Sodium-glucose co-transporter 2 inhibitors (SGLT2i), when combined with metformin (COMBI), offer multi-organ protective effects in patients with type 2 diabetes (T2D), particularly those at high risk of cardiovascular or renal complications. However, the underlying molecular mechanisms remain poorly understood. We profiled 303 targeted serum metabolites in 1494 participants of the KORA study, including T2D patients treated with COMBI therapy, metformin monotherapy, or no glucose-lowering medication. Additionally, metabolomic profiling was quantified on seven tissues (plasma, liver, adrenal glands, adipose tissue, testis, lung, and cerebellum), and related hepatic transcripts were evaluated in 40 mice. Multivariable linear regression analyses, adjusted for age, sex, BMI, lifestyle, glycemic, and cardiovascular risk factors, were applied to human data; tissue-specific regression analyses were conducted for murine samples. Identified metabolites were further investigated using biochemical pathway analyses and literature review. COMBI therapy was associated with significant changes in metabolite profiles. In humans, 10 metabolites were significantly altered compared to metformin monotherapy. In mice, 82 altered metabolites were identified in plasma, 52 in liver, 30 in adrenal glands, 12 in adipose tissue, seven in testis, seven in lung, and six in cerebellum. COMBI therapy lowered threonine concentrations in both human serum and murine plasma but raised threonine, glycine, and urea cycle metabolites (citrulline, asymmetric dimethyl arginine (ADMA), and ornithine) in murine liver. This was accompanied by enhanced hepatic expression of Slc38a2, a threonine transporter gene. In humans, urea cycle metabolites correlated strongly with the fibrosis-4 index, a marker of liver fibrosis. Additionally, COMBI therapy elevated ketone body markers, such as hydroxybutyrylcarnitine, across murine liver, plasma, adrenal glands, adipose tissue, and testis. COMBI therapy modulates amino acid metabolism, the urea cycle, and ketone body production, suggesting potential mechanisms underlying its protective effects against liver fibrosis and male subfertility. These findings provide novel insights into the systemic metabolic actions of COMBI therapy and highlight its translational potential to improve clinical outcomes in T2D patients.

Purpose To compare the plasma metabolic profile of patients with a CRB1-associated inherited retinal degeneration (CRB1-IRD) with healthy controls (HCs). Design A case-control study. Participants A cohort of 30 Dutch CRB1-associated IRD patients and 29 Dutch healthy controls. Methods The MxP® Quant 500 Kit was used for measuring metabolite concentrations. We fitted a linear regression model with adjustments for age and sex based on the concentration of metabolites in μM (μmol/L), or on the sums and ratios of metabolites, to determine differences between patients and controls. Main Outcome Measures Plasma concentration of 619 metabolites. Results Over-representation of pathways among metabolites associated strongest to CRB1-IRDs (P < 0.05, n = 62) identified amino acid pathways (such as beta-alanine, histidine, and glycine/serine) and bile acid biosynthesis, driven by a decrease in deoxycholic acid derivatives produced by gut microbiota. Enrichment analysis of metabolic classes across the plasma metabolic profile further identified significant positive enrichment for lipid metabolites glycerophospholipids, cholesterol esters, and ceramides, and significant depletion for bile acid metabolites. Further investigation of the sums and ratios (i.e., metabolism indicators) ascertained a significant decrease in intestinal microbial-dependent secondary bile acid classes. Conclusions Lipid metabolic alterations and decreased microbiota-related secondary bile acid concentrations indicate significant alterations in gut metabolism in patients with a CRB1-IRD.

