Metabolites In Fluids Research Articles

Mendelian randomization study of causal link from Cerebrospinal fluid metabolomics to neurodegenerative diseases.

To investigate the causal relationships between cerebrospinal fluid (CSF) metabolites and various neurodegenerative diseases (NDDs), we conducted a two-sample Mendelian randomization (MR) analysis. This study utilized summary statistics from genome-wide association studies (GWAS) of CSF metabolites and four common neurodegenerative diseases: Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS). MR methods were employed to determine causal associations, with the inverse variance weighted method as the primary approach. Additionally, different GWAS summary data for NDDs were used to validate the initial results and perform sensitivity analyses to enhance the robustness of the findings. Finally, reverse MR analyses were conducted to assess the possibility of reverse causation. Combining results from the initial and replication phases of MR analysis, we identified potential causal relationships between various CSF metabolites and different NDDs. Specifically, we found potential causal relationships between five CSF metabolites and AD, six CSF metabolites and MS, and thirteen CSF metabolites and ALS. Further sensitivity analyses confirmed the robustness of these associations. Reverse MR analysis indicated causal effects of AD on glucuronate and ALS on acetylcarnitine (C2). Our study, through genetic means, demonstrates close causal associations between the specific types of CSF metabolites and the risk of NDDS (AD, PD, MS, and ALS), providing useful guidance for future clinical researches.

Protracted maternal malnutrition induces aberrant changes in maternal uterine artery hemodynamics and the metabolic profiles of the dam and neonate

Malnutritional stress during gestation is a well-established driver of metabolic disfunction in offspring. Extended exposure to malnutrition requires metabolic plasticity as the animal shifts toward a catabolic state. In this paper we demonstrate the influence of malnutrition throughout gestation on uterine artery hemodynamics and the metabolism of the dam and neonate. We hypothesized that gestational malnutrition reduces blood flow of the maternal uterine artery and regulates the metabolic profile of the dam and offspring. Further, the combination of these factors consequently influences the concentration of metabolites in the cerebrospinal fluid of the neonate at birth. To test our hypotheses, pregnant cows caring a single female fetus were assigned to treatments by age and body condition score to one of three individually-fed dietary treatments: Underfed, Control, or Overfed throughout gestation. Uterine blood flow was measured via transrectal Doppler ultrasonography in late gestation. Blood samples were collected from dams throughout gestation, and blood and cerebrospinal fluid were collected from neonates at birth to analyze concentration of metabolites. In the current report, we reveal that maternal malnutrition regulates uterine artery hemodynamics and the maternal metabolic profile throughout gestation. This is the first report to demonstrate that maternal undernutrition leads to an increase in the concentration of urea nitrogen in neonates. Finally, a concentration gradient of metabolites from the dam to neonatal cerebrospinal fluid was observed, which may have potential implications for central nervous system development. These findings not only illustrate the complexity of the maternal-to-fetal interaction required to support the growth of the fetus and homeostasis of the dam but also reveals a novel avenue for investigating the influence of protracted maternal malnutrition on metabolic pathway preferences in offspring. Moreover, these findings are of paramount importance in the development of intervention strategies for morbid neonates.

Quantitative analysis of cannabinoids and metabolites in oral fluid by volumetric absorptive microsampling combined with UHPLC-HRMS.

With recent evolution of cannabis legalization around the world and multiplication of cannabis derived products, identifying and qualifying cannabis consumption has a proven interest. Although blood, plasma, and urine are common matrices widely used in toxicology laboratories, oral fluid presents specific advantages. In the context of doping tests, addiction consultation or roadside checks, where other matrices are impractical to collect or can be adulterated, oral fluid is a promising matrix that allows a non-invasive, rapid, and monitored self-sampling. However, available devices required a consequent volume of oral fluid,more than 250 µL, sometimes difficult to collect. We present here a fully optimized quantitative method for seven cannabinoids, including four metabolites, in oral fluid, Δ9-tetrahydrocannabinol, 11-hydroxy-Δ9-tetrahydrocannabinol and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol; cannabidiol, 7-hydroxy and 7-carboxycannabidiol; and cannabinol. After self-collection of 20 µL using an accurate and precise volumetric absorptive microsampling device (VAMS®), cannabinoids were derivatized with 2-fluoro-1-methylpyridinium p-toluenesulfonate to increase sensitivity. The successive steps of the proposed method, including biosampling, 1 h sample preparation with derivatization, and acquisition by ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry, were fully optimized. A limit of quantification of 0.5 ng/mL (≈10 pg per sampling) was thus targeted, adapted to the legal threshold required by the authorities and to clinical monitoring. Applied to six cannabis consumers, the proposed method made it possible to quantify in 20 µL oral fluid samples, Δ9-tetrahydrocannabinol ranging from 0.5 to 6236 ng/mL, cannabidiol from 0.6 to 190 ng/mL and cannabinol from 0.5 to 118 ng/mL.

