Plasma Metabolites Research Articles

Multiomic integration analysis identifies atherogenic metabolites mediating between novel immune genes and cardiovascular risk

BackgroundUnderstanding genetic-metabolite associations has translational implications for informing cardiovascular risk assessment. Interrogating functional genetic variants enhances our understanding of disease pathogenesis and the development and optimization of targeted interventions.MethodsIn this study, a total of 187 plasma metabolite levels were profiled in 4974 individuals of European ancestry of the GCAT| Genomes for Life cohort. Results of genetic analyses were meta-analysed with additional datasets, resulting in up to approximately 40,000 European individuals. Results of meta-analyses were integrated with reference gene expression panels from 58 tissues and cell types to identify predicted gene expression associated with metabolite levels. This approach was also performed for cardiovascular outcomes in three independent large European studies (N = 700,000) to identify predicted gene expression additionally associated with cardiovascular risk. Finally, genetically informed mediation analysis was performed to infer causal mediation in the relationship between gene expression, metabolite levels and cardiovascular risk.ResultsA total of 44 genetic loci were associated with 124 metabolites. Lead genetic variants included 11 non-synonymous variants. Predicted expression of 53 fine-mapped genes was associated with 108 metabolite levels; while predicted expression of 6 of these genes was also associated with cardiovascular outcomes, highlighting a new role for regulatory gene HCG27. Additionally, we found that atherogenic metabolite levels mediate the associations between gene expression and cardiovascular risk. Some of these genes showed stronger associations in immune tissues, providing further evidence of the role of immune cells in increasing cardiovascular risk.ConclusionsThese findings propose new gene targets that could be potential candidates for drug development aimed at lowering the risk of cardiovascular events through the modulation of blood atherogenic metabolite levels.

Retinol intake and PCOS management: a plasma metabolite and protein analysis via Mendelian randomization and NHANES 2011-2016.

Polycystic Ovary Syndrome (PCOS) represents a complex endocrine disorder characterized by a significant interplay with metabolic dysfunction and obesity. This research endeavors to elucidate the causal dynamics among plasma metabolites, proteins, and PCOS, alongside Body Mass Index (BMI), to pinpoint prospective therapeutic interventions. This investigation employed Mendelian randomization (MR) analyses combined with data derived from the National Health and Nutrition Examination Survey (NHANES) to explore the relationships between 1,400 plasma metabolites and PCOS, factoring in BMI adjustments. Additionally, the study examined the influence of plasma proteins and performed a retrospective cross-sectional analysis focusing on retinol consumption and testosterone levels. MR analyses showed metabolite Glycosyl-N-(2-hydroxynervonoyl)-sphingosine (GNS) and protein Keratin 19 (KRT19) were identified as significant markers in the context of PCOS and BMI adjustments. A Phenome-Wide Association Study (PheWAS) underscored the linkage between KRT19 and BMI, while gene-drug interaction findings demonstrated a connection between KRT19 and retinol. Analysis for NHANES data disclosed a negative correlation between retinol intake and testosterone levels, particularly within normal weight and obese cohorts, suggesting the feasibility of dietary interventions for PCOS management. The study sheds light on the intricate interactions between plasma metabolites, proteins, and PCOS, considering BMI variations, and highlights KRT19 protein as a promising therapeutic target. The outcomes support the integration of retinol consumption into dietary strategies to regulate testosterone levels and potentially alleviate PCOS symptoms, underscoring the necessity for personalized nutritional and therapeutic approaches in the effective management of PCOS.

Metabolic fate of the natural anticancer agent cucurbitacin B: an LC-MS/MS-enabled profiling of its major phase I and II conjugates in vivo.

Cucurbitacin B (CuB) is a natural triterpenoid with diverse pharmacological effects including potent anticancer activity. However, its oral bioavailability is hampered by limited metabolism in vivo. We characterized CuB's in vivo metabolism in rats to uncover bioactive metabolites retaining therapeutic potential, using a robust UHPLC-Q-TOF-MS/MS workflow. This workflow combined molecular networking, fragmentation filtering, and mass defect filtering to identify CuB metabolites in rat urine, plasma, and feces following oral administration. Thirteen metabolites were identified and seven were confirmed. Major phase I transformations involved hydrolysis, reduction, epoxidation, and amination. Phase II conjugation included cysteine, glutathione, glucuronide, and gluconic acid conjugates. Notably, one of the main metabolites formed was the cysteine conjugate CuB-Cys. CuB-Cys maintained similar in vitro antiproliferative activity to CuB on HepG2, MCF-7, and PANC-1 cancer cell lines. However, it demonstrated lower cytotoxicity towards non-cancerous L02 cells, highlighting improved therapeutic selectivity. Mechanistically, CuB-Cys induced greater apoptotic signaling in HepG2 cells than CuB via enhanced caspase activation and disrupted BAX-Bcl-2 balance. This represents the first systematic characterization of CuB's in vivo metabolic pathway. The identification and confirmation of CuB-Cys provide insight for drug development efforts aiming to maintain therapeutic efficacy while reducing toxicity, via metabolite-based approaches. Our findings shed light on strategies for improving CuB's clinical potential.

