Molecular composition of luteinizing hormone and folliclestimulating hormone in commercial gonadotropin preparations*

Transcription

1 FERTILITY AND STERILITY Cpyright e 1986 The American Fertility Sciety Vl. 46, N.6, December 1986 Printed in U.8A. Mlecular cmpsitin f luteinizing hrmne and flliclestimulating hrmne in cmmercial gnadtrpin preparatins* Jnas Harlin, M.D. Shafiq A. Khan, Ph.D.t Egn Diczfalusy, M.D.:j: Reprductive Endcrinlgy Research Unit, Department f Obstetrics and Gyneclgy, Karlinska Hspital, Stckhlm The bilgic (B) and immunlgic (l) prperties f luteinizing hrmne (LH) and fllicle-stimulating hrmne (FSH) were studied in three cmmercial urinary gnadtrpin preparatins and in the first internatinal standard preparatin f human urinary gnadtrpins befre and after fractinatin by iselectrfcusing (lef). Significant differences were fund in the IEF prfiles f bth biactive and immunreactive LH and FSH and in the BII ratis f the preparatins studied. The bserved differences in the mlecular cmpsitin f LH and FSH seem t be attributable t the purificatin prcedures emplyed. The pssible influence f these differences n the in viv ptencies, circulating half-lives, and clinical effects f gnadtrpin preparatins are discussed. Fertil SteriI46:155, 1986 Since Gemzell et al. 1 first published their study n the inductin f vulatin in amenrrheic wmen by human gnadtrpins, a wide variety f treatment regimens have been tried, with bth pituitary and urinary preparatins. 2 The schedules have ranged frm ne large single dse 3 f human menpausal gnadtrpin (hmg) t daily adjusted dses r t cntinuus infusin. 4 Because gnadtrpins f pituitary rigin are nt cmmercially available, urinary preparatins are used fr treatment by mst centers. With all regimens, Received April 25, 1986; revised and accepted August 13, *Supprted by Wrld Health Organizatin's Special Prgramme f Research in Human Reprductin, Geneva, Switzerland. tpresent address: Max-Planck Clinical Research Unit fr Reprductive Medicine, Steinfurter Str. 17, D-44 Munster, West Germany. :j:reprint requests: Prfessr E. Diczfalusy, Reprductive Endcrinlgy Research Unit, Karlinska Hspital, S-14 1 Stckhlm, Sweden. cmplicatins in the frm f verstimulatin and multiple gestatins are still ften seen, in spite f varius effrts t mnitr the dsage by different clinical and bichemical parameters, including ultrasngraphy.5, 6 Amng ther factrs believed t be f imprtance fr the clinical efficacy f the preparatins used, the fllicle-stimulating hrmne/luteinizing hrmne (FSH/LH) rati has been cnsidered. It was cncluded that a minimal threshld dse f LH accmpanying FSH is required t enable the latter t be effective, but abve this threshld level, LH des nt appear t influence the effect f FSH in terms f fllicle grwth. 7 Gnadtrpins, such as LH and FSH, exhibit a high degree f charge hetergeneity, predminantly due t differences in the amunt and/r nature f carbhydrate residues present in the different mlecular frms. 8 - l The relative abundance f different mlecular species f LH and FSH f pituitary glands and plasma has been shwn t depend n the sex and/r endcrine sta- Vl. 46, N.6, December 1986 Harlin et ai. IEF prfiles f human urinary LH and FSH 155

2 tus f the subject. 