The role of cerebrospinal fluid metabolites in mediating the impact of lipids on Late-Onset Alzheimer’s Disease: a two-step mendelian randomization analysis

BackgroundAlthough research has indicated correlations between lipids, cerebrospinal fluid (CSF) metabolites, and Late-Onset Alzheimer’s Disease (LOAD), the specific causal relationships among these elements, as well as the roles and mechanisms of the cerebrospinal fluid metabolites, remain unclear.MethodsStatistical datasets derived from Genome-Wide Association Studies (GWAS) were utilized to assess the bidirectional causal relationships between lipids and LOAD. Subsequently, genetic variants associated with CSF metabolites and established lipids underwent a two-step Mendelian randomization (MR) analysis to explore potential mediators and analyze mediation effects. Sensitivity analyses were employed to assess the robustness of the detection systems.ResultsGenetically predicted cholesterol (IVW OR = 0.989; 95% CI 0.982–0.996) was found to reduce the risk of LOAD, whereas Phosphatidylcholine (PC) (18:1_0:0) (IVW OR = 1.015; 95% CI 1.005–1.025) posed a risk factor. The potential mediator, CSF metabolite N-acetylneuraminate (NeuAC), was identified with a mediation proportion of 21.02% (3.25%, 45.50%). No pleiotropy or heterogeneity was detected across MR analyses.ConclusionsThe findings underscore the pivotal role of CSF metabolomics in elucidating the lipid-mediated pathogenesis of LOAD, highlighting potential diagnostic and preventative biomarkers.

A Comprehensive Review of Analytical Techniques for Quantifying Cyclin‐dependent Kinase 4 and 6 Inhibitors in Biological Samples, Bulk, and Pharmaceutical Samples

ABSTRACTCyclin‐dependent kinase 4 and 6 (CDK4/6) inhibitors are utilized as a remedy for advanced hormone receptor‐positive, HER2‐negative breast cancer (BC). By targeting CDK4/6, these drugs inhibit the phosphorylation of the retinoblastoma protein during the early G1 phase, leading to cell cycle arrest, disruption of deoxyribonucleic acid replication, and effective suppression of cancer cell proliferation. Recently, there has been an increasing focus on the advancement of analytical techniques for the determination of CDK4/6 inhibitors. These analytical methods are essential in the enhancement and refinement of CDK4/6 inhibitors for the management of advanced BC. This review provides a comprehensive overview of the various analytical techniques used for the analysis of palbociclib, ribociclib, and abemaciclib, along with their metabolites in biological fluids (such as blood and urine samples), commercial products, Ringer's solution, and simultaneous estimation. High‐performance liquid chromatography and liquid chromatography‐mass spectrometry are the primary analytical techniques commonly employed for the analysis of CDK4/6 inhibitors. This analytical review also provides insights into pharmacokinetics and stability studies, as well as the use of analytical techniques in detecting impurities and determining the structure of degraded products.

Cerebrospinal fluid metabolites as potential biomarkers for epilepsy: Insights from genome-wide association studies.

While metabolic imbalances have been observed in individuals with epilepsy, the direct involvement of specific metabolites in the development of the condition remains underexplored. A comprehensive analysis of the causality between cerebrospinal fluid metabolites (CSF) and epilepsy is pivotal in discovering innovative therapeutic interventions and prophylactic approaches. Summary data from genome-wide association studies (GWAS) of CSF metabolites and epilepsy subtypes were obtained separately. A total of 338 CSF metabolites were investigated as exposures, and 11 epilepsy phenotypes were examined as the outcomes. A two sample Mendelian randomization (MR) approach was utilized to explore the causal influence of these metabolites on epilepsy. Causality was primarily estimated through inverse variance weighted (IVW) analysis, complemented by a range of sensitivity analyses to ensure result stability. Additionally, reverse MR analysis was performed to explore the possibility of bidirectional causality. The IVW method, reinforced by sensitivity analyses, pinpointed 17 CSF metabolites with causal implications for six epilepsy phenotypes. After False Discovery Rate (FDR) multiple testing correction, two metabolites (Methylmalonate and Gamma-glutamyl-alpha-lysine) were found to have robust causal links to epilepsy (p < 0.05 and FDR<0.05). The other 15 metabolites exhibited suggestive evidence of a causal association (p < 0.05 and FDR>0.05). This study highlights CSF metabolites that could serve as valuable biomarkers and may be critical in developing targeted treatments and preventing epilepsy. This study explores how certain chemicals in the brain fluid might influence the development of epilepsy, aiming to find new ways to treat or prevent it. Researchers looked at the relationship between 338 cerebrospinal fluid metabolites and 11 types of epilepsy using genetic data. They found that 17 of these chemicals could potentially cause six types of epilepsy. Two of these chemicals were strongly linked to epilepsy, suggesting they could be important for creating specific treatments or prevention strategies.

