Radioimmunoassay of plasma equilin and estrone in postmenopausal women after the administration of premarin.

A RIA for the measurement of plasma equilin (3-hydroxy-1,3,5-(10)7-estratetraen-17-one) and estrone in postmenopausal women and other estrogen-deficient women on exogenous equine estrogen replacement therapy is described. Antiserum against estriol-3,16,17-trihemisuccinate-HSA and high specific activity [2,4-3H]equilin and [6,7-3H]estrone were used in the assay procedure. Specificity of the assay was achieved by separation of equilin from estrone by chromatography on micro-Celite partition columns using silver nitrate as the stationary phase. The sensitivity of the standard curves for both equilin and estrone was 10-20 pg, and the smallest amount of estrone and equilin that could be measured accurately in the plasma was 250 pg/ml for both steroids. The coefficient of variation for both steroids ranged from 1-8%, over a range of 250-5000 pg/ml plasma. The interassay coefficients of variation for equilin and estrone were 4.6% and 2.4%, respectively. After the administration of 10 mg Premarin iv, maximum concentrations of 4 and 11.2 ng/ml for equilin and estrone, respectively, were obtained after 20 min. Thereafter, both steroids disappeared from the plasma gradually. When 10 mg Premarin were administered orally, equilin and estrone appeared in the blood gradually, and maximum levels of 560 and 1400 pg/ml were reached after 3 and 5 h for equilin and estrone, respectively. Equilin gradually disappeared, and by 24 h, only small amounts (125 pg/ml) were detectable. The levels of estrone declined more rapidly, though it was still detectable after 24 h. These preliminary results indicate that equilin sulfate is converted to circulating unconjugated equilin in a manner similar to the conversion of circulating estrone sulfate to estrone.

Chronic ethanol treatment increases the circulating plasma levels of B-type natriuretic peptide (BNP-45) in the rat.

Chronic ethanol ingestion is associated with a number of cardiovascular disorders, including stroke, heart failure, and hypertension. Given that the regulation of A-type natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) is known to be altered in both congestive heart failure and essential hypertension, we have investigated the regulation of BNP under the influence of ethanol ingestion. Sprague-Dawley rats were given ethanol in drinking fluid for a 6-week period, while a weight-matched liquid-restricted group received an equivalent volume of ethanol-free solution. Plasma BNP levels were increased in ethanol-treated animals relative to both liquid-restricted and normal control groups. No changes in cardiac BNP gene expression were observed, but an increased trend in atrial tissue BNP levels was evident. No changes in either the mRNA, tissue, or plasma levels of ANP were evident. These results suggest a differential regulation of natriuretic peptides under the influence of ethanol, and implicate chronic ethanol ingestion as a further clinical condition under which the plasma levels of a natriuretic peptide may be elevated.

Hormonal studies in pregnancy. III. Unconjugated esterone and estradiol in mother and fetus at delivery in IUGR.

The levels of unconjugated estrone and estradiol in maternal vein serum during labor, in cord artery serum, and in cord vein serum were measured in normal pregnancy and in pregnancy complicated by intrauterine growth retardation. The mean level of unconjugated estrone and estradiol in maternal vein serum was significantly lower in the group with intrauterine growth retardation than in the normal patients (P less than 0.001 and 0.025, respectively). The mean unconjugated estrone and estradiol level in the cord vein was lower only in the most severely growth retarded subgroup. There was no difference seen in the cord artery values between the groups.

Immunoactive iso-ANP/BNP in plasma, tissues and atrial granules of the rat.

We have developed a specific radioimmunoassay (RIA) for iso-rANP(1-45)/rBNP(51-95) using antiserum produced against a peptide comprise of the first 20 amino acid sequence of this cardiac hormone. Using this RIA, we found that atria contained the highest amount of iso-rANP(1-45) (567.37 pmol/g) which is about 140-fold higher than ventricles (4.32 pmol/g). With the exception of the lung and kidney, all other tissues had negligible amounts. The plasma level was 1.4 fmol/ml and the only molecular form found was the 45 amino acid form. HPLC analysis of extracts of isolated, purified atrial granules revealed that, like atrial natriuretic peptide (ANP), iso-rANP/rBNP is also stored in these granules. However, while ANP is stored predominantly as pro-ANP(1-126) and cleaved during or after release, iso-ANP/BNP is stored as the 45 amino acid form and may be processed prior to storage in the granules.

Plasma clearance and tissue binding of rANP[99-126] and iso-rANP[1-45] in the rat.

Plasma clearance and tissue binding of atrial natriuretic peptide (ANP) and iso-ANP were compared in Inactin-anaesthetized rats. It was found that the plasma half-life of iso-ANP was comparable to ANP. Appearance of trichloroacetic acid-soluble radioactivity of iso-ANP in the plasma was considerably slower than that of ANP, suggesting that the metabolic process of these two peptides may be different. Although the binding distribution of these two peptides was similar, the total binding of iso-ANP to organs other than the kidney was much lower. The kidney, lung, heart and adrenal gland appeared to be major target organs for iso-ANP. Autoradiography showed that iso-ANP bound specifically to the renal glomerulus and proximal part of the proximal tubule. This latter binding site in the kidney was not apparent with ANP, suggesting that iso-ANP may exerts its physiological action at different sites in this organ.

