A method for determination of one hundred endogenous steroids in human serum by gas chromatography-tandem mass spectrometry.

Steroid profiling helps various pathologies to be rapidly diagnosed. Results from analyses investigating steroidogenic pathways may be used as a tool for uncovering pathology causations and proposals of new therapeutic approaches. The purpose of this study was to address still underutilized application of the advanced GC-MS/MS platform for the multicomponent quantification of endogenous steroids. We developed and validated a GC-MS/MS method for the quantification of 58 unconjugated steroids and 42 polar conjugates of steroids (after hydrolysis) in human blood. The present method was validated not only for blood of men and non-pregnant women but also for blood of pregnant women and for mixed umbilical cord blood. The spectrum of analytes includes common hormones operating via nuclear receptors as well as other bioactive substances like immunomodulatory and neuroactive steroids. Our present results are comparable with those from our previously published GC-MS method as well as the results of others. The present method was extended for corticoids and 17alpha-hydroxylated 5alpha/ß-reduced pregnanes, which are useful for the investigation of alternative "backdoor" pathway. When comparing the analytical characteristics of the present and previous method, the first exhibit by far higher selectivity, and generally higher sensitivity and better precision particularly for 17alpha-hydroxysteroids.

May circulating steroids reveal a predisposition to intrahepatic cholestasis of pregnancy in non-pregnant women?

Intrahepatic cholestasis of pregnancy (ICP) is a frequent liver disorder, mostly occurring in the third trimester. ICP is not harmful to the mothers but threatens the fetus. The authors evaluated steroid alterations in maternal and mixed umbilical blood to elucidate their role in the ICP development. Ten women with ICP were included in the study. Steroids in the maternal blood were measured by Gas Chromatography-Mass Spectrometry (GC-MS) (n=58) and RIA (n=5) at the diagnosis of ICP, labor, day 5 postpartum, week 3 postpartum and week 6 postpartum. The results were evaluated by ANOVA consisting of the subject factor, between subject factors ICP, gestational age at the diagnosis of ICP and gestational age at labor, within-subject factor Stage and ICP × Stage interaction. The 17 controls were firstly examined in the week 36 of gestation. ICP patients showed reduced CYP17A1 activity in the C17,20 lyase step thus shifting the balance between the toxic conjugated pregnanediols and harmless sulfated 5alpha/beta-reduced-17-oxo C19 steroids. Hence, more toxic metabolites originating in maternal liver from the placental pregnanes may penetrate backward to the fetal circulation. As these alterations persist in puerperium, the circulating steroids could be potentially used for predicting the predisposition to ICP even before next pregnancy.

Patients with IgA nephropathy have altered levels of immunomodulatory C19 steroids. Glucocorticoid therapy with addition of adrenal androgens may be the choice.

Glucocorticoid (GC) therapy is one of the methods of choices for treatment of autoimmune diseases (ADs). In addition, adrenal androgens are known as immunoprotective GC-antagonists. Adrenal steroids preferentially influence the Th1-components over the Th2 ones. We investigated steroid metabolome (using gas chromatography-mass spectrometry) in healthy controls (H), GC-untreated patients with ADs different from IgA nephropathy (U), GC-treated patients with ADs different from IgA nephropathy (T) and in patients with IgA nephropathy (IgAN), which were monitored on the beginning (N0), after one week (N1) and after one month (N2) of prednisolone therapy (60 mg of prednisolone/day/m(2) of body surface). Between-group differences were assessed by one-way ANOVA, while the changes during the therapy were evaluated by repeated measures ANOVA. The ANOVA testing was followed by Duncan's multiple comparisons. IgAN patients and patients with other ADs exhibited lack of adrenal androgens due to attenuated activity of adrenal zona reticularis (ZR). Androgen levels including their 7alpha-, 7beta-, and 16alpha-hydroxy-metabolites were further restrained by GC-therapy. Based on these results and data from the literature, we addressed the question, whether a combination of GCs with delta(5)-steroids or their more stable synthetic derivatives may be optimal for the treatment of antibodies-mediated ADs.

