Measurement of steroid hormones in plasma by isocratic high performance liquid chromatography coupled to radioimmunoassay.

The study of steroidal profiles requires simultaneous determinations of various steroid hormones that cannot be appropriately carried out with the conventional routine immunoassays. Moreover, there are several trials for which the assessment of multiple steroids from a single serum sample is mandatory. In this paper we describe a procedure for simultaneously measuring steroid hormones using a unified solid phase extraction which allows the measurement of both unconjugated and conjugated steroids from 1 ml of sample and a combination of HPLC with isocratic elution followed by RIA. The entire procedure was preliminary carried out for the measurement of testosterone, dehydroepiandrosterone and its sulphated conjugate, androstenedione and 17 hydroxyprogesterone. The use of this technique allows precise and accurate measurements of steroid profile with a single serum aliquot and could be helpful in the diagnosis of various form of endocrine disorders.

Estrone sulfate, estrone and estradiol concentrations in normal and cirrhotic postmenopausal women.

Circulating levels (mean +/- SD) of estrone sulfate (E1S), estrone (E1) and estradiol-17 beta (E2) were measured in normal and cirrhotic postmenopausal women matched for body weight and age. In cirrhotic postmenopausal women, the E1S concentrations (201 +/- 46 pg/ml), while both E1 and E2 levels showed an increase (46 +/- 7 and 30 +/- 8 pg/ml) compared to control subjects (32 +/- 6 and 18 +/- 7 pg/ml). These data suggest that the liver plays an important role on the control of estrogen sulfation.

Development of a rapid and ultrasensitive RIA method for estrogen (E2, E1, E1-S) determination with selective solid phase extraction.

The inhibition of the proliferative stimulation exercised by estrogens on neoplastic cells is the goal of all endocrine therapies in breast cancer. Under various circumstances, e.g. with the use of aromatase inhibitors, this result can be obtained by blocking the synthetic pathway and, consequently, by lowering the circulating levels of estradiol (E2), estrone (E1), and estrone sulfate (E1-S). The evaluation of these hormones in plasma could therefore represent a useful indicator of the biological efficacy of the therapy. However, the measurement of circulating steroids in a large series of patients is often a complicated procedure. Indirect methods of extraction are time consuming and expensive while the analytical sensitivity of direct methods is not sufficient to measure the residual levels of E2, E1, and E1-S. In this paper we describe a novel extraction method for the evaluation of plasma levels of E2, E1, and E1-S. This new method consists of solid phase extraction followed by a highly specific radioimmunoassay. The sensitivity of the assay is 0.6 pg/ml, 2.0 pg/ml and 7.0 pg/ml for E2, E1, and E1-S, respectively.

Metabolic clearance rate of oestrone sulphate in post-menopausal women.

The feasibility of using constant infusions of unlabelled oestrone sulphate (E1S) for the purposes of calculating its metabolic clearance rate (MCRE1S) and its conversion ratios to oestrone (E1) and oestradiol (E2) in post-menopausal women was exploited in this study. The results obtained by the infusion of unlabelled E1S were similar to those obtained by the infusion of labelled steroid. The MCRE1S values seen in our group of post-menopausal women fell within the range previously reported for fertile women. The contribution of E1S to circulating E1 averaged 18% (range 14-24%), indicating that the E1S-E1 equilibrium should be taken into account during studies on oestrogen balance in post-menopausal women.

Analysis of estrone sulphate levels in post-menopausal women with and without endometrial cancer.

In post-menopausal women, the role of Estrone sulphate (E1S) has not been clearly defined, and few data are available in the literature. The present study concerns the plasma E1S levels in post-menopausal women with and without endometrial cancer; the results showed that the E1S levels are related to body size but not to endometrial cancer as the free estrogens.

External quality assessment of assays for hormones and tumor markers in Italian laboratories.

External quality assessment (EQA) programs run by CNR/Tecnostandard for immunoassays of hormones and tumor markers, started in 1980, presently include as many as 20 analytes; about 300 laboratories are involved in these programs. For all immunoassays submitted to the EQA, the inspection of cumulative results allows the current situation to be documented for total variability and its within-kit and between-kit components (the former accounting for the reproducibility and robustness of the kits and the latter for their systematic differences of estimation). For 13 assays subjected to EQA for longer, the variability trends over time are depicted, and single factors affecting the overall quality of particular assays are identified. Among these, experimental simplification of kit structure, alignment of calibrators with an acknowledged reference material, and adoption of monoclonal-antibody based two-sites assays can be mentioned. On the contrary, neither automation of the procedures nor (more expectedly) increasing use of nonisotopic techniques has proved effective in significantly improving the analytical quality.

Quality of clinical information: a new approach of EQA in Emilia-Romagna, Italy.

