Molecular composition of human luteinizing hormone: biological and immunological profiles of highly purified preparations after electrofocusing

1982 ◽  
Vol 94 (1) ◽  
pp. 29-36 ◽  
Author(s):  
A. A. Zaidi ◽  
M. H. Qazi ◽  
E. Diczfalusy
Keyword(s):  
Biological Activity ◽  
Luteinizing Hormone ◽  
Sucrose Density Gradient ◽  
Molecular Composition ◽  
Immunological Reactivity ◽  
Human Pituitary ◽  
Before And After ◽  
Pituitary Glands ◽  
Ph Range

Five highly purified human pituitary LH preparations (candidate preparations for a new International Reference Preparation (IRP) and coded A to E) and three highly purified commercial preparations (referred to as Kabi I, II and III) were fractionated by electrofocusing on a sucrose density gradient with ampholytes in the pH range of 3·5–10·0 The LH activity was monitored in each of the eluted fractions by an in-vitro bioassay and a radioimmunoassay procedure and the profiles of biological activity and immunoreactivity were compared with those of the first IRP of Human Pituitary Luteinizing Hormone for Immunoassay (code no. 68/40). The overall recovery of the biological activity after electrofocusing of the nine preparations ranged between 75 and 92% (mean 86%) and was higher (P < 0·05) than that of the immunological reactivity which varied between 71 and 94% (mean 79%). The profiles of the biological activity and immunological reactivity were in close agreement with each other in all preparations. Significant differences were, however, observed in the distribution of various molecular species between the currently used standard and the eight other highly purified preparations. The ratios of biological activity (B) to immunoreactivity (I) of preparation B and of the three Kabi preparations were significantly higher than those of the other five preparations, both before and after electrofocusing. After electrofocusing, a slight but significant (P < 0·05) rise in the B/I ratios was observed, suggesting that some biologically inactive immunoreactive material was removed during fractionation. All purified preparations lacked the characteristic 'acidic' human LH species which accompanies FSH and which is abundant in the IRP (69/104) and is also present in aqueous extracts of postmenopausal pituitary glands. A comparison of the electrofocusing profiles of the nine highly purified preparations with those of human LH from plasma and individual pituitary glands revealed marked differences, suggesting that the methods used for the purification of the hormone significantly altered its molecular composition.

Studies on the biological and immunological properties of human follitropin: profiles of two international reference preparations and of an aqueous extract of pituitary glands after electrofocusing

1981 ◽  
Vol 97 (2) ◽  
pp. 157-165 ◽  
Author(s):  
A. A. Zaidi ◽  
D. M. Robertson ◽  
E. Diczfalusy
Keyword(s):  
Biological Activity ◽  
Aqueous Extract ◽  
Receptor Binding ◽  
Immunological Reactivity ◽  
Human Pituitary ◽  
International Reference ◽  
Pituitary Glands ◽  
The Mean ◽  
Ph Range

