SERIAL ASSAYS OF PLASMA GROWTH HORMONE IN TREATED AND UNTREATED ACROMEGALY*

Transcription

1 SERIAL ASSAYS OF PLASMA GROWTH HORMONE IN TREATED AND UNTREATED ACROMEGALY* W. M. HUNTER, F. J. GILLINGHAM, P. HARRIS, J. A. KANIS, F. M. McGURK, J. McLELLAND AND J. A. STRONG Medical Research Cuncil Radiimmunassay Team, 2 Frrest Rad, Edinburgh, EHI 2QW, and the University Departments f fsurgical Neurlgy, Raditherapy and % Medicine, Western General Hspital, Edinburgh, EH4 2XU (Received 25 September 1973) SUMMARY Assays fr human grwth hrmne (HGH) were carried ut n 89 acrmegalic patients, 81 f whm were studied befre any treatment had been given. Serial studies were undertaken, generally at 6-mnthly intervals, with the same test prcedure, using a 50 g ral glucse tlerance test (GTT) and identical assay cnditins ver a perid f 8 years. Twenty-three patients were assessed at intervals during perids f up t 4 years whilst they remained untreated. The general picture was ne f unchanging HGH levels. Ten patients were studied befre and after external irradiatin. HGH levels shwed a slw cntinuing fall fr as lng as 4 years and thereafter they stabilized at ne-third f pretreatment values. HGH levels in 12 patients treated with radiactive implants shwed an immediate fall t ne-third, and thereafter a further slw decline t ne-tenth f pretreatment levels. The respnse in eight patients treated by surgical remval f pituitary tissue and subsequent raditherapy varied cnsiderably. N patient, treated r untreated, shwed evidence f partial suppressin f HGH secretin during the GTT althugh three patients cnsistently respnded t glucse with paradxical hypersecretin. INTRODUCTION Plasma grwth hrmne (HGH) determinatins (Hunter & Greenwd, 1962, 1964; Glick, Rth, Yalw & Bersn, 1963) have been f value in cnfirming r refuting a clinical diagnsis f acrmegaly, but may als be used fr mnitring prgress and assessing the respnse t treatment, as well as prviding ne index f the endcrine activity respnsible fr the cnditin. There is still n single frm f treatment which is bth s effective and s free f risks as t be an autmatic chice fr all patients. The mst widely available frms f treatment at present include external irradiatin f the pituitary gland, the implantatin f radiactive surces * Reprint requests shuld be addressed t Dr W. M. Hunter.

2 in the gland r tumur, r the remval f the tumur by pen peratin. The respnse t treatment, as judged by HGH assay, is particularly imprtant. The bject f this study, in which identical test and assay prcedures were used ver a perid f 8 years, was t btain further infrmatin abut the changes in plasma HGH levels that ccurred in patients wh were: (1) untreated, and (2) treated by external irradiatin, radiactive implants, r surgical remval f tumur tissue, and t make cmparisns between these methds. The crrelatin f the clinical and ther bichemical data with the plasma HGH findings are the subject f a separate cmmunicatin (Kanis, Gillingham, Harris, Hrn, Hunter, Redpath & Strng, 1974). PATIENTS AND METHODS Eighty-nine patients with a clinical diagnsis f acrmegaly have been studied. These cmprised eight patients previusly treated and 81 wh were untreated when first seen. Of the 81 untreated patients, 58 had HGH assays perfrmed n ne ccasin nly, and 23 were tested n mre than ne ccasin fr perids f up t 4 years. Of the eight patients previusly treated, three had received external irradiatin nly, ne a radiactive implant, and fur transfrntal hypphysectmy fllwed by raditherapy. Thirty patients, untreated when first studied, were then treated by external irradiatin (10), radiactive implant (12) r hypphysectmy with raditherapy (8). The chice f treatment was based partly n clinical needs and partly n the methds available at the time that the patient required treatment. Hypphysectmy with raditherapy was favured fr patients with evidence f suprasellar extensins f the tumur. External irradiatin Methd f treatment A 4 MeV linear acceleratr was used; 16 equal daily dses were given ver a perid f 22 days using lateral fields frm left t right n alternate days. The ttal (tumur) dse was rad. Radiactive implants Yttrium-90 (gld-198 in the case f patient N. 81 nly) implants were placed by the trans-sphenidal apprach. The number f implants emplyed and the radiatin dses were calculated frm the area f the pituitary fssa determined radilgically. One r tw implants were used and the dse f each implant varied frm 1-5 t 8 mci. The minimum peripheral tumur dse ranged frm t rad. Hypphysectmy with raditherapy A transfrntal apprach t the sella was used t remve as much f the tumur as pssible. When the wund had healed, raditherapy was applied as described under 'External irradiatin'. T distinguish this cmbined treatment frm external irradiatin r internal irradiatin by radiactive implants, we shall refer t this hereafter by the title shwn.

