Untargeted Analysis Research Articles

#2194 Targeted metabolomics analysis identifies a specific metabolomic profile in patients with early chronic kidney disease

Abstract Background and Aims The complex interconnections between various metabolites and the kidney play a major role in the pathogenesis of chronic kidney disease (CKD). Hence, by the assessment of the relationship between metabolites and the kidney, more accurate biomarkers could be discovered to prevent and detect CKD at an early stage. The primary goal of this study was to find new serum and urine biomarkers for the diagnosis of early CKD by conducting a metabolomic profiling through targeted metabolomics of both blood and urine in patients with CKD. Method This cross-sectional study comprised 80 patients with CKD who were categorized into six subgroups (G1-G6) based on their eGFR according to the KDIGO Guidelines. Additionally, 20 healthy individuals were included as healthy control participants (group C). Ultra-high-performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry was used to perform serum and urine metabolomic profiling. The 6 metabolites evaluated by this study were selected based on the results of a previous untargeted analysis research. Both blood and urine samples underwent multivariate analysis, which was subsequently followed by univariate analysis and quantitative analysis. Initially, the PLSDA score plot and VIP score were utilized. The application of the cross-validation procedure to the initial set of 6 molecules provided outstanding results, including high accuracy, high R² values, and a significant Q2 value. Hence, the model might be deemed as predictive. Random Forest analysis was utilized to conduct biomarker analysis and prediction. The specificity and sensitivity of the compounds identified as possible biomarkers were assessed using the Receiver Operating Characteristic (ROC) curve and the area under the curve (AUC). Results There were strong positive correlations between eGFR and serum levels of Arginine (p < 0.05) and L-Acetylcarnitine (p < 0.05). Of note, the amounts of L-Methionine, L-Phenylalanine, Kynurenic acid, and Indoxyl sulfate in serum showed a progressive increase from group C to subgroups G1-G5. Furthermore, L-Methionine, L-Phenylalanine, Kynurenic acid, and Indoxyl sulfate exhibited a negative correlation with eGFR (p < 0.05). In addition, there was a positive correlation between the levels of urine Arginine, L-Methionine, and L-Phenylalanine and eGFR (p < 0.05). Urine levels of Kynurenic acid, Indoxyl sulfate, and L-Acetylcarnitine were higher in groups G4 and G5, as compared to groups G1-G3b and C, respectively. The urinary concentrations of Indoxyl sulfate, Kynurenic acid, Indoxyl sulfate, and L-Acetylcarnitine exhibited a negative correlation with eGFR (p < 0.05). Conclusion The current study shows that serum and urinary metabolites levels in patients with CKD display an alteration in production, secretion, and excretion in all CKD stages. There was demonstrated a distinct metabolomic profile in the course of CKD which points to novel biomarkers useful for the diagnosis and progression evaluation of CKD from its early stages.

Heat stress-induced dysbiosis of the gut microbiota impairs spermatogenesis by regulating secondary bile acid metabolism in the gut

Heat stress (HS) poses a substantial challenge to livestock. Studies have demonstrated that HS reduces fertility and leads to gut microbiota dysbiosis in bulls. However, the impact of the gut microbiota on fertility in bulls during HS is still unclear. Our research revealed that HS exposure decreased semen quality in bulls, and fecal microbiota transplantation (FMT) from heat-stressed bulls to recipient mice resulted in a significant decrease in number of testicular germ cells and epididymal sperm. Untargeted metabolomics methodology and 16S rDNA sequencing conjoint analysis revealed that Akkermansia muciniphila (A. muciniphila) seemed to be a key bacterial regulator of spermatogenesis after HS exposure. Moreover, the research indicated that A. muciniphila regulated secondary bile acid metabolism by promoting the colonization of bile salt hydrolase (BSH)-metabolizing bacteria, leading to increase of retinol absorption in the host gut and subsequently elevation of testicular retinoic acid level, thereby improving spermatogenesis. This study sheds light on the relationship between HS-induced microbiota dysbiosis and spermatogenesis, offering a potential therapeutic approach for addressing bull spermatogenic dysfunction triggered by HS exposure.

Differential impact of sex on regulation of skeletal muscle mitochondrial function and protein homeostasis by hypoxia-inducible factor-1α in normoxia.

