Plasma Metabolites Research Articles

Mediation effect of plasma metabolites on the relationship between immune cells and the risk of prostatitis: A study by bidirectional 2-sample and Bayesian-weighted Mendelian randomization.

According to the findings of multiple observational studies, immune disorder was a risk factor for prostatitis. However, it remained unknown whether there was a direct causal relationship between immune cells and prostatitis or whether this relationship was mediated by plasma metabolites. Based on the pooled data of a genome-wide association study (GWAS), a genetic variant was used to predict the effects of 731 immunophenotypes on the risk of prostatitis and determine whether the effects were mediated by 1400 metabolites. The bidirectional 2-sample Mendelian randomization (MR) method was adopted to uncover the causal relationship between immunophenotypes and prostatitis. Subsequently, a 2-step MR method was employed to evaluate whether the metabolites mediated this causal relationship and quantify the mediating effects and the corresponding ratios. In addition, the Bayesian-weighted Mendelian randomization (BWMR) method was employed to verify the results. Among the 731 immunophenotypes analyzed, 16 had causal relationships with the risk of prostatitis, including 11 with positive correlations (P < .05, beta > 0) and 5 with negative correlations (P < .05, beta < 0). The MR analysis screened out 9 metabolites related to the risk of prostatitis. The X - 24344 levels mediated the causal relationship between CD3 on CD39+ activated Treg and prostatitis (mediation effect: 0.01; ratio: 9.82%). Both histidine betaine (hercynine) levels and the proline-to-glutamate ratio mediated the causal relationship between CD14-CD16+ monocyte absolute count and prostatitis, with the mediation effects of -0.016 (14.20%) and -0.008 (7.24%), respectively. The glutamine degradant levels mediated the causal relationship between HLA DR+ CD4+ %T cells and prostatitis, with a mediation effect of -0.012, accounting for 8.07% of the total. The present study indicated that the immune cell subsets predicted based on gene expression profiles were potentially beneficial or harmful risk factors of prostatitis, and plasma metabolites may serve as the mediating factors of the relationship. The study thus shed light on deciphering the immunologic mechanism of prostatitis.

Targeted metabolomic profiling of acute ST-segment elevation myocardial infarction

Myocardial infarction is a major cause of morbidity and mortality worldwide. Metabolomic investigations may be useful for understanding the pathogenesis of ST-segment elevation myocardial infarction (STEMI). STEMI patients were comprehensively examined via targeted metabolomic profiling, machine learning and weighted correlation network analysis. A total of 195 subjects, including 68 STEMI patients, 84 patients with stable angina pectoris (SAP) and 43 non-CVD patients, were enrolled in the study. Metabolomic profiling involving the quantitative analysis of 87 endogenous metabolites in plasma was conducted. This study is the first to perform targeted metabolomic profiling in patients with STEMI. We identified 36 significantly altered metabolites in STEMI patients. Increased levels of four amino acids, eight acylcarnitines, six metabolites of the NO–urea cycle and neurotransmitters, and three intermediates of tryptophan metabolism were detected. The following metabolites exhibited decreased levels: six amino acids, three acylcarnitines, three components of the NO–urea cycle and neurotransmitters, and three intermediates of tryptophan metabolism. We found that the significant changes in tryptophan metabolism observed in STEMI patients—the increase in anthranilic acid and tryptophol and decrease in xanthurenic acid and 3-OH-kynurenine—may play important roles in STEMI pathogenesis. On the basis of the differences in the constructed weighted correlation networks, new significant metabolite ratios were identified. Among the 22 significantly altered metabolite ratios identified, 13 were between STEMI patients and non-CVD patients, and 17 were between STEMI patients and SAP patients. Seven of these ratios were common to both comparisons (STEMI patients vs. non-CVD patients and STEMI patients vs. SAP patients). Additionally, two ratios were consistently observed among the STEMI, SAP and non-CVD groups (anthranilic acid: aspartic acid and GSG (glutamine: serine + glycine)). These findings provide new insight into the diagnosis and pathogenesis of STEMI.

Causal relationship and mediation effects of immune cells and plasma metabolites in atopic dermatitis: A Mendelian randomization study.

Atopic dermatitis (AD) is a chronic inflammatory skin condition with complex etiology involving genetic, environmental, and immunological factors. This study employs Mendelian randomization to explore the causal relationships between immune cell phenotypes and AD, and the mediating effects of plasma metabolites. Using data from European cohorts, we identified 7 immune cell phenotypes significantly associated with AD. Mediation analysis revealed that the alpha-ketobutyrate to 4-methyl-2-oxopentanoate ratio negatively regulates CCR2 on monocytes, while the glycerol to carnitine ratio positively regulates HLA-DR on CD14- CD16- cells. These findings underscore the critical role of metabolic pathways in modulating immune responses and suggest potential dietary and therapeutic interventions for AD management. Further research should consider more diverse populations to validate these findings.

