Correction to: In Vitro Metabolic Profiling of 18 Semi-Synthetic Cannabinoids-Hexahydrocannabinol (HHC) and Its Analogs-with Identification in an Authentic Hexahydrocannabiphorol (HHCP) Urine Sample.

Saliva and urine metabolic profiling reveals altered amino acid and energy metabolism in male farmers exposed to pesticides in Madhya Pradesh State, India

Objective Metabolomic analysis is extensively applied to identify sensitive and specific biomarkers capable of reflecting pathological processes and physical responses or adaptations. Exercise training leads to profound metabolic changes, manifested as detectable alterations of metabolite levels and significant perturbations of metabolic pathways in sera, urine, and rarely, in saliva. Several metabolites have been exploited as biomarkers for generally evaluating physical states in almost all sports. However, alterations of metabolic profile caused by specific sports would be heterogeneous. Thus, developments of new techniques are eagerly required to identify characteristic metabolites as unique biomarkers for specifically accessing training stimulus and sports performances. In the present work, we conducted both metabolic profiling and a binary logistic regression model (BRM) of biological fluids derived from rowing ergometer test with the following aims: 1) to examine changes of metabolite profiles and identify characteristic metabolites in the samples of sera, urine, and saliva; 2) to screen out potential integrated biomarkers for sports-specific monitoring.
 Methods A total of 11 rowers (6 male, 5 female; aged 15±1 years; 4±2 years rowing training) underwent an indoor 6000m rowing ergometer test. Samples of sera, urine and saliva were collected before and immediately after the test. 1D 1H NMR spectra were recorded with a Bruker Avance III 650 MHz NMR spectrometer. NMR spectra were processed and aligned, resonances of metabolites were assigned and confirmed, and metabolite levels were calculated based on NMR integrals. Multivariate statistical analysis was carried out using partial least-squares discrimination analysis (PLS-DA) to distinguish metabolic profiles between the groups. The validated PLS-DA model gave the variable importance in the projection (VIP) for a given metabolite. Moreover, inter-group comparisons of metabolite levels were quantitatively conducted using the paired-sample t-test. Then, we identified characteristic metabolites with VIP>1 in PLS-DA and p<0.05 in t-test. Furthermore, we screened out potential biomarkers based on the characteristic metabolites identified from the three types of biological fluids using the BRM (stepwise).
 Results The rowing training induced profound changes of metabolic profiles in serum and saliva samples rather than in urine samples. Totally, 44 metabolites were assigned in which 19, 20, and 19 metabolites were identified from serum, urine and saliva samples, respectively. Seven metabolites were shared by the three types of samples. Moreover, five characteristic metabolites (pyruvate, lactate, succinate, N-acetyl-L-cysteine, and acetone) were identified from the serum samples. The elevated levels of pyruvate, lactate and succinate suggested that, the rowing training evidently promoted both oxidative phosphorylation and glycolysis pathways. Furthermore, three characteristic metabolites (tyrosine, formate, and methanol) were identified from the saliva samples. Given that tyrosine is the precursor of dopamine, the increased level of salivary tyrosine in all rowers experiencing the test, suggesting that salivary tyrosine could be explored as a potential indicator closely related to nervous fatigue in the test. On the other hand, PLS-DA did not show observable distinction of metabolic profiles between the urine samples before and immediately after the test. Moreover, 20 urinary metabolites did not display detectable altered levels. We then established the BRM with the identified characteristic metabolites, from which we selected one optimal regression model based on serum pyruvate and salivary tyrosine (adjusted R square was 0.935, P<0.001), indicating that the two selected metabolites would efficiently reflect the metabolic alterations in the test.
 Conclusions As far as the 6000m rowing ergometer test is concerned, serum samples could be a preferred resource for assessing the changes of energy metabolism in the test, while urine samples might have a relatively lower sensitivity to exercise-induced metabolic responses. Even though metabolite levels in saliva samples are generally lower than those in serum and urine samples, some salivary metabolites potentially have higher sensitivities to exercise-induced metabolic responses. Thus, the integration of multiple biomarkers identified from different type of species could potentially provide more sensitive and specific manners to monitor physical states in sports and exercise. This work may be of benefit to the exploration of integrated biomarkers for sports-specific monitoring.

