Time-resolved fluoroimmunoassay of plasma and urine O-desmethylangolensin.

We present a method for the determination of the phytoestrogen metabolite O-desmethylangolensin (O-DMA) in plasma (serum) and in urine. O-DMA is a metabolite of daidzein, which occurs in soybeans. It has been suggested that isoflavones may afford protection against breast and prostate cancer and therefore, also the metabolites are of interest. The method is based on time-resolved fluoroimmunoassay (TR-FIA) using a europium chelate as a label. After the synthesis of 4"-O-carboxymethyl-O-DMA, this compound is coupled to bovine serum albumin, and then used as antigen in immunization of rabbits. The tracers with the europium chelate are synthesized using the same 4"-O-derivative of the alpha-methyldeoxybenzoin. After enzymatic hydrolysis and ether extraction the immunoassay is carried out by time resolved fluoroimmunoassay (TR-FIA). Cross-reactivity was tested with angolensin, dihydrogenistein, dihydrodaidzein, equol, 6'-OH-angolensin, trans-4-OH-equol, 6'-OH-O-DMA, cis-4-OH-equol and 5-OH-equol. The antiserum cross-reacted only with angolensin. This cross-reactivity seems not to influence the results, which were highly specific. Plasma samples are hydrolyzed and extracted. Urine samples are analyzed directly after hydrolysis without extraction. The correlation coefficient between the plasma TR-FIA results and the GC-MS results was high; r value was 0.985. The correlation coefficient between the urine TR-FIA results and the GC-MS results was high over the entire range of concentrations (0-1500 nmol/l); r value was 0.976, but lower in the low concentration range (0-100 nmol/l), i.e. value was 0.631. The intra-assay coefficients of variation (CVs) for plasma O-DMA concentrations and for urine O-DMA concentrations at three different concentrations varied 2.8-7.7 and 3.0-6.0%, respectively and the inter-assay CVs varied 3.8-8.9 and 4.4-6.6%, respectively. The working range of the plasma and urine O-DMA assays was 0.5-512 nmol/l.

7-Hydroxydehydroepiandrosterone epimers in human serum and saliva. Comparison of gas chromatography-mass spectrometry and radioimmunoassay.

Recent reports demonstrated that 7-hydroxylated metabolites of dehydroepiandrosterone (DHEA) possess immunomodulatory and antiglucocorticoid properties. Increased 7alpha-OH-DHEA levels were found in patients with Alzheimer's disease. Hence, measurement of steroids in patients with autoimmune diseases or disturbances in the central nervous system could be of interest. A new sensitive GC-MS method for the determination of 7-hydroxydehydroepiandrosterone epimers was developed and compared with previously developed radioimmunoassays. Besides serum, these steroids were, for the first time, measured in saliva where their concentrations were about five times lower. 7alpha- and 7beta-epimer levels correlated well in both body fluids and they were larger in male.

Time-resolved fluoroimmunoassay of plasma daidzein and genistein.

We present a method for the determination of the phytoestrogens daidzein and genistein in plasma (serum). These weakly estrogenic isoflavones occur in soybeans and in smaller amounts in some other beans and plants. It has been suggested that they may afford protection against prostate and breast cancer. The method is based on time-resolved fluoroimmunoassay (TR-FIA) using a europium chelate as a label. After synthesis of 4'-O-carboxymethyl-daidzein and 4'-O-carboxymethyl-genistein the compounds are coupled to bovine serum albumin (BSA), then used as antigens to immunize rabbits. The tracers with the europium chelate are synthesized using the same 4'-O-derivative of the isoflavones. After enzymatic hydrolysis and ether extraction the immunoassay is carried out using the VICTOR 1420 multilabel counter (Wallac Oy, Turku, Finland). The antisera cross-reacted to some extent with some isoflavonoids but not with flavonoids. The cross-reactivity seems not to influence the results, which were highly specific for both compounds. The correlation coefficients between the TR-FIA methods and the reference method based on isotope dilution gas chromatography-mass spectrometry were high; r-values were about 0.95-0.99 depending on concentration. The intra-assay coefficients of variation (CV%) for daidzein and genistein at three different concentrations vary 3.2-4.5 and 3.2-4.1, respectively. The inter-assay CVs vary 5.0-6.3 and 4.5-5.3, respectively. The working ranges of the daidzein and genistein assays are 1.0-216 and 1.7-370 nmol/l, respectively. The plasma values (n = 80) of daidzein and genistein are very low in Finnish subjects (mean for daidzein, 3.8+/-6.8 and for genistein, 3.2+/-7.6 nmol/l; median value for daidzein 1.5 and for genistein 1.4 nmol/l).

