Twenty years of experience with the steroid receptor external quality assessment program - the paradigm for tumour biomarker EQA studies. On behalf of the EROTC Receptor and Biomarker Study Group.

Estrogen receptor (ER) assays have clinical relevance in selecting women who would benefit from endocrine intervention. As the degree of benefit from endocrine therapy is directly related to the quantity of receptor present in the tumour, the quality of the steroid receptor assays is important. Moreover, since patients entered in multi-centre trials often include stratification based on the receptor status, receptor assays should be comparable between different institutes. ER- and progesterone receptor (PgR)-assays have been evaluated in quality assessment studies for almost 20 years by the EORTC Receptor and Biomarker Study Group. This study analyses our findings over these years and concludes with a recommended minimum structure on which QA schemes for any biomarker could be based.

Female sex hormone replacement therapy increases serum free 1,25-dihydroxyvitamin D3: a 1-year prospective study.

OBJECTIVE: Currently, hormone replacement therapy is applied successfully to reduce post-menopausal bone resorption. However, the exact mechanism by which oestrogen exerts its effect has not yet been fully elucidated. In order to determine whether changes in the biologically active 1,25-dihydroxyvitamin D3 may be of importance in this process, the concentrations of both total and free 1,25-dihydroxyvitamin D3 in serum were assessed. DESIGN: In 36 post-menopausal women the effect of hormone replacement therapy, with a combination of 17 beta-oestradiol and norethisterone acetate, on the serum levels of total and free 1,25-dihydroxyvitamin D3 was studied after 0, 3, 6 and 12 cycles. MEASUREMENTS: The total concentration of 1,25-dihydroxyvitamin D3 in serum was assessed using a radioreceptor assay after diethylether extraction of the samples followed by paper chromatography. The free fraction of 1,25-dihydroxyvitamin D3 was measured using symmetric dialysis. The free 1,25 dihydroxyvitamin D3 concentration was calculated by multiplying the total concentration by the free fraction. RESULTS: During therapy, mean serum total 1,25-dihydroxyvitamin D3 concentrations (+/- SD) were 106.4 pmol/l (+/- 27.5), 155.0 pmol/l (+/- 49.5), 176.7 pmol/l (+/- 70.0) and 161.1 pmol/l (+/- 55.3) at 0, 3, 6 and 12 cycles, respectively. Serum free 1,25-dihydroxyvitamin D3 concentrations were 68 fmol/l (+/- 22), 107 fmol/l (+/- 35), 120 fmol/l (+/- 43) and 108 fmol/l (+/- 37), respectively. Baseline values of both total and free 1,25-dihydroxyvitamin D3 were significantly lower than those during therapy at all time (P < or = 0.001). CONCLUSION: Both the serum total 1,25-dihydroxyvitamin D3 and the serum free 1,25-dihydroxyvitamin D3 concentrations are increased during combined 17 beta-oestradiol and norethisterone acetate therapy for a year. Assuming that the free concentration 1,25-dihydroxyvitamin D3 reflects the biologically active fraction, this rise may in part explain the preventive effect of hormone replacement therapy on osteoporosis.

Prognostic value of tissue-type plasminogen activator (tPA) and its complex with the type-1 inhibitor (PAI-1) in breast cancer.

The prognostic value of tissue-type plasminogen activator (tPA) measured in samples derived from 865 patients with primary breast cancer using a recently developed enzyme-linked immunosorbent assay (ELISA) was evaluated. Since the assay could easily be adapted to the assessment of the complex of tPA with its type-1 inhibitor (PAI-1), it was investigated whether the tPA:PAI-1 complex also provides prognostic information. To this end, cytosolic extracts and corresponding detergent extracts of 100,000 g pellets obtained after ultracentrifugation when preparing the cytosolic fractions for routine steroid hormone receptor determination were assayed. Statistically significant correlations were found between the cytosolic levels and those determined in the pellet extracts (Spearman correlation coefficient r(s) = 0.75, P < 0.001 for tPA and r = 0.50, P < 0.001 for tPA:PAI-1 complex). In both Cox univariate and multivariate analysis elevated levels of (total) tPA determined in the pellet extracts, but not in cytosols, were associated with prolonged relapse-free (RFS) and overall survival (OS). In contrast, high levels of the tPA:PAI-1 complex measured in cytosols, but not in the pellet extracts, were associated with a poor RFS and OS. The prognostic information provided by the cytosolic tPA:PAI-1 complex was comparable to that provided by cytosolic (total) PAI-1. Furthermore, the estimated levels of free, uncomplexed tPA and PAI-1, in cytosols and in pellet extracts, were related to patient prognosis in a similar way as the (total) levels of tPA and PAI-1 respectively. Determination of specific forms of components of the plasminogen activation system, i.e. tPA:PAI-1 complex and free, uncomplexed tPA and/or PAI-1, may be considered a useful adjunct to the analyses of the separate components (tPA and/or PAI-1) and provide valuable additional prognostic information with respect to survival of breast cancer patients.