3 Methd f assay Human grwth hrmne was measured by radiimmunassay ; a single antiserum and the same incubatin cnditins were used thrughut these studies. During the first year f the study a labratry preparatin f HGH, extracted by the methd f Raben (1959), was used as the standard. In 1964 MRC-HGH Standard A, a stable ampuled preparatin (Bangham & Ctes, 1971) which was fund t be equiptent with the labratry standard, became available frm the MRC Divisin f Bilgical Standards, and this has been used as the standard in all subsequent assays. When the Internatinal Reference Preparatin 66/127 became available in 1966, MRC Standard A was fund t have a ptency f 0-96 i.u./mg in ur assay, i.e. fr practical purpses 1 ng MRC Standard A= 1 / u. In 1970 the prcedure fr separating bund and free labelled HGH was changed frm that using cellulse acetate electrphresis (Hunter & Greenwd, 1964) t a duble antibdy prcedure, first suggested by Mrgan & Lazarw (1962). This emplyed a single batch f dnkey anti-rabbit serum which was unaffected in its ability t precipitate antibdy-bund 125I-labelled HGH ver the range 0-25 % human plasma. Details f the ptimizatin f this system, with respect t dilutins f carrier nn-immune rabbit serum, and f secnd antibdy used have been reprted (Hunter, 1973). Befre the change in the separa tin prcedure was adpted, 110 samples f plasma were assayed n the same day in therwise identical incubates fr separatin by each f the tw prcedures. Thirty samples shwed undetectable levels f HGH ( < 1 ng Standard A/ml) by bth methds. The crrelatin cefficient fr the remaining samples which had HGH values ranging between 1 and 90 ng/ml was (P < 0-001); the intercept did nt differ significantly frm zer. The mean value by electrphresis was ng/ml and that by duble antibdy ng/ml. It was cncluded, therefre, that the tw methds were interchangeable. Plasma was assayed at tw r mre dilutins ver the range 1:4 t 1:128 as apprpriate and the mean f thse determinatins which fell mre than 10 (true) % bund belw the 0 Standard (crrespnding generally t 0-25 ng MRC Standard A/ml incubate) and were within the steep central prtin f the dse-respnse curve was used. When set up in this way the assay had a detectin limit f 1 ng MRC Standard A r 1 i.u./ml plasma. N departures frm parallelism between the respnse curves f individual plasma samples and standards were bserved. The within-assay cefficient f variatin calculated frm duplicate estimatins n 91 plasma samples selected at randm frm different assays carried ut during the 8 years cvered by this study was +6-8%. The between-assay cefficient f variatin frm the means f 110 plasma samples assayed in duplicate in each f tw separate assays was ±9-7%. Clinical test prcedure Patients were tested bth when first seen and n all subsequent assessments by means f an ral glucse (50 g) tlerance test (GTT). Between six and eight plasma samples were taken fr HGH measurement at 30-min intervals, glucse being given after the first tw (f six r seven samples) r the first three f eight samples.