For correct enforcement of an external quality assessment (EQA) scheme, suitable parameters are required for the assessment of analytical performance. Traditional EQA schemes have always been chiefly concerned with the agreement of analytical results between laboratories. Although we consider this concept to be important, we also believe that particular attention must be paid to the quality of the clinical information, in relation to correct use of the results. On the basis of this principle, we have developed an EQA model which, besides considering the absolute value, also take the reference limits (RLs) into consideration by means of the normalization procedure. The evaluation of clinical information is of vital importance, especially in relation to immunoassays, since the low degree of standardization between methods, and the ensuing phenomenon of relative inaccuracy, make the use of suitable RLs essential. Actually, analysis of the results reveals a high degree of heterogeneity in the RLs used by the laboratories, even within the same method-groups.

Target neurons of endogenous androgens immunocytochemically localized in male rat hypothalamus.

A polyclonal antiserum against androgens, i.e., testosterone, 5alpha-dihydrotestosterone and androstenedione, was tested to reveal target neurons of endogenous androgens in the hypothalamus of both intact and castrated male rats. Paraffin sections of hypothalamus and testis were immunostained by using Avidin-Biotin Complex method and 3-3' diaminobenzidine to visualize the immunoperoxidase complex. Conventional control experiments for method and antiserum specificity were performed. The antiserum proved to be specific for androgens, i.e., testosterone, 5alpha-dihydrotestosterone and androstenedione. The nuclear labeling observed in tissues stained by this procedure is consistent with the hypothesis that the labeled neurons contained DHT, which is the main testosterone metabolite active in the cell nucleus. The antiserum was effective in staining not only the hypothalamic neurons of intact males with normal serum levels of testosterone but also the hypothalamic neuron of castrated males with very low serum levels of testosterone. Evidence is presented indicating that the immunostaining technique represents a more specific and sensitive method to identify target neurons of endogenous androgens than autoradiography.

Seasonal changes in plasma prostaglandin F2 alpha and sex hormones in the male water frog, Rana esculenta.

Prostaglandin F2 alpha (PGF2 alpha), progesterone, androgens (testosterone + dihydrotestosterone), and 17 beta-estradiol were measured in the plasma of male frogs, Rana esculenta, by radioimmunoassays. Plasma concentrations of PGF2 alpha were higher from October to December and peaked in March (prereproduction) and in June (postreproduction). Plasma progesterone levels were relatively low but showed an increase from October to December and in June. Plasma androgen titres rapidly increased in early spring, started to fall during the reproductive period (May), and were lowest in July. 17 beta-Estradiol levels peaked in March and in June. The annual profile of the plasma PGF2 alpha levels was positively correlated with those of progesterone and androgens, while it was not correlated to the estradiol plasma pattern, except in March and June. The increase in plasma PGF2 alpha in the autumn may be related to gonadal recovery. The simultaneous increases in PGF2 alpha and 17 beta-estradiol, both in March and June, suggest a PGF2 alpha-dependent estradiol synthesis, a possibility also supported by the increased plasma 17 beta-estradiol previously observed in PGF2 alpha-treated postreproductive females. The effects of captivity and castration on plasma PGF2 alpha concentrations were also studied during the annual cycle. Captivity was associated with a reduced PGF2 alpha titre, while castration did not modify prostaglandin synthesis, which may point to an extragonadal source of plasma PGF2 alpha.

A solid phase chemiluminescent immunoassay (LIA) for testosterone glicuronide in diluted urines.

A chemiluminescent immunoassay (LIA) method in solid phase for the measurement of testosterone 17 beta-D-glicuronide (TG) in diluted urine is described, which utilizes as tracer a TG-isoluminol conjugate (TG-ABEI). An IgG fraction of antiserum of TG-BSA, has been passively adsorbed to the walls of polystyrene tubes. After the binding reaction the coated tubes were washed with buffer and the measure of chemiluminescence reaction was performed at high pH. The assay was validated in terms of specificity, accuracy, sensitivity and precision. The values obtained by chemiluminescence immunoassay were compared with that obtained by the RIA method, and the two methods agreed well (r = 0.95, n = 28). The assay method offers the advantage of speed and does not involve the use of radioisotopes or of a centrifugation step. Preliminary results show that the mean 24 h urinary TG excretion in a group of hirsute women is higher than in the control group, and decreases after suppression with dexamethasone for 1 month of therapy.

Validation of a high-performance liquid chromatographic-radioimmunoassay method for the determination of lacidipine in plasma.

A sensitive and reproducible method for the determination of lacidipine, a new potent antihypertensive dihydropyridine, is reported. The method involves solid-phase extraction, reversed-phase high-performance liquid chromatography and radioimmunoassay of the collected fraction. The assay provides a limit of detection of 20 pg/ml of plasma, allowing the determination of trough (24 h) plasma concentrations. The method is suitable for pharmacokinetic studies in man.