Maternal high fat diet exposure modifies amniotic fluid metabolites and expands group 3 innate lymphoid cells dependent on the maternal microbiome and MyD88-signaling.

Maternal high fat diet (mHFD) exposure expands IL-17 producing group 3 innate lymphoid cells (IL17+ve ILC3) in the small intestine of neonatal murine offspring and increases their susceptibility to intestinal inflammation. How mHFD modulates innate immunity in the fetal offspring remains unclear. Dams were exposed to 60% high fat diet or maintained on regular diet (RD) prior to and during mating. Amniotic fluid (AF) was collected during mid-pregnancy and metabolites examined by global non-targeted mass spectrometry in conventional wild-type (WT) and germ-free pregnant dams. Offspring were delivered by C-section or vaginally and fecal contents examined for major bacterial phyla and small intestinal lamina propria cells (LP) by flow cytometry. Susceptibility to intestinal inflammation was determined using a lipopolysaccharide and platelet-activating factor model (LPS/PAF) in WT, germ-free and MyD88 deficient offspring. Neonatal germ-free pups were exposed to HFD or RD AF by gavage and LP examined by flow cytometry. We identified differentially produced metabolites in mHFD AF when compared to RD AF in conventionally raised mice, with no difference seen in germ-free mice. C-section delivery maintained the mHFD phenotype of expansion of IL17+ve ILC3 and increased susceptibility to inflammation in neonatal offspring. In addition, mHFD offspring had expansion of IL17+ve ILC3 at birth and 2 weeks of life, which was not seen in germ-free and MyD88 KO mice exposed to mHFD. Germ-free and MyD88 KO mice were protected from mHFD induced LPS/PAF injury and IL17+ve ILC3 expansion, demonstrating that the maternal microbiome and MyD88 are prenatally necessary for the expansion of IL17+ve ILC3 in mHFD offspring. Furthermore, introduction of mHFD AF to neonatal germ-free pups by gavage was sufficient to expand IL17+ve ILC3 in the small intestine. Our findings indicate that mHFD interacts with the maternal microbiome to modify AF metabolites and signal via MyD88 to expand IL17+ve ILC3 in murine neonatal offspring.

Probe electrospray ionization coupled to a quadrupole time-Of-flight: A feasibility study for the detection of cocaine and its derivatives in oral fluid

Probe Electrospray Ionization (PESI) is an atmospheric pressure ionization method that can be directly coupled with a mass spectrometer to allow ultrafast analyses without chromatographic separation and with minimal sample preparation. Using the particular case of cocaine and its metabolites in human oral fluid, the main objective of the present study was to test the feasibility of a new hybrid system combining a PESI source and a quadrupole time-of-flight (QTOF). The best results were obtained for a sample preparation with a simple dilution of 100 µL of oral fluids in an ethanol / 10 mM ammonium formate buffer (50/50) and 10 µL deposited on a dedicated sample plate and introduced into the PESI source. For HRMS acquisition, an approach consisting in a full-mass scan (“untargeted approach” from 100 to 500 m/z; MS1) followed by a targeted scheduled MSMS acquisition (precursor ions of the 3 molecules of interest and their 3 internal standards; MS2) gave the best signals. The total time of analysis was 0.45 min and the method was validated according to ISO15189 standard for a 5–100 ng/mL range, including accuracy and precision (inter-day and intra-day precision and bias values were lower than 15 %), matrix effect, carryover and specificity (no interference with a mixture of 119 psychotropic drugs spiked at 1 mg/L). The LLOD values were 1 ng/mL for the 3 cocaine derivatives and 83 Driving Under the Influence of Drug (DUID) cases sent to our Lab for the determination of illicit drugs in oral fluid were analyzed using the PESI-QTOF method and compared to a LC-MS/MS method. A perfect agreement was observed between the 2 methods, whether the cases were positive or negative. Cocaine was detected in 51 out of these 83 real cases (61.6 %). BZE and EME were also simultaneously detected in 50 of them (98.5 %). This feasibility study reports the first analytical method based on a coupling of a PESI source to a QTOF mass spectrometer. Adding the major advantage of high specificity through HRMS is a step forward for PESI technology.

