RESUMO
1-Nitropyrene (1-NP) and 1,8-dinitropyrene (1,8-DNP) are diesel exhaust constituents and are classified by the International Agency for Research on Cancer as probable (Group 2A) or possible (Group 2B) human carcinogens. These nitroarenes undergo metabolic activation by nitroreduction to result in the formation of DNA adducts. Human aldo-keto reductases (AKRs) 1C1-1C3 catalyze the nitroreduction of 3-nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one, 3-NBA), but the extent of AKR contribution toward the nitroreduction of additional nitroarenes, including 1-NP and 1,8-DNP, is currently unknown. In the present study, we investigated the ability of human recombinant AKRs to catalyze 1-NP and 1,8-DNP nitroreduction by measuring the formation of the respective six-electron reduced amine products in discontinuous ultraviolet-reverse phase high-performance liquid chromatography enzymatic assays. We found that AKR1C1-1C3 were able to catalyze the formation of 1-aminopyrene (1-AP) and 1-amino-8-nitropyrene (1,8-ANP) in our reactions with 1-NP and 1,8-DNP, respectively. We determined kinetic parameters (Km, kcat, and kcat/Km) and found that out of the three isoforms, AKR1C1 had the highest catalytic efficiency (kcat/Km) for 1-AP formation, whereas AKR1C3 had the highest catalytic efficiency for 1,8-ANP formation. Use of ultra-performance liquid chromatography high-resolution mass spectrometry verified amine product identity and provided evidence for the formation of nitroso- and hydroxylamino-intermediates in our reactions. Our study expands the role of AKR1C1-1C3, which are expressed in human lung cells, in the metabolic activation of nitroarenes that can lead to DNA adduct formation, mutation, and carcinogenesis.
Assuntos
Aldo-Ceto Redutases , Pirenos , Humanos , Aldo-Ceto Redutases/química , Aldo-Ceto Redutases/metabolismo , Aminas , Pirenos/químicaRESUMO
3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen detected in diesel exhaust particulate and ambient air pollution. It requires metabolic activation via nitroreduction to promote DNA adduct formation and tumorigenesis. NAD(P)H:quinone oxidoreductase 1 (NQO1) has been previously implicated as the major nitroreductase responsible for 3-NBA activation, but it has recently been reported that human aldo-keto reductase 1C3 (AKR1C3) displays nitroreductase activity toward the chemotherapeutic agent PR-104A. We sought to determine whether AKR1C isoforms could display nitroreductase activity toward other nitrated compounds and bioactivate 3-NBA. Using discontinuous enzymatic assays monitored by UV-HPLC, we determined that AKR1C1-1C3 catalyze three successive two-electron nitroreductions toward 3-NBA to form the reduced product 3-aminobenzanthrone (3-ABA). Evidence of the nitroso- and hydroxylamino- intermediates were obtained by UPLC-HRMS. Km, kcat, and kcat/ Km values were determined for recombinant AKR1C and NQO1 and compared. We found that AKR1C1, AKR1C3, and NQO1 have very similar apparent catalytic efficiencies (8 vs 7 min-1 mM-1) despite the higher kcat of NQO1 (0.058 vs 0.012 min-1). AKR1C1-1C3 possess a Km much lower than that of NQO1, which suggests that they may be more important than NQO1 at the low concentrations of 3-NBA to which humans are exposed. Given that inhalation represents the primary source of 3-NBA exposure, we chose to evaluate the relative importance of AKR1C1-1C3 and NQO1 in human lung epithelial cell lines. Our data suggest that the combined activities of AKR1C1-1C3 and NQO1 contribute equally to the reduction of 3-NBA in A549 and HBEC3-KT cell lines and together represent approximately 50% of the intracellular nitroreductase activity toward 3-NBA. These findings have significant implications for the metabolism of nitrated polycyclic aromatic hydrocarbons and suggest that the hitherto unrecognized nitroreductase activity of AKR1C enzymes should be further investigated.
