Hippuric acid alleviates dextran sulfate sodium-induced colitis via suppressing inflammatory activity and modulating gut microbiota.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease associated with metabolic disorder and gut dysbiosis. Decreased abundance of hippuric acid (HA) was found in patients with IBD. HA, metabolized directly from benzoic acid in the intestine and indirectly from polyphenols, serves as a marker of polyphenol catabolism. While polyphenols and benzoic acid have been shown to alleviate intestinal inflammation, the role of HA in this context remains unknown. Herein, we investigated the effects and mechanism of HA on DSS-induced colitis mice. The results revealed that HA alleviated clinical activity and intestinal barrier damage, decreased pro-inflammatory cytokine production. Metagenomic sequencing suggested that HA treatment restored the gut microbiota, including an increase in beneficial gut bacteria such as Adlercreutzia, Eubacterium, Schaedlerella and Bifidobacterium_pseudolongum. Furthermore, we identified 113 candidate genes associated with IBD that are potentially under HA regulation through network pharmacological analyses. 10 hub genes including ALB, IL-6, HSP90AA1, and others were identified using PPI analysis and validated using molecular docking and mRNA expression analysis. Additionally, KEGG analysis suggested that the renin-angiotensin system (RAS), NF-κB signaling and Rap1 signaling pathways were important pathways in the response of HA to colitis. Thus, HA may provide novel biotherapy options for IBD.

Effect of methenamine hippurate shortage on antibiotic prescribing for urinary tract infections in Norway-an interrupted time series analysis.

BACKGROUND: Despite a lack of conclusive evidence of effect, methenamine hippurate is widely prescribed as preventive treatment for recurrent urinary tract infections (UTIs) in Norway. A national discontinuation of methenamine hippurate treatment due to a 4-month drug shortage in 2019 presented an opportunity to evaluate its preventive effect on UTIs among regular users. OBJECTIVE: To estimate the impact of the methenamine hippurate drug shortage on prescription frequency of UTI antibiotics. METHODS: Data from The Norwegian Prescription Database was analysed using an interrupted time series design. The time series consisted of 56 time periods of 14 days. The model included two naturally occurring interruptions: (i) the methenamine hippurate drug shortage, and (ii) reintroduction of the drug. The study population were 18 345 women ≥50 years receiving ≥2 prescriptions of methenamine hippurate in the study period before the shortage. Main outcome measure was number of prescriptions of UTI antibiotics per 1000 methenamine hippurate users. Prescription rates of antibiotics for respiratory tract infections were analysed to assess external events affecting antibiotic prescribing patterns. RESULTS: We found a significant increase of 2.41 prescriptions per 1000 methenamine hippurate users per 14-day period during the drug shortage (95%CI 1.39, 3.43, P < 0.001), followed by a significant reduction of -2.64 prescriptions after reintroduction (95%CI -3.66, -1.63, P < 0.001). CONCLUSIONS: During the methenamine hippurate drug shortage, we found a significant increase in prescribing trend for UTI antibiotics followed by a significant decrease in prescribing trend after reintroduction. This change in trend seems to reflect a preventive effect of the drug on recurrent UTIs.

Gamma-delta T cells suppress microbial metabolites that activate striatal neurons and induce repetitive/compulsive behavior in mice.

Intestinal γδ T cells play an important role in shaping the gut microbiota, which is critical not only for maintaining intestinal homeostasis but also for controlling brain function and behavior. Here, we found that mice deficient for γδ T cells (γδ-/-) developed an abnormal pattern of repetitive/compulsive (R/C) behavior, which was dependent on the gut microbiota. Colonization of WT mice with γδ-/- microbiota induced R/C behavior whereas colonization of γδ-/- mice with WT microbiota abolished the R/C behavior. Moreover, γδ-/- mice had elevated levels of the microbial metabolite 3-phenylpropanoic acid in their cecum, which is a precursor to hippurate (HIP), a metabolite we found to be elevated in the CSF. HIP reaches the striatum and activates dopamine type 1 (D1R)-expressing neurons, leading to R/C behavior. Altogether, these data suggest that intestinal γδ T cells shape the gut microbiota and their metabolites and prevent dysfunctions of the striatum associated with behavior modulation.