Genetic determinants of cerebrospinal fluid metabolites and risk of Guillain-Barré syndrome: A bidirectional two-sample Mendelian randomization study.

This study aims to investigate the potential causal relationship between cerebrospinal fluid (CSF) metabolites and Guillain-Barré syndrome (GBS) using a bidirectional two-sample Mendelian randomization (MR) approach. Publicly available summary data from genome-wide association studies (GWAS) were utilized for comprehensive analysis. The CSF metabolite GWAS summary data were extracted from a GWAS conducted by Panyard et al encompassing 338 CSF metabolites in European participants (n = 291). GWAS summary statistics for GBS were obtained from the FinnGen consortium (n = 215,931) comprising European populations. The primary method for MR analysis was the inverse variance weighted method. Various sensitivity analyses were conducted to assess the heterogeneity and pleiotropy of the findings. In the forward MR analysis, we identified a causal relationship between 15 CSF metabolites, including ribitol levels (odds ratio = 3.833, 95% confidence interval: 1.949-7.540, P = 9.87E-05), and the risk of developing GBS. In the reverse MR analysis, we found a causal relationship between GBS and 21 CSF metabolites, including gamma-glutamylphenylalanine levels (odds ratio = 0.934, 95% confidence interval: 0.904-0.966, P = 7.10E-05). No evidence of heterogeneity or horizontal pleiotropy was found in the MR analysis. Our findings suggest that the identified CSF metabolites and metabolic pathways can serve as valuable biomarkers for clinical screening and prevention of GBS. They may also be considered as candidate molecules for future research into the underlying mechanisms and for selecting drug targets.

Effect of L-Carnitine Level on the Risk of Neuromyelitis Optica Spectrum Disorders: A Two-Sample Mendelian Randomization Study.

Previous research has often focused on studying the CNS damage in neuromyelitis optica spectrum disorders (NMOSD), while the role of the peripheral blood in the development of NMOSD is also of significant importance. The relationship between metabolites in blood and cerebrospinal fluid (CSF) with neuroimmune is receiving increasing attention. L-carnitine, whose astrocytic accumulation is associated with neuroinflammation and demyelination, may participate in the pathogenesis of NMOSD. However, whether circulating L-carnitine level has a causal effect on NMOSD risk needs elucidation. With large data sets now available, we used two-sample Mendelian randomization (MR) to determine whether circulating L-carnitine level is causally associated with the risk of NMOSD. Genetic variants associated with circulating L-carnitine levels were derived from a genome-wide association study (GWAS) of 7797 individuals from TwinsUK and KORA F4 cohorts. NMOSD summary statistics, including 215 cases and 1244 controls, were obtained from a separate GWAS. Subgroup analyses included aquaporin-4 (AQP4)-IgG-seropositive NMOSD (132 cases) and AQP4-IgG-seronegative NMOSD (83 cases). We used two-sample MR to explore associations between circulating L-carnitine levels and NMOSD risk, as well as its seropositive and seronegative subtypes. 16 SNPs (single nucleotide polymorphisms) were significantly associated with circulating L-carnitine level (P < 5 × 10-8), all of which were independent and available in the NMOSD dataset, after 1 SNP removed for being palindromic with intermediate allele frequencies in harmonization. Finally, a high circulating L-carnitine level conferred a protective effect against combined NMOSD (OR = 2.216 × 10-4, 95% confidence interval [CI] = 2.335 × 10-7-2.104 × 10-1, P = 0.0161) as well as AQP4-IgG-seronegative NMOSD (OR = 7.678 × 10-7, 95% CI = 2.233 × 10-11-2.640 × 10-2, P = 0.0082). There is no causal effect on AQP4-IgG-seropositive NMOSD risk (OR = 5.471 × 10-3, CI = 1.090 × 10-6-27.465, P = 0.2798) by circulating L-carnitine. Results remained positive and robust after the horizontal pleiotropy test, heterogeneity test, and Bonferroni test. In the reverse MR analysis, there was no causal effect of NMOSD and its subtypes on circulating L-carnitine levels. Our findings suggest that higher circulating L-carnitine levels may reduce the risk of NMOSD, particularly in AQP4-IgG-seronegative patients. L-carnitine could serve as a valuable biomarker and potential therapeutic target for NMOSD, especially in cases without AQP4-IgG. The genetic evidence from this study supports further exploration of L-carnitine's role in managing NMOSD.