Hypoxia-inducible factor (HIF)-1α is continuously synthesized and degraded in normoxia. During hypoxia, HIF1α stabilization restricts cellular/mitochondrial oxygen utilization. Cellular stressors can stabilize HIF1α even during normoxia. However, less is known about HIF1α function(s) and sex-specific effects during normoxia in the basal state. Since skeletal muscle is the largest protein store in mammals and protein homeostasis has high energy demands, we determined HIF1α function at baseline during normoxia in skeletal muscle. Untargeted multiomics data analyses were followed by experimental validation in differentiated murine myotubes with loss/gain of function and skeletal muscle from mice without/with post-natal muscle-specific Hif1a deletion (Hif1amsd). Mitochondrial oxygen consumption studies using substrate, uncoupler, inhibitor, titration protocols; targeted metabolite quantification by gas chromatography-mass spectrometry; and post-mitotic senescence markers using biochemical assays were performed. Multiomics analyses showed enrichment in mitochondrial and cell cycle regulatory pathways in Hif1a deleted cells/tissue. Experimentally, mitochondrial oxidative functions and ATP content were higher with less mitochondrial free radical generation with Hif1a deletion. Deletion of Hif1a also resulted in higher concentrations of TCA cycle intermediates and HIF2α proteins in myotubes. Overall responses to Hif1amsd were similar in male and female mice, but changes in complex II function, maximum respiration, Sirt3 and HIF1β protein expression and muscle fibre diameter were sex-dependent. Adaptive responses to hypoxia are mediated by stabilization of constantly synthesized HIF1α. Despite rapid degradation, the presence of HIF1α during normoxia contributes to lower mitochondrial oxidative efficiency and greater post-mitotic senescence in skeletal muscle. In vivo responses to HIF1α in skeletal muscle were differentially impacted by sex. KEY POINTS: Hypoxia-inducible factor -1α (HIF1α), a critical transcription factor, undergoes continuous synthesis and proteolysis, enabling rapid adaptive responses to hypoxia by reducing mitochondrial oxygen consumption. In mammals, skeletal muscle is the largest protein store which is determined by a balance between protein synthesis and breakdown and is sensitive to mitochondrial oxidative function. To investigate the functional consequences of transient HIF1α expression during normoxia in the basal state, myotubes and skeletal muscle from male and female mice with HIF1α knockout were studied using complementary multiomics, biochemical and metabolite assays. HIF1α knockout altered the electron transport chain, mitochondrial oxidative function, signalling molecules for protein homeostasis, and post-mitotic senescence markers, some of which were differentially impacted by sex. The cost of rapid adaptive responses mediated by HIF1α is lower mitochondrial oxidative efficiency and post-mitotic senescence during normoxia.

Untargeted lipidomics analysis in women with morbid obesity and type 2 diabetes mellitus: A comprehensive study.

There is a phenotype of obese individuals termed metabolically healthy obese that present a reduced cardiometabolic risk. This phenotype offers a valuable model for investigating the mechanisms connecting obesity and metabolic alterations such as Type 2 Diabetes Mellitus (T2DM). Previously, in an untargeted metabolomics analysis in a cohort of morbidly obese women, we observed a different lipid metabolite pattern between metabolically healthy morbid obese individuals and those with associated T2DM. To validate these findings, we have performed a complementary study of lipidomics. In this study, we assessed a liquid chromatography coupled to a mass spectrometer untargeted lipidomic analysis on serum samples from 209 women, 73 normal-weight women (control group) and 136 morbid obese women. From those, 65 metabolically healthy morbid obese and 71 with associated T2DM. In this work, we find elevated levels of ceramides, sphingomyelins, diacyl and triacylglycerols, fatty acids, and phosphoethanolamines in morbid obese vs normal weight. Conversely, decreased levels of acylcarnitines, bile acids, lyso-phosphatidylcholines, phosphatidylcholines (PC), phosphatidylinositols, and phosphoethanolamine PE (O-38:4) were noted. Furthermore, comparing morbid obese women with T2DM vs metabolically healthy MO, a distinct lipid profile emerged, featuring increased levels of metabolites: deoxycholic acid, diacylglycerol DG (36:2), triacylglycerols, phosphatidylcholines, phosphoethanolamines, phosphatidylinositols, and lyso-phosphatidylinositol LPI (16:0). To conclude, analysing both comparatives, we observed decreased levels of deoxycholic acid, PC (34:3), and PE (O-38:4) in morbid obese women vs normal-weight. Conversely, we found elevated levels of these lipids in morbid obese women with T2DM vs metabolically healthy MO. These profiles of metabolites could be explored for the research as potential markers of metabolic risk of T2DM in morbid obese women.

To weight or not to weight? The effect of selection bias in 3 large electronic health record-linked biobanks and recommendations for practice.