Prognostic impact of urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1) in cytosols and pellet extracts derived from 892 breast cancer patients.

To evaluate the clinical relevance of urokinase-type plasminogen activator (uPA) and its type-1 inhibitor (PAI-1) measured by a recently developed enzyme-linked immunosorbent assay (ELISA), we analysed both components in samples derived from 892 patients with primary breast cancer (median follow-up 99 months). The assays were performed in cytosolic extracts as well as in corresponding detergent extracts of pellets obtained after ultracentrifugation, which was carried out when preparing the cytosolic fractions for routine steroid hormone receptor determination. Statistically significant correlations were found between the cytosolic levels and those determined in the pellet extracts (Spearman correlation coefficient r = 0.60, P < 0.0001 for uPA and r = 0.65, P < 0.0001 for PAI-1). Furthermore, strong correlations were found between the levels of both uPA (r = 0.85, P < 0.0001) and PAI-1 (r = 0.90, P< 0.0001) in the cytosols and their levels previously measured with ELISAs based on commercial reagents. In both Cox univariate and multivariate analysis, high cytosolic levels of uPA or PAI-1 were significantly associated with increased rates of relapse and death. The levels of uPA and PAI-1 in the pellet extracts also provided prognostic information, although to a lesser extent compared with the cytosolic extracts. The prediction of prognosis on the basis of uPA and PAI-1 assessed by an alternative ELISA once again emphasizes the established prognostic role and usefulness of these parameters in selection of breast cancer patients at high or low risk of recurrence.

External quality assessment of trans-European multicentre antigen determinations (enzyme-linked immunosorbent assay) of urokinase-type plasminogen activator (uPA) and its type 1 inhibitor (PAI-1) in human breast cancer tissue extracts.

High levels of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) in breast cancer tissue extracts have been associated with rapid disease progression. In these studies, different enzyme-linked immunosorbent assay (ELISA) kits have been applied for the quantification, and consequently the ranges of uPA and PAI-1 levels reported differ considerably. Therefore, the Receptor and Biomarker Study Group (RBSG) of the European Organization for Research and Treatment of Cancer (EORTC) and a consortium of the BIOMED-1 project 'Clinical Relevance of Proteases in Tumor Invasion and Metastasis' initiated three collaborative between-laboratory assessment trials aimed at controlling uPA and PAI-1 antigen analyses. For this purpose, two control preparations were produced from different sources: pooled human breast cancer specimens (QC-240893) and human breast cancer xenografts raised in nude mice (QC-101094). The lyophilized preparations were stable for prolonged times (at least 3 and 27 months respectively) at 4 degrees C. Furthermore, a good parallelism following dilution was found for uPA and PAI-1. The data from QC trial no. 1 clearly indicated that acceptable between-laboratory coefficients of variation (CVs) for uPA (<8.2%) and PAI-1 (<16.6%) in QC-240893 could be achieved when the same type of ELISA kit (American Diagnostica) was used. From the second trial, in which ten EORTC laboratories each received five identical lyophilized QC-101094 samples, it appeared that the within-laboratory variations for uPA and PAI-1 determinations obtained by 'experienced' laboratories were lower (<12.9%) than those from non-experienced laboratories (<36.4%). In a third QC trial, five BIOMED-1 laboratories, all of which employed ELISA procedures for uPA and PAI-1, participated in six subsequent quality assessment rounds receiving five samples of QC-101094. Although for each laboratory the within-run CVs for uPA as well as for PAI-1 were low (<7.8%), the between-run CVs were found to be considerably higher (up to 56.2% for uPA and to 27.6% for PAI-1). Consequently, because of the different ELISA formats used, the absolute analyte values measured in the different laboratories varied substantially. The use of 'common external standards' in the different ELISAs resulted in a significant reduction of the between-laboratory CVs from 61.3% to 15.7% (uPA) and from 42.1% to 19.1% (PAI-1). The present data demonstrate that in multicentre studies the same ELISA kit should be used, and that external quality assurance (QA) is mandatory. Furthermore, it appears from the present study that standardization of the protein assay as a tissular parameter is imperative.