4 RESULTS Pattern f HGH levels Human grwth hrmne has been measured in ver 300 glucse tlerance tests n 89 patients. In every case plasma HGH levels were clearly measurable, nt nly in the fasting state but thrughut the GTT. This picture cntrasted with that fund 300 h Patient n I 200 a» Patient n _L i 1 H 2 2i Time after glucse (h) Fig. 1. Plasma human grwth hrmne (HGH) levels (mean + s.e.m., between test) in three patients wh cnsistently shwed increased plasma HGH values after glucse. Patient N. 60 was studied ten times ver 5 years, N times in 4 years, and N times in 6 years. in nrmal subjects, wh withut exceptin had undetectable levels f HGH after glucse lading. Fr the acrmegalie patients tw patterns were fund. Three patients (Ns 36, 60 and 70) cnsistently shwed a paradxical rise in HGH after the glucse lad (Fig. 1). Fr the remaining patients the characteristic pattern was ne f unchanging HGH levels, n alteratin being seen after the glucse lad. N patient shwed partial suppressin f HGH secretin by glucse and any changes which might have been interpreted in this way did nt reappear in subsequent tests n the same patient. The values btained frm six t eight samples in a given test, therefre, represent the fluctuatins in secretin f an autnmus pituitary gland, tgether with assay variatin. Since bth variatins are randm the infrma tin prvided by an individual test may be represented as a mean + s.e.m. The data _L

5 25 frm the three cnsistent paradxical hypersecretrs have als been represented in this way fr purpses f cmparisn. Distributin f HGH values in patients when first tested The distributin f mean plasma HGH values (± s.e.m.) fr the 81 untreated patients at their first assessment is shwn in Fig. 2. This demnstrates a range vary ing 100-fld. When ranked in rder, ne-third f the patients shwed HGH values between 3-4 and 25 ng/ml, the middle third, ng/ml, and the upper third ranged frm 66 t 340 ng/ml. This distributin f HGH values was unrelated t the age r sex f the patients , J 100 b f» Ü E 20 I 104= ti.1* l.uh'iiihîji-*p*-tf* lì', '*1 I J_I_I_I_L,:I"i«tfl!, '. «'"in!_l_ Patient number (previusly treated patients), -I_ -1 _ _I_L Patient number (untreated series) = Fig. 2. Plasma human grwth hrmne (HGH) levels (means±s.e.m., calculated frm the arithmetic data) fr 6-8 samples cllected in the curse f an ral glucse tlerance test carried ut at first assessment. Patients Ns 1-81 had nt been treated, while patients Ns 82-8 had received sme treatment befre this assessment (see text fr details). The insert in Fig. 2 shws data fr the small grup f seven patients wh had already been treated befre their first assessment. An additinal patient (N. 89), nt shwn in Fig. 2, was first tested 3 years after hypphysectmy with raditherapy, and had a plasma HGH value f 500 ± 40 ng/ml, the highest level we have thus far encuntered. Serial plasma HGH determinatins in untreated patients Table 1 shws the HGH values frm 23 patients wh were assessed n tw r mre ccasins while still untreated. The initial HGH values d nt differ frm thse btained frm the ttal grup f 81 untreated patients, since eight fall int the range f the lwest third f the whle grup, ten int the middle third, while five are in the range f the upper third. Repeated assessments during prlnged perids withut treatment shwed little variatin. Plasma HGH levels in ten ut f the 23 patients remained within and + 50 % f their initial value, and a further eight 85 2

6 5~ «S. «* «ß S S +3 «CO " a c C -3-3 O,, y 'O. S S& S h-1 M O hh B 0 te B O! ß,2 ^ ^, b. S E ~ h-1 <5 M e Je S* S +» i *H i I I I I I c c I ti +i O O sm > Ó s d m i* tñ 00 CO l> ÏÏ + > s» 3 ti b c3 < s. TtiwlOC0-«00<íslTiHr-lOTt'iOs7Si0iO H^óArtMnñÑ«rt- n ci^nsc -*M -* ^«3îh^û0«OO!DOOOOOO10Q00ifflOOO10t-OOOOOOOOOOO v^ MHH-(^CsiíM<NtNrt^W^«Tttn^lO^^-stl0l0Cslns0Q0N0íOn-*O í < r~ c CO CO -* tji IO -^1 g h gg h hfeh g Pu g S fe g fe fcë g c tí ^2 H» H t- IN m 00 s «IÍ5 O CO CO