Cross-validation of methods for the quantitative determination of phenazepam and its active metabolite in human blood plasma at various extractions

Introduction. In conducting of therapeutic drug monitoring (TDM), often such situation arises where the drug concentration has measured by different methods or in different laboratories. To combine and analyze the data obtained with different methods, it is necessary to perform cross-validation procedure. Insufficient attention is paid to the statistical approaches used for this purpose.Aim. Performing cross-validation of different analytical methods for the quantitative determination of phenazepam (PHEN) and 3-hydroxyphenazepam (3-OH-PHEN) using the Bland – Altman analysis.Materials and methods. PHEN and 3-OH-PHEN concentrations in the blood plasma of patients (n = 100) with alcohol withdrawal syndrome were measured using high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The quantification of both analytes in each sample was measure twice by two different methods: solid phase extraction (SPE) and supported liquid extraction (SLE). Both methods have been fully validated before the experiment began. Cross-validation was performed at the end of the experiment using data from study samples. The Bland – Altman analysis was used to evaluate accuracy and precision. Deming regression was also used to identify a systematic error between measurement results.Results and discussion. The regression equations have been obtained between concentrations both analytes measured by different sample preparation methods. 95 % confidence intervals (CI) of the regression coefficients of both equations included one, and 95 % CI of the intercepts included zero. 95 % CI of the geometric mean of the individual SLE/SPE ratios was within the acceptable range (0.87; 1.15). These results confirm the absence of the influence of quantitative methods on the measurement of both analytes concentration. 66.7 % CI of the percent difference between two measurements was within acceptable limits (–0.2; 0.2), not exceeding 20 % of the range of their mean value. This confirms the acceptable precision between the methods. The estimated CIs were displayed in the Bland – Altman plots.Conclusion. The statistical approaches used in the work have confirmed the reproducibility of the results of different sample preparation methods. In addition to cross-validation, the statistical algorithm from this paper using Bland – Altman analysis can be successfully employed to assess accuracy and precision during bioanalytical method validation and evaluation of the acceptance of analytical runs, as well as to determine the level of reproducibility of incurred samples.

LC-MRM-MS-based lipidomics reveals differences in the digestion, absorption, and metabolism of phosphatidylcholines from Egg, Soy, and Krill in vivo

The acyl chain composition of dietary sources phosphatidylcholine (PC) is highly diverse. However, the effect of different PC intakes on the digestive products lysophosphatidylcholines (LPCs) and plasma metabolite species has not been elucidated. Here, LC-MRM-MS analysis was employed to identify and quantify 36 PC and 10 LPC molecular species in small intestinal contents, jejunum, plasma, and colonic contents within 12 h after administration of Egg-PC, Soy-PC, and Krill-PC. These results showed that the three PCs were rapidly hydrolyzed to LPCs within 0.5 h of reaching the small intestine, and the LPC species showed significant positive correlations with the corresponding fatty acid composition of the dietary PC. Notably, the abundance of plasma PUFA_PUFA PCs such as PC18:2_18:2 increased 5.8-fold after administration of Soy-PC for 2 h. Strikingly, administration of krill-PC for 2 h increased plasma LPC20:5 and PC16:0_20:5 by 23.6-fold and 26.6-fold, respectively, compared to 0 h, but the level of PC20:5_20:5 was low. In addition, correlation between the LPC species in small intestinal contents and plasma phospholipid species were analyzed, shedding light on the rules of re-esterification and absorption. These results could be useful for efficiently exploiting the nutritional functions of PCs with different acyl chains.

Quantitation of the DNA-dependent protein kinase inhibitor peposertib (M3814) and metabolite in human plasma by LC-MS/MS.

The DNA-dependent protein kinase (DNA-PK) is an abundant nuclear protein that mediates DNA double-strand break repair by nonhomologous end joining (NHEJ). As such, DNA-PK is critical for V(D)J recombination in lymphocytes and for survival in cells exposed to ionizing radiation and clastogens. Peposertib (M3814) is a small molecule DNA-PK inhibitor currently in preclinical and clinical development for cancer treatment. We have developed a high-performance liquid chromatography-mass spectrometry method for quantitating peposertib and its metabolite in 0.1 mL human plasma. After MTBE liquid-liquid extraction, chromatographic separation was achieved with a Phenomenex Synergi polar reverse phase (4μm, 2× 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and water over an 8min run time. Mass spectrometric detection was performed on an ABI SCIEX 4000 with electrospray, positive-mode ionization. The assay was linear from 10 to 3000 ng/mL for peposertib and 1-300 ng/mL for the metabolite and proved to be both accurate (97.3%-103.7%) and precise (<8.9%CV) fulfilling criteria from the Food and Drug Administration (FDA) guidance on bioanalytical method validation. This liquid chromatography-tandem mass spectroscopy (LC-MS/MS) assay will support several ongoing clinical studies by defining peposertib pharmacokinetics.