Background: Autism spectrum disorder (ASD) is defined as a pervasive developmental disorder which is caused by genetic and environmental risk factors. Besides the core behavioral symptoms, accumulated results indicate children with ASD also share some metabolic abnormalities.Objectives: To analyze the comprehensive metabolic profiles in both of the first-morning urine and plasma samples collected from the same cohort of autistic boys.Methods: In this study, 30 autistic boys and 30 tightly matched healthy control (HC) boys (age range: 2.4~6.7 years) were recruited. First-morning urine and plasma samples were collected and the liquid chromatography–mass spectrometry (LC-MS) was applied to obtain the untargeted metabolic profiles. The acquired data were processed by multivariate analysis and the screened metabolites were grouped by metabolic pathway.Results: Different discriminating metabolites were found in plasma and urine samples. Notably, taurine and catechol levels were decreased in urine but increased in plasma in the same cohort of ASD children. Enriched pathway analysis revealed that perturbations in taurine and hypotaurine metabolism, phenylalanine metabolism, and arginine and proline metabolism could be found in both of the plasma and urine samples.Conclusion: These preliminary results suggest that a series of common metabolic perturbations exist in children with ASD, and confirmed the importance to have a comprehensive analysis of the metabolites in different biological samples to reveal the full picture of the complex metabolic patterns associated with ASD. Further targeted analyses are needed to validate these results in a larger cohort.

Trofinetide is the first drug to be approved for the treatment of Rett syndrome, a neurodevelopmental disorder. The purpose of the study is to fully characterize the metabolic and excretion profiles of trofinetide in humans. This Phase 1, open-label, single-dose trial conducted in healthy male adults was designed to characterize the pharmacokinetics of trofinetide (absorption, metabolism, and excretion), mass balance of [14C]-trofinetide, and safety profile of trofinetide following administration of an oral 12-g dose administered as a mixture of trofinetide and [14C]-trofinetide. Blood, urine, and fecal samples were collected at prespecified timepoints. The pharmacokinetics of trofinetide were assessed in blood and urine samples using high-performance liquid chromatography (HPLC) with tandem mass spectrometric detection. Bioanalysis of radioactivity was conducted in blood, plasma, urine, and fecal samples using liquid scintillation counting. Metabolite profiling was conducted in blood, plasma, urine, and fecal samples using HPLC with liquid scintillation counting of chromatographic fractions. Safety and tolerability, including treatment-emergent adverse events (TEAEs), were assessed. Blood concentration-time profiles of trofinetide and total radioactivity were almost superimposable up to ~12 h after dosing. Urine concentration-time profiles of trofinetide and total radioactivity were similar. Trofinetide was rapidly absorbed into the circulation with an initial rapid decline (half-life [t½] alpha ~2.6 h), followed by a relatively slow terminal elimination phase (t½ beta ~20 h). The blood-to-plasma total radioactivity ratios were 0.529-0.592, indicating a lack of affinity for the cellular portion of blood. Renal excretion accounted for 83.8% of the administered radiochemical dose; 15.1% was recovered in feces. Urine and fecal recovery of radioactivity accounted for 99% of the administered dose at 168 h after dosing. Parent [14C]-trofinetide was the major radiolabeled entity in blood and plasma (88.4% and 93.1% in area under the concentration-time curves from 0 to 12 h [AUC0-12] in pooled blood and plasma samples, respectively) and the major entity excreted in urine (91.5% in 0-48-h pooled urine samples) and in feces (52.7% in 0-192-h pooled fecal samples). Only small levels of metabolites were present. In blood and plasma, only two minor metabolites were identified (each metabolite ≤ 2.24% of the AUC0-12 pool). These two metabolites were also observed in urine and fecal samples (≤ 2.41% of dose). In feces, one additional metabolite (0.84% of dose) was identified. Two mild TEAEs were reported in two participants and were not considered related to trofinetide. There were no clinically meaningful changes in individual laboratory parameters, vital signs, physical findings, or electrocardiogram results. Metabolic and excretion profiles confirm that trofinetide undergoes minimal hepatic or intestinal metabolism and is primarily excreted unchanged in the urine. Trofinetide containing radiolabeled [14C]-trofinetide was well tolerated.