1l -14 Due t differences in the relative ptencies in vitr, terminal half-life, and receptr-binding characteristics,14, 15 it is cnceivable that different mlecular species induce different respnses, when administered clinically fr vulatin inductin. Because hmg preparatins are extracted frm pstmenpausal urine, they culd be expected t pssess LH and FSH patterns characteristic fr pituitary glands and plasma f pstmenpausal wmen, in cntrast t the mlecular prfiles present in nrmally menstruating wmen. Such a difference culd playa certain rle in the simultaneus inductin f maturatin f several fllicles. Indeed, individual respnses t hmg vary greatly, nt nly between different subjects and different cycles in the same subject, but als when different preparatins are used.5, 6, 16 Several studies indicate that wmen with cmparatively high endgenus levels f LH (e.g., in plycystic varian disease) run a higher risk f verstimulatin and multiple gestatin than patients with lw r nrmal LH levels.6, 16 Whether r nt the mlecular cmpsitin f the gnadtrpins is in part respnsible fr the bserved verstimulatin has yet t be ascertained. As a first step in this directin, we carried ut an in-depth assessment f the electrfcusing prfiles f biactive and immunreactive LH and FSH in cmmercial preparatins; the data reprted in this paper indicate majr differences in the mlecular cmpsitin f bth LH and FSH in the available urinary gnadtrpin preparatins. MATERIALS AND METHODS HORMONE PREPARATIONS Cmmercially available purified urinary gnadtrpin preparatins (Humegn, batch G F 13392, N V Organn, Oss, Hlland; Pergnal, batch 276 I F, and Metrdin, Ch. -B 15 P, Industria Farmaceutica Sern S.p.A., Rme, Italy) were used. The LH ptency f Pergnal is stated t be 75 IUlampule and that f Metrdin <.1 IUlampule; the LH cntent f Humegn is nt stated. FSH ptency is stated t be 75 IUlampule in all three preparatins. The standard preparatin f hmg (lst Internatinal Standard [IS] f urinary FSH and LH/interstitial cell stimulating hrmne (lcsh), human, fr biassay, cde n. 7/45) was used as a reference preparatin. The cntents f each ampule were disslved in 2. ml f 5 mml phsphate buffer (ph 7.4, cntaining.8% NaCI and 1.% bvine serum albumin), distributed int.5-ml aliquts, and stred at - 7 C until analyzed. ISOELECTROFOCUSING PROCEDURE The fur hrmne preparatins (Humegn, Pergnal, Metrdin, and the hmg 1st IS) were fractined by iselectrfcusing (lef) in triplicate r duplicate runs in sucrse density gradient with the use f 11-ml clumn (N. 81, LKB Prdukter, Brmma, Sweden). Amphlytes (Amphline; LKB Prdukter) were used at a cncentratin f 1 % t create a ph gradient in the ph range f 2.5 t 1.. The details f the IEF prcedure have been describedp The pi value (iselectric ph) (mean ± standard deviatin) fr beef hemglbin (Sigma Chemicals, Lndn, UK), used t assess the reprducibility f the IEF prcedure, was 7.85 ±.6 (n = 11). Bth biactive and immunreactive LH and FSH were assayed in the individual 14 fractins frm each IEF experiment, with the exceptin f Metrdin, which in an earlier analysis was shwn t cntain undetectable LH activity. T assess the verall lsses f gnadtrpin activit)r..during IEF, we recmbined and analyzed equal aliquts frm individual fractins frm each experiment, alng with the starting materials and the standard in multiple pint parallel line assays. DETERMINATION OF LH Biactive LH in the individual IEF fractins and in the unfractinated preparatins was determined with the use f an in vitr biassay prcedure based n the LH-dependent andrgen prductin by muse Leydig cell preparatins.18 The mean index f precisin19 (X.) in eight multiplepint parallel line assays was.3. Immunreactive LH was estimated with the use f a radiimmunassay (RIA) prcedure.2 The mean index f precisin (n = 8) was.4. DETERMINATION OF FSH FSH biactivity was determined with the use f an in vitr biassay prcedure using as an endpint the armatizing capacity f immature rat Sertli cells.21 Immunreactive FSH was assayed with the use f a previusly described RIA methd Harlin et ai. IEF prfiles f human urinary LH and FSH Fertility and Sterility