Perturbations in the blood metabolome up to a decade before prostate cancer diagnosis in 4387 matched case-control sets from the European Prospective Investigation into Cancer and Nutrition.

Measuring pre-diagnostic blood metabolites may help identify novel risk factors for prostate cancer. Using data from 4387 matched case-control pairs from the European Prospective Investigation into Cancer and Nutrition (EPIC) study, we investigated the associations of 148 individual metabolites and three previously defined metabolite patterns with prostate cancer risk. Metabolites were measured by liquid chromatography-mass spectrometry. Multivariable-adjusted conditional logistic regression was used to estimate the odds ratio per standard deviation increase in log metabolite concentration and metabolite patterns (OR1SD) for prostate cancer overall, and for advanced, high-grade, aggressive. We corrected for multiple testing using the Benjamini-Hochberg method. Overall, there were no associations between specific metabolites or metabolite patterns and overall, aggressive, or high-grade prostate cancer that passed the multiple testing threshold (padj <0.05). Six phosphatidylcholines (PCs) were inversely associated with advanced prostate cancer diagnosed at or within 10 years of blood collection. metabolite patterns 1 (64 PCs and three hydroxysphingomyelins) and 2 (two acylcarnitines, glutamate, ornithine, and taurine) were also inversely associated with advanced prostate cancer; when stratified by follow-up time, these associations were observed for diagnoses at or within 10 years of recruitment (OR1SD 0.80, 95% CI 0.66-0.96 and 0.76, 0.59-0.97, respectively) but were weaker after longer follow-up (0.95, 0.82-1.10 and 0.85, 0.67-1.06). Pattern 3 (8 lyso PCs) was associated with prostate cancer death (0.82, 0.68-0.98). Our results suggest that the plasma metabolite profile changes in response to the presence of prostate cancer up to a decade before detection of advanced-stage disease.

Analyzing Paxlovid: Examining Its Properties, Characteristics, and Analytical and Bio‐Analytical Methods–A Comprehensive Review

ABSTRACTPaxlovid, a novel antiviral medication developed by Pfizer, has attracted considerable attention as a coronavirus disease 2019 therapeutic since it may be effective in treating the severe acute respiratory syndrome coronavirus type 2‐caused disease. Paxlovid is known as a combination therapy since it includes two agents, Nirmatrelvir and Ritonavir, which both are involved in the viral replication process inhibition. A further discussion of the molecular structure, pharmacokinetics, and pharmacodynamics of both components will help comprehend Paxlovid's mechanism of impact and effectiveness. High‐performance and spectroscopic methods, including high‐performance liquid chromatography (HPLC), infrared, and ultraviolet‐visible spectroscopy have been among the methods employed for compound identification, quantification, and structural elucidation. Bioanalytical methods have also made a significant contribution to the evaluation of Paxlovid's pharmacokinetic parameters, bioavailability, and metabolism in biological matrices. LC‐tandem mass spectrometry (LC‐MS/MS) is widely employed for the quantification of Paxlovid and its metabolites in plasma, serum, and other biological samples. This study brings forth an overview of the characteristics and properties of Paxlovid along with analytical and bioanalytical methods employed for its assessment and quantification.

Quantification of MDR-TB drug JBD0131 and its metabolite in plasma via UPLC-MS/MS: application in first-in-human study.

Aim: JBD0131, a novel anti-multidrug-resistant tuberculosis (MDR-TB) drug, can target and inhibit the synthesis of mycolic acids, which are crucial components of the cell wall of the Mycobacterium tuberculosis complex. To support the results of this clinical trial in healthy subjects, development of a specific and accurate quantification method for detecting JBD0131 and its metabolite DM131 in human plasma is needed.Materials & methods: Samples with prior added stabilizer were pretreated by protein precipitation method and the extracts weresubjected to ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The m/z transitions for the precursor/product ion pairs were 402.1/273 for JBD0131, 333.1/273 for DM131 and 386.1/257 for the internal standard (IS).Results: This method showed good linearity from 1 to 2000ng/ml for JBD0131 and 0.25 to 500ng/ml for DM131 and was validated in terms of selectivity, linearity, accuracy, precision, matrix effect, recovery of pretreament and stability.Conclusion: This method was sensitive and specific for measuring the plasma concentrations of JBD0131 and its metabolites. And it was applied for the investigation of the pharmacokinetics of JBD0131 and DM131 in a clinical trial.

Proximal Tubule Secretory Clearance, Injury, and Kidney Viability in Cirrhosis.

