Cocaine-derived hippuric acid activates mtDNA-STING signaling in alcoholic liver disease: Implications for alcohol and cocaine co-abuse.

The simultaneous abuse of alcohol-cocaine is known to cause stronger and more unpredictable cellular damage in the liver, heart, and brain. However, the mechanistic crosstalk between cocaine and alcohol in liver injury remains unclear. The findings revealed cocaine-induced liver injury and inflammation in both marmosets and mice. Of note, co-administration of cocaine and ethanol in mice causes more severe liver damage than individual treatment. The metabolomic analysis confirmed that hippuric acid (HA) is the most abundant metabolite in marmoset serum after cocaine consumption and that is formed in primary marmoset hepatocytes. HA, a metabolite of cocaine, increases mitochondrial DNA leakage and subsequently increases the production of proinflammatory factors via STING signaling in Kupffer cells (KCs). In addition, conditioned media of cocaine-treated KC induced hepatocellular necrosis via alcohol-induced TNFR1. Finally, disruption of STING signaling in vivo ameliorated co-administration of alcohol- and cocaine-induced liver damage and inflammation. These findings postulate intervention of HA-STING-TNFR1 axis as a novel strategy for treatment of alcohol- and cocaine-induced excessive liver damage.

The Puzzle of Aspirin and Iron Deficiency: The Vital Missing Link of the Iron-Chelating Metabolites.

Acetylsalicylic acid or aspirin is the most commonly used drug in the world and is taken daily by millions of people. There is increasing evidence that chronic administration of low-dose aspirin of about 75-100 mg/day can cause iron deficiency anaemia (IDA) in the absence of major gastric bleeding; this is found in a large number of about 20% otherwise healthy elderly (>65 years) individuals. The mechanisms of the cause of IDA in this category of individuals are still largely unknown. Evidence is presented suggesting that a likely cause of IDA in this category of aspirin users is the chelation activity and increased excretion of iron caused by aspirin chelating metabolites (ACMs). It is estimated that 90% of oral aspirin is metabolized into about 70% of the ACMs salicyluric acid, salicylic acid, 2,5-dihydroxybenzoic acid, and 2,3-dihydroxybenzoic acid. All ACMs have a high affinity for binding iron and ability to mobilize iron from different iron pools, causing an overall net increase in iron excretion and altering iron balance. Interestingly, 2,3-dihydroxybenzoic acid has been previously tested in iron-loaded thalassaemia patients, leading to substantial increases in iron excretion. The daily administration of low-dose aspirin for long-term periods is likely to enhance the overall iron excretion in small increments each time due to the combined iron mobilization effect of the ACM. In particular, IDA is likely to occur mainly in populations such as elderly vegetarian adults with meals low in iron content. Furthermore, IDA may be exacerbated by the combinations of ACM with other dietary components, which can prevent iron absorption and enhance iron excretion. Overall, aspirin is acting as a chelating pro-drug similar to dexrazoxane, and the ACM as combination chelation therapy. Iron balance, pharmacological, and other studies on the interaction of iron and aspirin, as well as ACM, are likely to shed more light on the mechanism of IDA. Similar mechanisms of iron chelation through ACM may also be implicated in patient improvements observed in cancer, neurodegenerative, and other disease categories when treated long-term with daily aspirin. In particular, the role of aspirin and ACM in iron metabolism and free radical pathology includes ferroptosis, and may identify other missing links in the therapeutic effects of aspirin in many more diseases. It is suggested that aspirin is the first non-chelating drug described to cause IDA through its ACM metabolites. The therapeutic, pharmacological, toxicological and other implications of aspirin are incomplete without taking into consideration the iron binding and other effects of the ACM.

Rats with Long-Term Cholestasis Have a Decreased Cytosolic but Maintained Mitochondrial Hepatic CoA Pool.

