4-hydroxyhippuric Acid Research Articles

Multiomics Analyses Explore the Immunometabolic Interplay in the Liver of White Crucian Carp (Carassius cuvieri) After Aeromonas veronii Challenge.

Aeromonas veronii is one of the predominant pathogenic species that can imperil the survival of farmed fish. However, the interactive networks of immune regulation and metabolic response in A. veronii-infected fish are still unclear. In this investigation, we aimed to explore immunometabolic interplay in white crucian carp (WCC) after the A. veronii challenge. Elevated levels of immune-related genes were observed in various tissues after A. veronii infection, along with the sharp alteration of disease-related enzymatic activities. Besides, decreased levels of antioxidant status were observed in the liver, but most metabolic gene expressions increased dramatically. Multiomics analyses revealed that metabolic products of amino acids, such as formiminoglutamic acid (FIGLU), L-glutamate (L-Glu), and 4-hydroxyhippuric acid, were considered the crucial liver biomarkers in A. veronii-infected WCC. In addition, A. veronii infection may dysregulate endoplasmic reticulum (ER) function to affect the metabolic process of lipids, carbohydrates, and amino acids in the liver of WCC. These results may have a comprehensive implication for understanding immunometabolic response in WCC upon A. veronii infection.

Urinary markers of Mycobacterium tuberculosis and dysbiosis in paediatric tuberculous meningitis cases undergoing treatment

BackgroundThe pathogenesis of tuberculous meningitis (TBM) involves infection by Mycobacterium tuberculosis in the meninges and brain. However, recent studies have shown that the immune response and inflammatory processes triggered by TBM can have significant effects on gut microbiota. Disruptions in the gut microbiome have been linked to various systemic consequences, including altered immunity and metabolic dysregulation. Inflammation caused by TBM, antibiotic treatment, and changes in host immunity can all influence the composition of gut microbes. This complex relationship between TBM and the gut microbiome is of great importance in clinical settings. To gain a deeper understanding of the intricate interactions between TBM and the gut microbiome, we report innovative insights into the development of the disease in response to treatment. Ultimately, this could lead to improved outcomes, management strategies and quality of life for individuals affected by TBM.MethodWe used a targeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) approach to investigate metabolites associated with gut metabolism in paediatric participants by analysing the urine samples collected from a control group (n = 40), and an experimental group (n = 35) with confirmed TBM, which were subdivided into TBM stage 1 (n = 8), stage 2 (n = 11) and stage 3 (n = 16).FindingsOur metabolomics investigation showed that, of the 78 initially selected compounds of microbiome origin, eight unique urinary metabolites were identified: 2-methylbutyrlglycine, 3-hydroxypropionic acid, 3-methylcrotonylglycine, 4-hydroxyhippuric acid, 5-hydroxyindoleacetic acid, 5-hydroxyhexanoic acid, isobutyrylglycine, and phenylacetylglutamine as urinary markers of dysbiosis in TBM.ConclusionThese results – which are supported by previous urinary studies of tuberculosis – highlight the importance of gut metabolism and of identifying corresponding microbial metabolites as novel points for the foundation of improved management of TBM patients.

Plasma metabolomic differences in early-onset compared to average-onset colorectal cancer

Deleterious effects of environmental exposures may contribute to the rising incidence of early-onset colorectal cancer (eoCRC). We assessed the metabolomic differences between patients with eoCRC, average-onset CRC (aoCRC), and non-CRC controls, to understand pathogenic mechanisms. Patients with stage I–IV CRC and non-CRC controls were categorized based on age ≤ 50 years (eoCRC or young non-CRC controls) or ≥ 60 years (aoCRC or older non-CRC controls). Differential metabolite abundance and metabolic pathway analyses were performed on plasma samples. Multivariate Cox proportional hazards modeling was used for survival analyses. All P values were adjusted for multiple testing (false discovery rate, FDR P < 0.15 considered significant). The study population comprised 170 patients with CRC (66 eoCRC and 104 aoCRC) and 49 non-CRC controls (34 young and 15 older). Citrate was differentially abundant in aoCRC vs. eoCRC in adjusted analysis (Odds Ratio = 21.8, FDR P = 0.04). Metabolic pathways altered in patients with aoCRC versus eoCRC included arginine biosynthesis, FDR P = 0.02; glyoxylate and dicarboxylate metabolism, FDR P = 0.005; citrate cycle, FDR P = 0.04; alanine, aspartate, and glutamate metabolism, FDR P = 0.01; glycine, serine, and threonine metabolism, FDR P = 0.14; and amino-acid t-RNA biosynthesis, FDR P = 0.01. 4-hydroxyhippuric acid was significantly associated with overall survival in all patients with CRC (Hazards ratio, HR = 0.4, 95% CI 0.3–0.7, FDR P = 0.05). We identified several unique metabolic alterations, particularly the significant differential abundance of citrate in aoCRC versus eoCRC. Arginine biosynthesis was the most enriched by the differentially altered metabolites. The findings hold promise in developing strategies for early detection and novel therapies.

