Jorge Pineda

Tumor necrosis factor in peritoneal fluid of women undergoing laparoscopic surgery

The level of tumor necrosis factor (TNF) in peritoneal fluid (PF-TNF) of 74 women undergoing laparoscopy was determined. The difference between the mean concentration of PF-TNF of women with normal pelvic anatomy and women with moderate or severe endometriosis was significant (P less than 0.01). The proportion of PF-TNF-positive women with PID and those with moderate or severe endometriosis was also significantly higher when compared to women with normal pelvic anatomy (P less than 0.05; P less than 0.02). The proportion of PF-TNF positive women among nulligravid and nulliparous women was significantly higher than that of women with two or more pregnancies (P less than 0.01) and two or more deliveries (P less than 0.005). These results indicate that the presence of PF-TNF is associated with primary infertility and endometriosis.

Serum levels of CA-125 in patients with endometriosis: a preliminary report

Seven out of 8 patients with endometriosis demonstrated levels of CA-125 antigen above 35 U/ml. None of 15 patients with other benign gynecologic diagnoses demonstrated elevated levels. This antigen has been proposed as a tumor marker for epithelial carcinoma and other gynecologic neoplasms. However, it cannot be used to differentiate clinically between cancer and endometriosis.

Therapeutic donor insemination: a prospective randomized study of scheduling methods.

OBJECTIVE To compare basal body temperature (BBT) graphs and urinary luteinizing hormone (LH) monitoring in scheduling therapeutic donor insemination. DESIGN Participants were prospectively randomized to the BBT or LH groups. SETTING Participants were private patients of the Reproductive Endocrine Division at Washington University School of Medicine. PATIENTS Inclusion criteria were designed to assure an isolated male factor. Seventy-four of 113 patients completed the study; 18 had ongoing treatment at the end of the study. INTERVENTIONS Basal body temperature graphs were physician interpreted and appointments prospectively chosen. Luteinizing hormone patients monitored daily urine samples and scheduled an appointment the day after the detected surge. MAIN OUTCOME MEASURES Fecundity rates, cumulative pregnancy rates, and cost per pregnancy were all prospectively evaluated. RESULTS Life table analysis yielded a 6-month cumulative probability of pregnancy of 36.3% in the LH group and 65.1% in the BBT group (P less than 0.025). The total cost per pregnancy was lower in the BBT group (+6,212 versus +3,997; P less than 0.001). CONCLUSIONS This randomized prospective study demonstrates significant therapeutic and economic advantages when therapeutic donor insemination is prospectively scheduled by BBT graphs.

Purification and characterization of 20α-hydroxysteroid dehydrogenase from bull testis

20α-Hydroxysteroid dehydrogenase (20α-HSD) from bull testis has been purified to homogeneity and characterized in terms of apparent molecular weight, lack of subunit composition, substrate and cofactor specificity and certain kinetic parameters. The enzyme activity is localized in the 105,000 g supernatant and is stable at 4°C in the presence of glycerol and dithiothreitol. Purification was achieved by ammonium sulfate precipitation followed by affinity chromatography on reactive red 120-agarose and subsequent gel fitration. The apparent molecular weight of the homogeneous enzyme, as determined by gel filtration on Sephacryl S-300 is 34,000. The mobility of the enzyme in sodium dodecyl sulfate (SDS) gel electrophoresis corresponds to a mol. wt of 40,000. These observations indicate that the enzyme is a single-stranded, monomeric polypeptide. The enzyme catalyzes the reduction of the 17-hydroxyprogesterone to 17,20α-dihydroxy-4-pregnene-3-one in the presence of NADPH, the preferred cofactor. Homogeneous 20α-HSD has an SA of 115 mIU/mg, and has been purified 14,000-fold with an overall 68% recovery. It exhibits a pH optimum at 5.6 and appears to be highly specific for 17-hydroxyprogesterone with an apparent Km-value of 7.3 × 10−5 M. Androstenedione and corticosterone do not serve as substrates under the described experimental conditions. The enzyme does not possess 17α-or 17β-HSD activity.

Changes in human amniotic fluid lecithin/sphingomyelin ratio and dipalmitoyl lecithin associated with maternal betamethasone therapy.