Previous studies showed that rats with long-term bile duct ligation have reduced coenzyme A stores per g of liver but maintained mitochondrial CoA stores. Based on these observations, we determined the CoA pool in the liver homogenate, liver mitochondria, and liver cytosol of rats with bile duct ligation for 4 weeks (BDL rats, n = 9) and sham-operated control rats (CON rats, n = 5). In addition, we tested the cytosolic and mitochondrial CoA pools by assessing the metabolism of sulfamethoxazole and benzoate in vivo and of palmitate in vitro. The hepatic total CoA content was lower in BDL than CON rats (mean ± SEM; 128 ± 5 vs. 210 ± 9 nmol/g), affecting all subfractions equally (free CoA (CoASH), short- and long-chain acyl-CoA). In BDL rats, the hepatic mitochondrial CoA pool was maintained, and the cytosolic pool was reduced (23.0 ± 0.9 vs. 84.6 ± 3.7 nmol/g liver; CoA subfractions were affected equally). The urinary excretion of hippurate after i.p. benzoate administration (measuring mitochondrial benzoate activation) was reduced in BDL rats (23.0 ± 0.9 vs. 48.6 ± 3.7% of dose/24 h), whereas the urinary elimination of N-acetylsulfamethoxazole after i.p. sulfamethoxazole administration (measuring the cytosolic acetyl-CoA pool) was maintained (36.6 ± 3.0 vs. 35.1 ± 2.5% of dose/24 h BDL vs. CON rats). Palmitate activation was impaired in the liver homogenate of BDL rats but the cytosolic CoASH concentration was not limiting. In conclusion, BDL rats have reduced hepatocellular cytosolic CoA stores, but this reduction does not limit sulfamethoxazole N-acetylation or palmitate activation. The hepatocellular mitochondrial CoA pool is maintained in BDL rats. Impaired hippurate formation in BDL rats is explained best by mitochondrial dysfunction.

Impaired Tubular Secretion of Organic Solutes in Advanced Chronic Kidney Disease.

BACKGROUND: The clearance of solutes removed by tubular secretion may be altered out of proportion to the GFR in CKD. Recent studies have described considerable variability in the secretory clearance of waste solutes relative to the GFR in patients with CKD. METHODS: To test the hypothesis that secretory clearance relative to GFR is reduced in patients approaching dialysis, we used metabolomic analysis to identify solutes in simultaneous urine and plasma samples from 16 patients with CKD and an eGFR of 7±2 ml/min per 1.73 m2 and 16 control participants. Fractional clearances were calculated as the ratios of urine to plasma levels of each solute relative to those of creatinine and urea in patients with CKD and to those of creatinine in controls. RESULTS: Metabolomic analysis identified 39 secreted solutes with fractional clearance >3.0 in control participants. Fractional clearance values in patients with CKD were reduced on average to 65%±27% of those in controls. These values were significantly lower for 18 of 39 individual solutes and significantly higher for only one. Assays of the secreted anions phenylacetyl glutamine, p-cresol sulfate, indoxyl sulfate, and hippurate confirmed variable impairment of secretory clearances in advanced CKD. Fractional clearances were markedly reduced for phenylacetylglutamine (4.2±0.6 for controls versus 2.3±0.6 for patients with CKD; P<0.001), p-cresol sulfate (8.6±2.6 for controls versus 4.1±1.5 for patients with CKD; P<0.001), and indoxyl sulfate (23.0±7.3 versus 7.5±2.8; P<0.001) but not for hippurate (10.2±3.8 versus 8.4±2.6; P=0.13). CONCLUSIONS: Secretory clearances for many solutes are reduced more than the GFR in advanced CKD. Impaired secretion of these solutes might contribute to uremic symptoms as patients approach dialysis.

Human and preclinical studies of the host-gut microbiome co-metabolite hippurate as a marker and mediator of metabolic health.

OBJECTIVE: Gut microbial products are involved in regulation of host metabolism. In human and experimental studies, we explored the potential role of hippurate, a hepatic phase 2 conjugation product of microbial benzoate, as a marker and mediator of metabolic health. DESIGN: In 271 middle-aged non-diabetic Danish individuals, who were stratified on habitual dietary intake, we applied 1H-nuclear magnetic resonance (NMR) spectroscopy of urine samples and shotgun-sequencing-based metagenomics of the gut microbiome to explore links between the urine level of hippurate, measures of the gut microbiome, dietary fat and markers of metabolic health. In mechanistic experiments with chronic subcutaneous infusion of hippurate to high-fat-diet-fed obese mice, we tested for causality between hippurate and metabolic phenotypes. RESULTS: In the human study, we showed that urine hippurate positively associates with microbial gene richness and functional modules for microbial benzoate biosynthetic pathways, one of which is less prevalent in the Bacteroides 2 enterotype compared with Ruminococcaceae or Prevotella enterotypes. Through dietary stratification, we identify a subset of study participants consuming a diet rich in saturated fat in which urine hippurate concentration, independently of gene richness, accounts for links with metabolic health. In the high-fat-fed mice experiments, we demonstrate causality through chronic infusion of hippurate (20 nmol/day) resulting in improved glucose tolerance and enhanced insulin secretion. CONCLUSION: Our human and experimental studies show that a high urine hippurate concentration is a general marker of metabolic health, and in the context of obesity induced by high-fat diets, hippurate contributes to metabolic improvements, highlighting its potential as a mediator of metabolic health.

