Steroid diagnostics of 21st century in the light of their new roles and analytical tools.

The determination of steroid hormones and subsequent interpretation of results is accompanied by a range of difficulties. The amount of information that current technology can provide on the circulating concentrations of more than a hundred various steroid compounds can lead to problems with interpretation. The aim of this study is to help provide orientation in this maze of data on steroid hormones. First we focus on specific aspects arising from the pre-analytical phase of steroid determination that need to be considered when planning sampling, whether for diagnostics or research. Then, we provide a brief summary of the characteristics and diagnostic relevance of several steroid hormones and/or their metabolites: pregnenolone, 17alpha-hydroxy-pregnenolone, dehydroepiandrosterone, hydroxyderivatives of dehydroepiandrosterone, androstenedione, testosterone, estrone, estradiol, estriol, cortisol, cortisone, which in our institute are determined with validated LC-MS/MS methods. For these steroids, we also provide newly calculated reference values in fertile women according to the phase of their menstrual cycle.

Parabens and their relation to obesity.

Parabens are a group of chemicals used as preservatives in the food, cosmetic and pharmaceutical industries. They are known to possess estrogenic effects, and therefore have been classified as endocrine disruptors. In addition to the classical endocrine organs, other tissues have endocrine activity, including adipose tissue. Several chemicals are known to cause obesogenic effects, and parabens are currently being studied in this context. The aim of this study was to investigate the possible connections of paraben exposure and obesity. Blood plasma from 27 healthy women was collected during their menstrual cycle. Basal anthropometric measures, levels of parabens (methylparaben, ethylparaben and propylparaben), adipokines (adiponectin, adipsin, leptin, resistin and visfatin) and hormones affecting energy balance and metabolic health (c-peptide, ghreline, GIP, GLP-1, glucagon, insulin, PAI-1) were measured. A Kolmogorov-Smirnov test showed higher methylparaben and propylparaben levels in women with BMI 25-34.9 compared to those with BMI 18.5-24.9. Plasma levels of methylparaben as well as the sum of parabens were positively associated with the plasma adipsin levels. Negative associations for methylparaben were found for glucagon, leptin and PAI-1. In accordance with other experimental studies we observed important associations of methylparaben and hormones affecting energy balance and metabolic health, indicating its obesogenic potential.

Can the gold standard be beaten? How reliable are various modifications of the Synacthen test compared to the insulin tolerance test.

Criteria for the evaluation of the insulin tolerance test (ITT) and Synacthen test are still a matter of debate. The objective of the study was to make a comparison of serum and salivary cortisol during four stimulation tests. Sixty four healthy volunteers underwent the ITT, the Synacthen test with 1 (LDST), 10 (MDST) and 250 (HDST) microg dose of ACTH. Maximum serum cortisol response was observed at the 90 min of the ITT (49 %), HDST (89 %) and MDST (56 %) and at the 40 min of the LDST (44 %). Results expressed as 95 % confidence intervals: 408.0-843.6 and 289.5-868.1 nmol/l in the IIT at 60 and 90 min. In the HDST and the MDST serum cortisol reached the maximum at 90 min 542.6-1245.5 and 444.2-871.3 nmol/l. Levels of salivary cortisol followed the same pattern as serum cortisol. Salivary cortisol reached the maximum response in the HDST and the MDST at 90 min and at 40 min in the LDST. We confirmed good reliability of all tests with respect to timing of response and maximum response compared to the ITT. We proved that the MDST test can provide the similar response in serum cortisol to the HDST. Measuring either salivary cortisol or ACTH levels did not provide any additional benefit then measuring serum cortisol by itself.

Antioxidant potential of essential oil from Lavandula angustifolia in in vitro and ex vivo cultured liver cells.

Lavender is a commonly used herb in traditional medicine in Asia and Europe. It has been reported to be an effective medical plant in treating inflammation, depression and stress, thanks to its sedative and anxiolytic action, thrombotic, and antimicrobial properties. In the present study we investigated the protective effects of essential oil from Lavandula angustifolia (LO) against hydrogen peroxide and tert-butyl hydroperoxide -induced DNA damage. Also the effects of LO on the levels of enzymatic and non-enzymatic antioxidants (SOD-superoxide dismutase, GPx-glutathione peroxidase, GSH-glutathione) were evaluated in in vitro (human hepatoma cell line HepG2) and in ex vivo (freshly isolated rat hepatocytes) systems. The results showed that the oxidant-induced DNA lesions were significantly reduced in both systems pre-treated with the Lavandula angustifolia. The observed DNA-protective activity could be explained by both elevation of GPx activity in cells pre-treated with LO and antioxidant activity of LO.