Simultaneous quantification of JWH-018, JWH-073, and their metabolites (4-OH JWH-018, 3-OH JWH-073) in human plasma and urine samples by LC-MS/MS

This study aims to develop and validate a method for the simultaneous quantification of JWH-073, JWH-018, and their two main metabolites 3-OH JWH-073 and 4-OH JWH-018 in human blood and urine samples by using liquid chromatography combined with a mass spectrometer (LC-MS/MS). The method was validated according to current FDA and EMA guidelines and was tested on 10 urine samples from suspected synthetic cannabinoid abusers and 10 plasma samples from drug-free volunteers. Experimental data showed that the limits of detection (LOD) for the analytes were very low at 0.01 ng/mL in plasma samples and 0.003 ng/mL in urine samples. The limits of quantification (LLOQ) were 0.03 ng/mL in plasma samples and 0.01 ng/mL in urine samples. The linear correlation coefficients (R 2) for these analytes ranged from 0.9914 to 0.9929 in plasma samples and 0.9820 to 0.9892 in urine samples. Among the 10 urine samples from suspected synthetic cannabinoid abusers, the parent JWH-073 and JWH-018 analytes were detected in six and three samples, with concentrations of 0.01 − 8.14 ng/mL and 7.16 − 17.50 ng/mL, respectively. At the same time, their two main metabolites, 3-OH JWH-073 and 4-OH JWH-018, were also found in the samples containing parent drugs, with concentrations of 32.13 − 84.24 ng/mL and 14.48–32.97 ng/mL, respectively. Three samples (T3, T5, and T6) contained only 4-OH JWH-018 metabolite (with concentrations of 25.69 − 51.87 ng/mL) and one sample (T10) contained only 3-OH JWH-073 metabolite (with concentration of 56.94 ng/mL) without the presence of their parent drugs. The plasma samples from drug-free volunteers were all negative for the analytes.

A systematic UHPLC-Q-TOF-MS/MS-based strategy for analysis of chemical constituents and related in vivo metabolites of Buyang Huanwu decoction

Ethnopharmacological relevanceBuyang Huanwu Decoction (BYHWD), a traditional Chinese medicine, is one of the classic prescriptions for the treatment of ischemic stroke in clinical practice. It has the effects of tonifying qi, activating blood circulation, and promoting meridian circulation. However, its chemical analysis has not been clarified, which greatly hinders its further clinical application. Therefore, it is necessary to clarify the chemical constituents and metabolites profile of BYHWD in vivo. Aim of the studyCharacterizing the chemical basis of BYHWD in vitro, and combing studies of related metabolism in vivo to screen out the potential active components of BYHWD with pharmacological effects in vivo. Materials and methodsTwelve male rats weighed 200 ± 20 each were selected for the experiments. According to the fragmentation of different structural types of components and diagnostic ions, UHPLC-Q-TOF-MS/MS was used to classify and clarify the unknown components of BYHWD and identify the material basis of BYHWD in vitro. Then, rat plasma, tissues, feces, and urine were collected for analysis. Based on the similarity of MS responses (accurate molecular weight and secondary fragmentation) and chromatographic behavior (retention time), the in vivo prototype and metabolites were analyzed. Through the phase I and phase II metabolism law, a metabolite library was established to analyze the prototype-matched metabolites. ResultsA total of 121 in vitro compounds and 55 in vivo prototypes of BYHWD were identified, corresponding to 123 matched prototypes. It was mainly composed of flavonoids, triterpene saponins, nucleosides and lactones both in vitro and in vivo. Quercetin, ligustilide, astragaloside IV, calycosin, paeoniflorin and ferulic acid were the main prototypes and metabolites in plasma and urine. ConclusionQuercetin, ligustilide, astragaloside IV, calycosin, paeoniflorin and ferulic acid were the main active ingredients of BYHWD.