Human endothelial cell migration is stimulated by urokinase plasminogen activator:plasminogen activator inhibitor 1 complex released from endometrial stromal cells stimulated with transforming growth factor beta1; possible mechanism for paracrine stimulation of endometrial angiogenesis.

Human endometrial stromal cell cultures, stimulated for two days with recombinant transforming growth factor beta1 (TGFbeta1; 10 ng/ml), contained conditioned medium concentrations of urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI1), and uPA:PAI1 complex. Since a number of cellular effects have been reported to follow a binding of enzymatically inactive uPA to the receptor in different cell types, we studied the influence of uPA:PAI1 complex on human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1). Increasing concentrations of uPA:PAI1 complex as well as free uPA resulted in a dose-dependent stimulation of endothelial cell migration. Stimulation by the complex was of the same magnitude as that of free uPA on a molar basis and reached its maximum at 1 nM in both cell types. PAI1 by itself, however, had no effect on cell migration. The migratory response to both uPA and the uPA:PAI1 complex was inhibited by antibody adhesion to the cell surface receptor for uPA. In addition, we found that TGFss1 had a direct stimulatory effect on migration in both HUVEC and HMEC-1. This response did not, however, involve the binding of uPA to the uPA receptor. Since TGFbetas are expressed in endometrial tissue and reportedly stimulate angiogenesis in other tissues in vivo, though not endothelial cell proliferation in vitro, they may engage in the regeneration of endometrial vasculature indirectly via perivascular cells. We found that the uPA:PAI1 complex, when released from endometrial stromal cells in response to TGFbeta1, stimulated endothelial cell migration. This suggests a possible mechanism for paracrine stimulation of endometrial angiogenesis.

Determination of non-protein-bound plasma 1,25-dihydroxyvitamin D by symmetric (rate) dialysis.

Most of the total circulating 1,25-dihydroxyvitamin D (1,25(OH)2D) is bound to plasma proteins, mainly vitamin D-binding protein (DBP) and albumin. Only a small fraction in plasma exists in the free form. It is widely assumed that the non-protein-bound free hormone reflects the biologically active fraction. We describe a dialysis method for the determination of plasma free 1,25(OH)2D which is relatively easy to perform. In this symmetric or "rate" dialysis method, identical samples are placed at both sides of a membrane. At one side, tritiated 1,25(OH)2D is added and the rate of transfer of this tritiated 1,25(OH)2D through a dialysis membrane is directly related to the free fraction of plasma 1,25(OH)2D. This method is much less susceptible toward tracer impurities than indirect equilibrium dialysis and centrifugal ultrafiltration. Moreover, it requires much less tracer. The intraassay coefficient of variation for the determination of the free fraction is 1.0%; the interassay variation is 7.7%. Comparison of the free fraction of 23 samples assessed with both centrifugal ultrafiltration and symmetric dialysis showed much higher values using the former method. No significant correlation between the two methods was found. The free fraction of 1,25(OH)2D in normal subjects as assessed with symmetric dialysis ranges from 0.049 to 0.103%.

p53 protein accumulation predicts poor response to tamoxifen therapy of patients with recurrent breast cancer.

