Inhibitory effect of plasma obtained from hypophysectomized and control women on the assay of bioactive luteinizing hormone

I. Galeraud-Denis1, P. Bouchard2, M. Herlicoviez1, E. Marie1 and S. Carreau3,4

1 Departments of Gynecologie–Obstétrique, CHU Clémenceau, Caen, 2 Endocrinologie, CHU Saint-Antoine, Paris and 3 Biochimie–IRBA, Université de Caen, 14032 Caen Cédex, France


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
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The purpose of this study was to determine the effect of components of female plasma on the value of bioactive luteinizing hormone (LH), especially in the presence of low immunological LH value. Using both an immunoradiometric assay (IRMA) and rat Leydig cell bioassay, immunoreactive (I) and bioactive (B) LH were assessed in plasma collected from women during a gonadotrophin releasing hormone (GnRH) test performed on day 7 of a spontaneous cycle. Two modes of response to an acute administration of GnRH were defined: normal production of gonadotrophins (group I) and excessive secretion (group II) associated with a significant difference in the B/I–LH ratio between the two groups. The B/I–LH ratio did not vary with sampling time during the test in either group. The addition of LH-free plasma collected from hypophysectomized women caused a 30% decrease in testosterone production compared to control values (in the presence or absence of hLH standard). A partial restoration of testosterone production was observed if plasma was first treated with PEG 12%. The inhibitory factor(s) was also present in plasma from ovulatory women, even after treatment by an antibody against the entire LH molecule. The effect of normal (A) or low I-LH plasma (B) on testosterone production varied strongly according to the plasma volume added to the bioassay, as well as to plasma treatments. Diethylether treatment caused a 50% decrease in testosterone secretion for plasma B (but not for A) whereas a diminution of the steroidogenesis is observed after a PEG treatment of plasma A (but not for B), suggesting that different inhibitory factors are present in plasmas A and B. Therefore the LH bioactivity measured in the rat Leydig cell assay, in terms of testosterone output, seems to represent a balance between the LH molecule and the presence of inhibitory factors in the plasma.

Key words: hypophysectomy/inhibitory factors/LH bioactivity/plasma


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Qualitative and quantitative changes in human plasma luteinizing hormone (LH) have been reported in various physiopathological situations, related to modulation of the hormonal environment by pulsatile secretion of gonadotrophin releasing hormone (GnRH) and sex steroids (Veldhuis and Dufau, 1993Go). Highly sensitive in-vitro cellular systems such as rodent Leydig cell preparations have been used for the determination of LH bioactivity (Dufau et al., 1974Go; Van Damme et al., 1974Go). The discordant and wide range of B/I (bioactive/immunoreactive) LH ratios reported in the literature could be explained by differences in the methods (Jaakkola et al., 1990Go) or the antibodies used for I-LH measurement (Imse et al., 1992Go), the animal species selected for the bioassay (Ding et al., 1991Go), or the molecular composition of the LH reference preparations (Zaidi et al., 1982Go). Discrepancies in the measurements of parallel serial dilutions between standard preparations and plasma samples have been described for male plasma samples when using the in-vitro mouse Leydig cell bioassay (Lichtenberg and Pahnke, 1976Go; Rajalakshmi et al., 1979Go). Indeed, we have reported a diminution of both basal and LH-stimulated testosterone biosynthesis by rat Leydig cells in the presence of serum obtained from hypophysectomized men or rams (Boujrad et al., 1991Go). Using an immature rat Leydig cell bioassay (Denis et al., 1994Go), we have studied the presence of inhibitory factors in plasma from hypophysectomized or ovulatory women. We have also investigated whether these factor(s) could interfere with the determination B-LH, particularly when I-LH values are low, with the object of defining the limits of the assay for LH bioactivity in humans.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reagents
Highly purified hLH (reference 80/552: 2nd International Standard LH Pituitary; sp.act.: 35 units/5.8 µg, in terms of 1st IRP 68/40) was purchased from the National Biological Standards Board (England). Ovine LH (NIH-oLH 25; sp.act.: 2.3 IU/mg) and three rabbit antibodies directed against either {alpha} or ß subunits of hLH or the whole hLH molecule were obtained from the NIDDK (NIH, Bethesda, MD, USA). Collagenase-dispase was purchased from Boehringer-Mannheim (Meylan, France). Soybean trypsin inhibitor, deoxyribonuclease I, 1-methyl-3-isobutyl xanthine (MIX), bovine serum albumin (BSA; Cohn fraction V and fatty-acid free, i.e. <0.005%), N6O2'-dibutyryl 3', 5' cyclic monophosphate (dbcAMP) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Sodium heparin was obtained from Roche Laboratory (France). Sheep serum anti-rabbit antibody was a gift from Dr Combarnous (INRA, Nouzilly, France). All other chemicals were obtained from Merck (Darmstadt, Germany) and reagents used for cell preparation and incubation were purchased as reported elsewhere (Papadopoulos et al., 1985Go). LH standard preparations were reconstituted in phosphate buffered saline (PBS; pH 7.4) and stored as aliquots at –20°C. Rabbit antibodies were respectively diluted (1/25, 1/50 and 1/100) in PBS supplemented by rabbit serum (2%). All other reagents and hormones were prepared in a Ham F12-DME (1:1; v/v) medium.