BACKGROUND:Childhood asthma has been linked to distinct metabolomic profiles. OBJECTIVE:We sought to identify phenotypes (metabotypes) in children with moderate to severe asthma through integrative fecal and serum metabolome analysis. METHODS:Children from the Systems Pharmacology Approach to Uncontrolled Pediatric Asthma cohort with Global Initiative for Asthma treatment step 3 or higher were recruited. Asthma control was defined by the Asthma Control Test and annual exacerbation history. Targeted metabolomic profiling of feces and serum was performed using liquid chromatography and flow injection electrospray ionization-triple quadrupole mass spectrometry. Similarity network fusion integrated fecal and serum metabolome profiles, followed by spectral clustering. Clusters were analyzed for differences in asthma characteristics, food diaries, fecal microbiota composition, and levels of serum inflammatory markers and blood cells. RESULTS:Integrative fecal and serum metabolome analysis of 92 children with moderate to severe asthma (median age, 11.5 years, 34% female) revealed 3 metabotypes. Metabotype 1 had the lowest percentage of allergic rhinitis, with elevated serum ceramides and triglycerides. Metabotype 2 had higher odds of asthma control, the highest percentage of children with 4 or more months of breast-feeding, reduced sugar intake, lowest levels of blood neutrophils and serum inflammatory markers, and elevated serum acylcarnitines and ω-3 fatty acids. Metabotype 3 included the highest percentage of uncontrolled asthma patients, with decreased serum cholesteryl esters, phosphatidylcholines, and sphingomyelins, elevated fecal amino acids, and reduced fecal microbiota diversity. CONCLUSIONS:Metabotypes in children with moderate to severe asthma are linked to asthma control, distinct fecal microbiota, and systemic inflammatory patterns. The findings suggest that metabotyping can be valuable in precision medicine approaches for asthma.

INTRODUCTION:Since the early 2000s, metabolomics has grown rapidly, becoming integral to fields like life sciences, health, and environmental research. This expansion has led to the formation of national and international societies, such as Germany's DGMet, to tackle emerging challenges. One of DGMet's goals is to improve measurement quality by assessing community needs for harmonization and standardization. A recent survey within the German-speaking community aimed to identify current practices and gaps in the use of chemical standards and reference materials, to guide future standardization efforts and collaborative initiatives. METHODS:An online survey was conducted between June 2023 and April 2024. The survey consisted of 38 key questions and was open to research institutions from Germany, Austria, and Switzerland. RESULTS:The survey was accessed by 68 laboratories, with 23 institutes providing complete or partial responses (34% response rate), which is comparable to rates reported in similar surveys within the metabolomics and lipidomics communities. Respondents were mainly experienced researchers from Germany, focusing mainly on health-related ("red") metabolomics, as indicated by 78% of the respondents, followed by microbial ("grey", 48%) and plant ("green", 39%) metabolomics (multiple answers possible). The use of targeted methods was reported more frequently (91%) than that of non-targeted methods (78%), whereas metabolite fractions studied were equally split between polar, midpolar and lipid fractions (83% each). Human (74%), mouse (61%) and Arabidopsis (30%) were the most frequently studied organisms. Most participants used synthetic chemical standards for instrument qualification (83%), calibration (78%), and metabolite identification (74%), while matrix reference materials were mainly applied for quality control (52%) and method validation (44%). There was a strong demand for more standards, especially for metabolite identification and quantification, with cost being a major barrier, particularly for isotopically labelled standards and certified reference materials. CONCLUSIONS:Valuable insights into the use of standards and reference materials within the German-speaking metabolomics community were obtained. Moving forward, the community should address critical gaps in metabolomics standardization. To achieve this, it must share its knowledge, articulate its needs clearly, and actively engage in joint efforts with national metrology institutes and international standardization initiatives.

