Serum binding of steroid tracers and its possible effects on direct steroid immunoassay.

We studied the serum protein binding of 3H-labelled progesterone, oestradiol and testosterone, and five 125I-labelled analogues of these steroids. All tracers investigated appeared to be bound by proteins in every serum sample tested. The addition of blocking agents caused a substantial reduction in serum protein binding of 3H-labelled steroids, but had relatively little effect on the binding of analogue steroid tracers. Use of analogue steroid tracers in conventional direct immunoassays for oestradiol and progesterone produced anomalous results for some patient samples when compared to extraction radioimmunoassays, but assays where tracer binding to serum constituents was prevented by adoption of two-step procedures appeared to avoid anomalous results. The results suggest that serum protein binding of steroid analogue tracers may be a source of interference in some direct steroid immunoassays.

Design of immunoassays for use in developing countries.

PIP: The applicability of both radioimmunoassay and non-radiometric assay methods for use in research and clinical laboratories in developing countries are reviewed. The WHO Special Program of Research in Human Reproduction has distributed radioimmunoassay reagents to laboratories collaborating on fertility research for 10 years, but it is predicted that non-radiometric methods will be used in the near future. Some of the qualities required by sites in developing countries are stability at high temperatures, low cost, simple procedures, simple and non-dedicated instrumentation or none at all, ease of local manufacture or practical distribution and supply, and small, practical storage requirements. Biological samples should not require invasive procedures. Assay time must be brief if patients are waiting, but it is usually not possible to handle large sample volumes to same time. Radioisotopes have the advantage of affecting the kinetics of the test reaction to a lesser extent than non-radiometric assays, but they do required separation of bound from free hormone. Radioimmunoassays also can be repeated, while many non-radiometric assays are destroyed in the assay procedure. Time-resolved fluorescence assays are a good choice for the medium term.^ieng

Qual o valor dos constituintes da saliva para predizer ovulaçäo? / What the value of saliva constituents to predict ovulation?

Foram relatados diferenças na concentraçäo de alguns constituintes da saliva (esteroides, eletrólitos, glicose e enzimas) que ocorrem durante o ciclo menstrual e que talvez poderiam ser usados para predizer a ovulaçäo. Nos medimos estradiol, progesterona, glicose, sódio, potássio e cálcio em amostras da mistura salivar colhidas diariamente durante todo o ciclo menstrual. O dia da ovulaçäo foi determinado por ultra-sonografia e medidas de LH em urina. O perfil de estradiol e progesterona em saliva refletiu aquele classicamente encontrado em sangue. Os níveis dos eletrólitos em saliva näo mostraram padräo ciclíco definido mesmo após a correçäo da taxa de produçäo da saliva. Foram observadas alteraçöes cíclicas na concentraçäo de glicose em saliva, sendo que os valores máximos (picos) foram encontrados no dia ou no dia após a aovulaçäo, ou seja, durante o período periovulatório. Pequenos picos foram observados durante as fases folicular e lútea sem, entretanto, mostrarem um padräo reprodutivel. Estas alteraçöes foram melhor vistas nas amostras de jejum. Amostras sem o jejum tiveram concentraçöes altas de glicose o que tornou difícil a identificaçäo dos picos. Este estudo confirma o valor da medida dos esteróides em saliva, mas entretanto, sugere que medidas de glicose e eletrólitos em saliva têm, provavelmente, pouco valor como teste preditivo para ovulaçäo

Inhibition by gossypol of cyclic AMP production in mouse Leydig cells.

Gossypol has been shown to inhibit steroidogenesis in leydig cells. This study examined the mechanism of this action by investigating the effect of gossypol on leydig cell hCG receptor binding, adenylate cyclase activity and cyclic AMP production in leydig cells. Gossypol had no effect on hCG binding to cell membranes but inhibited LH-stimulated cyclic AMP production in whole purified leydig cells. It also reduced the stimulation caused by LH, Gpp(NH)p, forskolin and fluoride in membranes from leydig cells and also from liver. It is therefore possible that gossypol affects cyclic AMP production at the level of ATP conversion to cyclic AMP, although effects on the G-protein and catalytic subunit cannot be ruled out.

