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

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

Vol. 46, No.6, December 1986 Printed in U.8A. FERTILITY AND STERILITY Copyright e 1986 The American Fertility Society Molecular composition of lutei...

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Vol. 46, No.6, December 1986 Printed in U.8A.

FERTILITY AND STERILITY Copyright e 1986 The American Fertility Society

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

Jonas Harlin, M.D. Shafiq A. Khan, Ph.D.t Egon Diczfalusy, M.D.:j: Reproductive Endocrinology Research Unit, Department of Obstetrics and Gynecology, Karolinska Hospital, Stockholm

The biologic (B) and immunologic (l) properties of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were studied in three commercial urinary gonadotropin preparations and in the first international standard preparation of human urinary gonadotropins before and after fractionation by isoelectrofocusing (lEF). Significant differences were found in the IEF profiles of both bioactive and immunoreactive LH and FSH and in the BII ratios of the preparations studied. The observed differences in the molecular composition of LH and FSH seem to be attributable to the purification procedures employed. The possible influence of these differences on the in vivo potencies, circulating half-lives, and clinical effects of gonadotropin preparations are discussed. Fertil SteriI46:1055, 1986

Since Gemzell et al. 1 first published their study on the induction of ovulation in amenorrheic women by human gonadotropins, a wide variety of treatment regimens have been tried, with both pituitary and urinary preparations. 2 The schedules have ranged from one large single dose3 of human menopausal gonadotropin (hMG) to daily adjusted doses or to continuous infusion. 4 Because gonadotropins of pituitary origin are not commercially available, urinary preparations are used for treatment by most centers. With all regimens, Received April 25, 1986; revised and accepted August 13, 1986. *Supported by World Health Organization's Special Programme of Research in Human Reproduction, Geneva, Switzerland. tPresent address: Max-Planck Clinical Research Unit for Reproductive Medicine, Steinfurter Str. 107, D-4400 Munster, West Germany. :j:Reprint requests: Professor E. Diczfalusy, Reproductive Endocrinology Research Unit, Karolinska Hospital, S-104 01 Stockholm, Sweden. Vol. 46, No.6, December 1986

complications in the form of overstimulation and multiple gestations are still often seen, in spite of various efforts to monitor the dosage by different clinical and biochemical parameters, including ultrasonography.5, 6 Among other factors believed to be of importance for the clinical efficacy of the preparations used, the follicle-stimulating hormone/luteinizing hormone (FSH/LH) ratio has been considered. It was concluded that a minimal threshold dose of LH accompanying FSH is required to enable the latter to be effective, but above this threshold level, LH does not appear to influence the effect of FSH in terms of follicle growth. 7 Gonadotropins, such as LH and FSH, exhibit a high degree of charge heterogeneity, predominantly due to differences in the amount and/or nature of carbohydrate residues present in the different molecular forms. 8 - lo The relative abundance of different molecular species of LH and FSH of pituitary glands and plasma has been shown to depend on the sex and/or endocrine sta-

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tus of the subject. 1l- 14 Due to differences in the relative potencies in vitro, terminal half-life, and receptor-binding characteristics,14, 15 it is conceivable that different molecular species induce different responses, when administered clinically for ovulation induction. Because hMG preparations are extracted from postmenopausal urine, they could be expected to possess LH and FSH patterns characteristic for pituitary glands and plasma of postmenopausal women, in contrast to the molecular profiles present in normally menstruating women. Such a difference could playa certain role in the simultaneous induction of maturation of several follicles. Indeed, individual responses to hMG vary greatly, not only between different subjects and different cycles in the same subject, but also when different preparations are used. 5, 6, 16 Several studies indicate that women with comparatively high endogenous levels of LH (e.g., in polycystic ovarian disease) run a higher risk of overstimulation and multiple gestation than patients with low or normal LH levels. 6, 16 Whether or not the molecular composition of the gonadotropins is in part responsible for the observed overstimulation has yet to be ascertained. As a first step in this direction, we carried out an in-depth assessment of the electrofocusing profiles of bioactive and immunoreactive LH and FSH in commercial preparations; the data reported in this paper indicate major differences in the molecular composition of both LH and FSH in the available urinary gonadotropin preparations.