Collection of plasma samples for LH bioassay
Control samples were obtained from healthy fertile men (mean age: 35.8 ± 3.5 years; n = 6), postmenopausal women (mean age: 53.6 ± 3.3 years; n = 14) and women on day 7 of their follicular phase (mean age: 32.3 ± 3.8 years; n = 10). Samples were also obtained from four hypophysectomized women (41–61 years), who had not received hormonal treatment; only plasma samples with undetectable concentrations of I-LH were used and these were defined as LH-free.

LH bioactivity was also determined in plasma samples from young women (mean age: 31.6 ± 1.2 years; n = 16) during the GnRH test. None of them had hyperprolactinaemia, signs of ovarian dystrophy, polycystic ovarian disease (PCOD) or hyperthyroidism. All I-LH plasma values were in the normal range and plasma samples with a high LH/follicle-stimulating hormone (FSH) ratio (>1.5) were excluded from the study. A GnRH test was performed on day 7 of the follicular phase because LH concentration is quite stable during this early part of the follicular phase (days 3–7). Blood samples were collected following a single i.v. GnRH injection (100 µg; StimuLH, Roussel-Uclaf, Paris) at 0 (T0), 15 (T15), 30 (T30), 60 (T60) and 90 (T90) min. All blood samples were collected in EDTA, centrifuged at 2500 g for 10 min at 4°C and stored as aliquots at –20°C until further use.

Treatment of plasma samples
Plasma aliquots obtained from control and hypophysectomized women were submitted to one of the following treatments:

(i) charcoal treatment: plasma was incubated with activated charcoal (1 mg/mg of protein) for 12 h at 4°C to eliminate endogenous free steroids and low molecular weight molecules;
(ii) diethylether treatment: unconjugated steroids were removed by two successive extractions of plasma with diethylether (v/v: 1/5) purified on an alumina column;
(iii) polyethylene glycol (PEG) treatment: plasma was treated with 12% PEG for 1 h at 4°C and centrifuged at 2000 g for 10 min at 4°C to precipitate proteins;
(iv) heat treatment: plasma was incubated at 50°C for 30 min and centrifuged at 2000 g for 10 min at 4°C to remove the denatured proteins;
(v) endogenous LH elimination: individual samples were incubated with antibodies raised against either the whole LH, or against {alpha}- or ß-LH. The final dilutions of the antibodies used to eliminate endogenous I-LH in the plasma samples were 1/10 000 for the whole LH or 1/1000 for the {alpha}-LH or the ß-LH. Dilutions of each antibody were incubated with plasma fractions for 24 h at 20°C. The LH-antibody complexes were precipitated by incubation for 12 h at 4°C with sheep antirabbit antiserum and 1% PEG, followed by centrifugation at 2500 g for 30 min at 4°C.

All treated samples were stored at 4°C, their I-LH values were measured and they were then subjected to in-vitro hLH bioassay.