Assuntos
Poluentes Atmosféricos/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase/metabolismo , Benzo(a)Antracenos/metabolismo , Células A549 , Ativação Metabólica , Poluentes Atmosféricos/análise , Membro C3 da Família 1 de alfa-Ceto Redutase/antagonistas & inibidores , Membro C3 da Família 1 de alfa-Ceto Redutase/genética , Benzo(a)Antracenos/análise , Biocatálise , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Massas , NAD(P)H Desidrogenase (Quinona)/antagonistas & inibidores , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Maternal diabetes and obesity induce marked abnormalities in glucose homeostasis and insulin secretion in the fetus, and are linked to obesity, diabetes, and metabolic disease in the offspring, with specific metabolic characterization based on offspring sex. Gestational diabetes (GDM) has profound effects on the intrauterine milieu, which may reflect and/or modulate the function of the maternalâ»fetal unit. In order to characterize metabolic factors that affect offspring development, we profiled the metabolome of second trimester amniotic fluid (AF) from women who were subsequently diagnosed with gestational diabetes (GDM) using a targeted metabolomics approach, profiling 459 known biochemicals through gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) assays. Using a nested case-control study design, we identified 69 total biochemicals altered by GDM exposure, while sex-specific analysis identified 44 and 58 metabolites in male and female offspring, respectively. The most significant changes were in glucose, amino acid, glutathione, fatty acid, sphingolipid, and bile acid metabolism with specific changes identified based on the offspring sex. Targeted isotope dilution LC/MS confirmatory assays measured significant changes in docosahexaenoic acid and arachidonic acid. We conclude that the sex-specific alterations in GDM maternalâ»fetal metabolism may begin to explain the sex-specific metabolic outcomes seen in offspring exposed to GDM in utero.
Assuntos
Líquido Amniótico/metabolismo , Diabetes Gestacional/metabolismo , Metabolômica/métodos , Segundo Trimestre da Gravidez/metabolismo , Adulto , Ácido Araquidônico/análise , Estudos de Casos e Controles , Cromatografia Líquida , Ácidos Docosa-Hexaenoicos/análise , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Recém-Nascido , Masculino , Espectrometria de Massas , Gravidez , Fatores SexuaisRESUMO
TET1/2/3 dioxygenases iteratively demethylate 5-methylcytosine, beginning with the formation of 5-hydroxymethylcytosine (5hmC). The post-mitotic brain maintains higher levels of 5hmC than most peripheral tissues, and TET1 ablation studies have underscored the critical role of TET1 in brain physiology. However, deletion of Tet1 precludes the disentangling of the catalytic and non-catalytic functions of TET1. Here, we dissect these functions of TET1 by comparing adult cortex of Tet1 wildtype (Tet1 WT), a novel Tet1 catalytically dead mutant (Tet1 HxD), and Tet1 knockout (Tet1 KO) mice. Using DNA methylation array, we uncover that Tet1 HxD and KO mutations perturb the methylation status of distinct subsets of CpG sites. Gene ontology (GO) analysis on specific differential 5hmC regions indicates that TET1's catalytic activity is linked to neuronal-specific functions. RNA-Seq further shows that Tet1 mutations predominantly impact the genes that are associated with alternative splicing. Lastly, we performed High-performance Liquid Chromatography Mass-Spectrometry lipidomics on WT and mutant cortices and uncover accumulation of lysophospholipids lysophosphatidylethanolamine and lysophosphatidylcholine in Tet1 HxD cortex. In summary, we show that Tet1 HxD does not completely phenocopy Tet1 KO, providing evidence that TET1 modulates distinct cortical functions through its catalytic and non-catalytic roles.
