Serum bioactive and immunoreactive LH and FSH were measured in clinical conditions with increased or decreased gonadotropin secretion. Gonadotropin immunoreactivity was measured using a conventional RIA (I) and an ultrasensitive immunofluorometric method (F). Bioactive (B) LH was assessed by the mouse interstitial cells in vitro bioassay, and B-FSH using the immature rat granulosa cell assay. Acute GnRH stimulation of adult men (n = 6) increased LH levels measured by the different methods 4.3- to 5.3-fold. The B/I ratio of LH increased from 2.34 +/- 0.21 to 3.71 +/- 0.36 (mean +/- SEM) at 120 min (P less than 0.05), but no change was found in the B/F ratio. After ovariectomy of premenopausal women (n = 6), the LH levels increased in 1 week 4- to 6-fold, the B/I ratio from 1.85 +/- 0.22 to 2.59 +/- 0.24, and the B/F ratio from 1.78 +/- 0.22 to 2.90 +/- 0.30 (P less than 0.05 for both). In addition, the LH levels were measured during GnRH agonist treatment of ovarian carcinoma (n = 8), endometriosis (n = 8), and prostatic carcinoma after orchiectomy (n = 8). In the two former groups, serum B-LH decreased in 1 month to undetectable levels (less than 0.5 IU/L), and in the prostate cancer patients to 1.2 (0.8-1.9) IU/L (log mean and range of +/- SEM). The concomitant decline of I-LH was to 1.5-1.9 IU/L in the agonist-treated female patients, and that of F-LH to 0.10-0.15 IU/L; in the prostate cancer patients, respectively, these values were 7-8 and 0.3-0.7 IU/L. The B/I and B/F ratios during the agonist treatments could only be calculated in the prostate cancer patients (in the others, B-LH became undetectable). The B/I ratio decreased from 2.34 +/- 0.5 to 0.14 +/- 0.03 (P less than 0.01), but no suppression was found in the B/F ratio from a pretreatment value of 3.6 +/- 0.8. B-, I-, and F-FSH levels were measured in the GnRH agonist-treated orchiectomized prostate cancer patients. The pretreatment level of B-FSH was 154 (137-175), that of I-FSH was 38.0 (34.4-42.0), and that of F-FSH was 39.8 (35.3-44.9) IU/L. The B/I ratio of FSH was 3.76 +/- 0.49, and the B/F ratio was 3.53 +/- 0.59. The mean B-FSH level decreased during treatment by 87-93.5%, that of I-FSH by 98%, and that of F-FSH by 91.5% (P less than 0.01 for all).(ABSTRACT TRUNCATED AT 400 WORDS)

An investigation was undertaken to characterize an immunological LH variant in a 31-yr-old healthy woman whose serum LH was either poorly or not at all recognized by two monoclonal antibodies. The two antibodies recognize epitopes present on the intact LH dimer, but not on the free subunits. It was found that the immunologically aberrant LH of the subject was bioactive, as evidenced by an in vitro bioassay for LH. Nothing in the personal history of the subject or in the results from a number of hormone analyses revealed any endocrine abnormalities. In a GnRH stimulation test, the increase in immunoreactive LH using two reference immunometric assays for LH was less than 10% of the mean response of five control subjects. In relative terms, the maximal increase in LH in the subject was only 60-100%, in contrast to 340-560% for the control subjects. The bio/immuno ratio of the LH in the subject was high and was further increased in the GnRH stimulation test. A low proportion of acid LH isoforms in basal and stimulated samples from the subject was in agreement with the high bio/immuno ratio. Gel filtration studies showed the presence of molecular species of apparently lower molecular size than the intact LH, but different from the free beta-subunit. The results suggest the presence of fragments of the alpha-beta-dimer where at least part of the beta-subunit has been lost. A pedigree analysis involving the parents, siblings, and children of the subject strongly suggests a genetic origin of the LH variant described with an autosomal mode of inheritance. This report on an immunological variant of LH illustrates the potential dangers of using monoclonal based immunoassays where a protein hormone with fully maintained biopotency may be partially or totally missed due to the monospecificity of the immunoreagent. This possibility should be kept in mind when inappropriately low levels of gonadotropins are detected in diagnostic routine.