Levels of adipokines and some steroids during the menstrual cycle.

The cyclical effects of hormones during the menstrual cycle (MC) are not just responsible for driving ovulation, but also have significant influence on dietary intake and appetite, as well as psychological and behavioral changes. The aim of our study was to describe changes and relationships between the MC and selected steroids, adipokines and food intake-related hormones. Twenty-seven women with regular menstrual cycles were included in the study, and their hormonal spectrum was measured in regular intervals starting from the first day of their cycle. Classical changes in gonadotropins, estrogens and progesterone during the menstrual cycle are accompanied by less striking but significant changes in 17-hydroxyprogesterone and testosterone. No significant changes show dehydroepiandrosterone and its 7-oxygenated metabolites. Adipokines show a tendency to increase during ovulation, while ghrelin and resistin decrease. There is also a remarkable association of sex hormone-binding globulin on the day of the cycle. Our results demonstrate that changes to adipokines during the menstrual cycle are not substantial, but nonetheless can play a role in the changes of food intake described in the literature. Precise descriptions of physiological changes in healthy women are important in helping us understand the significance of the changes accompanying various pathological states.

Daily profiles of steroid hormones and their metabolites related to food intake.

The aim of this study was to look for changes in the daily profile of steroid hormones after standardized food intake. Eight young women not taking contraceptives were followed from 5:30 a.m. till 9:30 p.m. before and 1 and 2 h after eating breakfast, snack, lunch, the second snack and dinner. The differences in steroid levels before and after meals were evaluated. As expected, glucose, C-peptide and ghrelin levels changed postprandially. The steroid hormones cortisol, progesterone, pregnenolone and dehydroepiandrosterone showed a decrease after main meals, whereas testosterone and dihydrotestosterone showed no significant dependence on food intake. Estrogen levels did not exhibit a significant nycthemeral rhythm, but estradiol decreased after main meals. In our study the known nycthemeral rhythm of LH, FSH, cortisol, progesterone and pregnenolone after food intake were confirmed, but significant changes after meals were also observed in the levels of cortisol, dehydroepiandrosterone, estradiol and SHBG.

The role of steroid hormones in the development of intrahepatic cholestasis of pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) is a disorder of liver function, commonly occurring in the third trimester but sometimes also as soon as the end of the second trimester of pregnancy. Symptoms of this disorder include pruritus, plus abnormal values of bile acids and hepatic transaminases. After birth, symptoms disappear and liver function returns to normal. Though ICP is relatively non-complicated and often symptomatically mild from the point-of-view of the mother, it presents a serious risk to the fetus, making this disease the subject of great interest. The etiology and pathogenesis of ICP is multifactorial and as yet not fully elucidated. Hormonal factors likely play a significant role, along with genetic as well as exogenous factors. Here we summarize the knowledge of changes in steroid hormones and their role in the development of intrahepatic cholestasis of pregnancy. In addition, we consider the role of exogenous factors as possible triggers of steroid hormone changes, the relationship between metabolic steroids and bile acids, as well as the combination of these factors in the development of ICP in predisposed pregnant women.

Response of cortisol metabolites in the insulin tolerance test and Synacthen tests.

Determination of response of cortisol and its metabolites to different stimuli may be important for adrenal gland disorders. To date, only one metabolite, cortisone, has been followed in stimulation tests of the adrenal gland. We aimed to describe a response of cortisol metabolites to the standard short Synacthen test (HDST), insulin tolerance test (ITT), low dose Synacthen test (LDST) and medium dose Synacthen test (MDST). Sixty healthy subjects were investigated: 30 men and 30 women. Plasma for measurements of cortisol and its metabolites was obtained before and 30th and 60th min after Synacthen and insulin administration. The cut-off 500 nmol/l of cortisol was reached after stimulation in all of tests, the maximal stimulation level was reached in 60th min in all of the tests except for LDST. The response of cortisol and its metabolites at 30th and 60th min strongly correlated in all of the tests except for LDST. Cortisol and its metabolites increased after stimulation; in contrast, cortisone and its metabolites decreased. We showed that the response of the cortisol metabolites during the Synacthen tests and ITT well correlated, and the MDST showed similar response compared to HDST. The decrease in cortisone metabolites may correspond to the regeneration of cortisol from cortisone in response to stimulation test.