Comorbidities confound metabolomics studies of human disease

The co-occurrence of multiple chronic conditions, termed multimorbidity, presents an expanding global health challenge, demanding effective diagnostics and treatment strategies. Chronic ailments such as obesity, diabetes, and cardiovascular diseases have been linked to metabolites interacting between the host and microbiota. In this study, we investigated the impact of co-existing conditions on risk estimations for 1375 plasma metabolites in 919 individuals from population-based Estonian Biobank cohort using liquid chromatography mass spectrometry (LC–MS) method. We leveraged annually linked national electronic health records (EHRs) data to delineate comorbidities in incident cases and controls for the 14 common chronic conditions. Among the 254 associations observed across 13 chronic conditions, we primarily identified disease-specific risk factors (92%, 217/235), with most predictors (93%, 219/235) found to be related to the gut microbiome upon cross-referencing recent literature data. Accounting for comorbidities led to a reduction of common metabolite predictors across various conditions. In conclusion, our study underscores the potential of utilizing biobank-linked retrospective and prospective EHRs for the disease-specific profiling of diverse multifactorial chronic conditions.

Integrating metabolomics and proteomics to identify novel drug targets for heart failure and atrial fibrillation

BackgroundAltered metabolism plays a role in the pathophysiology of cardiac diseases, such as atrial fibrillation (AF) and heart failure (HF). We aimed to identify novel plasma metabolites and proteins associating with cardiac disease.MethodsMendelian randomisation (MR) was used to assess the association of 174 metabolites measured in up to 86,507 participants with AF, HF, dilated cardiomyopathy (DCM), and non-ischemic cardiomyopathy (NICM). Subsequently, we sourced data on 1567 plasma proteins and performed cis MR to identify proteins affecting the identified metabolites as well as the cardiac diseases. Proteins were prioritised on cardiac expression and druggability, and mapped to biological pathways.ResultsWe identified 35 metabolites associating with cardiac disease. AF was affected by seventeen metabolites, HF by nineteen, DCM by four, and NCIM by taurine. HF was particularly enriched for phosphatidylcholines (p = 0.029) and DCM for acylcarnitines (p = 0.001). Metabolite involvement with AF was more uniform, spanning for example phosphatidylcholines, amino acids, and acylcarnitines. We identified 38 druggable proteins expressed in cardiac tissue, with a directionally concordant effect on metabolites and cardiac disease. We recapitulated known associations, for example between the drug target of digoxin (AT1B2), taurine and NICM risk. Additionally, we identified numerous novel findings, such as higher RET values associating with phosphatidylcholines and decreasing AF and HF. RET is targeted by drugs such as regorafenib which has known cardiotoxic side-effects. Pathway analysis implicated involvement of GDF15 signalling through RET, and ghrelin regulation of energy homeostasis in cardiac pathogenesis.ConclusionsThis study identified 35 plasma metabolites involved with cardiac diseases and linked these to 38 druggable proteins, providing actionable leads for drug development.

Effect of a Bacillus subtilis-based direct-fed microbial, on milk yield, milk components, feed intake and plasma hormones and metabolites in lactating Holstein cows

To investigate the effects of a Bacillus-based direct-fed microbial on milk production and related factors, a study was conducted on Holstein cows (n = 28) starting at 90 ± 11 DIM. The cows were divided into two dietary groups: a control group (CON) that received a total mixed ration (TMR), and a Bacillus-fed group (DFM) that received the same TMR along with a Bacillus subtilis product containing two strains (747 and 1781) in equal amounts. The study lasted for 25 wk and included both primiparous and multiparous cows. The cows were housed in a free-stall barn and were provided with ad libitum TMR, which was fed twice a day. Their daily meals and dry matter intake (DMI) were recorded during wk 1–4 and wk 19–25 using electronic feeders. Milk samples were collected weekly during morning and evening milkings and analyzed for milk fat, protein, lactose, and milk urea nitrogen (MUN). Blood samples were also collected weekly and analyzed for plasma glucose, insulin, cholesterol, insulin-like growth factor 1 (IGF1), and progesterone concentrations. Additionally, rumen fluid samples were collected and evaluated for various species of bacteria. One of the key findings was that daily 4 % fat-corrected milk production (FCM) was influenced by the interaction between treatment (DFM vs. CON) and parity (primiparous vs. multiparous), with multiparous DFM cows producing 11 % more FCM compared to CON. The DFM cows also had a higher milk fat percentage (4.41 % vs. 4.02 %) and MUN concentrations were 1.0 mg/dL higher in the DFM group. In terms of blood metabolites and hormones, multiparous DFM cows had lower plasma cholesterol, glucose, and insulin concentrations compared to CON multiparous cows. However, there were no significant differences between the groups in terms of plasma IGF1 concentrations, insulin-to-glucose ratios, and luteal phase plasma progesterone concentrations. The analysis of rumen fluid samples revealed that the abundance of Ruminococcus albus and Fibrobacter succinogenes group I was greater in the DFM cows compared to CON cows. Furthermore, during the periods when feed intake was measured, the DFM cows had a 9 % reduction in feed intake and a 14 % improvement in feed efficiency (FCM per DMI) compared to CON cows. In conclusion, the two-strain Bacillus product used in this study (Certillus) showed potential as an effective direct-fed microbial. It was found to increase feed efficiency and milk production by altering the composition of ruminal microbiota and metabolism in the cows.

