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Obesity, a global public health concern, is influenced by various factors, including genetic predispositions. Although many obesity-associated genes have been identified through genome-wide association studies (GWAS), the molecular mechanisms linking these genes to adipose tissue function remain largely unexplored. This study integrates proteomic data on adipocyte fat accumulation with GWAS data on obesity to unravel the roles of the identified key candidate genes — G protein subunit beta 1 ( GNB1 ) and scavenger receptor class B member 2 ( SCARB2 ) — involved in fat accumulation. We utilized RNA interference to knock down GNB1 and SCARB2 in human subcutaneous adipocytes, followed by lipidome and proteome analyses using mass spectrometry. Knockdown of these genes resulted in a reduction in lipid droplet accumulation, indicating their role in adipocyte lipid storage. Digital PCR confirmed effective gene knockdown, with GNB1 and SCARB2 mRNA levels significantly reduced. In total, the lipidomic analysis identified 96 lipid species with significant alterations. GNB1 knockdown resulted in a decrease in cholesterol esters and an increase in phosphatidylcholines, phosphatidylinositols, and ceramides. SCARB2 knockdown also led to an increase in phosphatidylcholines, with a trend towards decreased triacylglycerols. Proteomic analysis revealed significant changes in proteins involved in lipid metabolism and adipocyte function, including PLPP1 and CDH13, which were upregulated following GNB1 knockdown, and HSPA8, which was downregulated. Conversely, SCARB2 knockdown resulted in the downregulation of PLPP1 and METTL7A, and the upregulation of PLIN2, HSPA8, NPC2, and SQSTM1. Our findings highlight the significant roles of GNB1 and SCARB2 in lipid metabolism and adipocyte function, providing insights that could inform therapeutic strategies targeting these regulatory genes in obesity.

Purpose Quantification of olanzapine (OLZ) and its metabolites such as N -desmethylolanzapine (DM-O), 2-hydroxymethylolanzapine (2H-O) and olanzapine N -oxide (NO-O) in five kinds of human body fluids including whole blood by liquid chromatography (LC)–tandem mass spectrometry (MS/MS) has been presented; the quantification methods were carefully devised and validated using the matrix-matched calibration and standard addition methods. Methods OLZ and its three metabolites were extracted from 40 μL each of body fluids by two-step liquid–liquid separations. The samples and reagents were pre-cooled in a container filled with ice for the extraction because of the thermal instability of OLZ and its three metabolites especially in whole blood. Results The limits of quantification (LOQs) of OLZ and 2H-O were 0.05 ng/mL and those of DM-O and NO-O were 0.15 ng/mL in whole blood and urine, respectively. The concentrations of OLZ and its metabolites in heart whole blood, pericardial fluid, stomach contents, bile and urine were determined for two cadavers and those in whole blood and urine for the other two cadavers. The reduction from NO-O to OLZ was observed at 25 ℃ in whole blood in vitro. Conclusions To our knowledge, this is the first report on the quantification of metabolites of olanzapine in the authentic human body fluids by LC–MS/MS as well as on the confirmation of in vitro reduction from NO-O to OLZ in whole blood that seems to have induced the quick decrease of NO-O.

In patients with unresectable non-small cell lung cancer, histological diagnosis is frequently based on small biopsy specimens unsuitable for histological diagnosis when they are severely crushed and do not retain their morphology. Therefore, establishing a novel diagnostic method independent of tissue morphology or conventional immunohistochemistry (IHC) markers is required. We analyzed the lipid profiles of resected primary lung adenocarcinoma (ADC) and squamous cell carcinoma (SQCC) specimens using liquid chromatography-tandem mass spectrometry. The specimens of 26 ADC and 18 SQCC cases were evenly assigned to the discovery and validation cohorts. Non-target screening on the discovery cohort identified 96 and 13 lipid peaks abundant in ADC and SQCC, respectively. Among these 109 lipid peaks, six and six lipid peaks in ADC and SQCC showed reproducibility in target screening on the validation cohort. Finally, we selected three and four positive lipid markers for ADC and SQCC, demonstrating high discrimination abilities. In cases difficult to diagnose by IHC staining, [cardiolipin(18:2_18:2_18:2_18:2)-H] − and [triglyceride(18:1_17:1_18:1) + NH4] + showed the excellent diagnostic ability for ADC (sensitivity: 1.00, specificity: 0.89, accuracy: 0.93) and SQCC (sensitivity: 0.89, specificity: 0.83, accuracy: 0.87), respectively. These novel candidate lipid markers may contribute to a more accurate diagnosis and subsequent treatment strategy for unresectable NSCLC.