The steroid metabolome in men with mood and anxiety disorders.

The mood and behavior of individuals result from an orchestra of many factors. Among them steroids play an important role; however, only several common hormones have been investigated in this respect. It has been demonstrated that some steroid metabolites long considered merely the products of steroid hormone metabolism in fact possess considerable activity in the CNS. For this reason we studied the steroid metabolome including 50 analytes in 20 men with depression, 20 men with anxiety and 30 healthy controls. Significant differences were found not only between controls and men with either depression or anxiety, but also between men with depression and anxiety. Particularly striking were those steroids until now not generally associated with depression or anxiety, namely conjugated steroid forms, especially sulfates.

Steroid hormones in the development of postpartum depression.

Postpartum depression affects 10-15 % women after childbirth. There is no currently generally accepted theory about the causes and mechanisms of postpartum mental disorders. The principal hypothesis concerns the association with sudden changes in the production of hormones affecting the nervous system of the mother and, on the other hand, with the ability of receptor systems to adapt to these changes. We observed changes in steroidogenesis in the period around spontaneous delivery. We collected three samples of maternal blood. The first sampling was 4 weeks prior to term; the second sampling was after the onset of uterine contractions (the beginning of spontaneous labour); the third sampling was during the third stage of labour (immediately after childbirth). Additionally, we collected mixed umbilical cord blood. The almost complete steroid metabolome was analyzed by gas chromatography-mass spectrometry followed by RIA for some steroids. Mental changes in women in the peripartum period were observed using the Hamilton Depression Rating Scale. The local Ethics Committee approved the study. We found already the changes in androgens levels correlating with postpartum mood disorders four weeks prior to childbirth. The strongest correlations between steroid and postpartum mood change were found in venous blood samples collected from mothers after childbirth and from umbilical cord blood. The main role played testosterone, possibly of maternal origin, and estrogens originating from the fetal compartment. These results suggest that changes in both maternal and fetal steroidogenesis are involved in the development of mental changes in the postpartum period. Descriptions of changes in steroidogenesis in relation to postpartum depression could help clarify the causes of this disease, and changes in some steroid hormones are a promising marker of mental changes in the postpartum period.

The role of taste in cephalic phase of insulin secretion.

The effect of a short gustatory signal of a sweet solution was tested on 15 young male volunteers. The experiment consisted of mouth rinsing with either a sucrose or aspartate solution or pure water as a placebo. Blood was then taken in short intervals of 0, 5, 10, 15 and 20 min. Blood glucose, C-peptide, insulin and cortisol were determined. While C-peptide and glucose were unaffected, a short-term increase in insulin was observed after the sucrose, but not after the aspartate or placebo. The increase in insulin was significant, though it amounted to only 0.5 mIU/l and lasted approx. 15 min reaching then the starting value. The decline of cortisol level within 20 min of the experiment was approx. 40 nmol/l, although it was also observed after aspartate or placebo mouth rinsing and was probably caused by stress factors or anticipation. In conclusion, the contribution of taste to the cephalic phase of insulin secretion is small yet significant, and mouth rinsing with 5% sucrose causes an insulin increase of just under 1 IU/l, which returns to starting level within 15 min.

Effects of borneol on the level of DNA damage induced in primary rat hepatocytes and testicular cells by hydrogen peroxide.

