| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Service d’Endocrinologie et des Maladies de la Reproduction (B.C., P.C., J.Y., G.S.), Laboratoire de Biochimie Hormonale (S.B.), Hopital Bicêtre, 94275 Kremlin Bicêtre, France; Département de Biologie Clinique (J.P., J.M.B.), Institut Gustave-Roussy, 94805 Villejuif, France; and Department of Physiology, University of Turku (I.T.H.), Turku, 20520 Finland
Address all correspondence and requests for reprints to: Dr. Gilbert Schaison, Service d’Endocrinologie et des Maladies de la Reproduction, Hopital Bicêtre, 94275 Le Kremlin Bicêtre Cedex, France. E-mail: gilbert.schaison{at}bct.ap-hop-paris.fr
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
-subunit (FAS).
The LHß assay does not recognize the dimeric LH and cross-reacts only
with free hCG ß-subunit (CGß). Thus, all of the plasma samples were
also tested with a highly specific immunoradiometric assay for free
CGß. Molar concentrations (i.e. picomoles per L) were
used to compare the plasma levels of LH and its free subunits. Plasma
LH, LHß, FAS, and CGß levels were measured in five normally cycling
women during the early follicular phase and the ovulatory peak of LH.
The pulsatile profiles of LH, LHß, FAS, and CGß were studied in
five postmenopausal women before and 21 days after injection of a depot
preparation of the GnRH agonist D-Trp6 (3.75
mg, im) and in five women with functional hypothalamic amenorrhea
(FHA), i.e. low plasma LH levels, during pulsatile GnRH
administration (20 µg/pulse, 90 min, sc). Afterward, one of the
patients with FHA received a single sc injection of 1350 U recombinant
human LH, and plasma LH, LHß, FAS, and CGß levels were measured and
compared with the high plasma levels of one postmenopausal woman. In cycling women, basal plasma LHß and CGß levels were below the detection limit of the assays (1.34 and 0.65 pmol/L, respectively), and plasma FAS levels were 13.60 ± 0.13 pmol/L. During the LH surge, there was a parallel increase in LH, LHß, and FAS. Plasma CGß levels remained undetectable. In normal postmenopausal women, basal plasma dimeric LH, LHß, and FAS levels were increased in parallel, and their pulsatile profiles were similar, without measurable plasma CGß levels. After D-Trp6 administration, plasma LH and LHß levels were completely suppressed, whereas plasma FAS levels increased, and plasma CGß remained below 0.65 pmol/L. In FHA women, basal plasma levels of LH and FAS were low, without detectable LHß and CGß levels. During pulsatile GnRH administration, LHß became detectable, and pulses were synchronous with those of LH and FAS. The secretion of LH and LHß was almost equimolar. Plasma CGß levels remained undetectable. In the patient with FHA, administration of recombinant human LH increased only plasma LH levels, whereas plasma LHß and FAS levels remained very low.
In conclusion, when the production of dimeric LH increases, a
concomitant, parallel, and almost equimolar hypersecretion of
uncombined and biologically inactive LHß occurs. Like the
-subunit, LHß may be secreted in the dissociated free form. This
can lead to pitfalls during clinical investigations if assays of free
CGß display some cross-reaction with free LHß.