MATERIALS AND METHODS HORMONE PREPARATIONS

Commercially available purified urinary gonadotropin preparations (Humegon, batch G F 130392, N V Organon, Oss, Holland; Pergonal, batch 276 I F, and Metrodin, Ch. -B 105 P, Industria Farmaceutica Serono S.p.A., Rome, Italy) were used. The LH potency of Pergonal is stated to be 75 IUlampule and that of Metrodin < 0.1 IUlampule; the LH content of Humegon is not stated. FSH potency is stated to be 75 IUlampule in all three preparations. The standard preparation of hMG (lst International Standard [IS] of urinary FSH and LH/interstitial cell stimulating hormone (lCSH), human, for bioassay, code no. 70/45) was used as a reference preparation. 1056

The contents of each ampule were dissolved in 2.0 ml of 5 mmol phosphate buffer (pH 7.4, containing 0.8% NaCI and 1.0% bovine serum albumin), distributed into 0.5-ml aliquots, and stored at - 70°C until analyzed. ISOELECTROFOCUSING PROCEDURE

The four hormone preparations (Humegon, Pergonal, Metrodin, and the hMG 1st IS) were fractioned by isoelectrofocusing (lEF) in triplicate or duplicate runs in sucrose density gradient with the use of 110-ml column (No. 8100, LKB Produkter, Bromma, Sweden). Ampholytes (Ampholine; LKB Produkter) were used at a concentration of 1% to create a pH gradient in the pH range of 2.5 to 10.0. The details of the IEF procedure have been describedP The pI value (isoelectric pH) (mean ± standard deviation) for beef hemoglobin (Sigma Chemicals, London, UK), used to assess the reproducibility of the IEF procedure, was 7.85 ± 0.06 (n = 11). Both bioactive and immunoreactive LH and FSH were assayed in the individual 140 fractions from each IEF experiment, with the exception of Metrodin, which in an earlier analysis was shown to contain undetectable LH activity. To assess the overall losses of gonadotropin activit)r..during IEF, we recombined and analyzed equal aliquots from individual fractions from each experiment, along with the starting materials and the standard in multiple point parallel line assays. DETERMINATION OF LH

Bioactive LH in the individual IEF fractions and in the unfractionated preparations was determined with the use of an in vitro bioassay procedure based on the LH-dependent androgen production by mouse Leydig cell preparations. 18 The mean index of precision 19 (X.) in eight multiplepoint parallel line assays was 0.03. Immunoreactive LH was estimated with the use of a radioimmunoassay (RIA) procedure. 20 The mean index of precision (n = 8) was 0.04. DETERMINATION OF FSH

FSH bioactivity was determined with the use of an in vitro bioassay procedure using as an endpoint the aromatizing capacity of immature rat Sertoli cells. 21 Immunoreactive FSH was assayed with the use of a previously described RIA method. 22

Harlin et aI. IEF profiles of human urinary LH and FSH

Fertility and Sterility

Table 1. In Vitro LH and FSH Bioactivity and Immunoreactivity (RIA)" of Urinary Gonadotropin Preparations

LH Bioassay

RIA BII FSH Bioassay

RIA . BII FSHlLH Bioassay

RIA

Humegon

Pergonal

Metrodin

IUlampuk

IUlampule

IUlampule

42.5 (39.7-45.3) 29.9 (27.~2.4) 84.7 (76.0-95.6) 19.9 (17.6-22.4) 0.50 1.50

stated by the manufacturers on the basis of in vivo bioassays. Humegon contained significantly higher amounts of both bioactive and immunoreactive FSH than Pergonal and Metrodin. The BII ratios of FSH were close to unity in all preparations. Although the FSHlLH ratios were similar in the two commercial preparations, determined by in vitro bioassays, they were significantly lower in Humegon, compared with Pergonal (0.63 versus 2.03) according to immunoassay estimates.

< 0.008

57.8 (54.0-61.8) 36.7 (33.4-42.9) 32.5 (29.1-36.2) 53.0 (46.8-59.9) 40.4 (37.4-43.1) 30.9 (28.5-33.2) 1.09 0.91 1.05 1.36 0.63

1.23 2.03 ISOELECTROFOCUSING PROFILES

"Geometric mean values with 95% confidence limits. Assay results are expressed in terms of the 1st IS of urinary FSH and LH (ICSH) for bioassay (hMG, 1st IS).