To develop recommendations regarding the use of weights to reduce selection bias for commonly performed analyses using electronic health record (EHR)-linked biobank data. We mapped diagnosis (ICD code) data to standardized phecodes from 3 EHR-linked biobanks with varying recruitment strategies: All of Us (AOU; n = 244071), Michigan Genomics Initiative (MGI; n = 81243), and UK Biobank (UKB; n = 401167). Using 2019 National Health Interview Survey data, we constructed selection weights for AOU and MGI to represent the US adult population more. We used weights previously developed for UKB to represent the UKB-eligible population. We conducted 4 common analyses comparing unweighted and weighted results. For AOU and MGI, estimated phecode prevalences decreased after weighting (weighted-unweighted median phecode prevalence ratio [MPR]: 0.82 and 0.61), while UKB estimates increased (MPR: 1.06). Weighting minimally impacted latent phenome dimensionality estimation. Comparing weighted versus unweighted phenome-wide association study for colorectal cancer, the strongest associations remained unaltered, with considerable overlap in significant hits. Weighting affected the estimated log-odds ratio for sex and colorectal cancer to align more closely with national registry-based estimates. Weighting had a limited impact on dimensionality estimation and large-scale hypothesis testing but impacted prevalence and association estimation. When interested in estimating effect size, specific signals from untargeted association analyses should be followed up by weighted analysis. EHR-linked biobanks should report recruitment and selection mechanisms and provide selection weights with defined target populations. Researchers should consider their intended estimands, specify source and target populations, and weight EHR-linked biobank analyses accordingly.

Lipidomic studies revealing serological markers associated with the occurrence of retinopathy in type 2 diabetes

PurposeThe duration of type 2 diabetes mellitus (T2DM) and blood glucose levels have a significant impact on the development of T2DM complications. However, currently known risk factors are not good predictors of the onset or progression of diabetic retinopathy (DR). Therefore, we aimed to investigate the differences in the serum lipid composition in patients with T2DM, without and with DR, and search for potential serological indicators associated with the development of DR.MethodsA total of 622 patients with T2DM hospitalized in the Department of Endocrinology of the First Affiliated Hospital of Xi’an JiaoTong University were selected as the discovery set. One-to-one case–control matching was performed according to the traditional risk factors for DR (i.e., age, duration of diabetes, HbA1c level, and hypertension). All cases with comorbid chronic kidney disease were excluded to eliminate confounding factors. A total of 42 pairs were successfully matched. T2DM patients with DR (DR group) were the case group, and T2DM patients without DR (NDR group) served as control subjects. Ultra-performance liquid chromatography–mass spectrometry (LC–MS/MS) was used for untargeted lipidomics analysis on serum, and a partial least squares discriminant analysis (PLS-DA) model was established to screen differential lipid molecules based on variable importance in the projection (VIP) > 1. An additional 531 T2DM patients were selected as the validation set. Next, 1:1 propensity score matching (PSM) was performed for the traditional risk factors for DR, and a combined 95 pairings in the NDR and DR groups were successfully matched. The screened differential lipid molecules were validated by multiple reaction monitoring (MRM) quantification based on mass spectrometry.ResultsThe discovery set showed no differences in traditional risk factors associated with the development of DR (i.e., age, disease duration, HbA1c, blood pressure, and glomerular filtration rate). In the DR group compared with the NDR group, the levels of three ceramides (Cer) and seven sphingomyelins (SM) were significantly lower, and one phosphatidylcholine (PC), two lysophosphatidylcholines (LPC), and two SMs were significantly higher. Furthermore, evaluation of these 15 differential lipid molecules in the validation sample set showed that three Cer and SM(d18:1/24:1) molecules were substantially lower in the DR group. After excluding other confounding factors (e.g., sex, BMI, lipid-lowering drug therapy, and lipid levels), multifactorial logistic regression analysis revealed that a lower abundance of two ceramides, i.e., Cer(d18:0/22:0) and Cer(d18:0/24:0), was an independent risk factor for the occurrence of DR in T2DM patients.ConclusionDisturbances in lipid metabolism are closely associated with the occurrence of DR in patients with T2DM, especially in ceramides. Our study revealed for the first time that Cer(d18:0/22:0) and Cer(d18:0/24:0) might be potential serological markers for the diagnosis of DR occurrence in T2DM patients, providing new ideas for the early diagnosis of DR.

Relationship between Plasma Lipid Profile and Cognitive Status in Early Alzheimer Disease.

Alzheimer disease (AD) is a heterogeneous and complex disease in which different pathophysiological mechanisms are involved. This heterogenicity can be reflected in different atrophy patterns or clinical manifestations. Regarding biochemical pathways involved in early AD, lipid metabolism plays an important role; therefore, lipid levels have been evaluated as potential AD diagnosis biomarkers, and their levels could be related to different AD clinical manifestations. Therefore, the aim of this work is to study AD lipid profiles from early AD patients and evaluate their clinical significance. For this purpose, untargeted plasma lipidomic analysis was carried out in early AD patients (n = 31) diagnosed with cerebrospinal fluid (CSF) biomarkers. Cluster analysis was carried out to define early AD subgroups according to the lipid levels. Then, the clinical significance of each lipid profile subgroup was studied, analyzing differences for other variables (cognitive status, CSF biomarkers, medication, comorbidities, age, and gender). The cluster analysis revealed two different groups of AD patients. Cluster 1 showed higher levels of plasma lipids and better cognitive status than Cluster 2. However, no differences were found for the other variables (age, gender, medication, comorbidities, cholesterol, and triglycerides levels) between both groups. Plasma lipid levels could differentiate two early AD subgroups, which showed different cognitive statuses. However, further research with a large cohort and longitudinal study evaluating the clinical evolution of these patients is required. In general, it would involve a relevant advance in the knowledge of AD pathological mechanisms, potential treatments, and precision medicine.