Abstract. Two international reference preparations, the First International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH/ICSH) for Bioassay (identified hereafter by its code no.: 69/104) and the First International Standard of Urinary FSH and LH (ICSH) for Bioassay (hereafter: hMG 1st IS) and an aqueous extract of human pituitary glands (hPE) were fractionated in triplicate by isoelectric focusing on sucrose density gradient (Ampholine, pH range 2.5–10.0). The FSH activity was monitored in each fraction by an in vitro bioassay, a radioimmunoassay and a receptor binding assay. Unfractionated 69/104 was used as standard in each assay system. Compared with the hPE, the activity profiles of the 69/104 and hMG reference preparations were spread over a significantly wider pH range; the biological activity eluted in the pH range of 3.5–5.0 was of the order of 90% for hPE, 72% for 69/104 and less than 60% for hMG. Major variations in biological to immunological (B/I) and biological to receptor binding (B/R) activity ratios were observed in the individual electrofocusing fractions. The B/I ratios ranged from 0.8 to 2.2 (69/104), 1.0–5.7 (hMG) and 1.3–6.0 (hPE), respectively. The variation in the corresponding B/R ratios was: 0.5–1.7 (69/104), 0.58–4.0 (hMG) and 0.55–1.5 (hPE). When equal aliquots from each of the electrofocusing fractions were combined and this 're-constituted' pool was compared with the starting material, significant differences were observed in the B/I ratios: 1.63 instead of 1.0 (69/104), 2.58 vs 2.34 (hMG) and 2.72 vs 1.32 (hPE). There was no significant change in the mean B/R ratios before and after electrofocusing: 1.0 vs 1.02 (69/104), 1.09 vs 1.02 (hMG) and 1.05 vs 1.04 (hPE). The mean recovery of biological activity in each of the three reconstituted pools of the three preparations was 97% for 69/104, 88% for hMG and 98% for hPE, and the corresponding recoveries of receptor binding activities were 95%, 94% and 98%, respectively. In contrast, the mean recovery of immunological reactivity was only 55% (69/104), 80% (hMG) and 47% (hPE), respectively. The reduction in the immunological reactivity of the 69/104 preparation without any apparent loss of biological or receptor binding activities following electrofocusing indicates that its immunoreactivity is unstable under mild experimental conditions, which do not influence its biological and receptor binding activities. Hence, whereas this preparation might possibly be suitable as a standard for in vitro bioassays and receptor binding assays, it is unsuitable as a standard for the radioimmunoassay of hFSH.

BIOLOGICAL AND IMMUNOLOGICAL PROPERTIES OF DIFFERENT MOLECULAR SPECIES OF HUMAN FOLLICLE-STIMULATING HORMONE: ELECTROFOCUSING PROFILES OF EIGHT HIGHLY PURIFIED PREPARATIONS

1982 ◽  
Vol 92 (2) ◽  
pp. 195-204 ◽  
Author(s):  
A. A. ZAIDI ◽  
B. FRÖYSA ◽  
E. DICZFALUSY
Keyword(s):  
Biological Activity ◽  
Sucrose Density Gradient ◽  
Relative Distribution ◽  
Human Pituitary ◽  
Sialic Acid Content ◽  
Reference Preparation ◽  
Proportional Increase ◽  
Α And Β Subunits ◽  
Ph Range

Eight highly purified human pituitary FSH preparations and purified preparations of the α-and β-subunits of FSH were fractionated by an electrofocusing technique in the pH range of 2·5–10·0 on a sucrose density gradient. The human (h) FSH activity in each of the eluted fractions was monitored by an in-vitro bioassay and a radioimmunoassay procedure. After electrofocusing, the overall recovery of the biological activity of the eight preparations was between 80 and 94% (mean 88%). On the other hand, the recovery of immunoreactivity ranged between 30 and 84% (mean 71%). A loss of over 85% of hFSH immunoreactivity was observed when the α- and β-subunits of hFSH were fractionated by the same procedure. The specific loss of varying amounts of immunoreactivity in all preparations during electrofocusing was also reflected by a proportional increase in the ratios of biological activity (B) to immunoreactivity (I); preparation A, which exhibited a loss of 70% of the immunoreactivity, had a threefold increase in its B/I ratio after electrofocusing. Significant differences were observed in the electrofocusing profiles of the eight preparations both in terms of their pI values and of their spread. The disparity in the relative distribution of hFSH activities in different pH regions suggested major differences in the carbohydrate moieties (sialic acid content) of the preparations studied, probably as a result of the chemical manipulations involved in the purification of the hormone. It is suggested that a combination of several (but certainly not all) of the preparations might serve as a provisional International Reference Preparation for hFSH radioimmunoassays.