Purpose To compare the plasma metabolic profile of patients with a CRB1 -associated inherited retinal degeneration ( CRB1 -IRD) with healthy controls (HCs).Design A case-control study.Methods Plasma concentration of 619 metabolites was measured with the MxP® Quant 500 Kit in 30 patients with a CRB1 -IRD and 29 HCs. We fitted a linear regression model with adjustments for age and sex based on the concentration of metabolites in µM (µmol/L), or on the sums and ratios of metabolites, to determine differences between patients and controls.Results Over-representation of pathways among metabolites associated strongest to CRB1 -IRDs ( P < 0.05, n = 62) identified amino acid pathways (such as beta-alanine, histidine, and glycine/serine) and bile acid biosynthesis, driven by a decrease in deoxycholic acid derivatives produced by gut microbiota. Enrichment analysis of metabolic classes across the plasma metabolic profile further identified significant positive enrichment for lipid metabolites glycerophospholipids, cholesterol esters, and ceramides, and significant depletion for bile acid metabolites. Further investigation of the sums and ratios (i.e., metabolism indicators) ascertained a significant decrease in intestinal microbial-dependent secondary bile acid classes.Conclusions Lipid metabolic alterations and decreased microbiota-related secondary bile acid concentrations indicate significant alterations in gut metabolism in patients with a CRB1 -IRD.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementBartimeus Foundation### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:This study has the approval of the Institutional Review Board of the University Medical Center Utrecht (UMCU)I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

Metabolomics and lipidomics are pivotal in understanding phenotypic variations beyond genomics. However, quantification and comparability of mass spectrometry (MS)-derived data are challenging. Standardised assays can enhance data comparability, enabling applications in multi-center epidemiological and clinical studies. Here we evaluated the performance and reproducibility of the MxP® Quant 500 kit across 14 laboratories. The kit allows quantification of 634 different metabolites from 26 compound classes using triple quadrupole MS. Each laboratory analysed twelve samples, including human plasma and serum, lipaemic plasma, NIST SRM 1950, and mouse and rat plasma, in triplicates. 505 out of the 634 metabolites were measurable above the limit of detection in all laboratories, while eight metabolites were undetectable in our study. Out of the 505 metabolites, 412 were observed in both human and rodent samples. Overall, the kit exhibited high reproducibility with a median coefficient of variation (CV) of 14.3 %. CVs in NIST SRM 1950 reference plasma were below 25 % and 10 % for 494 and 138 metabolites, respectively. To facilitate further inspection of reproducibility for any compound, we provide detailed results from the in-depth evaluation of reproducibility across concentration ranges using Deming regression. Interlaboratory reproducibility was similar across sample types, with some species-, matrix-, and phenotype-specific differences due to variations in concentration ranges. Comparisons with previous studies on the performance of MS-based kits (including the AbsoluteIDQ p180 and the Lipidyzer) revealed good concordance of reproducibility results and measured absolute concentrations in NIST SRM 1950 for most metabolites, making the MxP® Quant 500 kit a relevant tool to apply metabolomics and lipidomics in multi-center studies.

Introduction: Omentin (intelectin-1) is an adipokine produced by the stromal vascular fraction of visceral adipose tissue and has been positively associated with insulin sensitivity. The underlying mechanism of action, however, is largely unknown. It has been described that omentin may increase insulin sensitivity and glucose uptake of adipocytes, but effects on other insulin-sensitive tissues such as skeletal muscle are unexplored. We therefore investigated effects of omentin on insulin sensitivity and metabolism of primary human myotubes. Methods: Primary human myotubes were treated with 0.5 or 2 µg/mL omentin and subsequently protein detection, glucose uptake assay, lactate assay, and lipidomics analysis were performed. Results: Omentin did not affect skeletal muscle insulin signaling, as assessed by basal and insulin-stimulated phosphorylation of IRS1 and AKT. Omentin increased basal, but not insulin-stimulated glucose uptake. While increased glycolytic activity was confirmed by elevated lactate release after omentin treatment, effects on cellular lipid composition were limited to an increase in total triacylglycerol concentration. Increased glucose uptake by omentin was counteracted by addition of extracellular lactotransferrin, which can bind to omentin. Conclusions: Overall, increased basal glucose uptake in skeletal muscle cells suggests differential effects of omentin on insulin-sensitive tissues. Moreover, an involvement of lactotransferrin in omentin’s mechanism of action may partially explain contradictory results of epidemiological studies on the role of omentin in different diseases.