Stable isotope profiling of cigarettes assisted with chemometrics for authenticity determination

The battle against tobacco fraud presents a formidable challenge, given the global proliferation of imitation and counterfeit cigarettes. This not only inflicts economic losses on governments but also poses significant health risks due to the prevalence of these fraudulent products. This research delves into the application of stable isotopes (δ13C, δ15N, δ2H, and δ18O) and bio-elements (C% and N%) to discern the authenticity of various cigarette brands. Differences in brand origin notably impacted the δ15N and δ2H values. The assessment of cigarette authenticity, as well as the specific brands, unveiled maximum variances in the δ15N and δ2H values, underscoring their potential as robust discriminators between genuine and counterfeit products. Unsupervised Principal Component Analysis results consistently corroborated the authenticity of the samples for the four brands. The integration of chemometric methods, such as Orthogonal Partial Least Squares-Discriminant Analysis, yielded highly accurate authentication for cigarettes across the four brand categories, achieving an accuracy of over 96.8 %. Additionally, this investigation revealed that the critical determinants in distinguishing authentic cigarettes were the isotopic values of δ15N, C%, and δ18O. This work paves the way for a more convenient avenue for the classification of cigarette authenticity compared to untargeted analysis that need to a comprehensive set of chemical fingerprints.

Inflammation-associated intramyocellular lipid alterations in human pancreatic cancer cachexia.

Cancer cachexia is a multifactorial metabolic syndrome characterized by systemic inflammation and ongoing skeletal muscle loss resulting in weakness, poor quality of life, and decreased survival. Whereas lipid accumulation in skeletal muscle is associated with cancer cachexia as well as the prognosis of cancer patients, surprisingly little is known about the nature of the lipids that accumulate in the muscle during cachexia, and whether this is related to inflammation. We aimed to identify the types and distributions of intramyocellular lipids in patients with and without cancer cachexia. Rectus abdominis muscle biopsies were collected during surgery of patients with pancreatic ductal adenocarcinoma (n=10 without cachexia, n=20 cachectic without inflammation (CRP<10mg/L), n=10 cachectic with inflammation (CRP≥10mg/L). L3-CT scans were analysed to assess body composition based on validated thresholds in Hounsfield units (HU). Muscle sections were stained with Oil-Red O and H&E to assess general lipid accumulation and atrophy. Untargeted lipidomic analyses were performed on laser-microdissected myotubes using LC-MS/MS. The spatial distribution of intramyocellular lipids with differential abundance between groups was visualized by mass-spectrometry imaging. Genes coding for inflammation markers and enzymes involved in de novo ceramide synthesis were studied by qPCR. Muscle radiation attenuation was lower in cachectic patients with inflammation (median 24.3 [18.6-30.8] HU) as compared with those without inflammation (34.2 [29.3-38.7] HU, P=0.033) or no cachexia (37.4 [33.9-42.9] HU, P=0.012). Accordingly, intramyocellular lipid content was lower in non-cachectic patients (1.9 [1.6-2.1]%) as compared with those with cachexia with inflammation (5.5 [4.5-7.3]%, P=0.002) or without inflammation (4.8 [2.6-6.0]%, P=0.017). Intramyocellular lipid accumulation was associated with both local IL-6 mRNA levels (rs=0.57, P=0.015) and systemic CRP levels (rs=0.49, P=0.024). Compared with non-cachectic subjects, cachectic patients had a higher relative abundance of intramyocellular glycerophospholipids and a lower relative abundance of glycerolipids. Furthermore, increases in several intramyocellular lipids such as SM(d36:1), PC(34:1), and TG(48:1) were found in cachectic patients with inflammation and correlated with specific cachexia features. Altered intramyocellular lipid species such as PC(34:1), LPC(18:2), and TG(48:1) showed an uneven distribution in muscle sections of cachectic and non-cachectic patients, with areas featuring abundance of these lipids next to areas almost devoid of them. Intramyocellular lipid accumulation in patients with cachexia is associated with both local and systemic inflammation, and characterized by changes in defined lipid species such as glycerolipids and glycerophospholipids.