In-vitro LH bioassay
The testes from Sprague Dawley rats (34 days old) were removed, decapsulated and then disrupted gently with scissors before incubation at 32°C for 10 min in a solution collagenase-dispase (0.05%), soybean trypsin inhibitor (0.005%) and deoxyribonuclease I (0.001%) in Ham's F12-DME medium. After sedimentation (twice) and filtration through nylon gauze (30 Mesh), the testicular cells were centrifuged and the pellet washed with fresh medium. From the final pellet, viability and 3ß-hydroxysteroid deshydrogenase activity (3ß-HSD) were performed (Papadopoulos et al., 1985Go). Incubations (106 testicular cells) were realized in duplicate at 32°C, under an air-CO2 (5%) atmosphere in Ham's F12-DME medium supplemented with fatty acid free BSA (2.5 g/l), sodium heparin (1 IU/l) and MIX (0.125 mM) (Denis et al., 1994Go). Diluted human plasma was added at four increasing concentrations to the Leydig cell incubation medium and the rate of testosterone synthesis was determined in the same assay. Biological value of LH was then calculated from two plasma dilutions (Revol et al., 1997Go). Results were expressed in percentages compared to two reference values X and Z [testosterone productions expressed in ng/106 Leydig cells/4h, X: in absence of LH and Z: after incubation with a saturating dose (1 IU/l) of human LH standard NBSB 80/552, which led to maximum stimulation of the Leydig cells in terms of testosterone output].

Hormone assays
After incubations, media were collected by centrifugation at 2000 g for 10 min at 4°C and testosterone measured by radioimmunoassay (Papadopoulos et al., 1985Go). Intra-and interassay coefficients of variation were 3 and 6% respectively and the sensitivity was 4 pg/tube. Plasma LH was measured by an immunoradiometric assay (IRMA) technique using 125I-hLH Coatria kit (bioMérieux); intra- and interassay coefficients of variation were 2.6 and 5.3%. Results were expressed in IU/l in terms of 1st IRP 68/40 (the normal LH range of values was 3.5–10 IU/l for men, 2.5–11 IU/l for women in follicular phase and 18–65 IU/l for postmenopausal women). The FSH concentration was evaluated by an 125I-FSH Coatria kit from bioMérieux (normal range: 1.5–8.0 IU/l for men, 2–13 IU/l for women in follicular phase; 25–145 IU/l for postmenopausal women).

Statistical analysis
Data are expressed as means ± SEM. Student's t-test was used to compare mean values obtained from at least three different experiments, performed in duplicate; a P value < 0.05 was considered to be significant. Correlation coefficients were determined by the method of least squares.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Validation of the LH bioassay
The addition of MIX, heparin and fatty acid free BSA to the culture medium improved the sensitivity (100 mIU versus 20 mIU of hLH NBSB 80/552) and the ED50 (0.40 versus 0.15 mIU) of the LH bioassay compared to the absence of these factors. The interassay coefficient of variation was 10% for both normal (I-LH = 5.9 IU/l, n = 15) and elevated (I-LH = 62 IU/l, n = 10) plasma LH values and the intra-assay coefficient of variation was 6%. In postmenopausal women, B and I-LH were respectively 95.8 ± 12.6 IU/l and 38.2 ± 5.9 IU/l, with a B/I LH ratio of 2.51 ± 0.15 (n = 14). In fertile men (n = 6), the B/I LH ratio was 3.30 ± 0.12 (B-LH: 17.5 ± 1.2 IU/l and I-LH: 5.3 ± 0.3 IU/l). In fertile women at day 7 of their follicular phase, the B/I LH ratio was 3.42 ± 0.34 (B-LH: 19.5 ± 2.8 IU/l and I-LH: 5.7 ± 0.8 IU/l).

