Low-polarity untargeted metabolomic profiling as a tool to gain insight into seminal fluid.

INTRODUCTION: A decrease in sperm cell count has been observed along the last several decades, especially in the most developed regions of the world. The use of metabolomics to study the composition of the seminal fluid is a promising approach to gain access to the molecular mechanisms underlying this fact. OBJECTIVES: In the present work, we aimed at relating metabolomic profiles of young healthy men to their semen quality parameters obtained from conventional microscopic analysis. METHODS: An untargeted metabolomics approach focusing on low- to mid-polarity compounds was used to analyze a subset of seminal fluid samples from a cohort of over 2700 young healthy men. RESULTS: Our results show that a broad metabolic profiling comprising several families of compounds (including acyl-carnitines, steroids, and other lipids) can contribute to effectively distinguish samples provided by individuals exhibiting low or high absolute sperm counts. CONCLUSION: A number of metabolites involved in sexual development and function, signaling, and energy metabolism were highlighted as being distinctive of samples coming from either group, proving untargeted metabolomics as a promising tool to better understand the pathophysiological processes responsible for male fertility impairment.

Monitoring caffeine intake in children with a questionnaire and urine collection: a cross-sectional study in a convenience sample in Switzerland.

PURPOSE: The objectives of this study were (1) to estimate caffeine intake and identify the main sources of intake using a dietary questionnaire, (2) to assess 24-h urinary excretion of caffeine and its metabolites, and (3) to assess how self-reported intake estimates correlates with urinary excretion among children in Switzerland. METHODS: We conducted a cross-sectional study of children between 6 and 16 years of age in one region of Switzerland. The participants filled in a dietary questionnaire and collected a 24-h urine sample. Caffeine intake was estimated with the questionnaire. Caffeine, paraxanthine, theophylline, and theobromine excretions were measured in the urine sample. Correlations between questionnaire-based intake and urinary excretion estimates were assessed using Spearman correlation coefficients. RESULTS: Ninety-one children were included in the analysis (mean age 10.6 years; 43% female). The mean daily caffeine intake estimate derived from the diet questionnaire was 39 mg (range 0-237), corresponding, when related to body weight, to 1.2 mg/kg (range 0.0-6.3). Seven children (8%) had a caffeine intake above the upper recommended level of 3 mg/kg per day. The main sources of caffeine intake were cocoa milk (29%), chocolate (25%), soft drinks (11%), mocha yogurt (10%), tea (8%), and energy drinks (8%). The 24-h urinary excretion of caffeine was 0.3 mg (range 0.0-1.5), paraxanthine 1.4 mg (range 0.0-7.1), theophylline 0.1 mg (range 0.0-0.6), and theobromine 14.8 mg (range 0.3-59.9). The correlations between estimates of caffeine intake and the 24-h urinary excretion of caffeine was modest (ρ = 0.21, p = 0.046) and with the metabolites of caffeine were weak (ρ = 0.09-0.11, p = 0.288-0.423). CONCLUSIONS: Caffeine intake in a sample of children in a region of Switzerland was relatively low. The major sources of intake were cocoa milk, chocolate and soft drinks. Self-reported caffeine intake correlated weakly with urinary excretion of caffeine and some of its main metabolites. TRIAL REGISTRATION NUMBER: NCT02900261.

Spot urine samples to estimate 24-hour urinary calcium excretion in school-age children.