The aim of this paper was to evaluate genotoxic effects of borneol and its ability to change DNA-damaging effects of H2O2 in rat hepatocytes and testicular cells. Both in vitro and ex vivo approaches were used in the case of hepatocytes. Testicular cells were tested only ex vivo, i.e. shortly after isolation from rats supplemented by borneol. Cytotoxicity of borneol increased in in vitro conditions in a concentration-dependent manner and it was associated with DNA-damaging effects at toxic concentrations. While non-toxic concentrations of borneol applied in vitro protected cells against H2O2-induced DNA damage and interfered only partly with rejoining of H2O2-induced DNA strand breaks, cytotoxic concentrations of borneol manifested synergy with H2O2, i.e. enhanced DNA-damaging effects of H2O2. On the other side, borneol given to rats in drinking water decreased the level of DNA damage induced by H2O2 in both hepatocytes and testicular cells. Our results show that though at higher concentrations (2-h treatment with >2 mM borneol >0.3084 mg/ml) borneol acts cytotoxically and genotoxically on primary hepatocytes cultured in vitro, if given to rats during 7 days in a daily concentration of 17.14 or 34.28 mg/kg it reduces genotoxicity of H2O2 in both hepatocytes and testicular cells.

Reduction of genotoxic effects of N-nitrosomorpholine in human hepatoma cells and hamster lung cells by carboxymethyl chitin-glucan.

N-nitrosomorpholine (NMOR) belongs to the group of N-nitrosamines and represents a known hepatocarcinogen. Exposure to this compound is considered to be a potential health hazard to humans. There is, however, considerable evidence that the effect of many carcinogenic agents can be markedly influenced or altered by various natural substances. The objective of this study was to assess the DNA-protective and anticlastogenic effects of the derivative of a natural compound, carboxymethyl chitin-glucan (CM-CG), against genotoxicity of N-nitrosomorpholine (NMOR) in human hepatoma cells HepG2 and hamster lung cells V79 cultured in vitro. The exponentially growing cells were pre-treated during 24 h with three different concentrations of CM-CG (150, 750 and 1500 mg/ml) and then treated with different concentrations of NMOR. DNAprotective effects of CM-CG were evaluated by single-cell gel electrophoresis (SCGE, comet assay) and anticlastogenic effects by chromosomal aberration assay. At the SCGE assay a short-term (30 min) and at the chromosomal aberration assay a continuous treatment with NMOR was used. In both HepG2 and V79 cells pre-treated with CM-CG, a significant decrease of the percentage of DNA lesions induced by NMOR was observed along with a reduction of NMOR-induced chromosomal aberrations. We did not find any substantial differences between the genotoxic effects of NMOR on HepG2 and V79 cells, which have different histopathological origins and different levels of metabolizing enzymes. Three different concentrations of CM-CG exerted a similar protective effect against NMOR-induced DNA lesions and chromosomal aberrations in both HepG2 and V79 cells.

DNA-protective effects of two components of essential plant oils carvacrol and thymol on mammalian cells cultured in vitro.

Many components of essential volatile oils show antioxidant activity and may serve e.g. as a natural replacement of synthetic antioxidant food additives. However, it is important to evaluate such compounds also for their pro-oxidant and toxic properties as their plant origin doesn't secure their safety for living beings, including humans. The aim of this study was therefore to investigate cytotoxic, genotoxic and DNA-protective effects of the long-term (24 h) incubation of mammalian cells with two components of essential plant oils (carvacrol and thymol) in in vitro conditions. Cytotoxicity testing was in all cell lines (human hepatoma cells HepG2, human colonic cells Caco-2 and hamster lung cells V79) performed on the basis of trypan blue exclusion. Plating efficiency was evaluated only in V79 cells which manifest a high colony forming ability. The amount of DNA lesions induced in cells treated with hydrogen peroxide, carvacrol, thymol or combinations of carvacrol or thymol with hydrogen peroxide was measured by standard alkaline single cell gel electrophoresis in human cells HepG2 and Caco-2. Trypan blue exclusion test showed that carvacrol was mildly more cytotoxic than thymol and that Caco-2 cells were mildly more resistant to both carvacrol and thymol than HepG2 and V79 cells. At concentrations = IC20-40, the compounds studied did not induce DNA strand breaks either in human cells HepG2 or in cells Caco-2. Incubation of HepG2 and Caco- 2 cells in the presence of the whole scale of concentrations of carvacrol or thymol led in both cases to a significant protection of the cells studied toward DNA strand breaks induced by a potent oxidant hydrogen peroxide.