PURPOSE: Mutations of the p53 gene are frequently observed in primary breast cancer and accumulation of p53 protein has been used as a surrogate marker of p53 inactivation. Previous studies have shown that p53 accumulation is related to poor prognosis in primary breast cancer. We studied whether p53 protein accumulation is a predictive factor for response to tamoxifen treatment in patients with recurrent breast cancer. PATIENTS AND METHODS: Levels of p53, estrogen receptor (ER), progesterone receptor (PgR), and urokinase-type plasminogen activator (uPA) were assayed in cytosolic extracts derived from primary tumors of 401 tamoxifen-naive patients who developed recurrent disease. All patients in the study received tamoxifen therapy upon relapse (median follow-up, 69 months). Association of tested factors with response to tamoxifen treatment was studied by logistic regression analysis, and with survival after the start of treatment by Cox univariate and multivariate regression analysis. RESULTS: p53 levels (median, 0.23 ng/mg protein) were not related to ER or PgR levels, but positively correlated with uPA (P < .0001). In a test for trend, we observed an association of p53 protein levels with response to tamoxifen therapy. When dichotomized (at the median value), 42% in the p53-high versus 56% in the p53-low group showed a response. In multivariate analysis, including patient and tumor characteristics, p53 accumulation retained significance with the rate of response (odds ratio [OR], 0.48; 95% confidence interval [CI], 0.31 to 0.74; P < .001). Also in multivariate analysis, reduced survival after the start of tamoxifen therapy was observed in the p53-high group (relative hazards rate [RHR], 1.56, 95% CI, 1.17 to 2.10; P = .002). A statistically significant association between p53 levels and decreased tamoxifen response was seen only in the subset of patients whose tumors expressed low levels of ER or PgR (<75 fmol/mg protein). CONCLUSION: Measurement of primary tumor p53 levels may be effective in predicting response to tamoxifen therapy in recurrent breast disease. However, more confirming studies on the association between p53 protein accumulation and response to antiestrogen therapy are needed before tumor p53 levels can be used in routine clinical practice.

Prognostic value of TP53 protein accumulation in human primary breast cancer: an analysis by luminometric immunoassay on 1491 tumor cytosols.

The tumor suppressor gene TP53 is implicated in the regulation of normal cell growth and division, DNA repair and apoptosis. Mutations in this gene usually give rise to a conformationally altered protein which is stably expressed at high levels. We have studied TP53 protein accumulation in routinely prepared cytosols from 1491 human primary breast cancer specimens (median follow-up of patients alive, 66 months), using a quantitative luminometric immunoassay (LIA). The TP53-LIA values varied between 0 and 153.53 (median 0.20 ng/mg protein). Median TP53 levels were significantly higher in ER- and PgR-negative tumors. In Cox univariate regression analysis, when analyzed as a continuous variable, increasing TP53 levels were related with a poor relapse-free survival (p < 0.01). In multivariate analysis for relapse-free survival, including age, menopausal status, tumor size, nodal status and steroid hormone receptor status, TP53 accumulation, when analyzed as a dichotomized variable, was an independent factor for predicting the rate of relapse with a relative relapse rate (95% confidence limits) of 1.39 (1.19-1.63). In conclusion, the LIA for the TP53 protein can easily be performed on cytosols routinely prepared for steroid hormone receptor analysis, it is a quantitative assay, and it may be useful in establishing the relation of TP53 accumulation and breast cancer prognosis.

Immunoassays (ELISA) of urokinase-type plasminogen activator (uPA): report of an EORTC/BIOMED-1 workshop.

The urokinase-type plasminogen activator (uPA) is considered to play a key role in the process of invasion and metastasis. In several independent studies, in a variety of cancer types (e.g. of the breast, colon, stomach, lung, ovary), high antigen levels of uPA in tumour extracts have been associated with rapid disease progression. In these studies, different sets of antibodies and standards (often as commercially available uPA ELISA kits) have been used. The standards provided with the different uPA ELISA kits are different from each other in both composition and source. In addition, the different uPA ELISA kits use antibodies which differ in specificity and affinity for the various forms of uPA including pro-uPA, HMW-uPA, LMW-uPA, the aminoterminal fragment (ATF) and complexes with inhibitors (PAI-1 and PAI-2) and the receptor (uPAR). Further, the composition of tumour tissue extraction buffers differ significantly among the published studies. Thus, it is not surprising that the ranges of cytosolic uPA levels reported differ considerably even when measured within the same tumour type. These discrepancies led the EORTC Receptor and Biomarker Study Group, in conjunction with the BIOMED-1 consortium on 'Clinical Relevance of Proteases in Tumour Invasion and Metastasis', to organise a workshop to study the characteristics associated with six different uPA immunoassays (ELISA) used in clinical studies reported in the literature. Although the absolute uPA antigen values measured with the respective uPA ELISA kits differed, high correlations were obtained for any two of the four uPA ELISA kits finally applied to sets of breast cancer cytosol preparations. The preparations used at present as standards in the various uPA ELISA kits are not representative of actual human breast cancer cytosols. Thus absolute standardisation is only possible by using a common reference sample (breast cancer cytosol) and similarly composed ELISA uPA kits. Then it will be possible to generate comparable data on clinical tissue as well as to check for batch-to-batch variations within particular ELISA kits.