7 50 patients were always within the range t % f their initial level. In the five remaining patients greater changes ccurred (Table 1, cases 15, 41, 50, 39 and 60). Serial determinatins f HGH in patients after external irradiatin Plasma HGH levels were measured in ten patients immediately befre external irradiatin and subsequently at intervals ver perids f up t 8 years (Table 2). The distributin f HGH values befre treatment cmpared with the whle grup f 81 patients shws three patients in the lwer, five in the middle, and tw in the upper range. There was n change in plasma HGH cncentratin during the first 6 mnths after treatment but subsequently a fall became apparent. This fall cntinued until the end f the 4th year after treatment, when the mean value had fallen t abut ne-third f the levels befre treatment. Thereafter n further change was seen in mean values. There were, hwever, quite marked differences between individual patients, ne f whm shwed little change (N. 36), while during the perid f bservatin thers shwed almst cmplete suppressin (Ns 66, 37 and 35). Althugh the degree f change varied between patients, the time-curse was fairly cnstant, a slw change ver a perid f 4 years, after which n striking change was apparent. Serial determinatins f HGH in patients treated by radiactive implants The data f the 12 patients treated in this way are shwn in Table 3. The distribu tin f their HGH levels befre treatment shwed three patients frm the lwer, fur frm the middle, and five frm the upper range fr the 81 patients. By cntrast with the results btained in the patients treated by external irradiatin, these patients, with ne exceptin (N. 21), shwed an immediate fall after implantatin. This was mst marked, with almst cmplete suppressin, in three patients. Cl lectively the levels immediately after the peratin fell t abut ne-third f thse befre the peratin. This change resembles that achieved 4 years after external irradiatin. Six patients were assessed during the first 6 mnths after the peratin and subsequently at intervals. Fur f these patients (Ns 46, 68, 60 and 81) shwed an immediate fall and thereafter a further decline in their plasma HGH levels. Serial determinatins f HGH in patients treated by hypphysectmy with raditherapy Eight patients were treated by this methd (Table 4). Mean levels f HGH were high in this grup; indeed nly ne patient was fund t have initial levels f HGH within the lwer range fr the series f 81 patients, three patients shwed values in the higher part f the middle range, while the remaining fur were within the higher part f the upper range. The respnse t treatment was variable, ranging frm cmplete and immediate suppressin f secretin f HGH (N. 55) t unchang ing levels within the perid 6 mnths after treatment. Of the fur patients studied fr 6 mnths r lnger after treatment, all shwed a delayed fall cmparable t that seen in the patients treated by external irradiatin alne.

8 I i -h i tn mi c M< c N so c~ c tn I fe 1 t r- CO # m< m -H CM (M IO sm IM -H Ort«rt Ml.-h t~ GO c ^ «ii CN l00t-00mi (N (N tn ~H c œ c tn in ( B s IN sn s«3«h aj Ml s* c IO Ml IN 2 s B< «ß si ti eg t; il I N -H c c œ IO -rt ( tn IO --H IO +i +i +i +i c > --H tn 00 M< 03 S =3 > *-t CN h CO -* 00 O CO I CO th c *-.» s2 * li œ IO ^H I M -H,-'- IN CO ~H t- tn -* 00 b- IO CO MI MI CO»O C0 Ml w B. " stí fe fe fe fe fe fe fe b I 3

9 e i IN IN CO CO Ml r~ c tn -* IO Csl tn Ml IO tn 00 tn tn CO 5 ß <N IO (N CO ( t- 00 CO -h CO Ml tn -^i cs -S s s IO Mi CO tn tn CO tn <N IO CO -* -O Ml tn 2 e- IO IO Csl + - S» e-... S I S - sn -* CO tn Cst tn c cs, 2- fe M s ~* tn CO CO sn +i +i +i +i +i IO 00 tn tn tn Csl tn -H CO - c Csl Csl Cl X -* IQ s-h rt œ tn IO Ml IO CO «S «es C b S < Ss mi œ Mi IO lo Ml fegfegfe Ml fefegfegfefe b I