Spatial metabolomics using mass-spectrometry imaging to decipher the impact of high red meat diet on the colon metabolome in rat

Colorectal cancer (CRC) is the third most common cancer in the world with a higher prevalence in the developed countries, mainly caused by environmental and lifestyle factors such as diet, particularly red meat consumption. The metabolic impact of high red meat consumption on the epithelial part of the colon was investigated using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MSI), to specifically analyze the epithelial substructure. Ten colons from rats fed for 100 days high red or white meat diet were subjected to untargeted MSI analyses using two spatial resolutions (100 μm and 10 μm) to evaluate metabolite changes in the epithelial part and to visualize the distribution of metabolites of interest within the epithelium crypts. Our results suggest a specific effect of red meat diet on the colonic epithelium metabolism, as evidenced by an increase of purine catabolism products or depletion in glutathione pool, reinforcing the hypothesis of increased oxidative stress with red meat diet. This study also highlighted cholesterol sulfate as another up-regulated metabolite, interestingly localized at the top of the crypts. Altogether, this study demonstrates the feasibility and the added value of using MSI to decipher the effect of high red meat diet on the colonic epithelium.

Assessment of Untargeted Metabolomics by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry to Define Breast Cancer Liquid Biopsy-Based Biomarkers in Plasma Samples.

An early diagnosis of cancer is fundamental not only in regard to reducing its mortality rate but also in terms of counteracting the progression of the tumor in the initial stages. Breast cancer (BC) is the most common tumor pathology in women and the second deathliest cancer worldwide, although its survival rate is increasing thanks to improvements in screening programs. However, the most common techniques to detect a breast tumor tend to be time-consuming, unspecific or invasive. Herein, the use of untargeted hydrophilic interaction liquid chromatography-mass spectrometry analysis appears as an analytical technique with potential use for the early detection of biomarkers in liquid biopsies from BC patients. In this research, plasma samples from 134 BC patients were compared with 136 from healthy controls (HC), and multivariate statistical analyses showed a clear separation between four BC phenotypes (LA, LB, HER2, and TN) and the HC group. As a result, we identified two candidate biomarkers that discriminated between the groups under study with a VIP > 1 and an AUC of 0.958. Thus, targeting the specific aberrant metabolic pathways in future studies may allow for better molecular stratification or early detection of the disease.

LC/MS untargeted lipidomics analysis in women with morbid obesity and type 2 diabetes mellitus: a comprehensive study

LC/MS untargeted lipidomics analysis in women with morbid obesity and type 2 diabetes mellitus: a comprehensive study

Serum and urine lipidomic profiles identify biomarkers diagnostic for seropositive and seronegative rheumatoid arthritis.

Seronegative rheumatoid arthritis (RA) is defined as RA without circulating autoantibodies such as rheumatoid factor and anti-citrullinated protein antibodies; thus, early diagnosis of seronegative RA can be challenging. Here, we aimed to identify diagnostic biomarkers for seronegative RA by performing lipidomic analyses of sera and urine samples from patients with RA. We performed untargeted lipidomic analysis of sera and urine samples from 111 RA patients, 45 osteoarthritis (OA) patients, and 25 healthy controls (HC). These samples were divided into a discovery cohort (n = 97) and a validation cohort (n = 84). Serum samples from 20 patients with systemic lupus erythematosus (SLE) were also used for validation. The serum lipidome profile of RA was distinguishable from that of OA and HC. We identified a panel of ten serum lipids and three urine lipids in the discovery cohort that showed the most significant differences. These were deemed potential lipid biomarker candidates for RA. The serum lipid panel was tested using a validation cohort; the results revealed an accuracy of 79%, a sensitivity of 71%, and a specificity of 86%. Both seropositive and seronegative RA patients were differentiated from patients with OA, SLE, and HC. Three urinary lipids showing differential expression between RA from HC were identified with an accuracy of 84%, but they failed to differentiate RA from OA. There were five lipid pathways that differed between seronegative and seropositive RA. Here, we identified a panel of ten serum lipids as potential biomarkers that can differentiate RA from OA and SLE, regardless of seropositivity. In addition, three urinary lipids had diagnostic utility for differentiating RA from HC.

Abstract PO5-13-07: Prediction of recurrence in triple negative breast cancer patients after receiving neoadjuvanttreatment using plasma metabolomics