Urinary calcium/creatinine ratio (UCa/Cr) on a single spot urine sample is frequently used in children to evaluate calciuria, but its accuracy to estimate 24-h urinary calcium excretion (24hUCa) has not been properly assessed. We analyzed the correlation between UCa/Cr in various spot samples and 24hUCa among healthy children. A 24-h urine specimen and three spot urine samples (evening, first, and second morning) were collected in a convenience sample of children aged 6 to 16 years (n = 101). Measured 24hUCa was compared with UCa/Cr in each of the three spot samples. The ability of UCa/Cr to discriminate between children with and without hypercalciuria (calciuria > 4 mg/kg/24 h, 1 mmol/kg/24 h) and optimal timing of the spot sample were determined. Eighty-five children completed an adequate 24-h urine collection. Pearson correlation coefficients between the UCa/Cr on the spot sample and 24hUCa were 0.64, 0.71, and 0.52 for the evening, first, and second morning spot samples, respectively. Areas under the ROC curve were 0.90, 0.82, and 0.75, respectively, for the corresponding spot samples.Conclusion: The relatively strong correlation between 24hUCa and UCa/Cr in evening and first morning spot urine samples suggests that these spots could be preferred in clinical practice.Trial registration: ClinicalTrials.gov , NCT02900261, date of trial registration 14 September 2016. What is Known: •Urinary calcium/creatinine ratio on a single spot urine sample is frequently used as a proxy for 24-h urinary calcium excretion. •Correlation of these indicators, including the best timing for spot urine sampling, has not been properly assessed. What is New: •Relatively strong correlations were found between the calcium/creatinine ratio on a single spot urine sample and 24-h urinary calcium excretion in healthy children. •Evening and first morning spot samples had the highest correlation.

Estimation of salt intake and excretion in children in one region of Switzerland: a cross-sectional study.

PURPOSE: Salt intake among children in Switzerland is unknown. The objectives of this study were to determine salt excretion and to identify the main dietary sources of salt intake among children in one region of Switzerland. METHODS: We conducted a cross-sectional study using a convenient sample of children 6-16 years of age in Valais, Switzerland, between 2016 and 2018. All children visiting several regional health care providers and without any clinical condition that could affect sodium intake or excretion were eligible. Each child completed a 24-h urine collection to assess salt excretion and two dietary questionnaires to assess dietary sources of salt intake. Weight and height were measured. RESULTS: Data were available on 94 children (55 boys and 39 girls; mean age 10.5 years; age range 6-16 years). The mean 24-h salt urinary excretion was 5.9 g [SD 2.8; range 0.8-16.0; 95% confidence interval (CI) 5.3-6.5]. Two-thirds (62%) of the children had salt excretions above recommendations of maximum intake (i.e., ≥ 2 g per day for children up to 6 years of age and ≥ 5 g per day for children 7-16 years of age). The salt excretion tended to be higher during the week-end (6.0 g, 95% CI 5.4-6.6) than during the week (5.4 g, 95% CI 4.3-6.7). The main sources of salt intake were pastas, potatoes, and rice (23% of total salt intake), pastries (16%), bread (16%), and cured meats (10%). One child out of three (34%) added salt to their plate at the table. CONCLUSIONS: Salt intake in children in one region of Switzerland was high. Our findings suggest that salt intake in children could be reduced by lowering salt content in commonly eaten foods. TRIAL REGISTRATION NUMBER: NCT02900261.

Urine Spot Samples Can Be Used to Estimate 24-Hour Urinary Sodium Excretion in Children.

Background: The gold standard to assess salt intake is 24-h urine collections. Use of a urine spot sample can be a simpler alternative, especially when the goal is to assess sodium intake at the population level. Several equations to estimate 24-h urinary sodium excretion from urine spot samples have been tested in adults, but not in children. Objective: The objective of this study was to assess the ability of several equations and urine spot samples to estimate 24-h urinary sodium excretion in children. Methods: A cross-sectional study of children between 6 and 16 y of age was conducted. Each child collected one 24-h urine sample and 3 timed urine spot samples, i.e., evening (last void before going to bed), overnight (first void in the morning), and morning (second void in the morning). Eight equations (i.e., Kawasaki, Tanaka, Remer, Mage, Brown with and without potassium, Toft, and Meng) were used to estimate 24-h urinary sodium excretion. The estimates from the different spot samples and equations were compared with the measured excretion through the use of several statistics. Results: Among the 101 children recruited, 86 had a complete 24-h urine collection and were included in the analysis (mean age: 10.5 y). The mean measured 24-h urinary sodium excretion was 2.5 g (range: 0.8-6.4 g). The different spot samples and equations provided highly heterogeneous estimates of the 24-h urinary sodium excretion. The overnight spot samples with the Tanaka and Brown equations provided the most accurate estimates (mean bias: -0.20 to -0.12 g; correlation: 0.48-0.53; precision: 69.7-76.5%; sensitivity: 76.9-81.6%; specificity: 66.7%; and misclassification: 23.0-27.7%). The other equations, irrespective of the timing of the spot, provided less accurate estimates. Conclusions: Urine spot samples, with selected equations, might provide accurate estimates of the 24-h sodium excretion in children at a population level. At an individual level, they could be used to identify children with high sodium excretion. This study was registered at clinicaltrials.gov as NCT02900261.