3 Table 1. In Vitr LH and FSH Biactivity and Immunreactivity (RIA)" f Urinary Gnadtrpin Preparatins LH RIA BII FSH RIA. BII FSHlLH RIA Humegn IUlampuk Pergnal IUlampule 42.5 ( ) 29.9 (27.~2.4) 84.7 ( ) 19.9 ( ) Metrdin IUlampule < ( ) 36.7 ( ) 32.5 ( ) 53. ( ) 4.4 ( ) 3.9 ( ) "Gemetric mean values with 95% cnfidence limits. Assay results are expressed in terms f the 1st IS f urinary FSH and LH (ICSH) fr biassay (hmg, 1st IS). HORMONE POTENCIES RESULTS When the LH cntent f the cmmercial ampules was assayed, bth the biactivity and immunreactivity f Humegn was fund t be significantly (P <.5) higher than thse f Pergnal (Table 1). Als, the rati f bilgic activity t immunreactivity (BII) f the tw preparatins was markedly different. Cmpared with the 1st IS, the frmer had an excess f immunreactivity and the latter an excess f biactivity. The highly purified FSH preparatin, Metrdin, did nt have detectable LH activity. The FSH cntent f the three preparatins was significantly lwer, accrding t bth in vitr biassay and immunassay prcedures, than thse stated by the manufacturers n the basis f in viv biassays. Humegn cntained significantly higher amunts f bth biactive and immunreactive FSH than Pergnal and Metrdin. The BII ratis f FSH were clse t unity in all preparatins. Althugh the FSHlLH ratis were similar in the tw cmmercial preparatins, determined by in vitr biassays, they were significantly lwer in Humegn, cmpared with Pergnal (.63 versus 2.3) accrding t immunassay estimates. ISOELECTROFOCUSING PROFILES LH The recveries f LH activity after IEF are presented in Table 2. Recveries f immunreactivities were cnsistently lwer than thse f the crrespnding biactivities. The IEF prfiles f Humegn and hmg exhibited tw majr areas f LH biactivity, lcated in the ph regins f 4. t 5.5 and 6.5 t 8.5, respectively (Fig. 1). The predminant activity was fund in the regin ph 6.5, in which at least three distinct peaks culd be identified. In the regin < ph 6.5, a smaller part f the ttal activity was lcated, withut any distinct peaks. In cntrast, biactive LH in Pergnal was exclusively cncentrated in the acidic regin f the ph gradient (between ph 4. and 5.5), and n LH biactivity was recvered in the ph regin 5.5. The prfiles f immunreactive LH in Humegn and hmg exhibited a pattern similar t that seen in the biassay, with a majr part f activity lcated in the alkaline regin and cmparatively Table 2. Percentage Recveries f LH and FSH Activities After IEF f Three Urinary Gnadtrpins, as Determined by In Vitr and RIA Prcedures hmg 1st IS Mean" Humegn Mean Pergnal Mean Metrdin Mean "Mean f three experiments. bonly duplicate runs b LH FSH Immunassay Immunassay 67.9 b Vl. 46, N.6, December 1986 Harlin et al. IEF prfiles f human urinary LH and FSH 157

4 LH-BIQ z 25 t; :.!:.. 2 e E ISO 1 5 ' LH-BIO z 6 E ~ - " E lh-bio 2 z t; i " E 1,,, i,r--t---r---, ,----" 4 1 ph LH-RIA z 11 ~ 1 : ~ E 8 Figure 1 Representative IEF prfiles fbiactive (LH-BIO) and immunreactive (LH-RIA) human-lh in different urinary gnadtrpin preparatins. hmg indicates the 1st IS preparatin f hmg LH-RIA z 225 :"' 2..!!:. E " 15 lh-ria z t; i " E 4 2 ph, 1 small amunts f immunreactive material at ph < 5.5. Like the distributin f biactive LH, the bulk fthe LH immunreactivity fpergnal was fund in the acidic regin (ph 4. t 5.5); nly a small prprtin f it was recvered frm the alkaline regin (Fig. 1). T assess quantitatively the distributin f LH activity in different ph regins, we frmed segments crrespnding t.5 ph units frm the data f each experiment. Humegn and hmg cntained 83% and 62% f the ttal biactivity, respectively, in the alkaline ph segments (calculated frm an arbitrarily chsen pint f ph 6.5), whereas Pergnal exhibited 1% f its biactivity in the acidic regin. The assay f immunreactivity revealed the same distributin pattern in Humegn and hmg, with alkaline material amunting t 73% and 6%, respectively. In cntrast, nly 18% f the immunreactive material f Pergnal was fund in the alkaline regin, and 82% in the acidic regin. The