Diabetologia 9 Springer-Verlag 1982

Transcription

1 Dabetoloa (1982) 22: Dabetoloa 9 Sprner-Verla 1982 Glucaon Immunoreactvty n Plasma from Normal and Dystrophc Mce B. Ahr6n and I. Lundqust Department of Pharmacoloy, Unversty of Lund, Lund, Sweden Summary. The present nvestaton was undertaken to determne and characterze lucaon mmunoreactvty n plasma from normal NMRI mce and from dystrophc mce and ther unaffected lttermates of the 129/ReJ stran. Very youn dystrophc mce (6 weeks old) dsplayed much hher basal levels of plasma lucaon mmunoreactvty than normal mce. In contrast, plasma concentratons of nsuln and lucose were lower n these dystrophc mce than n normal NMRI mce. The plasma lucaon levels declned wth ae n both strans durn the tme-perod studed (1.5-5 months). Gel fltraton of plasma from dystrophc as well as normal mce on Sephadex G-2 revealed that a lare part of the total lucaon mmunoreactvty was eluted n fractons contann the mmunolobulns. The amount of the 'true' lucaon part was lower n plasma from normal mce (about.2 ~t/1) than n plasma from mce of the dystrophc stran (.4-.5 ~t/1) ). Ths fndn was ndrectly corroborated by the observaton that a lare ntravenous lucose load decreased plasma lucaon by approxmately.2 Ix/1 n the non-dystrophc NMRI stran and by about.4-.6 ~t/l n the dystrophc stran. Thus, the ablty of lucose to suppress lucaon secreton appeared unaffected n the dystrophc mce. Glucose-nduced nsuln release, however, was consderably mpared n these anmals. It s concluded that mce of the dystrophc 129/ReJ stran have hher plasma levels of'true' lucaon than mce of the non-dystrophc NMRI stran. Whether the abnormally hh plasma lucaon levels n the dystrophc stran, partcularly n very youn dystrophc mce, mht contrbute to the development of the muscular dystrophy remans to be elucdated. Key words: Plasma lucaon, mce, muscular dystrophy, el fltraton, mmunolobulns, lucose, nsuln. A lare number of studes n dfferent speces have been undertaken n order to measure pancreatc lucaon n plasma by radommunolocal technques [1-9]. However, t has been dffcult to evaluate results from varous laboratores because of the dfferent specfcty of the varous lucaon antsera, crossreacton wth extrapancreatc lucaon and wth certan lobulns present n normal plasma and speces dfferences. Data from the mouse are lmted [8, 9]. In an earler study t was observed that the pancreas of a dystrophc mouse stran (129/Re J) dsplayed hh concentratons of lucaon as measured by radommunolocal and mmunohstochemcal methods [1]. Moreover, blood lucose levels were low n these dystrophc mce and the release of nsuln was dsturbed [1, 11]. To study further the abnormaltes of the carbohydrate metabolsm n these mce, we have attempted to measure and characterze crculatn lucaon mmunoreactvty n the dystrophc mce and ther unaffected lttermates n comparson wth normal non-dystrophc NMRI mce. Materals and Methods Anmals Female 129/ReJ dystrophc mce and ther unaffected lttermate controls were obtaned from the Jackson Laboratory, Bar Harbor, Mane, USA. The dystrophc anmals were homozyous for the dystrophc ene (dy/dy), whereas ther clncally unaffected lttermates were ether heterozyous (dy/dy) or homozyous normals (Dy/Dy). ach dystrophc mouse was housed wth ts lttermate control n an ndvdual cae and fed on a standard pellet det (Astra-wos, S6dert~lje, Sweden) supplemented wth wheat erm and dred mlk. A smlar det was ven to female mce of the NMRI stran (a non-dystrophc stran, Laboratory Anmal Breedn, Laven, Denmark). M1 anmals had free access to food and tap water before and throuhout the study X/82/22/258/$1.2

2 B. Ahr6n and I. Lundqust: Glucaon n Mouse Plasma c 15 O 2 o~ 1.. f".5- ""._,... I I I I Ae (months) F. 1. Basal plasma lucaon mmunoreactvty n relaton to ae n non-dystrophc NMRI mce (n = 1) ( ), n dystrophc mce (n = 19) (... ) and ther unaffected lttermates (n = 18) (... ) of the 129/ReJ stran. All data are expressed as mean + SM. There are snfcant dfferences between the NMRI mce and the other roups of mce at all tme ponts recorded Determnaton of Glucaon Immunoreactvty n Mouse Plasma Blood (1 l) was collected by orbtal puncture n unanaesthetsed mce nto ced tubes contann aprotnn 1 ~tl (15, kallkren nactvator unts, KIU/ml). After centrfuaton, plasma (25 lxl) was ppetted nto chlled lass tubes contann phosphate buffer 75 p3 (.4 tool/l, ph 7.4) wth human