P-cresol Glucuronide Research Articles

A high-throughput liquid chromatography-tandem mass spectrometry assay for the simultaneous quantification of p-cresol sulfate, p-cresol glucuronide, indoxyl sulfate, and indoxyl glucuronide in HepaRG culture medium and the demonstration of mefenamic acid as a potent and selective detoxifying agent

ABSTRACT Background p-cresol and indole are uremic compounds which undergo sulfonation to generate the highly toxic p-cresol sulfate (pCS) and indoxyl sulfate (IxS). They are also subjected to glucuronidation to produce the less toxic p-cresol glucuronide (pCG) and indoxyl glucuronide (IG). We developed and validated an assay to quantify these metabolites in HepaRG cells. We also tested the effects of mefenamic acid on their in-situ formations in relation to the development of cellular necrosis. Research design and methods HepaRG cells were exposed to p-cresol or indole (0-1 mM) with mefenamic acid (0-3000 nM) for 24 hours to generate uremic metabolites. Cells were also exposed to 0.5 mM p-cresol or indole with/without 30 nM mefenamic acid to characterize lactate dehydrogenase (LDH) release. Results The assay exhibited high sensitivity and wide calibration ranges covering human concentrations. HepaRG cells also generated physiologically-relevant concentrations of each metabolite. Mefenamic acid inhibited pCS formation in a concentration-dependent manner without affecting pCG, IxS, or IG. Mefenamic acid also reduced LDH release from p-cresol (by 50.12±5.86%) or indole (56.26±3.58%). Conclusions This novel assay is capable of quantifying these metabolites in HepaRG cells. Our novel findings suggest that mefenamic acid can be potentially utilized therapeutically to attenuate pCS-associated toxicities.

Circulating Gut Microbe-Derived Metabolites Are Associated with Hepatocellular Carcinoma.

Forty-one patients with HCC and 96 healthy controls were enrolled from surgical clinics at the Cleveland Clinic from 2016 to 2020. Gut microbiota-derived circulating metabolites detectable in plasma were compared between patients with HCC and healthy controls. Hierarchical clustering was performed for generating heatmaps based on circulating metabolite concentrations using ClustVis, with Euclidean and Ward settings and significant differences between metabolite concentrations were tested using a binary logistic regression model. In patients with HCC, 25 (61%) had histologically confirmed cirrhosis. Trimethylamine (TMA)-related metabolites were found at higher concentrations in those with HCC, including choline (p < 0.001), betaine (p < 0.001), carnitine (p = 0.007), TMA (p < 0.001) and trimethylamine N-oxide (TMAO, p < 0.001). Notably, concentrations of P-cresol glucuronide (p < 0.001), indole-lactic acid (p = 0.038), 5-hydroxyindoleacetic acid (p < 0.0001) and 4-hydroxyphenyllactic acid (p < 0.001) were also increased in those with HCC compared to healthy controls. Hierarchical clustering of the metabolite panel separated patients based on the presence of HCC (p < 0.001), but was not able to distinguish between patients with HCC based on the presence of cirrhosis (p = 0.42). Gut microbiota-derived metabolites were differentially abundant in patients with HCC versus healthy controls. The observed perturbations of the TMAO pathway in HCC seem particularly promising as a target of future research and may have both diagnostic and therapeutic implications.

Reduction in Serum Concentrations of Uremic Toxins Driven by Bifidobacterium Longum Subsp. Longum BL21 is Associated with Gut Microbiota Changes in a Rat Model of Chronic Kidney Disease.

Gut microbiota dysbiosis and consequent impairment of gut barrier function, culminating in elevated levels of uremic toxins, are prevalent in chronic kidney disease (CKD) patients. These toxins, notably indoxyl sulphate (IS), indole-3-acetic acid (IAA), and trimethylamine oxide (TMAO), are implicated in a spectrum of CKD-related complications, including cardiovascular disease, bone and mineral disorders, and inflammation. The specific impacts of various probiotics on these CKD manifestations remain unexplored. This study delved into the potential of dietary probiotic interventions, particularly Bifidobacterium longum subsp. longum BL21, to modulate gut microbiota and mitigate metabolic disorders in a CKD rat model. Over a six-week period, we administered a dietary regimen of BL21 and conducted comprehensive analyses, including serum uremic toxin quantification and 16S rRNA gene sequencing, to systematically profile gut microbial alterations at the phylogenetic level. Our findings reveal that BL21 intervention significantly ameliorated CKD-induced disruptions in gut microbial populations, enhancing both microbial richness and the relative abundance of key taxa. Importantly, BL21 appeared to exert its beneficial effects by modulating the abundance of crucial species such as Barnesiella and Helicobacter. Functionally, the intervention markedly normalized serum levels of IS, IAA, and TMAO, while potentially attenuating p-cresol sulphate (PCS) and p-cresol glucuronide (PCG) concentrations. Consequently, BL21 demonstrated efficacy in regulating gut microbiota and curtailing the accumulation of uremic toxins. Our results advocate for the utilization of BL21 as a dietary intervention to diminish serum uremic toxins and re-establish gut microbiota equilibrium at the phylogenetic level, underscoring the promise of probiotic strategies in the management of CKD.