Circulating metabolites modulated by diet are associated with depression.

Metabolome reflects the interplay of genome and exposome at molecular level and thus can provide deep insights into the pathogenesis of a complex disease like major depression. To identify metabolites associated with depression we performed a metabolome-wide association analysis in 13,596 participants from five European population-based cohorts characterized for depression, and circulating metabolites using ultra high-performance liquid chromatography/tandem accurate mass spectrometry (UHPLC/MS/MS) based Metabolon platform. We tested 806 metabolites covering a wide range of biochemical processes including those involved in lipid, amino-acid, energy, carbohydrate, xenobiotic and vitamin metabolism for their association with depression. In a conservative model adjusting for life style factors and cardiovascular and antidepressant medication use we identified 8 metabolites, including 6 novel, significantly associated with depression. In individuals with depression, increased levels of retinol (vitamin A), 1-palmitoyl-2-palmitoleoyl-GPC (16:0/16:1) (lecithin) and mannitol/sorbitol and lower levels of hippurate, 4-hydroxycoumarin, 2-aminooctanoate (alpha-aminocaprylic acid), 10-undecenoate (11:1n1) (undecylenic acid), 1-linoleoyl-GPA (18:2) (lysophosphatidic acid; LPA 18:2) are observed. These metabolites are either directly food derived or are products of host and gut microbial metabolism of food-derived products. Our Mendelian randomization analysis suggests that low hippurate levels may be in the causal pathway leading towards depression. Our findings highlight putative actionable targets for depression prevention that are easily modifiable through diet interventions.

Associations of personal urinary volatile organic compounds and lung function in children.

BACKGROUND: We investigated the correlation between urine VOC metabolites and airway function in children exposed to anthropogenic volatile organic compounds (VOCs), notable pollutants impacting respiratory health. METHODS: Out of 157 respondents, 141 completed skin prick tests, spirometry, IOS, and provided urine samples following the International Study of Asthma and Allergies in Childhood (ISAAC)-related questions. Allergic sensitization was assessed through skin prick tests, and airway functions were evaluated using spirometry and impulse oscillometry (IOS). Forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC) was recorded and FEV1/FVC ratio was calculated. Airway mechanics parameters including respiratory resistance at 5 Hz (Rrs5) mean respiratory resistance between 5 Hz and 20 Hz (Rrs5-20), were also recorded. Urine concentrations of metabolites of benzene, ethylbenzene, toluene, xylene, styrene, formaldehyde, carbon-disulfide were analyzed by gas chromatography/tandem mass spectroscopy. RESULTS: The median age at study participation was 7.1 (SD 0.3) years. Muconic acid (benzene metabolites) and o-methyl-hippuric acid (xylene metabolites) above medians were associated with a significant increase in Rrs5 (muconic acid: aß = 0.150, p = .002; o-methyl-hippuric acid: aß = 0.143, p = .023) and a decrease in FEV1/FVC (o-methyl-hippuric acid: aß = 0.054, p = .028) compared to those below median. No associations were observed for Rrs5-20 and FEV1 between the groups categorized as above and below the median (all parameter p values > .05). CONCLUSIONS: Elevated levels of benzene and xylene metabolites were associated with a significant increase in Rrs5 and a decrease in FEV1/FVC, related to increased resistance and restrictive lung conditions compared to individuals with concentrations below the median.

In vitro Removal of Protein-Bound Retention Solutes by Extracorporeal Blood Purification Procedures.