B/I ratis in varius ph segments were clse t unity in hmg and in Humegn, whereas in Pergnal these ratis were cnsiderably higher than unity (arund 3.) in all segments, except the segment between ph 5. and 5.5. FSH The recveries f FSH biactivities and immunreactivities after IEF are shwn in Table 2. There was a cnsistent lss f 25% t 4% f bth activities during the fractinatin prcedure. The FSH activity exhibited a hetergeneus prfile in a wide ph range (3.5 t 6.5). Significant differences were bserved amng the fur preparatins in the distributin ffsh species fvarius pi values. The hmg 1st IS exhibited a relatively extended IEF prfile, with significant activity lcated in the ph regin at the alkaline side f ph 5.5, in cmparisn t the three ther preparatins, which lacked such mlecular species. The cmmercial preparatins seem t be cmpsed f mre r less similar mlecular frms f mre acidic pi values, althugh significant differences were evident amng these preparatins with respect t varius FSH species f higher pi values. The prfiles f immunreactive FSH were fund t be similar t thse f biacti ve FSH in all fur preparatins. When the data frm each individual experiment were pled int segments crrespnding t.5 ph units (Fig. 2) and the FSH biactivity f these segments was cmpared, significant differences were bserved amng the fur preparatins. Abut 3% f the ttal biactivity f hmg 1st IS was recvered in segments mre alkaline than ph 5., whereas < 15% f the activity f the three cmmercial preparatins was fund in this regin. On the ther hand, nly 2% f the biactivity f the hmg 1st IS and Metrdin was recvered in the regin < ph 4., cmpared with 4% in the case f Pergnal and Humegn. The bulk ( 7% f the ttal) f the FSH activity f the three cmmercial preparatins exhibited pi values < 4.5, cmpared with abut 5% in the case f the hmg 1st IS. The recveries f immunreactive 158 Harlin et a1. IEF prfiles f human urinary LH and FSH Fertility and Sterility

5 FSH -BIO SO "I. '" 4 FSH-BIO ~ ] ~ 3 JiL '" 4 ~ 3 SO 'I. ~ ~,O~ ", J, ph J t ph J ph!::rl FSH-BIO ~ 3 j 1 J, 5 FSH-RIA 5.,. '" J1L ~ ' 8 ~ 3D 1 1..:: I a6w I, FSH- RIA 5 'J. Figure 2 Percentage recveries f biactive and immunreactive FSH and the ratis f bi- t immunptencies (BII) in different ph regins. Clumns represent mean values, with ranges f three experiments. '" ~ 4 8 ~ , FSH-BIO 5 'I. '" 4 ~ 3D 1 1 FSH-RIA SO 'I. '" ~ ' u ~ ::...., 1 J t 5 [. 7 ph 3, ph 3 " ph METRODIN METRODIN..:r~ METRO DIN ~,, 5 FSH exhibited distributin prfiles similar t thse f biactive FSH; hwever, there was a gradual decrease in immunreactivity in relatin t the crrespnding biactivity, with increasing ph values in all preparatins except Pergnal. The FSH activity in unfractinated preparatins exhibits BII ratis clse t unity. Hwever, cmparisn f the B/I ratis f different segments revealed that there was a gradual increase f the ratis with increasing ph in the case fhmg 1st IS, Humegn, and Metrdin. The B/I ratis f Pergnal were the lwest and similar in all segments. The increase f BII with increasing ph was especially marked in the case f Metrdin. DISCUSSION These data shw that the LH and FSH activity f cmmercial urinary gnadtrpins exhibit a marked hetergeneity. This was expected, n the basis f earlier studies n varius pituitary and urinary preparatins. 