EFFECT OF STORAGE ON THE BIOACTIVITY OF TWO INTERNATIONAL REFERENCE PREPARATIONS OF HUMAN PITUITARY LUTEINIZING HORMONE IN VITRO

1979 ◽  
Vol 81 (1) ◽  
pp. 153-155 ◽  
Author(s):  
D. M. ROBERTSON ◽  
BERIT FRÖYSA
Keyword(s):  
Biological Activity ◽  
Luteinizing Hormone ◽  
Interstitial Cell ◽  
Saline Solution ◽  
Plasma Albumin ◽  
Human Pituitary ◽  
International Reference ◽  
Bovine Plasma ◽  
Reference Preparation

There was no loss of biological activity in vitro when the 1st International Reference Preparation (IRP) for human pituitary gonadotrophins [FSH and LH/interstitial cell stimulating hormone (ICSH) for bioassay] code no. 69/104 and the 1st IRP for human pituitary LH/ICSH [for immunoassay] code no. 68/40 were stored for 1 year at −70 °C in a buffered 0·8% saline solution containing 1% bovine plasma albumin (BPA). However, storage of the 69/104 preparation at −20 °C in either 0·1 or 1% BPA, or at −70 °C in the presence of 0·1% BPA showed a small but significant decrease (∼ 10%) in activity over the same period. It is, therefore, advantageous to store these reference preparations at −70 °C in the presence of 1% BPA.

Effect of desialylation of highly purified isoforms of human luteinizing hormone on their bioactivity in vitro, radioreceptor activity and immunoactivity

1997 ◽  
Vol 9 (5) ◽  
pp. 501 ◽  
Author(s):  
Patrick G. Burgon ◽  
Peter G. Stanton ◽  
Kim Pettersson ◽  
David M. Robertson
Keyword(s):  
Sialic Acid ◽  
Luteinizing Hormone ◽  
Acid Content ◽  
Specific Activity ◽  
Sialic Acid Residue ◽  
In Vitro Bioactivity ◽  
Human Pituitary ◽  
Sialic Acid Content ◽  
Before And After

To establish whether sialic acid content is responsible for an observed 7–8-fold variability in bioactivity in vitro of highly purified human pituitary luteinizing hormone (hLH) isoforms, the bioactivity in vitro, radioreceptor activity and immunoactivity of hLH isoforms were determined before and after enzymatic desialylation. Three immunofluorometric assays with different hLH specificities allowed characterization of 13–24 pituitary hLH isoform preparations of pI 7·03–8·98 in terms of sialic acid content (1–5 sialic acid residues per LH molecule), bioactivity in vitro (4030–30 000 I.U. mg-1), radioreceptor activity (6420–25 400 I.U. mg-1) and hLH immunoactivity (2900–4400 to 18 300–27 300 I.U. mg-1). Significant positive correlations between sialic acid content and either immunoactivity or in vitro bioactivity were observed, whereas radioreceptor activity showed a curvilinear response. Following more than 90% removal of sialic acid, both in vitro bioactivity and radioreceptor activity were increased, although specific activity still differed between isoforms; immunoactivities were unaffected. It is concluded that the presence of the sialic acid residue(s) on hLH isoforms does partially contribute to the in vitro bioactivity and radioreceptor activity of the isoforms, but that hLH immunoactivity is independent of sialic acid content.

Polymorphism of human pituitary FSH: analysis of immunoreactivity and in vitro bioactivity of different molecular species

1994 ◽  
Vol 141 (2) ◽  
pp. 359-367 ◽  
Author(s):  
M Simoni ◽  
F Jockenhövel ◽  
E Nieschlag
Keyword(s):  
Molecular Species ◽  
Assay Method ◽  
Aromatase Activity ◽  
Human Pituitary ◽  
Pituitary Glands ◽  
Reference Preparation ◽  
Commercial Kits ◽  
Ph Range ◽  
The Individual