Background To reduce disease recurrence after radical surgery for lung squamous cell carcinomas (SQCCs), accurate prediction of recurrent high-risk patients is required for efficient patient selection for adjuvant chemotherapy. Because treatment modalities for recurrent lung SQCCs are scarce compared to lung adenocarcinomas (ADCs), accurately selecting lung SQCC patients for adjuvant chemotherapy after radical surgery is highly important. Predicting lung cancer recurrence with high objectivity is difficult with conventional histopathological prognostic factors; therefore, identification of a novel predictor is expected to be highly beneficial. Lipid metabolism alterations in cancers are known to contribute to cancer progression. Previously, we found that increased sphingomyelin (SM)(d35:1) in lung ADCs is a candidate for an objective recurrence predictor. However, no lipid predictors for lung SQCC recurrence have been identified to date. This study aims to identify candidate lipid predictors for lung SQCC recurrence after radical surgery. Methods Recurrent ( n  = 5) and non-recurrent ( n  = 6) cases of lung SQCC patients who underwent radical surgery were assigned to recurrent and non-recurrent groups, respectively. Extracted lipids from frozen tissue samples of primary lung SQCC were analyzed by liquid chromatography-tandem mass spectrometry. Candidate lipid predictors were screened by comparing the relative expression levels between the recurrent and non-recurrent groups. To compare lipidomic characteristics associated with recurrent SQCCs and ADCs, a meta-analysis combining SQCC ( n  = 11) and ADC ( n  = 20) cohorts was conducted. Results Among 1745 screened lipid species, five species were decreased (≤ 0.5 fold change; P  < 0.05) and one was increased (≥ 2 fold change; P  < 0.05) in the recurrent group. Among the six candidates, the top three final candidates (selected by AUC assessment) were all decreased SM(t34:1) species, showing strong performance in recurrence prediction that is equivalent to that of histopathological prognostic factors. Meta-analysis indicated that decreases in a limited number of SM species were observed in the SQCC cohort as a lipidomic characteristic associated with recurrence, in contrast, significant increases in a broad range of lipids (including SM species) were observed in the ADC cohort. Conclusion We identified decreased SM(t34:1) as a novel candidate predictor for lung SQCC recurrence. Lung SQCCs and ADCs have opposite lipidomic characteristics concerning for recurrence risk. Trial registration This retrospective study was registered at the UMIN Clinical Trial Registry ( UMIN000039202 ) on January 21, 2020.

Purpose The quantification of parent molecules of pyrethroids tetramethrin and resmethrin in human specimens by a mass spectrometry (MS) technique has not been reported yet. A woman in her 60s was found dead in a wasteland. At the scene, an empty beer can and a spray for insecticides containing tetramethrin and resmethrin were found. Therefore, the concentrations of tetramethrin and resmethrin in postmortem specimens and the methanol solution used for rinsing the inside of the beer can were determined using liquid chromatography (LC)–tandem mass spectrometry (MS/MS). Methods The quantification method by LC–MS/MS for intact parent molecules of tetramethrin and resmethrin in whole blood and urine has been devised and validated in this work. The method was applied to the quantification of tetramethrin and resmethrin in whole blood, urine and stomach contents obtained from a cadaver at autopsy. Results The limits of detection of tetramethrin and resmethrin were 0.06 and 0.03 ng/mL; limits of quantification were 0.2 and 0.1 ng/mL in blood and urine, respectively. The concentrations of tetramethrin of the deceased were 11.1 ± 1.2 and 0.425 ± 0.017 ng/mL for stomach contents and urine, respectively; the concentration of resmethrin in stomach contents was 1.77 ± 0.18 ng/mL. The tetramethrin and resmethrin were unstable in blood and urine at room temperature; they should be kept at not higher than 4 ℃. Conclusions To our knowledge, this is the first report for quantification of unchanged tetramethrin and resmethrin in human specimens obtained in a fatal case.

