Evaluation of neuroactive steroid levels by liquid chromatography-tandem mass spectrometry in central and peripheral nervous system : effect of diabetes

The nervous system is a target for physiological and protective effects of neuroactive steroids. Consequently, the assessment of their levels in nervous structures under physiological and pathological conditions is a top priority. To this aim, identification and quantification of pregnenolone (PREG), progesterone (PROG), dihydroprogesterone (DHP), tetrahydroprogesterone (THP), testosterone (T), dihydrotestosterone (DHT), 5aandrostan- 3a, 17b-diol (3a-diol), 17a- and 17b-estradiol (17a-E and 17b-E) by liquid chromatography and tandem mass spectrometry (LC\u2013MS/ MS) has been set up. After validation, this method was applied to determine the levels of neuroactive steroids in central (i.e., cerebral cortex, cerebellum and spinal cord) and peripheral (i.e., brachial nerve) nervous system of control and diabetic rats. In controls only the brachial nerve had detectable levels of all these neuroactive steroids. In contrast, 17a-E in cerebellum, 17a-E, 17b-E, DHP and THP in cerebral cortex, and 17a-E, 17b-E and DHP in spinal cord were under the detection limit. Diabetes, induced by injection with streptozotocin, strongly affected the levels of some neuroactive steroids. In particular, the levels of PREG, PROG and T in cerebellum, of PROG, T and 3a-diol in cerebral cortex, of PROG, DHTand 3a-diol in spinal cord and of PREG, DHP, THP, T, DHTand 3a-diol in brachial nerve were significantly decreased. In conclusion, the data here reported demonstrate that the LC\u2013MS/MS method allows the assessment of neuroactive steroids in the nervous system with high sensitivity and specificity and that diabetes strongly affects their levels, providing a further basis for new therapeutic tools based on neuroactive steroids aimed at counteracting diabetic neuropathy

Selective Serotonin Reuptake Inhibitor (SSRI) Antidepressants in Pregnancy and Congenital Anomalies: Analysis of Linked Databases in Wales, Norway and Funen, Denmark

Background: Hypothesised associations between in utero exposure to selective serotonin reuptake inhibitors (SSRIs) and congenital anomalies, particularly congenital heart defects (CHD), remain controversial. We investigated the putative teratogenicity of SSRI prescription in the 91 days either side of first day of last menstrual period (LMP). Methods and Findings: Three population-based EUROCAT congenital anomaly registries- Norway (2004–2010), Wales (2000–2010) and Funen, Denmark (2000–2010)—were linked to the electronic healthcare databases holding prospectively collected prescription information for all pregnancies in the timeframes available. We included 519,117 deliveries, including foetuses terminated for congenital anomalies, with data covering pregnancy and the preceding quarter, including 462,641 with data covering pregnancy and one year either side. For SSRI exposures 91 days either side of LMP, separately and together, odds ratios with 95% confidence intervals (ORs, 95%CI) for all major anomalies were estimated. We also explored: pausing or discontinuing SSRIs preconception, confounding, high dose regimens, and, in Wales, diagnosis of depression. Results were combined in meta-analyses. SSRI prescription 91 days either side of LMP was associated with increased prevalence of severe congenital heart defects (CHD) (as defined by EUROCAT guide 1.3, 2005) (34/12,962 [0.26%] vs. 865/506,155 [0.17%] OR 1.50, 1.06–2.11), and the composite adverse outcome of 'anomaly or stillbirth' (473/12962, 3.65% vs. 15829/506,155, 3.13%, OR 1.13, 1.03–1.24). The increased prevalence of all major anomalies combined did not reach statistical significance (3.09% [400/12,962] vs. 2.67% [13,536/506,155] OR 1.09, 0.99–1.21). Adjusting for socio-economic status left ORs largely unchanged. The prevalence of anomalies and severe CHD was reduced when SSRI prescriptions were stopped or paused preconception, and increased when >1 prescription was recorded, but differences were not statistically significant. The dose-response relationship between severe CHD and SSRI dose (meta-regression OR 1.49, 1.12–1.97) was consistent with SSRI-exposure related risk. Analyses in Wales suggested no associations between anomalies and diagnosed depression. Conclusion: The additional absolute risk of teratogenesis associated with SSRIs, if causal, is small. However, the high prevalence of SSRI use augments its public health importance, justifying modifications to preconception care