10 r I I M I I I S e* ûo tn s - ce S 11 Ml CO IO tn fl is ^ IO IO +i I +i +i +i 'Ì S S <a c S c tn Csl go -ß A a a s s ta sa C* ^> s; J. «I i +i CO 2. O S, Ss s -s ß CO M* CO. -H Sj r.3eíh & S. «stn«)») PQtütNM<-* c fe a a a. <J ^ tî< e -h» s» tggggfefefeg g O MI IO Ml CO l> <N p; Cl IO IO IO t- O t-

11 DISCUSSION Diagnsis and assessment f acrmegaly The measurement f plasma HGH can prvide definite diagnstic evidence f state cannt be used fr this acrmegaly. Islated samples cllected in the fasting purpse, hwever, since nrmal subjects may shw intermittent HGH secretin with plasma levels ccasinally reaching as high as 100 ng/ml (Hunter & Greenwd, 1964; Sukkar, Hunter & Passmre, 1968). Thus the ntin f acrmegalie levels as ppsed t nrmal levels is miscnceived. Hwever, HGH secretin in nrmal sub jects is suppressed rapidly after a glucse lad, while in patients with armegaly nn-suppressible autnmus secretin cntinues (Rth, Glick, Yalw & Bersn, 1963). Sme authrs (Glick, 1967; Lawrence, Tbias, Linft, Brn, Lyman, Chng, Manugian & Wei, 1970) have described a stage f partial suppressin by glucse. In the curse f mre than 300 tests in 89 patients we have nt bserved partial suppressin. It is pssible that partial suppressin may ccur when the larger dse (100 g) f glucse emplyed fr the ral GTT in Nrth America is used. Three f ur patients shwed an increase in HGH levels in respnse t glucse; this rise was fund n all ccasins n which these patients were tested. Such a paradxical respnse t glucse with hypersécrétin f HGH was first described by Beck, Schalch, Parker, Kipnis & Daughaday (1965). Serial plasma determinatins in untreated patients Little is knwn abut the lng-term changes in plasma HGH levels in untreated acrmegalics. Rth, Grden & Brace (1970) studied three patients twice and fur patients three times ver perids f 1-4 years. Five patients shwed increases, ne a decrease and the ther unchanged levels f HGH. Our findings prvide additinal data f this type. Twenty-three f ur patients were tested n tw t seven ccasins, ver perids extending t 4 years. The results suggest that while cnsiderable changes in HGH levels may ccur spntaneusly, many patients shw essentially unvarying levels ver perids f many mnths r even years. Riglsi, Schwartz & Glick (1968) reprted a spntaneus cure in acrmegaly, apparently because f sudden haemrrhage int a pituitary tumur. This has nt been bserved by us and we believe must be an infrequent ccurrence. Plasma HGH in treated patients Serial assays f HGH prvide an index f the respnse t a particular methd f treatment, and culd indicate a need fr further interventin. The patterns f change in plasma HGH after treatment in ur patients may be summarized as fllws. External irradiatin prduces n immediate respnse but a steady delayed fall in HGH levels. Rth et al. (1970) reprted this delayed respnse when measuring HGH levels immediately after, 1 year, and 3 years after similar treatment. Lawrence, Pinsky & Gldfine (1971) als emphasized the delay in respnse which may ccur after external irradiatin f the pituitary gland, and described the results f serum HGH assays shwing striking reductins t nrmal levels in selected patients within 2-5 years f treatment. Our patients were assessed repeatedly fr perids f up t 7 years after treatment. The results cnfirmed that mst patients shw this delayed 3-2