Abstract Background Early-stage triple-negative breast cancer (TNBC) is usually treated with neoadjuvant chemotherapy (NADC), but prognosis remains poor compared with other BC subtypes. Unfortunately, less than 30% of BC patients achieve pathological complete response (pCR) to NADC and the overall survival of TNBC when cancer spreads is estimated at 10-13 months. Complete response is associated with improved progression-free and overall survival rates, but more accurate prognostic markers are needed. In this study, we assessed the prognostic value of blood circulating metabolites by using targeted-based metabolomics. Methods Patients received standard neoadjuvant chemotherapy with epirubicin plus cyclophosphamide followed by paclitaxel. Blood samples were obtained upon completion of chemotherapy and before surgery. Pathological response to treatment was recorded according to the Miller &amp; Payne system, and categorized as partial (MP 1, 2, &amp; 3) or complete response (MP 4 &amp; 5). Blood samples were collected in EDTA tubes, centrifuged and plasma was stored at -80°C. After QC standard addition and methanol extraction of proteins, four fractions of the resulting extract were analysed: two by reverse-phase/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one by reverse phase/UPLC-MS/MS with negative ion mode ESI, and one by hydrophilic interaction liquid chromatography/UPLC-MS/MS) by Metabolon using a Thermo Scientific Q-Exactive mass spectrometer with a heated electrospray ionization source and an Orbitrap mass analyser. Using Metabolon's hardware and software, raw data were extracted, identified, QC processed and quantified using the area under the curve (AUC). Identified compounds were compared with library entries of purified standards or recurrent unknown entities. The prognostic capacity of each metabolite was assessed using BRB ArrayTools. Selected metabolites were validated using a targeted approach using compound standards as follows. Plasma samples were re-analysed by UPLC/MS using a Vanquish LC coupled to an Orbitrap Exploris 240 MS (Thermo Fisher Scientific) with positive and negative ionization mode ESI under polarity switching. Identification of compounds were performed by comparison of retention times (against in-house authentic standards), accurate mass (with an accepted deviation of 3ppm), and MS/MS spectra. These analyses were carried out by MS-Omics (Denmark). Results Twenty patients treated in the Medical Oncology Unit of the University Hospital of Jaén were recruited. Median age was 50 (31-76). Twelve patients obtained a pathological complete response. Median follow-up after surgery was 25 months. Seven patients eventually had distant metastases (2 in the lungs, 1 in the brain, 1 in the bones and 3 multi-site). Metabolon untargeted analysis identified and quantified 985 metabolites, 789 after xenobiotics exclusion. Survival analysis identified 16 metabolites related with distant relapse (p&amp;lt; 0.05). The ten metabolites with lower p-value were selected for targeted validation. Ms-Omics pipeline allowed absolute quantification of 5 metabolites, 3 of them showing high correlation (R &amp;gt;0.7) between both measurements (targeted and untargeted). Validation of the prognostic value of the candidate metabolites is ongoing in a larger TNBC cohort. Conclusions Metabolomics is a useful tool for the detection of simply assessable and cost-effective prognostic biomarkers in TNBC. Easily monitoring the presence of minimal residual disease is worth to be settled up in the clinical practice for the most aggressive molecular subtype of breast cancer. In this work we have defined a set of metabolites that could predict distant relapse events in TNBC patients treated with neoadjuvant chemotherapy. Further analyses are being carried out to validate the prognostic value of the proposed candidate metabolites. Citation Format: Pedro Sánchez-Rovira, Rocio Urbano-Cubero, Angelo Gamez Pozo, Carmen González-Olmedo, Alicia Cano-Jimenez, Andrea Zapater- Moros, Leticia Díaz-Beltrán, Lucia Trilla-Fuertes, Mariana Díaz-Almirón, Enrique Espinosa, Pilar Zamora, Juan Angel Fresno Vara. Prediction of recurrence in triple negative breast cancer patients after receiving neoadjuvanttreatment using plasma metabolomics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-13-07.

Exploration of circulating metabolic signature of erythrodermic psoriasis based on LC-MS metabolomics.

Erythrodermic psoriasis (EP) is a rare and life-threatening disease, the pathogenesis of which remains to be largely unknown. Metabolomics analysis can provide global information on disease pathophysiology, candidate biomarkers, and potential intervention strategies. To gain a better understanding of the mechanisms of EP and explore the serum metabolic signature of EP, we conducted an untargeted metabolomics analysis from 20 EP patients and 20 healthy controls. Furthermore, targeted metabolomics for focused metabolites were identified in the serum samples of 30 EP patients and 30 psoriasis vulgaris (PsV) patients. In the untargeted analysis, a total of 2992 molecular features were extracted from each sample, and the peak intensity of each feature was obtained. Principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA) revealed significant difference between groups. After screening, 98 metabolites were found to be significantly dysregulated in EP, including 67 down-regulated and 31 up-regulated. EP patients had lower levels of L-tryptophan, L-isoleucine, retinol, lysophosphatidylcholine (LPC), and higher levels of betaine and uric acid. KEGG analysis showed differential metabolites were enriched in amino acid metabolism and glycerophospholipid metabolism. The targeted metabolomics showed lower L-tryptophan in EP than PsV with significant difference and L-tryptophan levels were negatively correlated with the PASI scores. The serum metabolic signature of EP was discovered. Amino acid and glycerophospholipid metabolism were dysregulated in EP. The metabolite differences provide clues for pathogenesis of EP and they may provide insights for therapeutic interventions.