serum albumn 1 /l, NaCI 13 mmol/1; aprotnn 2 KIU/ml and sodum mertholate.59 mmol/l. The tubes were stored at - 2 ~ C untl analysed. The concentraton of lucaon mmunoreactvty was measured wth alcohol-precptaton of the antbody-bound lucaon mmunoreactvty [3] usn antserum K 964 (Novo Research Insttute, Copenhaen, Denmark). Ths antserum s rased n rabbts and drected aanst the C-termnal resdue of porcne pancreatc lucaon. The antserum was stored n a phosphate buffer (.4 tool/l, ph 7.4) wth the addton of human serum albumn 1 /1 (Behrnwerke AG, Marbur, Germany) and used n a fnal dluton of 1:2. Twce crystallzed pork lucaon, used as standard, and J25I-labelled porcne lucaon were from Novo Research Insttute, Copenhaen, Denmark. They were stored n the phosphate buffer; two standard solutons were also added NaCl 13 mmol/1 and aprotnn.2 KIU/ml. In the assay, 25 ~tl samples of ths buffer soluton, contann human serum albumn 6 /l, were added to lucaon 75 ~tl standard soluton. The coeffcents of varaton were: 7.3% ntra-assay (n = 8) and 12.9% nterassay (n = 8) at the medum rane of the standard curve. The lower lmt of detecton was 1 n/l. Determnaton of Glucaon Immunoreactvty n Gel Fltrated Mouse Plasma Pooled plasma (.5 ml) from NMRI mce, dystrophc mce and unaffected lttermates was appled to a Sephadex G-2 column (1.6 x 85 cm). The plasma was eluted wth buffer consstn of lycne sodum hydroxde (.2 tool/l, ph 8.8), human serum albumn 2.5 /l and aprotnn 5 KIU/ml. The flow rate was 7.2 ml/h. Fltraton was performed at 4 ~ C, and the fractons (3.7 ml) were stored at -2~ untl analysed for content of lucaon mmunoreactvty. Absorbance at 28 nm was recorded for dentfcaton of the presence of proten. For calbraton of the column, Blue Dextran (Pharmaca, Uppsala, Sweden), human mmunolobuln (Kab, Stockholm, Sweden) and 125I-lucaon were used. 125I-Glucaon was appled twce, frst toether wth plasma (.5 ml) from NMRI mce and then toether wth a mxture of human mmunolobuln 12.5 m and human serum albumn 1 m, n manntol.4 mol/l, the fnal volume ben.5 ml. On both occasons 125I-lucaon 7.4 n (specfc actvty 228 ~tc/~t) was used and the lzsi-lucaon was recovered n the same eluton poston. The concentraton of lucaon mmunoreactvty n the fractons from the el fltraton was measured by radommunoassay usn antserum K 964. The antserum, standards and 125I-labelled lucaon were from Novo, Copenhaen, Denmark and dentcal to those used for measurn the lucaon mmunoreactvty n plasma. For the purpose of determnn lucaon n the el fltrated fractons, however, they were stored n the lycne buffer. When assayn the fractons, 1 l-d of a soluton of NaCI 1.2 mol/1 was added to 9 ~1 of the fracton content, and to ths soluton the antserum and the tracer were added. Tubes wthout antserum served as controls for the non-specfc precptaton. Determnaton of Plasma Immunoreactve Insuln and Plasma Glucose Plasma concentratons ofmmunoreactve nsuln were determned by radommunoassay [12]. Concentratons of plasma lucose were determned enzymatcally [13]. xperments n Vvo D-lucose (Brtsh Dru Houses, Poole, UK) was njected IV nto a tal ven;.22 mmol/mouse was ven and the volume load was 1 ~tl/ body weht. Blood was sampled by orbtal puncture n conscous mce [14] mmedately before and at dfferent ntervals after the njecton. In ths partcular experment hormone analyss was performed on two 1 ~tl plasma samples. L-Arnne hydrochlorde (Brtsh Dru Houses, Poole, UK) was njected IV (2.38 mmol/k) and blood was sampled mmedately before and 2 mn after the njecton (peak level of lucaon concentraton n mouse plasma follown a rapd IV njecton of L-arnne). Results Determnaton of Glucaon n Plasma from the Dfferent Strans Blood samples were taken from unanaesthetzed mce at 1.5, 3.5 and 4.5 months of ae and the plasma concentratons of lucaon mmunoreactvty were measured. Fure 1 shows the values n the three roups of mce durn ths tme perod. It s seen that the plasma concentratons of lucaon were hher n the youner mce than n adults. It can also be seen that the concentratons of lucaon were hher n the 129/ReJ stran than n non-dystrophc NMRI mce, and that the dystrophc mce had values above ther lttermate controls. At 1.5 months of ae the plasma levels of lucaon, nsuln and lucose were recorded (Table 1). It appears that dystrophc mce had low levels of lucose and nsuln and hh levels oflucaon compared wth normal non-dystrophc NMRI mce.