Cirrhosis affects all structures of the kidney, in particular the tubules which are responsible for secretion of protein-bound metabolites and electrolyte/water homeostasis. Yet, prevailing assessments of kidney function focus solely on glomerular filtration rate (GFR) which may incompletely reflect these processes. We sought to characterize markers of tubular function, injury, and viability in patients with and without cirrhosis. We recruited outpatients undergoing liver transplantation evaluation for a collection of plasma and 24-hour urine, matching by GFR to control participants without cirrhosis. We measured urinary kidney injury molecule-1 (KIM-1), a marker of proximal tubular injury, as well as epidermal growth factor (EGF), a marker of viability necessary for tubular epithelial cell proliferation after injury. We also estimated secretory clearance by measuring several highly secreted endogenous metabolites in urine and plasma. We recruited 39 patients with cirrhosis (mean MELD-Na 17±4, Child-Pugh 8±2, eGFR 66±20 ml/min/1.73m2) and 58 GFR-matched controls without cirrhosis (eGFR 66±21 ml/min/1.73m2). Urinary KIM-1 was 4.4-fold higher than controls (95% CI:2.9-6.5), and EGF averaged 7.41-fold higher than controls (95% CI:2.15-25.53). We found that of 8 solutes, 5 had significantly greater kidney clearance in cirrhosis (1.3-2.1-fold higher): indoxyl sulfate, p-cresol sulfate, pyridoxic acid, tiglylglycine, and xanthosine. Cirrhosis was characterized by molecular signs of tubular injury in stable outpatients without acute kidney injury, accompanied by largely preserved tubular secretory clearance and greater signs of tubular viability. Within the limitations of the study, this suggests a phenotype of chronic ischemic injury but with initial preservation of tubular function in cirrhosis.

8679 Case: The Sexual, Physical and Psychological Effects of Post-Finasteride Syndrome

Abstract Disclosure: E.M. Lonergan: None. S.M. Maher: None. I. Yaseen: None. D.B. O'Shea: None. Post-finasteride syndrome (PFS) is an increasingly recognised disorder with a myriad of physical and neuropsychiatric symptoms persisting for greater than three months in a small proportion of patients treated with Finasteride. We present the case of a 45-year-old male who was taking oral Finasteride for hair loss. He first presented to endocrinology services having stopped Finasteride after twenty years reporting new onset reduced libido, penile shrinkage, fatigue, new food intolerances and agitated depression which was the predominant symptom in this case. He described the emergence of gynaecomastia, which was evident on examination, as well as muscle atrophy. Biochemistry was unremarkable including testosterone 24.6 nmol/L (8.6-29.0), oestradiol 100 pmol/L (50 – 159), LH 7.1 IU/L (1.7-8.6), FSH 16.8 IU/L (1.5-12.4), urinary free cortisol 177 nmol/24h (12-486) and urinary steroid profile. The patient was offered a trial of IM testosterone for 3 months followed by a course of hCG which he declined after discussing with patients online via a PFS patient forum citing anecdotal pseudo immune downregulation in this cohort. GLP-1 receptor agonist therapy was considered as a second-line treatment option. The patient expressed a preference for hydrocortisone treatment after conducting online research which has not been commenced due to a lack of evidence. The inhibition of 5-alpha-reductase in Finasteride-treated patients has been documented to cause reversible sexual dysfunction. However, with increasing case reports of PFS, it was included in the list of Rare and Genetic Diseases of the NIH in 2015. The syndrome encompasses symptoms and signs seen in our case, as well as erectile dysfunction, decreased ejaculatory volume, infertility, Peyronie’s disease, testicular pain, dry skin, memory problems and insomnia. Suicidal ideation was included in the product label as per FDA mandate in 2022. The literature is largely inconclusive regarding the underlying aetiology, duration, and severity of the disorder. A large retrospective study revealed 0.8% of patients developed sexual dysfunction with persistence in 33% of these cases after drug suspension, the main independent risk factor being drug treatment for longer than 7 months. Neuropsychiatric symptoms are likely related to the presence of 5-alpha-reductase isoforms in the brain with resulting alterations in CSF and plasma neuroactive steroid levels and metabolites. Evidence-based treatment options for PFS are limited. PFS is a rare but debilitating syndrome to which a subset of patients appears to be predisposed, but of which the pathophysiology is poorly understood. Further studies in this regard, as well as an exploration of effective treatment options are required. Presentation: 6/1/2024

