Frédérique Courant

Assessment of circulating sex steroid levels in prepubertal and pubertal boys and girls by a novel ultrasensitive gas chromatography-tandem mass spectrometry method.

CONTEXT Estrogens and androgens play key roles for pubertal onset and sexual maturation. Most currently used immunoassays are not sensitive enough to accurately measure the low circulating levels of sex steroids in children without any signs of puberty. However, this does not exclude that sex steroids have important biological roles in prepubertal children. OBJECTIVES To accurately determine levels of sex steroid hormones and their metabolites in serum of healthy children before any physical signs of puberty and to evaluate possible sex differences. MAIN OUTCOME MEASURES Total (unconjugated plus conjugated) serum levels of 17beta-testosterone, 17alpha-testosterone, 5alpha-dihydrotestosterone, 5beta-dihydrotestosterone, androsterone, etiocholanolone, estradiol, and estrone measured by an ultrasensitive method based on gas chromatography-tandem mass spectrometry in samples from 81 healthy schoolchildren (42 boys) without any signs of puberty. For comparison, 48 pubertal children were studied. RESULTS 17beta-Estradiol levels in prepubertal boys were undetectable or extremely low (median < 3.7 pmol/liter), whereas levels in prepubertal girls were significantly higher (median 9.6 pmol/liter, P < 0.001). Among the older prepubertal children (>8 yr), girls had significantly higher androsterone (4.07 vs. 1.45 nmol/liter, P < 0.05), etiocholanolone (5.45 vs. 1.95 nmol/liter, P < 0.0001), 5alpha-dihydrotestosterone (0.11 vs. <0.10 nmol/liter, P < 0.01), and 17beta-testosterone concentrations (0.69 vs. 0.47 nmol/liter, P < 0.05) compared with similarly aged prepubertal boys. CONCLUSION Using an accurate and sensitive method, we found significantly higher levels of estrogens as well as androgen metabolites in prepubertal girls compared with age-matched boys. The higher prepubertal sex steroid levels in girls may contribute to their earlier onset of puberty including pubic hair development.

Exposure Assessment of Prepubertal Children to Steroid Endocrine Disruptors. 2. Determination of Steroid Hormones in Milk, Egg, and Meat Samples

In the present study, the occurrence of the main sex steroid hormones in milk, egg, and meat was evaluated on the basis of a highly specific gas chromatography-tandem mass spectrometry measurement method. Globally, the results indicated that targeted estrogens and androgens occurred at similar levels (concentration levels in the 10-100 ng kg (-1) range) in the analyzed muscle and milk samples. The same compounds occurred at about 10-fold higher concentrations (i.e., in the 100-1000 ng kg (-1) range) in eggs and kidney samples. More precisely, egg and milk appeared as a non-negligible sources of estradiol (i.e., 2.2 +/- 0.8 and 3.1 +/- 2.0 ng day (-1), respectively), whereas testosterone exposure is caused by ingestion of meat and/or egg (i.e., 12.2 +/- 48.2 and 5.2 +/- 2.3 ng day (-1), respectively). The provided exposure data will be further exploited in the scope of a risk assessment study regarding endocrine disruption associated with these molecules.

Maternal and cord blood LC-HRMS metabolomics reveal alterations in energy and polyamine metabolism, and oxidative stress in very-low birth weight infants.

To assess the global effect of preterm birth on fetal metabolism and maternal-fetal nutrient transfer, we used a mass spectrometric-based chemical phenotyping approach on cord blood obtained at the time of birth. We sampled umbilical venous, umbilical arterial, and maternal blood from mothers delivering very-low birth weight (VLBW, with a median gestational age and weight of 29 weeks, and 1210 g, respectively) premature or full-term (FT) neonates. In VLBW group, we observed a significant elevation in the levels and maternal-fetal gradients of butyryl-, isovaleryl-, hexanoyl- and octanoyl-carnitines, suggesting enhanced short- and medium chain fatty acid β-oxidation in human preterm feto-placental unit. The significant decrease in glutamine-glutamate in preterm arterial cord blood beside lower levels of amino acid precursors of Krebs cycle suggest increased glutamine utilization in the fast growing tissues of preterm fetus with a deregulation in placental glutamate-glutamine shuttling. Enhanced glutathione utilization is likely to account for the decrease in precursor amino acids (serine, betaine, glutamate and methionine) in arterial cord blood. An increase in both the circulating levels and maternal-fetal gradients of several polyamines in their acetylated form (diacetylspermine and acetylputrescine) suggests an enhanced polyamine metabolic cycling in extreme prematurity. Our metabolomics study allowed the identification of alterations in fetal energy, antioxidant defense, and polyamines and purines flux as a signature of premature birth.

Identification and quantification of 5α-dihydrotestosterone in the teleost fathead minnow (Pimephales promelas) by gas chromatography-tandem mass spectrometry.