Assuntos
5-Metilcitosina , Córtex Cerebral , Metilação de DNA , Proteínas Proto-Oncogênicas , Animais , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análogos & derivados , Córtex Cerebral/metabolismo , Camundongos Knockout , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ilhas de CpG , MutaçãoRESUMO
Background & Aims: Non-alcoholic fatty liver disease (NAFLD) is characterised by the accumulation of lipid droplets (LDs) within hepatocytes. Perilipin 2 (PLIN2) is the most abundant protein in hepatic LDs and its expression correlates with intracellular lipid accumulation. A recently discovered PLIN2 coding variant, Ser251Pro (rs35568725), was found to promote the accumulation of small LDs in embryonic kidney cells. In this study, we investigate the role of PLIN2-Ser251Pro (PLIN2-Pro251) on hepatic LD metabolism in vivo and research the metabolic phenotypes associated with this variant in humans. Methods: For our animal model, we used Plin2 knockout mice in which we expressed either human PLIN2-Pro251 (Pro251 mice) or wild-type human PLIN2-Ser251 (Ser251 mice) in a hepatocyte-specific manner. We fed both cohorts a lipogenic high-fat, high-cholesterol, high-fructose diet for 12 weeks. Results: Pro251 mice were associated with reduced liver triglycerides (TGs) and had lower mRNA expression of fatty acid synthase and diacylglycerol O-acyltransferase-2 compared with Ser251 mice. Moreover, Pro251 mice had a reduction of polyunsaturated fatty acids-TGs and reduced expression of epoxygenase genes. For our human study, we analysed the Penn Medicine BioBank, the Million Veteran Program, and UK Biobank. Across these databases, the minor allele frequency of PLIN2-Pro251 was approximately 5%. There was no association with the clinical diagnosis of NAFLD, however, there was a trend toward reduced liver fat in PLIN2-Pro251 carriers by MRI-spectroscopy in UK Biobank subjects. Conclusions: In mice lacking endogenous Plin2, expression of human PLIN2-Pro251 attenuated high-fat, high-fructose, high-cholesterol, diet-induced hepatic steatosis compared with human wild-type PLIN2-Ser251. Moreover, Pro251 mice had lower polyunsaturated fatty acids-TGs and epoxygenase genes expression, suggesting less liver oxidative stress. In humans, PLIN2-Pro251 is not associated with NAFLD. Impact and Implications: Lipid droplet accumulation in hepatocytes is the distinctive characteristic of non-alcoholic fatty liver disease. Perilipin 2 (PLIN2) is the most abundant protein in hepatic lipid droplets; however, little is known on the role of a specific polymorphism PLIN2-Pro251 on hepatic lipid droplet metabolism. PLIN2-Pro251 attenuates liver triglycerides accumulation after a high-fat-high-glucose-diet. PLIN2-Pro251 may be a novel lipid droplet protein target for the treatment of liver steatosis.
RESUMO
Androgen receptor signaling inhibitors (ARSI) are used to treat castration-resistant prostate cancer (CRPC) to stop a resurgence of androgen receptor (AR) signaling. Despite early success, patients on ARSIs eventually relapse, develop drug resistance, and succumb to the disease. Resistance may occur through intratumoral steroidogenesis mediated by upregulation of aldo-keto reductase family 1C member 3 (AKR1C3). Patients treated with leuprolide (castrate) and those treated with leuprolide plus abiraterone (post-Abi) harbor a reservoir of DHEA-S which could fuel testosterone (T) biosynthesis via AKR1C3 to cause a resurgence of prostate cancer cell growth. We demonstrate that concentrations of DHEA-S found in castrate and post-Abi patients are (i) converted to T in an AKR1C3-dependent manner in prostate cancer cells, and (ii) in amounts sufficient to stimulate AKR1C3-dependent cell growth. We observed this in primary and metastatic prostate cancer cell lines, CWR22PC and DuCaP, respectively. Androgen measurements were made by stable isotope dilution LC-MS/MS. We demonstrate AKR1C3 dependence using stable short hairpin RNA knockdown and pharmacologic inhibitors. We also demonstrate that free DHEA is reduced to 5-androstene-3ß,17ß-diol (5-Adiol) by AKR1C3 and that this is a major metabolite, suggesting that in our cell lines 5-Adiol is a predominant precursor of T. We have identified a mechanism of ARSI resistance common to both primary and metastatic cell lines that is dependent on the conversion of DHEA to 5-Adiol on route to T catalyzed by AKR1C3. SIGNIFICANCE: We show that reservoirs of DHEA-S that remain after ARSI treatment are converted into T in primary and metastatic prostate cancer cells in amounts sufficient to stimulate cell growth. Pharmacologic and genetic approaches demonstrate that AKR1C3 is required for these effects. Furthermore, the route to T proceeds through 5-Adiol. We propose that this is a mechanism of ARSI drug resistance.