3 26 B. Ahr6n and I. Lundqust: Glucaon n Mouse Plasma Table 1. Basal plasma levels of lucose, nsuln and lucaon n non-dystrophc NMRI mce and n dystrophc mce and ther unaffected lttermates of the 129/ReJ stran at 6 weeks of ae No Plasma lucose p Plasma nsuln p Plasma lucaon p of mce (retool/l) (mu/1) (~/1) NMRI _ _ Dystrophc mce _+.1 Lttermates _+.7 All data are expressed as mean + SM. NS = not snfcant <.5 <.1 < _+.18 NS NS < _+.5 Influences of Aprotnn, Temperature, and Dluton on Determnaton of Glucaon n Mouse Plasma Plasma samples taken wth or wthout the addton of aprotnn and then stored for 6 h at room temperature had vrtually the same lucaon concentraton (.59 _+.2 and.55 _+.2 lx/1) as samples taken n cechlled tubes wth aprotnn (.56 +_.2 lx/1) (mean + SM). In addton, L-arnne (2.38 mmol/k) was njected IV to stmulate the secreton of lucaon. Plasma samples were taken 2 mn after the njecton and analysed for lucaon. Arnne nduced an ncrease n plasma lucaon by.59.8 tx/1 above basal. When the 2 mn samples were taken wthout aprotnn, the ncrease n plasma concentratons of lucaon was.55 _+.1 ~/1 (mean + SM). The effect of plasma dluton on the apparent lucaon concentraton n mouse plasma s seen n Fure 2. Plasma samples wth both hh (arnne-stmulated) and low lucaon levels were chosen. The observed values do not devate snfcantly from the expected values. ffects of Glucose on Plasma Concentratons of Glucaon Immunoreactvty, Immunoreactve Insuln, and Glucose. D-Glucose (.22 mmol/mouse) was njected IV to mce at the ae of 5 months. Blood samples were taken mmedately before the njecton and at dfferent tme ntervals thereafter. The ntal plasma lucose levels were mmol/1 n dystrophc mce, mmol/1 n lttermates, and mmol/1 (mean + SM) n NMRI mce. Fure 3 shows the depresson of plasma lucaon by lucose n all three roups of mce to about half of the pre-njecton level. The values decreased durn the frst 3 mn and thereafter stablsed. Also shown are the chanes n the levels of plasma nsuln and lucose n response to the lucose njecton: nsuln release was reatest n the non-dystrophc NMRI stran. It should o.5-.3 o.1 O, =2 1:~ 118 Plasma Dluton F.2. ffect of dluton on apparent lucaon concentraton n plasma from non-dystrophc NMRI mce. Basal (... ) and arnne-stmulated ( ) plasma pools are used. ach pont represents the mean + SD of fve determnatons be noted that ths lare nsuln release n the NMRI mce was obtaned wth a lower total dose of lucose (F. 3), snce each mouse receved the same lucose load (.22 mmol/mouse) and the body-weht of the NMRI mce was enerally somewhat hher than that of the 129/ReJ mce. Calculaton of the lucose elmnaton rate (k-value) n the dystrophc anmals (k = )and ther lttermates (k = ) (mean _+ SM) demonstrated a snfcant mparment of lucose tolerance n the dystrophc mce (p <.25). The k-value for NMRI mce could not be accurately calculated n the present experment because n some of these mce ntal plasma lucose levels had already been reached after 15 mn (F.3). Gel Fltraton of Plasma Pooled plasma from the three dfferent roups of mce,.5 ml from each, was appled to a Sephadex G- 2 column and eluted wth lycne buffer (F. 4). All three roups dsplayed mmunoassayable lucaon