Antifungal compound, methyl hippurate from Bacillus velezensis CE 100 and its inhibitory effect on growth of Botrytis cinerea.

Botrytis cinerea, the causal agent of gray mold is one of the major devastating fungal pathogens that occurs in strawberry cultivation and leads to massive losses. Due to the rapid emergence of resistant strains in recent years, an ecofriendly disease management strategy needs to be developed to control this aggressive pathogen. Bacillus velezensis CE 100 exhibited strong antagonistic activity with 53.05% against B. cinerea by dual culture method. In the present study, 50% of culture filtrate supplemented into PDA medium absolutely inhibited mycelial growth of B. cinerea whereas the highest concentration (960 mg/L) of different crude extracts including ethyl acetate, chloroform, and n-butanol crude extracts of B. velezensis CE 100, strongly inhibited mycelial growth of B. cinerea with the highest inhibition of 79.26%, 70.21% and 69.59% respectively, resulting in severe damage to hyphal structures with bulging and swellings. Hence, the antifungal compound responsible was progressively separated from ethyl acetate crude extract using medium pressure liquid chromatography. The purified compound was identified as methyl hippurate by nuclear magnetic resonance and mass spectrometry. The inhibitory effect of methyl hippurate on both spore germination and mycelial growth of B. cinerea was revealed by its dose-dependent pattern. The spore germination rate was completely restricted at a concentration of 3 mg/mL of methyl hippurate whereas no mycelial growth was observed in agar medium supplemented with 4 mg/mL and 6 mg/mL of methyl hippurate by poisoned food method. Microscopic imaging revealed that the morphologies of spores were severely altered by long-time exposure to methyl hippurate at concentrations of 1 mg/mL, 2 mg/mL and 3 mg/mL and hyphae of B. cinerea were severely deformed by exposure to methyl hippurate at concentrations of 2 mg/mL, 4 mg/mL and 6 mg/mL. No significant inhibition on tomato seed germination was observed in treatments with methyl hippurate (2 mg/mL) for both 6 h and 12 h soaking period as compared to the controls. Based on these results, B. velezensis CE 100 could be considered a potential agent for development of environmentally friendly disease control strategies as a consequence of the synergetic interactions of diverse crude metabolites and methyl hippurate.

Molecular Typing of Campylobacter jejuni and Campylobacter coli of Human Strains Isolated in Turkey Over an Eight-Year Period.

BACKGROUND: Campylobacter spp. is one of the leading causes of bacterial foodborne infections worldwide. In this study, we aimed to investigate the genetic diversity of 341 Campylobacter strains isolated in Turkey. METHODS: Campylobacter spp. was identified by phenotypical methods and PCR. Species level identification was carried out by the hippurate hydrolysis test and PCR. C. jejuni and C. coli strains were typed by using flaA-RFLP and PFGE. RESULTS: Of 341 strains, 300 (88%), 37 (10.8%), and four were identified as C. jejuni, C. coli, and non-jejuni/non-coli, respectively. The hippurate hydrolysis test misidentified 12% of 341 strains. The typeabilities of flaA-RFLP and PFGE were 100% for C. coli, whereas those of flaA-RFLP and PFGE for C. jejuni were 99.3% and 99%, respectively. The discriminatory power of the combination of PFGE and flaA-RFLP was determined to be higher than either method alone for both C. jejuni and C. coli. Both of the strains were so diverse that 80% and 64% of C. jejuni and C. coli genotypes included only one strain, respectively. In two patients, Campylobacter strains that were isolated from the first stool samples were C. jejuni where as those isolated from the second samples, collected eight and 20 days after the collection of the first samples, were C. coli. C. jejuni strains that were recovered from two different stool samples of two patients, collected 1 - 2 days apart, were found to be genetically different. CONCLUSIONS: Species identification of Campylobacter strains should be done using molecular methods. Combination of two methods is prerequisite for increasing the accuracy of molecular typing. Mixed or subsequent infection by different Campylobacter species and C. jejuni of different genotypes should not be underestimated.