Metabolic profiles and screening tactics for MDMB-4en-PINACA in human urine and serum samples

Adverse physiology and antibiotic exposure devastate the intestinal microbiome in critical illness. Time and cost implications limit the immediate clinical potential of microbial sequencing to identify or treat intestinal dysbiosis. Here, we examined whether metabolic profiling is a feasible method of monitoring intestinal dysbiosis in critically ill children. Prospective multicenter cohort study. Three U.K.-based PICUs. Mechanically ventilated critically ill (n = 60) and age-matched healthy children (n = 55). Collection of urine and fecal samples in children admitted to the PICU. A single fecal and urine sample was collected in healthy controls. Untargeted and targeted metabolic profiling using 1H-nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry or urine and fecal samples. This was integrated with analysis of fecal bacterial 16S ribosomal RNA profiles and clinical disease severity indicators. We observed separation of global urinary and fecal metabolic profiles in critically ill compared with healthy children. Urinary excretion of mammalian-microbial co-metabolites hippurate, 4-cresol sulphate, and formate were reduced in critical illness compared with healthy children. Reduced fecal excretion of short-chain fatty acids (including butyrate, propionate, and acetate) were observed in the patient cohort, demonstrating that these metabolites also distinguished between critical illness and health. Dysregulation of intestinal bile metabolism was evidenced by increased primary and reduced secondary fecal bile acid excretion. Fecal butyrate correlated with days free of intensive care at 30 days (r = 0.38; p = 0.03), while urinary formate correlated inversely with vasopressor requirement (r = -0.2; p = 0.037). Disruption to the functional activity of the intestinal microbiome may result in worsening organ failure in the critically ill child. Profiling of bacterial metabolites in fecal and urine samples may support identification and treatment of intestinal dysbiosis in critical illness.

In 2020, 5F-MDMB-PICA (5F-MDMB-2201) was one of the most common synthetic cannabinoids (SCs) identified in drugs seized by the Beijing Drug Control Agency, and it was categorized as Schedule II by the United Nations Office on Drugs and Crime in March 2020. It is difficult to detect 5F-MDMB-PICA in biological matrices due to its fast metabolic rate in vivo. In this work, 5F-MDMB-PICA metabolic profiles were investigated by liquid chromatography--quadrupole exactive high field orbitrap mass spectrometer (LC-Q Exactive HF MS), with accurate mass measurements in human urine, serum and pubic hair. To obtain intact metabolites, solid-phase extraction for urine and serum and direct ultrasonic extraction for pubic hair were applied to clean the samples without enzymatic hydrolysis. The differences in 5F-MDMB-PICA metabolism in the three different matrices were compared for the first time to determine the best detection biomarkers for monitoring 5F-MDMB-PICA misuse. Urine samples were determined to be the preferred biological material for identifying 5F-MDMB-PICA abuse. Forty-seven intact metabolites were detected in human urine, the ester hydrolyzed with glucuronidated metabolite in urine samples can be used as the primary biomarker to identify drug misuse. Fifteen metabolites were found in serum samples. Ester hydrolysis was considered to be the major metabolic pathway, and a large number of metabolites were involved with it. Zero metabolites apart from the parent drug were detected in pubic hair samples. Twenty-eight new metabolites and their metabolic pathways were characterized and tentatively identified by LC-QE-HF-MS, and a new potential biomarker (M5 ester hydrolysis + propionic acid) was also identified.