Identification of urine biomarkers associated with early puberty in children: An untargeted metabolomics analysis

A trend toward earlier pubertal maturation in both sexes has been shown in many countries. Early puberty affects an increasing proportion of children for reasons that remain obscure. Novel candidate biomarkers are strongly needed. We sought to apply untargeted metabolomic profiling to identify triggering mechanisms and candidate biomarkers in children with early puberty. Participants aged 7 – 12 years old were recruited directly from two elementary schools of Bengbu, Anhui Province, China, from Feb 2021 to May 2021. Early puberty was determined by breast and testicular development at baseline (May 2021) and 6-month later. Ultra-high-performance liquid chromatography-based untargeted metabolomic profiling was performed on urine samples of children with early puberty and control subjects. Metabolomic profiling for early puberty in a sex dependent manner. For boys, we identified several perturbed pathways, including histidine metabolism, glycine, serine and threonine metabolism, and selenoamino acid metabolism, associated with early puberty. In contrast, there were differences in pyruvate metabolism, one carbon pool by folate, and D-glutamine and D-glutamate metabolism pathways in girls with early puberty compared with controls. In addition, 4-hydroxyhippuric acid and 5-methoxytryptophol were shown as potential independent diagnostic biomarker for early puberty in boys, 3-hydroxybenzoic acid and glutaminylproline were shown as early biomarker for early puberty in girls, achieving area under the ROC curve of 0.71 and 0.72 in discriminating early puberty boys, and 0.70 and 0.74 in discriminating early puberty girls from controls. Through metabolomic analysis, we have identified metabolic perturbations and potential biomarkers of early puberty.

Metabolomic differences in young-onset versus average-onset colorectal adenocarcinoma.

3510 Background: Novel deleterious effects of environmental exposures may play a role in the rising incidence of young-onset colorectal cancer (yoCRC). We used metabolomics to assess differences between yoCRC and average-onset CRC (aoCRC), in comparison to healthy controls, which may provide insight into the pathogenesis and suggest any exposure risks. Methods: Patients with stage I-IV CRC and healthy controls were identified from prospective biobanks and categorized based on age &lt; 50 years (yoCRC or young controls) or age &gt; 60 years (aoCRC or older controls). Plasma metabolites were profiled using GC-TOF mass spectrometry. Differential abundance of metabolites was investigated using unadjusted and adjusted logistic regression. Metabolic pathway analysis was performed using Metaboanalyst 5.0. All p-values were adjusted for multiple testing (false-discovery rate, FDR p &lt; 0.20 considered significant). Results: The study population comprised 170 CRC patients (66 yoCRC and 104 aoCRC) and 49 healthy controls (34 young and 15 old). Association analyses revealed four differentially abundant metabolites: citrate (FDR p = 0.04), cholesterol (0.14), and two unidentified metabolites (UM). Metabolic pathways significantly altered in yoCRC vs. aoCRC included: carbohydrate metabolism (citrate cycle, FDR p = 0.04), carbohydrate biosynthesis (glyoxylate and dicarboxylate metabolism, FDR p = 0.004), amino-acid metabolism (alanine, aspartate, and glutamate metabolism, FDR p = 0.01, arginine biosynthesis, FDR p = 0.02, and amino-acid t-RNA biosynthesis, FDR p = 0.03). There were no significant metabolomic differences between young and old controls. Significant associations on survival analysis included: adipic acid with aoCRC (HR = 3.1, 95% CI = , 1.7-5.6, FDR p = 0.13, unadjusted analysis) suggesting worse survival with higher levels and 4-hydroxyhippuric acid with the whole cohort (HR = 0.4, 95% CI = 0.3-0.7, FDR p = 0.05, adjusted analyses) indicating better survival with higher levels. Conclusions: We identified significant differences in the citrate cycle - a core pathway of cellular metabolism associated with colorectal cancer. Metabolomic differences in pathways of carcinogenic significance (aspartate) and environmental exposures (arginine and dietary red meat) were also noted, suggesting potential relationships with younger age of CRC onset. [Table: see text]