[Basal values and circadian variations of testosterone, 17beta-oestradiol, FSH and LH in the plasma of normal adult males].

The Authors have determined the concentrations of Testosterone, 17beta-oestradiol, FSH and LH by of radioimmunologic assay in blood of seventy normal adult men blood drawn between eight and eleven a.m.) and in blood of thirteen normal adult men blood drawn at eight and twelve a.m., four and eight p.m.). Average values under basal conditions, and circadian variations have been discussed and confronted with those obtained by other authors.

Prostaglandins and corticosterone in the oviparous female lizard, Podarcis sicula sicula, during reproduction.

The in vitro effects of prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E2 (PGE2) on corticosterone release by ovarian follicles, corpora lutea (CL), and interrenals were studied in the female lizard, Podarcis sicula sicula, during reproduction. Follicles and CL studied in the female lizard, Podarcis sicula sicula, during reproduction. Follicles and CL were divided according to their different developmental stages; follicles: previtellogenic, early-vitellogenic, mid-vitellogenic and fully-grown; CL: CL1 (unshelled eggs in the oviducts), CL2 (shelled eggs in the oviducts), CL3 (eggs laid 6 h previously) and CL4 (eggs laid 48 h previously). Interrenals were divided according to the reproductive stages: pre-vitellogenesis, vitellogenesis, ovulation, post-ovulation, and post-deposition. PGF2 alpha release was highest in fully-grown follicles and PGE2 in early-vitellogenic follicles, corticosterone was highest in pre-vitellogenic and lowest in early-vitellogenic follicles. PGE2 decreased corticosterone in pre-vitellogenic, mid-vitellogenic and fully-grown follicles. PGF2 alpha release was highest in CL4, and PGE2 in CL1 and CL2, corticosterone was highest in CL4. PGF2 alpha increased corticosterone in CL1, CL2 and CL3. In interrenals, PGF2 alpha release was highest and PGE2 lowest during ovulation, corticosterone was highest during ovulation. PGF2 alpha increased and PGE2 decreased interrenal corticosterone during vitellogenesis, ovulation, and post-ovulation. In the plasma, PGF2 alpha levels were highest and PGE2 lowest during ovulation, corticosterone was highest during ovulation. These results suggest that corticosterone, modulated by PGF2 alpha and PGE2, is implied in the reproductive processes with different roles. In fact this steroid could favour ovulatory and luteolytic processes. In addition the hypothesis of an anti-vitellogenic role of corticosterone is discussed.

Seminal estrone, estrone sulfate, and estradiol-17 beta levels in fertile and infertile males.

The seminal levels of estrone (E1), estrone sulphate (E1S), and estradiol-17 beta (E2) were measured simultaneously after a chromatographic step in the semen samples of 79 men, including fertile volunteers, vasectomized subjects, and patients with oligozoospermia and secretory azoospermia. E1S concentrations in seminal plasma were higher than in serum (with a semen/serum ratio of approximately 2). Seminal E1 and E1S levels in oligozoospermic subjects were significantly decreased compared to controls (p less than 0.02 and p less than 0.03, respectively). The seminal E1S concentration was significantly reduced in azoospermic patients (p less than 0.02) and to a greater extent in vasectomized subjects (p less than 0.001). As seminal E1S is likely to be mainly of testicular origin, the decreased seminal E1S levels in oligoazoospermia are an index of impaired testicular function.

Hypothalamic target cells of endogenous sexual steroids in female rat. An immunocytochemical approach utilizing antiestradiol antiserum.

The immunostaining proposed in this study identifies cells containing endogenous estrogens in the Central Nervous System, in the presence of both very high and very low serum estrogen levels. Ten adult rats, seven normal and three ovariectomized females, were used for this study. Paraffin sections of hypothalamus and ovary of each female were stained using Avidin-Biotin Complex method and antiestradiol antiserum. Two different antiestradiol antisera were tested in this work: tests for method and antisera specificity are described. The immunostaining used here shows to be specific and sensitive revealing a higher number of labeled hypothalamic areas than those revealed by other techniques.

Rapid and specific RIA of serum estrone sulfate with selective solid phase extraction.

The methods commonly used to evaluate conjugated steroids require hydrolysis and chromatographic purification. To avoid these steps, a simple method involving selective solid phase extraction and RIA using a highly specific antiserum for estrone sulfate (E1S) has been evolved. A Bond-Elut C2 cartridge was used for solid phase extraction of estrone (E1) and E1S; recoveries were 80 and 90% respectively. The intra- and inter assay precision of the assay at 3 serum levels, were 6.5, 10.4 and 4.4 and 12.7, 13.9 and 7.4% respectively. Accuracy, tested by linearity and recovery tests, was acceptable. A good correlation exists between a conventional enzymatic method and the proposed method. The latter is less time consuming and more reliable, thus providing a rapid assay to evaluate E1 and E1S in the same serum sample.