<b>Abstract ID 24507</b> <b>Poster Board 187</b> Endogenous metabolites can be used as phenotypic biomarkers of solute carrier (SLC) transporters to predict transporter-mediated drug-drug interactions (DDIs). The SLC22 subfamily members organic anion transporter (OAT) 1 and 3 are expressed on the basolateral membrane of kidney proximal tubule cells. Both facilitate the uptake of several drugs and endogenous metabolites for renal secretion. Probenecid-mediated inhibition of OAT1/3 increases plasma concentrations of endogenous metabolites, including pyridoxic acid and kynurenic acid.^1,2 The objective of this study was to assess the effects of OAT1/3 inhibition on the metabolic profile in human plasma and urine using targeted and untargeted metabolomics approaches. Plasma and urine samples from 7 healthy adults who participated in an ongoing clinical pharmacokinetic DDI study (n=16) involving oral furosemide (OAT1/3 substrate, 5 mg) and probenecid (OAT1/3 inhibitor, 1000 mg) were selected for untargeted analysis. Plasma (pool of 1, 1.5, and 2 h collections) and urine (0-4 h) samples representing timepoints that captured maximum probenecid plasma concentrations were analyzed <i>via</i> liquid chromatography-mass spectrometry. Using XCMS Online software (xcmsonline.scripps.edu), &gt;10,000 individual features (<i>m/z</i> values) were detected in the pooled plasma samples that were either elevated or repressed in the presence of probenecid (Fig 1). Co-administration of probenecid increased furosemide plasma concentrations by 1.5- to 3-fold (p &lt; 0.05), indicating a DDI. However, pyridoxic acid and kynurenic acid, known biomarkers of OATs, showed no statistically significant differences due to high interindividual variability. This variability is likely because the precursors of these biomarkers are derived from diet.¹ More sensitive, selective, and less variable putative OAT1/3 biomarkers were identified by shortlisting the detected features that 1) were elevated in plasma by 2- to 10-fold in presence of probenecid (p &lt; 0.05) and 2) showed acceptable chromatographic peak signal to noise ratios. Correlations between the fold increase in furosemide plasma concentration and fold increase in plasma concentrations of the putative hits were next evaluated. Of these hits, 16 putative biomarkers (<i>m/z</i> 100-505) that correlated with furosemide (r &gt; 0.50) were selected for a METLIN database search. The identified putative biomarkers were mostly anionic compounds with variable hydrophilicity (LogP: -1.0 to 6.5). Five of the putative biomarkers were carboxylic acids, two were glucuronide conjugates, three belonged to a steroidal pathway, and one each belonged to the bile acid and tryptophan metabolism pathways. A detailed pharmacokinetic analysis (0-24 h) of furosemide, pyridoxic acid, and kynurenic acid concentrations measured in plasma and urine samples from 2 participants revealed a 2.2-, 2.3-, and 1.7-fold increase in the AUC, respectively, in the presence of probenecid. Targeted metabolomics analysis of the identified metabolites in all study participants will validate their utility as biomarkers of OAT1/3 for predicting transporter-mediated DDIs. Ref: Shen <i>et&nbsp;al</i>., 2019. <i>JPET</i> 368:136-45. Tang <i>et&nbsp;al</i>., 2021. <i>DMD</i> 49:1063-69. This work was funded by NIH/NICHD R01HD081299.