Analysis of Relationships between Metabolic Changes and Selected Nutrient Intake in Women Environmentally Exposed to Arsenic.

Nutrients involved in the metabolism of inorganic arsenic (iAs) may play a crucial role in mitigating the adverse health effects associated with such exposure. Consequently, the objective of this study was to analyze the association between the intake levels of nutrients involved in iAs metabolism and alterations in the metabolic profile during arsenic exposure. The study cohort comprised environmentally exposed women: WL (lower total urinary arsenic (As), n = 73) and WH (higher As, n = 73). The analysis included urinary untargeted metabolomics (conducted via liquid chromatography-mass spectrometry) and the assessment of nutrient intake involved in iAs metabolism, specifically methionine, vitamins B2, B6, and B12, folate, and zinc (based on 3-day dietary records of food and beverages). In the WL group, the intake of all analyzed nutrients exhibited a negative correlation with 5 metabolites (argininosuccinic acid, 5-hydroxy-L-tryptophan, 11-trans-LTE4, mevalonic acid, aminoadipic acid), while in the WH group, it correlated with 10 metabolites (5-hydroxy-L-tryptophan, dihyroxy-1H-indole glucuronide I, 11-trans-LTE4, isovalerylglucuronide, 18-oxocortisol, 3-hydroxydecanedioic acid, S-3-oxodecanoyl cysteamine, L-arginine, p-cresol glucuronide, thromboxane B2). Furthermore, nutrient intake demonstrated a positive association with 3 metabolites in the WL group (inosine, deoxyuridine, glutamine) and the WH group (inosine, N-acetyl-L-aspartic acid, tetrahydrodeoxycorticosterone). Altering the intake of nutrients involved in iAs metabolism could be a pivotal factor in reducing the negative impact of arsenic exposure on the human body. This study underscores the significance of maintaining adequate nutrient intake, particularly in populations exposed to arsenic.

Untargeted serum metabolomics reveals novel metabolite associations and disruptions in amino acid and lipid metabolism in Parkinson’s disease

BackgroundUntargeted high-resolution metabolomic profiling provides simultaneous measurement of thousands of metabolites. Metabolic networks based on these data can help uncover disease-related perturbations across interconnected pathways.ObjectiveIdentify metabolic disturbances associated with Parkinson’s disease (PD) in two population-based studies using untargeted metabolomics.MethodsWe performed a metabolome-wide association study (MWAS) of PD using serum-based untargeted metabolomics data derived from liquid chromatography with high-resolution mass spectrometry (LC-HRMS) using two distinct population-based case-control populations. We also combined our results with a previous publication of 34 metabolites linked to PD in a large-scale, untargeted MWAS to assess external validation.ResultsLC-HRMS detected 4,762 metabolites for analysis (HILIC: 2716 metabolites; C18: 2046 metabolites). We identified 296 features associated with PD at FDR<0.05, 134 having a log2 fold change (FC) beyond ±0.5 (228 beyond ±0.25). Of these, 104 were independently associated with PD in both discovery and replication studies at p<0.05 (170 at p<0.10), while 27 were associated with levodopa-equivalent dose among the PD patients. Intriguingly, among the externally validated features were the microbial-related metabolites, p-cresol glucuronide (FC=2.52, 95% CI=1.67, 3.81, FDR=7.8e-04) and p-cresol sulfate. P-cresol glucuronide was also associated with motor symptoms among patients. Additional externally validated metabolites associated with PD include phenylacetyl-L-glutamine, trigonelline, kynurenine, biliverdin, and pantothenic acid. Novel associations include the anti-inflammatory metabolite itaconate (FC=0.79, 95% CI=0.73, 0.86; FDR=2.17E-06) and cysteine-S-sulfate (FC=1.56, 95% CI=1.39, 1.75; FDR=3.43E-11). Seventeen pathways were enriched, including several related to amino acid and lipid metabolism.ConclusionsOur results revealed PD-associated metabolites, confirming several previous observations, including for p-cresol glucuronide, and newly implicating interesting metabolites, such as itaconate. Our data also suggests metabolic disturbances in amino acid and lipid metabolism and inflammatory processes in PD.