Increased serum levels of C21 steroids in female patients with multiple sclerosis.

Multiple sclerosis (MS) is one of the most common neurological diseases. This neurodegenerative autoimmune disease manifests as inflammatory and demyelinating impairment of the central nervous system (CNS). Although some studies demonstrated associations between altered steroidogenesis and pathophysiology of MS as well as the importance of steroids in the pathophysiology of MS, the knowledge concerning the steroid metabolome in female patients is limited. Hence, 51 steroids and steroid polar conjugates were measured in the serum of 12 women with MS, untreated with steroids and 6 age-corresponding female controls with the use of gas chromatography - mass spectrometry (GC-MS). The data were processed using age adjusted ANCOVA, receiver operating characteristics (ROC) analysis and orthogonal projections to latent structures (OPLS). Our data show higher levels of circulating C21 steroids including steroid modulators of ionotropic type A gamma-aminobutyric acid (GABA A) receptors and glutamate receptors. Furthermore, the levels of GABAergic androsterone and 5-androsten-3beta,7alpha,17beta-triol were also higher in the female MS patients. In conclusion, the data demonstrate higher levels of circulating C21 steroids and their polar conjugates and some bioactive C19 steroids in women with MS, which may influence neuronal activity and affect the balance between neuroprotection and excitotoxicity.

Reduced sulfotransferase SULT2A1 activity in patients with Alzheimer's disease.

Steroids are important components in the pathophysiology of Alzheimer's disease (AD). Although their role has been studied, the corresponding metabolomic data is limited. In the present study we evaluate the role of steroid sulfotransferase SULT2A1 in the pathophysiology of AD on the basis of circulating steroids (measured by GC-MS), in which the sulfation catalyzed by SULT2A1 dominates over glucuronidation (pregnenolone/sulfate, DHEA/sulfate, androstenediol/sulfate and 5alpha-reduced pregnane and androstane catabolites). To estimate a general trend of SUL2A1 activity in AD patients we compared the ratios of steroid conjugates to their unconjugated counterparts (C/U) in controls (11 men and 22 women) and AD patients (18 men and 16 women) for individual circulating steroids after adjustment for age and BMI using ANCOVA model including the factors AD status and gender. Decreased C/U ratio for the C19 steroids demonstrate an association between attenuated sulfation of C19 steroids in adrenal zona reticularis and the pathophysiology of AD.

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.

Steroid metabolome in the umbilical cord: is it necessary to differentiate between arterial and venous blood?

Steroids are important markers in pregnancy. Although estimating their levels separately in umbilical arterial (UA) and venous blood (UV) enable more precise insights into the functioning fetoplacental unit compared to using mixed umbilical blood (UM), selective aspiration of UA and UV is technically more demanding than collecting UM. We measured the levels of 67 unconjugated steroids and steroid polar conjugates in UA and UV using GC-MS in 80 women giving birth within weeks 28 to 42 of gestation. The samples were sorted into three groups: women entering labor within weeks 28-32 (group A, n=19), weeks 33-37 (group B, n=19), and weeks 38-42 (group C, n=42) of gestation, respectively. The preterm labors were due to pathologies unrelated to steroid status. Most unconjugated steroids exhibited pronounced arteriovenous differences (AVD). The AVD were less distinct in more stable steroid conjugates. Most steroids positively correlate with gestational age, but unconjugated 5beta-reduced pregnanes show negative correlations, as do testosterone and androstenediol, substrates for the placental synthesis of estrogens. Tight correlations between steroids in UA and UV indicate that steroid measurements in UA, UV and UM can be accurately derived from each other, which is important for the diagnostics of steroid related diseases in newborns.

Chronic cigarette smoking alters circulating sex hormones and neuroactive steroids in premenopausal women.