Hepatocellular carcinoma (HCC) is a common malignancy in the world with high morbidity and mortality rate. Identification of novel biomarkers in HCC remains impeded primarily because of the heterogeneity of the disease in clinical presentations as well as the pathophysiological variations derived from underlying conditions such as cirrhosis and steatohepatitis. The aim of this study is to search for potential metabolite biomarkers of human HCC using serum and urine metabolomics approach. Sera and urine samples were collected from patients with HCC (n = 82), benign liver tumor patients (n = 24), and healthy controls (n = 71). Metabolite profiling was performed by gas chromatography time-of-flight mass spectrometry and ultra performance liquid chromatography-quadrupole time of flight mass spectrometry in conjunction with univariate and multivariate statistical analyses. Forty three serum metabolites and 31 urinary metabolites were identified in HCC patients involving several key metabolic pathways such as bile acids, free fatty acids, glycolysis, urea cycle, and methionine metabolism. Differentially expressed metabolites in HCC subjects, such as bile acids, histidine, and inosine are of great statistical significance and high fold changes, which warrant further validation as potential biomarkers for HCC. However, alterations of several bile acids seem to be affected by the condition of liver cirrhosis and hepatitis. Quantitative measurement and comparison of seven bile acids among benign liver tumor patients with liver cirrhosis and hepatitis, HCC patients with liver cirrhosis and hepatitis, HCC patients without liver cirrhosis and hepatitis, and healthy controls revealed that the abnormal levels of glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, and chenodeoxycholic acid are associated with liver cirrhosis and hepatitis. HCC patients with alpha fetoprotein values lower than 20 ng/ml was successfully differentiated from healthy controls with an accuracy of 100% using a panel of metabolite markers. Our work shows that metabolomic profiling approach is a promising screening tool for the diagnosis and stratification of HCC patients.

Mass spectrometric characterization and identification of new methcathinone metabolites in human blood (plasma), urine and hair by liquid chromatography-high field quadrupole exactive orbitrap mass spectrometer

Globally, breast cancer (BC) is leading at the top of women's diseases and, as a multifactorial disease, there is the need for the development of new approaches to aid clinicians on monitoring BC treatments. In this sense, metabolomic studies have become an essential tool allowing the establishment of interdependency among metabolites in biological samples. The combination of nuclear magnetic resonance (NMR) and gas chromatography-quadrupole mass spectrometry (GC-qMS) based metabolomic analyses of urine and breast tissue samples from BC patients and cancer-free individuals was used. Multivariate statistical tools were used in order to obtain a panel of metabolites that could discriminate malignant from healthy status assisting in the diagnostic field. Urine samples (n = 30), cancer tissues (n = 30) were collected from BC patients, cancer-free tissues were resected outside the tumor margin from the same donors (n = 30) while cancer-free urine samples (n = 40) where obtained from healthy subjects and analysed by NMR and GC-qMS methodologies. The orthogonal partial least square discriminant analysis model showed a clear separation between BC patients and cancer-free subjects for both classes of samples. Specifically, for urine samples, the goodness of fit (R2Y) and predictive ability (Q2) was 0.946 and 0.910, respectively, whereas for tissue was 0.888 and 0.813, revealing a good predictable accuracy. The discrimination efficiency and accuracy of tissue and urine metabolites was ascertained by receiver operating characteristic curve analysis that allowed the identification of metabolites with high sensitivity and specificity. The metabolomic pathway analysis identified several dysregulated pathways in BC, including those related with lactate, valine, aspartate and glutamine metabolism. Additionally, correlations between urine and tissue metabolites were investigated and five metabolites (e.g. acetone, 3-hexanone, 4-heptanone, 2-methyl-5-(methylthio)-furan and acetate) were found to be significant using a dual platform approach. Overall, this study suggests that an improved metabolic profile combining NMR and GC-qMS may be useful to achieve more insights regarding the mechanisms underlying cancer.

PurposeThe metabolic pathways of APP-CHMINACA were characterized to select the markers of intake for implementation into analytical assays used by the clinical and forensic communities. We have combined the evidences obtained by both in vitro experiments and administration studies on mice.MethodsAPP-CHMINACA was incubated with either human or mouse liver microsomes. Urine and blood samples were collected at different time points from mice after injection of a 3 mg/kg dose of the test compound. Samples were analyzed using liquid chromatography-tandem mass spectrometry.ResultsThe in vitro studies allowed to isolate eight different metabolic reactions, formed by two metabolic routes, with no differences between human and mouse liver microsomes. The main biotransformation route involved the hydrolysis of the distal amide group and the subsequent hydroxylation on the cyclohexyl-methyl ring. The second route involved multiple hydroxylation of the parent compound, followed by reduction to generate minor metabolites. In blood samples, the most abundant substances identified were APP-CHMINACA unchanged and the metabolites formed by the hydrolysis of the distal amide together with its hydroxylated products. In urine samples, four metabolites formed following the hydroxylation of the distal amide hydrolysis metabolite were detected as the most abundant and long-term metabolites.ConclusionsThe outcomes of our study showed that the most suitable markers to detect the intake of APP-CHMINACA in blood and urine samples in the framework of toxicological, clinical and forensic investigations were the metabolite formed by the hydrolysis of the distal amide and its hydroxylated products.