4 B. Ahr6n and I. Lundqust: Glucaon n Mouse Plasma t > ~c oo 49 3 ~ 2 o 1, r~ r~ o 6 ].4 ~ c 2 o, ~3 < 15 IVo IV1 A ' 45 5' I-lucaon ~ 15~ Plasma Proten,,-,[ I / Fracton Number 25- >' 4._> IV ~ IV ~ B o :#,,',....[ t 6 15! 3 6 Glucose njecton Tme (ran) F. 3. ffects of IV lucose (.22 mmol/mouse) on plasma concentratons of lucaon mmunoreactvty, mmunoreactve nsuln, and lucose, n non-dystrophc NMRI mce ( ), n dystrophc mce (... ) and ther unaffected lttermates (... ) of the 129/ ReJ stran. All data are expressed as mean _+ SM. (n = 1 for each roup) n the fractons where lucaon should be eluted, accordn to the eluton profle of 125I-lucaon. The total lucaon content n these fractons was 98 p for non-dystrophc NMRI mce (F. 4A), 257 p for the dystrophc mce (F. 4 B) and 27 p for ther unaffected lttermates (F. 4 C). All three roups also had a lare quantty of lucaon mmunoreactvty n the fractons where the mmunolobulns were eluted. The total amount of lucaon mmunoreactvty n these fractons was 696 p for NMRI mce, 527 p for dystrophc mce, and 453 p for ther unaffected lttermates. Determnaton of Glucaon Immunoreactvty n Solutons of Human Immunolobuln Snce lucaon mmunoreactvty was found n fractons contann mmunolobulns, solutons wth known concentratons of human mmunolobulns were made n lycne buffer and lucaon mmunoreactvty was recorded. Fure 5 shows that human mmunolobulns dsplay lucaon mmunoreactv- ~r 3 ~ 2 o_ 1 9 =s 15 o o.6j 4 2 o < 1'5 >. 4 -.>_ ~6 = 3 - ~s ~ lo- N O- O4 ~ 2 xz P, 2 Proteo 2s 3' 35 L--/X'' 4'o 45 5'o 71 & t2,_oucaon OJ o Fracton Number {V IV 1 C Plasm~rot en 1251" ~ On 1::::t ~....,.-I < Fracton Number F. 4. Sephadex -2 el chromatorams of lucaon mmunoreactvty n plasma from non-dystrophc NMRI mce (A), from dystrophc mce (B) and ther unaffected lttermates (C) of the 129/ ReJ stran. Arrows ndcate the eluton volume of blue dextran (V) and human mmunolobuln (V1). Fltraton profles of plasma protens and 125I-lucaon are also ndcated ty and that ths mmunoreactvty s dependent upon mmunolobuln concentraton. However, after addton of smlar amounts of human mmunolobutns to mouse plasma, recovery of the lucaon mmunoreactvty was reduced (F. 5). Apparently crculatn concentratons of mmunolobulns (2 m/ml) may contrbute to basal assayable lucaon mmunoreactvty n plasma.

5 262 B. Ahr6n and I. Lundqust: Glucaon n Mouse Plasma 4. "5 O2- } o- ( Human Immunolobuln (/I) F.5. Dose related ncrease n lucaon mmunoreactvty by reactants n human mmunolobulns added to lycne buffer ( ) or added to mouse plasma n the routne assay (... ) Dscusson The present study shows that t s possble to assay lucaon n small samples of plasma (1-25 ~tl) from ndvdual small anmals such as the mouse. Snce unknown reactants n plasma mmunolobulns apparently nterfere n the assay of the basal plasma lucaon levels, t seems advsable to measure and calculate the acute chanes n plasma lucaon from basal values rather than the absolute values when studyn lucaon release mechansms n these small anmals n vvo. Chromatoraphy of mouse plasma revealed that a lare part of the lucaon mmunoreactvty was eluted n fractons contann the mmunolobulns, and only a mnor part n the fractons where the 125Ilucaon was eluted. Thus, the chromatoram suests that the mmunolobulns n mouse plasma may cross-react wth the lucaon antserum. However, the data do not exclude the possblty that moetes dfferent from the mmunolobulns, althouh of smlar sze, could be responsble for ths lare lucaon mmunoreactve fracton. An nterference of human mmunolobulns wth lucaon antsera has been reported prevously [6], and s further demonstrated by the 'standard curves' obtaned when measurn lucaon mmunoreactvty n solutons contann human mmunolobulns (F. 5). Snce only a certan fracton of the lucaon mmunoreactvty n mmunolobulns added to plasma was recovered n the assay, t s concevable that when plasma lucaon s assayed, the rato: "'true' lucaon/lucaon mmunoreactvty n mmunolobulns", s not as small as would be suested by the el fltraton studes,. e. part of the lucaon mmunoreactvty n the mmunolobulns seems to be masked n plasma. The fractons contann mmunolobulns may consst of 'b plasma