Renal energy excretion of horses depends on renal hippuric acid and nitrogen excretion.

The prediction of renal energy excretion is crucial in a metabolizable energy system for horses. Phenolic acids from forage cell walls may affect renal energy losses by increasing hippuric acid excretion. Therefore, the relationships were investigated between renal energy, nitrogen (N) and hippuric acid excretion of four adult ponies (230-384 kg body weight (BW)) consuming diets based on fresh grass, grass silage, grass cobs (heat-dried, finely chopped, pressed grass), alfalfa hay, straw, extruded straw and soybean meal. Feed intake was measured; urine and faeces were quantitatively collected for three days. Feed was analysed for crude nutrients, gross energy, amino acids and neutral-detergent-insoluble crude protein (CP); faeces were analysed for crude nutrients and cross energy; urine was analysed for N, hippuric acid, creatinine and gross energy. Renal energy excretion (y; kJ/kg BW0.75 ) correlated with renal N excretion (x1 ; g/kg BW0.75 ) and renal hippuric acid excretion (x2 ; g/kg BW0.75 ): y = 14.4 + 30.2x1 +20.7x2 (r = .95; n = 30; p < .05). Renal hippuric acid excretion was highest after intake of fresh grass and lowest after intake of soybean meal. The ratio of hippuric acid to creatinine in urine and the excretion of hippuric acid per gram of dry matter intake was significantly higher for fresh grass than for all other rations. There was no relationship between aromatic amino acid intake and renal hippuric acid excretion. The results of the present study and literature data suggest that feed can be categorized into four groups with regard to the energy losses per gram CP intake: (i) protein supplements (e.g., soybean meal): 4.2-4.9 kJ/g CP intake (ii) alfalfa hay, grains, dried sugar beet pulp: 6.4 kJ/g CP intake, (iii) hay, preserved grass products, straw: 5.2-12.3 kJ/g CP intake (mean 8) and (iv) fresh grass. For group (iii) a negative relationship was observed between renal energy losses per gram of CP and the content of CP or neutral-detergent-insoluble CP in dry matter.

Increased Plasma Exposures of Conjugated Metabolites of Morinidazole in Renal Failure Patients: A Critical Role of Uremic Toxins.

Morinidazole is a 5-nitroimidazole drug. Its sulfate conjugate M7 was a sensitive substrate of organic anion transporter 1 (OAT1) and OAT3, whereas N+-glucuronides M8-1 and M8-2 were only OAT3 substrates. In chronic renal failure (CRF) patients, plasma exposures of the three conjugates increased by 15-fold, which were also found in 5/6 nephrectomized (5/6 Nx) rats in this study. Although the transcriptions of Oat1 and Oat3 in 5/6 Nx rat kidneys decreased by 50%, no difference was observed on the three conjugate uptakes between control and 5/6 Nx rat kidney slices. Thus, the highly elevated endogenous uremic toxins in 5/6 Nx rats and humans, namely, 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), hippuric acid (HA), and indoxyl sulfate (IS), were considered as influential factors. In rat kidney slices, the uptake of M7, M8-1, and M8-2 was dose dependently reduced by HA and IS, whose plasma concentrations were elevated 5 times in 5/6 Nx rats. In OAT3-overexpressed cells, the three conjugate uptakes were inhibited by CMPF, HA, and IS with IC50 values of 19.2, 87.4, and 222 µM (M7); 8.53, 39.4, and 161 µM (M8-1); and 6.75, 24.1, and 78.3 µM (M8-2), respectively. In OAT1-overexpressed cells, CMPF, HA, and IS showed weak inhibition on M7 uptake with IC50 values of 187, 162, and 200 µM, correspondingly. Results suggest that the reduced mRNA expression of renal transporters in CRF patients may not influence the activities of these transporters. However, accumulated uremic toxins may inhibit the transporters, particularly OAT3, leading to plasma exposure changes of relevant substrates.