Chronic smoking alters the circulating levels of sex hormones and possibly also the neuroactive steroids. However, the data available is limited. Therefore, a broad spectrum of free and conjugated steroids and related substances was quantified by GC-MS and RIA in premenopausal smokers and in age-matched (38.9+/-7.3 years of age) non-smokers in the follicular (FP) and luteal phases (LP) of menstrual cycle (10 non-smokers and 10 smokers, in the FP, and 10 non-smokers and 8 smokers in the LP). Smokers in both phases of the menstrual cycle showed higher levels of conjugated 17-hydroxypregnenolone, 5alpha-dihydroprogesterone, conjugated isopregnanolone, conjugated 5alpha-pregnane-3beta,20alpha-diol, conjugated androstenediol, androstenedione, testosterone, free testosterone, conjugated 5alpha-androstane-3alpha/beta,17beta-diols, and higher free testosterone index. In the FP, the smokers exhibited higher levels of conjugated pregnenolone, progesterone, conjugated pregnanolone, lutropin, and a higher lutropin/follitropin ratio, but lower levels of cortisol, allopregnanolone, and pregnanolone. In the LP, the smokers exhibited higher levels of free and conjugated 20alpha-dihydropregnenolone, free and conjugated dehydroepiandrosterone, free androstenediol, 5alpha-dihydrotestosterone, free and conjugated androsterone, free and conjugated epiandrosterone, free and conjugated etiocholanolone, 7alpha/beta-hydroxy-dehydroepiandrosterone isomers, and follitropin but lower levels of estradiol and sex hormone binding globulin (SHBG) and lower values of the lutropin/follitropin ratio. In conclusion, chronic cigarette smoking augments serum androgens and their 5alpha/beta-reduced metabolites (including GABAergic substances) but suppresses the levels of estradiol in the LP and SHBG and may induce hyperandrogenism in female smokers. The female smokers had pronouncedly increased serum progestogens but paradoxically suppressed levels of their GABA-ergic metabolites. Further investigation is needed concerning these effects.

[Progesterone neuroactive metabolites in human pregnancy]. / Progesteron a jeho neuroaktivní metabolity v mechanismu porodu.

OBJECTIVE: Review of the physiological role of neuroactive and neuroprotective steroids in human pregnancy. DESIGN: A review article. SETTING: Gynecological-Obstetrical Clinic, 1st Medical Faculty, Charles University and General Hospital, Prague. CONCLUSION: Human parturition is a multi-factorial process. Various mechanisms related to the onset of labor were suggested. Estrogens show accelerating increase in late pregnancy, which probably reflect the increasing activity of fetal zone of the fetal adrenal. This zone is stimulated by progressive increase of placental CRH resulting in excessive production of conjugated 3beta-hydroxy-5-en-steroids, which are transported by circulation to placenta and further metabolized to active hormones. Some progesterone metabolites probably participate in pregnancy sustaining via modulation of ligand-gated ion channels in the CNS and periphery. In this review, the question was addressed whether the catabolism of pregnancy sustaining progesterone metabolites accelerate like the estrogen formation.

Is maternal progesterone actually independent of the fetal steroids?