Background The risk of postoperative recurrence is higher in lung cancer patients who smoke than non-smokers. However, objective evaluation of the postoperative recurrence risk is difficult using conventional pathological prognostic factors because of their lack of reproducibility. Consequently, novel objective biomarkers that reflect postoperative risk in lung cancer patients who smoke must be identified. Because cigarette smoking and oncogenesis alter lipid metabolism in lung tissue, we hypothesized that the lipid profiles in lung cancer tissues are influenced by cigarette smoking and can reflect the postoperative recurrence risk in smoking lung cancer patients. This study aimed to identify lipid biomarkers that reflect the smoking status and the postoperative recurrence risk. Methods Primary tumor tissues of lung adenocarcinoma (ADC) ( n  = 26) and squamous cell carcinoma (SQCC) ( n  = 18) obtained from surgery were assigned to subgroups according to the patient’s smoking status. The ADC cohort was divided into never smoker and smoker groups, while the SQCC cohort was divided into moderate smoker and heavy smoker groups. Extracted lipids from the tumor tissues were subjected to liquid chromatography-tandem mass spectrometry analysis. Lipids that were influenced by smoking status and reflected postoperative recurrence and pathological prognostic factors were screened. Results Two and 12 lipid peaks in the ADC and SQCC cohorts showed a significant positive correlation with the Brinkman index, respectively. Among them, in the ADC cohort, a higher lipid level consisted of three phosphatidylcholine (PC) isomers, PC (14:0_18:2), PC (16:1_16:1), and PC (16:0_16:2), was associated with a shorter recurrence free period (RFP) and a greater likelihoods of progressed T-factor (≥ pT2) and pleural invasion. In the SQCC cohort, a lower m/z 736.5276 level was associated with shorter RFP and greater likelihood of recurrence. Conclusions From our data, we propose three PC isomers, PC (14:0_18:2), PC (16:1_16:1), and PC (16:0_16:2), and a lipid peak of m/z 736.5276 as novel candidate biomarkers for postoperative recurrence risk in lung ADC and SQCC patients who are smokers.

Purpose The aim of this study is to investigate the stabilities of the 24 synthetic cannabinoid metabolites (SCMs) in blood and urine at various temperatures from − 30 to 37 ℃ stored for 1–168 days. In addition, experiments of stabilities at lower temperatures and for much longer duration have been performed as described below. Methods The quantification was performed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The blank blood and urine spiked with SCMs and non-spiked real case (authentic) specimens were incubated at 37 ℃ up to 56 days and at 22, 4 or – 30 ℃ up to 168 days. The non-spiked authentic blood and urine specimens were also stored at − 30 or – 80 ℃ for 1, 3 or 5 years to investigate stabilities during very long time frames. Results All the 24 SCMs were much more stable in urine than in blood at 37, 22 or 4 ℃. All 24 SCMs spiked into blood or urine were stable at – 30 ℃ for up to 168 days. The 6 SCMs in the authentic specimens exhibited long stabilities at − 30 or – 80 ℃ for 3–5 years. Some tendencies were observed according to the relation between the structures of SCMs and their stabilities. Conclusions The long-term stabilities of 24 SCMs in spiked samples and those of 6 SCMs in the authentic specimens were examined using LC–MS/MS. SCMs were largely very stable and usable several years after storage at − 30 or – 80 ℃.