INTRODUCTION: When the kidneys or liver fail, toxic metabolites accumulate in the patient's blood, causing cardiovascular and neurotoxic complications and increased mortality. Conventional membrane-based extracorporeal blood purification procedures cannot remove these toxins efficiently. The aim of this in vitro study was to determine whether commercial hemoperfusion adsorbers are suitable for removing protein-bound retention solutes from human plasma and whole blood as well as to compare the removal to conventional hemodialysis. METHODS: For in vitro testing of the removal of protein-bound substances, whole blood and plasma were spiked with uremic retention solutes (homocysteine, hippuric acid, indoxyl sulfate, 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid) and the toxins of liver failure (bilirubin, cholic acid, tryptophan, phenol). Subsequently, the protein binding of each retention solute was determined. The adsorption characteristics of the hemoperfusion adsorbers, Jafron HA and Biosky MG, both approved for the adsorption of protein-bound uremic retention solutes and Cytosorb, an adsorber recommended for adsorption of cytokines, were tested by incubating them in spiked whole blood or plasma for 1 h. Subsequently, the adsorption characteristics of the adsorbers were tested in a dynamic system. For this purpose, a 6-h in vitro hemoperfusion treatment was compared with an equally long in vitro hemodialysis treatment. RESULTS: Hippuric acid, homocysteine, indoxyl sulfate, and tryptophan were most effectively removed by hemodialysis. Bilirubin and cholic acid were removed best by hemoperfusion with Cytosorb. A treatment with Jafron HA and Biosky MG showed similar results for the adsorption of the tested retention solutes and were best for removing phenol. 3-Carboxy-4-methyl-5-propyl-2-furanpropionic acid could not be removed with any treatment method. DISCUSSION/CONCLUSION: A combination of hemodialysis with hemoperfusion seems promising to improve the removal of some toxic metabolites in extracorporeal therapies. However, some very strongly protein-bound metabolites cannot be removed adequately with the adsorbers tested.

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.

A fast and accurate colorimetric assay for quantifying hippuric acid in human urine.

Hippuric acid is an abundant metabolite in human urine. Urinary hippuric acid levels change with toxic exposure to aromatic compounds, consumption of fruits and vegetables, cancers, chronic kidney disease, schizophrenia and Crohn's disease. While urinary hippuric acid can be detected and quantified via mass spectrometry or nuclear magnetic resonance spectroscopy, a colorimetric assay would be preferable for a low-cost, point-of care clinical assay. Two colorimetric methods, that use p-dimethylaminobenzaldehyde (DMAB) or benzenesulfonyl chloride (PhSO2Cl), respectively, have been previously developed to detect hippuric acid but these assays have many limitations. We replaced PhSO2Cl with p-toluenesulfonyl chloride (p-TsCl), to create a simpler, faster and more accurate method that works with human urine. This modified colorimetric assay detects from 60 µM to 1000 µM hippuric acid in urine in 2 min. We also corrected for the effects of interfering compounds present in urine such that the assay works across many urine backgrounds. We validated this improved assay on multiple hippurate-spiked urine samples, observing an excellent correlation (R2 > 0.94) between observed and known hippurate concentrations. These data suggest that this colorimetric assay is accurate and should greatly facilitate the measurement of hippuric acid in urine to detect a variety of human conditions.

The resurgence of methenamine hippurate in the prevention of recurrent UTIs in women- a systematic review.

PURPOSE OF REVIEW: Recurrent urinary tract infections (rUTIs) in women are prevalent and difficult to manage. The rise of antimicrobial resistance makes it prudent to re-investigate the role of nonantimicrobial agents in the prevention of RUTIs. We wanted to evaluate randomised controlled trials (RCTs) that employed methenamine hippurate as a therapy or prophylactic in adult women with rUTIs. RECENT FINDINGS: Relevant databases were searched for RCTs using Cochrane methodology and reporting items for systematic reviews and meta-analyses (PRISMA) checklist, comparing the efficacy of methenamine hippurate to either an antibiotic or a placebo for the prophylaxis of rUTI in women.Six trials involving 322 patients taking methenamine and 419 patients receiving antibiotics in total were evaluated. The duration of the trials ranged from 12-24 months. Studies reported that methenamine was effective in extending the mean period between symptomatic episodes of urinary tract infections (UTIs), keeping the patient symptom- and infection-free, and reducing the number of UTI episodes. The newer studies reported that methenamine reduced the incidence rates of recurrent UTIs and was not inferior to the antibiotic in this regard. SUMMARY: The outcomes of methenamine hippurate were found to be at par with the antibiotic prophylaxis. It might serve as a suitable alternative nonantibiotic prophylaxis for females with rUTIs.