New insights into the catalytic mechanism of human glycine N-acyltransferase.

Even though the glycine conjugation pathway was one of the first metabolic pathways to be discovered, this pathway remains very poorly characterized. The bi-substrate kinetic parameters of a recombinant human glycine N-acyltransferase (GLYAT, E.C. 2.3.1.13) were determined using the traditional colorimetric method and a newly developed HPLC-ESI-MS/MS method. Previous studies analyzing the kinetic parameters of GLYAT, indicated a random Bi-Bi and/or ping-pong mechanism. In this study, the hippuric acid concentrations produced by the GLYAT enzyme reaction were analyzed using the allosteric sigmoidal enzyme kinetic module. Analyses of the initial rate (v) against substrate concentration plots, produced a sigmoidal curve (substrate activation) when the benzoyl-CoA concentrations was kept constant, whereas the plot with glycine concentrations kept constant, passed through a maximum (substrate inhibition). Thus, human GLYAT exhibits mechanistic kinetic cooperativity as described by the Ferdinand enzyme mechanism rather than the previously assumed Michaelis-Menten reaction mechanism.

The Use of p-Aminobenzoic Acid as a Probe Substance for the Targeted Profiling of Glycine Conjugation.

Glycine conjugation facilitates the metabolism of toxic aromatic acids, capable of disrupting mitochondrial integrity. Owing to the high exposure to toxic substrates, characterization of individual glycine conjugation capacity, and its regulatory factors has become increasingly important. Aspirin and benzoate have been employed for this purpose; however, adverse reactions, aspirin intolerance, and Reye's syndrome in children are substantial drawbacks. The goal of this study was to investigate p-aminobenzoic acid (PABA) as an alternative glycine conjugation probe. Ten human volunteers participated in a PABA challenge test, and p-aminohippuric acid (PAHA), p-acetamidobenzoic acid, and p-acetamidohippuric acid were quantified in urine. The glycine N-acyltransferase gene of the volunteers was also screened for two polymorphisms associated with normal and increased enzyme activity. All of the individuals were homozygous for increased enzyme activity, but excretion of PAHA varied significantly (16-56%, hippurate ratio). The intricacies of PABA metabolism revealed possible limiting factors and the potential of PABA as an indicator of Phase 0 biotransformation.

Metabolism and elimination of methyl, iso- and n-butyl paraben in human urine after single oral dosage.

Parabens are used as preservatives in personal care and consumer products, food and pharmaceuticals. Their use is controversial because of possible endocrine disrupting properties. In this study, we investigated metabolism and urinary excretion of methyl paraben (MeP), iso-butyl paraben (iso-BuP) and n-butyl paraben (n-BuP) after oral dosage of deuterium-labeled analogs (10 mg). Each volunteer received one dosage per investigated paraben separately and at least 2 weeks apart. Consecutive urine samples were collected over 48 h. In addition to the parent parabens (free and conjugated) which are already used as biomarkers of internal exposure and the known but non-specific metabolites, p-hydroxybenzoic acid (PHBA) and p-hydroxyhippuric acid (PHHA), we identified new, oxidized metabolites with hydroxy groups on the alkyl side chain (3OH-n-BuP and 2OH-iso-BuP) and species with oxidative modifications on the aromatic ring. MeP represented 17.4 % of the dose excreted in urine, while iso-BuP represented only 6.8 % and n-BuP 5.6 %. Additionally, for iso-BuP, about 16 % was excreted as 2OH-iso-BuP and for n-BuP about 6 % as 3OH-n-BuP. Less than 1 % was excreted as ring-hydroxylated metabolites. In all cases, PHHA was identified as the major but non-specific metabolite (57.2-63.8 %). PHBA represented 3.0-7.2 %. For all parabens, the majority of the oral dose captured by the above metabolites was excreted in the first 24 h (80.5-85.3 %). Complementary to the parent parabens excreted in urine, alkyl-chain-oxidized metabolites of the butyl parabens are introduced as valuable and contamination-free biomarkers of exposure.

Wide expression of type I Na+-phosphate cotransporter 3 (NPT3/SLC17A2), a membrane potential-driven organic anion transporter.