Decreased plasma nicotinamide and altered NAD+ metabolism in glial cells surrounding Aβ plaques in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disease and a leading cause of senile dementia. Amyloid-β (Aβ) accumulation triggers chronic neuroinflammation, initiating AD pathogenesis. Recent clinical trials for anti-Aβ immunotherapy underscore that blood-based biomarkers have significant advantages and applicability over conventional diagnostics and are an unmet clinical need. To further advance ongoing clinical trials and identify novel therapeutic targets for AD, developing additional plasma biomarkers closely associated with pathogenic mechanisms downstream of Aβ accumulation is critically important. To identify plasma metabolites reflective of neuroinflammation caused by Aβ pathology, we performed untargeted metabolomic analyses of the plasma by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) and analyzed the potential roles of the identified metabolic changes in the brain neuroinflammatory response using the female App knock-in (AppNLGF) mouse model of Aβ amyloidosis. The CE-TOFMS analysis of plasma samples from female wild-type (WT) and AppNLGF mice revealed that plasma levels of nicotinamide, a nicotinamide adenine dinucleotide (NAD+) precursor, were decreased in AppNLGF mice, and altered metabolite profiles were enriched for nicotinate/nicotinamide metabolism. In AppNLGF mouse brains, NAD+ levels were unaltered, but mRNA levels of NAD+-synthesizing nicotinate phosphoribosyltransferase (Naprt) and NAD+-degrading Cd38 genes were increased. These enzymes were induced in reactive astrocytes and microglia surrounding Aβ plaques in the cortex and hippocampus of female AppNLGF mouse brains, suggesting neuroinflammation increases NAD+ metabolism. This study suggests plasma nicotinamide could be indicative of the neuroinflammatory response and that nicotinate and nicotinamide metabolism are potential therapeutic targets for AD, by targeting both neuroinflammation and neuroprotection.

6870 Cardiac Imaging And Metabolomics Studies Suggest Differential Risk And Protective Biomarkers For Cardiovascular Disease In Renoprotected, Long-Duration Type 1 Diabetes

Abstract Disclosure: M. Yu: None. S. Jangolla: None. N. Ziemniak: None. E. Viebranz: None. T. Chokshi: None. K. Park: None. I. Wu: None. H. Shah: None. G.L. King: None. Cardiovascular disease (CVD) is a major cause of mortality in people with Type 1 diabetes (T1D), especially in those with nephropathy (DN). However, in long-duration T1D, a substantial proportion of individuals without DN still exhibit excessive CVD risks. The Joslin 50-Year “Medalist” Study (n=1043), composed of people with T1D≥50 years, are ideal for studying CVD in a renoprotected T1D cohort, as only 13% have DN, and yet 40% have CVD, despite excellent control of lipids and blood pressure. Stratifying the Medalists by DN revealed that exercise was cardioprotective in the group with DN (OR 0.60, p=0.04); while age at T1D diagnosis (OR 1.03, p=0.01), HbA1c (OR 1.25, p=0.01), C-reactive protein (OR 1.14, p=0.01), and interleukin-6 (OR 1.13, p=0.01) all increased CVD risk in Medalists without DN. A subset of Medalists also underwent coronary artery calcification scans (CAC; n=153) and cardiac magnetic resonance imaging of left ventricular (LV) structure and function (CMR; n=111). The majority of these Medalists (97.8%) had detectable CAC&amp;gt;0; furthermore, the median CAC score of 1000 was higher at any cutoff compared to similar age-matched T1D and Type 2 diabetes (T2D) populations with higher DN prevalence. In contrast, mean CMR values in this Medalist subset were comparable to other cohorts. Continuous glucose monitoring (CGM) metrics for hyperglycemia (mean glucose, β=0.002, p=0.03; glucose management indicator, β=0.09, p=0.03; and time above range&amp;gt;180 mg/dL, β=0.003, p=0.02), but not glucose variability or hypoglycemia, were associated with LV remodeling in the Medalists, even after adjusting for eGFR. Likewise, plasma levels of advanced glycation end-products (AGEs) N-carboxyethyl-lysine (CEL), N-carboxymethyl-lysine (CML), and methylglyoxal-derived hydroimidazolone (MGH1) were associated with CAC (β=16.22, p=0.05; β=15.21, p=0.01; and β=10.18, p=0.01, respectively) and LV remodeling (β=0.001, p=0.09; β=0.001, p=0.05; and β=0.001, p=0.04, respectively) in the Medalists. The association of CEL with CVD replicated in a separate Medalist subset (n=311) with self-reported CVD data (OR 1.26, p=0.01). Plasma global metabolomics studies (n=140) revealed that amino acid activation pathways, with lower levels of branched chain amino acids, were associated with CAC (FDR=0.002); while amino acid metabolism pathways were associated with less favorable LV volumes (FDR=0.03). In this comprehensive study of aging T1D people with no substantial DN, hyperlipidemia, or hypertension, cardiac imaging and metabolomics studies suggest disproportionate advanced CAC, as compared to subclinical LV remodeling or dysfunction, and highlight the importance of targeting hyperglycemia for CVD risk reduction. Plasma metabolites associated with CVD in T1D are different from those in T2D, and may provide novel information on potential therapeutic targets for CVD in T1D. Presentation: 6/1/2024

Causal effects of plasma metabolites on autoimmune hepatitis (AIH): a bidirectional two-sample mendelian randomization study