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
- and ß-subunits encoded
by two different genes (1). The -subunit is common to the four
glycoprotein hormones LH, FSH, TSH, and CG. The secretion of free
-subunit (FAS) has been studied extensively under physiological and
pharmacological conditions (2, 3, 4, 5, 6, 7, 8, 9). The ß-subunit of LH confers
hormonal specificity, but a strong homology exists between CG and LH,
explaining the cross-reactivity in immunoassays. Little is known about
the physiological secretion of the free ß-subunit of LH, which until
recently was considered negligible (10). Using a very specific (i.e. noncross-reactive with the intact hormones) and sensitive assay, we have previously shown the production of free LH ß-subunit (LHß) in postmenopausal women and its increase after GnRH stimulation in premenopausal women (10). The aim of the present study was to compare LH, FAS, and LH ß-subunit secretion in normally cycling women during the ovulatory peak of LH. The pulsatile profile of LH, FAS, and LH ß-subunit and the response to a long-acting GnRH agonist were studied in postmenopausal women. The pulsatility of LH, FAS, and LH ß-subunits was also studied during pulsatile GnRH administration in patients with functional hypothalamic amenorrhea (FHA) chosen for their low endogenous LH levels. Finally, one FHA woman received recombinant LH to assess plasma levels of LH, LHß, and FAS in the absence of endogenous LH. As the present assay of LHß also recognizes CGß, we used simultaneously a specific and sensitive immunoradiometric assay (IRMA) for measurement of the free form of plasma CGß in all subjects. The present study demonstrates that LHß, like FAS, may be secreted as a free (noncombined) component.
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Five normally cycling women, aged 23–28 yr, with a history of regular ovulatory cycles, as judged by basal body temperature charts, LH surge, and plasma progesterone (P) values observed during two spontaneous cycles preceding the study, participated. Five healthy postmenopausal women, aged 50–58 yr, were previously studied and described (3). Five women with functional hypothalamic amenorrhea (FHA) of nutritional origin (body mass index, 18.5 ± 0.5 kg/m2), aged 25–30 yr, with plasma estradiol (E2) levels below 37 pmol/L and a negative classical GnRH test (100 µg, iv) were studied because of the absence of endogenous LH secretion. These women with FHA desired pregnancy and were willing to enter the program of ovulation induction with pulsatile administration of GnRH. None of the subjects had received sex steroids for at least 2 months before the study. All women had normal plasma PRL and TSH levels. Each woman signed an informed consent. The study was approved by the investigation committee of the University Paris-Sud.
Protocol
The five normally cycling women included in this study were asked to record basal body temperature and to determine the LH surge by a daily urine assay during the study cycle. Plasma samples were collected for E2, P, LH, FAS, LHß, and CGß measurements, daily from days 3–5 of the follicular phase until day 6 after the LH surge.
The plasma samples of five postmenopausal women were retrieved from long-term storage at the clinical research center serum bank. The following protocol was performed (3). An indwelling iv catheter was placed in a forearm vein at 0700 h, and blood was sampled every 10 min for 4 h (from 0800–1200 h). Each postmenopausal woman had received at 0800 h a single im injection of 3.75 mg D-Trp6 GnRH, delivering a daily dose of 100 µg/day for 30 days (3). Study of pulsatile LH, FSH, FAS, LHß, and CGß secretion was performed on days 0 and 21 days after this injection. Blood samples were immediately centrifuged, and plasma was separated and kept in aliquot (-70 C).
The five women with FHA were studied 10 days after a negative P test (absence of uterine bleeding). Intermittent GnRH pulses were administered sc at a dose of 20 µg/pulse every 90 min. A portable autoinfusion pump was used (Zyklomat, Ferring Pharmaceuticals Ltd., Paris, France). On days 0 and 10 of pulsatile GnRH administration, a study of pulsatile LH, FSH, FAS, LHß, and CGß secretion was performed. An indwelling iv catheter was placed in a forearm vein at 0700 h, and blood was sampled every 10 min for 4 h (from 0800–1200 h).
One ampule of recombinant human LH (rhLH; Serono, Aubonne, Switzerland; 75 IU) was tested to confirm the absence of measurable LHß. One patient with FHA, who was not pregnant after the first cycle with pulsatile GnRH, received a single sc injection of 1350 IU rhLH (18 vials of 75 IU) to obtain menopausal plasma LH levels (Schaison, G., unpublished personal data). No discomfort was reported after the injection. Plasma LHß, LH, and FAS levels were measured every hour for 8 h in this patient. The peak was compared to the level in one of the postmenopausal women with similar plasma LH levels.