Occurrence of Polyphenols, Isoflavonoids, and Their Metabolites in Milk Samples from Different Cow Feeding Regimens

In this work, milk samples collected in a cohort of intensive dairy farms of the Po Valley (Italy) were screened for their (poly)-phenolic profile to check the occurrence of phenolic metabolites of biological interest. The selected dairy farms were previously classified on the basis of their cow feeding system, considering the utilization of corn silage as the main ingredient of the rations. Overall, ultra-high-pressure liquid chromatography coupled with mass spectrometry using an Orbitrap analyzer, followed by unsupervised and supervised statistics, allowed identifying clear different phenolic distributions in the milk samples. Accordingly, a great variability in the phenolic profiles of the different milk samples was observed, with two main phenolic clusters outlined by the unsupervised hierarchical clustering approach and not fully correlated to the nutritional strategy considered. The variables’ importance in the projection approach allowed selecting the most important metabolites, resulting in samples’ discrimination. Among the most discriminative compounds, we found phenolic metabolites (such as hippuric acid and 4-hydroxyhippuric acid), followed by lignans (such as enterolactone) and isoflavonoids (such as equol and O-desmethylangolensin). Taken together, our findings suggested that both the feeding systems and the ability of dairy cows to process parent phenolic compounds were the main factors providing the final (poly)-phenolic profile of the milk samples. Future targeted and ad hoc studies appear of great interest to evaluate the potential biological effects of these compounds on cow health.

Metabolic profiling of organic acids in urine samples of Cri Du Chat syndrome individuals by gas chromatography-mass spectrometry

Cri Du Chat (CDC) syndrome is a rare genetic condition caused by the deletion of genetic material on the small arm (the p arm) of chromosome 5. A high-pitched cry that sounds like that of a cat, dysmorphic characteristics, and cytogenetic methods are often used for diagnosing the syndrome. In this study, we applied GC–MS analysis for determining organic acids in urine from 17 control volunteers without CDC syndrome, and from 16 individuals with the CDC syndrome in order to determine the profile of organic acids and biochemical pathways alterations resulting from this genetic condition. First, performing multivariate data analysis selected the best method for extracting organic acids with greater signal intensities and good reproducibility. After selection, multivariate (PLS-DA) and univariate (Mann-Whitney test) data analysis discriminated the metabolites responsible for separation between groups. Nine organic acid metabolites had values of VIP ≥ 1.0 and p-values ≤ 0.05, with highest intensities in the samples from CDC individuals, indicating the strongest discriminative power (tricarballylic acid, indoleacetic acid, anthranilic acid, 4-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, 4-hydroxyhippuric acid, pantothenic acid, homovanillic acid, and vanillylmandelic acid). These metabolites are involved in several biochemical pathways like in the tyrosine and phenylalanine metabolism, as well as the tryptophan metabolism, which could be associated (i) to some neuropsychiatric alterations commonly observed in CDC individuals, (ii) to exogenous compounds related to transformation products by intestinal microbial, and (iii) to a possible deficiency in enzyme activity due to the syndrome.

A rapid and sensitive method for simultaneous determination of eight protein-bound uremic toxins in human serum by UHPLC-MS/MS: application in assessing peritoneal dialysis

A simple, rapid, reliable and sensitive ultra-high performance liquid chromatography tandem spectrometry (UHPLC-MS/MS) method was established for determination of eight serum protein-bound uremic toxins (hippuric acid, indoxyl sulfate, indole-3-acetic acid, kynurenic acid, L-kynurenine, melatonin, 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid, 4-hydroxyhippuric acid) in serum from chronic kidney disease (CKD) dialysis patients. The chromatographic separation was achieved on an Atlantis T3 column (3 μm, 2.1 mm × 100 mm) using a gradient elution with acetonitrile (phase B) and 0.1% formic acid and 10 mmol/L ammonium acetate aqueous solution (phase A). The flow rate was 0.3 mL/min with analytical time of 5 min. The pretreatment procedure was developed with a simple protein precipitation and the hydrochlorothiazide was used as internal standard. The calibration ranges were set as 156.250-20000.000 ng/mL for indoxyl sulfate, hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid; 78.125-10000.000 ng/mL for L-kynurenine, indole-3-acetic acid and 4-hydroxyhippuricacid; 1.562-200.000 ng/mL for kynurenic acid; 0.078-10.000 ng/mL for melatonin. The UHPLC-MS/MS method for quantification of eight protein-bound uremic toxins was successfully developed and validated, and its clinical practicability was assessed on 81 serum samples from CKD patients.