LH

The recoveries of LH activity after IEF are presented in Table 2. Recoveries of immunoreactivities were consistently lower than those of the corresponding bioactivities. The IEF profiles of Humegon and hMG exhibited two major areas of LH bioactivity, located in the pH regions of 4.0 to 5.5 and 6.5 to 8.5, respectively (Fig. 1). The predominant activity was found in the region > pH 6.5, in which at least three distinct peaks could be identified. In the region < pH 6.5, a smaller part of the total activity was located, without any distinct peaks. In contrast, bioactive LH in Pergonal was exclusively concentrated in the acidic region of the pH gradient (between pH 4.0 and 5.5), and no LH bioactivity was recovered in the pH region > 5.5. The profiles of immunoreactive LH in Humegon and hMG exhibited a pattern similar to that seen in the bioassay, with a major part of activity located in the alkaline region and comparatively

RESULTS HORMONE POTENCIES

When the LH content of the commercial ampules was assayed, both the bioactivity and immunoreactivity of Humegon was found to be significantly (P < 0.05) higher than those of Pergonal (Table 1). Also, the ratio of biologic activity to immunoreactivity (BII) of the two preparations was markedly different. Compared with the 1st IS, the former had an excess of immunoreactivity and the latter an excess of bioactivity. The highly purified FSH preparation, Metrodin, did not have detectable LH activity. The FSH content of the three preparations was significantly lower, according to both in vitro bioassay and immunoassay procedures, than those

Table 2. Percentage Recoveries of LH and FSH Activities After IEF of Three Urinary Gonadotropins, as Determined by In Vitro Bioassay and RIA Procedures LH

Bioassay

hMG 1st IS Mean" Range Humegon Mean Range Pergonal Mean Range Metrodin Mean Range

FSH Immunoassay

Bioassay

Immunoassay

84.9 b

67.9 b

78.8--91.0

57.5-80.0

74.8 64.7--86.4

74.8 68.6--80.3

81.4 76.9--87.8

62.1 54.4-69.8

74.4 68.2--81.3

60.8 54.1--69.0

68.1 61.1-72.6

54.1 44.8--65.0

68.6 60.0-77.7

60.2 57.1--65.4

66.0 58.5-74.4

65.4 60.6--71.0

"Mean of three experiments. bOnly duplicate runs. Vol. 46, No.6, December 1986

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1 ity in the acidic region. The assay of immunoreactivity revealed the same distribution pattern in Humegon and hMG, with alkaline material amounting to 73% and 60%, respectively. In contrast, only 18% of the immunoreactive material of Pergonal was found in the alkaline region, and 82% in the acidic region. The B/I ratios in various pH segments were close to unity in hMG and in Humegon, whereas in Pergonal these ratios were considerably higher than unity (around 3.0) in all segments, except the segment between pH 5.0 and 5.5.

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110

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10 pH

10 pH

Figure 1 Representative IEF profiles ofbioactive (LH-BIO) and immunoreactive (LH-RIA) human-LH in different urinary gonadotropin preparations. hMG indicates the 1st IS preparation of hMG.

small amounts of immunoreactive material at pH < 5.5. Like the distribution of bioactive LH, the bulk ofthe LH immunoreactivity ofPergonal was found in the acidic region (pH 4.0 to 5.5); only a small proportion of it was recovered from the alkaline region (Fig. 1). To assess quantitatively the distribution of LH activity in different pH regions, we formed segments corresponding to 0.5 pH units from the data of each experiment. Humegon and hMG contained 83% and 62% of the total bioactivity, respectively, in the alkaline pH segments (calculated from an arbitrarily chosen point of pH 6.5), whereas Pergonal exhibited 100% of its bioactiv1058

The recoveries of FSH bioactivities and immunoreactivities after IEF are shown in Table 2. There was a consistent loss of 25% to 40% of both activities during the fractionation procedure. The FSH activity exhibited a heterogeneous profile in a wide pH range (3.5 to 6.5). Significant differences were observed among the four preparations in the distribution ofFSH species ofvarious pI values. The hMG 1st IS exhibited a relatively extended IEF profile, with significant activity located in the pH region at the alkaline side of pH 5.5, in comparison to the three other preparations, which lacked such molecular species. The commercial preparations seem to be composed of more or less similar molecular forms of more acidic pI values, although significant differences were evident among these preparations with respect to various FSH species of higher pI values. The profiles of immunoreactive FSH were found to be similar to those of bioacti ve FSH in all four preparations. When the data from each individual experiment were pooled into segments corresponding to 0.5 pH units (Fig. 2) and the FSH bioactivity of these segments was compared, significant differences were observed among the four preparations. About 30% of the total bioactivity of hMG 1st IS was recovered in segments more alkaline than pH 5.0, whereas < 15% of the activity of the three commercial preparations was found in this region. On the other hand, only 20% of the bioactivity of the hMG 1st IS and Metrodin was recovered in the region < pH 4.0, compared with> 40% in the case of Pergonal and Humegon. The bulk (> 70% of the total) of the FSH activity of the three commercial preparations exhibited pI values < 4.5, compared with about 50% in the case of the hMG 1st IS. The recoveries of immunoreactive