[Selected phytoestrogens with potential beneficial effects on risk of certain environmental diseases]. / Vybrané fytoestrogeny jako mozné príznivé faktory z hlediska rizika nekterých civilizacních onemocnení.

Evidence has been accumulated that some phytoestrogens act as protective factors against development of cancer and also cardiovascular diseases. These are phytoestrogens of isoflavone and lignane series, found especially in soy. Beneficial effect of these compounds may be explained by a complexity of their actions at various levels: they interact with estrogen receptors, some of them are inhibitors of the key enzymes responsible in the final effect for cell growth and proliferation, and, due to their chemical nature they are scavengers of free radicals. In the presented work the authors, in collaboration with Finnish partners from the University of Helsinki, developed original immunoassays for determination of main phytoestrogens of isoflavone series--daidzein, genistein, formononoetin, biochanin A, their metabolite equal and lignane enterolactone. The methods are sensitive enough for follow-up of actual levels of phytoestrogens in serum. By using these methods, the levels of phytoestrogens in Czech population have been established. The group of patients suffering from osteoporosis has been investigated, too. Significantly lower levels of isoflavonoids in comparison with sex- and age-matched healthy subject have been found in the patients. The methods have also enabled us to follow up the dynamics of these compounds in the organism, as well as to determine their content in food and its sources. The original detection and quantification of four above mentioned isoflavonoids in beer is an example.

Epitestosterone in human blood and prostatic tissue.

Epitestosterone, a C19-steroid with anti-androgenic activity, was determined in the plasma of 234 boys and men from the ages of 6-86 years, and in the prostate tissue of 15 men 55-82 years of age. It was documented that, while in adulthood the concentration of epitestosterone is about ten times lower than the concentration of testosterone, in the pre-pubertal period the level of epitestosterone is similar or even higher than that of testosterone. In the hyperplastic prostate tissue the content of epitestosterone is comparable to that of androstenedione, it is about twice as high as the content of testosterone and approximately half that of the content of dihydrotestosterone. At least in the case of pre-pubertal boys and in the prostatic tissue it is therefore possible to include epitestosterone into consideration as a regulatory factor for the androgen-dependent events.

Evaluation and separation of steroid-bovine serum albumin conjugates by high-performance liquid chromatography.

The conventional methods for characterization of steroid immunogens are based on the determination of the total amount of hapten bound to the protein carrier either by the UV spectroscopy or titration of unsubstituted amino groups. These methods do not allow more detailed insight into the immunogen composition. HPLC of the immunogen combined with UV detection is a relatively rapid and convenient method enabling determination of the hapten content in each fraction and, eventually, separation of individual fractions differing in the hapten content or purification of crude product.

[Epitestosterone as an endogenous antiandrogen in men]. / Epitestosteron jako endogenní antiandrogen u muzu.

The authors assessed in the plasma of 156 boys and men aged 6 - 65 years and in prostatic tissues of 15 men aged 55 - 82 years epitestosterone, C19-steroid with anti-androgenic properties. It was revealed that during the prepubertal period the epitestosterone level is higher than that of testosterone, in adult age the epitestosterone is about ten times lower than the testosterone concentration. In tissue of hyperplastic prostates the epitestosterone level is comparable with the androstendione level; it is about twice as high as the testosterone level and approximately half as high as the dihydrotestosterone level. Thus at least during the prepubertal period in boys and in the androgen-dependent prostate tissue a more significant participation of epitestosterone in the regulation of androgen-dependent processes must be taken into consideration.

A novel radioimmunoassay of allopregnanolone.

A radioimmunoassay for determination of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) in serum or plasma has been developed and evaluated. The method employs rabbit antiserum to 3 alpha-hydroxy-5 alpha-pregnane-11,20-dione-11-O-carboxymethyloxime bovine serum-albumin conjugate and tritiated radioligand. The main cross-reactant interfering in the assay, progesterone, is eliminated by permanganate oxidation. Two assay variants were compared, with and without a micro-column chromatography. The simplified variant appeared to be reliable enough for determination of allopregnanolone in normally menstruating women at luteal phase, whereas the column-chromatography step is necessary when analyzing samples of expected low analyte concentration as in women in follicular phase, postmenopausal women, or in men. The levels of allopregnanolone in healthy women correlated excellently with progesterone in agreement with previous findings.

The effect of epitestosterone on gonadotrophin synthesis and secretion.