In an effort to understand the biochemical changes responsible for the decreased incidence of neonatal RDS observed in premature infants delivered after maternal glucocorticoid therapy we have compared quantitative changes in L/S ratio and qualitative changes in dipalmitoyl lecithin in serial amniotic fluid samples from 20 pregnant women treated with betamethasone and 11 patients who had repeated amniocentesis at similar gestational age but did not receive glucocorticoids. Initial L/S ratio values were not significantly different between treated (1.24 +/- 0.24 [S.D.]) and control (1.09 +/- 0.29 [S.D.]) groups but the values obtained within 1 week after therapy (1.49 +/- 0.21 [S.D.]) were significantly higher (t=3.8; p less than 0.01) than those obtained after a similar period in the untreated group (1.24+/- 0.29 [S.D.]). However, only one of the treated patients reached a mature L/S ratio (2.0 or more). Simultaneously, 10 of 17 treated patients exhibited increases in the amount of dipalmitoyl lecithin within the first week after treatment, wheras no change was detected in serial samples from six control patients (p = 0.016). These results indicate that qualitative changes in amniotic fluid lecithin composition are a part of the fetal response to glucocorticoids which may be responsible for the decreased incidence of RDS observed after treatment.

Laser vaporization of endometriosis in an infertile population: the role of complicating infertility factors

One hundred twenty-two infertility patients with endometriosis were evaluated and treated using laparoscopy and laser vaporization to provide immediate elimination of all intraperitoneal disease. Ninety-five couples (77.8%) had one or more infertility factors (other than endometriosis) contributing to their infertility. Cervical factors, male factors, and luteal defects were associated with significantly decreased pregnancy rates, despite the use of laser vaporization. The number of contributing factors seemed to be unrelated to the likelihood of success in achieving pregnancy. The authors emphasize the need for total evaluation of other infertility factors in these patients. Such factors should be corrected prior to the use of laser laparoscopy, if possible, and dealt with on a continuing basis following use of this technique.

Estradiol 17β-dehydrogenase : full enzymatic activity in the absence of zinc

The precise catalytic mechanism of the steroid interconverting enzyme, human placental estradiol 17β-dehydrogenase (EC 1.1.1.62, estradiol-17β:NAD+17-oxidoreductase), is not known. Two general models for the catalytic mechanism of dehydrogenase have been defined. One model requires Zn2+ metal for the catalytic event, as has been shown for horse liver alcohol dehydrogenase (EC 1.1.1.1, alcohol: NAD+ oxidoreductase). Another model has been demonstrated for the 2-hydroxy acid dehydrogenases in which histidine residues are necessary for enzyme activity, without participation of a metal ion. In order to define which mechanism might be operative for the placental enzyme, it became important to determine whether Zn2+, or another metal ion, is associated with the macromolecule. Several homogeneous enzyme preparations, having protein concentrations from 5–80 μM, were extensively dialyzed in a buffer containing EDTA. Atomic absorption analysis of each sample demonstrated that no Zn2+ was present, although the enzymatic activity was maintained. In addition, there was no significant detection of Mg2+ or Mn2+ above background levels. When the isolated enzyme was dialyzed against buffer containing added 0.01–20 μM ZnCl2, no increase in specific activity of the enzyme was seen. The data indicate that the presence of zinc is not required for the catalytic event. These results, together with our previous affinity-labeling studies, which demonstrate a histidine residue in the catalytic region of the active site, allow us to propose that the catalytic mechanism of the human placental estradiol 17β-dehydrogenase is similar to that of the 2-hydroxy acid dehydrogenases.