Assuntos
Neoplasias da Próstata , Testosterona , Masculino , Humanos , Testosterona/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Congêneres da Testosterona , Androstenos , Sulfato de Desidroepiandrosterona , Membro C3 da Família 1 de alfa-Ceto RedutaseRESUMO
Vitamin D deficiency is associated with an increased risk of prostate cancer mortality and is hypothesized to contribute to prostate cancer aggressiveness and disparities in African American populations. The prostate epithelium was recently shown to express megalin, an endocytic receptor that internalizes circulating globulin-bound hormones, which suggests regulation of intracellular prostate hormone levels. This contrasts with passive diffusion of hormones that is posited by the free hormone hypothesis. Here, we demonstrate that megalin imports testosterone bound to sex hormone-binding globulin into prostate cells. Prostatic loss of Lrp2 (megalin) in a mouse model resulted in reduced prostate testosterone and dihydrotestosterone levels. Megalin expression was regulated and suppressed by 25-hydroxyvitamin D (25D) in cell lines, patient-derived prostate epithelial cells, and prostate tissue explants. In patients, the relationships between hormones support this regulatory mechanism, as prostatic DHT levels are higher in African American men and are inversely correlated with serum 25D status. Megalin levels are reduced in localized prostate cancer by Gleason grade. Our findings suggest that the free hormone hypothesis should be revisited for testosterone and highlight the impact of vitamin D deficiency on prostate androgen levels, which is a known driver of prostate cancer. Thus, we revealed a mechanistic link between vitamin D and prostate cancer disparities observed in African Americans. Significance: These findings link vitamin D deficiency and the megalin protein to increased levels of prostate androgens, which may underpin the disparity in lethal prostate cancer in African America men.
Assuntos
Androgênios , Calcifediol , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Neoplasias da Próstata , Deficiência de Vitamina D , Animais , Humanos , Masculino , Camundongos , Negro ou Afro-Americano , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Próstata/metabolismo , Testosterona , Vitamina D/metabolismoRESUMO
Polycystic ovary syndrome (PCOS) is the most prevalent endocrinopathy in women. A common symptom of PCOS is hyperandrogenism (AE); however, the source of these androgens is uncertain. Aldo-keto reductase family 1 member C3 (AKR1C3) catalyzes the formation of testosterone (T) and 5α-dihydrotestosterone (DHT) in peripheral tissues, which activate the androgen receptor (AR). AKR1C3 is induced by insulin in adipocytes and may be central in driving the AE in PCOS. We elucidated the conversion of both classical and 11-oxygenated androgens to potent androgens in a model of PCOS adipocytes. Using high-performance liquid chromatography (HPLC) discontinuous kinetic assays to measure product formation by recombinant AKR1C3, we found that the conversion of 11-keto-Δ4-androstene-3,17-dione (11K-4AD) to 11-ketotestosterone (11K-T) and 11-keto-5α-androstane-3,17-dione (11K-5AD) to 11-keto-5α-dihydrotestosterone (11K-DHT) were superior to the formation of T and DHT. We utilized a stable isotope dilution liquid chromatography high resolution mass spectrometric (SID-LC-HRMS) assay for the quantification of both classical and 11-oxygenated androgens in differentiated Simpson-Golabi-Behmel syndrome adipocytes in which AKR1C3 was induced by insulin. Adipocytes were treated with adrenal derived 11ß-hydroxy-Δ4-androstene-3,17-dione (11ß-OH-4AD), 11K-4AD, or Δ4-androstene-3,17-dione (4AD). The conversion of 11ß-OH-4AD and 11K-4AD to 11K-T required AKR1C3. We also found that once 11K-T is formed, it is inactivated to 11ß-hydroxy-testosterone (11ß-OH-T) by 11ß-hydroxysteroid dehydrogenase type 1 (HSD11B1). Our data reveal a unique role for HSD11B1 in protecting the AR from AE. We conclude that the 11-oxygenated androgens formed in adipocytes may contribute to the hyperandrogenic profile of PCOS women and that AKR1C3 is a potential therapeutic target to mitigate the AE of PCOS.