Autoimmune hepatitis(AIH) is a chronic progressive inflammatory liver disease induced by loss of immune tolerance. The role of circulating metabolites in disease pathogenesis is unclear. This study aimed to investigate potential causal links between plasma metabolites and AIH risk by employing a two-sample Mendelian randomization approach. A comprehensive bidirectional two-sample Mendelian randomization analysis was conducted using genome-wide significant variant-metabolite and variant-AIH associations in European ancestry individuals. Various methods assessed causal relationships among 1400 metabolites and AIH, incorporating sensitivity analyses to evaluate pleiotropy and heterogeneity. Fifty-eight metabolites displayed possible associations, including increased AIH risk with genetically predicted higher kynurenine (p = 2.79 × 10− 5, OR: 1.64, 95% CI 1.30–2.07) and a protective effect for the dopamine sulfate ratio (p = 1.06 × 10− 5,OR: 0.62, 95% CI 0.49–0.79). Reciprocal analysis revealed a causal effect of AIH on kynurenine( p = 2.79 × 10− 5, OR: 1.64, 95% CI 1.30–2.07), but not on the dopamine sulfate ratio(p = 0.691, OR: 1.05, 95% CI 0.67–1.64). Our genetics-based approach provides evidence supporting a causal role for specific metabolite levels in AIH risk. The results deliver evidence supporting a causal effect of a specific metabolite ratio(dopamine 4-sulfate/dopamine 3-O-sulfate) on AIH risk. Experimental validation and mechanistic examinations are warranted to confirm findings.

Stocking density affects growth, feed utilisation, metabolism, welfare and associated mRNA transcripts in liver and muscle of rainbow trout more pronouncedly than dietary fish meal inclusion level

In aquaculture, stocking density and diet composition are critical factors that influence the performance and welfare of the farmed animal. However, there is limited information on their interactive effects. In this context, applying a 2 × 2 factorial design, we conducted a 12-week feeding trial to evaluate the effect of stocking density (low, LSD: 3.3–15 kg m−3 and high, HSD: 5.8–25 kg m−3; initial-final values) and dietary fish meal content (high, HFM: 30 % and low, LFM: 15 %) on growth, feed utilisation, metabolic oxygen consumption, tissue indices, carcass composition, plasma metabolites, and transcriptional regulation of growth, metabolism and welfare-related biomarker mRNAs in liver and muscle of the juvenile rainbow trout. At the whole-animal level, the HSD group showed significantly higher routine metabolic rate and feed intake indicating higher energy demand; but growth and feed efficiency was lower in HSD than the LSD group. Morpho-anatomically, HSD fish had lower relative gut length and higher intra-peritoneal fat index than LSD fish. Lower plasma free amino acids and glucose levels reiterated high energy expenditure in HSD, and elevated albumin levels indicated potential oxidative stress in the HSD group. Correspondingly, at the transcriptional level, hepatic mRNA levels of antioxidative enzymes (sod, cat, gst, g6pd) and stress-related molecular chaperone hsp90 were up-regulated in HSD; while the transcript abundance of growth-axis markers (ghr, igf1 igf2) and nutrient-sensor mtor were decreased. In muscle, proteolytic pathway components fbx32 and murf1 were upregulated in HSD, and concurrently mtor was also upregulated suggesting higher protein turnover in this group. With respect to the effect of dietary fish meal inclusion levels, LFM showed decrease in growth, hepatic igf2 expression, and lower plasma triglycerides and free amino acid levels than HFM group. However, the sensory appearance of flesh was found to be slightly better in LFM. Significant diet-husbandry interactions were not observed phenotypically, but the mRNA abundance of hepatic markers (ampk1, hsp70, mhc1 and tnfa) linked to energy-sensing, stress and immune response were differentially regulated. Overall, the study shows that high stocking density has a greater degree of impact on fish growth and welfare than reduction in dietary fishmeal inclusion, with limited interactional effects.

B-298 Quantitation and Abundance of Xylazine and Xylazine Metabolites in Human Urine by LC-MS/MS