Effects of interpersonal violence-related post-traumatic stress disorder (PTSD) on mother and child diurnal cortisol rhythm and cortisol reactivity to a laboratory stressor involving separation.

Women who have experienced interpersonal violence (IPV) are at a higher risk to develop posttraumatic stress disorder (PTSD), with dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and impaired social behavior. Previously, we had reported impaired maternal sensitivity and increased difficulty in identifying emotions (i.e. alexithymia) among IPV-PTSD mothers. One of the aims of the present study was to examine maternal IPV-PTSD salivary cortisol levels diurnally and reactive to their child's distress in relation to maternal alexithymia. Given that mother-child interaction during infancy and early childhood has important long-term consequences on the stress response system, toddlers' cortisol levels were assessed during the day and in response to a laboratory stressor. Mothers collected their own and their 12-48month-old toddlers' salivary samples at home three times: 30min after waking up, between 2-3pm and at bedtime. Moreover, mother-child dyads participated in a 120-min laboratory session, consisting of 3 phases: baseline, stress situation (involving mother-child separation and exposure to novelty) and a 60-min regulation phase. Compared to non-PTSD controls, IPV-PTSD mothers - but not their toddlers, had lower morning cortisol and higher bedtime cortisol levels. As expected, IPV-PTSD mothers and their children showed blunted cortisol reactivity to the laboratory stressor. Maternal cortisol levels were negatively correlated to difficulty in identifying emotions. Our data highlights PTSD-IPV-related alterations in the HPA system and its relevance to maternal behavior. Toddlers of IPV-PTSD mothers also showed an altered pattern of cortisol reactivity to stress that potentially may predispose them to later psychological disorders.

Gonadotropin axis and semen quality in young Swiss men after cannabis consumption: Effect of chronicity and modulation by cannabidiol.

BACKGROUND: While cannabis is the most widely used recreational drug in the world, the effects of phytocannabinoids on semen parameters and reproductive hormones remain controversial. Cannabinoid receptors are activated by these compounds at each level of the hypothalamus-pituitary-gonadotropic axis. OBJECTIVES: To assess the impact of the consumption of Δ-9-tetrahydrocannabinol and cannabidiol on semen parameters, as well as on male reproductive hormone and endocannabinoid levels, in a cohort of young Swiss men. MATERIALS AND METHODS: The individuals in a Swiss cohort were divided according to their cannabis consumption. In the cannabis user group, we determined the delay between the last intake of cannabis and sample collection, the chronicity of use and the presence of cannabidiol in the consumed product. Urinary Δ-9-tetrahydrocannabinol metabolites were quantified via gas chromatography-mass spectrometry. Blood phytocannabinoids, endocannabinoids and male steroids were determined via liquid chromatography-mass spectrometry/mass spectrometry, and other hypothalamus-pituitary-gonadotropic axis hormones were determined via immunoassays. Semen parameters such as sperm concentration and motility were recorded using computer-assisted sperm analysis. RESULTS: Anandamide, N-palmitoyl ethanolamide, androgens, estradiol and sex hormone binding globulin levels were all higher in cannabis users, particularly in chronic, recent and cannabidiol-positive consumers. Gonadotropin levels were not significantly different in these user subpopulations, whereas prolactin and albumin concentrations were lower. In addition, cannabis users had a more basic semen pH and a higher percentage of spermatozoa with progressive motility. However, the two latter observations seem to be related to a shorter period of sexual abstinence in this group rather than to the use of cannabis. CONCLUSIONS: Because both cannabidiol and Δ-9-tetrahydrocannabinol are frequently used by men of reproductive age, it is highly relevant to elucidate the potential effects they may have on human reproductive health. This study demonstrates that the mode of cannabis consumption must be considered when evaluating the effect of cannabis on semen quality.

Effect of a four-week isocaloric ketogenic diet on physical performance at very high-altitude: a pilot study.

