Toxicant exposure during pregnancy increases protective proteins in the dam and a sexually dimorphic response in the fetus.

Endocrine disrupting compounds (EDCs) are ubiquitous environmental pollutants that alter endocrine system function, induce birth defects, and a myriad of other negative health outcomes. Although the mechanism of toxicity of many EDCs have been studied in detail, little work has focused on understanding the mechanisms through which pregnant dams and fetuses protect themselves from EDCs, or if those protective mechanisms are sexually dimorphic in fetuses. In this study, we examined proteomic alterations in the livers of mouse dams and their male and female fetuses induced by vinclozolin, a model antiandrogenic EDC. Dam livers upregulated nine phase I and phase II detoxification pathways and pathway analysis revealed that more pathways are significantly enriched in dam livers than in fetal livers. Phase I and II detoxification proteins are also involved in steroid and steroid hormone biosynthesis and vinclozolin likely alters steroid levels in both the dam and the fetus. The response of the fetal liver proteome to vinclozolin exposure is sexually dimorphic. Female fetal livers upregulated proteins in xenobiotic metabolism pathways, whereas male fetal livers upregulated proteins in oxidative phosphorylation pathways. These results suggest that female fetuses increase protective mechanisms, whereas male fetuses increase ATP production and several disease pathways that are indicative of oxidative damage. Females fetuses upregulate proteins and protective pathways that were similar to the dams whereas males did not. If this sexually dimorphic pattern is typical, then males might generally be more sensitive to EDCs.

Maternal obesity alters placental lysophosphatidylcholines, lipid storage, and the expression of genes associated with lipid metabolism‡.

Dyslipidemia is a characteristic of maternal obesity and previous studies have demonstrated abnormalities in fatty acid oxidation and storage in term placentas. However, there is little information about the effect of pre-pregnancy obesity on placental lipid metabolism during early pregnancy. The objective of this study was to determine the relationship between lipid profiles and markers of metabolism in placentas from obese and lean dams at midgestation. Mice were fed a western diet (WD) or normal diet (ND) and lysophosphatidylcholines (LPCs) and/or phosphatidylcholines (PCs) were measured in dam circulation and placenta sections using liquid chromatography-tandem mass spectrometry and mass spectrometry imaging, respectively. In WD dam, circulating LPCs containing 16:1, 18:1, 20:0, and 20:3 fatty acids were increased and 18:2 and 20:4 were decreased. In WD placenta from both sexes, LPC 18:1 and PC 36:1 and 38:3 were increased. Furthermore, there were moderate to strong correlations between LPC 18:1, PC 36:1, and PC 38:3. Treatment-, spatial-, and sex-dependent differences in LPC 20:1 and 20:3 were also detected. To identify genes that may regulate diet-dependent differences in placenta lipid profiles, the expression of genes associated with lipid metabolism and nutrient transport was measured in whole placenta and isolated labyrinth using droplet digital PCR and Nanostring nCounter assays. Several apolipoproteins were increased in WD placentas. However, no differences in nutrient transport or fatty acid metabolism were detected. Together, these data indicate that lipid storage is increased in midgestation WD placentas, which may lead to lipotoxicity, altered lipid metabolism and transport to the fetus later in gestation.

Steroid analysis by ion mobility spectrometry.

Steroids are an important biomolecule class for analysis due to their promise as biomarkers for various diseases and their abuse as performance enhancers in sports. Current analytical methods, including chromatography and nuclear magnetic resonance spectroscopy, fall short of being able to confidently analyze steroids, partly due to the large number of steroid isomers. Ion mobility spectrometry (IMS), a gas-phase ion separator, has shown potential for steroid analysis both in conjunction with liquid chromatography (LC) and as a stand-alone technique. This review will examine the current literature on IMS analysis of steroids. Analysis by LC-IMS will include examination of steroids and steroid glucuronides in human urine and serum samples for enhanced signal-to-noise ratios and higher confidence of identification. The stand-alone IMS analysis will examine the use of derivatization of steroids and formation of multimers to enhance resolution for steroid isomers analysis, where both methods have been shown to greatly increase the separation of steroid isomer species. However, these methods have not been applied to biological mixtures to assess their applicability to medical and forensic applications, which should be a future direction of this field.

Liquid chromatography-ion mobility spectrometry-mass spectrometry analysis of multiple classes of steroid hormone isomers in a mixture.

Methods for the analysis of steroids have long been of interest due to the multiple uses for such methods in medical applications, sports monitoring, and environmental science. The analysis of steroids involves inherent analytical hurdles due to their low biological concentrations, poor ionization efficiencies, and frequent occurrence of isomerism. One analytical technique that has been recently applied to steroid analysis is ion mobility spectrometry (IMS). While previous work has focused on the use of metal adduction and multimer formation to enhance separation through IMS analysis coupled to mass spectrometry (MS), this work furthers this approach by coupling IMS-MS with liquid chromatography (LC). Three different LC methods with varying tradeoffs between chromatographic resolution and run time were developed, with one of these achieving a resolution above 1.5 for all steroid isomers. These results also indicate that the coupling of LC to IMS-MS can increase the overall resolution of steroid isomers relative to what can be achieved by either LC or IMS alone. Furthermore, the use of LC and IMS in concert can allow for a more rapid analysis of steroid isomers than can be achieved by LC-MS alone. Finally, the IMS dimension provided for measurements of ion-neutral collision cross sections (CCSs), which were found to be in good agreement with previously reported measurements. Thus, this approach provides three complementary quantitative parameters (retention time, CCS, and mass-to-charge ratio) that can contribute the identification of analytes. Overall, the work presented here demonstrates the potential of coupling LC, IMS, and MS for the analysis of isomeric steroid hormones.