Hypertensive disorders of pregnancy, including preeclampsia, are major contributors to maternal morbidity. The goal of this study was to evaluate the potential of metabolomics to predict preeclampsia and gestational hypertension from urine and serum samples in early pregnancy, and elucidate the metabolic changes related to the diseases. Metabolic profiles were obtained by nuclear magnetic resonance spectroscopy of serum and urine samples from 599 women at medium to high risk of preeclampsia (nulliparous or previous preeclampsia/gestational hypertension). Preeclampsia developed in 26 (4.3%) and gestational hypertension in 21 (3.5%) women. Multivariate analyses of the metabolic profiles were performed to establish prediction models for the hypertensive disorders individually and combined. Urinary metabolomic profiles predicted preeclampsia and gestational hypertension at 51.3% and 40% sensitivity, respectively, at 10% false positive rate, with hippurate as the most important metabolite for the prediction. Serum metabolomic profiles predicted preeclampsia and gestational hypertension at 15% and 33% sensitivity, respectively, with increased lipid levels and an atherogenic lipid profile as most important for the prediction. Combining maternal characteristics with the urinary hippurate/creatinine level improved the prediction rates of preeclampsia in a logistic regression model. The study indicates a potential future role of clinical importance for metabolomic analysis of urine in prediction of preeclampsia.

Brown seaweeds such as Ascophyllum nodosum are a rich source of phlorotannins (oligomers and polymers of phloroglucinol units), a class of polyphenols that are unique to Phaeophyceae. At present, there is no information on the bioavailability of seaweed polyphenols and limited evidence on their bioactivity in vivo. Consequently, we investigated the gastrointestinal modifications in vitro of seaweed phlorotannins from A. nodosum and their bioavailability and effect on inflammatory markers in healthy participants. In vitro, some phlorotannin oligomers were identified after digestion and colonic fermentation. In addition, seven metabolites corresponding to in vitro-absorbed metabolites were identified. Urine and plasma samples contained a variety of metabolites attributed to both unconjugated and conjugated metabolites (glucuronides and/or sulphates). In both urine and plasma, the majority of the metabolites were found in samples collected at late time points (6-24 h), suggesting colonic metabolism of high-molecular-weight phlorotannins, with three phlorotannin oligomers (hydroxytrifuhalol A, 7-hydroxyeckol, C-O-C dimer of phloroglucinol) identified in urine samples. A significant increase of the cytokine IL-8 was also observed. Our study shows for the first time that seaweed phlorotannins are metabolised and absorbed, predominantly in the large intestine, and there is a large inter-individual variation in their metabolic profile. Three phlorotannin oligomers present in the capsule are excreted in urine. Our study is the first investigation of the metabolism and bioavailability of seaweed phlorotannins and the role of colonic biotransformation. In addition, IL-8 is a possible target for phlorotannin bioactivity.

In Belgium, to control the abuse of anabolic steroids in cattle, urine samples have been gradually replaced by feces samples, because the latter can be obtained more easily from living animals. Urine and feces samples were collected from heifers after administration of boldenone, norethandrolone or ethylestrenol. Metabolites present in feces or urine were determined by GC-MS. Large qualitative and quantitative differences in the metabolic profiles were observed. In feces, in contrast to urine, the parent compounds or their major metabolites were detectable only shortly after administration. On the other hand, metabolites resulting from the reduction of the 3-oxo group and the unsaturated carbon-carbon bonds, present on the A-ring, allow for long-term detection in feces. A-ring reduced metabolites have been identified in samples found positive for norgestrel, boldenone, methylboldenone and methyltestosterone, respectively. These results are in agreement with concomitant in vivo experiments.