Abstract Follicle-stimulating hormone is known to be highly heterogeneous in serum and in the pituitary. In the present study, we have partially separated different molecular species of human pituitary FSH and characterized their immunoreactivity and in vitro bioactivity. Pooled extracts of male (n=15) and female (n=9) human pituitary glands were chromatographed on a column of Sephacryl S-200 and FSH-containing fractions were fractionated by chromatofocusing in the pH range 4–6. FSH was measured in the individual fractions by an in vitro bioassay, based on the FSH-dependent aromatase activity of immature rat Sertoli cells, and by the following methods based on commercial kits: radioimmunoassay (RIA), immunofluorimetric assay (IFMA), immunoradiometric assay (IRMA), immunoenzymometric assay (IEMA). In each assay, the kit standard, calibrated against the 2nd International Reference Preparation (IRP) 78/549, and the International Standard (IS) 83/575 were run in parallel. The relative potencies of the kit standards in terms of IS 83/575 were: IFMA 3·08, IRMA 1·62, RIA 2·42, IEMA 1·45 and bioassay 1·14. After chromatofocusing, pituitary FSH eluted mostly in fractions with pH≈4·5, without sex-related differences. In both sexes ≈25% of bioactive material showed a pI<4 and eluted with 1 m NaCl. Although the same IS 83/575 was used in the various assays, the profiles of immunoreactive FSH were significantly different. The highest intermethod variability was observed in the case of male pituitary FSH. The relative biopotency of the different molecular species of FSH did not appear to change according to their pI but, rather, varied with the assay method and the standard. In terms of 2nd IRP 78/549 the activity profiles were similar but not identical to those obtained in terms of IS 83/575 and the ratios between the two values (IS:IRP ratios) were significantly different among the methods. No significant correlation was found between IS:IRP ratios and FSH concentrations or pH. These data suggest that: (1) all the methods have different affinities for standard and unknown and/or for different molecular isoforms of FSH; (2) overall, they perform more accurately when assaying female pituitary FSH, perhaps because of a closer resemblance between standard and unknown; (3) the variability of the IS:IRP ratios indicates a different affinity of the antibodies for IS 83/575 and the kit standard, highlighting the importance of the molecular composition of the reference preparations for the final result; and (4) the results do not support the commonly accepted concept of a higher in vitro biopotency of less acidic species of pituitary FSH. Journal of Endocrinology (1994) 141, 359–367

Effects of androgens on bioactivity and immunoreactivity of pituitary FSH in GnRH antagonist-treated male rats

1990 ◽  
Vol 122 (2) ◽  
pp. 168-174 ◽  
Author(s):  
Om P. Sharma ◽  
Shafiq A. Khan ◽  
Gerhard F. Weinbauer ◽  
Mohammed Arslan ◽  
Eberhard Nieschlag
Keyword(s):  
Isoelectric Focusing ◽  
Gnrh Antagonist ◽  
Molecular Composition ◽  
Male Rats ◽  
Sprague Dawley ◽  
Sprague Dawley Rats ◽  
Combined Administration ◽  
Ph Range ◽  
Fsh Bioactivity

Abstract The effects of androgens on the bioactivity and molecular composition of pituitary FSH were examined in intact and GnRH antagonist-suppressed male rats. Eight groups of adult Sprague-Dawley rats were subjected to the following treatments: antagonist (75 μg/day by osmotic minipumps; sc), testosterone-filled Silastic implants (3×5 cm, sc), dihydrotestosterone-filled Silastic implants (3×5 cm, sc), E2 benzoate (15 μg/day, sc), and combined administration of antagonist with either steroid for 3 weeks. At the end of the treatment period, pituitaries were dissected out and homogenised. FSH content was determined in the pituitary extracts by an in vitro bioassay and a radioimmunoassay. Individual pituitary extracts from rats treated with vehicle, testosterone and testosterone + antagonist were subjected to isoelectric-focusing on sucrose density gradients performed in the pH range from 3.5 to 7.0. Individual isoelectric-focusing fractions (100-120) were analysed for bioactive and immunoreactive FSH. Treatment with antagonist, E2 or antagonist + E2 caused a significant decrease in pituitary FSH, whereas testosterone and dihydrotesterone alone or in combination with antagonist prevented the decrease in pituitary FSH. The effects of all treatments on both bioactive and immunoreactive FSH were similar. Testosterone treatment not only maintained FSH synthesis but also altered the molecular composition of pituitary FSH. Following treatment with testosterone there was a shift of maximal FSH bioactivity to the more acidic pH range. On the other hand, less bioactivity was recovered than corresponding immunoreactivity in the higher pH region, resulting in significantly reduced ratios of bioactivity to immunoreactivity of FSH. No significant differences were found in the isoelectric-focusing profiles or bioactivity to immunoreactivity ratios of pituitary FSH in animals treated with testosterone alone or in combination with antagonist. The results demonstrate that testosterone not only maintained the synthesis of both bioactive and immunoreactive FSH in male rats, but also influences the molecular composition of pituitary FSH. These effects of testosterone on pituitary FSH appear not to be mediated through hypothalamic GnRH.