12 fall, up t the furth year after treatment. The mean level at this time was abut ne-third f the levels befre treatment, althugh individual respnses varied widely. Treatment with radiactive implants by cntrast is usually effective immediately and HGH levels fall rapidly t abut ne-third f pretreatment values. Kaufman, Pearsn, Shealy, Chernat, Samaan & Strasli (1966) describe HGH cncentratins in nine patients befre and after such treatment. Single samples fr HGH assay were taken frm 2 weeks t 2 years after treatment. A mean depressin f HGH secretin f 63 % was btained in a ppulatin f patients whse initial mean HGH values were nly 34 ng/ml. Larger series f samples (Elkeles, Wright, Lwy & Fraser, 1969; Wright, Hartg, Patter, Tevaarwerk, Dyle, Arnt, Jplin & Fraser, 1970) shwed a mean depressin f HGH f 58 and 54 % respectively, after 1 year, whilst Frrest, Thmas, Richards, Wd, Stewart & Gleave (1970) achieved less satisfactry results. The respnse t radiactive implantatin is clearly dse-dependent (Hartg, Dyle, Fraser & Jplin, 1965; Wright et al. 1970). In these reprts less than rad were usually received by the peripheral part f the tumur. Our patients shwed a mderate immediate fall after radiactive implants f similar dses and ften a further delayed fall resembling that seen in the first 4 years after external irradiatin. As a result, HGH levels n average ultimately fell by 90 % frm the levels befre treatment. This cntinuing fall after radiactive implantatin des nt appear t have been reprted previusly. The respnse t hypphysectmy with raditherapy varied widely in the first year after the peratin, but all patients wh were restudied 2-6 years after treatment shwed further falls in plasma HGH levels. This delayed effect resembled clsely that seen after external irradiatin alne. The immediate respnse f HGH secretin after hypphysectmy, hwever, cmpared unfavurably with that after radiactive implantatin. A further methd f treating acrmegaly was described by Lawrence et al. (1970) wh reprted the HGH levels f 67 acrme galie patients befre and after treatment with an -particle beam frm a cycltrn. After this treatment HGH levels fell by 60% after 1 year, 65% after 2-3 years, 75 % after 4 years, with a steady further fall t 88 % in the few patients studied fr as lng as 8 years. Their results are smewhat better than thse f ur patients wh were treated with radiactive implants. General cnclusins Our data n untreated patients indicate clearly that HGH secretin in acrmegaly cannt be expected t diminish spntaneusly. The wide range f plasma HGH readings btained verall, and their failure t crrelate with the clinical features f the disrder, wuld suggest that these assays shuld be used fr diagnsis primarily, and then t assess the respnse t treatment, but nt fr the purpse f deciding whether t intervene. The chice f treatment fr the individual patient with acrmegaly will cntinue t be based n several criteria, including plasma HGH assays. The slw but steady fall in HGH secretin seen after external irradiatin in this series f patients is ntewrthy, and the nn-invasive nature f the technique is a cnsiderable advan tage. These and related cnsideratins are discussed in further detail in anther publicatin (Kanis et al. 1974). Befre HGH assays became widely available, the literature attested t the

