Oldřich Lapčík

Immunoassay of 7-hydroxysteroids : 2. Radioimmunoassay of 7α-hydroxy-dehydroepiandrosterone

High sensitivity radioimmunoassay of 3beta, 7alpha-dihydroxy-5-androsten-17-one (7alpha-OH-DHEA) has been developed and evaluated. The method is based on polyclonal rabbit antisera raised against 19-O-(carboxymethyl)oxime bovine serum albumin conjugate and bridge- and position homologous [(125)I]iodotyrosine methyl ester as a tracer. Sensitivity of the assay amounted to 3.12 fmol (0.95 pg)/tube, precision as a mean intra- and interassay coefficient of variation was 7.1 and 10.6%, respectively, and the average recovery of the analyte added to steroid-free serum was 110%. Out of the steroids occurring in human serum which may interfere with the assay, the only important cross-reactants were dehydroepiandrosterone and 3beta, 7beta-dihydroxy-5-androsten-17-one (7beta-OH-DHEA) with cross-reactivities of 1.95 and 1.16%, respectively. The levels of free (unconjugated) 7alpha-OH-DHEA have been determined in 29 sera from healthy volunteers (23 females and 6 males), and from 48 patients (43 females and 5 males) in which dehydroepiandrosterone and its sulfate (DHEA/S) had been measured for various endocrinopathies. The levels in healthy subjects ranged from 0.21 to 6.57 (mean 2.33+/-1.50) nM, while those of the patients from 0 to 5. 99 (mean 1.46+/-1.52) nM. The levels of 7alpha-OH-DHEA in patients significantly correlated with those of DHEA and its sulfate.

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.

Immunoassay of 7-hydroxysteroids : 1. Radioimmunoassay of 7β-hydroxy dehydroepiandrosterone

High sensitivity radioimmunoassay of 3beta,7beta-dihydroxy-5-androsten-17-one (7beta-OH-DHEA) has been developed and evaluated. The method is based on polyclonal rabbit antisera raised against its 19-O-(carboxymethyl)oxime bovine serum albumin conjugate and bridge- and position homologous [125I]iodotyrosine methyl ester as a tracer. Alternatively, [3H]tracer has been prepared, which was recognized by the antiserum as well, but the assay sensitivity was lower. The identity of measured immunoreactive material was confirmed by high performance liquid chromatography which separated 7beta-OH-DHEA from its 7alpha-isomer. Using radioiodinated tracer, the sensitivity of the method was 3.48 fmol (1.06 pg) per tube, the mean recovery of standard added to steroid-free serum was 98.5%. Free (unconjugated and not-esterified) 7beta-OH-DHEA amounted in average 5.8% of the total 7beta-OH-DHEA present in human serum. It was measured in 42 normal subjects (28 females and 14 males) and in 92 randomly selected patients with various endocrinopathies. The mean values +/- SD in normals were 2.05 +/- 1.02 nmol l(-1), the broad range of values from undetectable levels to 10.3 nmol l(-1) was found in the patients. Serum levels of free 7beta-OH-DHEA in the patients significantly correlated with DHEA and its sulfate.

Immunoanalysis of isoflavonoids in Pisum sativum and Vigna radiata

Abstract Radioimmunoassays (RIAs) combined with liquid chromatography were used for study of isoflavonoids in seeds of pea Pisum sativum and mung bean Vigna radiata. Radioimmunoassays with these specifities were used: (1) daidzein and its 4′-derivatives (e.g. formononetin); (2) daidzein and its 7-derivatives (e.g. daidzin, isoformononetin); (3) genistein and its 4′-derivatives (e.g. biochanin A); and (4) genistein and its 7-derivatives (e.g. genistin, prunetin). Dormant or germinating seeds were extracted with 80% ethanol. Immunoreactivities were measured either in crude extracts or after chromatographic fractionation by HPLC (reversed phase, octadecylsilica). Chromatographic mobilities of immunoreactive fractions were compared to those of daidzein, daidzin, formononetin, isoformononetin, genistein, genistin biochanin A and prunetin standards. Extracts from Vigna radinfa contained daidzein, genistein and their 7- O glucosides, daidzin and genistin, respectively. No immunoreactivity was recorded in HPLC fractions corresponding to glycosides in extracts from P. sativum, but the methods sensitive to 7-derivatives of daidzein and genistein showed peaks with chromatographic mobilities identical to those of the 7-methoxyderivatives, isoformononetin and prunetin, respectively. In additional experiments, the pea extracts were fractionated either by thin layer chromatography (TLC) on silica or by ion-exchange TLC on aminosilica. Identity of the daidzein-7 and genistein-7 immunoreactive entities with isoformononetin and prunetin, respectively, was confirmed by the identical chromatographic behavior in all these different chromatographic systems.