Harlin et a1. IEF profiles of human urinary LH and FSH

Fertility and Sterility

HMG

FSH -BIO

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HMG

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FSH exhibited distribution profiles similar to those of bioactive FSH; however, there was a gradual decrease in immunoreactivity in relation to the corresponding bioactivity, with increasing pH values in all preparations except Pergonal. The FSH activity in unfractionated preparations exhibits BII ratios close to unity. However, comparison of the B/I ratios of different segments revealed that there was a gradual increase of the ratios with increasing pH in the case ofhMG 1st IS, Humegon, and Metrodin. The B/I ratios of Pergonal were the lowest and similar in all segments. The increase of BII with increasing pH was especially marked in the case of Metrodin.

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Figure 2 Percentage recoveries of bioactive and immunoreactive FSH and the ratios of bio- to immunopotencies (BII) in different pH regions. Columns represent mean values, with ranges of three experiments. Vol. 46, No.6, December 1986

These data show that the LH and FSH activity of commercial urinary gonadotropins exhibit a marked heterogeneity. This was expected, on the basis of earlier studies on various pituitary and urinary preparations. 8 - 10,23 However, the three commercial preparations studied differed from each other, in terms of both their BII and FSHlLH ratios and isoelectrofocusing profiles. All commercialpreparations exhibited lower in vitro biopotencies of FSH and LH, compared with those stated by the manufacturers based on in vivo bioassays. These low in vitro biopotencies can on no account be taken as an indication that the ampules of commercial preparations contained less gonadotropin activity than stated. The relationship between in vivo and in vitro activities of gonadotropins is .a complicated one; the former, whiCh is recommended by the WHO Expert Committee on Biological Standardization,24 is influenced by several factors, such as the amount and! or nature of carbohydrate residues, terminal halflife, the nature of receptor binding in the target tissue, and possible conformational changes in the circulation. A few systematic studies with highly purified preparations of pituitary FSH and LH indicate some of the complexities involved in the establishment of the potency of gonadotropin preparations, with the use of different in vivo and in vitro techniques. 25 , 26 On the other hand, significant differences in potencies between two preparations, when established by the same method in the same assay, are likely to reflect true differences. One important aspect, which was beyond the scope of this study, is the lack of information on the batch-to-batch variation in the relative com-

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position of the various molecular species carrying the LH and FSH activities. Obviously, this is an important aspect that requires further investigation. The differences in the B/I ratios of LH in unfractionated gonadotropin preparations seem to indicate that a significant amount of LH immunoreactivity in Humegon is not associated with in vitro bioactivity, pointing to the possible presence of free subunits in this preparation. On the other hand, the B/I ratios of hMG in various pH segments after isofocusing were found to be close to unity, which indicates a composition yielding immunologic and biologic properties in all fractions, similar to those of the unfractioned standard. Humegon also exhibited B/I ratios close to unity in the pH segments> pH 6.5 (containing 80% of the total activity), but higher B/I ratios were found in the pH region of 5.0 to 6.0. In contrast, the assay of Pergonal revealed high B/I ratios (around 3) in > 90% of the total bioactive material; in addition, there was some immunoreactive material in the alkaline region, with undetectable bioactivity. These high BII ratios indicate that the biologic and immunologic composition of LH in Pergonal is dissimilar to that in Humegon and hMG. However, as shown by Robertson and Diczfalusy,17 the B/I ratios could be influenced by several factors, such as impurities of the standard preparations, giving different results in different regions of the pH gradient. In B/I ratios of FSH in the hMG 1st IS, Humegon, and Metrodin showed a gradual increase with increasing pI of the molecular species. A similar relationship has been observed following IEF of pituitary FSH preparations from various speciesY' 17, 23 Pergonal, with its unchanged B/I ratio, is an exception in this regard. We do not completely understand the reason for this. Metrodin, on the other hand, exhibited a very marked increase in B/I ratios, with increasing pH values, which suggests the selective removal of biologically inactive immunoreactive material from this particular preparation. The FSH/LH ratios of Pergonal and Humegon were significantly different when immunoassays were used, but similar on the basis of in vitro bioassay estimates. Whether or not these differences have any significance for the clinical effects of these preparations remains to be established. The major part of LH bioactivity in Humegon and hMG was in the pH range of 6.0 to 9.0, which is in agreement with the findings reported in uri1060