Progesterone and estradiol are the foremost steroid hormones in human pregnancy. However, the origin of maternal progesterone has still not been satisfactorily explained, despite the generally accepted opinion that maternal LDL-cholesterol is a single substrate for placental synthesis of maternal progesterone. The question remains why the levels of progesterone are substantially higher in fetal as opposed to maternal blood. Hence, the role of the fetal zone of fetal adrenal (FZFA) in the synthesis of progesterone precursors was addressed. The FZFA may be directly regulated by placental CRH inducing an excessive production of sulfated 3beta-hydroxy-5-ene steroids such as sulfates of dehydroepiandrosterone (DHEAS) and pregnenolone (PregS). Due to their excellent solubility in plasma these conjugates are easily transported in excessive amounts to the placenta for further conversion to the sex hormones. While the significance of C19 3beta-hydroxy-5-ene steroid sulfates originating in FZFA for placental estrogen formation is mostly recognized, the question "Which maternal and/or fetal functions may be served by excessive production of PregS in the FZFA?" - still remains open. Our hypothesis is that, besides the necessity to synthesize de novo all the maternal progesterone from cholesterol, it may be more convenient to utilize the fetal PregS. The activities of sulfatase and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) are substantially higher than the activity of cytochrome P450scc, which is rate-limiting for the placental progesterone synthesis from LDL-cholesterol. However, as in the case of progesterone synthesis from maternal LDL-cholesterol, the relative independence of progesterone levels on FZFA activity may be a consequence of substrate saturation of enzymes converting PregS to progesterone. Some of the literature along with our current data (showing no correlation between fetal and maternal progesterone but significant partial correlations between fetal and maternal 20alpha-dihydroprogesterone (Prog20alpha) and between Prog20alpha and progesterone within the maternal blood) indicate that the localization of individual types of 17beta-hydroxysteroid dehydrogenase is responsible for a higher proportion of estrone and progesterone in the fetus, but also a higher proportion of estradiol and Prog20alpha in maternal blood. Type 2 17beta-hydroxysteroid dehydrogenase (17HSD2), which oxidizes estradiol to estrone and Prog20alpha to progesterone, is highly expressed in placental endothelial cells lining the fetal compartment. Alternatively, syncytium, which is directly in contact with maternal blood, produces high amounts of estradiol and Prog20alpha due to the effects of type 1, 5 and 7 17?-hydroxysteroid dehydrogenases (17HSD1, 17HSD5, and 17HSD7, respectively). The proposed mechanisms may serve the following functions: 1) providing substances which may influence the placental production of progesterone and synthesis of neuroprotective steroids in the fetus; and 2) creating hormonal milieu enabling control of the onset of labor.

Effects of the hormones of the thyroid axis on the vitamin K-dependent plasma factors of blood coagulation (II, VII, IX, and X).

The hormonal regulation of haemostasis is a problem which has not received much attention. The data concerning the influence of hormones from the hypothalamic-pituitary-thyroid axis are scarce, contradictory and based mainly on clinical observations. The objective of the current research is to study the influence of the Thyrotropin releasing hormone (TRH), Thyroid stimulating hormone (TSH), Triiodothyronine (T3) and Thyroxin (T4) on the activity level of the vitamin K-dependent plasma factors of blood coagulation--factor II (F II), factor VII (F VII), factor IX (F IX) and factor X (F X). This study was carried out on 40 male Wistar rats. The necessary quantity of blood was obtained by cardiac puncture under ether narcosis. The indicators studied were activated partial thromboplastin time (aPTT), protothrombin time (PT), F II, F VII, F IX and F X, and were determined by means of Diagnostica Stago tests and with the help of an automatic coagulometer. The hormones studied were: TRH (0.06 mg/kg b.w.), TSH (1 MU/kg b.w.), T3 (0.08 mg/kg b.w.) T4 (0.08 mg/kg b.w.) prolonged aPTT (p<0.001) and PT (p<0.001). TRH and T3 significantly reduced the activity level of factors II, VII, IX and X; T4 only reduced the level of F II (p<0.05), and TSH did not induce significant changes in the haemocoagulation factors studied. The TRH, TSH, T3 and T4 hormones, although elements of one and the same axis, have an ambiguous effect on the vitamin K-dependent factors of blood coagulation. The results obtained show that the determined changes in the activity levels of the vitamin K-dependent plasma factors of blood coagulation are undoubtedly related to the hypocoagulation observed in the intrinsic and extrinsic pathways under the influence of the hormones of the thyroid axis.

Acute heart failure after treatment with 5-fluorouracil.