Membrane potential (Δψ)-driven and Cl(-)-dependent organic anion transport is a primary function of the solute carrier family 17 (SLC17) transporter family. Although the transport substrates and physiological relevance of the major members are well understood, SLC17A2 protein known to be Na(+)-phosphate cotransporter 3 (NPT3) is far less well characterized. In the present study, we investigated the transport properties and expression patterns of mouse SLC17A2 protein (mNPT3). Proteoliposomes containing the purified mNPT3 protein took up radiolabeled p-aminohippuric acid (PAH) in a Δψ- and Cl(-)-dependent manner. The mNPT3-mediated PAH uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDs) and Evans blue, common inhibitors of SLC17 family members. The PAH uptake was also inhibited by various anionic compounds, such as hydrophilic nonsteroidal anti-inflammatory drugs (NSAIDs) and urate. Consistent with these observations, the proteoliposome took up radiolabeled urate in a Δψ- and Cl(-)-dependent manner. Immunohistochemistry with specific antibodies against mNPT3 combined with RT-PCR revealed that mNPT3 is present in various tissues, including the hepatic bile duct, luminal membranes of the renal urinary tubules, maternal side of syncytiotrophoblast in the placenta, apical membrane of follicle cells in the thyroid, bronchiole epithelial cells in the lungs, and astrocytes around blood vessels in the cerebrum. These results suggested that mNPT3 is a polyspecific organic anion transporter that is involved in circulation of urate throughout the body.

Gastrointestinal Symptoms and Altered Intestinal Permeability Induced by Combat Training Are Associated with Distinct Metabotypic Changes.

Physical and psychological stress have been shown to modulate multiple aspects of gastrointestinal (GI) physiology, but its molecular basis remains elusive. We therefore characterized the stress-induced metabolic phenotype (metabotype) in soldiers during high-intensity combat training and correlated the metabotype with changes in GI symptoms and permeability. In a prospective, longitudinal study, urinary metabotyping was conducted on 38 male healthy soldiers during combat training and a rest period using gas chromatography-mass spectrometry. The urinary metabotype during combat training was clearly distinct from the rest period (partial least-squares discriminant analysis (PLSDA) Q(2) = 0.581), confirming the presence of a unique stress-induced metabotype. Differential metabolites related to combat stress were further uncovered, including elevated pyroglutamate and fructose, and reduced gut microbial metabolites, namely, hippurate and m-hydroxyphenylacetate (p < 0.05). The extent of pyroglutamate upregulation exhibited a positive correlation with an increase in IBS-SSS in soldiers during combat training (r = 0.5, p < 0.05). Additionally, the rise in fructose levels was positively correlated with an increase in intestinal permeability (r = 0.6, p < 0.005). In summary, protracted and mixed psychological and physical combat-training stress yielded unique metabolic changes that corresponded with the incidence and severity of GI symptoms and alteration in intestinal permeability. Our study provided novel molecular insights into stress-induced GI perturbations, which could be exploited for future biomarker research or development of therapeutic strategies.

Alterations of urinary metabolite profile in model diabetic nephropathy.

Countering the diabetes pandemic and consequent complications, such as nephropathy, will require better understanding of disease mechanisms and development of new diagnostic methods. Animal models can be versatile tools in studies of diabetic renal disease when model pathology is relevant to human diabetic nephropathy (DN). Diabetic models using endothelial nitric oxide synthase (eNOS) knock-out mice develop major renal lesions characteristic of human disease. However, it is unknown whether they can also reproduce changes in urinary metabolites found in human DN. We employed Type 1 and Type 2 diabetic mouse models of DN, i.e. STZ-eNOS(-/-) C57BLKS and eNOS(-/-) C57BLKS db/db, with the goal of determining changes in urinary metabolite profile using proton nuclear magnetic resonance (NMR). Six urinary metabolites with significantly lower levels in diabetic compared to control mice have been identified. Specifically, major changes were found in metabolites from tricarboxylic acid (TCA) cycle and aromatic amino acid catabolism including 3-indoxyl sulfate, cis-aconitate, 2-oxoisocaproate, N-phenyl-acetylglycine, 4-hydroxyphenyl acetate, and hippurate. Levels of 4-hydroxyphenyl acetic acid and hippuric acid showed the strongest reverse correlation to albumin-to-creatinine ratio (ACR), which is an indicator of renal damage. Importantly, similar changes in urinary hydroxyphenyl acetate and hippurate were previously reported in human renal disease. We demonstrated that STZ-eNOS(-/-) C57BLKS and eNOS(-/-) C57BLKS db/db mouse models can recapitulate changes in urinary metabolome found in human DN and therefore can be useful new tools in metabolomic studies relevant to human pathology.