Eu(III)-Functionalized MOF-Based Dual-Emission Ratiometric Sensor Integrated with Logic Gate Operation for Efficient Detection of Hippuric Acid in Urine and Serum.

With the introduction of Eu3+ ions as the secondary fluorescent signal reporter and sensing active sites, a dual-emission ratiometric sensor of Eu3+@NiMOF (Eu3+ functional NiMOF) for hippuric acid (HA) detection in urine and serum was fabricated via the postsynthetic encapsulating strategy. Based on the two emission signals at 441 nm (turn-on) and 628 nm (turn-off), the produced Eu3+@NiMOF ratiometric sensor provided enhanced sensitivity, higher selectivity, and 9.7 times lower limits of detection (LOD) for the detection of HA (2.38 µM, 0.42 µg·mL-1) than that of the pristine NiMOF. Considering the high sensitivity and visualization results, further exploration of intelligent applications in the HA sensing process was carried out by constructing a tandem combinational logic gate to improve the practicability and convenience with the help of a smartphone. This work provides a promising approach for developing MOF-based ratiometric sensors to detect biomarkers.

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.

The management of recurrent urinary tract infections within a nurse-led urology team.

The aim of this article is to share experience and learning of managing recurrent urinary tract infections (UTIs) within a specialist urology nurse-led team based at a district general hospital. It looks at current practice and supporting evidence for how to manage and treat recurrent UTIs in both male and female patients. Two case studies are presented to illustrate the management strategies and outcomes, demonstrating a planned approach that informs the design of a local management guideline to organise patients' care.

Comparative Analysis of Host Metabolic Alterations in Murine Malaria Models with Uncomplicated or Severe Malaria.

Malaria varies in severity, with complications ranging from uncomplicated to severe malaria. Severe malaria could be attributed to peripheral hyperparasitemia or cerebral malaria. The metabolic interactions between the host and Plasmodium species are yet to be understood during these infections of varied pathology and severity. An untargeted metabolomics approach utilizing the liquid chromatography-mass spectrometry platform has been used to identify the affected host metabolic pathways and associated metabolites in the serum of murine malaria models with uncomplicated malaria, hyperparasitemia, and experimental cerebral malaria. We report that mice with malaria share similar metabolic attributes like higher levels of bile acids, bile pigments, and steroid hormones that have been reported for human malaria infections. Moreover, in severe malaria, upregulated levels of metabolites like phenylalanine, histidine, valine, pipecolate, ornithine, and pantothenate, with decreased levels of arginine and hippurate, were observed. Metabolites of sphingolipid metabolism were upregulated in experimental cerebral malaria. Higher levels of 20-hydroxy-leukotriene B4 and epoxyoctadecamonoenoic acids were found in uncomplicated malaria, with lower levels observed for experimental cerebral malaria. Our study provides insights into host biology during different pathological stages of malaria disease and would be useful for the selection of animal models for evaluating diagnostic and therapeutic interventions against malaria. The raw data files are available via MetaboLights with the identifier MTBLS4387.

Legionella bononiensis sp. nov., isolated from a hotel water distribution system in northern Italy.

Legionella-like isolates, strains 27fs60, 30fs61 and 30cs62T, were isolated from a hotel water distribution system in the Emilia-Romagna region, Italy. Isolates were Gram- and Ziehl Neelsen-stain-negative, rod-shaped, with transitory flagella presence and able to grow at 32-37 °C (with an optimum at 32 °C) on buffered charcoal-yeast extract agar with l-cysteine, glycine-vancomycin-polymyxin B-cycloheximide agar and Wadowsky-Yee medium agar. The strains showed positive reactions for oxidase, hippurate and gelatinase and a weakly positive reaction for catalase. Based on the EUCAST cut-off, strain 30cs62T was resistant to ciprofloxacin (5 mg l-1). The mip and rpoB gene sequences of the three strains showed close matches to those of Legionella quateirensis ATCC 49507T with similarity values of 98.2 and 94.5 %, respectively. Whole genome sequencing of the three strains was performed, resulting in G+C contents of 39.0, 39.1 and 39.0 mol%, respectively. The identity percentage measured by average nucleotide identity between the three strains and their respective closest strains were: 91.32 % L. quateirensis NCTC 12376T, 91.45 % L. quateirensis ATCC 49507T and 91.45 % L. quateirensis ATCC 49507T, respectively. The digital DNA-DNA hybridization analysis demonstrated how the isolates were separated from the most related phylogenetic Legionella species (L. quateirensis ATCC 49507T, ≤40.10 % DNA-DNA relatedness). The concatenated phylogenetic tree based on 16S rRNA, mip, rpoB and rnpB genes, shows a close relationship with L. quateirensis ATCC 49507T. The results obtained confirm the status of an independent species. The name proposed for this species is Legionella bononiensis sp. nov. with 30cs62T (=ATCC TSD-262T=DSM 112526T) as the type strain.