Plasma metabolites of aromatic amino acids associate with clinical severity and gut microbiota of Parkinson’s disease

Gut microbial proteolytic metabolism has been reportedly altered in Parkinson’s disease (PD). However, the circulating aromatic amino acids (AAA) described in PD are inconsistent. Here we aimed to investigate plasma AAA profiles in a large cohort of PD patients, and examine their correlations with clinical severity and gut microbiota changes. We enrolled 500 participants including 250 PD patients and 250 neurologically normal controls. Plasma metabolites were measured using liquid chromatography mass spectrometry. Faecal samples were newly collected from 154 PD patients for microbiota shotgun metagenomic sequencing combined with data derived from 96 PD patients reported before. Data were collected regarding diet, medications, and motor and non-motor symptoms of PD. Compared to controls, PD patients had higher plasma AAA levels, including phenylacetylglutamine (PAGln), p-cresol sulfate (Pcs), p-cresol glucuronide (Pcg), and indoxyl sulfate (IS). Multivariable linear regression analyses, with adjustment for age, sex, and medications, revealed that the plasma levels of PAGln (coefficient 4.49, 95% CI 0.40–8.58, P = 0.032) and Pcg (coefficient 1.79, 95% CI 0.07–3.52, P = 0.042) positively correlated with motor symptom severity but not cognitive function. After correcting for abovementioned potential confounders, these AAA metabolites were also associated with the occurrence of constipation in PD patients (all P < 0.05). Furthermore, plasma levels of AAA metabolites were correlated with the abundance of specific gut microbiota species, including Bacteroides sp. CF01-10NS, Bacteroides vulgatus, and Clostridium sp. AF50-3. In conclusion, elevated plasma AAA metabolite levels correlated with disease characteristics in PD, suggesting that upregulated proteolytic metabolism may contribute to the pathophysiology of PD.

Atlas of gut microbe-derived products from aromatic amino acids and risk of cardiovascular morbidity and mortality.

Precision microbiome modulation as a novel treatment strategy is a rapidly evolving and sought goal. The aim of this study is to determine relationships among systemic gut microbial metabolite levels and incident cardiovascular disease risks to identify gut microbial pathways as possible targets for personalized therapeutic interventions. Stable isotope dilution mass spectrometry methods to quantitatively measure aromatic amino acids and their metabolites were used to examine sequential subjects undergoing elective diagnostic cardiac evaluation in two independent cohorts with longitudinal outcome data [US (n = 4000) and EU (n = 833) cohorts]. It was also used in plasma from humans and mice before vs. after a cocktail of poorly absorbed antibiotics to suppress gut microbiota. Multiple aromatic amino acid-derived metabolites that originate, at least in part, from gut bacteria are associated with incident (3-year) major adverse cardiovascular event (MACE) risks (myocardial infarction, stroke, or death) and all-cause mortality independent of traditional risk factors. Key gut microbiota-derived metabolites associated with incident MACE and poorer survival risks include: (i) phenylacetyl glutamine and phenylacetyl glycine (from phenylalanine); (ii) p-cresol (from tyrosine) yielding p-cresol sulfate and p-cresol glucuronide; (iii) 4-OH-phenyllactic acid (from tyrosine) yielding 4-OH-benzoic acid and 4-OH-hippuric acid; (iv) indole (from tryptophan) yielding indole glucuronide and indoxyl sulfate; (v) indole-3-pyruvic acid (from tryptophan) yielding indole-3-lactic acid and indole-3-acetyl-glutamine, and (vi) 5-OH-indole-3-acetic acid (from tryptophan). Key gut microbiota-generated metabolites derived from aromatic amino acids independently associated with incident adverse cardiovascular outcomes are identified, and thus will help focus future studies on gut-microbial metabolic outputs relevant to host cardiovascular health.

Association of the gut microbiome with kidney function and damage in the Hispanic Community Health Study/Study of Latinos (HCHS/SOL)

ABSTRACT Background The gut microbiome is altered in chronic kidney disease (CKD), potentially contributing to CKD progression and co-morbidities, but population-based studies of the gut microbiome across a wide range of kidney function and damage are lacking. Methods In the Hispanic Community Health Study/Study of Latinos, gut microbiome was assessed by shotgun sequencing of stool (n = 2,438; 292 with suspected CKD). We examined cross-sectional associations of estimated glomerular filtration rate (eGFR), urinary albumin:creatinine (UAC) ratio, and CKD with gut microbiome features. Kidney trait-related microbiome features were interrogated for correlation with serum metabolites (n = 700), and associations of microbiome-related serum metabolites with kidney trait progression were examined in a prospective analysis (n = 3,635). Results Higher eGFR was associated with overall gut microbiome composition, greater abundance of species from Prevotella, Faecalibacterium, Roseburia, and Eubacterium, and microbial functions related to synthesis of long-chain fatty acids and carbamoyl-phosphate. Higher UAC ratio and CKD were related to lower gut microbiome diversity and altered overall microbiome composition only in participants without diabetes. Microbiome features related to better kidney health were associated with many serum metabolites (e.g., higher indolepropionate, beta-cryptoxanthin; lower imidazole propionate, deoxycholic acids, p-cresol glucuronide). Imidazole propionate, deoxycholic acid metabolites, and p-cresol glucuronide were associated with prospective reductions in eGFR and/or increases in UAC ratio over ~6 y. Conclusions Kidney function is a significant correlate of the gut microbiome, while the relationship of kidney damage with the gut microbiome depends on diabetes status. Gut microbiome metabolites may contribute to CKD progression.