3 Alpha-hydroxysteroid oxidoreductase activities in dihydrotestosterone degradation and back-formation in rat prostate and epididymis.

The metabolism of dihydrotestosterone (DHT) and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-Adiol) was assessed in full homogenates of rat prostate and epididymis. The major degradational route of DHT was catalysed by the enzyme(s) 3 alpha-hydroxysteroid oxidoreductase (HSOR). Enzyme kinetic characteristics Vmax, Km and Vmax/Km ratio, were obtained for the NADP(H)- and NAD(H)-dependent interconversion of DHT and 3 alpha-Adiol at pH 7.0 and at saturated co-factor concentration. For both the reduction of DHT and the oxidation of 3 alpha-Adiol, NAD(H) was the preferred co-factor when activities were rated by their Vmax and Vmax/Km ratio. Combining the data with the earlier established Vmax/Km ratios for the 5 alpha-reductase isozyme type I and II activities in rat prostate and epididymis indicated that DHT, at saturated co-factor concentrations, would not be sustained in either tissue considering the reported enzyme characteristics. The reported exclusive bioavailability of the co-factors NADPH and NAD+ in vivo, however, will direct the metabolic pathways in these tissues to sustain the formation of DHT.

Prognostic significance of TP53 accumulation in human primary breast cancer: comparison between a rapid quantitative immunoassay and SSCP analysis.

TP53 accumulation in human primary breast carcinomas was studied by a quantitative luminometric immunoassay (LIA), and TP53 gene alterations, exons 5-8, were examined by single-strand conformation polymorphism (SSCP) analysis. In 48 of 142 breast tumor samples, a TP53 gene alteration was identified. In tumor samples without a TP53 gene alteration, the median cytosolic TP53 protein level, as determined by LIA, was 0.4 ng/mg protein (range 0-70.8 ng/mg protein), whereas the median TP53 protein level for tumor samples with a TP53 gene alteration was 10 times higher, i.e., 4.1 ng/mg protein (range 0.1-176.0 ng/mg protein). Despite a significant correlation between the outcome of LIA and SSCP, a disagreement was found in 22% of cases analyzed. Significant correlations were found between TP53 protein accumulation and low estrogen receptor content, and with a shorter relapse-free as well as overall survival, with a median duration of follow-up of 100 months. Due to its rapid and easy performance on routinely prepared cytosols, the LIA for TP53 protein may be useful in evaluating the prognostic impact of TP53 protein accumulation in human primary breast cancer.

Differential subcellular distribution of rat prostatic steroid 5alpha-reductase isozyme activities.

The rat prostate, a classical androgen-target tissue, contains both known isozymes of steroid 5alpha-reductase. i.e. type I and type II. So far, the role of the type I isozyme has been proposed as catabolic. The abundant expression of type I 5alpha-reductase in an androgen-target tissue is therefore puzzling. Assessment of the subcellular localization of 5alpha-reductase isozymes in rat prostate might contribute in elucidating their possibly distinct roles. After obtaining crude subcellular fractions by differential centrifugation, both isozyme activities were measured at neutral pH by plotting according to Eadie-Scatchard. The observations were extended by assessment of pH-dependent velocity ratios and type II 5alpha-reductase inhibitor sensitivities in these subcellular fractions. The results indicated a preferentially--although not exclusively--nuclear localization for the type I and a predominantly microsomal localization for the type II isozyme activity in the rat prostate. In conclusion, the nuclear localization of the type I isozyme seems not to concur with its proposed catabolic role.

Thyroid function studies in normal pregnant Tanzanian women.