BACKGROUND: A ketogenic diet (KD) reduces daily carbohydrates (CHOs) ingestion by replacing most calories with fat. KD is of increasing interest among athletes because it may increase their maximal oxygen uptake (VO2max), the principal performance limitation at high-altitudes (1500-3500 m). We examined the tolerance of a 4-week isocaloric KD (ICKD) under simulated hypoxia and the possibility of evaluating ICKD performance benefits with a maximal graded exercise bike test under hypoxia and collected data on the effect of the diet on performance markers and arterial blood gases. METHODS: In a randomised single-blind cross-over model, 6 recreational mountaineers (age 24-44 years) completed a 4-week ICKD followed or preceded by a 4-week usual mixed Western-style diet (UD). Performance parameters (VO2max, lactate threshold [LT], peak power [Ppeak]) and arterial blood gases (PaO2, PaCO2, pH, HCO3-) were measured at baseline under two conditions (normoxia and hypoxia) as well as after a 4-week UD and 4-week ICKD under the hypoxic condition. RESULTS: We analysed data for all 6 participants (BMI 19.9-24.6 kg m-2). Mean VO2max in the normoxic condition was 44.6 ml kg-1 min-1. Hypoxia led to decreased performance in all participants. With the ICKD diet, median values for PaO2 decreased by - 14.5% and VO2max by + 7.3% and Ppeak by + 4.7%. CONCLUSION: All participants except one could complete the ICKD. VO2max improved with the ICKD under the hypoxia condition. Therefore, an ICKD is an interesting alternative to CHOs dependency for endurance performance at high-altitudes, including high-altitude training and high-altitude races. Nevertheless, decreased PaO2 with ICKD remains a significant limitation in very-high to extreme altitudes (> 3500 m). Trial registration Clinical trial registration Nr. NCT05603689 (Clinicaltrials.gov). Ethics approval CER-VD, trial Nr. 2020-00427, registered 18.08.2020-prospectively registered.

Anti-apolipoprotein A-1 IgG as an independent cardiovascular prognostic marker affecting basal heart rate in myocardial infarction.

AIMS: To assess the prognostic value of anti-apolipoprotein A-1 (anti-apoA-1) IgG after myocardial infarction (MI) and its association with major cardiovascular events (MACEs) at 12 months and to determine their association with resting heart rate (RHR), a well-established prognostic feature after MI. Anti-apoA-1 IgG have been reported in MI without autoimmune disease, but their clinical significance remains undetermined. METHODS AND RESULTS: A total of 221 consecutive patients with MI were prospectively included, and all completed a 12-month follow-up. Major cardiovascular events consisted in death, MI, stroke, or hospitalization either for an acute coronary syndrome or heart failure. Resting heart rate was obtained on Holter the day before discharge under the same medical treatment. Neonate rat ventricular cardiomyocytes (NRVC) were used in vitro to assess the direct anti-apoA-1 IgG effect on RHR. During follow-up, 13% of patients presented a MACE. Anti-apoA-1 IgG positivity was 9% and was associated with a higher RHR (P = 0.0005) and higher MACE rate (adjusted OR, 4.3; 95% CI, 1.46-12.6; P = 0.007). Survival models confirmed the significant nature of this association. Patients with MACE had higher median anti-apoA-1 IgG values at admission than patients without (P = 0.007). On NRVC, plasma from MI patients and monoclonal anti-apoA-1 IgG induced an aldosterone and dose-dependent positive chronotropic effect, abrogated by apoA-1 and therapeutic immunoglobulin (IVIG) pre-incubation. CONCLUSIONS: In MI patients, anti-apoA-1 IgG is independently associated with MACE at 1-year, interfering with a currently unknown aldosterone-dependent RHR determinant. Knowing whether anti-apoA-1 IgG assessment could be of interest to identify an MI patient subset susceptible to benefit from apoA-1/IVIG therapy remains to be demonstrated.

The Cardiac Mineralocorticoid Receptor (MR): A Therapeutic Target Against Ventricular Arrhythmias.