Components of variance analysis of data produced in a national quality-control survey of radioimmunoassays of thyroxin, triiodothyronine, thyrotropin, prolactin, and progesterone.

Data collected in a collaborative survey for radioimmunoassays have been studied by using analysis of variance to estimate the within-kit (CVw.kit) and the between-kit (CVb.kit) components of the total variability (CVT). This analysis has been applied to the results for triiodothyronine, thyroxin, thyrotropin, prolactin, and progesterone produced by 80-150 laboratories that assayed blind, replicate samples. Total variability was lowest in the thyroxin assay (CVT = 10.9%), associated with a very close between-kit agreement (CVb.kit = 4.0%); in the triiodothyronine assay, on the other hand, the large between-kit component (CVb.kit = 10.1%) increased the total variability to 16.1%. In the prolactin assay the CVT of 19.3% included 17.5% CVw.kit and 8.1% CVb.kit. Assays for thyrotropin and progesterone involve analyses of two pools at different hormone concentrations. The CVb.kit component was very high in the low-concentration pool, both for thyrotropin (25.1%) and progesterone (45.2%); in the higher-concentration pool it decreased to 8.3% for thyrotropin but remained high (21.6%) for progesterone. Applying analysis of variance to the triiodothyronine and thyroxin data obtained by different laboratories using the same kit showed that most kits yielded significantly different measurements when used in different laboratories.

Homogeneous luminescence immunoassay for total estrogens in urine.

We describe an homogeneous luminescence immunoassay for "total" estrogens in enzymically hydrolyzed urine from nonpregnant women. The antiserum, raised against estriol-16,17- dihemisuccinate conjugated to bovine serum albumin, specifically bound the C-19 steroids carrying the estrogen-characteristic phenolic group. 17 beta-Estradiol conjugated with aminobutylethyl isoluminol was used to monitor the immunological reaction; this conjugate was stable for at least two years. Because binding to the antiserum markedly enhances the light-producing efficiency of the tracer, no separation of bound and free antigen is necessary. Results (microgram/24 h) by this method (y) correlated well (r = 0.958) with those by a conventional fluorometric (x) method (y = 2. 51x - 2.83). The sensitivity (detection limit) is 4 micrograms/L and the precision compares well with that of commonly used RIA methods. The method appears suited to large numbers of samples, as in menstrual cycle monitoring.

Measurement of estrone-3-glucuronide and pregnanediol-3 alpha-glucuronide in early morning urine samples to monitor ovarian function.

The determination of the concentration of estrone-3-glucuronide and pregnanediol-3 alpha-glucuronide has been performed by a chemiluminescent immunoassay in early morning urine samples of 14 normal menstruating women and 11 women affected by luteal phase defect. The early morning urine samples were daily collected for an entire menstrual cycle. We have employed a timed and measured volume collection procedure as correction factor. The integrated values of the hormonal data in definite time intervals were used to create a nomogram. By means of this method, it was possible to completely separate normal from luteal insufficiency subjects and to distinguish two different types of luteal phase defects. Moreover, the same approach was applied to the study of the role and the frequency of luteal phase defect in 15 patients affected by habitual abortion and in 17 premenopausal women who had undergone quadrantectomy for T1a No Mo breast cancer. A luteal phase defect was detected in nine of the aborting patients (60%) and in eight women affected by breast cancer (47%). Finally estrone-3-glucuronide was measured in early morning urine samples of 96 prepubertal and pubertal girls in different pubertal stages and in one patient affected by precocious puberty, before and during an agonist GnRH treatment. The urinary test of ovarian function seems to be suitable for diagnostic purposes and for clinical studies.

Effects of transportation and delay in processing on the stability of nutritional and metabolic biomarkers.

The effects of transportation and delay in processing of blood samples on the concentration of biomarkers are significant in epidemiological studies for which specimens are collected from participants at locations other than a designated center or laboratory. These sources of variability in measurement were studied by collecting two sets of blood samples from 51 men between 26 and 50 years of age. The first set was sent immediately to the laboratory for processing. The second set was transported by car for one hour and then returned to the laboratory for processing. Both sets were stored together at -80 degrees C until the end of the study. Several blood constituents were evaluated. Vitamins, liver enzymes, and electrolytes showed no changes in concentration after transport by car for one hour. There were decreases in the concentrations of red and white blood cells, high-density-lipoprotein cholesterol, glucose, and creatinine after transportation. The transported total cholesterol, total testosterone, free testosterone, alkaline phosphatase, total bilirubin, and thiobarbituric acid-reactive substances increased in concentration. Although transportation and delay in processing of blood samples do not appear to greatly impact relative risk estimates, epidemiologists should be aware of these potential sources of variability in measurement and consider the consequences in their particular study.