Iodine deficiency is well known as a cause of several disorders such as endemic goiter and cretinism, along with a wide spectrum of psychoneurologic development disorders including endemic mental deficiency, which are generally correlated with damage to the fetus. Since as much as 40% of the Tanzanian population is at risk for iodine deficiency disorders (IDD) because they live in iodine-deficient areas, and although the effects of iodine deficiency on human reproduction in Tanzania have not been objectively studied, it is estimated that there are approximately 600,000 cretins and cretinoids in the country as a result of IDD. As a baseline study for future research on iodine deficiency and its effects on human reproduction in Tanzania, we assayed serum thyroxine (T4), triiodothyronine (T3), thyrotropin (TSH), and free thyroxine (FT4) in 93 clinically euthyroid pregnant women and 34 nonpregnant women as controls. Pregnancy was accompanied by significantly increased levels of total T3 and T4, decreased FT4, and increased TSH concentration in serum. However, biochemical euthyroidism (assessed by FT4 and basal TSH) was demonstrated in almost all (99%) of the pregnant subjects in conformity with most of the previous findings elsewhere. We conclude that pregnant Tanzanian women residing in areas without iodine deficiency experience changes in biochemical parameters of thyroid function similar to their counterparts in other places.

Highly sensitive and specific HPLC with fluorometric detection for determination of plasma epinephrine and norepinephrine applied to kinetic studies in humans.

An HPLC separation method combined with fluorometric detection was extended to enable simultaneous assessment of plasma 3H-labeled and endogenous epinephrine (E) and norepinephrine (NE). Forearm fractional extraction (FFE) of 3H-labeled E and NE and of endogenous E was measured in 40 healthy volunteers who were receiving a continuous infusion of 3H-labeled E and NE. Concentrations of arterial and venous E were 26.8 +/- 1.95 (mean +/- SE) and 6.8 +/- 0.75 ng/L, respectively. Arterial and venous NE and dopamine (DA) were also measured, with respective values of 140.7 +/- 8.5 and 192.1 +/- 15.1 for NE, and 13.1 +/- 0.78 and 11.3 +/- 0.70 ng/L for DA. The FFE of 3H-labeled E was slightly but significantly higher (0.790 +/- 0.016) than the that of either 3H-labeled NE or endogenous E (0.748 +/- 0.0146 and 0.745 +/- 0.0185, respectively; P < 0.001), the correlations being highly significant (r = 0.80, P < 0.001) in both cases. The small difference between the FFE of E and of 3H-labeled E allows the calculation of the apparent spillover of E. However, this spillover was negligible compared with forearm NE spillover (0.0112 +/- 0.0031 vs 1.369 +/- 0.128 ng/L per minute. The high sensitivity of this measurement of venous E widens the possibilities for studying E kinetics under physiological conditions.

Rat steroid 5 alpha-reductase kinetic characteristics: extreme pH-dependency of the type II isozyme in prostate and epididymis homogenates.

Reevaluating the assay for rat steroid 5 alpha-reductase isozymes in prostate and epididymis homogenates we encountered an extreme pH-dependency of the type II isozyme. The time-course of the metabolism of testosterone (T) to 17 beta-hydroxy-5 alpha-androstan-3-one (DHT) at acidic pH shows an initial burst when the homogenate is not brought to pH before the start of the incubation. Therefore, the rat type II 5 alpha-reductase isozyme does not follow Michaelian law under these conditions making a single time point measurement invalid. Assessing the pH-optimum of 5 alpha-reduction in both rat prostate and epididymis homogenates we found a strong substrate dependency: at high substrate concentrations a pH-optimum for the type II isozyme of pH 5.0 was found, whereas at lower concentrations pH 5.5 is optimal. Establishing Vmax (maximum velocities) and Km (affinity constants) for the 5 alpha-reduction of T at pH 4.5-8.0, the efficiency optimum Vmax/Km appeared to be pH 5.5 in both prostate and epididymis homogenates. Specifically at acidic pH these kinetic characteristics of the type II isozyme vary many-fold. Discrepancies in literature concerning 5 alpha-reductase characteristics can, at least in part, be attributed to the choice of optimal pH, or to pH shifts during the assay.

Hormone replacement therapy increases serum 1,25-dihydroxyvitamin D: A 2-year prospective study.