Abstract Background 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 associated with xylazine as an emerging threat to the US, and a national response plan will include xylazine testing. Xylazine, a clonidine analog 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 investigate the presence and abundance of xylazine metabolites in remnant urine samples submitted for clinical testing. Methods Remnant urine samples were identified when tested positive for xylazine during routine clinical LC-MS/MS testing. All experiments were performed using an Acquity HPLC coupled to a Waters TQS-micro triple quadrupole mass spectrometer. Urines were diluted with xylazine-D6 internal standard, hydrolyzed using β-glucuronidase and separated on a 100mm x 2.1mm, 2.6μm Kinetex C18 column (Phenomenex) with a 4 minute step 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. Xylazine and 4-OH-xylazine were detected in positive mode using multiple reaction monitoring, with transitions optimized from standards (Cayman Chemical and/or Sigma Aldrich), resulting in the following transitions: xylazine: 221&amp;gt;90, 221&amp;gt;164; 4-OH xylazine: 237&amp;gt;90, 237&amp;gt;137. Xylazine and 4-OH-xylazine were quantified using calibration curves prepared from 1 - 5,000 ng/mL in negative urine. Product ion scanning (PIS) was used to detect four additional xylazine metabolites, based on spectral matching to previously published full scan high resolution product ion spectra. The highest abundance fragment ions and identified retention times from PIS experiments were used to perform targeted detection for sulfone-xylazine (253&amp;gt;181, 253&amp;gt;147), oxo-xylazine (235&amp;gt;122, 235&amp;gt;114), OH-sulfone-xylazine (269&amp;gt;197, 269&amp;gt;163), and OH-oxo-xylazine (251&amp;gt;197, 251&amp;gt;148). Results Xylazine was detected at &amp;gt;=1 ng/mL in 38% (54/138) of fentanyl positive urines. A subset (n= 362) of urines that screened positive for opiates, amphetamines, or cocaine metabolite, were also tested but xylazine was detected in only 1 urine that did not contain fentanyl.Xylazine concentrations quantified in 61 remnant urines ranged from &amp;lt;1 to &amp;gt;5,000 ng/mL. 4-OH-xylazine concentrations ranged from &amp;lt;1 to 509 ng/mL. We observed five previously reported xylazine metabolites: 4-OH-xylazine, oxo-xylazine, sulfone-xylazine, OH-oxo-xylazine, OH-sulfone-xylazine. The mean concentration ratio of 4-OH-xylazine-to-xylazine was 0.11. The mean peak area ratios (metabolite-to-xylazine) for sulfone-xylazine, OH-oxo-xylazine, OH-sulfone-xylazine, and oxo-xylazine were 3.5, 3.8, 2.1, and &amp;lt;0.1, respectively. The proposed sulfone-xylazine and OH-oxo-xylazine metabolites were observed to have the most abundant signal of all detected metabolites, although there is currently no commercially available standard to facilitate quantification. Conclusions Xylazine and 4-OH-xylazine were quantitated in 61 patient urines. Four additional xylazine metabolites were observed and relative abundance to xylazine was estimated using peak areas. Future studies include quantitation of xylazine and xylazine metabolites in plasma and association of xylazine detection with clinical presentation and patient outcomes.

Multi-omics analysis reveals the key factors involved in the severity of the Alzheimer’s disease

Alzheimer’s disease (AD) is a debilitating neurodegenerative disorder with a global impact, yet its pathogenesis remains poorly understood. While age, metabolic abnormalities, and accumulation of neurotoxic substances are potential risk factors for AD, their effects are confounded by other factors. To address this challenge, we first utilized multi-omics data from 87 well phenotyped AD patients and generated plasma proteomics and metabolomics data, as well as gut and saliva metagenomics data to investigate the molecular-level alterations accounting the host-microbiome interactions. Second, we analyzed individual omics data and identified the key parameters involved in the severity of the dementia in AD patients. Next, we employed Artificial Intelligence (AI) based models to predict AD severity based on the significantly altered features identified in each omics analysis. Based on our integrative analysis, we found the clinical relevance of plasma proteins, including SKAP1 and NEFL, plasma metabolites including homovanillate and glutamate, and Paraprevotella clara in gut microbiome in predicting the AD severity. Finally, we validated the predictive power of our AI based models by generating additional multi-omics data from the same group of AD patients by following up for 3 months. Hence, we observed that these results may have important implications for the development of potential diagnostic and therapeutic approaches for AD patients.

A-001 The Postprandial Levels of the Metabolites in the Metaorganismal Trimethylamine N-Oxide (TMAO) Pathway are Altered in A Diet-, Microbe-, and Sex-Specific Manner

Abstract Background Cardiovascular disease (CVD) remains the leading cause of death in developed countries. Elevated levels of the gut-microbe-associated metabolite trimethylamine-N-oxide (TMAO) have been associated with increased risk for CVD mortality in many large independent studies. In fact, large laboratory corporations such as Labcorp and Quest Diagnostic now offer TMAO diagnostic tests for the assessment of CVD risk and as a marker for disease-associated dysbiosis, using samples obtained from fasting patients. Although the strong association between TMAO levels and CVD risk has been established in fasting blood samples, here we are investigating the potential for a single meal to dynamically alter TMAO-related metabolites based on sex, diverse dietary substrates, and microbial influences. Methods 35 healthy participants were randomized to one of four study groups with approximately 8-9 subjects in each arm. Half of the participants were randomly assigned to receive a three-day broad spectrum antibiotic regimen (ciprofloxacin, metronidazole, and vancomycin) while the others received no antibiotics. These two groups were further subdivided into a group consuming a highly processed meal (originating from local fast food restaurants) or alternatively a whole food meal (containing a variety of fruits, vegetables, and healthy fiber). Blood samples were taken at baseline (after an overnight fast), and then postprandially at 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 6 hours after meal ingestion. Targeted plasma metabolites were quantified using a stable isotope dilution, liquid chromatography - tandem mass spectrometry (LC-MS/MS) method Results While plasma TMAO levels between food groups did not change significantly within the total cohort, there were clear individualized responses to either highly processed or whole food meals. Separation based on sex showed that TMAO levels were significantly reduced in females at 6 hours but remained steady in males for both food groups. Particularly, in the processed food group, TMAO levels were significantly lower in females than males at the 6-hour time point. Neither of these observations held true for TMAO precursors choline, carnitine, or betaine. However, plasma levels of a dietary precursor for TMAO, γ-butyrobetaine, showed clear diet-microbe-host interactions. For males in the processed food group, plasma γ-butyrobetaine levels were significantly increased in subjects on broad spectrum antibiotics. Conclusions Our results show the postprandial levels of TMAO, and its nutrient precursors dynamically change in a diet-, microbe-, and sex-dependent manner. These findings provide new insights into the postprandial levels of TMAO-related metabolites and may inform precision nutritional approaches in those who could benefit from TMAO-lowering strategies.