Low blood S-methyl-5-thioadenosine is associated with postoperative delayed neurocognitive recovery

Elderly individuals display metabolite alterations that may contribute to development of cognitive impairment following surgery and anesthesia. However, these relationships remain largely unexplored. The study aims to assess the S-methyl-5-thioadenosine (MTA) is associated with postoperative delayed neurocognitive recovery (dNCR). We assess altered metabolites following anesthesia/surgery in both mice and patients to identify blood biomarkers of dNCR. Preoperative and postoperative plasma metabolites are determined by widely targeted metabolomics. The brains of mice with anesthesia/surgery show decreased MTA and activated MTA phosphorylase. Mice also show that preoperative administration of MTA can prevent inflammation and cognitive decline. In clinical patients, we detect lower preoperative serum MTA levels in those who developed dNCR. Both low preoperative and postoperative blood MTA levels are associated with increased risk of postoperative dNCR. These results suggest that anesthesia/surgery induces cognitive decline through methionine synthesis pathways and that MTA could be a perioperative predictor of dNCR.

EphA2 blockage ALW-II-41-27 alleviates atherosclerosis by remodeling gut microbiota to regulate bile acid metabolism.

Coronary artery disease (CAD), a critical condition resulting from systemic inflammation, metabolic dysfunction, and gut microbiota dysbiosis, poses a global public health challenge. ALW-II-41-27, a specific inhibitor of the EphA2 receptor, has shown anti-inflammatory prosperities. However, the impact of ALW-II-41-27 on atherosclerosis has not been elucidated. This study aimed to examine the roles of pharmacologically inhibiting EphA2 and the underlying mechanism in ameliorating atherosclerosis. ALW-II-41-27 was administered to apoE-/- mice fed a high-fat diet via intraperitoneal injection. We first discovered that ALW-II-41-27 led to a significant reduction in atherosclerotic plaques, evidenced by reduced lipid and macrophage accumulation, alongside an increase in collagen and smooth muscle cell content. ALW-II-41-27 also significantly lowered plasma and hepatic cholesterol levels, as well as the colonic inflammation. Furthermore, gut microbiota was analyzed by metagenomics and plasma metabolites by untargeted metabolomics. ALW-II-41-27-treated mice enriched Enterococcus, Akkermansia, Eggerthella and Lactobaccilus, accompanied by enhanced secondary bile acids production. To explore the causal link between ALW-II-41-27-associated gut microbiota and atherosclerosis, fecal microbiota transplantation was employed. Mice that received ALW-II-41-27-treated mouse feces exhibited the attenuated atherosclerotic plaque. In clinical, lower plasma DCA and HDCA levels were determined in CAD patients using quantitative metabolomics and exhibited a negative correlation with higher monocytes EphA2 expression. Our findings underscore the potential of ALW-II-41-27 as a novel therapeutic agent for atherosclerosis, highlighting its capacity to modulate gut microbiota composition and bile acid metabolism, thereby offering a promising avenue for CAD.

Metabolic characteristics of ischaemic preconditioning induced performance improvement in Taekwondo athletes using LC‒MS/MS-based plasma metabolomics.