Bioactive LH concentrations in normal ovulatory women
The effects of a single GnRH injection on B-LH and I-LH concentrations are shown in Figure 1Go. Two groups were determined according to the intensity of the release of the plasma gonadotrophins: group I (50 < FSH < 150% and 150 < LH < 300%, n = 9) and group II (FSH > 150% and LH > 3 00%, n = 7). The basal I-LH values were not significantly different in the two groups (5.8 ± 0.8 IU/l in group I versus 4.4 ± 0.8 IU/l in group II). The B-LH concentrations at T0 were significantly lower in group II than in group I (12.1 ± 2.2 IU/l versus 21.4 ± 3.2 IU/l; P < 0.05). According to their respective control values and taking into account that the Tm for groups I and II were not significantly different from each other, there was a greater increase in B-LH in group II (6.7-fold) compared to group I (2.9-fold) between T0 and Tm respectively. A close correlation between I-LH and B-LH values in group I (r = 0.96) and in group II (r = 0.98) was noted. Within the two groups, the profile of the B/I–LH ratios according to the sampling time following GnRH administration did not change. Group I was characterized by a significantly higher B/I LH ratio at T0 (P < 0.001), T15, T30, T60 and T90 when compared to group II (P < 0.01). The B/I LH ratio remained stable during the GnRH test, although a slight decrease was observed at T15 (12 versus 16%; group I versus group II).



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Figure 1. Determination of immunoreactive (I) luteinizing hormone (LH) and bioactive (B)-LH concentrations during a GnRH test performed in women on day 7 of their follicular phase. Two groups were defined according to the intensity of the gonadotrophin response: group I (n = 9) with a normal increase in LH and group II (n = 7) with an amplified increase in LH. Results are expressed as means ± SEM at two time-points: T0 (just before the i.v. injection) and Tm (time of maximal production of gonadotrophins). *P < 0.05 when group I was compared to group II.

 
LH bioactivity measurement in human plasma samples
Effect of LH-free plasma
To mimic the effect of plasma components on testosterone production in the cellular system, we evaluated the effects of the inclusion in the culture system of LH-free plasma from hypophysectomized women on basal and hLH-stimulated steroidogenesis in immature rat Leydig cells. Under basal conditions, the addition of increasing proportions of plasma (5–20%, v/v) induced a dose-dependant decrease of steroidogenesis. At a plasma concentration of 5%, the amount of inhibition differed according to the source of plasma used. Regardless of the concentration of purified hLH (NBSB 80/552), the addition of 5% plasma led to a decrease in testosterone production (36.5 ± 10.6% at 0.075 IU/l and 27.8 ± 2.6% at 0.15 IU/l; n = 3). This inhibition increased with 10% addition although it still varied according to the plasma source. Complete inhibition of testosterone output occurred when the plasma volume was increased to 20%. In an attempt to eliminate the inhibitory factors, the plasma samples were subjected to various treatments prior to the testosterone assay (Figure 2Go). Treatment by charcoal did not modify testosterone production when compared to that of untreated plasma (inhibition percentage of testosterone production: 25.7 ± 4.6 versus 23.3 ± 5.9). On the other hand, diethylether extraction increased the inhibition of testosterone production (53.5 ± 6.9 versus 23.3 ± 5.9 % for untreated plasma, P < 0.05). Only the treatment of plasma with PEG was able to decrease its level of inhibition (inhibition: 9.2 ± 8.7%).



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Figure 2. Percentage inhibition of hLH stimulated testosterone production (hLH NBSB 80/552: 0.15 IU/l) when 5% luteinizing hormone (LH)-free plasma collected from hypophysectomized women (n = 4) was included in the culture medium. Each plasma was either untreated or treated as indicated before addition to the in-vitro bioassay. A representative experiment is shown in which all plasma samples (n = 4) were examined in the same bioassay.

 
Effects of LH + plasma
Table IGo summarizes the effects of various treatments on the measurement of LH bioactivity in human plasma samples with normal I-LH values. For each plasma the treatment volume represented less than 1.25% of the final volume in the bioassay. The B-LH concentrations were unchanged compared with controls when samples were treated by diethylether or by heating. After charcoal adsorption, a loss of biological activity of LH was observed, associated with a significant decrease in I-LH of 85%. A significant reduction of I-LH but not of B-LH occurred when plasma samples were treated with PEG.


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Table 1. Luteinizing hormone (LH) immunoreactivity (I) and bioactivity (B) of untreated plasma or plasma subjected to different treatments as outlined in Methods, obtained from women (n = 3) with normal I-LH concentrations. Results are expressed as means ± SEM, each one of the three plasma samples (one from each woman) (treated or not) was tested in duplicate in three different experiments
 
Figure 3Go shows the results of treating plasma containing normal LH concentrations from ovulatory women with LH antibodies. Only the addition of an antibody directed against the complete molecule induced a decrease in testosterone production compared with controls. No effects were seen with antibodies against the {alpha} or ß subunits.