Stereospecificity of hydrogen transfer by bovine testicular 20α-hydroxysteroid dehydrogenase

The stereospecificity of hydrogen transfer between steroid (17-hydroxyprogesterone) and both natural cofactors by bovine testicular 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) has been determined. Cofactors used in these studies, [4-pro-S-3H]NADH ([4B-3H]NADH) and [4-pro-S-3H]NADPH ([4B-3H]NADPH) were generated with human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) utilizing [17 alpha-3H]estradiol-17 beta and NAD+ or NADP+, respectively. The resulting [4B-3H]NADH and [4B-3H]NADPH were purified by ion-exchange chromatography and separately incubated with molar excess of 17-hydroxyprogesterone as substrate in the presence of 20 alpha-HSD. Following incubation, steroid reactant and product were extracted, separated by HPLC and quantitated as to mass and content of tritium. The oxidized and reduced cofactors were separated by ion-exchange chromatography and quantitated as to mass and tritium content. In all incubations, equimolar amounts of 17,20 alpha-dihydroxy-4-pregnen-3-one and oxidized cofactor were obtained. Further, all recovered radioactivity remained with cofactor and none was found in the steroid product. In additional experiments, both reduced cofactors were separately incubated with glutamate dehydrogenase, an enzyme known to transfer from the B-side of the nicotinamide ring. Here radioactivity was present only in the unreacted cofactor fractions and in the product, glutamic acid. The results indicate that bovine testicular 20 alpha-HSD catalyzes transfer of the 4A-hydrogen from the dihydronicotinamide moiety of the reduced cofactor. Finally, this work described modifications that represent considerable improvement in the purification and assay of bovine 20 alpha-HSD as originally described.

Tumor Necrosis Factor Alpha Is Elevated in the Peritoneal Fluid from Women with Ruptured Ectopic Pregnancies

The purpose of this study was to determine whether exposure of the peritoneum to fetal tissue is associated with elevated tumor necrosis factor alpha (TNF alpha) levels in the peritoneal cavity. We measured TNF alpha levels in the peritoneal fluids and serum from women with ruptured ectopic, unruptured ectopic and intrauterine pregnancies, as well as nonpregnant women undergoing tubal ligation. The results showed that patients with ruptured ectopic pregnancies were more likely to have TNF alpha levels in the peritoneal fluid greater than 40 units/ml (68%), compared with women with unruptured ectopic or intrauterine pregnancies (21%) (p less than 0.05, Fisher exact test). No elevation of peritoneal fluid TNF alpha levels was found in nonpregnant patients. Because TNF alpha is primarily a product of activated macrophages, it is likely that elevated TNF alpha levels in the peritoneal fluid of women with ruptured ectopic pregnancies reflects activation of peritoneal macrophages.

Stereospecificity of hydrogen transfer between progesterone and cofactor by human placental estradiol-17β dehydrogenase

We have previously shown that human placental estradiol-17 beta dehydrogenase (EC 1.1.1.62; 17 beta-EDH) catalyzes the conversion of estradiol-17 beta to estrone and stereospecifically reduces NAD+ to [4-pro-S]NADH, [( 4-B]NADH). Subsequently, this enzyme was found to reduce the ketone function at C-20 of progesterone, and evidence indicates that both activities reside at the same active site. This study was done to further elucidate spatial arrangements of cofactor and the 21-carbon substrate as they bind at the active site. The cofactor, [4B-3H]NADPH, was generated with homogeneous 17 beta-EDH from term human placenta, utilizing [17 alpha-3H]estradiol-17 beta and NADP+. The resulting [4B-3H]NADPH was then purified by ion exchange chromatography and was separately incubated (24.4 microM) with a large molar excess of progesterone (150 microM) as substrate in the presence of the enzyme. Following incubation, the steroid reactants and products were extracted, separated by high-performance liquid chromatography and quantitated as to mass and tritium content. Oxidized and reduced cofactor were separated by ion-exchange chromatography and similarly quantitated. In all incubations, equimolar amounts of 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OHP) and NADP+ were obtained. Radioactivity was stoichiometrically transferred from [4B-3H]NADPH to the steroid product [( 3H]20 alpha-OHP). These results further substantiate a single active site for both 17 beta- and 20 alpha-dehydrogenation enzyme activities. In addition, the enzyme is B-side specific, catalyzing the transfer of the 4B-hydrogen from the dihydronicotinamide moiety of the cofactor, for both C-18 and C-21 steroid substrates. Since the 20 alpha-dehydrogenation by other enzyme sources has always been demonstrated to be an A-side specific reaction, this observation represents an important exception to the Alworth-Bentley rules of enzyme stereospecificity.