E2 Assay Research Articles

A randomized controlled trial of interleukin-1 receptor antagonist in a rabbit model of ascending infection in pregnancy.

Objective: To determinewhether treatment with interleukin-1 receptor antagonist (IL1-ra) would affect amniotic fluid concentrations of tumor necrosis factor alpha (TNF-α) and prostaglandins or clinical or microbiological outcomes in a model of ascending bacterial infection in pregnancy. Methods: Timed pregnant New Zealand white rabbits at 70% of gestation underwent endoscopic inoculation of the cervices with 106–106 cfu Escherichia coli . Animals were randomly assigned in a blinded manner to a 5-h intravenous infusion of human IL1-ra (10 mg/kg) or placebo beginning 1 – 2 h after inoculation. Blood was drawn fromthe does for assay of serum IL1-ra concentration before inoculation, at mid-infusion, after the infusion ended and at necropsy. At necropsy, temperature and cultures were taken, and aspirated amniotic fluid was pooled for assays of TNF-α, prostaglandin E2 (PGE2) and IL1-ra. Results: Serum IL1-ra concentrations rose to a mean of 2 mg/ml at mid-infusion and fell markedly after the infusion to concentrations barely detectable at necropsy. Between the two groups, there were no significant differences in the rates of fever or positive cultures or in amniotic fluid concentrations of PGE2 or TNF-α.One unique finding was the demonstration that administration of human IL1-ra to the does resulted inmeasurable concentrationsof human IL1-ra in the amniotic fluid. Conclusions: Treatment with an intravenous infusion of human IL1-ra after cervical inoculation with E. coli did not affect clinical or microbiological outcomes or amniotic fluid concentrations of TNF-α or PGE2. This experiment provides the first demonstration of passageof human IL1-ra from the maternal bloodstreamto the amniotic fluid.

Core domain mutation (S86Y) selectively inactivates polyubiquitin chain synthesis catalyzed by E2-25K.

The mammalian ubiquitin conjugating enzyme known as E2-25K catalyzes the synthesis of polyubiquitin chains linked exclusively through K48-G76 isopeptide bonds. The properties of truncated and chimeric forms of E2-25K suggest that the polyubiquitin chain synthesis activity of this E2 depends on specific interactions between its conserved 150-residue core domain and its unique 50-residue tail domain [Haldeman, M. T., Xia, G., Kasperek, E. M., and Pickart, C. M. (1997) Biochemistry 36, 10526-10537]. In the present study, we provide strong support for this model by showing that a point mutation in the core domain (S86Y) mimics the effect of deleting the entire tail domain: the ability to form an E2 approximately ubiquitin thiol ester is intact, while conjugation activity is severely inhibited (>/=100-fold reduction in kcat/Km). The properties of E2-25K enzymes carrying the S86Y mutation indicate that this mutation strengthens the interaction between the core and tail domains: both free and ubiquitin-bound forms of S86Y-25K are completely resistant to tryptic cleavage at K164 in the tail domain, whereas wild-type enzyme is rapidly cleaved at this site. Other properties of S86Y-26K suggest that the active site of this mutant enzyme is more occluded than the active site of the wild-type enzyme. (1) Free S86Y-25K is alkylated by iodoacetamide 2-fold more slowly than the wild-type enzyme. (2) In assays of E2 approximately ubiquitin thiol ester formation, S86Y-25K shows a 4-fold reduced affinity for E1. (3) The ubiquitin thiol ester adduct of S86Y-25K undergoes (uncatalyzed) reaction with dithiothreitol 3-fold more slowly than the wild-type thiol ester adduct. One model to accommodate these findings postulates that an enhanced interaction between the core and tail domains, induced by the S86Y mutation, causes a steric blockade at the active site which prevents access of the incoming ubiquitin acceptor to the thiol ester bond. Consistent with this model, the S86Y mutation inhibits ubiquitin transfer to macromolecular acceptors (ubiquitin and polylysine) more strongly than transfer to small-molecule acceptors (free lysine and short peptides). These results suggest that unique residues proximal to E2 active sites may influence specific function by mediating intramolecular interactions.

Analytical and clinical evaluation of the Immulite® estradiol assay in serum from patients undergoing in vitro fertilization: estradiol increase in mature follicles

We examined an immunoassay for estradiol (E2) on the Immulite-an automated, random access chemiluminescent immunoassay system-to determine its accuracy and precision required for in vitro fertilization (IVF) studies. The assay, which has a reportable range from 73 to 7300 pmol/L, demonstrated good linearity under dilution, a detection limit of 44 pmol/L, and interassay CVs of 12.6% and 7.6% at 466 and 6164 pmol/L, respectively. In a retrospective analysis of 545 serum samples, the assay showed adequate agreement with an antibody-coated-tube RIA. The two E2 assays showed good agreement, even on samples from patients receiving a variety of different estrogen replacement therapies. Longitudinal studies of individual IVF cycles showed good parallelism between the automated system and the RIA, and results by the automated assay correlated well with the total number of follicles.