Mineralocorticoid antagonists have been shown to be useful in the treatment of severe heart failure and may even save lives in this context. However, the reason for the beneficial action of these drugs, as well as the physiological role played by the cardiac mineralocorticoid receptor (MR), are still poorly understood. While the proinflammatory action of aldosterone on the heart and the resulting fibrosis partly explain the improvement due to the anti-mineralocorticoid therapy, the reduction in sudden death is probably related to a lower occurrence of ventricular arrhythmias. In this review, the author explains the physiological mechanism linking the positive chronotropic response induced by aldosterone observed in vitro with isolated ventricular cardiomyocytes and the increased risk of ventricular arrhythmias reported in vivo in hyperaldosteronism. He describes the molecular steps involved between MR activation and acceleration of spontaneous myocyte contractions, including expression of a specific micro RNA (miR204), down-regulation of a silencing transcription factor (NRSF), and re-expression of a fetal gene encoding a low threshold voltage-gated calcium channel (CaV3.2). Finally, he provides evidence suggesting aldosterone-independent and redox-sensitive mechanisms of MR activation in cardiac myocytes. Taken together, this information suggests that the use of anti-mineralocorticoid therapy could benefit the heart by preventing ventricular arrhythmias, not only in established hyperaldosteronism, but also in various pathological situations such as Cushing's disease, oxidative stress, or even diabetes mellitus.

Semen endocannabinoids are correlated to sperm quality in a cohort of 200 young Swiss men.

BACKGROUND: A role for endocannabinoids in the male and female reproductive systems has been highlighted during the recent decades. Some of these compounds bind the cannabinoid CB1 receptor, which is abundantly expressed in the central nervous system but also present in the reproductive system, while others act as 'entourage compounds' modulators. OBJECTIVES: The present study aimed at evaluating the relationship between sperm quality and endocannabinoid profiles in a cohort of 200 young Swiss men and whether the presence of specific xenobiotics could influence these profiles. MATERIALS AND METHODS: Semen analysis was performed according to WHO guidelines. Endocannabinoid profiles in blood and semen, as well as bisphenol A and S in urine, were determined by LC-MSMS methods. The presence of selected drugs was tested in urine by immunological screening, and urinary tetrahydrocannabinol (THC) metabolites were quantified by GC-MS. RESULTS: Anandamide concentrations in seminal fluid and oleoylethanolamide (OEA) concentrations in blood serum appeared inversely correlated with sperm motility, while semen palmytoylethanolamide (PEA) was positively linked to sperm concentration. Moreover, OEA and PEA in seminal fluid were associated with better sperm morphology. Interestingly, the concentrations of the same endocannabinoids measured in both blood and semen were not correlated, and the presence of THC metabolites in some individuals was linked to lower concentrations of endocannabinoids. CONCLUSIONS: In the context of the general decline of the sperm count observed within the male population, endocannabinoids in semen constitute a class of promising biochemical markers that open new perspectives as a complement for the usual evaluation of semen quality or for the toxicological screening of individuals' exposure to putative endocrine disruptors.

Steroid profile analysis by LC-HRMS in human seminal fluid.

Steroids are essential hormones that play a crucial role in homeostasis of many biological processes including sexual development, spermatogenesis, sperm physiology and fertility. Although steroids have been largely studied in many biological matrices (such as urine and plasma), there is very limited information of the steroid content and their study as potential indicators of the quality of the seminal fluid. In this study, a LC-HRMS (liquid chromatography-high resolution mass spectrometry) strategy has been developed in order to obtain the extended steroid profile of human seminal fluid. A comparison between supported liquid extraction (SLE) and solid liquid extraction (SPE) was carried out and the chosen SPE method was further optimized to evidence the largest possible number of compounds. Steroids were automatically annotated by using DynaStI, a publicly available retention time prediction tool developed in our lab, to match the experimental data (i.e. accurate mass and tR). Altogether, these resources allowed us to develop a post-targeted approach able to consistently detect 41 steroids in seminal fluid (with half of them being androgens). Such steroid pattern was found to be stable across different extraction times and injection days. In addition to accurate mass and retention time, the identity of 70% of the steroids contained in such steroid profile was confirmed by comparing their fragmentation patterns in real samples to those of pure commercial standards. Finally, the workflow was applied to compare and distinguish the steroid profile in seminal fluid from healthy volunteers (n = 7, with one of them being a vasectomized subject). In all, the developed steroidomics strategy allows to reliably monitor an extended panel of 41 steroids in human seminal fluid.

Hint2 is expressed in the mitochondria of H295R cells and is involved in steroidogenesis.