Actual levels of soy phytoestrogens in children correlate with thyroid laboratory parameters

Abstract Thyroid hormones and thyroid autoantibodies, along with serum concentrations of two phytoestrogens of the isoflavone series, daidzein and genistein, were measured in 268 children without overt thyroid diseases, screened for iodine deficiency in one region of the Czech Republic. Since both phytoestrogens have been reported to inhibit thyroid hormone biosynthesis and in high concentrations to exert goitrogenic effects, we investigated whether their presence in the circulation could influence thyroid hormone function in a population where soy consumption is not common. Correlation analysis revealed a significant positive association of genistein with thyroglobulin autoantibodies and a negative correlation with thyroid volume. Multiple regression analysis of the relationships between actual phytoestrogen levels and measured thyroid parameters revealed only a weak but significant association between genistein and thyroid variables. Higher levels of free thyroxine were found in a subgroup of 36 children who ate soy food in the previous 24h. In conclusion, only modest association was found between actual phytoestrogen levels and parameters of thyroid function. On the other hand, even small differences in soy phytoestrogen intake may influence thyroid function, which could be important when iodine intake is insufficient.

Elimination of cross-reactivity by addition of an excess of cross-reactant for radioimmunoassay of 17α-hydroxypregnenolone

Polyclonal antiserum against 3beta,17alpha-dihydroxypregn-5-en-20-one-19-O-(carboxymethyl )-oxime bovine serum albumin (17alpha-hydroxypregnenolone-19-CMO:BSA), was raised in rabbits. Its main structural determinants were the substituents on D-ring as demonstrated by its 107% cross-reaction with 17alpha-hydroxyprogesterone. This unspecificity was almost completely eliminated by addition of the excess of the cross-reactant directly to the analytical system. The contribution of the cross-reactant from the sample in such a system became negligible due to saturation of the populations of polyclonal antibodies recognizing the analyte as well as the cross-reactant. The possible interference of 17alpha-hydroxypregnenolone-3-sulfate was avoided by inserting ether extraction. The analytical system appeared to be stable to differences in cross-reactant concentrations even in samples from patients with pathologically elevated serum levels of 17alpha-hydroxyprogesterone. The radioimmunoassay was compared with the system using the unspecific antiserum alone, but after separation of the cross-reactants by HPLC. As demonstrated by parallel measurement of 125 samples of human plasma from both sexes and various ages either before and/or after adrenocorticotropin stimulation and 17 samples with elevated basal of human plasma 17alpha-hydroxyprogesterone levels, an excellent correlation was achieved between both methods. The method, based on a simple addition of the cross-reactant, avoids the time-consuming chromatographic separation and, in comparison with the other approaches for improving the specificity of polyclonal antisera, is efficient and rapid. Mathematical analysis of the relations in equilibrium demonstrates that such a simple approach is an efficient way for improvement of immunoassay specificity using some polyclonal antisera.

Determination of 17α-hydroxypregnenolone sulfate and its application in diagnostics

New combined radioimmunoassay for determination of 17-hydroxypregnenolone sulfate (17-PregS) involving the hydrolysis of analyte by methanolysis was developed. 17-PregS, in addition to being secreted by the adrenals, is also formed by peripheral sulfoconjugation of 17-hydroxypregnenolone (17-Preg) or directly by hydroxylation of pregnenolone sulfate with 17alpha-hydroxylase/C17-20lyase. The measurement of 17-PregS can be used as a tool for detection of enzymatic deficiency particularly in pregnancy and for detection of congenital adrenal hyperplasia or gonadal dysfunction. The serum levels of 17-PregS, 17-Preg, dehydroepiandrosterone, dehydroepiandrosterone sulfate, pregnenolone and pregnenolone sulfate were measured in different age groups of human and in pregnant women respecting the age of gestation. The levels of 17-PregS are approximately three times higher than the levels of free 17-Preg in all subject groups. The levels of 17-PregS during pregnancy reached the local minimum in the 3rd month of gestation. The ratio of 17-PregS to free 17-Preg showed increasing profile during pregnancy with a maximum in the 8th month of gestation. These findings indicate that, the conversion of pregnenolone sulfate to 17-PregS is the major metabolic pathway for biosynthesis of 17-PregS.