8-1,23 Hwever, the three cmmercial preparatins studied differed frm each ther, in terms f bth their BII and FSHlLH ratis and iselectrfcusing prfiles. All cmmercialpreparatins exhibited lwer in vitr biptencies f FSH and LH, cmpared with thse stated by the manufacturers based n in viv biassays. These lw in vitr biptencies can n n accunt be taken as an indicatin that the ampules f cmmercial preparatins cntained less gnadtrpin activity than stated. The relatinship between in viv and in vitr activities f gnadtrpins is.a cmplicated ne; the frmer, which is recmmended by the WHO Expert Cmmittee n Bilgical Standardizatin,24 is influenced by several factrs, such as the amunt and! r nature f carbhydrate residues, terminal halflife, the nature f receptr binding in the target tissue, and pssible cnfrmatinal changes in the circulatin. A few systematic studies with highly purified preparatins f pituitary FSH and LH indicate sme f the cmplexities invlved in the establishment f the ptency f gnadtrpin preparatins, with the use f different in viv and in vitr techniques.25, 26 On the ther hand, significant differences in ptencies between tw preparatins, when established by the same methd in the same assay, are likely t reflect true differences. One imprtant aspect, which was beynd the scpe f this study, is the lack f infrmatin n the batch-t-batch variatin in the relative cm- Vl. 46, N.6, December 1986 Harlin et al. IEF prfiles f human urinary LH and FSH 159

6 psitin f the varius mlecular species carrying the LH and FSH activities. Obviusly, this is an imprtant aspect that requires further investigatin. The differences in the B/I ratis f LH in unfractinated gnadtrpin preparatins seem t indicate that a significant amunt f LH immunreactivity in Humegn is nt assciated with in vitr biactivity, pinting t the pssible presence f free subunits in this preparatin. On the ther hand, the B/I ratis f hmg in varius ph segments after isfcusing were fund t be clse t unity, which indicates a cmpsitin yielding immunlgic and bilgic prperties in all fractins, similar t thse f the unfractined standard. Humegn als exhibited B/I ratis clse t unity in the ph segments ph 6.5 (cntaining 8% f the ttal activity), but higher B/I ratis were fund in the ph regin f 5. t 6.. In cntrast, the assay f Pergnal revealed high B/I ratis (arund 3) in 9% f the ttal biactive material; in additin, there was sme immunreactive material in the alkaline regin, with undetectable biactivity. These high BII ratis indicate that the bilgic and immunlgic cmpsitin f LH in Pergnal is dissimilar t that in Humegn and hmg. Hwever, as shwn by Rbertsn and Diczfalusy,17 the B/I ratis culd be influenced by several factrs, such as impurities f the standard preparatins, giving different results in different regins f the ph gradient. In B/I ratis f FSH in the hmg 1st IS, Humegn, and Metrdin shwed a gradual increase with increasing pi f the mlecular species. A similar relatinship has been bserved fllwing IEF f pituitary FSH preparatins frm varius speciesy' 17, 23 Pergnal, with its unchanged B/I rati, is an exceptin in this regard. We d nt cmpletely understand the reasn fr this. Metrdin, n the ther hand, exhibited a very marked increase in B/I ratis, with increasing ph values, which suggests the selective remval f bilgically inactive immunreactive material frm this particular preparatin. The FSH/LH ratis f Pergnal and Humegn were significantly different when immunassays were used, but similar n the basis f in vitr biassay estimates. Whether r nt these differences have any significance fr the clinical effects f these preparatins remains t be established. The majr part f LH biactivity in Humegn and hmg was in the ph range f 6. t 9., which is in agreement with the findings reprted in urinary preparatins23 and in pstmenpausal plasma and pituitary preparatins.27 The relatively acidic LH biactivity recvered «ph 6.5) amunted t 17% in Humegn and t 38% in hmg, which are f the same rder as that reprted fr pituitary preparatins frm fertile and pstmenpausal wmen. 11 The crrespnding immunreactivities were 26% and 38%, respectively. On the ther hand, in Pergnal all biactivity was cncentrated in a very narrw ph range f nly 1.5 ph unit (frm ph 4. t 5.5), and 75% f the ttal immunreactive