Hint2 belongs to the superfamily of histidine triad hydrolase enzymes. Recently, it has been shown to influence the mitochondria-dependent apoptosis occurring in hepatocytes, but its mechanism of action is still obscure. Here, we demonstrate that Hint2 is expressed in the mitochondria of H295R cells and in normal adrenals, and that this protein is involved in steroidogenesis. The presence of Hint2 in H295R cells was revealed by RT-PCR and by immunoblot analysis of subcellular fractions. The protein appeared associated with mitochondrial membranes, probably facing the interior of the organelle. Hint2 overexpression in H295R cells had no effect on pregnenolone secretion elicited by angiotensin II or K+, whereas protein silencing with specific small interfering RNA resulted in a marked reduction of the steroidogenic response. The duration of the mitochondrial calcium signal induced by angiotensin II was also reduced upon Hint2 down-regulation with small interfering RNA, but not affected after its overexpression, suggesting that under basal conditions, Hint2 is optimally expressed, and not rate limiting in steroidogenesis. Moreover, Hint2 also appeared involved in Ca2+-independent pathways leading to steroid formation. Indeed, pregnenolone formation in response to either forskolin or a hydroxyl analog of cholesterol was markedly reduced after Hint2 silencing. Calcium-dependent and calcium-independent actions of Hint2 on steroidogenesis could be related to its ability to maintain a favorable mitochondrial potential. In conclusion, these data suggest that, in H295R cells, Hint2 is required for an optimal steroidogenic response, possibly because of a particular signalling function exerted within the mitochondria and that still remains to determine at the molecular level.

CRP-Based Cardiovascular Risk Assessment: New Conventional CRP Assay Fit for Purpose?

BACKGROUND: Subclinical inflammation was shown to play a role in the context of cardiovascular disorder processes. American College of Cardiology/American Heart Association guidelines on cardiovascular risk assessment in specific clinical contexts recommend the use of C-reactive protein (CRP) measurement with high sensitive (hs)-CRP assays that meet the precision requirements for values <2 mg/L. Until now, only hs-CRP assays reached the required limit of quantification. However, new regular CRP assays allow measuring CRP down to 0.6 mg/L. METHODS: A multisite comparative study between hs-CRP and a new conventional CRP assay (Tina-quant) was performed to evaluate the possibility of using regular CRP assays for cardiovascular risk assessment. RESULTS: A satisfactory concordance was observed between regular CRP assays and the hs-CRP assay. Both assays met the analytical precision requirements at the different cutpoints tested (1.00, 2.00, and 3.00 mg/L). CONCLUSION: These results suggest that this new regular CRP assay can be used for cardiovascular risk assessment, which is expected to provide substantial operational and financial advantages when compared with hs-CRP assays.

Steroid profiles in both blood serum and seminal plasma are not correlated and do not reflect sperm quality: Study on the male reproductive health of fifty young Swiss men.

Steroids play an important role in sperm production and quality. These hormones have been extensively studied in blood, but poorly investigated in semen. The purpose of our study was to evaluate the relationship between sperm quality and steroid profiles in blood and semen in a small cohort of young Swiss men. Another objective was to determine whether the presence of xenobiotics or drugs could influence these profiles. Semen analysis was performed according to WHO guidelines, and steroid profiles in blood serum and seminal plasma were determined by two complementary approaches: a targeted investigation involving the quantification of a limited number of relevant steroids for testing putative correlations with sperm parameters and a global "steroidomic" analysis highlighting their complex metabolic relationship. Results showed that steroid profiles are distinct within blood and seminal fluid. No significant correlation was found between individual steroids measured in blood and in semen, demonstrating the relevance of assessing hormone levels in both fluids. Moreover, testosterone and androstenedione levels were significantly correlated in semen but not in blood. None of the evaluated spermiogram parameters was linked to steroid levels measured in any medium. The steroidomic analyses confirmed that the steroids present in both fluids are different and that there is no correlation with spermiogram parameters. Finally, upon toxicological screening, we observed that all the three samples positive for tetrahydrocannabinol, which is known to act as an endocrine disruptor, displayed low seminal testosterone concentrations. In conclusion, we did not find any evidence suggesting using steroid profiles, neither in blood nor in semen, as surrogates for sperm analyses. However, steroid profiles could be useful biomarkers of individual exposure to endocrine disruptors.