Abstract 24: Association of the Gut Microbiome With Kidney Function and Damage in the Hispanic Community Health Study/Study of Latinos (HCHS/SOL)

Background: The gut microbiome is altered in chronic kidney disease (CKD), potentially contributing to CKD progression and co-morbidities such as cardiovascular disease, but population-based studies of the gut microbiome across a wide range of kidney function and damage are lacking. Methods: In the Hispanic Community Health Study/Study of Latinos, gut microbiome was assessed by shotgun sequencing of stool (n=2,438; 292 with suspected CKD). We examined cross-sectional associations of estimated glomerular filtration rate (eGFR), urinary albumin:creatinine (UAC) ratio, and CKD with gut microbiome features, adjusting for covariates, and also stratified by diabetes status. Kidney trait-related microbiome features were interrogated for correlation with serum metabolites (n=700), and associations of microbiome-related serum metabolites with kidney trait progression were examined in a prospective analysis (n=3,635) ( Figure 1a ). Results: Higher eGFR was significantly associated with overall gut microbiome composition, greater abundance of species from Prevotella , Faecalibacterium , Roseburia , and Eubacterium , and microbial functions related to synthesis of long-chain fatty acids and carbamoyl-phosphate. Higher UAC ratio and CKD were related to lower gut microbiome diversity and altered overall microbiome composition only in participants without diabetes. Microbiome features related to better kidney health were associated with many serum metabolites (e.g., higher indolepropionate, beta-cryptoxanthin; lower imidazole propionate, deoxycholic acids, p-cresol glucuronide) ( Figure 1b ). Imidazole propionate, deoxycholic acid metabolites, and p-cresol glucuronide were associated with prospective reductions in eGFR and/or increases in the UAC ratio ( Figure 1c ). Conclusions: Kidney function is a significant correlate of the gut microbiome, while the relationship of kidney damage with the gut microbiome depends on diabetes status. Gut microbiome metabolites may contribute to CKD progression.

S-57-4: THE ROLE OF ORAL L-TYROSINE, METABOLISED BY THE GUT MICROBIOTA INTO P-CRESOL DERIVED METABOLITES, IN BLOOD PRESSURE REGULATION

Objective: Low fibre intake has emerged as an important risk factor for hypertension through changes in the gut microbiota, but the specific metabolites involved in this phenotype are unknown. We identified two novel secondary metabolites, p-Cresol glucuronide (PCG) and p-Cresol sulfate (PCS) that were elevated in plasma samples in mice on a low fibre diet. The precursor of PCG and PCS, p-Cresol, is a microbial metabolite derived from tyrosine fermentation by the gut microbiota. We hypothesized p-cresol-derived metabolites would trigger immune-activation to promote the development of hypertension. We aimed to assess whether oral L-tyrosine increases circulating PCG and PCS levels, leading to a worsened cardiovascular phenotype via immune-dependent mechanisms. Design and method: Male C57BL/6 mice (n = 8/group) were infused with saline or angiotensin II (Ang II, 0.5 mg/kg/day) for 4 weeks. Mice were treated with or without L-tyrosine in their drinking water accompanied by a normal chow diet for the duration of the experiment. Blood pressure (BP) was measured using tail-cuff and a targeted metabolomic approach was applied to identify the levels of PCG and PCS in the circulation. Additionally, liver enzymatic activities, immune cell activation and infiltration in the bone marrow, spleen and kidney were examined. Results: L-tyrosine treated hypertensive mice had significantly reduced systolic and diastolic BP compared to the control hypertensive mice (-16.4 and -14.2 mmHg, respectively, p &lt; 0.01). Flow cytometric analysis revealed that L-tyrosine did not alter the numbers of the immune cells, including monocytes, neutrophils or T cells in bone marrow, spleen or kidneys in hypertensive or normotensive conditions. Furthermore, immune cell activation and production of cytokines, including TNF-alpha and IL17, did not change. Preliminary data suggests L-tyrosine increased the levels of PCS, but not PCG, in the circulation of both hypertensive and normotensive mice. The expression of the hepatic enzymes responsible for the biotransformation of p-Cresol to PCS and PCG were not differentially expressed, suggesting that conversion of p-Cresol to PCS occurs outside the liver. Conclusions: This study provides novel insights into the roles of L-tyrosine and PCS in hypertension. When delivered orally, L-tyrosine is digested by the gut microbiota, and leads to the production of PCS, lowering BP through immune-cell independent mechanisms. However, the role of PCG, a low-fibre driven-metabolite, in BP is still under investigation.