High gastrointestinal permeability and local metabolism of naringenin: influence of antibiotic treatment on absorption and metabolism.

The present study aims to determine the permeability of naringenin in the stomach, small intestine and colon, to evaluate intestinal and hepatic first-pass metabolism, and to study the influence of the microbiota on the absorption and disposition of naringenin (3.5 µg/ml). A single-pass intestinal perfusion model in mice (n 4-6) was used. Perfusate (every 10 min), blood (at 60 min) and bile samples were taken and analysed to evaluate the presence of naringenin and its metabolites by an HPLC-MS/MS method. To study the influence of the microbiota on the bioavailability of naringenin, a group of animals received the antibiotic rifaximin (50 mg/kg per d) for 5 d, and naringenin permeability was determined in the colon. Naringenin was absorbed well throughout the gastrointestinal tract but mainly in the small intestine and colon (mean permeability coefficient 7.80 (SD 1.54) × 10(-4) cm/s and 5.49 (SD 1.86) × 10(-4) cm/s, respectively), at a level similar to the highly permeable compound, naproxen (6.39 (SD 1.23) × 10(-4) cm/s). According to the high amounts of metabolites found in the perfusate compared to the bile and plasma, naringenin underwent extensive intestinal first-pass metabolism, and the main metabolites excreted were sulfates (84.00 (SD 12.14)%), followed by glucuronides (8.40 (SD 5.67)%). Phase II metabolites were found in all perfusates from 5 min of sampling. Mice treated with rifaximin showed a decrease in naringenin permeability and in the amounts of 4-hydroxyhippuric acid and hippuric acid in the lumen. Naringenin was well absorbed throughout the gastrointestinal tract and its poor bioavailability was due mainly to high intestinal metabolism.

Effects of uremic solutes on reactive oxygen species in vitro model systems as a possibility of support the renal function management.

BACKGROUND: In view of the prevalence of oxidative stress in chronic kidney disease (CKD) patients, the loss of low-molecular-weight biomolecules by hemodialysis and the antioxidant potential of some uremic solutes that accumulate in CKD, we used in vitro model systems to test the antioxidant potential of the following uremic solutes: uric acid, hippuric acid, p-cresol, phenol, methylguanidine, L-arginine, L-tyrosine, creatinine and urea. METHODS: The in vitro antioxidant efficiencies of the uremic solutes, isolated or in mixtures, were tested with the following assays: i) ABTS radical cation decolorization assay; ii) hypochlorous acid (HOCl/OCl(-)) scavenging activity; iii) superoxide anion radical (O2(•-)) scavenging activity; iv) crocin bleaching assay (capture of peroxyl radical, ROO(•)); v) hydrogen peroxide (H2O2) scavenging activity. RESULTS: Four of the tested uremic solutes (p-cresol, phenol, L-tyrosine, uric acid) were effective antioxidants and their IC50 were found in three model systems: ABTS(•+), HOCl/OCl(-) and crocin bleaching assay. In the 4-solutes mixtures, each one of the solute captured 12.5% for the IC50 of the mixture to ABTS(•+) or HOCl/OCl(-), exhibiting a virtually exact additive effect. In the 2-solutes mixtures, for ROO(•) capture, it was observed the need of more mass of uremic solutes to reach an IC50 value that was higher than the projected IC50, obtained from the IC50 of single solutes (25% of each, in the binary mixtures) in the same assay. In model systems for O2(•-) and H2O2, none of the uremic solutes showed scavenging activity. CONCLUSIONS: The use of the IC50 as an analytical tool to prepare and analyze mixtures allows the determination of their scavenging capacities and may be useful for the assessment of the antioxidant status of biological samples under conditions of altered levels of the endogenous antioxidant network and/or in the employment and monitoring of exogenous antioxidant therapy.

Fecal metabolomics: assay performance and association with colorectal cancer.