The effects of 3-week treatment with increasing doses of epitestosterone (ET) on gonadotrophin gene expression and secretion, on testosterone and 5 alpha-dihydrotestosterone (DHT) levels, and on the weight of testes and prostates, were studied in intact adult male rats. The hormones were delivered by means of silastic capsules of different lengths filled with the steroid. One group of rats received testosterone (T) instead of ET, to compare the results with previous studies concerning the testosterone effect. The controls were given capsules with glucose only. Treatment with ET, as well as with T, significantly reduced the weights of prostates. When the data from ET-treated rats and controls were combined, a significant negative correlation (P < 0.001) was found between the weight of prostates and serum ET. T, in contrast to ET, also decreased significantly the weights of testes, ET treatment caused a significant reduction of serum T levels but only an insignificant decline of DHT levels, independent of the dose. Serum and pituitary (p) luteinizing hormone (LH) levels in the ET-treated rats did not change. Pituitary mRNA contents for the beta LH subunit (beta LH-mRNA) showed a dose-dependent significant increase, up to 170% (P < 0.01), with ET treatment. pFSH decreased with the lowest ET (2 cm) dose (P < 0.05), but no change was observed with the other doses. The mRNA for the common alpha-subunit also increased with the ET load. In conclusion, ET acts at several sites in the regulation of gonadotrophin formation and release. It enhances the steady-state mRNA levels of both gonadotrophins in the pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of long-term glucocorticoid therapy on glucocorticoid receptor content and on steroid response to ACTH.

The effect of long-term glucocorticoid therapy for systemic diseases on glucocorticoid receptor (GR) content and on basal and ACTH-stimulated levels of plasma and salivary cortisol 17 alpha-hydroxy-progesterone, androstenedione, 11 beta-hydroxyandrostenedione, DHEA, its sulfate and sex hormone-binding globulin (SHBG), as well as on basal levels of aldosterone, was investigated in a group of 24 children treated with prednisone for at least 8 months. The therapy was interrupted 24 h before the ACTH test and before plasma and saliva sampling. The control group consisted of 21 healthy children of corresponding age and sex. The patients were divided into two subgroups with normal and subnormal basal cortisolemia, they also differed in their response to ACTH. The GR levels in patient groups were indistinguishable from those found in controls. No correlation was found between GR content and basal levels of the above steroids or their response to ACTH. The best markers, apart from basal cortisolemia, for evaluation of the degree of suppression of adrenal function appeared to be the response of salivary (but not of plasma) cortisol and 17 alpha-hydroxy-progesterone to ACTH. Surprisingly, significantly lower levels of SHBG levels, which rose markedly after ACTH, were found in all the patients.

The effect of epitestosterone on the plasma levels of LH and FSH in ovariectomized immature rats.

Immature ovariectomized female rats primed with estradiol or without estrogen priming were treated with epitestosterone i.p. After 7 h blood was collected and LH and FSH levels were determined. The dose-response relationship was a biphasic one. LH and less markedly FSH levels decreased under epitestosterone treatment with doses up to 10 mg, whereas at higher doses an increase of gonadotrophins was observed.

[Determination of bone mineral content in vivo in dialyzed patients using photon radiation absorptiometry]. / Stanovení obsahu minerálu v kostech dialyzovaných nemocných in vivo merením absorpce fotonového zárení.

In a group of 47 women and 54 men with chronic renal failure included in a regular haemodialyzation programme the mineral content of bone was assessed using the method of radiation absorption of the radioisotope americium (241Am) on a Bone mineral detector of Gambro Co. The bone mineral content was reduced in men and women by 22%, as compared with healthy subjects of the same age and sex. The reduction was more marked in patients dialyzed for longer periods and when there were lamoratory signs of activity of the process.

[Accumulation of aluminum in the dialyzed patient: incidence, causes and possibilities of prevention]. / Akumulace aluminia u dialyzovaných pacientu: incidence, príciny a moznosti prevence.

In a group of 60 patients dialyzed for more than three years laboratory signs of aluminum accumulation were found in 30%. Serum Al values correlated with the increment in the desferal test. Orally administered aluminium hydroxide is a significant source of Al accumulation, as has been demonstrated by the decline of the blood aluminium level after its discontinuation and a negative desferal test in patients to whom it was not prescribed. The passage of Al across the dialyzation membrane depends on the difference between the ultrafiltrable ratio of the blood value and the Al concentration in the dialyzate. Therefore even a concentration of 10 micrograms/l Al in the dilyzate, so far recommended as "safe", can lead to a rise of the Al blood level during haemodialysis and to Al accumulation in tissues. Effective removal of Al from the organism is possible only during haemodialysis after previous administration of Desferal. Unless an adequate substitute of aluminium hydroxide will be available and a completely aluminium free dialyzate, Al accumulation can be prevented by preventive administration of Desferal.