Stable Cd Metal-Organic Framework as a Multiresponsive Luminescent Biosensor for Rapid, Accurate, and Recyclable Detection of Hippuric Acid, Nucleoside Phosphates, and Fe3+ in Urine and Serum.

Detecting biomarkers associated with diseases has significant meaning for early prevention, diagnosis, and treatment of diseases. The development of luminescent biosensors for rapid and accurate detection in real urine and serum is urgently desired for human health monitoring. Herein, a luminescent cadmium metal-organic framework, {[Cd(L)(bpbix)]·x(solv)}n (1), was successfully prepared by using a urea-functionalized dicarboxylate ligand, 5-(3-(pyridin-4-yl)ureido)isophthalic acid (H2L), 4,4'-bis((1H-imidazol-1-yl)methyl)biphenyl (bpbix), and the Cd2+ ion. The structure of 1 presents a 2-fold interpenetrating three-dimensional pillared-layer framework. The complex 1 exhibits good stability in different-pH aqueous solutions and physiological fluids. Strikingly, the complex 1 shows quick response, high sensitivity, good anti-interference performance, and a recyclable ability for simultaneous sensing of hippuric acid (HA), nucleoside phosphates, and Fe3+ in water. More significantly, this sensor can realize the sensitive and accurate detection of HA, nucleoside phosphates, and Fe3+ in real urine and serum and meet the practical detection needs in clinical diagnosis. These results indicate that the complex 1 as a multiresponsive luminescent biosensor possesses great potential for practical detection of HA, nucleoside phosphates, and Fe3+ in biological samples.

Methenamine hippurate compared with trimethoprim for the prevention of recurrent urinary tract infections: a randomized clinical trial.

INTRODUCTION AND HYPOTHESIS: The objective was to find an alternative treatment to a low-dose antibiotic for the prevention of recurrent urinary tract infections (UTI) and to evaluate the difference in rates of reinfection within 1 year when treated with methenamine hippurate for prophylaxis compared with trimethoprim. METHODS: We present a non-blinded randomized trial comparing methenamine hippurate with trimethoprim for the prevention of recurrent UTI at 12 months after starting treatment. Women over 18 who had at least two culture-positive UTI in the prior 6 months or three in the prior year were included. Ninety-two patients met enrollment criteria and were randomized to receive daily prophylaxis with methenamine hippurate or trimethoprim for a minimum of 6 months. Both intent-to-treat and per-protocol analyses if patients received the alternative drug after randomization were analyzed using Student's t test, Mann-Whitney U test, Kaplan-Meier curves, log-rank test, and a logistic and multivariate regression model. The primary outcome of this study was culture-proven UTI recurrence by 12 months after initiating prophylaxis. RESULTS: In the intent-to-treat analysis, we found no difference between groups in recurrent UTI, with a 65% (28 out of 43) recurrence in the trimethoprim group versus 65% (28 out of 43) in the methenamine hippurate group (p = 1.00). In the per-protocol analysis, 65% (26 out of 40) versus 65% (30 out of 46) of patients had UTI recurrences in the trimethoprim group versus the methenamine hippurate group (p = 0.98). CONCLUSIONS: Methenamine hippurate may be an alternative for the prevention of recurrent UTI, with similar rates of recurrence and adverse effects to trimethoprim.