In recent years, ischemic preconditioning (IPC) has garnered significant attention in sports research. While IPC has demonstrated positive effects in high-intensity sports such as judo and swimming, its potential benefits for enhancing the performance of Taekwondo athletes have not been extensively studied. This study aimed to investigate the effects of IPC on taekwondo performance and to observe the metabolic characteristics associated with enhancing sports performance via LC‒MS/MS-based plasma metabolomics. Seventeen participants underwent the repeated frequency speed of kick test (FSKT) after IPC, along with pre- and post-exercise plasma metabolite analysis. Differential abundance metabolite analysis, enriched pathway analysis, and weighted gene coexpression network analysis (WGNCA) were employed to delve into metabolic characteristics. The findings highlighted a significant enhancement in FSKT performance in the experimental group. Metabolomic analysis revealed 109 differentially abundant metabolites, including Dl-lactate, hypoxanthine, acetylcarnitine, and acetylsalicylic acid. Enriched pathway analysis revealed pathways such as pentose and glucuronic acid interconversion, ascorbic acid and aldonic acid metabolism, the pentose phosphate pathway (PPP), and the Warburg effect. In conclusion, IPC can significantly increase the specific athletic abilities of Taekwondo athletes, with enhancements linked to anaerobic metabolism, PPP utilization, the Warburg effect for energy production, redox system stability, reduced muscle fatigue, and pain alleviation.

Potential Impacts of Dried Green Coleus forskolin Leaves on Thermo-tolerance Parameters, Blood and Plasma Profiles in Anesthetized Mice

Background: Transient negative side effects of hypothermia, hyperglycemiaand bradycardia due to general anesthesia using diazepam and xylazine drugs has been reported in mice. Methods: Twenty albino male mice of 31.52±0.25 g body weight were classified into two groups; control group (G1) fed basal control diet versus C. forskolin group fed basal control diet containing C. forskolin leaves (G2; 1.5%) for four weeks. The mice were general anesthetized using xylazine and diazepam (XD) drugs (13.3 mg/kg BW diazepam and 26.6 mg/kg BW xylazine). Changes of rectal temperature (°C), glucose (mg/dl), pulse rate (pulse/min.) and partial pressure of oxygen (SPO2) were recorded at 0.0, 0.5h, 1.0h, 2.0h, 3.0h, 5.0h and 7.0h over general anesthesia. In addition, blood samples were collected and were subjected to blood and plasma analyses. Result: The results indicated that dietary C. forskolin caused significant decrease (P less than 0.05) in glucose values in addition to significant increase (P less than 0.05) in rectal temperature, pulse rate and SPO2 during the period hypothermia, hyperglycemiaand bradycardia over general anesthesia. In addition, values of blood (RBCs, WBCs, PCV and Hb) and plasma parameters (TP, BUN, creatinine, AST and ALT, LDH) were normal due to C. forskolin supplementation compared to control diet. It could be concluded that 1.5% C. forskolin supplementation modulates thermo-tolerance responses, blood glucose indices, blood and plasma metabolites over general anesthesia.

The potential mediating role of the gut microbiome and metabolites in the association between PFAS and kidney function in young adults: A proof-of-concept study

BackgroundChronic kidney disease (CKD) affects over 10 % of the global population and can lead to kidney failure and death. Exposure to per- and polyfluoroalkyl substances (PFAS) is associated with increased risk of CKD, yet studies examining the mechanisms linking PFAS and kidney function are lacking. In this exploratory study, we examined longitudinal associations of PFAS exposure with kidney function, and tested if associations were mediated by altered gut bacterial taxa or plasma metabolites using a multi-omics mediation analysis. MethodsSeventy-eight young adults from the Children's Health Study were included in this longitudinal cohort study. At baseline, seven plasma PFAS and untargeted plasma metabolomics were measured using liquid chromatography/mass-spectrometry. Baseline gut bacterial abundance was characterized using 16S rRNA sequencing and examined at the genus level. At follow-up, serum creatinine and cystatin-C concentrations were quantified to estimate glomerular filtration rate (eGFR). High-dimensional multi-omics analyses were conducted to assess the association between baseline PFAS exposure with follow-up eGFR, mediated by gut microbiome and circulating metabolite levels. ResultsPFAS burden score, a variable developed to estimate exposure to chemical mixtures, was associated with kidney function. Each standard deviation increase in baseline PFAS burden score was associated with a 2.4 % lower eGFR at follow-up (95 % CI:[0.1 %,4.8 %]). Following high-dimensional mediation analyses with the microbiome and circulating metabolites, a joint component (characterized by reduced Lachnospiraceae and 17b-estradiol and increased succinate, retinoate and dodecanoic acid) and a metabolite component (characterized by increased hypotaurine and decreased D-pinitol and ureidopropionate) mediated 38 % and 50 % of the effect between PFAS burden score and eGFR, respectively. ConclusionOur proof-of-concept analysis provides the first evidence that reduced short-chain fatty acid-producing bacteria and anti-inflammatory metabolites may link PFAS exposure with impaired kidney function. This study raises the possibility of future targeted interventions that can alter gut microbiome or circulating metabolite profiles to prevent PFAS induced kidney damage.