Until menopause, women have a lower propensity to develop metabolic diseases than men, suggestive of a protective role for sex hormones. Although a functional synergy between central actions of estrogens and leptin has been demonstrated to protect against metabolic disturbances, the underlying cellular and molecular mechanisms mediating this crosstalk have remained elusive. By using a series of embryonic, adult-onset, and tissue/cell-specific loss-of-function mouse models, we document an unprecedented role of hypothalamic Cbp/P300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 1 (Cited1) in mediating estradiol (E2)-dependent leptin actions that control feeding specifically in pro-opiomelanocortin (Pomc) neurons. We reveal that within arcuate Pomc neurons, Cited1 drives leptin's anorectic effects by acting as a co-factor converging E2 and leptin signaling via direct Cited1-ERα-Stat3 interactions. Together, these results provide new insights on how melanocortin neurons integrate endocrine inputs from gonadal and adipose axes via Cited1, thereby contributing to the sexual dimorphism in diet-induced obesity.

Adverse health effects driven by airborne particulate matter (PM) are mainly associated with reactive oxygen species formation, pro-inflammatory effects, and genome instability. Therefore, a better understanding of the underlying mechanisms is needed to evaluate health risks caused by exposure to PM. The aim of this study was to compare the genotoxic effects of two oxidizing agents (menadione and 3-chloro-1,2-propanediol) with three different reference PM (fine dust ERM-CZ100, urban dust SRM1649, and diesel PM SRM2975) on monocytic THP-1 and alveolar epithelial A549 cells. We assessed DNA oxidation by measuring the oxidized derivative 8-hydroxy-2'-deoxyguanosine (8-OHdG) following short and long exposure times to evaluate the persistency of oxidative DNA damage. Cytokinesis-block micronucleus cytome assay was performed to assess chromosomal instability, cytostasis, and cytotoxicity. Particles were characterized by inductively coupled plasma mass spectrometry in terms of selected elemental content, the release of ions in cell medium and the cellular uptake of metals. PM deposition and cellular dose were investigated by a spectrophotometric method on adherent A549 cells. The level of lipid peroxidation was evaluated via malondialdehyde concentration measurement. Despite differences in the tested concentrations, deposition efficiency, and lipid peroxidation levels, all reference PM samples caused oxidative DNA damage to a similar extent as the two oxidizers in terms of magnitude but with different oxidative DNA damage persistence. Diesel SRM2975 were more effective in inducing chromosomal instability with respect to fine and urban dust highlighting the role of polycyclic aromatic hydrocarbons derivatives on chromosomal instability. The persistence of 8-OHdG lesions strongly correlated with different types of chromosomal damage and revealed distinguishing sensitivity of cell types as well as specific features of particles versus oxidizing agent effects. In conclusion, this study revealed that an interplay between DNA oxidation persistence and chromosomal damage is driving particulate matter-induced genome instability.

Fragestellung Verschiedene humane Studien zeigten Assoziationen zwischen den Plasma-Konzentrationen des Adipokins Omentin mit Typ-2-Diabetes und kardiovaskulären Erkrankungen. Die zugrundeliegenden molekularen Mechanismen sind weitgehend unklar; jedoch konnte gezeigt werden, dass Omentin die Insulinsensitivität und die Insulin-stimulierte Glukoseaufnahme von Adipozyten erhöht. Da der Effekt von Omentin auf andere Insulin-sensitive Gewebe bisher weitgehend unbekannt ist, untersuchten wir den Effekt von Omentin auf die Insulin-Sensitivität und den Stoffwechsel von Skelettmuskelzellen.