Osteoporosis is a common disorder in postmenopausal women, which is probably due to decreased ovarian function. Currently, hormone replacement therapy (HRT), involving administration of estrogen and progestogen, is successfully applied to reduce bone resorption. We studied the effect of HRT on 23 postmenopausal women. This consisted of a combination of 17 beta-estradiol and dydrogesterone, on the serum level of 1,25-dihydroxyvitamin D (1,25(OH)2D) after 0, 6, 12, and 24 months. We found mean serum concentrations (+/- SD) of 1,25(OH)2D of 130.5 pmol/liter (46.1), 152.7 pmol/liter (45.1), 170.8 pmol/liter (64.0), and 155.2 pmol/liter (59.7), respectively. The baseline values in these women were found to be significantly lower than those during therapy (P < or = 0.005). No statistically significant differences were observed when comparing the estrogen-only phase with the combined estrogen-progestogen phase. It is concluded that HRT results in an increase in the serum 1,25(OH)2D concentration which lasts for at least 2 years. This increase may partly explain the preventive effect of HRT on osteoporosis. Furthermore, these results suggest that dydrogesterone does not influence the estrogen-induced changes in serum 1,25(OH)2D concentration.

Predictive value of luteinizing hormone releasing hormone (LHRH) bolus testing before and after 36-hour pulsatile LHRH administration in the differential diagnosis of constitutional delay of puberty and male hypogonadotropic hypogonadism.

Intravenous LHRH bolus testing (100 micrograms) after 36 h of pulsatile LHRH administration (5 micrograms/90 min) preliminary has been reported to allow complete differentiation between constitutional delay of puberty (DP) and hypogonadotropic hypogonadism (HH) in a small group of sexually immature patients. So far, these data have never been confirmed. To assess the discriminatory power of the test, 33 patients with a presumptive diagnosis of either DP (n = 17) or HH (n = 16), confirmed by clinical follow-up, were studied accordingly. Both groups of patients had similar mean basal LH and FSH levels (P > 0.10). The mean basal plasma testosterone level was three times higher in DP than in HH (4.2 +/- 1.0 vs. 1.4 +/- 0.2 nmol/L, P* < 0.001), but there was a wide overlap. In response to the first LHRH bolus test, the mean LH increment was significantly lower in HH than in DP patients (P < 0.001), but, in 44% of the patients, the values overlapped. The FSH increments were similar in HH and DP. Pulsatile LHRH administration for 36 h similarly increased LH levels in HH and DP to values (2.7 +/- 0.4 and 3.8 +/- 0.5, respectively) slightly higher than before (P < 0.01), but again, not statistically significantly different from each other. The mean testosterone levels increased 2-fold in both groups and remained significantly higher in DP than in HH (7.6 +/- 2.1 vs. 2.8 +/- 0.5 nmol/L P* < 0.05). The mean FSH levels after priming also rose, however, to levels significantly higher in HH than in DP (5.2 +/- 0.8 vs. 3.5 +/- 0.4, P* < 0.05). In HH the ratio of FSH to LH almost doubled, whereas it virtually remained unchanged in DP. LHRH bolus testing after LHRH priming evoked a significantly lower LH response in both HH and DP than before priming despite only slightly higher baseline LH values. The LH increment in HH was five times lower in HH than in DP. In any of the 16 HH patients, the LH increment was < or = 3 IU/L, whereas in 15 out of 17 DP patients the increase was higher (sensitivity of the test 100%, specificity 88%, and diagnostic efficiency 94% after LHRH priming against 56%, 94%, and 75% respectively, before LHRH priming.(ABSTRACT TRUNCATED AT 400 WORDS)

Advantages of paper chromatography as a preparative step in the assay of 1,25-dihydroxyvitamin D.

Ether extraction and paper chromatography were used to separate the main metabolites of vitamin D in plasma [25-(OH), 24,25-(OH)2 and 1,25-(OH)2 vitamin D]prior to radio receptor-assay. The overall procedural loss of the 1,25-(OH)2 vitamin D was 58 +/- 5% (n = 40), corrected for by tracer addition. The sensitivity of the assay was 0.5 fmol/tube, corresponding to 4 pmol/l, and the intra- and inter-assay coefficients of variation were 10.5% and 11.5%, respectively. The range of values measured in healthy controls was 80-200 pmol/l (n = 60), which is in agreement with findings reported in the literature. A comparison of the results of the present procedure with those obtained with a procedure employing C18 purification, disclosed a correlation coefficient of 0.92 (p < or = 0.0001), a slope of 0.89 (p < or = 0.0001) and a small non-significant intercept of 5.0 pmol/l (n = 53).