nary preparations 23 and in postmenopausal plasma and pituitary preparations. 27 The relatively acidic LH bioactivity recovered « pH 6.5) amounted to 17% in Humegon and to 38% in hMG, which are of the same order as that reported for pituitary preparations from fertile and postmenopausal women. 11 The corresponding immunoreactivities were 26% and 38%, respectively. On the other hand, in Pergonal all bioactivity was concentrated in a very narrow pH range of only 1.5 pH unit (from pH 4.0 to 5.5), and 75% of the total immunoreactive material was found in the same pH range. The remaining 25% of the immunoreactive substances were evenly distributed in the alkaline region and exhibited little, if any, bioactivity. These results indicate that the purification of Humegon and Pergonal resulted in the selection of predominantly alkaline LH activity in the former and of predominantly acidic LH in the latter. Both types of molecular species are present in unextracted urine, hMG preparations, pituitary extracts, and blood of postmenopausal women. 11 Similar differences, although of a lesser magnitude, in the FSH profiles were observed among the various preparations. Compared with the hMG standard, the commercial preparations lacked significant amounts of relatively alkaline molecular species, but they were rich in species with more acidic pI values. It has been shown that the purification procedures employed to obtain highly purified pituitary FSH and LH preparations result in major changes in their IEF profiles. 8 , 10 The results of the present study demonstrate that also in the case of urinary preparations, it is mainly the purification procedures employed that are responsible for the differences in IEF profiles. These data lead to the conclusion that Humegon and hMG show considerable similarity, with respect to the IEF profiles of LH activity, although the IEF profile of Humegon is closer to the pattern of LH in the pituitary gland and the peripheral blood of fertile women. On the other hand, Pergonal seems to contain LH isohormones characterized by a rather uniform species with a definite acidic composition, previously shown to be present only as a minor constituent of the LH profile in the pituitary gland and plasma of postmenopausal women. 11 , 12 The data also indicate differences in the IEF profiles of FSH in the three commercial preparations compared with the hMG standard, which we interpret as a consequence of the purification procedure employed. Hence, this

Harlin et al. IEF profiles of human urinary LH and FSH

Fertility and Sterility

study highlights the need to study the extent of batch-to-batch variation in the molecular composition of gonadotropin preparations used in therapy. The results also point to a need to assess the properties of urinary gonadotropins following their administration to women for the induction of ovulation, to clarify the effects of different physicochemical properties on the in vivo halflives of injected urinary LH, and particularly FSH. Acknowledgments. We are grateful to Mrs. Berit Froysa and Mrs. Monica Lindberg for their expert technical assistance. The highly purified urinary FSH preparation, Metrodin, was a gift from Svenska AB Serono, Stockholm, Sweden. REFERENCES 1. Gernzell CA, Diczfalusy E, Tillinger G: Clinical effect of

2.

3.

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11.

12.

human pituitary follicle-stimulating hormone (FSH). J Clin Endocrinol Metab 28:1333, 1958 Thompson CR, Hansen LM: Pergonal (menotropins): a summary of clinical experience in the induction of ovulation and pregnancy. Fertil Steril 21:844, 1970 Seddon RJ: The ovarian response in women to large single injections of human menopausal gonadotrophin and human chorionic gonadotrophin. J Reprod Fertil 23:299, 1970 Kemmann E, Brandeis VT, Shelden RM, Nosher JL: The initial experience with the use of a portable infusion pump in the delivery of human menopausal gonadotropins. Fertil Steril 40:448, 1983 Wang CF, Gernzell C: The use of human gonadotropins for the induction of ovulation in women with polycystic ovarian disease. Fertil Steril 33:479, 1980 Schwartz M, Jewelewicz R: The use of gonadotropins for induction of ovulation. Fertil Steril 35:3, 1981 Louwerens B: The clinical significance of the FSHlLH ratio in gonadotropin preparations of human origin. Acta Obstet Gynecol Scand (Suppll) 48:31,1969 Zaidi AA, Qazi MH, Diczfalusy E: Molecular composition of human luteinizing hormone: biological and immunological profiles of highly purified preparations after electrofocusing. J Endocrinol 94:29, 1982 Loeber JG: Human luteinizing hormone: structure and function of some preparations. Acta Endocrinol (Copenh) (Suppl 210) 85:1, 1977 Zaidi AA, Froysa B; Diczfalusy E: Biological and immunological properties of different molecular species of human follicle-stimulating hormone: electrofocusing profiles of eight highly purified preparations. J Endocrinol 92:195, 1982 Reader SCJ, Robertson WR, Diczfalusy E: Microheterogeneity of luteinizing hormone in pituitary glands from women of pre- and postmenopausal age. Clin Endocrinol 19:355, 1983 Strollo F, Harlin J, Hernandez-Montes H, Robertson DM, Zaidi AA, Diczfalusy E: Qualitative and quantitative differences in the isoelectrofocusing profile of biologically active lutropin in the blood of normally menstruating and post-menopausal women. Acta Endocrinol (Copenh) 97: 166, 1981