Radioimmunoassay of three deoxycorticoids in human plasma following HPLC separation.

A radioimmunoassay of three deoxycorticoids, namely 11 beta,17 alpha-dihydroxy-4-pregnene-3,20-dione (21-deoxycortisol), 17 alpha,21-dihydroxy-4-pregnene-3,20-dione (11-deoxycortisol), and 21-hydroxy-4-pregnene-3,20-dione (11-deoxycorticosterone) which are important for differential diagnosis of congenital adrenal disorders, is described and evaluated. Antisera against 3-(O-carboxymethyl)oximes conjugated to bovine serum albumin were raised in rabbits. The radioligands were prepared by radioiodination of previously synthesized homologous tyrosine methyl ester derivatives. Following diethyl ether extraction, the steroids were separated from each other and from cross-reactants by HPLC using a Nucleosil C8 reverse-phase column and a methanol-water mixture (7:5, v/v) as an eluent. Normal levels of analyzed steroids ranged from 0.02 to 0.348, 0.185 to 3.80, and 0.013 to 0.299 nmol/l, for 21-deoxycortisol, 11-deoxycortisol and 11-deoxycorticosterone, respectively. The levels of both deoxycortisols rose significantly after ACTH treatment. Data are given with respect to the concentrations of these steroids in some pathological situations such as 21-hydroxylase and 11 beta-hydroxylase block, hyperaldosteronism, and polycystic ovary syndrome.

Neuroactive steroids, their precursors and polar conjugates during parturition and postpartum in maternal and umbilical blood: 3.3β-hydroxy-5-ene steroids

Five 3beta-hydroxy-5-ene steroids involved in the metabolic route from pregnenolone sulfate to dehydroepiandrosterone and its sulfate, of which three are known allosteric modulators of neurotransmitter receptors, were monitored in the serum of 20 women around parturition. In addition, their levels in maternal and umbilical serum were compared at delivery. On the basis of these data, a scheme of steroid biosynthesis in maternal organism during the critical stages around parturition is proposed. In maternal serum, all the steroids except dehydroepiandrosterone sulfate decreased during labor and even first day after delivery, although their changes were less distinct the more distant from pregnenolone sulfate (PregS) in the metabolic pathway. Calculation of product/immediate precursor ratios in maternal serum over all stages around parturition enabled identification of the respective changes in the activities of the relevant enzymes. The ratio of 17-hydroxypregnenolone/pregnenolone did not change significantly, while that of dehydroepiandrosterone/17-hydroxypregnenolone grew, indicating increased C17,20 side chain cleavage on the account of C17-hydroxylation both catalyzed by C17-hydroxylase-C17,20-lyase. As was shown by factor analysis, the changes in the maternal steroids were associated with a single common factor, which strongly correlated with all the steroids except dehydroepiandrosterone sulfate. The lack of change in the pregnenolone sulfate/pregnenolone ratio and a marked increase of the ratio dehydroepiandrosterone sulfate to unconjugated dehydroepiandrosterone indicate a different means of formation of both steroid sulfates. On the basis of these data, a scheme of steroid biosynthesis in maternal organism during the critical stages around parturition is proposed.

The presence of monoiodinated derivates of daidzein and genistein in human urine and its effect on thyroid gland function.

Dietary supplements used by women during menopause are usually based on plant extracts containing isoflavonoids, daidzein and genistein. Genistein is a known inhibitor of many enzymes, including thyroid peroxidase (TPO). In the thyroid follicle, genistein acts as its alternate substrate for the formation of genistein iodinated derivatives. The aim of this study was to search for daidzein- and genistein-iodinated derivatives in urine of isoflavonoid-supplemented women. Additionally, selected phytoestrogens, steroid and thyroid hormones before and after three months of phytoestrogen supplementation were estimated. Urinary levels of free phytoestrogen increased significantly after therapy. They ranged between 0.3-1600, 0.6-670 and 0-206 nmol/L for daidzein, genistein and S-equol, respectively. Monoiodinated derivatives of genistein were observed (0-504 pmol/L) in 60% of the investigated samples. Steroid and thyroid hormone levels were within the normal range and were not significantly altered. The presence of monoiodinated derivates in human urine confirmed that genistein and daidzein may enter human thyroid follicles and influence TPO. Since the levels of the free thyroid hormones were not affected, we propose that the use of phytoestrogen dietary supplements is not associated with the development of thyroid-gland disorders in subjects with adequate iodine intake.