Effects of cooking on the phytochemical profile of breadfruit as revealed by high-resolution UPLC-MSE.

This study evaluated the impact of cooking on the profile of bioactive compounds in unripe breadfruit. To this end, the accessibility of bioactive compounds by various solvents was assessed through total phenolic content and antioxidant capacity analyses. The most efficient solvent was applied to extract the metabolites, which were evaluated by ultra-performance liquid chromatography coupled with high-resolution quadrupole time-of-flight mass spectrometry in MSE mode. Cooked and raw breadfruit presented total phenolic content and antioxidant capacities in almost all extracts, and pure water proved to be the best extractor. Globally, 146 bioactive compounds have been identified for both raw and cooked fruits' aqueous extracts. Most of these compounds were stable to the heat treatment applied (121 °C/10 min). However, results revealed that 22 metabolites contributed to significantly distinguishing the raw from the cooked samples. Among those, 15 compounds, such as pyrogallol, 1-acetoxypinoresinol, and scopolin, evidenced higher relative abundance in the cooked extracts. On the other hand, only seven metabolites, such as 4-hydroxyhippuric acid, epicatechin, and leptodactylone, decreased post-heating. Cooking promoted little alteration in the bioactive compounds profile of immature breadfruit and thus appears to be an exploitation alternative for this perishable fruit, which seems to be a source of a large range of bioactive compounds. © 2019 Society of Chemical Industry.

A Preliminary Study of Uric Metabolomic Alteration for Postpartum Depression Based on Liquid Chromatography Coupled to Quadrupole Time-of-Flight Mass Spectrometry.

Postpartum depression affects about 10-20% of newly delivered women, which is harmful for both mothers and infants. However, the current diagnosis of postpartum depression depends on the subjective judgment of a practitioner, which may lead to misdiagnosis. Hence, an appended objective diagnosis index may help the practitioner to improve diagnosis. A metabolomic study can find biomarkers as an objective index to facilitate disease diagnosis. Forty-nine postpartum depressed patients and 50 healthy controls were recruited into this study. The metabolites in urine were scanned with LC-Q-TOF-MS. The metabolomic data were analyzed with a multivariate statistical analysis method. Data from 40 patients and 40 controls were used for partial least square-discriminate analysis (PLS-DA). The urine metabolomic profiles of patients were different from those of controls. The PLS-DA model was validated by a permutation test, and the model could accurately classify the other 9 patients and 10 controls in T-prediction. Ten differentiating metabolites were found as main contributors to this difference, which are involved in amino acid metabolism, neurotransmitter metabolism, bacteria population, etc. Some of these potential biomarkers, such as 4-hydroxyhippuric acid, homocysteine, and tyrosine, showed relatively high sensitivities and specificities. The metabolic profile alteration induced by postpartum depression was found, and some of the differentiating metabolites may serve as biomarkers to facilitate the diagnosis of postpartum depression.

Hemodialysis Decreases the Concentration of Accumulated Plant Phenols in the Plasma of Patients on Maintenance Dialysis: Influence of Residual Renal Function.

Plant phenols may accumulate in end-stage kidney disease. The effect of hemodialysis on their plasma concentration remains poorly determined. Contingent on concentration, health-promoting or noxious effects occur; therefore, we assessed plasma concentration in hemodialyzed patients. In total, 21 maintenance hemodialyzed patients with diuresis < 500 mL per day (with oliguria), nine hemodialyzed patients with diuresis ≥ 500 mL per day (without oliguria) and 31 healthy volunteers were included. Nine phenolic acids were identified with high-performance liquid chromatography and total polyphenol concentration was determined with the Folin-Ciocalteu method in pre- or post-hemodialysis plasma and pre- or intra-hemodialysis dialysate. The concentration of total polyphenols was 27% higher in pre-hemodialysis plasma than in that of controls (0.95 ± 0.18 mmol/L [P < 0.0001]). The concentration of total polyphenols was higher in patients with oliguria (1.01 ± 0.17) than in those without (0.84 ± 0.13 mmol/L), despite the former having more intense hemodialysis (Kt/V 1.29 ± 0.31 and 0.77 ± 0.25, respectively). Pre-hemodialysis phenolic acid concentration in patients undergoing dialysis exceeded reference values by 3 to 34 times (3-hydroxyphenylacetic acid and vanillic acid, respectively), from 0.69 (dihydrocaffeic acid) to 169.3 μmol/L (hippuric acid). The concentration of six phenolic acids (3-hydroxyhippuric, caffeic, dihydrocaffeic, hippuric, homovanillic, and vanillic acid) was 1.1 (homovanillic) to 11.3 (3-hydroxyhippuric) times higher in patients with oliguria than in those without. 4-hydroxyhippuric acid occurred more in the plasma of patients with oliguria than in those without oliguria. A single hemodialysis session decreased total polyphenol concentration by 16% and phenolic acids from 30% (caffeic) to 58% (vanillic and 3-hydroxyphenylacetic acid) and these compounds appeared in the dialysate. The percentage decrease (Δ%) of creatinine concentration correlated with the Δ% of total polyphenols and five phenolic acids (3-hydroxyphenylacetic, dihydrocaffeic, hippuric, homovanillic, and vanillic acid). Urea Δ% and Kt/V correlated only with the Δ% of homovanilic acid. The results demonstrate that phenols accumulate variably in hemodialyzed patients and are differently eliminated during hemodialysis. Residual renal function ensures a lower concentration of plasma phenols.