High-altitude hypoxia exposure alters follicular metabolome and oocyte developmental potential in women.

To explore the effects of high-altitude hypoxia on the microenvironment of oocyte development and fertilization potential, we compared the metabolomic patterns of follicular fluid from women living in different altitude areas and traced their oocyte maturation and subsequent development. A total of 315 clinical cases were collected and divided into three groups according to their residence altitudes: 138 cases in low-altitude (< 2300m) group, 100 cases in middle-altitude (2300-2800m) group and 77 cases in high-altitude (> 2800m) group. The clinical outcomes were statistically estimated, including hormonal level, oocyte maturation, in vitro fertilization, and embryo development. Meanwhile, a metabolomic analysis was performed on the follicular fluid of women from different groups using ultra-high-performance liquid chromatography and high-resolution mass spectrometry and differential metabolites were analyzed through the KEGG pathway. The clinical data indicated that the physical condition and reproductive hormone secretion were similar among different groups. Although personalized gonadotropin-releasing hormone strategies were applied, the numbers of antral follicles and obtained oocytes were not impacted by the residence altitude change. In in vitro culture, the maturing rate, fertility rate and cleavage rate of high-altitude group were compared with the other groups. However, the rates of high-quality embryo, formative blastocyst, and available blastocyst were gradually decreased with the rise of residence altitude. Metabolome analysis identified 1193 metabolites in female follicular fluid. Differential analysis indicated that metabolic components in follicular fluid were remarkably changed with the elevation of residence altitude. These differential metabolites were closely related with amino acid metabolism, protein digestion and absorption, oocyte meiosis and steroid biosynthesis. The residence altitude alters the microenvironment of follicular fluid, which could damage the oocyte developmental potential. This study provides diagnostic basis and therapeutic targets for research on female oocyte and embryo development.

Metabolomic Profiling of Cerebrospinal Fluid Reveals Metabolite Biomarkers in Tick-Borne Encephalitis Patient.

Tick-borne encephalitis virus (TBEV) can cause life-threatening central nervous system infection. Changes in cerebrospinal fluid (CSF) metabolites may reflect critical aspects of host responses and end-organ damage in neuro infection and neuroinflammation. In this study, we applied an untargeted metabolomics screen of CSF samples to investigate the metabolites profile and explore biomarkers for TBEV infection. By analyzing CSF samples from 77 patients with TBEV infection and 23 without TBEV infection, tryptophan metabolism and Citrate cycle were found to be the top important metabolic pathways in differentiating the control and case groups; acetoacetate, 5'-deoxy-5'-(methylthio)-adenosine, 3-methyl-2-oxobutanoic acid, and so forth. were identified to be metabolic biomarkers (| FC|> 1, VIP > 1, FDR < 0.05) in CSF and clearly separated the TBEV infection from the noninfected samples. Moreover, four metabolites were identified to be associated with fatal outcome, including kynurenic acid, 5-hydroxyindole-3-acetic acid, DL-tryptophan, indole-3-acrylic acid, demonstrating the potential predictive biomarkers for severe TBEV infection. This study explored the metabolic profile of TBEV infection in CSF samples and identified candidate biomarkers for TBEV infection, which might be useful in target screening for differential diagnosis and therapeutic inter-vention.

Current Analytical Methods and Their Limitations in Quantifying Antidiabetic Drug Nateglinide and Repaglinide in Pharmaceutical Formulations and Biological Fluids

Pharmaceutical growth dominated a transformation in human health. These drugs need to attend to their target only, so they must be free from impurities and appropriately controlled. Due to that, diverse instrumental techniques were advanced at steady intervals to accomplish their intention to quantify the limits per the regulatory. NTG and RPG pharmaceuticals might generate impurities during the development phases, packing, and shipping, which could be risky to administer. Hence, detecting and quantifying them using various analytical techniques at multiple stages is necessary. This review highlights the function of different analytical methods, including UV–Vis, HPLC, HPTLC, UPLC, HPLC/MS and UPLC/MS, in quantifying drugs, impurities and metabolites in bulk, pharmaceutical formulations and biological fluids. Also, it discussed the specific advantages and limitations of individual techniques. It compared them regarding sensitivity, specificity, cost, time consumption, efficacy, and the practical challenges of implementing these analytical techniques in real-world settings to determine pharmaceuticals.