Metabolomic analysis of feces may provide insights on colorectal cancer (CRC) if assay performance is satisfactory. In lyophilized feces from 48 CRC cases, 102 matched controls, and 48 masked quality control specimens, 1043 small molecules were detected with a commercial platform. Assay reproducibility was good for 527 metabolites [technical intraclass correlation coefficient (ICC) >0.7 in quality control specimens], but reproducibility in 6-month paired specimens was lower for the majority of metabolites (within-subject ICC ≤0.5). In the CRC cases and controls, significant differences (false discovery rate ≤0.10) were found for 41 of 1043 fecal metabolites. Direct cancer association was found with three fecal heme-related molecules [covariate-adjusted 90th versus 10th percentile odds ratio (OR) = 17-345], 18 peptides/amino acids (OR = 3-14), palmitoyl-sphingomyelin (OR = 14), mandelate (OR = 3) and p-hydroxy-benzaldehyde (OR = 4). Conversely, cancer association was inverse with acetaminophen metabolites (OR <0.1), tocopherols (OR = 0.3), sitostanol (OR = 0.2), 3-dehydrocarnitine (OR = 0.4), pterin (OR = 0.3), conjugated-linoleate-18-2N7 (OR = 0.2), N-2-furoyl-glycine (OR = 0.3) and p-aminobenzoate (PABA, OR = 0.2). Correlations suggested an independent role for palmitoyl-sphingomyelin and a central role for PABA (which was stable over 6 months, within-subject ICC 0.67) modulated by p-hydroxy-benzaldehyde. Power calculations based on ICCs indicate that only 45% of metabolites with a true relative risk 5.0 would be found in prospectively collected, prediagnostic specimens from 500 cases and 500 controls. Thus, because fecal metabolites vary over time, very large studies will be needed to reliably detect associations of many metabolites that potentially contribute to CRC.

The impact of obesity-associated glycine deficiency on the elimination of endogenous and exogenous metabolites via the glycine conjugation pathway.

Background: Glycine is an integral component of the human detoxification system as it reacts with potentially toxic exogenous and endogenously produced compounds and metabolites via the glycine conjugation pathway for urinary excretion. Because individuals with obesity have reduced glycine availability, this detoxification pathway may be compromised. However, it should be restored after bariatric surgery because of increased glycine production. Objective: To examine the impact of obesity-associated glycine deficiency on the glycine conjugation pathway. We hypothesize that the synthesis rates of acylglycines from endogenous and exogenous sources are significantly reduced in individuals with obesity but increase after bariatric surgery. Methods: We recruited 21 participants with class III obesity and 21 with healthy weight as controls. At baseline, [1,2-13C2] glycine was infused to study the glycine conjugation pathway by quantifying the synthesis rates of several acylglycines. The same measurements were repeated in participants with obesity six months after bariatric surgery. Data are presented as mean ± standard deviation, and p-value< 0.05 is considered statistically significant. Results: Baseline data of 20 participants with obesity were first compared to controls. Participants with obesity were significantly heavier than controls (mean BMI 40.5 ± 7.1 vs. 20.8 ± 2.1 kg/m2). They had significantly lower plasma glycine concentration (168 ± 30 vs. 209 ± 50 µmol/L) and slower absolute synthesis rates of acetylglycine, isobutyrylglycine, tigylglycine, isovalerylglycine, and hexanoylglycine. Pre- and post-surgery data were available for 16 participants with obesity. Post-surgery BMI decreased from 40.9 ± 7.3 to 31.6 ± 6.0 kg/m2. Plasma glycine concentration increased from 164 ± 26 to 212 ± 38 µmol/L) and was associated with significantly higher rates of excretion of acetylglycine, isobutyrylglycine, tigylglycine, isovalerylglycine, and hexanoylglycine. Benzoic acid (a xenobiotic dicarboxylic acid) is excreted as benzoylglycine; its synthesis rate was significantly slower in participants with obesity but increased after bariatric surgery. Conclusion: Obesity-associated glycine deficiency impairs the human body's ability to eliminate endogenous and exogenous metabolites/compounds via the glycine conjugation pathway. This impairment is ameliorated when glycine supply is restored after bariatric surgery. These findings imply that dietary glycine supplementation could treat obesity-associated metabolic complications due to the accumulation of intramitochondrial toxic metabolites. Clinical trial registration: https://clinicaltrials.gov/study/NCT04660513, identifier NCT04660513.