Dietary and Animal Strategies to Reduce the Environmental Impact of Pastoral Dairy Systems Result in Altered Nutraceutical Profiles in Milk.

The objective of this study was to evaluate and provide further insights into how dairy cows genetically divergent for milk urea N breeding values [MUNBV, high (2.21 ± 0.21) vs. low (-1.16 ± 0.21); µ ± SEM], consuming either fresh cut Plantain (Plantago lanceolata L., PL) or Ryegrass (Lolium perenne L., RG) herbage, impacted the nutraceutical profile of whole milk by investigating amino and fatty acid composition and applying metabolomic profiling techniques. Both diet and MUNBV, and their interaction term, were found to affect the relative abundance of alanine, glycine, histidine, and phenylalanine in the milk (p &lt; 0.05), but their minor absolute differences (up to ~0.13%) would not be considered biologically relevant. Differences were also detected in the fatty acid profile based on MUNBV and diet (p &lt; 0.05) with low MUNBV cows having a greater content of total unsaturated fatty acids (+16%) compared to high MUNBV cows and cows consuming PL having greater content of polyunsaturated fatty acids (+92%), omega 3 (+101%) and 6 (+113%) compared to RG. Differences in the metabolomic profile of the milk were also detected for both MUNBV and dietary treatments. Low MUNBV cows were found to have greater abundances of choline phosphate, phosphorylethanolamine, N-acetylglucosamine 1-phosphate, and 2-dimethylaminoethanol (p &lt; 0.05). High MUNBV cows had a greater abundance of methionine sulfoxide, malate, 1,5-anhydroglucitol (1,5-AG), glycerate, arabitol/xylitol, 3-hydroxy-3-methylglutarate, 5-hydroxylysine and cystine (p &lt; 0.05). Large differences (p &lt; 0.05) were also detected as a result of diet with PL diets having greater abundances of the phytochemicals 4-acetylcatechol sulfate, 4-methylcatechol sulfate, and p-cresol glucuronide whilst RG diets had greater abundances of 2,6-dihydroxybenzoic acid, 2-acetamidophenol sulfate, and 2-hydroxyhippurate. The results of this study indicate the potential to alter the nutraceutical value of milk from dietary and genetic strategies that have been previously demonstrated to reduce environmental impact.

LC-MS metabolomics of urine reveals distinct profiles for non-muscle-invasive and muscle-invasive bladder cancer.

Bladder cancer (BC) is among the most frequent malignancies worldwide. Novel non-invasive markers are needed to diagnose and stage BC with more accuracy than invasive procedures like cystoscopy. To date, no study has identified urine metabolites characteristic of all BC stages. To discover novel urine metabolomic profiles to diagnose and stage non-muscle-invasive (NMIBC) and muscle-invasive (MIBC) patients using mass spectrometry-based metabolomics. We prospectively recruited 198 BC patients and 98 age- and sex-matched healthy volunteers without evidence of renal or bladder condition confirmed by ultrasound, from whom we collected a first morning urine sample (before surgery in patients). In a discovery stage, an untargeted metabolomic analysis was conducted in urine samples of a selection of 64 BC patients (19 TaG1, 11 TaG3, 20 T1G3, 12 T2G3, 1 T2G2, 1 T3G3) and 20 controls to identify dysregulated metabolites. Next, after exhaustive multivariate analysis, confirmed dysregulated metabolites were validated in an independent cohort of 134 BC patients (19 TaG1, 62 TaG2, 9 TaG3, 15 T1G2, 16 T1G3, 4 T2G2, 9 T2G3) and 78 controls. We validated p-cresol glucuronide as potential diagnostic biomarker for BC patients compared to controls (AUC = 0.79). For NMIBC, p-cresol glucuronide was valuable as staging biomarker (AUC = 0.803). And among NMIBCs, p-coumaric acid may be a potential specific staging biomarker for the TaG1 NMIBC; however, future validation experiments should be conducted once the precise version of the standard is commercially available. Remarkably, for MIBC we validated spermine as potential specific staging biomarker (AUC = 0.882). Ours is the first metabolomics study conducted in urine of a thoroughly characterized cohort comprising all stages of NMIBC, MIBC and healthy controls in which we identified non-invasive diagnostic and staging biomarkers. These may improve BC management, thus reducing the use of current harmful diagnostic techniques.