Chamomile infusions inhibit proteases involved in gastric inflammation

Chamomile, prepared with dried flowers from Matricaria recutita L., is one of the most commonly consumed herbal tea. The drug is used for the treatment of gastrointestinal complaints (minor spasms, epigastric distensions, gastritis and gastric inflammation). Several classes of bio-active compounds have been identified in the extracts of chamomile including phenolic acids, coumarins, and flavonoids such as the glycosides of apigenin, quercetin, patuletin, luteolin and several derivatives. Several studies showed that chamomile infusions possess a protective effect on gastritis and gastric ulcer, but the mechanisms involved in this effect are not completely established. Matrix metalloproteinases (MMPs) and neutrophils elastase (NE) are proteases that degrade extracellular matrix in physiological and pathological conditions. Since MMP-9 and NE are involved in gastric inflammation, the aim of this work was the evaluation of the effects of chamomile infusions of dried capitula (CFI) and sifted (SFI) flowers on MMP-9 and NE, and the identification of the compounds responsible for the observed effect. Each infusion was analyzed by LC-MS/MS in order to verify whether compositional differences affected biological activity. Analysis of CFI and SFI by LC-MS showed a complex profile. The compounds unequivocally identified by LC-MS/MS were the flavonoids apigenin-7-O-glucoside (api7glu), luteolin-7-O-glucoside (lut7glu), patuletin-7-O-glucoside (pat7glu) and hyperoside (hyp). Api7glu was more abundant in CFI than in SFI whereas the opposite was for lut7glu (5.2 \ub5g/ml vs 8.1 \ub5g/ml). Pat7glu was the most abundant in both the infusions, whereas Hyp was the lowest. CFI and SFI inhibited enzymatic activity of MMP-9 catalytic domain in a concentration-dependent manner. At 1500 microg/ml the inhibition was 28 % and 55 % for CFI and SFI, respectively. Api7glu and lut7glu (10 \ub5M) showed an inhibitory activity of 40% and 30%, respectively, demonstrating their contribute to the effect of the infusions. The inhibitory effect of CFI and SFI was confirmed on MMP-9 released by human adenocarcinoma cells (AGS cells). CFI was able to inhibit MMP-9 secretion from AGS cells (85% at 1500 microg/ml). The inhibitory effect of the infusions on NE was also tested. Concentration-response curves were performed and IC50 of CFI and SFI on NE were 369.2 microg/ml and 536.7 microg/ml, respectively. The individual compounds that showed an inhibitory effect on NE activity were api7glu (IC50 74.3 microM), lut7glu (IC50 8.6 microM), pat7glu (IC50 10.4 microM), and chlorogenic acid (IC50 31.3 microM). In conclusion, the present study shows some biochemical mechanism of action for the effect of chamomile infusions and supports the use of chamomile in the treatment of gastrointestinal inflammation

Dihydroprogesterone Increases the Gene Expression of Myelin Basic Protein in Spinal Cord of Diabetic Rats

Alterations in myelin membranes, as well as in the expression of myelin proteins have been reported in experimental models of diabetes. Data here reported show for the first time that the mRNA levels of two isoforms of myelin basic protein (MBP), 18.5 and 21.5 kDa, are decreased in the spinal cord of streptozotocin-treated rats and that treatment with a neuroactive steroid, such as progesterone (P), may counteract this effect. Interestingly, metabolism of progesterone into dihydroprogesterone (DHP) by the enzyme 5 alpha-reductase seems to exert an important role in such an effect. As here demonstrated, 5 alpha-reductase mRNA and DHP levels are reduced by diabetes in spinal cord, but treatment with P, is able to counteract these effects. Moreover, treatment with DHP is able to mimic the effect of P on MBP gene expression. Thus, the effects of P here observed are due to its enzymatic conversion into DHP. Because DHP, like P, interacts with P receptor (PR), the present results may suggest the importance to analyze the effects of PR modulators as tools of therapeutic strategies for diabetic complications occurring in nervous system

Inhibition of human cAMP-phosphodiesterase as a mechanism of the spasmolytic effect of Matricaria recutita L.