The present study aimed at investigating the nature and causes of non-parallelism in testosterone responses to serial dilutions of peripheral serum and standard LH preparations in the mouse Leydig cell in vitro bioassay of LH. Immunoadsorption with monoclonal antibody to the \g=b\-subunit of LH was used to obtain LH\x=req-\ free serum; the procedure removed more than 98% of the immunoassayable LH. When a constant amount of the LH-free serum was added to standard dilutions, the bioassay dose-response curves to serum dilutions and standards became parallel, i.e. the well-known source of error of this assay system was eliminated. When standard curves prepared in medium and LH-free serum (final concentration 10%) were compared, no effect of the serum was found on basal cAMP and testosterone production.However, the LH-stimulated testosterone and cAMP production were suppressed by serum by a rather constant factor of 40%. Mild heating (60\s=deg\C,15 min) or treatment with dextran-coated charcoal, but not ether extraction, was able to eliminate the inhibitory activity of the LH\x=req-\ free serum. Binding studies demonstrated that [125I]hCG interaction with mouse Leydig cell homogenates was inhibited by LH-free serum in a fashion indicative of reduced LH receptor number, but not of reduced binding affinity. In conclusion, these data show that human serum contains LH inhibitor(s) which affect the LH-receptor interaction and LH stimulated testosterone production in mouse Leydig cell in vitro. The effect is marked in serum concentration over 1.5% and it shows only minor variation between individual sera. This source of error can be effectively removed from the LH in vitro bioassay by using LH-free serum for preparation of dilutions of LH standards.Human luteinizing hormone has stimulatory and trophic effects on gonadal steroidogenic cells via specific receptors. In the male, Leydig cell androgen production is regulated by LH. The inter¬ action of LH with its receptors on the cell surface initially increases intracellular cAMP by activation of adenylyl cyclase, which event subsequently acti¬ vates cholesterol transport and steroidogenesis.On the basis of this mode of LH action, therefore, the testosterone response of Leydig cells to gonadotropins has provided a useful model for in vitro bioassays of LH. The most widely used of these methods are those employing mouse ( 1 ) and rat (2) testicular interstitial cell suspensions.There is increasing evidence from the last de¬ cade that the human peripheral serum may con¬ tain inhibitors of LH action. For instance, it is a common finding in the in vitro bioassay of LH (2-5) that the maximum testosterone response to serum dilutions is always lower than the response to standard LH preparations, which non-parallel¬ ism of the responses brings an error to the assay system. As an explanation for such an event, some studies (3,4,6) have suggested the presence of inter¬ fering factor(s) in human serum, but no informa¬ tion on their nature is available. This study aimed at identification ...

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In an attempt to correlate serum LH isoform distribution with its bio/immuno ratio, follicular phase blood samples from four normally cycling women and four patients with polycystic ovarian disease (PCOD) were studied. Chromatofocusing of peripheral serum samples across a pH gradient of 9.5-4.5 yielded a broad area of LH immunoreactivity comprising several peaks in the pH range of 7.2-9.0, and six other major peaks at pH values of 9.4, 6.8, 6.4, 5.1, 4.7 and less than 4.0. In addition, the bioactive and immunoreactive levels of LH were measured in the unfractionated serum samples. In three out of four PCOD patients and one healthy woman, the majority of LH species (approximately 70%) were distributed at a pI value greater than 7.0. All of them had a high bio/immuno ratio (greater than 2.0) and an elevated bioactive level of serum LH (greater than 40 IU/l). Conversely, fewer alkaline pI isoforms of LH were found in the other three normal women and one PCOD patient, who had low levels of the bio/immuno ratio of LH (less than 2.0) and bioactive LH (less than 40 IU/l). A significant (P less than 0.05) direct correlation was observed between the bio/immuno ratio of serum LH and the proportion of alkaline LH eluted. In conclusion, as demonstrated previously in the pituitary, the bio/immuno ratio of serum LH also correlates well with the charge distribution of LH isoforms. The results indicate that an altered isoform distribution with more basic LH forms is associated with a high biological activity of serum LH.