13 benefits f external radiatin. Subsequently HGH assays shwed that raditherapy resulted in a variable and ften incmplete reductin f HGH secretin and this in turn led t the develpment f ther frms f treatment. If, hwever, the results f external irradiatin are assessed 4 years after treatment, the degree f suppressin f HGH secretin is cmparable with that achieved by ther methds f treatment. The results f treatment als require assessment in terms f mrbidity, mrtality and the need fr pituitary hrmne replacement therapy. Nn-invasive techniques such as external irradiatin r prtn beam bmbardment are clearly advantageus fr sme f these reasns. Further clinical studies are required in rder t develp a ratinal basis fr treatment strategy, nt nly in terms f HGH suppressin, but als in preventing r reversing sme f the metablic abnrmalities assciated with the acrmegalie state. It remains t be shwn that treatment can be cnsidered successful when the plasma HGH has fallen by (say) 70 r 80 per cent, and when the end result in the majrity f patients is still a nn-suppressible elevated fasting HGH. Furthermre, it is nt clear that an elevated fasting HGH f say 50 ng/ml, whether prer pst-perative, is equally disadvantageus t all patients. Cntinuing lng-term assessment with serial HGH assays is still required. We are grateful t a number f clinical clleagues in varius hspitals fr permis sin t use data frm their patients. REFERENCES Bangham, D. R. & Ctes, P. M. (1971). Reference standards fr radi-immunassays. In Radiimmunassay methds, pp Eds K. E. Kirkham & W. M. Hunter. Edinburgh: Churchill Livingstne. Beck, P., Schalch, D. S., Parker, M. L., Kipnis, D. M. & Daughaday, W. H. (1965). Crrelative studies f grwth hrmne and insulin plasma cncentratins with metablic abnrmalities in acrmegaly. J. Lab. clin. Med. 66, Elkeles, R. S., Wright, A. D., Lwy, C. & Fraser, T. R. (1969). Serum insulin in acrmegaly. Lancet ii, Frrest, A. P. M., Thmas, J. P., Richards, S. H., Wd, R. G., Stewart, H. J. & Gleave, E. N. (1970). Radiactive implants f the pituitary fr endcrine disease. Prc. R. Sc. Med. 63, Glick, S. M. (1967). Physilgy f grwth hrmne secretin. Ann. intern. Med. 66, Glick, S. M., Rth, J., Yalw, R. S. & Bersn, S. A. (1963). Immunassay f human grwth hrmne in plasma. Nature, Lnd. 199, Hartg, M., Dyle, F., Fraser, T. R. & Jplin, G. F. (1965). Partial pituitary ablatin with implants f gld-198 and yttrium-90 fr acrmegaly. Br. med. J. ii, Hunter, W. M. (1973). The radiimmunassay. In Handbk f experimental immunlgy, chap. 17, pp Ed. D. M. Weir. Lndn: Blackwell. Hunter, W. M. & Greenwd, F. C. (1962). A radiimmunelectrphretic assay fr human grwth hrmne. Bichem. J. 85, 39P. Hunter, W. M. & Greenwd, F. C. (1964). A radiimmunelectrphretic assay fr human grwth hrmne. Bichem. J. 91, Kanis, J.., Gillingham, F. J., Harris, P., Hrn, D. B., Hunter, W. M., Redpath, A. T. & Strng, J. A. (1974). Clinical and labratry study f acrmegaly : assessment befre and ne year after treatment. Q. Jl Med. (In Press.) Kaufman, B., Pearsn, O. H., Shealy, C. N., Chernat, E. S., Samaan, N. & Strasli, J. P. (1966). Transnasal transsphenidal yttrium-90 pituitary implantatin in the therapy f acrmegaly. Radilgy 86, Lawrence, A. M., Pinsky, S. M. & Gldfine, I. D. (1971). Cnventinal radiatin therapy in acrmegaly. Archs intern. Med. 128, Lawrence, J. H., Tbias, C.., Linft, J.., Brn, J. L., Lyman, J. T., Chng, C. Y., Manugian, E. & Wei, W. C. (1970). Successful treatment f acrmegaly : metablic and clinical studies in 145 patients. J. clin. Endcr. Metab. 31, Mrgan, C. R. & Lazarw, A. (1962). Immunassay f insulin using a tw-antibdy system. Prc. Sc. exp. Bil. Med. 110,

14 Raben, M. S. (1959). Human grwth hrmne. Recent Prg. Hrm. Res. 15, Riglsi, R. S., Schwartz, E. & Glick, S. M. (1968). Occurrence f grwth-hrmne deficiency in acr megaly as a result f pituitary applexy. New. Engl. J. Med. 279, Rth, J., Glick, S. M., Yalw, R. S. & Bersn, S. A. (1963). Secretin f human grwth hrmne: physilgic and experimental mdificatin. Metablism 12, Rth, J., Grden, P. & Brace, K. (1970). Efficacy f cnventinal pituitary irradiatin in acrmegaly. New Engl. J. Med. 282, Sukkar, M. Y., Hunter, W. M. & Passmre, R. (1968). The effect f a prtein meal with and withut subsequent exercise n plasma insulin and grwth hrmne. Q. Jl exp. Physil. 53, Wright, A. D., Hartg, M., Patter, H., Tevaarwerk, G., Dyle, F. H., Arnt, R., Jplin, G. F. & Fraser, T. R. (1970). The use f yttrium-90 implantatin in the treatment f acrmegaly. Prc. R. Sc. Med. 63,