Molecular landscape of the overlap between Alzheimer’s disease and somatic insulin-related diseases

Alzheimer’s disease (AD) is a multifactorial disease with both genetic and environmental factors contributing to its etiology. Previous evidence has implicated disturbed insulin signaling as a key mechanism that plays a role in both neurodegenerative diseases such as AD and comorbid somatic diseases such as diabetes mellitus type 2 (DM2). In this study, we analysed available genome-wide association studies (GWASs) of AD and somatic insulin-related diseases and conditions (SID), i.e., DM2, metabolic syndrome and obesity, to identify genes associated with both AD and SID that could increase our insights into their molecular underpinnings. We then performed functional enrichment analyses of these genes. Subsequently, using (additional) GWAS data, we conducted shared genetic etiology analyses between AD and SID, on the one hand, and blood and cerebrospinal fluid (CSF) metabolite levels on the other hand. Further, integrating all these analysis results with elaborate literature searches, we built a molecular landscape of the overlap between AD and SID. From the landscape, multiple functional themes emerged, including insulin signaling, estrogen signaling, synaptic transmission, lipid metabolism and tau signaling. We also found shared genetic etiologies between AD/SID and the blood/CSF levels of multiple metabolites, pointing towards “energy metabolism” as a key metabolic pathway that is affected in both AD and SID. Lastly, the landscape provided leads for putative novel drug targets for AD (including MARK4, TMEM219, FKBP5, NDUFS3 and IL34) that could be further developed into new AD treatments.

Environmental Heat Stress Decreases Sperm Motility by Disrupting the Diurnal Rhythms of Rumen Microbes and Metabolites in Hu Rams.

Heat stress (HS) has become a common stressor, owing to the increasing frequency of extreme high-temperature weather triggered by global warming, which has seriously affected the reproductive capacity of important livestock such as sheep. However, little is known about whether HS reduces sperm motility by inducing circadian rhythm disorders in rumen microorganisms and metabolites in sheep. In this study, the year-round reproduction of two-year-old Hu rams was selected, and the samples were collected in May and July 2022 at average environmental temperatures between 18.71 °C and 33.58 °C, respectively. The experiment revealed that the mean temperature-humidity index was 86.34 in July, indicating that Hu rams suffered from HS. Our research revealed that HS significantly decreased sperm motility in Hu rams. Microbiome analysis further revealed that HS reshaped the composition and circadian rhythm of rumen microorganisms, leading to the circadian disruption of microorganisms that drive cortisol and testosterone synthesis. Serum indicators further confirmed that HS significantly increased the concentrations of cortisol during the daytime and decreased the testosterone concentration at the highest body temperature. Untargeted metabolomics analysis revealed that the circadian rhythm of rumen fluid metabolites in the HS group was enriched by the cortisol and steroid synthesis pathways. Moreover, HS downregulated metabolites, such as kaempferol and L-tryptophan in rumen fluid and seminal plasma, which are associated with promotion of spermatogenesis and sperm motility; furthermore, these metabolites were found to be strongly positively correlated with Veillonellaceae_UCG_001. Overall, this study revealed the relationship between the HS-induced circadian rhythm disruption of rumen microorganisms and metabolites and sperm motility decline. Our findings provide a new perspective for further interventions in enhancing sheep sperm motility with regard to the circadian time scale.

GnRH Immunocastration in Male Xizang Sheep: Impacts on Rumen Microbiome and Metabolite Profiles for Enhanced Health and Productivity.

Castration is a prevalent and indispensable practice in sheep husbandry, aiding in enhancing meat quality, mitigating aggressive behavior, and managing unwanted reproduction. Nevertheless, the conventional surgical castration procedure poses several challenges, including heightened stress and pain, detrimental impacts on animal welfare, and diminished economic efficacy in farming operations. Consequently, immunocastration methods, serving as substitutes for surgical castration, are progressively finding application in livestock. The rumen, an essential and distinctive digestive and absorptive organ in ruminants, has been associated with enhanced meat quality and productive performance following castration in previous research studies, albeit fewer investigations have explored the potential impacts of GnRH immunization on the rumen's internal milieu in sheep post-de-escalation. Hence, the present study delved into evaluating the impact of GnRH immunocastration on the rumen microbiome and metabolomics in male Xizang sheep. This was achieved through the establishment of a GnRH immunocastration animal model and the collection of rumen fluid for microbiological and comprehensive metabolomics investigations. The outcomes of this investigation unveiled that the impact of GnRH immunocastration on body weight gain was more pronounced during the achievement of the castration objective. In addition, the Firmicutes-to-Bacteroidota ratio in the immune male (IM) group exceeded that of the control group (EM), suggesting that GnRH immunodeficiency may enhance the digestion and absorption of feed in male Xizang sheep. At the taxonomic level, the elevated presence of Prevotella and Quinella bacteria in the IM group compared to the EM group indicated that castration influenced a segment of the rumen microbiota in male Xizang sheep, thereby bolstering the digestive and metabolic efficacy of the rumen concerning nutrient utilization, particularly in the breakdown and absorption of proteins, carbohydrates, and lipids, ultimately expediting the fattening process and weight gain in male Xizang sheep following castration. Moreover, analysis of ruminal fluid metabolomics revealed that GnRH immunization had notable impacts on certain metabolites in the ruminal fluid of male Xizang sheep, with metabolites like 5-hydroxyindole acetic acid and 3-hydroxyindole acetic acid showing significant downregulation in the IM group compared to the EM group, while niacin and tyramine exhibited significant upregulation. These findings indicate a profound influence of GnRH immunization on the maintenance of ruminal equilibrium and ruminal health (including the health of ruminal epithelial cells). This study validates that GnRH immunocastration not only achieves the objectives of castration but also enhances ruminal health in male Xizang sheep, thus laying a foundational theoretical basis for the application and dissemination of GnRH immunocastration technology.