Intrinsic Pulsatility of Luteinizing Hormone Release from the Human Pituitary in Vitro

1988 ◽  
Vol 43 (1) ◽  
pp. 49-50
Author(s):  
MARCO GAMBACCIANI ◽  
JAMES H. LIU ◽  
WILLIAM H. SWARTZ ◽  
VICKI S. TUEROS ◽  
SAMUEL S. C. YEN ◽  
...  
Keyword(s):  
Luteinizing Hormone ◽  
Hormone Release ◽  
Human Pituitary ◽  
Luteinizing Hormone Release

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

1981 ◽  
Vol 91 (2) ◽  
pp. 353-362 ◽  
Author(s):  
P. L. STORRING ◽  
A. A. ZAIDI ◽  
Y. G. MISTRY ◽  
BERIT FRÖYSA ◽  
BRIDGET E. STENNING ◽  
...  
Keyword(s):  
Biological Activity ◽  
Follicle Stimulating Hormone ◽  
In Vitro Bioassay ◽  
Human Pituitary ◽  
International Reference ◽  
Biological Potency ◽  
Reference Preparation ◽  
In Vivo Bioassay

The FSH potencies of 12 preparations of highly purified human pituitary FSH, originating from six different laboratories, were determined by in-vivo and in-vitro bioassays and by immunoassay in terms of the First International Reference Preparation of Human Pituitary Gonadotrophins (FSH and LH) for Bioassay (IRP; coded 69/104). The contamination of these FSH preparations with LH was also determined. Estimates of protein content were based on the absorbance at 280 nm of solutions of the preparations, assuming that A1%1 cm 280 = 10. The FSH potencies varied between different preparations from 827 i.u./mg to 13 100 i.u./mg by in-vivo bioassay; from 2930 to 14 600 i.u./mg by in-vitro bioassay and from 1680 to 5690 i.u./mg by immunoassay. The ratios of in-vivo biological activity relative to in-vitro biological activity and to immunoreactivity respectively varied between preparations from 0·06 to 2·3 and from 0·15 to 4·1, and there was a significant positive correlation between each of these ratios and the in-vivo biological potency of the preparations; such differences could be due to varying degrees of sialylation between preparations. On the other hand the ratios of in-vitro biological activity to immunoreactivity between preparations were fairly constant (approx. 2). The excess biological activity relative to immunoreactivity observed, in terms of the IRP, in all these materials is consistent with recent findings of some immunoreactive FSH in the IRP unassociated with biological activity. These data did not demonstrate any significant advantage, in terms of FSH in-vivo biological potency, from the use of fresh-frozen rather than acetone-dried pituitary glands for the isolation of FSH. Contamination of all these preparations with LH appeared to be less than 3% (w/w), as determined by in-vitro bioassay and by immunoassay. The results of this study are discussed in relation to the selection of material for an international reference preparation for immunoassay and attention is drawn to the value of high in-vivo biological FSH potency as a criterion of the identity of a preparation as well as of its freedom from contaminants without FSH biological activity.

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