Site of gossypol inhibition of steroidogenesis in purified mouse Leydig cells.

Gossypol, a phenolic compound that has been studied as a potential male contraceptive, inhibits basal and LH-stimulated testosterone release from Leydig cells in vitro. The present study investigates the mechanism of this inhibition using preparations of purified mouse Leydig cells. Gossypol inhibited LH-stimulated progesterone, 17-hydroxyprogesterone and androstenedione production by mouse Leydig cells incubated in vitro. It also inhibited dibutyryl cyclic AMP-stimulated production of testosterone but was without effect in the presence of added pregnenolone or 25-hydroxycholesterol. These results suggest gossypol exerts its major inhibitory effect on Leydig cell function at a point between LH-dependent stimulation of cyclic AMP dependent protein kinase activity and increased availability of cholesterol for side chain cleavage.

The viability of mouse Leydig cells in vitro following incubation with gossypol.

Gossypol, a phenolic compound that has been studied as a potential male contraceptive, has also been shown to inhibit basal and LH-stimulated testosterone production by mouse Leydig cells in vitro. This inhibition persists even when cells treated with gossypol are washed prior to reincubation with LH. While gossypol has been reported to be cytotoxic, its effect on steroidogenesis does not appear to be a consequence of cytotoxicity as there was no reduction in the number of viable cells after incubations in which inhibition of testosterone production was observed.

Studies on the nature of prolactin-like immunoreactivity in bromelin-treated cervical mucus.

Prolactin has been reported to be present in cervical mucus at concentrations higher than those found in blood. Our initial findings appeared to confirm this and the material fulfilled criteria of validity generally applied when an immunoassay is employed on a new biological matrix, i.e. parallelism and chromatographic identity. Further experiments demonstrated that prolactin concentrations in cervical mucus were less than 40 mU/L and the prolactin-like immunoreactivity originally detected was due to the action of the enzyme bromelin which was used to liquefy the mucus. Bromelin has a similar molecular weight to prolactin and appeared to digest prolactin tracer and reduce its ability to bind antiserum in a manner paralleling the effect of adding pituitary prolactin.

Inhibition by (+) and (-) isomers of gossypol of testosterone release from mouse Leydig cells in vitro.

Racemic (+/-) mixtures of gossypol isomers exert an antifertility effect by inhibiting sperm motility and spermatogenesis. Purified (+) gossypol has been shown to be without these actions. In this study pure preparations of both (+) and (-) gossypol were found to inhibit, in a similar manner, both basal and LH-stimulated release of testosterone by isolated Leydig cells at concentrations down to 21 microM. It is possible that use of low doses of pure (-) gossypol could inhibit fertility with less endocrine side effects.

Inhibition by gossypol of testosterone production by mouse Leydig cells in vitro.

Gossypol, a polyphenolic compound prepared from cotton seed, has potential uses as a male contraceptive. It inhibits spermatogenesis and sperm motility but has toxic side effects and furthermore may have effects on endocrine function though evidence for this is contradictory. In this study, gossypol had a dose-dependent inhibitory action on release of testosterone from dispersed mouse Leydig cells. Doses down to 20 microM and 10 microM significantly inhibited unstimulated and LH-stimulated release, respectively.

Multicenter evaluation of assays for estradiol and progesterone in saliva.

Blood samples can be difficult to obtain in studies involving serial sampling, especially in developing countries where there may also be logistic, ethical, and cultural constraints that make frequent blood collection impractical. Assays for steroids in saliva may avoid some of these difficulties. A multicenter study involving laboratories in five countries was carried out to compare the results of assays for salivary estradiol and progesterone performed with centrally provided reagents and assay protocols. Concentrations of salivary steroid as obtained by all but one center were comparable with those reported in the literature. We conclude that assays of hormones in saliva are useful adjuncts to those performed on other body fluids.