Multi-omics approach to reveal follicular metabolic changes and their effects on oocyte competence in PCOS patients.

Polycystic ovary syndrome (PCOS) is a common heterogeneous disorder linked with endocrine and metabolic disturbances. The underlying mechanism of PCOS, especially its effect on oocyte competence, remains unclear. The study aimed to identify abnormal follicular metabolic changes using a multi-omics approach in follicular fluid from PCOS patients and to determine their effects on oocyte competence. A total of 36 women with PCOS and 35 women without PCOS who underwent in vitro fertilization and embryo transfer were included in the study. Cumulus cells and follicular fluid samples were collected. Follicular fluid samples underwent metabolomic analysis, while cumulus cell clusters from the same patients were assessed using transcriptomic analysis. Clinical information of patients and assisted reproductive technology (ART) results were recorded. Transcriptomics and metabolomics were integrated to identify disrupted pathways, and receiver operation characteristics (ROC) analysis was conducted to identify potential diagnostic biomarkers for PCOS. Pearson correlation analysis was conducted to assess the relationship between metabolites in follicular fluid and oocyte competence (fertilization and early embryo development potential). Through multi-omics analysis, we identified aberrantly expressed pathways at both transcriptional and metabolic levels, such as the citrate cycle (TCA cycle), oxidative phosphorylation, the cAMP signaling pathway, the mTOR signaling pathway, and steroid hormone biosynthesis. Ten candidate metabolites were identified based on metabolic profiling data from these altered pathways. Phytic acid, succinic acid, 2'-deoxyinosine triphosphate, and 4-trimethylammoniobutanoic acid in the follicular fluid exhibited high specificity and sensitivity in distinguishing PCOS. Among these metabolites, L-arginine showed a negative correlation with the 2PN fertilization rate and cleavage rate, while estrone sulfate showed a negative correlation with the high-quality embryo rate in the in-vitro fertilization (IVF) cycle. We have conducted a preliminary study of a novel metabolic signature in women with PCOS using a multi-omics approach. The alterations in key metabolic pathways may enhance our understanding of the pathogenesis of PCOS.

Development and Validation of a New HPLC Bio Analytical Internal Standard Method for the Analysis of Two Immunology Drugs (Lamivudine and Dolutegravir) in Human Plasma

Bioanalytical methods are useful for the quantitative analysis of drugs and their metabolites in biological fluids. Bio-analytical internal standards methods plays vital role in areas of human clinical pharmacology. The aim of this work is to development and validation of bio-analytical internal standard method for determination of two immunology drugs Lamivudine (LMVD) and Dolutegravir (DTGR) in plasma with Abacavir(ABVR) drug as internal standard (IS). Liquid-liquid extraction with Diethyl ether and methanol in the ratio of 50:50 (v/v) was used for the extraction of drugs from the biological matrix. The optimized chromatography conditions consist of Water, Acetonitrile and Methanol in the ratio of 40:25:35 (v/v) as a mobile phase with Ascentis, C18 Column (250 X 4.6 mm, 5μ) as stationery phase. Isocratic elution with 1.2 ml flow separates theLMVD at 3.5 min, DTGR at 9.8 min and ABVR at 6.8 min. The method was validated as per ICH guidelines. The relative standard deviation (%RSD) were found to be &lt;5%for precision studies. Hence the method was found to be suitable fort the analysis of LMVD and DTGR in spiked human plasma.