A host–gut microbial amino acid co-metabolite, p-cresol glucuronide, promotes blood–brain barrier integrity in vivo

ABSTRACT The sequential activity of gut microbial and host processes can exert a powerful modulatory influence on dietary components, as exemplified by the metabolism of the amino acids tyrosine and phenylalanine to p-cresol by gut microbes, and then to p-cresol glucuronide (pCG) by host enzymes. Although such glucuronide conjugates are classically thought to be biologically inert, there is accumulating evidence that this may not always be the case. We investigated the activity of pCG, studying its interactions with the cerebral vasculature and the brain in vitro and in vivo. Male C57Bl/6 J mice were used to assess blood–brain barrier (BBB) permeability and whole-brain transcriptomic changes in response to pCG treatment. Effects were then further explored using the human cerebromicrovascular endothelial cell line hCMEC/D3, assessing paracellular permeability, transendothelial electrical resistance and barrier protein expression. Mice exposed to pCG showed reduced BBB permeability and significant changes in whole-brain transcriptome expression. Surprisingly, treatment of hCMEC/D3 cells with pCG had no notable effects until co-administered with bacterial lipopolysaccharide, at which point it was able to prevent the permeabilizing effects of endotoxin. Further analysis suggested that pCG acts as an antagonist at the principal lipopolysaccharide receptor TLR4. The amino acid phase II metabolic product pCG is biologically active at the BBB, antagonizing the effects of constitutively circulating lipopolysaccharide. These data add to the growing literature showing glucuronide conjugates to be more than merely metabolic waste products and highlight the complexity of gut microbe to host communication pathways underlying the gut–brain axis.

Effects of p-Cresol on Oxidative Stress, Glutathione Depletion, and Necrosis in HepaRG Cells: Comparisons to Other Uremic Toxins and the Role of p-Cresol Glucuronide Formation.

The toxicological effects of p-cresol have primarily been attributed to its metabolism products; however, very little human data are available in the key organ (i.e., liver) responsible for the generation of these metabolites. Experiments were conducted in HepaRG cells utilizing the following markers of cellular toxicity: 2′-7′-dichlorofluorescein (DCF; oxidative stress) formation, total cellular glutathione (GSH) concentration, and lactate dehydrogenase (LDH; cellular necrosis) release. Concentrations of p-cresol, p-cresol sulfate, and p-cresol glucuronide were determined using validated assays. p-Cresol exposure resulted in concentration- and time-dependent changes in DCF (EC50 = 0.64 ± 0.37 mM at 24 h of exposure) formation, GSH (EC50 = 1.00 ± 0.07 mM) concentration, and LDH (EC50 = 0.85 ± 0.14 mM) release at toxicologically relevant conditions. p-Cresol was also relatively more toxic than 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, indole-3-acetic acid, indoxyl sulfate, kynurenic acid, and hippuric acid on all markers. Although the exogenous administration of p-cresol sulfate and p-cresol glucuronide generated high intracellular concentrations of these metabolites, both metabolites were less toxic compared to p-cresol at equal-molar conditions. Moreover, p-cresol glucuronide was the predominant metabolite generated in situ from p-cresol exposure. Selective attenuation of glucuronidation (without affecting p-cresol sulfate formation, while increasing p-cresol accumulation) using independent chemical inhibitors (i.e., 0.75 mM l-borneol, 75 µM amentoflavone, or 100 µM diclofenac) consistently resulted in further increases in LDH release associated with p-cresol exposure (by 28.3 ± 5.3%, 30.0 ± 8.2% or 27.3 ± 6.8%, respectively, compared to p-cresol treatment). These novel data indicated that p-cresol was a relatively potent toxicant, and that glucuronidation was unlikely to be associated with the manifestation of its toxic effects in HepaRG cells.

MO622IMPAIRED PROTEIN-BOUND UREMIC TOXIN EXCRETION SUGGESTS TUBULAR DYSFUNCTION IN DIABETIC NEPHROPATHY

Abstract Background and Aims Kidney tubular damage is an important prognostic determinant in diabetic nephropathy (DN) (1). Proximal tubular secretion is a vital homeostatic function that is responsible for excretion of waste, such as protein bound uremic toxins (PBUTs) that are minimally eliminated via glomerular filtration. PBUTs are potentially harmful waste products of endogenous metabolism that are efficiently excreted by tubular secretion via organic anion transporters (OATs). Currently available diagnostic methods cannot accurately detect tubular dysfunction. We hypothesize that renal PBUT clearance may be a sensitive tubular function marker. Here, we measured PBUTs in long-term streptozotocin (STZ)-induced murine diabetic nephropathy (DN), which was characterized by severe tubular atrophy and interstitial fibrosis (2). Method Diabetes mellitus was induced in C57Bl/6 mice by a single intraperitoneal injection of 200 mg/kg STZ which resulted in substantial kidney damage after 6 months evaluated by histopathology and conventional markers (2). Indoxyl sulfate (IS), hippuric acid (HA), kynurenic acid (KA), kynurenine (Kyn), p-cresol glucuronide (pCG), p-cresol sulfate (pCS) and indole acetic acid (IAA) were measured in plasma and urine after 6 and 8 months by LC-MS/MS. Results Among the PBUTs with the highest OAT affinity, viz. IS, HA and KA, plasma concentrations were 2.2-, 2.3- and 1.5-fold higher (p=0.005, 0.0006, 0.03; Fig 1A-C) after 6 months and 1.9-, 2.1- and 2-fold higher (p=0.008, 0.0005, 0.001; Fig 2A-C) after 8 in DN, and urinary excretions (normalized for plasma concentrations) were 3.3-, 2.3- and 3.0-fold lower (p=0.012, 0.16, 0.03; Fig 1G-I) after 6 months and 2.5-, 1.6-, 2.3-fold lower (p=0.028, 0.046, 0.0005; Fig 2G-I) after 8 months in DN. Other PBUTS, viz. IAA, Kyn, pCS and pCG, were not significantly affected. Conclusion Our findings suggest that OAT function is compromised in murine long-term DN. Renal clearance of IS, HA and KA may be a marker of tubular function in DN. Future studies should focus on correlations with histology and validation in other species.