Anthracycline cardiotoxicity is cumulative and can cause congestive heart failure. The cardiotoxicity caused by 5-fluorouracil (5-FU) is acute and is usually observed during the first cycle of chemotherapy. We present the case of a female patient operated on for colorectal cancer and receiving her first postoperative chemotherapy cycle. Three hours after the initiation of continuous 5-FU infusion she developed signs of acute heart failure (AHF) and pulmonary edema. The patient did not have any previous history of heart disease. Symptoms resolved 24 hours from the onset of the episode after the initiation of the relevant emergency therapy. One of the most common symptoms related to 5-FU cardiotoxicity is chest pain. In case of such a toxicity treatment should be switched to another antineoplastic agent.

Screening study of the effects of the hypothalamic-pituitary-thyroid axis on hemostasis in rats.

The hormonal regulation of hemostasis has had little attention in research, and the existing literature data are relatively contradictory. The possible effects of the hormones of the hypothalamic-pituitary-thyroid axis on hemocoagulation and the fibrinolytic system are studied here. The study was conducted on 80 white male rats of the Wistar breed. The necessary blood quantity was obtained by cardiac puncture realized under ether narcosis. The basic parameters of the hemocoagulation and fibrinolytic activity of the plasma were determined by Diagnostica Stago tests (France), using an automatic coagulometer (Italy). The hormones employed in the study: Thyreotropin releasing hormone (0.06 mg/kg bw), Thyroid stimulating hormone (1 MU/kg bw), Triiodothyronine (0.08 mg/kg bw), and Thyroxin (0.08 mg/kg bw) applied s.c. for three consecutive days, extended the activated partial thromboplastine time (p less than 0.001), proto-thromboplastine time (p less than 0.001), thrombin time (p less than 0.001), reptilase time (p less than 0.001), and shortened the euglobin clot lysis time of (p less than 0.001). These data indicate that each of the hormones used causes significant changes in hemostasis by suppressing the coagulability by the intrinsic and extrinsic system pathways, and transformation of fibrinogen into fibrin. The shortened euglobim clot lysis time may be recognized as a manifestation of increased levels of plasma plasminogen activators. The results obtained show that hypothalamic-pituitary-thyroid hormones are significant regulators of hemostasis, since they cause an expressed hypocoagulation and increase plasma fibrinolytic activity.

Chemical composition and biological activity of propolis from Brazilian meliponinae.

Twenty-one propolis samples produced by 12 different Meliponinae species were analyzed by GC-MS. Several chemical types of stingless bees' propolis could be grouped, according to the prevailing type of compounds like: 'gallic acid", "diterpenic" and "triterpenic" types. The results confirm that neither the bee species nor the geographical location determine the chemical composition of Meliponinae propolis and the choice of its plant source, respectively. This could be explained by the fact that Meliponinae forage over short distances (maximum 500 m) and thus use as propolis source the first plant exudate they encounter during their flights. The antibacterial, antifungal and cytotoxic activities of the samples were also investigated. Most samples had weak or no activity against E. coli, weak action against Candida albicans. Some of them showed significant activity against St. aureus., presumably connected to the high concentration of diterpenic acids. Samples rich in diterpenic acids possessed also high cytotoxic activity (Artemia salina test).

Propolis from the Mediterranean region: chemical composition and antimicrobial activity.

The chemical composition of propolis from Bulgaria, Turkey, Greece and Algeria was investigated by GC-MS. All of them contained mainly flavonoids and esters of caffeic and ferulic acids, which indicated that their main source are buds of poplars of the taxonomic section Aegieros. Some Turkish samples contained a low percent of diterpenic acids, while in Algerian samples significant amounts of a hydroxyditerpenic acid (M=322, its structure not determined by its MS) were found. All samples showed significant antibacterial and weak to moderate antifungal activity.

Antibacterial ent-kaurene from Brazilian propolis of native stingless bees.

Three ent-kaurene diterpenoids, not previously described as constituents of propolis, were isolated from a sample collected by Brazilian native bees Melipona quadrifasciata anthidioides. One of them, kaurenoic acid, as well as the total extract, displayed moderate antibacterial activity.