T channels and steroid biosynthesis: in search of a link with mitochondria.

The activity of T-type Ca2+ channels has been associated for a long time with steroid biosynthesis in adrenal cortical cells. Because Ca2+-dependent, rate-limiting steps of steroidogenesis have been shown to occur within the mitochondria, a functional link between these organelles and T-type channels has been thoroughly investigated. Based on several experimental data, a model has been proposed in which plasma-membrane-embedded T channels specifically bring calcium entering the cell in proximity of a pumping site of the endoplasmic reticulum. The quasi direct transfer of Ca2+ from the extracellular medium into the lumen of the e.r. would be a specific feature insured by T channels, not by other voltage-operated calcium channels. The e.r. would then act as a sort of Ca2+ pipeline, carrying the cation to the proximity of mitochondria, where it would be released, upon activation of the inositol 1,4,5-trisphosphate receptor, before being immediately and avidly taken up by the organelle. A strict structural organization must be maintained at each extremity of the pipeline in order to optimize the specificity and the efficacy of this signal transduction. Both functional and structural evidences supporting this model of calcium transport within steroidogenic glomerulosa cells are reviewed in the present article.

Aldosterone increases T-type calcium channel expression and in vitro beating frequency in neonatal rat cardiomyocytes.

OBJECTIVE: Although aldosterone has been implicated in the pathogenesis of cardiac hypertrophy and heart failure, its cellular mechanism of action on cardiomyocyte function is not yet completely elucidated. This study was designed to investigate the effect of aldosterone on calcium channel expression and cardiomyocyte contraction frequency. METHODS: Cultured neonatal rat ventricular cardiomyocytes were stimulated in vitro with 1 micromol/L aldosterone for 24 h. Calcium currents were then measured with the patch clamp technique, while calcium channel expression was assessed by real-time RT-PCR. RESULTS: In the present study, we show that aldosterone increases Ca2+ currents by inducing channel expression. Indeed, aldosterone led to a substantial increase of L- and T-type Ca2+ current amplitudes, and we found a concomitant 55% increase of the mRNA coding for alpha1C and beta2 subunits of cardiac L channels. Although T-type currents were relatively small under control conditions, they increased 4-fold and T channel alpha1H isoform expression rose in the same proportion after aldosterone treatment. Because T channels have been implicated in the modulation of membrane electrical activity, we investigated whether aldosterone affects the beating frequency of isolated cardiomyocytes. In fact, aldosterone dose-dependently increased the spontaneous beating frequency more than 4-fold. This effect of aldosterone was prevented by actinomycin D and spironolactone and reduced by RU486, suggesting a mixed mineralocorticoid/glucocorticoid receptor-dependent transcriptional mechanism. Moreover, inhibition of T currents with Ni2+ or mibefradil significantly reduced beating frequency towards control values, while conditions affecting L-type currents completely blocked contractions. CONCLUSION: Aldosterone modulates the expression of cardiac voltage-operated Ca2+ channels and accelerates beating in cultured neonatal rat ventricular myocytes. This chronotropic action of aldosterone appears to be linked to increased T channel activity and could contribute to the deleterious effect of an excess of this steroid in vivo on cardiac function.

T-Type Calcium Channel: A Privileged Gate for Calcium Entry and Control of Adrenal Steroidogenesis.