Assuntos
Androgênios , Síndrome do Ovário Policístico , Adipócitos , Membro C3 da Família 1 de alfa-Ceto Redutase , Androstenos , Di-Hidrotestosterona/farmacologia , Feminino , Humanos , Insulina , TestosteronaRESUMO
The mechanisms leading to adrenal cortex development and steroid synthesis in humans remain poorly understood due to the paucity of model systems. Herein, we recapitulate human fetal adrenal cortex specification processes through stepwise induction of human-induced pluripotent stem cells through posterior intermediate mesoderm-like and adrenocortical progenitor-like states to ultimately generate fetal zone adrenal-cortex-like cells (FZLCs), as evidenced by histomorphological, ultrastructural, and transcriptome features and adrenocorticotropic hormone (ACTH)-independent Δ5 steroid biosynthesis. Furthermore, FZLC generation is promoted by SHH and inhibited by NOTCH, ACTIVIN, and WNT signaling, and steroid synthesis is amplified by ACTH/PKA signaling and blocked by inhibitors of Δ5 steroid synthesis enzymes. Finally, NR5A1 promotes FZLC survival and steroidogenesis. Together, these findings provide a framework for understanding and reconstituting human adrenocortical development in vitro, paving the way for cell-based therapies of adrenal insufficiency.
Assuntos
Córtex Suprarrenal , Células-Tronco Pluripotentes Induzidas , Humanos , Via de Sinalização Wnt , Hormônio Adrenocorticotrópico , EsteroidesRESUMO
Alterations in gut microbiota impact the pathophysiology of several diseases, including cancer. Radiotherapy (RT), an established curative and palliative cancer treatment, exerts potent immune modulatory effects, inducing tumor-associated antigen (TAA) cross-priming with antitumor CD8+ T cell elicitation and abscopal effects. We tested whether the gut microbiota modulates antitumor immune response following RT distal to the gut. Vancomycin, an antibiotic that acts mainly on gram-positive bacteria and is restricted to the gut, potentiated the RT-induced antitumor immune response and tumor growth inhibition. This synergy was dependent on TAA cross presentation to cytolytic CD8+ T cells and on IFN-γ. Notably, butyrate, a metabolite produced by the vancomycin-depleted gut bacteria, abrogated the vancomycin effect. In conclusion, depletion of vancomycin-sensitive bacteria enhances the antitumor activity of RT, which has important clinical ramifications.
Assuntos
Apresentação de Antígeno/efeitos da radiação , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Microbioma Gastrointestinal , Neoplasias Experimentais , Animais , Apresentação de Antígeno/genética , Antígenos de Neoplasias/genética , Butiratos/imunologia , Linfócitos T CD8-Positivos/patologia , Células Dendríticas/patologia , Feminino , Microbioma Gastrointestinal/imunologia , Microbioma Gastrointestinal/efeitos da radiação , Camundongos , Camundongos Knockout , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/radioterapiaRESUMO
During alcohol consumption, the esophageal mucosa is directly exposed to high concentrations of ethanol (EtOH). We therefore investigated the response of normal human esophageal epithelial cell lines EPC1, EPC2 and EPC3 to acute EtOH exposure. While these cells were able to tolerate 2% EtOH for 8 h in both three-dimensional organoids and monolayer culture conditions, RNA sequencing suggested that EtOH induced mitochondrial dysfunction. With EtOH treatment, EPC1 and EPC2 cells also demonstrated decreased mitochondrial ATPB protein expression by immunofluorescence and swollen mitochondria lacking intact cristae by transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) was decreased in a subset of EPC1 and EPC2 cells stained with ΔΨm-sensitive dye MitoTracker Deep Red. In EPC2, EtOH decreased ATP level while impairing mitochondrial respiration and electron transportation chain functions, as determined by ATP fluorometric assay, respirometry, and liquid chromatography-mass spectrometry. Additionally, EPC2 cells demonstrated enhanced oxidative stress by flow cytometry for mitochondrial superoxide (MitoSOX), which was antagonized by the mitochondria-specific antioxidant MitoCP. Concurrently, EPC1 and EPC2 cells underwent autophagy following EtOH exposure, as evidenced by flow cytometry for Cyto-ID, which detects autophagic vesicles, and immunoblots demonstrating induction of the lipidated and cleaved form of LC3B and downregulation of SQSTM1/p62. In EPC1 and EPC2, pharmacological inhibition of autophagy flux by chloroquine increased mitochondrial oxidative stress while decreasing cell viability. In EPC2, autophagy induction was coupled with phosphorylation of AMP activated protein kinase (AMPK), a cellular energy sensor responding to low ATP levels, and dephosphorylation of downstream substrates of mechanistic Target of Rapamycin Complex (mTORC)-1 signaling. Pharmacological AMPK activation by AICAR decreased EtOH-induced reduction of ΔΨm and ATP in EPC2. Taken together, acute EtOH exposure leads to mitochondrial dysfunction and oxidative stress in esophageal keratinocytes, where the AMPK-mTORC1 axis may serve as a regulatory mechanism to activate autophagy to provide cytoprotection against EtOH-induced cell injury.