Assays
Molar substance concentrations were used to compare plasma levels of LH and subunits. Indeed, it is well known that the concentrations measured by immunoprocedures correlate better with substance concentrations than with bioactivity or mass. On the basis of an absolutely pure preparation, the specific activity of LH is about 1 IU, equal to 2.1 pmol. The substance concentration of FAS was calculated on the basis that 1 IU is represented by 1 µg or 68 pmol of the pure subunit preparation. A highly purified preparation of LH ß-subunit was used on the basis that 1 µg of preparation corresponds to approximately 67 pmol.
Plasma LH concentrations were measured in duplicate with a monoclonal antibody (mAb) IRMA (International Cis Reagents, Gif-sur-Yvette, France) as previously described (11). The mean detection limit of the assay was 0.05 pmol/L. The intra- and interassay coefficients of variation were, respectively, 10% and 13% for a concentration of 0.21 pmol/L, 4% and 6% for 13.12 pmol/L, and 2% and 3% for 63 pmol/L.
FAS was measured using a commercial IRMa based on two monoclonal antibodies (m-IRMA; Immunotech, Marseilles, France). The cross-reactivity of this immunoassay is less than 0.1% for human dimeric hormones (including CG, LH, FSH, and TSH) and 0% for the free ß- subunits of these hormones. Results were expressed as picomoles per L, and the detection limit was 1.7 pmol/L. Within-run and coefficients of variation were, respectively, 6% and 12% at a concentration of 20.4 pmol/L and 3% and 5% at a concentration of 136 pmol/L. Normal ranges for basal plasma FAS levels were 0–40.8 pmol/L in premenopausal women during the early follicular phase and 61.2–129.2 pmol/L in postmenopausal women.
To detect free LHß, we developed a 2-site in-house m-IRMA based on monoclonal mAb BLH01 as the capture antibody. This antibody was selected for its specificity for free hLHß, and the design of the assay has been previously reported (12). The epitope recognized by BLH01 encompassed a region of the ß-subunit including amino acids within the Cys93 and Cys100 loop. Purified hLHß (AFP-3282B), a gift from the NIDDK and the National Hormone and Pituitary Program, was used as the standard. The sensitivity of this assay is defined by the least detectable concentration, i.e. the free LHß concentration resulting in an increase (in counts per min bound) that was 2 SD higher than the mean in 20 replicates, and was 1.34 pmol/L. The within-run coefficient of variation, determined by assaying 20 replicates of a sample containing 33.5 pmol/L free LHß, was 6.5%. The between-run coefficient of variation, determined by assaying a serum sample (53.6 pmol/L) 10 times, was 7.6%. We also performed a recovery test on 1 mix using equal volumes of samples and standards containing increasing levels of free hLHß (0, 8.4, 16.8, 33.5, 83.8, 167.5, and 335.0 pmol/L). Regression analysis confirmed that the free LH ß-subunit concentration, when corrected for dilution, was not significantly affected by the dilution factor (r = 0.99). The relative cross-reactivity with hLH (1.5%) was due not to the recognition of LH by BLH01, but to cross-contamination of hLH material by free LHß (12). As this m-IRMA displayed a cross-reactivity of about 65% with the free hCGß, we used a second immunoassay specific for free CGß and based on FBT11 mAb (13). This m-IRMA had a sensitivity of 0.65 pmol/L for free CGß, and cross-reactivity of less than 0.2% with free LHß.
Data analysis
The data are presented as the mean ± SEM. Statistical significance was assumed for P < 0.05. The LH surge was compared to the LHß, FAS, and CGß in the normally cycling women using the Wilcoxon rank sum test. In postmenopausal women and in women with FHA, LH, FAS, LHß, and CGß, pulsatile secretions were analyzed with the program of Thomas et al. (14). Pulses were defined as concordant if they occurred less than 10 min apart. Comparisons between baseline and agonist values in postmenopausal women were made with the Wilcoxon rank-sum test for matched pairs. In the FHA patient, the peaks of LH, LHß, and FAS obtained after rhLH were compared to basal levels in one of the postmenopausal women with similar LH levels.