The steroid hormone 5α-dihydrotestosterone (DHT) is one of the most physiologically important androgens in male vertebrates, with the exception of teleost fish, in which it is generally assumed that DHT does not play any major physiological role. However, this assumption is challenged by the fact that all the components involved in DHT biosynthesis and action are present and evolutionary conserved in teleost fish. In fact, testosterone (T) is converted into DHT by two isoforms of the enzyme steroid-5-alpha-reductase (5αR), and both 5αRs gene expression and enzymatic activity have been detected in several tissues of different teleost species, which also have an androgen receptor with high binding affinity to DHT. This body of evidence strongly suggest that DHT is synthesised by teleost fish. We investigated this hypothesis using the cyprinid fathead minnow (Pimephales promelas) as the experimental model. The study of the evolutionary and functional conservation of 5αRs in teleost fish was used to support the experimental approach, based on an ultrasensitive gas chromatography-tandem mass spectrometry (GC-MS/MS) method to identify and measure simultaneously T and DHT in fathead minnow biological fluids and tissues. The analyses were performed using plasma samples collected from both male and female adult fish and samples of testicular tissue collected from sexually mature males. Both T and DHT were identified and quantified in all the samples analysed, and in particular, the high concentrations of DHT quantified in the testes suggested that these organs are a likely site of synthesis of DHT in the teleost fathead minnow, as they are in mammals. These results may represent the basis for future studies aimed at elucidating the physiological role, if any, of DHT in teleost fish.

Metabolomics as a Potential New Approach for Investigating Human Reproductive Disorders

Metabolomics has been emerging for several years as a global chemical phenotyping approach offering fascinating descriptive capabilities for addressing life complexity. It facilitates the understanding of the mechanisms of biological and biochemical processes in complex systems and promises new insights into specific research questions. The objective of this study was to use for the first time a metabolomic approach based on liquid chromatography high resolution mass spectrometry for characterizing an alteration of the testicular function, namely impaired semen quality. Metabolomic fingerprints were generated from serum samples collected from Danish young men presenting low, intermediate, or high sperm concentrations. Serum metabolic profiles were found to be significantly different among the three groups of volunteers. The developed methodology permitted to correlate the studied clinical parameter (i.e., sperm concentration) with the metabolite profiles generated. Peptides related to the Protein Complement C3f were identified as putative markers associated with this clinical parameter. The biological interpretation and further robustness linked to this observation remain to be further investigated, in particular to address the inter- and intraindividual variabilities.

How metabolomics can contribute to bio-processes: a proof of concept study for biomarkers discovery in the context of nitrogen-starved microalgae grown in photobioreactors

Microalgae appear to be one of the most promising sustainable resources as alternative crops for the production of renewable transport fuel. The exploitation of this bioresource requires, however, a fine monitoring of the culture conditions, for example by using more relevant control variables than usual macroscopic indicators (biomass or pigment estimation). In this proof of concept study, we propose to search potential biomarkers of progressive nitrogen regime culture conditions using an untargeted metabolomic approach based on LC-HRMS combined to a non-invasive analysis based on FTIR spectroscopy. One microalgae model was investigated i.e. Chlamydomonas reinhardtii to characterize the effect of progressive nitrogen regime in batch culture conditions on its metabolome. FTIR allowed assessing the intracellular macrometabolic perturbations, highlighting the over-accumulation of carbohydrates. LC-HRMS complemented the macromolecular information by revealing the dependence of microalgae metabotypes on nitrogen regime conditions tested for cells culture. Patterns of significantly modulated metabolites were also detected during those slight contrasted nitrogen regimes and interesting features were structurally elucidated. This included metabolites belonging to the pantothenate, branched chain and aromatic amino acids pathways. In the last step of this proof of concept study, amino acid targets proposed by metabolomic investigations were assessed on nitrogen-limited continuous culture on photobioreactors. This was performed to test the validity of proposed targets in real small-scale industrial production conditions. Results were very encouraging and suggested the possibility of using potentially relevant metabolites as intracellular biomarkers only (tryptophan) or as both intra and extracellular biomarkers (e.g. 2-methylbutyric acid and ketoleucine).

Statistical strategies for relating metabolomics and proteomics data: a real case study in nutrition research area

The current investigations were carried out in the context of a nutritional case study aiming at assessing the postnatal impact of maternal dietary protein restriction during pregnancy and lactation on rat offspring plasma metabolome and hypothalamic proteome. Although data generated by different “Omics” technologies are usually considered and analyzed separately, their interrelation may offer a valuable opportunity for assessing the emerging ‘integrated biology’ concept. The overall strategy of analysis first investigated data pretreatment and variable selection for each dataset. Then, three multivariate analyses were applied to investigate the links between the abundance of metabolites and the expression of proteins collected on the same samples. Unfold principal component analysis and regularized canonical correlation analysis did not take into account the presence of groups of individuals related to the intervention study. On the contrary, the predictive MultiBlock Partial Least Squares method used this information. Regularized canonical correlation analysis appeared as a relevant approach to investigate of the relationships between the two datasets. However, in order to highlight the molecular compounds, proteins and metabolites, associated in interacting or common metabolic pathways for the experimental groups, MultiBlock partial least squares was the most appropriate method in the present nutritional case study.