material was fund in the same ph range. The remaining 25% f the immunreactive substances were evenly distributed in the alkaline regin and exhibited little, if any, biactivity. These results indicate that the purificatin f Humegn and Pergnal resulted in the selectin f predminantly alkaline LH activity in the frmer and f predminantly acidic LH in the latter. Bth types f mlecular species are present in unextracted urine, hmg preparatins, pituitary extracts, and bld f pstmenpausal wmen. 11 Similar differences, althugh f a lesser magnitude, in the FSH prfiles were bserved amng the varius preparatins. Cmpared with the hmg standard, the cmmercial preparatins lacked significant amunts f relatively alkaline mlecular species, but they were rich in species with mre acidic pi values. It has been shwn that the purificatin prcedures emplyed t btain highly purified pituitary FSH and LH preparatins result in majr changes in their IEF prfiles. 8, 1 The results f the present study demnstrate that als in the case f urinary preparatins, it is mainly the purificatin prcedures emplyed that are respnsible fr the differences in IEF prfiles. These data lead t the cnclusin that Humegn and hmg shw cnsiderable similarity, with respect t the IEF prfiles f LH activity, althugh the IEF prfile f Humegn is clser t the pattern f LH in the pituitary gland and the peripheral bld f fertile wmen. On the ther hand, Pergnal seems t cntain LH ishrmnes characterized by a rather unifrm species with a definite acidic cmpsitin, previusly shwn t be present nly as a minr cnstituent f the LH prfile in the pituitary gland and plasma f pstmenpausal wmen. 11, 12 The data als indicate differences in the IEF prfiles f FSH in the three cmmercial preparatins cmpared with the hmg standard, which we interpret as a cnsequence f the purificatin prcedure emplyed. Hence, this 16 Harlin et al. IEF prfiles f human urinary LH and FSH Fertility and Sterility

7 study highlights the need t study the extent f batch-t-batch variatin in the mlecular cmpsitin f gnadtrpin preparatins used in therapy. The results als pint t a need t assess the prperties f urinary gnadtrpins fllwing their administratin t wmen fr the inductin f vulatin, t clarify the effects f different physicchemical prperties n the in viv halflives f injected urinary LH, and particularly FSH. Acknwledgments. We are grateful t Mrs. Berit Frysa and Mrs. Mnica Lindberg fr their expert technical assistance. The highly purified urinary FSH preparatin, Metrdin, was a gift frm Svenska AB Sern, Stckhlm, Sweden. REFERENCES 1. Gernzell CA, Diczfalusy E, Tillinger G: Clinical effect f human pituitary fllicle-stimulating hrmne (FSH). J Clin Endcrinl Metab 28:1333, Thmpsn CR, Hansen LM: Pergnal (mentrpins): a summary f clinical experience in the inductin f vulatin and pregnancy. 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WHO Technical Reprt Series N. 626, Strring PL, Zaidi AA, Mistry YG, Frysa B, Stenning BE, Diczfalusy E: A cmparisn f preparatins f highly purified human pituitary fllicle-stimulating hrmne: differences in the fllicle-stimulating hrmne ptencies as determined by in viv biassay, in vitr biassay and immunassay. J Endcrin191:353, Strring PL, Zaidi AA, Mistry YG, Lindberg M, Stenning BE, Diczfalusy E: A cmparisn f preparatins f highly purified human pituitary luteinizing hrmne: difference in the luteinizing hrmne ptencies as determined by in viv biassays, in vitr biassay and immunassay. Acta Endcrinl (Cpenh) 11:339, Rbertsn DM, Van Damme M-P, Diczfalusy E: Bilgical and immunlgical characterizatin f human luteinizing hrmne. I. Bilgical prfile in pituitary and plasma samples after electrfcusing. Ml Cell Endcrinl 9:45, 1977 Vl. 46, N.6, December 1986 Harlin et ai. IEF prfiles f human urinary LH and FSH 161