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
). Plasma CGß levels remained
undetectable (<0.65 pmol/L).
|
). On day 21 of GnRH agonist
administration, plasma FAS levels increased significantly to 189.0
± 8.2 pmol/L (P < 0.001), but the pulsatile secretory
pattern was abolished (Fig. 3, A and B).
Plasma CGß remained below 0.65 pmol/L. In contrast, the suppression
of the mean plasma LH and LHß levels was almost complete (93%;
4.8 ± 0.3 and 4.0 ± 0.2 pmol/L, respectively) after 3 weeks
of D-Trp6 GnRH
administration (Fig. 3, A and B).
|
|
). During pulsatile GnRH
administration, LHß became detectable, and the secretory patterns of
LH closely paralleled those of LHß and FAS, as shown in Fig. 4B.
Plasma LH and LHß levels were almost equimolar, whereas plasma FAS
levels were parallel, but significantly higher (P <
0.01). Plasma CGß levels remained below the detection limit of the
assay.
|
In the FHA woman, rhLH administration at a dose of 1350 IU increased
plasma LH levels from 0.6 to 19.6 pmol/L 7 h after the injection
(Fig. 5A), comparable to the basal plasma
LH levels in one of the postmenopausal women. In contrast, plasma LHß
and FAS levels remained very low compared to those in the same
postmenopausal woman (Fig. 5B).
|
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Previous studies have shown that FAS secretion is pulsatile and follows
with a high concordance LH pulses (2, 3, 4, 5, 6, 7, 8, 9). In addition, we previously
reported that free LHß was measurable in postmenopausal women and in
men and premenopausal women after a GnRH test (10). Using this
sensitive and specific assay for LHß, we demonstrate in the present
study that in normally cycling women, a peak of LHß was superimposed
with the LH surge and concordant with FAS, whereas basal plasma LHß
levels were undetectable during the follicular phase. It is noteworthy
that both intact LH and the ß-subunit were secreted in similar molar
amounts during the ovulatory peak and pulsatile GnRH administration. In
postmenopausal women with spontaneously elevated gonadotropin levels, a
similar high concordance of LH, LHß, and FAS pulses was observed. In
women with FHA with low plasma LH and FAS levels and without detectable
LHß levels, pulsatile GnRH replacement increased plasma LH, LHß,
and FAS levels. A high concordance of LHß and FAS pulses was found,
confirming that GnRH stimulates both FAS and free LHß secretion (15).
However, the regulation of LHß and FAS was different. In
postmenopausal women treated with GnRH agonist, there was a parallel
suppression of LH and LHß, whereas plasma FAS levels increased. This
has been previously demonstrated by measuring the subunit’s messenger
ribonucleic acid (mRNA). ß-Subunit mRNA decreased and -subunit
mRNA increased during GnRH agonist administration (3, 16). The distinct
secretion pattern of free subunits gives evidence that their
biosynthesis is different. LHß, not FAS, is entirely controlled by
pulsatile GnRH.
To control that the measured LHß was secreted directly from the pituitary and was not a product of LH proteolysis, we compared free LHß levels in one of the FHA woman after the administration of recombinant LH (1350 IU) to obtain high plasma levels of LH and in one of the postmenopausal woman with similar LH levels. In the FHA patient, plasma LHß and FAS levels remained very low compared to those in the postmenopausal woman, demonstrating that the measured LHß in postmenopausal women is of pituitary origin.
During basal, low LH secretion (follicular and luteal phases of the cycle), the sensitivity of our LHß assay was not sufficient to detect ß-subunit secretion, whereas FAS was already measurable, as previously reported (17). In the absence of LH stimulation, the ß-subunit may be the limiting step of the dimer formation. When the synthesis or release of LH is increased (ovulatory peak, postmenopause, or GnRH stimulation), plasma LHß levels become measurable. Additional studies are needed to explain why the gonadotrophs are able to secrete the dimeric hormone as well as its bioinactive, unassembled subunits.