lucaon' [15] or the 'nterference factor' [16]. These are both hh molecular weht factors reactn wth lucaon antsera, but ther relaton to lucaon and lucaon metabolsm or to mmunolobulns s not fully establshed. It should also be noted that mmunolobulns nterfere wth the radommunoassay of astrn [17]. Dystrophc mce, and ther lttermate controls, had hher values of plasma lucaon mmunoreactvty than normal, non-dystrophc NMRI mce. The results from the el fltraton studes were smlar to those n the non-dystrophc NMRI mce, n that more than half of the total plasma lucaon was obtaned n fractons contann mmunolobulns. However, the dystrophc mce and ther lttermates had larer amounts of lucaon than NMRI mce. Althouh t s stll possble that these fractons may contan mmunoreactve peptdes of about the same sze as lucaon, t s lkely that the hher values of mmunoreactve lucaon n the dystrophc mce reflect truly reater levels of crculatn lucaon. Such an ncrease n the dystrophc mce may be consstent wth our recent observatons of a hher concentraton of lucaon n the pancreas and a reater number oflucaon cells n the slets of the 129/ReJ stran than n the non-dystrophc NMRI stran [1]. Moreover t s noteworthy that the apparent lucaon mmunoreactvty of the mmunolobulns n the non-dystrophc NMRI stran was hher than n the dystrophc 129/ ReJ stran althouh the total amount of serum protens, as revealed by the proten eluton profle, seemed to be smlar to the normal non-dystrophc NMRI mce (F.4). It s smlarly of nterest that muscular dystrophc chckens have reduced levels of IG but mantan normal levels of total serum protens, IA and IM when compared wth normal nondystrophc chckens [18]. The dystrophc mce were shown to have lower plasma lucose concentratons than normal NMRI mce [1, 11], and very youn dystrophc mce also dsplayed lower plasma nsuln. Apparently the dystrophc mce have an unusual nsuln/lucaon balance wth a low nsuln and a hh lucaon level n plasma and, despte ths, have a low plasma lucose level. Furthermore, the dystrophc stran had a low and delayed nsuln release n response to lucose (F. 3). In earler studes, other abnormaltes n the nsuln releasn mechansms of the dystrophc mce have been demonstrated [1, 11]. Apart from lucose-nduced nsuln release, nsuln secreton stmulated by the sulphonylurea aent lbenclamde s low, whereas nsuln release nduced by the fl-adrenoceptor aonst sopropylnoradrenalne s enhanced [1, 11]. Whether stmulaton of lucaon secreton n these mce wll

6 B. Ahr6n and I. Lundqust: Glucaon n Mouse Plasma 263 reveal any abnormaltes n lucaon release remans to be establshed. However, the very hh plasma lucaon levels measured n youn dystrophc anmals do ndeed suest abnormal reulaton of basal lucaon secreton. Glucose nhbts the release of lucaon [19], and we showed that, 3-6 mn after an IV njecton of a lare dose of lucose, the concentratons of plasma lucaon were depressed n all three roups of mce (F. 3). Ths decrease was about.2 ~t/1 n normal mce and.4-.6 ~t/1 n dystrophc mce. These values are n far areement wth the el fltraton lucaon values of approxmately.2 tx/1 plasma, eluted n the lucaon zone n normal mce, and.4-.5 tx/l n the dystrophc stran. It cannot be excluded that the remann part of the ntal basal lucaon mmunoreactvty s the cross-reactn factor(s) found n fractons contann mmunolobulns n the chromatoraphc study, snce n vtro studes wth the perfused pancreas have shown that ntroducton of a lucose concentraton as small as 5 mmol/1 causes total nhbton of lucaon secreton [2]. In summary, the dystrophc mouse stran 129/ReJ dsplays abnormally hh levels of plasma mmunoreactve lucaon. Mce wth overt muscular dystrophy have hher plasma lucaon levels than