Chlorella and vegetable oil inclusion in diets for growing rabbits: effects on growth, digestibility, plasma metabolites, and caecal fermentations and microbiota

The inclusion of microalgae in livestock diets has been shown to enhance animal productivity, immune response, and meat quality. However, the role of chlorella (Chlorella vulgaris) in growing rabbit nutrition has been scarcely explored, with available studies focusing on low inclusion levels (<1%) and their effects on rabbit growth and immune response. This study evaluated the growth performance, nutrient digestibility, plasma metabolites, caecal fermentative activity, and caecal microbiota composition of growing rabbits fed diets with different inclusion levels of chlorella and crude fat. A total of 648 mixed-sex Grimaud crossbred rabbits (33 d of age; 841 ± 140 g live weight) were fed 6 experimental diets (96 rabbits per diet for the growth trial) based on a bifactorial design with three dietary inclusion levels of chlorella (0%, 1%, and 2%) and two levels of crude fat (3% and 5%) obtained by the inclusion of soybean oil (1% and 3%, respectively). The trial lasted 38 days until slaughter. From 47 to 51 days of age, 72 rabbits (12 per diet) were submitted to a digestibility trial. At 51 days of age, samples of plasma and caecal content were collected from 36 rabbits (6 rabbit per diet) to analyse plasma metabolites, caecal fermentations, and caecal microbiota. Rabbit live weight at 71 days of age (2 700 g, on average), weight gain (48.8 g/d) and feed conversion ratio (3.27) were unaffected by chlorella inclusion, while feed conversion ratio improved (−5%; P < 0.001) with an increase of crude fat from 3% to 5%. The digestibility of ADF (23.2% vs. 20.9%; P < 0.05) and crude fat (83.8% vs. 85.6%; P < 0.01) improved with the inclusion of chlorella at 2%, as well as the digestibility of crude fat (82.4% vs. 86.9%; P < 0.001) and gross energy (57.3% vs. 58.7%; P < 0.001) with crude fat inclusion at 5%. Plasma non-esterified fatty acids decreased (−19%; P < 0.05) in diets with 5% crude fat. Neither chlorella nor crude fat inclusion levels affected other plasma metabolites, caecal fermentations, or caecal microbiota. Overall, the inclusion of chlorella up to 2% in diets for growing rabbits did not significantly affect diet nutritional value, animal performance, or caecal activity. On the other hand, increasing crude fat to 5% improved the overall feed efficiency.

Novel pilot study on plasma metabolites and biomarkers in a rat model of silica-induced lung inflammation and fibrosis

Silica-induced lung damage may be associated with changes in distinct metabolites potentially serving as biomarkers. Due to the lack of metabolomic data from animal models, this pilot study aimed to evaluate changes in markers of inflammation and fibrosis, as well as plasma metabolites in rats at 14 and 28 days after silica instillation.Adult male Wistar rats were administered a single oropharyngeal intratracheal dose of silica suspension or sterile saline in controls. Selected markers of inflammation, oxidative stress, fibrosis, and cell counts in blood and bronchoalveolar lavage fluid have been evaluated. Finally, plasma metabolites were detected using a targeted metabolomics approach with an MxP® Quant 500 kit.Silica instillation induced noticeable inflammatory, oxidative, and fibrotic changes in lung tissue within the first 14 days. During the next two weeks, the shifts in some markers were further accentuated. After exposure to silica, the metabolomic analysis identified significant changes in metabolites associated with lipid metabolism, biogenic amines, amino acid derivatives, carboxylic acids, bile acids, putrescine, glycosylceramides, and acylcarnitines.This pilot study provides initial evidence that significant alterations in plasma metabolite profiles accompany silica-induced lung injury in rats. These findings suggest a possible systemic impact, particularly on lipid metabolism, and indicate the urgent need for a deeper understanding of the metabolic reprogramming associated with silica-induced lung injury to pave the way for the discovery of novel biomarkers.