Anti-HCV envelope prevalence in blood donors from Baden-Württemberg.

We assess the diagnostic value of the recombinant antigens E1/E2 (heterodimer) and E2 for the screening of blood donors. For this purpose an ELISA assay was developed which used these recombinant glycoproteins as solid antigens on the microtiter plates. The anti-E1/E2 assay was able to detect 80% of the positive samples by Matrix and 87% of the PCR-positive samples. Anti-E2 was less sensitive. Fifteen percent of the indeterminate samples were anti-E1/E2 and 8% anti-E2 positive. In the case of the indeterminate samples there was no coincidence of results between these assays and the PCR. Fifty percent of seroconverters showed an anti-E1/E2-positive result with a previous indeterminate donation. We conclude that the recombinant envelope proteins, mainly anti-E1/E2, might be used in future in the diagnostics of HCV infection to increase the sensitivity of the currently used assays.

Bioavailability of nasally administered progesterone

8 healthy women volunteers between the ages of 22-38 participated in a study designed to explore the relationship between endogenous estradiol (E2) levels and progesterone (P) absorption. Physical and pelvic examinations and laboratory screening tests revealed no abnormalities. All women had regular menstrual cycles, at 24-32-day intervals, and all were free of a significant menstrual cycle symptoms. 2 studies were performed at least 72 hours apart in the follicular phase of the menstrual cycle. The following medications were administered in random order: nasal P (Pronasone), 20 mg and nasal P (Pronasone), 30 mg. Serum P levels were drawn at the following times: 0, 3, 6, 10, 20, 30, 60, 120, 180, 240, 360, and 480 minutes. Serum for E2 assay was taken from the 0 time sample. The women were examined with a nasal speculum after each nasal absorption study. Serum was separated and frozen for subsequent assay. The data were analyzed using the CLINFO system from the National Institutes of Health. All of the women complained of a mildly unpleasant taste within several minutes following Pronasone administration. No evidence of nasal irritation was observed in any woman. The similar absorption curves obtained with Pronasone 20 mg and 30 mg doses and the aberrantly high values and delayed peaks ob tained in 2 subjects with the 30 mg dose imply that further work on dosage range, ointment formulation, and the method of application may be necessary before dependable clinical utility can be demonstrated. The peak levels of P that were reached compare favorably with results using similar doses (25 mg) in cocoa butter rectal or vaginal suppositories but are somewhat lower than those seen with polyethylene glycol base suppositories. The apparent inverse relationship between serum E2 levels and P levels obtained with Pronasone in the 20-mg dose was not expected. Alterations of nasal vascularity, interstitial hydration, and mucous blanket production all might influence absorption. The study demonstrates that the intranasal route is a potentially useful approach for the administration of unmodified sex steroid medications and is likely to be clinically safe and acceptable to patients.

Comparison of intermediate-dose purified urinary follicle-stimulating hormone with and without human chorionic gonadotropin for ovulation induction in polycystic ovarian disease

Urinary FSH is capable of inducing ovulation in PCOD. The duration of treatment can be reduced by administering an intermediate dose. However, it appears that prospective monitoring with E2 assays and ultrasound, combined with hCG, is required to optimize outcome and minimize complications. Studies comparing urinary FSH with similar doses of hMG with and without hCG are needed to determine the most effective form of gonadotropin therapy in PCOD.

Antihypertensive Therapy With Triamterene-Hydrochlorothiazide vs Amiloride-Hydrochlorothiazide

Amiloride hydrochloride has now been recognized as a safe and effective potassium-sparing diuretic alternative to triamterene with a similar mechanism of pharmacologic activity. Studies were undertaken to assess the difference between therapy with the triamterene-hydrochlorothiazide combination (Dyazide) and an amiloride hydrochloride-hydrochlorothiazide combination (Moduretic) on renal prostaglandin production, since an increase in renal prostaglandin synthesis has been shown to mediate or enhance the pharmacologic action of certain diuretic drugs. Eight subjects treated for four weeks with triamterene-hydrochlorothiazide were compared with nine patients similarly treated with amiloride-hydrochlorothiazide. A 24-hour urine sample for prostaglandin E2 (PGE2) assay was collected under control conditions and after six weeks of therapy with either diuretic in all patients. The PGE2 excretion increased in the amiloride-hydrochlorothiazide-treated group; in the other group PGE2 excretion actually declined. It is concluded from these studies that therapy with amiloride-hydrochlorothiazide enhanced renal PGE2 production, whereas that with triamterene-hydrochlorothiazide actually decreased renal PGE2 production. This difference is an important renal consequence of the use of either drug and should be considered in the choice between these diuretic combinations.