The high degree of sequence similarity (85%) between LHß and CGß
explains why our assay of LHß cross-reacts with free CGß, implying
simultaneous determination of both free ß-subunits (18). In the
present study all plasma samples were tested for the presence of free
CGß using a very specific IRMA (19, 20, 21). No detectable CGß could be
found in any of the women studied. Odell and Griffin demonstrated
in 1987 the pulsatile secretory pattern of hCG and its production by
the pituitary (22). Since then, evidence has accumulated for the
production of CG by the pituitary. mRNA for CGß was found in human
pituitaries (23), and CG secretion by pituitary cells in culture was
detected. However, in all of the previous studies it was surprising
that the secretion of pituitary CG was always parallel to the secretion
of dimeric LH throughout the menstrual cycle. A midcycle ovulatory peak
of CG coincident with the LH peak has been observed (24). Distinct
pulses of CG were detected in parallel with those of LH in
postmenopausal women (22). Finally, the regulation of CG levels was
similar to that of LH: stimulated by GnRH and inhibited by a combined
estrogen-progestin administration (24). In the present study the free
ß-subunit of LH presented all of these characteristics, whereas CGß
was undetectable. Even if CG is present in the pituitary, the parallel
levels of LH and - and ß-subunits of LH and the similar regulation
of LH and ßLH by pulsatile GnRH and GnRH agonist administration raise
some uncertainty about the nature of the previously measured
glycoprotein hormone.
In conclusion, when the production of dimeric LH increases, i.e. during the ovulatory peak, after menopause, or after pulsatile GnRH administration, a concomitant and parallel secretion of free LHß occurs. The assembly of the newly synthesized ß-subunit with FAS to form the heterodimer is incomplete. Uncombined and biologically inactive LHß may be, like FAS, secreted in dissociated free form. These results have to be taken into account for the diagnosis of patients with pituitary gonadotropin-secreting tumors (10). Specific measurement of the free ß-subunit of CG is also important in the follow-up of postmenopausal patients with nontrophoblastic malignancies (25). An ultraspecific assay of CGß, as presented here, is necessary to measure only the ectopic production of free CGß and avoid the pitfall of recognizing the free LHß normally secreted by the pituitary.
|
Acknowledgments |
|---|
Received December 30, 1999.
Revised March 13, 2000.
Accepted March 17, 2000.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
-subunit secretion from the
gonadotrope by gonadotropin-releasing hormone (GnRH): evidence from the
use of two GnRH antagonists. J Clin Endocrinol Metab. 70:328–335.[Abstract]
-subunit in man. J Clin Endocrinol Metab. 60:344–348.[Abstract]
-subunit pulses after LH-releasing hormone antagonist administration
in normal men. J Clin Endocrinol Metab. 70:1742–1748.
-subunit in the neonate and in prepubertal and
pubertal children: effects of luteinizing hormone-releasing hormone. J Clin Endocrinol Metab. 50:450–455.[Medline]
-subunit
secretion. J Clin Endocrinol Metab. 62:102–108.[Abstract]
-Subunit secretion in
men with idiopathic hypogonadotropic hypogonadism. J Clin
Endocrinol Metab. 66:338–342.[Abstract]
-subunit secretion after
administration of a luteinizing hormone-releasing hormone antagonist in
normal men. J Clin Endocrinol Metab. 70:1219–1224.[Abstract]
hCG, and ßhCG as measured by specific monoclonal
immunoradiometric assays. Endocrinology. 120:549–558.[Abstract]
-subunit levels during pulsatile
gonadotropin-releasing hormone replacement in women with idiopathic
hypogonadotropic hypogonadism. J Clin Endocrinol Metab. 83:241–247.
and rat luteinizing hormone-ß messenger ribonucleic acids during
gonadotropin-releasing hormone agonist treatment in vivo in
the male rat. Endocrinology. 123:2111–16.[Abstract]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