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13. Wide L: Male and female forms of human follicle stimulating hormone in serum. J Clin Endocrinol Metab 55: 682, 1983 14. Khan SA, Syed V, Froysa B, Lindberg M, Diczfalusy E: Influence of gonadectomy on isoelectrofocusing profiles of pituitary gonadotropins in rhesus monkeys. J Med PrimatoI14:174,1985 15. Wide L, Wide M: Higher plasma disappearance rate in the mouse for pituitary follicle-stimulating hormone of young women compared to that of men and elderly women. J Clin Endocrinol Metab 58:426, 1984 16. Raj SG, Berger MJ, Grimes EM, Taymor ML: The use of gonadotropins for the induction of ovulation in women with polycystic ovarian disease. Fertil Steril 28:1280, 1977 17. Robertson DM, Diczfalusy E: Biological and immunological characterization of human luteinizing hormone. II. A comparison of the immunological and biological activities of pituitary extracts after electrofocusing using different standard preparations. Mol Cell Endocrinol 9:57 r 1977 18. Van Damme M-P, Robertson DM, Diczfalusy E: An improved in vitro bioassay method for measuring luteinizing hormone (LH) activity using mouse Leydig cell preparations. Acta Endocrinol (Copenh) 77:655, 1974 19. Gaddum JH: Reports on biological standards. III. Methods of biological assay depending on a quantal response. Medical Research Council Special Report Series 183, 1933 20. Bartfai G, Robertson DM, Diczfalusy E: Biologically active luteinizing hormone (LH) in plasma. IV. Comparison with immunologically active LH in plasma of men. Acta Endocrinol (Copenh) 90:599, 1979 21. Van Damme M-P, Robertson DM, Marana R, Ritzen EM, Diczfalusy E: A sensitive and specific in vitro bioassay method for the measurement of follicle-stimulating hormone activity. Acta Endocrinol (Copenh) 91:224, 1979 22. Marana R, Robertson DM, Suginami H, Diczfalusy E: The assay of human follicle stimulating hormone preparations: the choice of a suitable standard. Acta Endocrinol 92:599, 1979 23. Van Damme M-P, Robertson DM, Diczfalusy E: Biological and immunological characterization of human luteinizing hormone. III. Biological and immunological profiles of urinary preparations after electrofocusing. Mol Cell Endocrinol 9:69, 1977 24. WHO Expert Committee on Biological Standardization. Twenty-Ninth Report. WHO Technical Report Series No. 626, 1978 25. Storring PL, Zaidi AA, Mistry YG, Froysa B, Stenning BE, Diczfalusy E: A comparison of preparations of highly purified human pituitary follicle-stimulating hormone: differences in the follicle-stimulating hormone potencies as determined by in vivo bioassay, in vitro bioassay and immunoassay. J Endocrino191:353, 1981 26. Storring PL, Zaidi AA, Mistry YG, Lindberg M, Stenning BE, Diczfalusy E: A comparison of preparations of highly purified human pituitary luteinizing hormone: difference in the luteinizing hormone potencies as determined by in vivo bioassays, in vitro bioassay and immunoassay. Acta Endocrinol (Copenh) 101:339, 1982 27. Robertson DM, Van Damme M-P, Diczfalusy E: Biological and immunological characterization of human luteinizing hormone. I. Biological profile in pituitary and plasma samples after electrofocusing. Mol Cell Endocrinol 9:45, 1977

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