Assuntos
Autofagia , Esôfago/citologia , Queratinócitos/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Quinases Proteína-Quinases Ativadas por AMP , Animais , Linhagem Celular , Células Cultivadas , Etanol/farmacologia , Feminino , Queratinócitos/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Bisphenol A (BPA) is an endocrine disrupting chemical with ubiquitous environmental exposure. Animal studies have demonstrated that in utero BPA exposure leads to increased adult body weight. Our aim was to characterize human fetal BPA exposure by measuring BPA concentration in second trimester amniotic fluid (AF) samples and to study its relationship with birth weight (BW) in full term infants. To achieve these goals, we developed a total BPA assay utilizing derivatization with pentafluorobenzyl followed by analysis with LC-ECAPCI-MS/MS with a limit of detection of 0.08ng/mL and limit of quantification (LOQ) of 0.25ng/mL. The mean BW of infants with AF BPA 0.40-2.0ng/mL was 241.8g less than infants with AF BPA less than the LOQ after controlling for covariates (p=0.049). No effect was seen outside this range indicating a non-monotonic effect. Our data suggest that low level BPA exposure in utero decreases BW and needs further study.
Assuntos
Líquido Amniótico/química , Compostos Benzidrílicos/análise , Disruptores Endócrinos/análise , Recém-Nascido de Baixo Peso , Fenóis/análise , Efeitos Tardios da Exposição Pré-Natal/etiologia , Cromatografia Líquida , Feminino , Humanos , Limite de Detecção , Gravidez , Segundo Trimestre da Gravidez , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Espectrometria de Massas em TandemRESUMO
A multiplexed quantitative method for the analysis of three major unconjugated steroids in human serum by stable isotope dilution liquid chromatography-high resolution mass spectrometry (LC-HRMS) was developed and validated on a Q Exactive Plus hybrid quadrupole/Orbitrap mass spectrometer. This quantification utilized isotope dilution and Girard P derivatization on the keto-groups of testosterone (T), androstenedione (AD) and dehydroepiandrosterone (DHEA) to improve ionization efficiency using electrospray ionization. Major isomeric compounds to T and DHEA; the inactive epimer of testosterone (epiT), and the metabolite of AD, 5α-androstanedione (5α-AD) were completely resolved on a biphenyl column within an 18min method. Inter- and intra-day method validation using LC-HRMS with qualifying product ions was performed and acceptable analytical performance was achieved. The method was further validated by comparing steroid levels from 100µL of serum from young vs older subjects. Since this approach provides high-dimensional HRMS data, untargeted analysis by age group was performed. DHEA and T were detected among the top analytes most significantly different across the two groups after untargeted LC-HRMS analysis, as well as a number of other still unknown metabolites, indicating the potential for combined targeted/untargeted analysis in steroid analysis.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Esteroides/análise , Androstenodiona/análise , Androstenodiona/química , Desidroepiandrosterona/análise , Desidroepiandrosterona/química , Humanos , Soro/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Esteroides/química , Testosterona/análise , Testosterona/químicaRESUMO
The 5-lipoxygenase product 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is the most powerful human eosinophil chemoattractant among lipid mediators and could play a major pathophysiological role in eosinophilic diseases such as asthma. Its actions are mediated by the OXE receptor, orthologs of which are found in many species from humans to fish, but not rodents. The unavailability of rodent models to examine the pathophysiological roles of 5-oxo-ETE and the OXE receptor has substantially hampered progress in this area. As an alternative, we have explored the possibility that the cat could serve as an appropriate animal model to investigate the role of 5-oxo-ETE. We found that feline peripheral blood leukocytes synthesize 5-oxo-ETE and that physiologically relevant levels of 5-oxo-ETE are present in bronchoalveolar lavage fluid from cats with experimentally induced asthma. 5-Oxo-ETE (EC50, 0.7nM) is a much more potent activator of actin polymerization in feline eosinophils than various other eicosanoids, including leukotriene (LT) B4 and prostaglandin D2. 5-Oxo-ETE and LTB4 induce feline leukocyte migration to similar extents at low concentrations (1nM), but at higher concentrations the response to 5-oxo-ETE is much greater. Although high concentrations of selective human OXE receptor antagonists blocked 5-oxo-ETE-induced actin polymerization in feline granulocytes, their potencies were about 200 times lower than for human granulocytes. We conclude that feline leukocytes synthesize and respond to 5-oxo-ETE, which could potentially play an important role in feline asthma, a common condition in this species. The cat could serve as a useful animal model to investigate the pathophysiological role of 5-oxo-ETE.