The antidiuretic effect of pneumadin requires a functional arginine vasopressin system.

Pneumadin is an antidiuretic decapeptide, recently isolated from rat and human lung. Bolus intravenous injection of 5 nmol of pneumadin into water-loaded rats caused a rapid and significant antidiuresis and a reduction in Na+ and Cl- excretion. Pneumadin administration did not alter mean arterial pressure, right atrial pressure, heart rate or haematocrit. Bolus intravenous injection of 20 nmol of pneumadin into non-water-loaded rats caused a significant increase in arginine vasopressin (AVP) within 10 min. Pneumadin administration also increased circulating atrial natriuretic peptide (ANP) but did not alter aldosterone or plasma renin activity levels. Injection of pneumadin into water-loaded Brattleboro rats, which genetically lack circulating AVP, did not change urine flow, confirming that the pneumadin induced antidiuresis is AVP dependent. Radioactive pneumadin was cleared from the circulation with a t1/2 beta of 480.3 s. Radioactive pneumadin, isolated from plasma, eluted at an altered position on reverse phase HPLC, which indicated that the peptide was modified in vivo. This modification was also observed when synthetic pneumadin was incubated in rat plasma in vitro. Purification and sequencing of the modified synthetic peptide indicated that the modification is not a proteolytic cleavage. These results indicate that pneumadin injected into the rat caused an antidiuresis by altering circulating AVP levels.

Optimization of atrial natriuretic factor radioimmunoassay.

The development and long-term performance of a radioimmunoassay method for cardionatrin (C I = ANF 99-126) is described. The method was evaluated for accuracy, specificity and precision using different protocols and in various species. The antiserum raised against C I cross-reacts 100% with both human and rat C I and 122% with cardionatrin IV (C IV = ANF 1-126). Dextran-coated charcoal (DCC) and double antibody (DA) disequilibrium protocols were used for the separation of free from antibody-bound radioactivity. The sensitivity, coefficient of variation within assay and between assay for DCC was 4.6 pg/tube, 3.1% and 13.8% respectively, and 1.5 pg/tube, 3.8% and 9.1% for the DA. The mean normal plasma C I levels in human, rat and dog after plasma acidification and extraction using the DA method was 49.7 +/- 4.0; 253.6 +/- 19.6 and 59.9 +/- 3.4 pg/mL respectively.

Effect of the suppository base on progesterone delivery from the vagina.

Inadequate levels of circulating progesterone (P) seen in clinical practice can be increased by P supplementation. When supplementation is via the vaginal route, its presentation may be in many different suppository bases. We have measured circulating P in the follicular phase to compare delivery from the vagina after P was presented in three different bases (glycerinated gelatin, cocoa butter, and polyethylene glycol). The mean peak level achieved and the area under the curve were highest after supplementation from polyethylene glycol-based suppositories. The duration of elevation above baseline was similar with all three suppositories. It is concluded that the suppository base is important in controlling the amount of this steroid absorbed from a given amount presented by this route.

Acute ethanol ingestion modifies the circulating plasma levels of atrial natriuretic peptide.

Since ethanol ingestion is associated with a disruption of water and electrolyte balance in a variety of species, we sought to evaluate the regulatory control of atrial natriuretic peptide (ANP) in response to acute doses of ethanol. Male Sprague-Dawley rats were administered a 5-g/kg dose of ethanol (40% w/v) via a gastric tube, while control animals received an equivalent volume of water. Expressed as a percentage of control, plasma ANP levels were 39.0%, 28.5%, and 23.6% in the ethanol-treated animals at 30, 60, and 120 min postintubation, respectively. Ethanol-treated animals displayed blood alcohol concentrations of 89.0, 137.6, and 214.1 mg/dl at the same time periods. After 120 min, plasma renin activity was elevated from 8.7 to 20.3 ng/ml/h in conjunction with an increase in the levels of circulating aldosterone from 16.3 to 42.5 ng/dl and an increase in plasma vasopressin from 2.2 to 3.6 pg/ml. Levels of atrial ANP mRNA remained consistent over the time course of the experiment, and no changes in the amount of ventricular ANP transcript were observed. Tissue ANP levels were similar between ethanol-treated and water-loaded control animals. In vitro experiments using cultured cardiac myocytes suggest that ethanol exposure may not directly affect ANP secretion. We propose that acute ethanol treatment may inhibit atrial distension and subsequently modify the control of ANP release under volume loading conditions.

[Prognostic factors for postoperative mortality in cancer of the esophagus. Analysis of 46 cases]. / Factores pronósticos de la mortalidad postoperatoria del cáncer de esófago. Análisis de 46 casos.