Metabolomics of Dietary Acid Load and Incident Chronic Kidney Disease.

OBJECTIVE: Blood biomarkers of dietary intake are more objective than self-reported dietary intake. Metabolites associated with dietary acid load were previously identified in 2 chronic kidney disease (CKD) populations. We aimed to extend these findings to a general population, replicating their association with dietary acid load, and investigating whether the individual biomarkers were prospectively associated with incident CKD. METHODS: Among 15,792 participants in the Atherosclerosis Risk in Communities cohort followed up from 1987 to 1989 (baseline) to 2019, we evaluated 3,844 black and white men and women with dietary and metabolomic data in cross-sectional and prospective analyses. We hypothesized that a higher dietary acid load (using equations for potential renal acid load and net endogenous acid production) was associated with lower serum levels of 12 previously identified metabolites: indolepropionylglycine, indolepropionate, N-methylproline, N-δ-acetylornithine, threonate, oxalate, chiro-inositol, methyl glucopyranoside, stachydrine, catechol sulfate, hippurate, and tartronate. In addition, we hypothesized that lower serum levels of these 12 metabolites were associated with higher risk of incident CKD. RESULTS: Eleven out of 12 metabolites were significantly inversely associated with dietary acid load, after adjusting for demographics, socioeconomic status, health behaviors, health status, and estimated glomerular filtration rate: indolepropionylglycine, indolepropionate, N-methylproline, threonate, oxalate, chiro-inositol, catechol sulfate, hippurate, methyl glucopyranoside (α + ß), stachydrine, and tartronate. N-methylproline was inversely associated with incident CKD (hazard ratio: 0.95, 95% confidence interval: 0.91, 0.99, P = .01). The metabolomic biomarkers of dietary acid load significantly improved prediction of elevated dietary acid load estimated using dietary data, beyond covariates (difference in C statistics: 0.021-0.077, P ≤ 1.08 × 10-3). CONCLUSION: Inverse associations between candidate biomarkers of dietary acid load were replicated in a general population. N-methylproline, representative of citrus fruit consumption, is a promising marker of dietary acid load and could represent an important pathway between dietary acid load and CKD.

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.

Exploring GPR109A Receptor Interaction with Hippuric Acid Using MD Simulations and CD Spectroscopy.

Previous research has indicated that various metabolites belonging to phenolic acids (PAs), produced by gut microflora through the breakdown of polyphenols, help in promoting bone development and protecting bone from degeneration. Results have also suggested that G-protein-coupled receptor 109A (GPR109A) functions as a receptor for those specific PAs such as hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA). Indeed, HA has a molecular structural similarity with nicotinic acid (niacin) which has been shown previously to bind to GPR109A receptor and to mediate antilipolytic effects; however, the binding pocket and the structural nature of the interaction remain to be recognized. In the present study, we employed a computational strategy to elucidate the molecular structural determinants of HA binding to GPR109A and GPR109B homology models in understanding the regulation of osteoclastogenesis. Based on the docking and molecular dynamics simulation studies, HA binds to GPR109A similarly to niacin. Specifically, the transmembrane helices 3, 4 and 6 (TMH3, TMH4 and TMH6) and Extracellular loop 1 and 2 (ECL1 and ECL2) residues of GRP109A; R111 (TMH3), K166 (TMH4), ECL2 residues; S178 and S179, and R251 (TMH6), and residues of GPR109B; Y87, Y86, S91 (ECL1) and C177 (ECL2) contribute for HA binding. Simulations and Molecular Mechanics Poisson-Boltzmann solvent accessible area (MM-PBSA) calculations reveal that HA has higher affinity for GPR109A than for GPR109B. Additionally, in silico mutation analysis of key residues have disrupted the binding and HA exited out from the GPR109A protein. Furthermore, measurements of time-resolved circular dichroism spectra revealed that there are no major conformational changes in the protein secondary structure on HA binding. Taken together, our findings suggest a mechanism of interaction of HA with both GPR109A and GPR109B receptors.

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.