Phytochemical study and cytotoxic activity of alkaloids from Uvaria chamae P. Beauv.

Phytochemical study of leaves of Uvaria chamae resulted in the isolation for the first time for the genus Uvaria of benzylisoquinoline alkaloids (+)-armepavine (1) and racem. O,O-dimethylcoclaurine (2). The aporphines nornantenine (3), nantenine (4) and corydine (7) are new for the species. The alkaloids were found to express cytotoxic activity against L 929 transformed cells. The highest activity was shown by 1, 3, and 5. At a concentration corresponding to their IC50 against L929 cells, they were nontoxic against mouse thymocytes.

Influence of reserpine on iron absorption in rats.

The aim of our investigations was to define the influence of reserpine on iron absorption in rats. Iron absorption was determined in vivo at the 24th hour and in situ in a tied-off intestinal segment. Five-days treatment with reserpine (1 mg/kg b.wt) led to suppressed bone marrow erythropoiesis, manifested a considerable decrease in reticulocyte count and in a percentage of 59Fe-incorporation into newly formed erythrocytes. Increased plasma iron and decreased plasma ferritin levels were found. Iron absorption was found to be significantly increased, in spite of the suppressed erythropoiesis. The major conclusions of the experiments are, that reserpine interferes with the regulation of iron metabolism and erythropoiesis.

Granisetron in repeated cycles of chemotherapy with platinum.

The aim of the present study was to assess the antiemetic efficacy of granisetron in repeated cycles of chemotherapy with platinum derivatives. The study included 50 patients (28 females, 22 males; aged 17-72, mean age 51 years). From 2 to 5 cycles of chemotherapy with cisplatin or carboplatin were performed. Granisetron was administered intravenously at a dose of 3 mg, 5 minutes before commencement of cytostatic chemotherapy. In case of 2 episodes of vomiting and severe nausea 2 additional doses of granisetron were given. Total control of emesis was achieved in 60% of patients after the first cycle of chemotherapy, and this percentage did not change significantly over the 5 cycles of chemotherapy. There were no differences in the antiemetic efficacy of granisetron in relation to patient sex up to cycle III, while in cycles IV and V a tendency towards less efficacy in females was observed. The adverse effects (headache, dizziness) were observed with the same frequency in the first 3 cycles of chemotherapy, while these were absent in cycles IV and V. Severe side effects were recorded only in cycle I, after that they were less expressed. In conclusion, granisetron is highly effective in prevention of emesis, induced by platinum derivatives and its efficacy is maintained over repeated cycles of chemotherapy. The toxicity of granisetron is mostly expressed in the first cycle, while after that it decreases significantly.

Influence of aluminium on erythropoiesis, iron metabolism and some functional characteristics of erythrocytes in rats.

The increased aluminium (Al) levels in serum of patients with chronic renal failure on hemodialysis are associated with impaired erythropoiesis and iron metabolism. The long term Al loading of rats (20 to 90 days) has similar effect. Data are still lacking about the effects after short-term aluminium treatment. The 7 day's treatment with Al2(SO4)3 in a dose 67.5 mg/kg b. w., i. m. m. significantly decreased hemoglobin, hematocrit, incorporation of 59Fe in newly formed erythrocytes and increased reticulocytes in absolute and relative counts. We observed a mild degree hypochromic, ferropenic, microcytic anemia and polychromazia in the available macrocytes. The immature erythroblasts were predominant forms in the erythroblastogram while the number of mature erythroblasts was decreased. Index of maturation of erythroblasts was lower, indicating inhibited erythroblast maturation. Plasma iron, TIBC, transferrin saturation and 59Fe absorption in the experimental group were significantly decreased. Spontaneous and mechanical hemolysis of erythrocytes were lower while erythrocyte deformability was increased. Obviously, Al treatment inhibits erythropoiesis and iron metabolism, probably hinders hemoglobin synthesis and erythroid cell maturation but does not affect the studied functional characteristics of mature erythrocytes negatively.