A green and sensitive electromembrane extraction (EME) method for the determination of dialkylphosphates in maternal urine and amniotic fluid

Organophosphates compounds are extensively used pesticides, whose metabolites, dialkyl phosphates, are considered as bioindicators of human exposure. Recently, research has indicated that they can affect thyroid endocrine function. Pregnant women are a particularly vulnerable cohort to these effects due to concerns about prenatal exposure to environmental pollutants. For the first time an electromembrane microextraction method is proposed for the simultaneous determination of seven dialkylphosphate metabolites in maternal urine and amniotic fluid, which is compatible with mass spectrometry detection and achieves highly sensitive levels of quantitation. Dialkylphosphates were extracted from a 10 mL donor solution at pH 7 into a 50 µL acceptor solution at pH 12 using 1-octanol as the supported liquid membrane and applying 60 V for 10 min at 600 rpm. The obtained extracts were analysed by ion pair liquid chromatography coupled to a triple quadrupole mass spectrometer, using an Acquity UPLC® BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm particle size) at 30 °C. Under optimal extraction conditions, the preconcentration factors were achieved were up to 178-fold, allowing for high sensitivity levels, in the order of ng mL−1. The limits of quantitation were within 0.075 ng mL−1 (diethyl thiophosphate) and 1.67 ng mL−1 (dimethyl phosphate) in maternal urine samples and within 0.015 ng mL−1 (diethyl thiophosphate) and 0.5 ng mL−1 (dimethyl dithiophosphate) in amniotic fluid samples. The comparative study revealed a significant improvement in terms of analysis time, sensitivity, greenness, and practicality. Finally, the applicability of the method for the determination of selected dialkylphosphates was demonstrated in paired maternal urine and amniotic fluid samples. These results suggest potential placental transfer to the offspring.

7 Role of the preovulatory follicle in oocyte metabolic and developmental competence for pregnancy

Abstract A majority of bovine pregnancy loss occurs in the first 7 d of gestation. The oocyte is a key driver of embryo viability during this time, and its competence for embryo development and pregnancy establishment is greatly influenced by the surrounding microenvironment during oocyte maturation. Assisted reproductive technologies allow for more efficient propagation of superior genetics and improved reproductive management of the cattle herd; however, many assisted reproductive technologies alter the normal microenvironment of the maturing oocyte and negatively impact oocyte competence for early embryo development. For example, oocytes induced to ovulate from preovulatory follicles of lesser physiological maturity (decreased estradiol production and smaller diameter) have reduced blastocyst development, poorer embryo quality, and a hastened rate of embryo development compared with those ovulated from follicles of greater maturity. Our overarching hypothesis is that the intrafollicular milieu of physiologically mature preovulatory follicles provides the necessary microenvironment to metabolically program maturing oocytes for successful embryo development. Studies by our team have demonstrated that the follicular fluid metabolite milieu during in vivo oocyte maturation differs between preovulatory follicles of greater and lesser maturity and that such differences have direct effects on the maturing oocyte. Numerous follicular fluid metabolites involved in glucose and amino acid metabolism were more abundant in preovulatory follicles of greater versus lesser physiological maturity. Additionally, abundance of transcripts encoding glycolytic enzymes was higher in cumulus cells from follicles of greater maturity, and both ATP and levels of key transcripts in the mitochondrial respiratory chain were higher in oocytes collected from preovulatory follicles of greater versus lesser maturity. We supplemented in vitro maturing oocytes with follicular fluid from preovulatory follicles of greater or lesser maturity to determine causality of differing follicular fluid milieu on cumulus-oocyte metabolism and oocyte competence for early embryo development. Follicle maturity status had robust effects on cumulus-oocyte complex consumption or production of key metabolites in glucose, purine, and amino acid metabolism pathways during oocyte maturation. We also observed hastened development rate in embryos arising from in vitro matured oocytes supplemented with lesser maturity follicular fluid, mirroring past in vivo findings by others. Furthermore, RNA-sequencing of resulting blastocysts of identical stage and quality suggested more efficient metabolism and Wnt/β-catenin signaling in blastocysts derived from oocytes matured in preovulatory follicular fluid from greater maturity follicles. In contrast, transcriptome profiles of blastocysts from oocytes matured in preovulatory follicular fluid from lesser maturity follicles indicated oxidative stress and dysregulated cell division. These recent findings by our lab indicate that impacts of follicle maturity and the resulting follicular fluid microenvironment on the maturing oocyte withstand throughout early embryo development and hold promising roles to improve reproductive efficiency in cattle.