Background: Exposure to polycyclic aromatic hydrocarbons (PAHs) has been linked to acute and chronic health effects through the suggested pathways of oxidative stress and inflammation. However, evidence is still limited. We aimed to investigate jointly the relationship of PAHs, oxidative stress, and inflammation.Methods: We measured 13 biomarkers of PAH exposure (n = 6: hydroxylated polycyclic aromatic hydrocarbons, [OH-PAHs]), oxidative stress (n = 6: malondialdehyde (MDA); 8-hydroxy-2 '-deoxyguanosine (8-OHdG); and 4 representatives of the compound class of F2 alpha-isoprostanes) in urine, and inflammation (n = 1: high-sensitivity Creactive protein, [hs-CRP]) in serum from 400 participants at the second follow-up (2013/2014) of the German KORA survey S4. Multiple linear regression models were applied to investigate the interplay between biomarkers.Results: Concentrations of biomarkers varied according to sex, age, smoking status, season, and a history of obesity, diabetes, or chronic kidney disease. All OH-PAHs were significantly and positively associated with oxidative stress biomarkers. An interquartile range (IQR) increase in sum OH-PAHs was associated with a 13.3% (95% CI: 9.9%, 16.9%) increase in MDA, a 6.5% (95% CI: 3.5%, 9.6%) increase in 8-OHdG, and an 8.4% (95% CI: 6.6%, 11.3%) increase in sum F2 alpha-isoprostanes. Associations were more pronounced between OH-PAHs and F2 alpha isoprostanes but also between OH-PAHs and 8-OHdG for participants with potential underlying systemic inflammation (hs-CRP >= 3 mg/L). We observed no association between OH-PAHs and hs-CRP levels. While 8OHdG was significantly positively associated with hs-CRP (13.7% [95% CI: 2.2%, 26.5%] per IQR increase in 8-OHdG), F2 alpha-isoprostanes and MDA indicated only a positive or null association, respectively.Conclusion: The results of this cross-sectional study suggest, at a population level, that exposure to PAHs is associated with oxidative stress even in a low exposure setting. Oxidative stress markers, but not PAHs, were associated with inflammation. Individual risk factors were important contributors to these processes and should be considered in future studies. Further longitudinal studies are necessary to investigate the causal chain of the associations.

Pharmaceutically active compounds have increasingly been detected in groundwater worldwide. Despite constituting a risk for human health and ecosystems, their fate in the environment has still not been exhaustively investigated. This study characterizes the transport behavior of five selected pharmaceutically active compounds (antipyrine, atenolol, caffeine, carbamazepine and sulfamethoxazole) in two sediments (coarse quartz sand and sandy loam) using column experiments with long-term injection of spiked groundwater. Transport parameters were estimated using an analytical reactive transport model. When five selected compounds were injected simultaneously, transport behavior of antipyrine, carbamazepine and the antibiotic sulfamethoxazole were similar to the conservative tracer in both sediments and under varying redox conditions. Atenolol and caffeine were retarded significantly stronger in the sandy loam sediment than in the coarse quartz sand. Biodegradation of caffeine was observed in both sediments after an adaption period and depended on dissolved oxygen. The identification of biodegradation processes was supported by monitoring of intracellular adenosine triphosphate (ATPitc) as a measure for microbial activity. ATPitc was present in varying concentrations in all sediments and was highest when biodegradation of pharmaceuticals, especially caffeine, was observed. When only caffeine and sulfamethoxazole were injected simultaneously, sulfamethoxazole was degraded while caffeine degradation was reduced. The latter seemed to be influenced by low concentrations in dissolved oxygen rather than the presence of the antibiotic sulfamethoxazole. Results of these experiments emphasize the impact on pharmaceutical sorption and (bio)degradation of sediment type and redox conditions, as well as available time for microbial adaption and the combination of pharmaceuticals that are released together into groundwater.