Variables affecting the yield and developmental potential of embryos following superstimulation and in vitro fertilization in rhesus monkeys

AbstractThree follicular‐stimulation protocols were compared to evaluate the yield and quality of oocytes obtained from rhesus monkeys. Five animals received a high‐dose regimen of PMSG (protocol I), three received a lower‐dose regimen (protocol II), and two received Clomid‐Pergonal (protocol III). Oocytes were recovered at laparoscopy after HCG injection, fertilized in vitro, and cultured up to the blastocyst stage. Yields of mature oocytes were 17.2 ± 13.0 (80% of total recovered), 6.7 ± 6.6 (41%), and 4.5 ± 2.5 (90%) per stimulated cycle for protocols I, II, and III, respectively. Of mature oocytes, 72%, 45%, and 89% were fertilized for protocols I, II and III, respectively. Protocol I produced the most fertilized oocytes per stimulated cycle (11.6 ± 11.6) and the greatest E2 production (approximately fourfold that maximally expected for an unstimulated cycle). For the combined protocol I and II results, there was a significant correlation (P ⩽ 0.05) between mean embryo development score and E2 production. Fertilized oocytes from protocol I yielded 7.8 ± 8.0 morulae and 6.8 ± 7.2 early zonal blastocysts per cycle. After transfer of nine singleton embryos to surrogate recipients, one live birth resulted. We conclude that our high‐dose PMSG regimen offers the best means at present for obtaining susbstantial numbers of developmentally competent oocytes in rhesus monkeys and that more extensive use of rapid serum E2 assays for monitoring both stimulated cycles and those of potential surrogate recipients could help to predict the success of in vitro fertilization and embryonic development following embryo transfer in rhesus monkeys.

Behavior of preovulatory markers in normal and abnormal cycles.

Various ovulation markers in natural cycle (ultrasound measurement of follicular diameter, E2, progesterone, and LH assays) have been studied in 18 women. The patients were divided into two groups. Group 1: women who became pregnant during the same cycle; group 2: women who showed evidence of follicular growth, E2 and a LH midcycle rise, but low P levels in the luteal phase. A weaker increase in these parameters was found in group 2, especially the LH rise. Moreover, an asynchronicity between the endocrinological and morphological events has been observed in the suboptimal cycles. These findings suggest a process of follicular atresia at the end of the maturation phase, as is commonly found in otherwise normal women.

Prediction of ovulation by urinary estrogen assays

In order to investigate which assay of a number of urinary estrogen metabolite methods could be used for the prediction of ovulation in normal women, urine was collected over 29 cycles in 21 women. Seven women collected 2 cycles with about a 1 year interval. Estnol-16α-glucuronde (E3-16G) was assayed in day, night and 24-h samples in 29 cycles, estriol-3-glucuronide (E3-3G) in day, night and 24-h collections in 10 cycles, estrone-3-glucuronide (E1-3G) in day, night and 24-h samples in 20 cycles and estrone (E1) and estradiol (E2) following hydrolysis in 24-h samples in 15 cycles. The day of highest LH excretion in urine followed by an increase in urinary pregnanediol-3-glucuronide and/or plasma progesterone was regarded as day 0 of the cycle. Mathematical analyses of the total material of about 16,000 hormone assays revealed that the increase in excretion starting from day −6 was best fitted by a linear regression function. The use of six different mathematical procedures in order to find out the day on which ovulation could be predicted revealed that the best methods were E2, E1-3G and E1 assays. With the mathematical methods giving the best results the average day of prediction of ovulation was day −3. If a method could not predict the ovulation by day −2 or earlier it was regarded as a failure. The failure rate varied from 0–15% for the best assays and mathematical methods used. It is concluded that measurement of estrone and/or estradiol in urine preferably by a direct assay of the conjugate(s), will probably give the most satisfactory prediction of impending ovulation.

A non-chromatographic radioimmunoassay of estrone and estradiol-17β in serum

A sensitive and efficient non-chromatographic procedure employing the Girard reagent and solvent-partitioning has been developed for the accurate radioimmunoassay (RIA) of estrone (E1) and estradiol-17β(E2) in a single 1.0 ml specimen of male or female serum. Using standard curves which permitted the discrimination of zero from 0.75–1.5 pg (p=0.05), the following mean procedural blanks (pg ± S.D.) were determined (1.0 ml water, n= 24): estrone, 2. 1 ± 1.1 (range 0–4.1); estradiol 1.0± 1.1 (range 0–3.6). A comparison of RIA of estrogens (1) in serum after separation by the Girard procedure and by TLC yielded correlation coefficients of 0.99 and 0.98 for E1 and E2 respectively. The following results (pg/ml ± S.D.) were obtained on RIA of E1 and E2 in 12 different 1.0 ml specimens of male and female serum using the Girard procedure: male. E1 (32.0 ± 9.2), E2 (24.1 ± 10.9); female, E1 (108.5 ± 60.8), E2 (126.4 ± 63.2). The intra-assay variability (c.v.) was found to be 12.6% for E1 and 9.4% for E2. The interassay variability was 14.2% for both estrogens. Twenty-four assays of E1 and E2 can be completed by one person in 2 working days.