Intracellular calcium plays a crucial role in modulating a variety of functions such as muscle contraction, hormone secretion, gene expression, or cell growth. Calcium signaling has been however shown to be more complex than initially thought. Indeed, it is confined within cell microdomains, and different calcium channels are associated with different functions, as shown by various channelopathies. Sporadic mutations on voltage-operated L-type calcium channels in adrenal glomerulosa cells have been shown recently to be the second most prevalent genetic abnormalities present in human aldosterone-producing adenoma. The observed modification of the threshold of activation of the mutated channels not only provides an explanation for this gain of function but also reminds us on the importance of maintaining adequate electrophysiological characteristics to make channels able to exert specific cellular functions. Indeed, the contribution to steroid production of the various calcium channels expressed in adrenocortical cells is not equal, and the reason has been investigated for a long time. Given the very negative resting potential of these cells, and the small membrane depolarization induced by their physiological agonists, low threshold T-type calcium channels are particularly well suited for responding under these conditions and conveying calcium into the cell, at the right place for controlling steroidogenesis. In contrast, high threshold L-type channels are normally activated by much stronger cell depolarizations. The fact that dihydropyridine calcium antagonists, specific for L-type channels, are poorly efficient for reducing aldosterone secretion either in vivo or in vitro, strongly supports the view that these two types of channels differently affect steroid biosynthesis. Whether a similar analysis is transposable to fasciculata cells and cortisol secretion is one of the questions addressed in the present review. No similar mutations on L-type or T-type channels have been described yet to affect cortisol secretion or to be linked to the development of Cushing syndrome, but several evidences suggest that the function of T channels is also crucial in fasciculata cells. Putative molecular mechanisms and cellular structural organization making T channels a privileged entry for the "steroidogenic calcium" are also discussed.

Evaluation of steroidomics by liquid chromatography hyphenated to mass spectrometry as a powerful analytical strategy for measuring human steroid perturbations.

This review presents the evolution of steroid analytical techniques, including gas chromatography coupled to mass spectrometry (GC-MS), immunoassay (IA) and targeted liquid chromatography coupled to mass spectrometry (LC-MS), and it evaluates the potential of extended steroid profiles by a metabolomics-based approach, namely steroidomics. Steroids regulate essential biological functions including growth and reproduction, and perturbations of the steroid homeostasis can generate serious physiological issues; therefore, specific and sensitive methods have been developed to measure steroid concentrations. GC-MS measuring several steroids simultaneously was considered the first historical standard method for analysis. Steroids were then quantified by immunoassay, allowing a higher throughput; however, major drawbacks included the measurement of a single compound instead of a panel and cross-reactivity reactions. Targeted LC-MS methods with selected reaction monitoring (SRM) were then introduced for quantifying a small steroid subset without the problems of cross-reactivity. The next step was the integration of metabolomic approaches in the context of steroid analyses. As metabolomics tends to identify and quantify all the metabolites (i.e., the metabolome) in a specific system, appropriate strategies were proposed for discovering new biomarkers. Steroidomics, defined as the untargeted analysis of the steroid content in a sample, was implemented in several fields, including doping analysis, clinical studies, in vivo or in vitro toxicology assays, and more. This review discusses the current analytical methods for assessing steroid changes and compares them to steroidomics. Steroids, their pathways, their implications in diseases and the biological matrices in which they are analysed will first be described. Then, the different analytical strategies will be presented with a focus on their ability to obtain relevant information on the steroid pattern. The future technical requirements for improving steroid analysis will also be presented.

Mineralocorticoid receptor antagonism prevents the electrical remodeling that precedes cellular hypertrophy after myocardial infarction.

BACKGROUND: Cardiac hypertrophy underlies arrhythmias and sudden death, for which mineralocorticoid receptor (MR) activity has recently been implicated. We sought to establish the sequence of ionic events that link the initiating insult and MR to hypertrophy development. METHODS AND RESULTS: Using whole-cell, patch-clamp and quantitative reverse transcription-polymerase chain reaction techniques on right ventricular myocytes of a myocardial infarction (MI) rat model, we examined the cellular response over time. One week after MI, no sign of cellular hypertrophy was found, but action potential duration (APD) was lengthened. Both an increase in Ca2+ current (I(Ca)) and a decrease in K+ transient outward current (I(to)) underlay this effect. Consistently, the relative expression of mRNA coding for the Ca2+ channel alpha1C subunit (Ca(v)1.2) increased, and that of the K+ channel K(v)4.2 subunit decreased. Three weeks after MI, AP prolongation endured, whereas cellular hypertrophy developed. I(Ca) density, Ca(v)1.2, and K(v)4.2 mRNA levels regained control levels, but I(to) density remained reduced. Long-term treatment with RU28318, an MR antagonist, prevented this electrical remodeling. In a different etiologic model of abdominal aortic constriction, we confirmed that APD prolongation and modifications of ionic currents precede cellular hypertrophy. CONCLUSIONS: Electrical remodeling, which is triggered at least in part by MR activation, is an initial, early cellular response to hypertrophic insults.