ther unaffected lttermates. Gel fltraton studes suest that the dystrophc stran ndeed has hher plasma concentratons of true lucaon than the non-dystrophc NMRI stran. A lare part of the basal plasma lucaon n both strans conssts of unknown reactants n the mmunolobuln fracton. Whether the observed abnormaltes of plasma lucaon values n the dystrophc 129/ReJ stran, partcularly n very youn dystrophc mce, may contrbute, drectly or ndrectly, to the development of ther muscular dystrophc dsease remans to be elucdated. Acknowledements: The authors express ther apprecaton to L. Kvst and P. Okmark for techncal assstance and to. Bj6rkbom for secretaral help. Ths study was supported by the Swedsh Medcal Research Councl (14P4289, 14X-4286) and by rants from the Medcal Faculty, Unversty of Lurd, Sweden and Nordsk Insulnfond, Gentofte, Denmark. References 1. Uner RH, sentraut AM, McCall MS, Keller S, Lanz H-C, Madson LL (1959) Glucaon antbodes and ther use for mmunoassay for lucaon. Proc Soc xp Bol Med 12: sentraut A, Ohneda A, Panada, Uner RH (1968) Immunoloc dscrmnaton between pancreatc lucaon and enterc lucaon-lke mmunoreactvty (GLI) n tssue and plasma. Dabetes 17: Hedn LG (1971) Radommunolocal determnaton of pancreatc and ut lucaon n plasma. Dabetoloa 7: Alford P, Bloom SR, Nabarro JDN (1977) Glucaon levels n normal and dabetc subjects: use of a specfc mmunoabsorbent for lucaon radommunoassay. Dabetoloa 13 : 1~6 5. Host J J, Aasted B (1974) Producton and evaluaton of lucaon antbodes for radommunoassay. Acta ndocrnol (Copenh) 77: von Schenk H (1977) Producton and characterzaton of an antserum aanst pancreatc lucaon. Cln Chm Acta 8: Wer G (1977) Assessment of lucaon mmunoreactvty n plasma. In: Fo/t PP, Bajaj JS, Fo NL (eds) Glucaon: ts role n physoloy and clncal medcne, Sprner-Verla, New York Hedelber Berln pp Cuendet GS, Loten G, Cameron DP, Renold A, Marlss B (1975) Hormone-substrate responses to total fastn n lean and obese mce. Am J Physo1228: Lavne RL, Voyles N, Perrno PV, Recant L (1975) The effect of fastn on tssue cyclc AMP and plasma lucaon n the obese hyperlycemc mouse. ndocrnoloy 97: /. Lundclust I, Hfkanson R, Harrs JB, Lbelus R, Sundler F (1979) ndocrne pancreas n the dystrophc mouse. Ann NY Acad Sc 317: Lundqust I, Harrs JB (1979) Insuln secreton and carbohydrate metabolsm n the dystrophc mouse. urop J Pharmacol 53 : Hedn L (1966) A smplfed nsuln radommunoassay method. In: Donato L, Mlhaud G, Srchs J (eds) Labelled protens n tracer studes. uratom, Brussel pp Bruss ML, Black AL (1978) nzymatc mcrodetermnaton of lycoen. Anal Bochem 84: Rerup C, Lundqust I (1966) Blood lucose level n mce I. valuaton of a new technque of multple seral sampln. Acta ndocrnol (Copenh) 52: Valverde I, Vllanueva ML, Lozano I, Marco J (1974) Presence of lucaon mmunoreactvty n the lobuln fracton of human plasma ("b plasma lucaon"). J Cln ndocrnol Metab 39: Wer GC, Knowlton SD, Martn DB (1975) Hh molecular weht lucaon-lke mmunoreactvty n plasma. J Cln ndocrnol Metab 4: Rehfeld JF, Schwartz TW, Stadl F (1977) Immunochemcal studes on macromolecular astrns. vdence that "b b astrns" are artfacts n blood and mucosa but truly present n some lare astrnomas. Gastroenteroloy 73: Sanders BG, Klne K (1977) IG mmunolobuln defcency n muscular dystrophc chckens. J Heredty 68: Uner RH, Orc L (1976) Physoloy and pathophysoloy of lucaon. Physol Rev 56: Grodsky GM, Fanska R, Lundqust I (1975) Interrelatonshps between a and fl-anomers of lucose affectn both nsuln and lucaon secreton n the perfused rat pancreas. ndocrnoloy 97: Receved: 24 February 1981 and n revsed form: 17 November 1981 Dr. Bo Ahr6n Department of Pharmacoloy S61veatan 1 S Lund, Sweden