Unraveling a Novel Mechanism of Altered Glomerular Hemodynamics in Pre-diabetic Obesity

Background: In pre-diabetic obesity, disrupted glomerular autoregulation leads to increased glomerular capillary pressure and renal blood flow, causing potential barotrauma-induced glomerular damage. A decrease in Afferent arteriole ( Af-Art) resistance causes higher glomerular capillary pressure but the underlying mechanism behind this, specifically in pre-diabetic obesity, remains unclear. Hyperinsulinemia is common in pre-diabetic obesity, but its role in reducing Af-Art resistance in the kidneys is unknown. Kidney blood flow is auto-regulated by vasoconstriction via tubuloglomerular feedback (TGF) and vasodilation through the Distal convoluted tubule-2/connecting tubule (DCT-2/CNT) mechanism. Our earlier studies indicate increased Af-Art dilation in pre-diabetic obese rats through the DCT-2/CNT mechanism without affecting TGF. This DCT-2/CNT induced Af-Art dilation requires initiation from epithelial sodium channels (ENaC) present at this tubular segment and interestingly insulin is a known ENaC activator. Hypothesis: We hypothesize that in the context of pre-diabetic obesity, hyperinsulinemia contributes to an increase in renal cortical blood flow (CBF) by increasing DCT-2/CNT-induced Af-Art dilation, subsequently leading to glomerular injury. Methods: We assessed the effects of insulin on Af-Art dilation by perfusing juxtaposed DCT-2/CNT segments with insulin in normal rabbits. Contribution of DCT-2/CNT induced Af-Art dilation was measured by co-administration of the ENaC blocker benzamil. To study hyperinsulinemia's role in an in-vivo setting without altering blood glucose levels, we used a hyperinsulinemic-euglycemic (HI-EUG) clamp in obese rats with intravenous (IV) glucose and insulin infusions. Renal CBF and blood pressure were monitored using a laser Doppler probe and femoral artery catheter, respectively, both with and without benzamil administration (400 μg/kg, IV). To evaluate insulin's potential role in causation of renal damage, we infused insulin directly into one kidney of lean rats for 6 weeks via a renal sub-capsular catheter, with the contralateral kidney serving as control. Glomerular basement membrane (GBM) thickness, a marker of renal damage, was measured via electron microscopy. Untargeted lipidomic analysis of the renal cortex was performed to identify potential renal damage pathways in insulin infused kidneys. Results: Insulin perfusion in rabbits' DCT-2/CNT potentiated the dilation of adherent Af-Art compared to control infusions with artificial tubular fluid. ENaC inhibition reversed the insulin-induced Af-Art dilation, confirming that insulin increases Af-Art dilation through the ENaC-initiated DCT-2/CNT mechanism. In obese rats, insulin delivery using a HI-EUG clamp resulted in a significant 20.8±4.9% increase in CBF, which was negated by the administration of the ENaC blocker benzamil, indicating the potential involvement of DCT-2/CNT-mediated vasodilation. Neither the HI-EUG clamp nor benzamil infusion affected blood pressure. Additionally, continuous direct insulin infusion into the kidneys of lean rats led to an increase in glomerular basement membrane (GBM) thickness and ceramide accumulation in the renal cortex, both indicative of renal damage. Conclusion: Our study demonstrates that insulin directly augments DCT-2/CNT-mediated Af-Art dilation and increases renal cortical blood flow in obese rats. Chronic renal insulin infusion led to structural changes and damage markers in the kidney, independent of blood glucose and blood pressure levels. Henry Ford Health System Internal Funding. Pilot and Feasibility grant from southeast center for integrated metabolomics. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Proteomic Changes during Human Heat Stress and Heat Acclimation

INTRODUCTION. To thrive in a warmer climate, humans will need to adapt physiologically. Improved thermoregulatory and cardiovascular responses during heat acclimation have been widely described. However, the cellular and molecular mechanisms mediating these improvements remain understudied in humans. The aim of this study was to perform a broad-spectrum, non-targeted proteomic analysis to determine how plasma protein levels change in response to moderate heat stress and heat acclimation. METHODS. Ten healthy adults (4F/6H, age: 25 ± 3 years) attended two laboratory visits performed before and after a 7-day heat acclimation protocol (hot water immersion, 60 min at a rectal temperature ≥38.5°C). During the visits, blood samples were drawn before and during passive heat stress (~1.5°C increase in esophageal temperature, water-perfused suit). Proteomic analyses were performed on 2938 plasma proteins using the Olink Explore 3072 assay. We quantified the number of proteins that changed by more than one log2(fold change) in concentration. RESULTS. Prior to acclimation, ~1% of the proteins (n=29) changed in concentration during heat stress. The change in concentration for 9 of these proteins reached the p value threshold. After acclimation, 54 proteins changed in concentration under a normothermic state. During subsequent heat stress, 175 proteins (~6%) increased in concentration and 19 (~0.6%) decreased. For 180 of these proteins, the change in concentration during heat stress was unique to the post-acclimation visit. The genes for proteins that changed in concentration during heat stress prior to acclimation are more highly expressed in brain (FGFBP3), arterial (TIMP4), skin (OBP2B), lung/salivary gland/esophageal (CXCL17) tissue. The genes for proteins that changed concentration in response to heat acclimation are highly expressed in skin (MMP7, CA6, KLK14) and brain tissue (DNER). The genes for proteins that only changed in concentration during heat stress post-acclimation are more highly expressed in brain (MOG, SMOC1, PTPRN2), and arterial (TNC, C1QTNF1, MATN2, NTF3, SMOC1) tissue. CONCLUSION. This untargeted and large-scale proteomic analysis identified novel plasma proteins that change in concentration during human heat stress and subsequent heat acclimation. The individual protein results can be utilized as external validation for other studies, as the basis of further data analysis explorations, and/or to guide targeted investigations of the biological responses and adaptations to heat exposure. Natural Sciences and Engineering Research Council of Canada. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Organic acid and aromatic compounds create distinctive flavor in the blackening process of jujube