Mechanisms underlying the spasmolytic activity of chamomile still remain unclear. Inhibition of cAMP-and cGMP-phosphodiesterases (PDE) is one of the mechanisms operated by spasmolytic drugs. In this study, the effect of chamomile on PDE was investigated. Human platelet cAMP-PDE and recombinant PDE5A1 were assayed in the presence of infusions prepared from sifted flowers and capitula. LC-ESI-MS/MS analysis showed different compositions in infusions made with sifted flowers and capitula. Chamomile inhibited cAMP-PDE activity (IC 50 = 17.9-40.5 μg/mL), while CGMP-PDE5 was less affected (-15% at 50 μg/mL). Among the individual compounds tested, only flavonoids showed an inhibitory effect (IC50 = 1.3-14.9 μM), contributing to around 39% of the infusion inhibition; other compounds responsible for cAMP-PDE inhibition still remain unknown. Although experimental evidence supporting the use of chamomile for gastrointestinal minor spasms dates back to the fifties, cAMP-PDE inhibition as a likely mechanism underlying the spasmolytic activity is reported for the first time

Sex differences in neuroactive steroid levels in the nervous system of diabetic and non-diabetic rats

Neuropathy and encephalopathy represent important complications of diabetes. Recent observations obtained in experimental models have suggested that, in male rats, neuroactive steroids are protective agents and that their levels in peripheral (PNS) and central (CNS) nervous system are strongly affected by the disease. It is interesting to highlight that incidence, progression and severity of diabetic neuropathy and diabetic encephalopathy are different in the two sexes. Consequently, it is important to determine the changes in neuroactive steroid levels in the PNS and the CNS of both males and females. To this aim, we have evaluated the levels of neuroactive steroids such as, pregnenolone, progesterone and its metabolites, testosterone and its metabolites, and dehydroepiandrosterone in different CNS regions (i.e., cerebral cortex, cerebellum and spinal cord) and in the sciatic nerve of control and diabetic (i.e., induced by streptozotocin) male and female rats. Data obtained by liquid chromatography-tandem mass spectrometry indicate that the levels of neuroactive steroids show sex and regional differences in control animals. Streptozotocin-induced diabetes resulted in a strong general decrease in neuroactive steroid levels, in both the PNS and the CNS. In addition, the effects of diabetes on neuroactive steroid levels also show sex and regional differences. These findings may have strong implications for the development of new sex-oriented therapies for the treatment of diabetic neuropathy and diabetic encephalopathy, based on the use of neuroactive steroids

Effect of short-and long-term gonadectomy on neuroactive steroid levels in the central and peripheral nervous system of male and female rats

Significant levels of neuroactive steroids are still detected in the nervous system of rodents after the removal of peripheral steroidogenic glands. However, the influence of the plasma levels of gonadal steroids on the levels of neuroactive steroids in the nervous system has not so far been clarified in detail. Accordingly, by liquid chromatography tandem mass spectrometry, we have analysed the levels of neuroactive steroids in the sciatic nerve, in three central nervous system (CNS) regions (i.e. cerebellum, cerebral cortex and spinal cord) and in the plasma of male and female animals. The levels present in gonadally intact animals were compared with those present in short- and long-term gonadectomised animals. We observed that: (i) changes in neuroactive steroid levels in the nervous system after gonadectomy do not necessarily reflect the changes in plasma levels; (ii) long-term gonadectomy induces changes in the levels of neuroactive steroids in the peripheral nervous system (PNS) and the CNS that, in some cases, are different to those induced by short-term gonadectomy; (iii) the effect of gonadectomy on neuroactive steroid levels is different between the PNS and the CNS and within different CNS regions; and (iv) the effects of gonadectomy on neuroactive steroid levels in the nervous system show sex differences. Altogether, these observations indicate that the nervous system adapts its local levels of neuroactive steroids in response to changes in gonadal hormones with sex and regional specificity and depending on the duration of the peripheral modifications