The aim of this study was to explore the impact of three different standard reference particulate matter (ERMCZ100, SRM-1649, and SRM-2975) on epigenetic DNA modifications including cytosine methylation, cytosine hydroxymethylation, and adenine methylation. For the determination of low levels of adenine methylation, we developed and applied a novel DNA nucleobase chemical derivatization and combined it with liquid chromatography tandem mass spectrometry. The developed method was applied for the analysis of epigenetic modifications in monocytic THP-1 cells exposed to the three different reference particulate matter for 24 h and 48 h. The mass fraction of epigenetic active elements As, Cd, and Cr was analyzed by inductively coupled plasma mass spectrometry. The exposure to fine dust ERM-CZ100 and urban dust SRM-1649 decreased cytosine methylation after 24 h exposure, whereas all 3 p.m. increased cytosine hydoxymethylation following 24 h exposure, and the epigenetic effects induced by SRM-1649 and diesel SRM-2975 were persistent up to 48 h exposure. The road tunnel dust ERM-CZ100 significantly increased adenine methylation following the shorter exposure time. Twodimensional scatters analysis between different epigenetic DNA modifications were used to depict a significantly negative correlation between cytosine methylation and cytosine hydroxymethylation supporting their possible functional relationship. Metals and polycyclic aromatic hydrocarbons differently shapes epigenetic DNA modifications.

Polycyclic aromatic hydrocarbons (PAHs) are a group of > 100 carcinogenic compunds emitted during combustion of fuels and other materials. In this work, distribution, sources and fate of twelve PAHs: phenanthrene (Phe), anthracene (Ant), fluoranthene (Fla), pyrene (Pyr), benz[a]anthracene (Baa), chrysene (Cry), benzo[b]fluoranthene (Bbf), benzo[k]fluoranthene (Bkf), benzo[a]pyrene (Bap), dibenz[a,h]anthracene (Dba), benzo[ghi]perylene (Bgh) and indeno[1,2,3-cd]pyrene (Ind) in the air, road dust and sediment of the most industrilized area of central India are described. The PAHs contents in the ambient air (n = 24) during year 2007-08 were ranged from 0.04 – 0.17 μg/m3 with mean value of 0.09±0.02 μg/m3, respectively. The highest mass concentration was observed in the winter season, December – January. The concentration of the PAHs in the PM10, road dust (n = 8) and sediment (n = 10) of Raipur city was ranged from 238 – 467, 8.7 – 21.7 and 6.8 – 10.9 mg/kg with mean value of 342, 12.7 and 9.2 mg/kg. The vehicular emissions and coal/biomass comustion were apportioned as main sources for relase of the PAHs in the environment. The spatial (residential, commercial and industrial), seasonal (summer, rainy, autumn and winter) and temporal (2007-2015) variations of the PAHs in the environment of the central India are discussed. Biography: Khageshwar Singh Patel has completed his PhD from Pt. Ravishankar Shukla University, Raipur, India and postdoctoral studies from TU, Darmstadt, Germany. He is a Professor at the Amity University, Raipur. He has published more than 150 papers in reputed journals, and supervised 34 PhD students.

Steroids regulate many physiological processes. Hydroxysteroid dehydrogenases (HSDs) modulate the levels of steroids in pre- and post-receptor metabolism. The subfamily of 20β-HSD type 2 currently comprises six members from six different species. The zebrafish ortholog converts cortisone to 20β-dihydrocortisone and is involved in the catabolism of the stress hormone cortisol. Here, we elucidated the substrate preferences of all 20β-HSD type 2 enzymes towards a selected panel of steroids. For quantification of the substrates and their respective 20β-reduced products, we first developed and validated a liquid chromatography-mass spectrometry based method. Applying this method to activity assays with recombinantly expressed enzymes, our findings indicate that the 20β-HSD type 2 enzymes catalyze the 20β-reduction of a plethora of steroids of the glucocorticoid biosynthesis pathway. The observed multispecificity among the homologous 20β-HSD type 2 enzymes implies different physiological roles in different species.