Previous studies demonstrated jujube blackening effectively increased cyclic adenosine phosphate and triterpene acid levels, improving its nutritional value. However, compositional changes during this process require further elucidation. The objective aimed to analyze compositional transformations during this process with SEM, TPA, UPLC- MS, E-nose. Results showed decreased hardness, springiness, and chewiness coupled with increased gumminess over blackening durations. Untargeted omics analysis revealed increases of 2-aminooctadec-8-ene-1,3,4-trioland carbendazim. Targeted organic acid analysis showed initial citric acid accumulation (1481.62 to 1645.78 mg/kg) in the first 24 h, then declines to 1072.96 mg/kg. Meanwhile, oxalic and lactic acids steadily rose, peaking at 96–120 h before slightly decreasing. E-nose analysis implied alterations in organic sulfide aromatics engendered the characteristic flavors. Organic acid fluctuations likely resulted from sugar biotransformation and thermal degradation. These comprehensive analyses demonstrate jujube blackening imparts a rich and unique flavor, providing theoretical support for investigating the mechanisms and products underlying this process.

Rapid evaporative ionization mass spectrometry: A survey through 15 years of applications.

Rapid evaporative ionization mass spectrometry (REIMS) is a relatively recent MS technique explored in many application fields, demonstrating high versatility in the detection of a wide range of chemicals, from small molecules (phenols, amino acids, di- and tripeptides, organic acids, and sugars) to larger biomolecules, that is, phospholipids and triacylglycerols. Different sampling devices were used depending on the analyzed matrix (liquid or solid), resulting in distinct performances in terms of automation, reproducibility, and sensitivity. The absence of laborious and time-consuming sample preparation procedures and chromatographic separations was highlighted as a major advantage compared to chromatographic methods. REIMS was successfully used to achieve a comprehensive sample profiling according to a metabolomics untargeted analysis. Moreover, when a multitude of samples were available, the combination with chemometrics allowed rapid sample differentiation and the identification of discriminant features. The present review aims to provide a survey of literature reports based on the use of such analytical technology, highlighting its mode of operation in different application areas, ranging from clinical research, mostly focused on cancer diagnosis for the accurate identification of tumor margins, to the agri-food sector aiming at the safeguard of food quality and security.

Plasma-based lipidomics reveals potential diagnostic biomarkers for esophageal squamous cell carcinoma: a retrospective study.

Esophageal squamous cell carcinoma (ESCC) is highly prevalent and has a high mortality rate. Traditional diagnostic methods, such as imaging examinations and blood tumor marker tests, are not effective in accurately diagnosing ESCC due to their low sensitivity and specificity. Esophageal endoscopic biopsy, which is considered as the gold standard, is not suitable for screening due to its invasiveness and high cost. Therefore, this study aimed to develop a convenient and low-cost diagnostic method for ESCC using plasma-based lipidomics analysis combined with machine learning (ML) algorithms. Plasma samples from a total of 40 ESCC patients and 31 healthy controls were used for lipidomics study. Untargeted lipidomics analysis was conducted through liquid chromatography-mass spectrometry (LC-MS) analysis. Differentially expressed lipid features were filtered based on multivariate and univariate analysis, and lipid annotation was performed using MS-DIAL software. A total of 99 differential lipids were identified, with 15 up-regulated lipids and 84 down-regulated lipids, suggesting their potential as diagnostic targets for ESCC. In the single-lipid plasma-based diagnostic model, nine specific lipids (FA 15:4, FA 27:1, FA 28:7, FA 28:0, FA 36:0, FA 39:0, FA 42:0, FA 44:0, and DG 37:7) exhibited excellent diagnostic performance, with an area under the curve (AUC) exceeding 0.99. Furthermore, multiple lipid-based ML models also demonstrated comparable diagnostic ability for ESCC. These findings indicate plasma lipids as a promising diagnostic approach for ESCC.