Flexibility of equine bioenergetics and muscle plasticity in response to different types of training: An integrative approach, questioning existing paradigms.

Equine bioenergetics have predominantly been studied focusing on glycogen and fatty acids. Combining omics with conventional techniques allows for an integrative approach to broadly explore and identify important biomolecules. Friesian horses were aquatrained (n = 5) or dry treadmill trained (n = 7) (8 weeks) and monitored for: evolution of muscle diameter in response to aquatraining and dry treadmill training, fiber type composition and fiber cross-sectional area of the M. pectoralis, M. vastus lateralis and M. semitendinosus and untargeted metabolomics of the M. pectoralis and M. vastus lateralis in response to dry treadmill training. Aquatraining was superior to dry treadmill training to increase muscle diameter in the hindquarters, with maximum effect after 4 weeks. After dry treadmill training, the M. pectoralis showed increased muscle diameter, more type I fibers, decreased fiber mean cross sectional area, and an upregulated oxidative metabolic profile: increased β-oxidation (key metabolites: decreased long chain fatty acids and increased long chain acylcarnitines), TCA activity (intermediates including succinyl-carnitine and 2-methylcitrate), amino acid metabolism (glutamine, aromatic amino acids, serine, urea cycle metabolites such as proline, arginine and ornithine) and xenobiotic metabolism (especially p-cresol glucuronide). The M. vastus lateralis expanded its fast twitch profile, with decreased muscle diameter, type I fibers and an upregulation of glycolytic and pentose phosphate pathway activity, and increased branched-chain and aromatic amino acid metabolism (cis-urocanate, carnosine, homocarnosine, tyrosine, tryptophan, p-cresol-glucuronide, serine, methionine, cysteine, proline and ornithine). Trained Friesians showed increased collagen and elastin turn-over. Results show that branched-chain amino acids, aromatic amino acids and microbiome-derived xenobiotics need further study in horses. They feed the TCA cycle at steps further downstream from acetyl CoA and most likely, they are oxidized in type IIA fibers, the predominant fiber type of the horse. These study results underline the importance of reviewing existing paradigms on equine bioenergetics.

Thrombolome and Its Emerging Role in Chronic Kidney Diseases.

Patients with chronic kidney disease (CKD) are at an increased risk of thromboembolic complications, including myocardial infarction, stroke, deep vein thrombosis, and pulmonary embolism. These complications lead to increased mortality. Evidence points to the key role of CKD-associated dysbiosis and its effect via the generation of gut microbial metabolites in inducing the prothrombotic phenotype. This phenomenon is known as thrombolome, a panel of intestinal bacteria-derived uremic toxins that enhance thrombosis via increased tissue factor expression, platelet hyperactivity, microparticles release, and endothelial dysfunction. This review discusses the role of uremic toxins derived from gut-microbiota metabolism of dietary tryptophan (indoxyl sulfate (IS), indole-3-acetic acid (IAA), kynurenine (KYN)), phenylalanine/tyrosine (p-cresol sulfate (PCS), p-cresol glucuronide (PCG), phenylacetylglutamine (PAGln)) and choline/phosphatidylcholine (trimethylamine N-oxide (TMAO)) in spontaneously induced thrombosis. The increase in the generation of gut microbial uremic toxins, the activation of aryl hydrocarbon (AhRs) and platelet adrenergic (ARs) receptors, and the nuclear factor kappa B (NF-κB) signaling pathway can serve as potential targets during the prevention of thromboembolic events. They can also help create a new therapeutic approach in the CKD population.

Simple Determination of p-Cresol in Plasma Samples Using Fluorescence Spectroscopy Technique.