Differentiation of preadipocytes into mature adipocytes is a highly complex cellular process. At lipidome level, the adipogenesis remains poorly characterized. To investigate the lipidomic changes during human adipogenesis, we used the LipidyzerTM assay, which quantified 743 lipid species from 11 classes. The undifferentiated human SGBS cell strain showed a heterogeneous lipid class composition with the most abundant classes, phosphatidylethanolamines (PE), phosphatidylcholines (PC), and sphingomyelins (SM). The differentiation process was accompanied by increased ceramide concentrations. After completion of differentiation around day 4, massive lipid remodeling occurred during maturation, characterized by substantial synthesis of diacylglycerols (DAG), lysophosphatidylethanolamines (LPE), PC, PE, SM, and triacylglycerols (TAG). Lipid species composition became more homogeneous during differentiation to highly concentrated saturated and monounsaturated long-chain fatty acids (LCFA), with the four most abundant being C16:0, C16:1, C18:0, and C18:1. Simultaneously, the amount of polyunsaturated and very long-chain fatty acids (VLCFA) markedly decreased. High negative correlation coefficients between PE and PC species containing VLCFA and TAG species as well as between ceramides and SM imply that PE, PC, and ceramides might have served as additional sources for TAG and SM synthesis, respectively. These results highlight the enormous remodeling at the lipid level over several lipid classes during adipogenesis.

The development of chronic obstructive pulmonary disease (COPD) pathogenesis remains unclear, but emerging evidence supports a crucial role for inducible bronchus-associated lymphoid tissue (iBALT) in disease progression. Mechanisms underlying iBALT generation, particularly during chronic CS exposure, remain to be defined. Oxysterol metabolism of cholesterol is crucial to immune cell localization in secondary lymphoid tissue. Here, we demonstrate that oxysterols also critically regulate iBALT generation and the immune pathogenesis of COPD. In both COPD patients and cigarette smoke (CS)-exposed mice, we identified significantly upregulated CH25H and CYP7B1 expression in airway epithelial cells, regulating CS-induced B-cell migration and iBALT formation. Mice deficient in CH25H or the oxysterol receptor EBI2 exhibited decreased iBALT and subsequent CS-induced emphysema. Further, inhibition of the oxysterol pathway using clotrimazole resolved iBALT formation and attenuated CS-induced emphysema invivo therapeutically. Collectively, our studies are the first to mechanistically interrogate oxysterol-dependent iBALT formation in the pathogenesis of COPD, and identify a novel therapeutic target for the treatment of COPD and potentially other diseases driven by the generation of tertiary lymphoid organs.

A high-performance liquid chromatographic (HPLC) method with integrated solid-phase extraction for the determination of 1-hydroxypyrene and 1-, 2-, 3-, 4- and 9-hydroxyphenanthrene in urine was developed and validated. After enzymatic treatment and centrifugation of 500 μL urine, 100 μL of the sample was directly injected into the HPLC system. Integrated solid-phase extraction was performed on a selective, copper phthalocyanine modified packing material. Subsequent chromatographic separation was achieved on a pentafluorophenyl core-shell column using a methanol gradient. For quantification, time-programmed fluorescence detection was used. Matrix-dependent recoveries were between 94.8 and 102.4%, repeatability and reproducibility ranged from 2.2 to 17.9% and detection limits lay between 2.6 and 13.6 ng/L urine. A set of 16 samples from normally exposed adults was analyzed using this HPLC-fluorescence detection method. Results were comparable with those reported in other studies. The chromatographic separation of the method was transferred to an ultra-high-performance liquid chromatography pentafluorophenyl core-shell column and coupled to a high-resolution time-of-flight mass spectrometer (HR-TOF-MS). The resulting method was used to demonstrate the applicability of LC-HR-TOF-MS for simultaneous target and suspect screening of monohydroxylated polycyclic aromatic hydrocarbons in extracts of urine and particulate matter.