Forty six patients with esophageal cancer underwent surgery between January 1986 and January 1990. In 14 patients (30.4%) distant metastases were recognized before surgery, whereas in 29 cases (63%) regional neoplastic lymph node infiltration was observed during surgery. Complications during and after surgery occurred in 32 (69.6%) patients and in 30 cases (65.2%) respectively. During the first 30 days after surgery 12 patients died. This represents a postoperative mortality of 26.1%. Among a total number of 51 variables analyzed in this study, 11 influenced the postoperative mortality: duration of intubation, previous history of toxic syndrome, presence of distant metastases before surgery, presence of neoplastic node involvement during surgery, tumor size greater than 4 cm, localization of the tumor at the middle third of the esophagus, respiratory insufficiency, cardiac failure, septic shock, and suture failure during the postoperative phase. However, multivariate analysis revealed that only three of these variables had an independent prognostic value on postoperative mortality: tumor size, presence of distant metastases, and development of respiratory insufficiency during the postoperative period.

Functional-morphological studies on in vitro cardionatrin release.

Atrial natriuretic peptides (ANP) are hormones produced by the heart atrial cardiocytes in mammals. The main function of these peptides appears to be that of modulating the actions of the renin-angiotensin-aldosterone system. In the rat ANP are stored within specific atrial granules, mainly as a 126-amino acid peptide (cardionatrin IV) which is processed to a 28-amino acid peptide (cardionatrin I) just prior to or during release from cardiocytes. Development of an isolated perifused rat atria preparation has allowed quantitative and qualitative studies on ANP release. Increasing mechanical load in this preparation gives an increased rate of release of immunoreactive cardionatrin. This finding suggests that there is an intrinsic 'stretch-secretion coupling' in the atria.

Unconjugated serum oestriol levels in mother and baby with meconium staining of the amniotic fluid.

To examine the effect on oestrogen metabolism of the stimulus which causes the passage of meconium, we measured maternal venous and cord artery and vein serum unconjugated oestriol levels in 46 patients with meconium staining of the amniotic fluid and 19 controls. Mean cord vein oestriol levels were significantly lower than in controls in the presence of meconium stained amniotic fluid and no fetal acidosis (cord artery buffer base of 36.2 meq/l or more) (P less than 0.02). The same tendency was seen with acidotic infants (P less than 0.05). There is thus evidence of a change in oestrogen metabolism associated with meconium staining of the amniotic fluid.

Hormonal studies in pregnancy. II. Unconjugated estriol in maternal peripheral vein, cord vein, and cord artery serum at delivery in pregnancies complicated by intrauterine growth retardation.

The levels of unconjugated estriol in maternal vein serum during labor, cord artery serum, and cord vein serum were measured in normal pregnancy and in pregnancy complicated by intrauterine growth retardation. The mean level of unconjugated estriol in maternal vein serum was significantly lower in the intrauterine growth-retarded group than in the normals (p less than 0.001). The mean estriol level in cord vein was lower only in the most severely growth-retarded subgroup as compared to the normal. There was no difference seen in the cord artery values among the groups.

Hormonal studies in pregnancy. I. Total unconjugated estrogens in maternal peripheral vein, cord vein, and cord artery serum at delivery.

The concentration of unconjugated total estrogens (primarily estrone, estradiol-17beta, and estriol) were measured by rapid radioimmunoassay method in serum samples from maternal peripheral vein, cord vein, and cord artery in normal pregnancies at delivery. The serum concentration of total free estrogens found in cord vein was four to five times higher than in maternal peripheral vein and cord artery. Levels obtained in maternal peripheral vein, cord vein, and cord artery were 47.8+/-1.8, 140.1+/-8.57, 31.8+/-2.15 ng. per millilitre (+/-standard error of the mean), respectively. These values are compared with those reported by others.

Heterotopic columnar epithelium and adenosis in the vagina of the mouse after neonatal treatment with clomiphene citrate.

Physiologically, the epithelium of the mouse vagina undergoes conversion during early postnatal life from columnar to stratified squamous. A similar process in the human occurs in the late first and early second trimesters of pregnancy. The mouse vagina has been identified as a good developmental model of the human. Previous work in the mouse has shown that this process is affected by neonatal administration of diethylstilbestrol. We have administered diethylstilbestrol and clomiphene citrate to parallel groups of BALB/c neonatal mice. They were followed up to 24 weeks. In both groups, persistent columnar or heterotopic columnar epithelium, not seen in the control mice, was identified and associated with adenosis. This effect of diethylstilbestrol and clomiphene citrate appears to be similar to the biologic response to transplacental diethylstilbestrol in the human. After transplacental diethylstilbestrol, malignant vaginal tumors rarely develop. If clomiphene citrate, given to the human prior to pregnancy to induce ovulation or by inadvertence during pregnancy, were to circulate into the critical time of vaginal differentiation, a similar biologic potential may exist. The first situation seems to be unlikely. The second is of more concern.