Causal Metabolomic and Lipidomic Analysis of Circulating Plasma Metabolites in Autism: A Comprehensive Mendelian Randomization Study with Independent Cohort Validation.

The increasing prevalence of autism spectrum disorder (ASD) highlights the need for objective diagnostic markers and a better understanding of its pathogenesis. Metabolic differences have been observed between individuals with and without ASD, but their causal relevance remains unclear. Bidirectional two-sample Mendelian randomization (MR) was used to assess causal associations between circulating plasma metabolites and ASD using large-scale genome-wide association study (GWAS) datasets-comprising 1091 metabolites, 309 ratios, and 179 lipids-and three European autism datasets (PGC 2015: n = 10,610 and 10,263; 2017: n = 46,351). Inverse-variance weighted (IVW) and weighted median methods were employed, along with robust sensitivity and power analyses followed by independent cohort validation. Higher genetically predicted levels of sphingomyelin (SM) (d17:1/16:0) (OR, 1.129; 95% CI, 1.024-1.245; p = 0.015) were causally linked to increased ASD risk. Additionally, ASD children had higher plasma creatine/carnitine ratios. These MR findings were validated in an independent US autism cohort using machine learning analysis. Utilizing large datasets, two MR approaches, robust sensitivity analyses, and independent validation, our novel findings provide evidence for the potential roles of metabolomics and circulating metabolites in ASD diagnosis and etiology.

Associations between pre-diagnostic plasma metabolites and biliary tract cancer risk in the prospective UK Biobank cohort.

Biliary tract cancer (BTC) is potentially influenced by metabolic dysregulation yet previous metabolomic evaluations are limited. To address this gap, we prospectively investigated associations of blood metabolites and BTC risk in the UK biobank cohort study. We used Cox proportional hazards models to estimate hazard ratios (HR) and 95% confidence intervals (95% CI) between 249 plasma metabolites per standard deviation and BTC risk in 232,781 participants. We implemented exploratory factor analyses and evaluated associations between factors and BTC risk. Associations at p-value<0.001 were considered statistically significant after multiple comparison adjustments. In a median follow-up of 11.8 years, we identified 268 first primary incident BTC cases. Of 49 biomarkers significantly associated with BTC risk, 12% were fatty acids, and 49%, 31%, and 8% were cholesterol, triglyceride, and phospholipid to total lipids ratios, respectively. Multiple cholesterol ratios were inversely associated with BTC with HRs (95% CIs) of 0.74 (0.65-0.84), p<6.0x10-6. Conversely, a triglyceride ratio was positively associated with BTC with an HR (95% CI) of 1.40 (1.22-1.61), p=2.5x10-6. Congruently, a factor high in cholesterol measures and low in triglyceride measures was inversely associated with BTC. Multiple metabolite biomarkers were associated with BTC risk, suggesting metabolism has a substantial role in BTC etiology.

Quantitation and Abundance of Xylazine and Xylazine Metabolites in Human Urine and Plasma by LC-MS/MS