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Figure 3. Determination of testosterone production after plasma treatment with an antibody directed against either the entire luteinizing hormone (LH), or the {alpha}-LH or the ß-LH subunits. Plasma was obtained from normal ovulatory women (n = 2). Horizontal lines represent basal and hLH stimulated testosterone production.

 
As shown in Figure 4Go, at low I-LH concentrations (I-LH < 1.0 IU/l), the volume of plasma required to measure the LH bioactivity in the rat Leydig cells bioassay reached 2–12% of the final volume. An inhibitory effect on testosterone production was observed when plasma samples were treated with either activated charcoal or diethylether, leading to a decrease in B-LH of >50%, but not when plasma samples were treated with 12% PEG.



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Figure 4. Determination of testosterone production when plasma from two women with low immunoreactive (I) luteinizing hormone (LH) concentrations (I-LH from plasma E: 0.9 IU/l and from plasma F: 0.4 IU/l) was added to culture medium at different volumes. The plasma samples were either untreated or subjected to different treatment as indicated. Each point is the mean of triplicate incubations.

 

    Discussion
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 Materials and methods
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As previously reported (Denis et al., 1994Go), the addition of fatty acid free albumin, heparin and MIX to the incubation medium of 34 day old rat Leydig cells improves the sensitivity of the LH bioassay in terms of testosterone production. We studied LH bioactivity following GnRH administration during the follicular phase of normal ovulatory women. A close correlation between immunological and bioactive LH values, and therefore a stable B/I–LH ratio, was obtained; these results are in agreement with those of Dufau et al. (1976). The important point of our study is the quantitative change observed in the B/I LH ratio in normally menstruating women according to the increase in gonadotrophin output during the GnRH test: a low B/I–LH ratio (<3) was associated with an exaggerated response, and a B/I–LH ratio >3 with a normal response. An excessive elevation in immunoreactive gonadotrophins during the GnRH test is generally observed in PCOD women, characterized by a high basal I-LH, an increased basal LH bioactivity (Lobo et al., 1983Go) associated with a greater proportion of basic isoforms (Ding and Huhtaniemi, 1991Go). In our study, the group with an excessive response of gonadotrophins was composed of non-PCOD patients with normal ovulatory cycles, normal basal LH concentrations and a decreased basal B/I–LH ratio. The data we have obtained both in patients and controls could be compared to those of Fauser et al (1991): non-PCOD patients showed a greater decrease in the B/I–LH ratio compared to PCOD and normal groups; however, in that study, women with abnormalities of the ovulatory cycles were included.

The variations of LH bioactivity in normally cycling women might be explained by the existence of either structural alterations in the LH molecule (variants) or variations in glycosylation (isoforms). Recently, polymorphic variants resulting from single substitutions of Trp8/Arg and Ile15/Thr, inserting a new potential glycosylation site into the LHß-chain, have been isolated from the serum of women (Suganuma et al., 1996Go). Functional differences of the LH variant from wild-type LH have been observed such as a higher B/I–LH ratio, a shorter half-life in peripheral circulation and an increased bioactivity (Haavisto et al., 1995Go). The frequency of LH variants seems to differ widely in healthy women from various populations: 28% in Finland, 15% in UK and 7.5% in North American Hispanics (Haavisto et al., 1995Go; Rajhkowa et al., 1995). The consequences for fertility of these LH variants in healthy women are still being discussed (Furui et al., 1994Go; Haavisto et al., 1995Go).

The qualitative modification of LH bioactivity could be also explained by the existence of numerous isoforms with a wide range of biological potencies in vitro and different half-lives in vivo (Stanton et al., 1996Go). The isoform profile can be modified according to physiopathological situations: change of relative proportions of plasma HCG isoforms at the 13th week of pregnancy (Wide et al., 1994Go), enrichment of basic LH isoforms in PCOD patients (Ding and Huhtaniemi, 1991Go), appearance of more basic isoforms in pubertal children during chronic GnRH agonist therapy (Wide et al., 1996Go).