Ion Mobility Spectrometry and Tandem Mass Spectrometry Analysis of Estradiol Glucuronide Isomers.

Estradiol is an estrogenic steroid that can undergo glucuronidation at two different sites, which results in two estradiol glucuronide regioisomers. These isomers can be produced by different enzymes and can have different biological activities before being eliminated from the body. Although there have been previous methods that can distinguish the two isomers, these methods often have long acquisition times or high cost per analysis. In this study, traveling wave ion mobility spectrometry (TWIMS) coupled with mass spectrometry (MS) was employed to separate estradiol glucuronides using alkali metal adduction in positive ion mode, where the sodiated dimer adduct provided adequate separation both in single-component standards and in two-component mixtures. Additionally, in negative ion mode, tandem mass spectrometry (MS/MS) was used to quantitatively determine the relative composition of the two isomers. This was possible due to differences in the energetic requirements for loss of the glucuronic acid, which was characterized by energy-resolved collision-induced dissociation (CID). This work demonstrated that the intensity of the glucuronic acid neutral loss product as compared with the intensity of the intact precursor ion can be used to determine the percentage of each isomer present in a mixture. Overall, TWIMS successfully separated estradiol glucuronide isomers in positive ion mode and MS/MS via CID enables relative quantitation of each isomer in negative ion mode.

Formation of multimeric steroid metal adducts and implications for isomer mixture separation by traveling wave ion mobility spectrometry.

Steroid analysis is essential to the fields of medicine and forensics, but such analyses can present some complex analytical challenges. While chromatographic methods require long acquisition times and often provide incomplete separation, ion mobility spectrometry (IMS) as coupled to mass spectrometry (MS) has demonstrated significant promise for the separation of steroids, particularly in concert with metal adduction and multimerization. In this study, traveling wave ion mobility spectrometry (TWIMS) was employed to separate multimer steroid metal adducts of isomers in mixtures. The results show the ability to separate steroid isomers with a decrease in resolution compared with single component standards because of the formation of heteromultimers. Additionally, ion-neutral collision cross sections (CCS) of the species studied were measured in the mixtures and compared with CCSs obtained in single component standards. Good agreement between these values suggests that the CCS may aid in identification of unknowns. Furthermore, a complex mixture composed of five sets of steroid isomers were analyzed, and distinct features for each steroid component were identified. This study further demonstrated the potential of TWIMS-MS methods for the rapid and isomer-specific study of steroids in biological samples for use either in tandem with or without chromatographic separation.

Application of Group I Metal Adduction to the Separation of Steroids by Traveling Wave Ion Mobility Spectrometry.

Steroids represent an interesting class of small biomolecules due to their use as biomarkers and their status as scheduled drugs. Although the analysis of steroids is complicated by the potential for many isomers, ion mobility spectrometry (IMS) has previously shown promise for the rapid separation of steroid isomers. This work is aimed at the further development of IMS separation for the analysis of steroids. Here, traveling wave ion mobility spectrometry (TWIMS) was applied to the study of group I metal adducted steroids and their corresponding multimers for five sets of isomers. Each set of isomers had a minimum of one dimeric metal ion adduct that exhibited a resolution greater than one (i.e., approaching baseline resolution). Additionally, ion-neutral collision cross sections (CCSs) were measured using polyalanine as a calibrant, which may provide an additional metric contributing to analyte identification. Where possible, measured CCSs were compared to previously reported values. When measuring CCSs of steroid isomers using polyalanine as the calibrant, nitrogen CCS values were within 1.0% error for monomeric sodiated adducts and slightly higher for the dimeric sodiated adducts. Overall, TWIMS was found to successfully separate steroids as dimeric adducts of group I metals. Graphical Abstract ᅟ.

MICROSCALE SERUM EXTRACTION METHOD FOR THE SIMULTANEOUS ANALYSIS OF CORTICOSTERONE AND LIPIDS.

Corticosterone is an important steroid for the regulation of metabolism and stress response. Existing methods for the measurement of corticosterone include radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA), and liquid chromatography-mass spectrometry (LC-MS). While each of these approaches have their advantages, RIAs use radioactive isotopes that necessitate specially regulated usage and disposal. Furthermore, both ELISAs and RIAs require expensive kits and can only measure a single analyte. In this study, we establish a new sample preparation technique based on a modified Folch extraction that allows for the simultaneous isolation of corticosterone and lipids from serum. The extract is then analyzed by LC-MS. Using only 5 µL of serum, quantification of corticosterone was achieved with coefficients of variation at 3% or less and a detection limit of 0.12 µM. Overall, the results of this study should be beneficial to the measurement of circulating corticosterone and lipids for a variety of studies using small volumes of samples.