Abstract The most common cause of drug overdose fatality in the US involves synthetic opioids such as fentanyl. Recently the Director of the White House Office of National Drug Control Policy designated fentanyl adulterated or associated with xylazine as an emerging threat to the US, and a national response plan will include xylazine testing. Xylazine is a clonidine analog and is not FDA-approved for use in humans but has been increasingly detected in unregulated illicit drugs supplies. Our understanding of xylazine detection, metabolism, and clinical impact on patients, however, is limited. The objectives of this study are to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect xylazine and xylazine metabolites in urine and plasma, and to investigate the presence and abundance of xylazine metabolites in remnant urine and plasma samples submitted for clinical testing. This is achieved by first collecting paired urine and serial plasma samples when urine tests positive for xylazine during routine clinical testing. Plasma samples were prepared for LC-MS/MS analysis by protein precipitation with methanol and xylazine-D6 internal standard. Urines were prepared by dilution with xylazine-D6, followed by a hydrolysis with beta-glucuronidase. All experiments were performed using an Aquity HPLC coupled to a Waters TQS-micro triple quadrupole mass spectrometer. LC separation utilized a C18 column with a 4-minute gradient from 2% to 98% mobile phase B (0.1% formic acid and 2mM ammonium acetate in A: water; B: methanol) with a flow rate of 400 μl/min at 50°C. Multiple reaction monitoring for xylazine and 4-OH-xylazine was optimized with commercial standards. Calibration curves for both analytes were prepared from 0.5 – 2,000 ng/mL in plasma, and 5 – 5,000 ng/mL in urine. Product ion scanning was used to detect four additional xylazine metabolites by spectral matching with previously published full scan tandem mass spectra. The highest intensity fragment ions observed in the product ion spectra were used to develop multiple reaction monitoring experiments for four additional xylazine metabolites, previously described as: sulfone-xylazine, oxo-xylazine, OH-sulfone-xylazine, and OH-oxo-xylazine, in 50 remnant urines and 70 individual plasmas. In remnant urines, xylazine concentrations ranged from less than 5 to greater than 5,000 ng/mL. In remnant plasmas, xylazine ranged from less than 0.5 to 158 ng/mL. Previously reported xylazine metabolites were detected in both urine and plasma, and relative peak area ratios were calculated as metabolite-to-xylazine in both matrices. In urine, the highest average peak area ratio (3.1) was observed for sulfone xylazine, after hydrolysis. In plasma, the highest average peak area ratio (1.4) was observed for oxo-xylazine. We were unable to absolutely quantitate either metabolite due to lack of available standards. Future studies include investigation on the association of xylazine exposure and plasma concentrations with clinical presentation and patient outcomes.

LITERATURE REVIEW: THE OMICS STUDY FOR DETERMINING BIOMARKERS IN HUMAN SERUM AND PLASMA WITH DIFFERENT COVID-19 SEVERITY

The severity of COVID-19 provides information on various stages of changes in the body's normal state in various parameters called biological markers (biomarkers) as the initial identification that facilitates management, selection, and total outcomes in therapy. These biomarkers were selected from the selection of samples that are often used in the advanced diagnosis of COVID-19, serum and plasma. This study aims to determine what biomarkers are measured in serum and plasma samples of COVID-19 patients. This literature review is classified as non-experimental, qualitative, and descriptive research. The inclusion criteria are the full-text journals published within the last two years regarding biomarkers in the serum and plasma of COVID-19 patients. Based on these criteria, 49 relevant articles were obtained. The results show that changes occur in the protein, lipid, and metabolite in serum and plasma by the omics approach. These alterations can be in the form of increasing or decreasing levels of each parameter determined through various analytical methods. The biomarkers profile correlates with the severity of COVID-19 as well as with host cells.

Lifelong impacts of puberty timing on human plasma metabolic profiles: A metabolome-wide Mendelian randomization study.

The aim was to investigate the causal relationship between puberty timing and plasma metabolites, accounting for birth weight, childhood and adulthood adiposity. The meta-analysis of genome-wide association studies (GWAS) for puberty timing was extracted from the ReproGen Consortium, involving 329 345 women of European ancestry. Summary data for 174 plasma metabolites were retrieved from a recently conducted cross-platform GWAS that involved a meta-analysis of three cohort studies (i.e. the Fenland, European Prospective Investigation into Cancer-Norfolk and INTERVAL studies) and three publicly available studies and included up to 86 507 participants. We conducted a two-sample Mendelian randomization (MR) analysis to infer the causal relationship of puberty timing on 174 plasma metabolites, complemented by a two-step and multivariable Mendelian randomization (MVMR) analysis to assess direct and indirect effects. Additionally, summary-level data from the UK Biobank were used for our replication analysis. The results of the two-sample MR provide moderate evidence supporting a causal relationship between puberty timing and 23 of 174 plasma metabolites (i.e. 7 acylcarnitines, 8 amino acids, 2 biogenic amines and 6 lysophosphatidylcholines). Even after single-nucleotide polymorphisms associated with birth weight and childhood adiposity were excluded, causal effects persisted for 16 metabolites (i.e. 8 acylcarnitines, 4 amino acids, 2 biogenic amines and 2 lysophosphatidylcholines). The two-step MR analysis provided evidence that the relationship between puberty timing and plasma metabolites was mediated by adulthood adiposity. Additionally, moderate evidence emerged for an independent causal effect of puberty timing on 10 metabolites through an MVMR analysis (i.e. 5 acylcarnitines, 2 amino acids, 1 biogenic amine, 1 lysophosphatidylcholine and 1 phosphatidylcholine). Furthermore, the replication analysis suggested the robustness of our results. In summary, our study provides compelling evidence that puberty timing has a causal influence on certain plasma metabolites, although this influence is largely mediated by adulthood adiposity.