For plasmas with low or nil I-LH values, the significance of the concentration of bioactive LH is more difficult to evaluate, because of the proportion of plasma volume added to the in-vitro bioassay. It is well known that plasma contains numerous factors which interfere with Leydig cell steroidogenesis (Papadopoulos et al., 1990Go). Discrepancies between the parallelism of male serum and standard LH curves have been observed for LH determination in a mouse in-vitro bioassay: the addition of serum exceeding 5% of the total volume assay results in a lower testosterone response (Lichtenberg and Pahnke, 1976Go; Rajalakshmi et al., 1979Go). However, the above observation has not been shown when male serum is used with a rat Leydig cell bioassay (Dufau et al., 1976Go). It has been reported that factors present in the serum of infertile stallions (Whitcomb et al., 1991Go) and in testicular and peripheral blood of rams, either healthy, hypophysectomized or castrated (Papadopoulos et al., 1990Go), inhibit the binding of LH to its receptor in vitro. Indeed, we have shown that plasma collected from hypophysectomized women leads to a decrease of both basal and hLH-induced testosterone production. The concentration of inhibitory factors differs between samples, whereas a complete inhibition of testosterone output has been observed in the presence of 20% (v/v) plasma. Together with other data, this demonstrates that inhibitory factors are present in samples of plasma obtained from hypophysectomized men (Lichtenberg et al., 1982Go; Boujrad et al., 1991Go), young women using oral contraception (Lichtenberg et al., 1982Go), and healthy men and postmenopausal women whose serum was treated with a ß-LH antibody (Ding and Huhtaniemi, 1989Go). The presence of inhibitory factors was also observed when plasma from normal ovulatory women were treated with an antibody directed against the whole LH molecule, but not when treated with antibodies against either {alpha}- or ß-LH subunits, suggesting the existence of LH agonist or antagonist molecules in normal I-LH plasma. Our data are different from those published by Ding and Huhtaniemi (1989) showing an interference of postmenopausal plasma with the LH bioassay which may be explained by the age and the hormonal status of the patients studied.

The inhibitory effect on steroidogenesis was not modified when LH-free plasma was treated with activated charcoal although it was increased after diethylether extraction which may unmask other inhibitory factors. Only treatment by 12% PEG partially restored the testosterone production through precipitation of inhibitory factors. These factors were also eliminated by heating at 50°C for 15 min (Lichtenberg and Pahnke, 1976Go), 60°C for 15 min (Ding and Huhtaniemi, 1989Go) or 50°C for 30 min (data not shown). The effects of blood sample treatments, particularly 12% PEG or diethylether, gave different values for the I-LH concentration. The consequences of these treatments on the testosterone production were similar in LH free and low I-LH plasmas (decrease with diethylether and partial restoration with 12% PEG) suggesting that inhibitory factor activities induce similar effects whatever their origin but differ from those in normal ovulatory women. The nature of these inhibitory factors remains unknown: the diverse effects of diethyl ether and PEG suggest that lipids are not likely to be involved, although plasma cholesterol and triglycerides are decreased by these two treatments (data not shown). Perhaps these inhibitory factors should be compared to the truncated LH molecules able to bind or mask LH receptors.

In conclusion, this data demonstrates that the value of bioactive LH determined by assay represents probably a balance between the bioactive LH molecule and inhibitory(s) factor(s) present in the plasma matrix. In plasmas with low I-LH concentrations (< 1 IU/l), the determination of LH bioactivity is strongly related to the plasma volume added to the bioassay and, consequently, to the proportion of inhibitory factors present. Nevertheless, the measurement of LH bioactivity remains useful, especially in physiopathological situations associated with an increased or normal immunoreactive gonadotrophin concentration.


    Acknowledgments
 
The authors would like to express their gratitude to Pr. Leymarie and the staff of the Hormone Laboratory (CHRU Clemenceau, Caen) for the hormonal measurements and to Mrs Edine for her technical assistance. The authors wish also to thank Dr T.N. Ledger for revision of the English in the manuscript.


    Notes
 
4 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on May 11, 1998; accepted on November 17, 1998.





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