The development of simple, fast, cheap and reliable analytical methods for tracing biological indicators is demanded through clinical investigations. Herein, we developed, for the first time, a cheap and specific method for the extraction and quantification of p-cresol (pC) in real plasma samples of chronic kidney disease (CKD). Plasma samples were prepared by hydrolyzing in an acidic medium to convert pCS (p-cresol sulfate) and p-Cresol glucuronide (pCG) to pC. Next, proteins of plasma samples were precipitated and then pC was extracted by acetonitrile (ACN) and saturated NaCl (as salting-out agent). Finally, fluorescence emissions were measured at λex/λem = 280/310 nm. The specificity of the method was checked by testing various possible interfering agents. The obtained results revealed a specific determination of pC. Under optimal conditions, a linear range was detected from 0.5 to 30 µg/mL of pC with a lower limit of detection (LLOQ) of 0.5 µg/mL. The reliability of the method was checked by calculating the repeatability, selectivity, and accuracy of the developed method for pC determination in plasma samples. The application of the developed method was investigated for the detection of pC in a number of CKD patients. Due to the simplicity and selectivity, the developed method could be applied for routine analysis of pC concentrations in the plasma samples of CKD patients. In addition, the developed method showed great potential for developing a point-of-care testing (POCT) device.

A targeted ultra performance liquid chromatography – Tandem mass spectrometric assay for tyrosine and metabolites in urine and plasma: Application to the effects of antibiotics on mice

Tyrosine plays a key role in mammalian biochemistry and defects in its metabolism (e.g., tyrosinemia, alkaptonuria etc.) have significant adverse consequences for those affected if left untreated. In addition, gut bacterially-derived p-cresol and its metabolites are of interest as a result of various effects on host xenobiotic metabolism. A fit-for-purpose quantitative ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay was developed to target and quantify tyrosine and eleven metabolites in urine and plasma. Dansylation, using dansyl chloride, was used to improve chromatographic and mass spectral properties for tyrosine and nine phenolic metabolites, with detection using positive electrospray ionisation (ESI). The sulfate and glucuronide conjugates of p-cresol, where the phenol group was blocked, were quantified intact, using negative ESI via polarity switching during the same run. Sample preparation for urine and plasma involved deproteinization by solvent precipitation (of acetonitrile:isopropyl alcohol (1:1 v/v)) followed by in situ dansylation in 96 well plates. To minimize sample and solvent usage, and maximize sensitivity, analysis was performed using microbore reversed-phase gradient UPLC on a C8 phase with a 7.5 min. cycle time. The coefficients of variation obtained were <15%, with lower limits of quantification ranging from 5 to 250 nM depending upon the analyte. The method was applied to plasma and urine samples obtained from mice placed on a high tyrosine diet with one subgroup of animals subsequently receiving antibiotics to suppress the gut microbiota. Whilst plasma profiles were largely unaffected by antibiotic treatment clear reductions in the amount of p-cresol sulfate and p-cresol glucuronide excreted in the urine were observed for these mice.

Abstract MP28: A New Gut Microbial Metabolite Derived From Low Fibre Intake

Low fermentable fibre intake has emerged as an important risk factor for hypertension through changes in the gut microbiota, but the biological pathways and specific metabolites involved are unknown. We performed untargeted liquid-chromatography mass spectrometry metabolomic profiling on plasma samples from 16 mice fed low or high fibre diets and 70 participants with ambulatory blood pressure recordings. Mouse gut microbiome was analysed using 16S rRNA gene sequencing . In vitro experiments were performed in human peripheral blood mononuclear cells (PBMCs) to assess the role of a new metabolite in inflammatory responses. The phenylalanine, tyrosine and tryptophan biosynthesis pathway was upregulated in hypertensive compared to normotensive participants, as well as in low compared to high fibre fed mice (both q &lt;0.05, pathway impact factor=1.0). In particular, p-cresol glucuronide (pCG), an end-product of tyrosine metabolism, was higher in mice fed with low compared to high fibre in both angiotensin II-treated ( q =4.83 x10 -4 , fold change= 88) and sham groups ( q =1.56 x10 -4 , fold change= 297). pCG is derived from p-Cresol, which is produced by the gut microbiota. β-diversity analyses showed that distinct gut microbiome compositions were associated with levels of pCG in plasma (q&lt;0.05). While diet had a major effect on the gut microbiome (~30%), we found that pCG levels were associated with 5.7-6.3% of the total gut microbiome variation. This is relevant as pCG’s precursor, p-cresol, may inhibit the growth of certain types of bacteria. To understand the possible role of pCG in hypertension, human PBMCs obtained commercially were treated with physiological levels of pCG for 24 hours. This resulted in an increase in proinflammatory IL-17A mRNA ( P =0.01, fold change= 1.79) and a decrease in anti-inflammatory IL-10 mRNA ( P =0.014, fold change= -2.32) when compared to untreated and mock-treated cells. In conclusion, tyrosine biotransformation was associated with both human hypertension and low fibre intake. pCG, a key microbial metabolite derived from the metabolism of tyrosine, was associated with distinct gut microbiome compositions and modulated inflammatory response, suggesting it may be involved in the genesis of hypertension.