ, Smith SS, Vyles NR, Penhs JC, Recant L (1977) Studies n submaxillary gland immunreactive glucagn. Bichem Biphys Res Cmmun 74: Smith S, Mazur A, Vyles N, Bhathena S, Recant L (1977) Is submaxillary gland immunreactive glucagn imprtant in carbhydrate hmestasis? Metablism 28: Perez-Castill A, Blazques E (1980) Synthesis and release f glucagn by human salivary glands. Diabetlgia 19: Perez-Castill A, Blazques E (1980) Tissue distributin f glucagn, glucagn-like immunreactivity, and insulin in rat. Am J Physi1238:E258-E Hattn TW, Kvacevic N, Dutzak M, Vranic M (1982) Glucagn-like immunreactants in extracts f the rat hypthalamus. Endcrinlgy 111 : Tahara Y, Shima K, Hirta H, Ikegami H, Tanaka A, Kumahara Y (1983) Salivary gland glucagn is a fictitus substance due t tracer-degrading activity resistant t prtease inhibitrs. Bichem Biphys Res Cmmun 113: Kenny AM (1955) Extractable glucagn f the human pancreas. J Clin Endcrinl Metab 15: Tminaga M, Ebitani I, Marubasbi S, Kamimura T, Katagiri T, Sasaki H (1981) Species difference f glucagn-like materials in the brain. Life Sci 29: Falna GR, Unger RH (1974) Glucagn: In: Jaffe BM, Behrman HR (eds) Methds f hrmne radiimmunassay. Academic Press, New Yrk, pp Yanaihara N, Nishin T, Kdaira K, Imagawa T, Nishida S, Mihara S, Yanaihara C (1979) Characterizatin f anti-glucagn sera elicited against a C-terminal fragment f pancreatic glucagn and their use in glucagn radiimmunassay. In: Baba S, Kanek T, Yanaihara N (eds) Prinsulin, insulin, C-peptide. Elsevier, Nrth- Hlland Amsterdam, pp Heding LG, Frandesn EK, Jacbsen H (1976) Structure-functin relatinship: immunlgic. Metablism 25 (Suppl 1): Nishin T, Kdaira T, Shin S, Imagawa K, Yanaihara N, Shima K, Kumahara Y (1981) Prductin f antisera t des- Asn28-Thrzg-HmserZT-glucagn; the develpment f radiimmunassay fr ttal glucagn-like immunreactivity in human plasma. Endcrinl Jpn 28: Sugan T, Suda T, Shimada M, Oshin N (1978) Bichemical and ultrastructural evaluatin f islated rat liver systems perfused with a hemglbin-free medium. J Bichem 83: Kunitada S, Hnma M, Ui M (1978) Increase in plasma cyclic AMP dependent n endgenus catechlamine. Eur J Pharmacl 48: Hunter WM, Greenwd FC (1962) Preparatin f idine-131 labelled human grwth hrmne f high specific activity. Nature 194: Jrgensen KH, Larsn UD (1972) Purificatin f 12SI-glucagn by anin exchange chrmatgraphy. Hrm Metab Res 4: Tager HS, Patzelt C, Asscian RK, Chan S J, Duguid JR, Steiner DF (1980) Bisynthesis f islet cell hrmnes. Ann NY Acad Sci 343: Thim L, Mdy AM (1981) The primary structure f prcine glycentin (prglucagn). Regul Pept 2: Reichlin M, Schnure J J, Vance VK (1968) Inductin f antibdies t prcine ACTH in rabbits with nnsteridgenic plymers f BSA and ACTH. Prc Sc Exp Bil Med 128: Received: 31 August 1983 and in revised frm: 13 June 1984 Dr. M. Tminaga Third Department f Internal Medicine Schl f Medicine Yamagata University Za-Iida Yamagata Japan

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