Assuntos
Ácidos Araquidônicos/farmacologia , Asma/metabolismo , Eosinófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Actinas/genética , Actinas/metabolismo , Alérgenos/imunologia , Animais , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/metabolismo , Ácidos Araquidônicos/biossíntese , Asma/induzido quimicamente , Asma/genética , Asma/imunologia , Benzenoacetamidas/farmacologia , Benzotiazóis/farmacologia , Líquido da Lavagem Broncoalveolar/citologia , Gatos , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/imunologia , Cynodon/química , Cynodon/imunologia , Modelos Animais de Doenças , Eosinófilos/metabolismo , Eosinófilos/patologia , Feminino , Expressão Gênica , Humanos , Leucotrieno B4/farmacologia , Masculino , Neutrófilos/metabolismo , Neutrófilos/patologia , Polimerização , Cultura Primária de Células , Prostaglandina D2/farmacologia , Receptores Eicosanoides/antagonistas & inibidores , Receptores Eicosanoides/genética , Receptores Eicosanoides/metabolismoRESUMO
Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.
Assuntos
Extratos Celulares/análise , Cromatografia Líquida de Alta Pressão/métodos , Metabolômica/métodos , Espectrometria de Massas em Tandem/métodos , Extratos de Tecidos/análise , Biomarcadores/análise , Análise Química do Sangue/métodos , Linhagem Celular Tumoral , Glioblastoma/química , HumanosRESUMO
Oxidized (ox) phospholipids are receiving growing recognition as important messengers in oxidative stress signaling pathways and as endogenous electrophilic toxins that interfere with protein function through covalent modifications. Phosphatidylcholine lipids predominate in low-density lipoproteins (LDL). Our previous studies of oxLDL identified a family of biologically active oxidatively truncated phosphatidylcholines that are also present in atherosclerotic plaques. In contrast, phosphatidylethanolamine (PE) lipids are extraordinarily abundant in retina. Because photoreceptors contain the most highly unsaturated fatty acids found in vertebrate tissues, these membranes are expected to be especially susceptible to oxidative damage. Here, we report that oxidatively truncated ethanolamine phospholipids (oxPEs) are present in retina. As expected, the most abundant oxPEs, succinyl (2.2 +/- 0.8 pmol/retina) and omega-oxobutyryl (1.5 +/- 1.0 pmol/retina) esters of 2-lysophosphatidylethanolamine, are derived from the docosahexaenoyl ester, the most abundant polyunsaturated PE in retina. However, a large amount of the omega-oxononanoyl ester (1.3 +/- 0.6 pmol/retina), derived from linoleyl-PE, is also present even though linoleate is an order of magnitude less abundant than docosahexenoate in retina. There is a notable trend for the presence in retina of greater amounts, relative to the levels of their precursors, of longer chain homologous aldehydes and alkanedioate monoesters. We considered the possibility that this trend results from differences in the proclivities of various polyunsaturated fatty acyl (PUFA)-PEs to generate these homologous products. Therefore, we examined oxidative cleavage of various PUFA-PEs in small unilamellar vesicles. Alkanedioate monoesters are the major stable end products. Particularly notable is the fact that omega-oxononanoyl-PE levels either do not decline or decline less than those of the analogous aldehydes omega-oxobutyryl-PE or omega-oxovaleryl-PE during autoxidation for 33 h. The resistance of omega-oxononanoyl-PE, as compared with omega-oxobutyryl-PE and omega-oxovaleryl-PE, to further oxidation may contribute to the greater amount of this oxPE relative to its precursor, linoleyl-PE, in retina.