Purpose A synthetic cannabinoid BB-22 and its metabolite BB-22 3-carboxyindole have not yet been quantified in human urine. The aim of this study is to establish a sensitive analytical method for the quantification of BB-22 and its 3-carboxyindole in human serum and urine specimens, and the characterization of the unreported metabolites of BB-22 in authentic urine specimens from three individuals. Methods These compounds were extracted from β-glucuronide-hydrolyzed and unhydrolyzed urine and/or serum via liquid–liquid extraction. The identification and quantification were performed using liquid chromatography (LC)–QTRAP-tandem mass spectrometry (MS/MS) and the characterization of the new metabolites was made by high-resolution LC–MS/MS. Results The limits of detection of BB-22 and BB-22 3-carboxyindole were 3 and 30 pg/mL in urine, respectively. The devised method was applied to quantify these compounds in authentic serum and urine obtained from two drug abusers and in urine from one drug abuser. The serum levels of BB-22 were 149 and 6680 pg/mL, and those of BB-22 3-carboxyindole were 0.755 and 38.0 ng/mL in cases 1 and 2, respectively. The urine levels of BB-22 were 5.64, 5.52 and 6.92 pg/mL and those of BB-22 3-carboxyindole were 0.131, 21.4 and 5.15 ng/mL in cases 1, 2 and 3, respectively. New monohydroxyl metabolites retaining the structure of BB-22 were found in the urine specimens. Conclusions The synthetic cannabinoid BB-22 and its metabolite BB-22 3-carboxyindole were identified and quantified in authentic human serum and urine specimens for the first time, and new metabolites of BB-22 were tentatively identified in authentic urine specimens obtained from three drug users in this study.

The genomic regions containing PNPLA3, SAMM50 and PARVB are susceptibility loci for the development and progression of nonalcoholic fatty liver disease (NAFLD). In order to search for all common variations in this region, we amplified the genomic DNA of 28 NAFLD patients by long-range PCR, covering the entire susceptibility region and sequenced the DNA using indexed multiplex next-generation sequencing. We found 329 variations, including four novel variations. Fine mapping of variations including insertion/deletions was performed for 540 NAFLD patients (488 with nonalcoholic steatohepatitis (NASH) and 52 with simple steatosis) and 1012 control subjects. HaploView analysis showed that linkage disequilibrium (LD) block 1 and 2 occurred in PNPLA3, block 3 in SAMM50 and block 4 in PARVB. Variations in LD blocks 1-4 were significantly associated with NAFLD as compared with control subjects (P<1 × 10(-8)). Variations in LD block 2 were significantly associated with the NAFLD activity score (NAS), aspartate aminotransferase and alanine aminotransferase. Variations in LD block 1 were significantly associated with the fibrosis stage. The strongest associations were observed for variations in LD block 4, with NASH as compared with simple steatosis (P=7.1 × 10(-6)) and NAS (P=3.4 × 10(-6)). Our results suggested that variations, including insertion/deletions, in PARVB, as well as those in PNPLA3, are important in the progression of NAFLD.

Purpose The aim of this study is to characterize the metabolites of EAM-2201 in human hepatocytes obtained in vitro and those in liver and urine specimens obtained in vivo from the autopsy of an EAM-2201 abuser. Methods EAM-2201 was incubated with human hepatocytes for 3 h in a CO 2 incubator and the metabolites of EAM-2201 were produced. The human liver specimen was homogenized and the metabolites were extracted. The urine specimen was hydrolyzed first with β-glucuronidase and the metabolites were extracted. The tentative detection and identification of those metabolites were performed using liquid chromatography (LC)–quadrupole-ion trap tandem mass spectrometry (MS/MS) and high-resolution LC–Orbitrap-MS/MS, respectively. Results Twelve metabolites of EAM-2201 could be characterized in hepatocytes, liver and urine. They were produced by defluorination, hydroxylation, carboxylation, dehydrogenation, N -dealkylation and/or glucuronidation of EAM-2201. The most detectable metabolite obtained from the in vitro hepatocyte incubation was that monohydroxylated at the 4-ethylnaphthalene moiety of EAM-2201 after dehydrogenation. However, only a trace amount of the same metabolite was detected in the liver tissue obtained postmortem from an EAM-2201 abuser. In the liver tissue, the highest metabolite was 5-carboxypentyl derivative of EAM-2201, which was monohydroxylated at the 4-ethylnaphthalene moiety, followed by monohydroxylpentyl and monohydroxy-4-ethylnaphthalenyl derivative of EAM-2201 without dehydrogenation. Conclusions Twelve metabolites of a synthetic cannabinoid, EAM-2201, were tentatively identified in human hepatocytes in vitro, and in human liver and urine specimens in vivo for the first time. There was a distinct difference in metabolism profile between the in vitro and in vivo results.