Safety Assessment of Camelid-Derived Single-Domain Antibody as Feed Additive for Juvenile Whiteleg Shrimp (Litopenaeus vannamei) Against White Spot Syndrome Virus.

A six-week feeding trial was conducted to assess the safety of single-domain antibodies (sdAbs) derived from camelids against the white spot syndrome virus (WSSV) (WSSVvp28 was used as the antigen), focusing on the whole-organism responses and molecular-level changes in juvenile whiteleg shrimp (Litopenaeus vannamei). Five experimental diets with varying levels of sdAbs were formulated: CON (no sdAb supplementation); SDA8.2 (8.20% of sdAbs); SDA16.4 (16.40% of sdAbs); SDA24.6 (24.60% of sdAbs); and SDA32.8 (32.80% of sdAbs). In the CON diet, 450 mL of water per kg of diet (45%) was used to form a feed dough, while sdAbs were used to replace the water in the treatment diets. A total of 450 shrimp, with an initial body weight of 3.27 ± 0.02 g (mean ± SEM), were randomly distributed in 15 tanks (30 shrimp per tank; three tanks per treatment). Each tank was filled with 30 L of seawater (77 L capacity) in an indoor semi-recirculating system with a constant water flow rate of 1.2 L min-1. The photoperiod was maintained at 12 h of light and 12 h of dark. The water temperature, pH, salinity, and dissolved oxygen were 27.3 ± 0.1 °C, 7.61 ± 0.01, 34 ± 1 ppt, and 5.94 ± 0.04 mg L-1, respectively. During the feeding trial, the shrimp were fed the experimental diet (40% protein and 11% lipid) three times a day for six weeks. Following the feeding trial, an acute cold-water-temperature stress test was conducted by abruptly exposing the shrimp from each treatment to 15 °C for 4 h, down from 27 °C. The results showed no significant differences in the growth performance (weight gain, feed utilization efficiency, survival, etc.), plasma metabolites (aspartate aminotransferase activity, alanine aminotransferase activity, total protein, and glucose), or antioxidant enzymes (superoxide dismutase and glutathione peroxidase) among all the experimental diets (p > 0.05). In the acute cold-temperature stress test, there was no significant interaction between sdAb supplementation and temperature stress, nor any main effect from either factor, except for the main effect of temperature stress on the glucose levels, which was significantly higher in shrimp exposed to cold-temperature stress (p < 0.05). The next-generation sequencing of differentially expressed genes (DEGs) in the hepatopancreases of shrimp fed the CON, SDA16.4, and SDA32.8 diets, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, indicated that DEGs were significantly enriched in signaling pathways associated with growth, cold stress, and antioxidant systems. Overall, the results from conventional measurements suggest that the use of sdAbs against the WSSV may be safe for juvenile whiteleg shrimp. However, findings from the sophisticated analysis indicate that further research is needed to understand the molecular mechanisms underlying the observed changes, and to evaluate the long-term effects of sdAb supplementation in shrimp diets.

Maternal Diet during Pregnancy Alters the Metabolites in Relation to Metabolic and Neurodegenerative Diseases in Young Adult Offspring

Maternal nutrition during the critical period of pregnancy increases the susceptibility of offspring to the development of diseases later in life. This study aimed to analyze metabolite profiles to investigate the effect of maternal diet during pregnancy on changes in offspring plasma metabolites and to identify correlations with metabolic parameters. Pregnant Sprague-Dawley rats were exposed to under- and overnutrition compared to controls, and their offspring were fed a standard diet after birth. Plasma metabolism was profiled in offspring at 16 weeks of age using liquid chromatography–mass spectrometry (LC-MS/MS) and gas chromatography–tandem mass spectrometry (GC-MS/MS). We analyzed 80 metabolites to identify distinct metabolites and metabolic and neurodegenerative disease-associated metabolites that were sex-differentially altered in each group compared to controls (p &lt; 0.05, VIP score &gt; 1.0). Specifically, changes in 3-indolepropionic acid, anthranilic acid, linoleic acid, and arachidonic acid, which are involved in tryptophan and linoleic acid metabolism, were observed in male offspring and correlated with plasma leptin levels in male offspring. Our results suggest that fatty acids involved in tryptophan and linoleic acid metabolism, which are altered by the maternal diet during pregnancy, may lead to an increased risk of metabolic and neurodegenerative diseases in the early life of male offspring.