Single nucleotide polymorphisms of ABCC2 modulate renal secretion of endogenous organic anions

The ATP-binding cassette family transporter MRP2 (multidrug resistance-associated protein 2), encoded by the ABCC2 gene, is involved in the renal excretion of numerous xenobiotics and it is likely that it also transports many endogenous molecules arising from not only normal essential metabolic processes but also from environmental toxins or food intake. We used a targeted gas chromatography-mass spectrometry metabolomics analysis to study whether endogenous organic anions are differentially excreted in urines of healthy volunteers according to their genotype for three functional single nucleotide polymorphisms (SNPs) in ABCC2. This was the case for 35 of the 108 metabolites analyzed. Eight of them are most likely substrates of MRP2 since they are the most contributive to the difference between carriers of a decreasing function allele vs those carrying an increasing function one. Seven out of 8 metabolites are fatty acids (dodecanoic acid; 3-hydroxypropanoic acid) or metabolites of polyphenols (caffeine; resorcinol; caffeic acid; 2-(3,4-dihydroxyphenyl) acetic acid; and 4-hydroxyhippuric acid). Most of them were structurally similar to a series of substances previously shown to interact with MRP2 function in vitro. Interestingly, coproporphyrin isomer I, a prototypical substrate of MRP2, also belonged to our final list although it was not significantly discriminant on its own. This suggests that the simultaneous measurement of a set of endogenous metabolites in urine, rather than that of unique metabolites, has the potential to provide a phenotypic measure of MRP2 function in vivo. This would represent an innovative tool to study the variability of the transport activity of MRP2 under a physiological or pathological condition, especially in pharmacokinetic studies of its substrates.

Urinary excretion of Citrus flavanones and their major catabolites after consumption of fresh oranges and pasteurized orange juice: A randomized cross-over study.

Orange juice contains flavanones including hesperidin and narirutin, albeit at lower concentrations as compared to orange fruit. Therefore, we compared bioavailability and colonic catabolism of flavanones from orange juice to a 2.4-fold higher dose from fresh oranges. Following a randomized two-way cross-over design, 12 healthy subjects consumed a test meal comprising either fresh oranges or pasteurized orange juice, delivering 1774 and 751 μmol of total Citrus flavanones, respectively. Deglucuronidated and desulfated hesperetin, naringenin, and the flavanone catabolites 3-(3'-hydroxy-4'-methoxyphenyl)propionic acid, 3-(3'-hydroxyphenyl)hydracrylic acid, 4-hydroxyhippuric acid, and hippuric acid were quantitated in 24-h urine by UHPLC-MS/MS. Differences in urinary hesperetin excretion were found to be nonsignificant (p = 0.5209) both after consumption of orange fruit (21.6 ± 8.0μmol) and juice (18.3 ± 7.2μmol). By analogy, postprandial flavanone catabolite excretions were highly similar between treatments. Excretion of 3-(3'-hydroxy-4'-methoxyphenyl)propionic acid was inversely related to that of hesperetin, illustrating the catabolite/precursor relationship. Despite 2.4-fold higher doses, excretion of flavanones from ingested fresh orange fruit did not differ from that following orange juice consumption, possibly due to a saturation of absorption or their entrapment in the fiber-rich matrix of the fruit.

Addition of strawberries to the usual diet increases postprandial but not fasting non-urate plasma antioxidant activity in healthy subjects.

Strawberries can augment plasma antioxidant activity, but this may be confounded by selection of methods, time of blood sampling and concomitant dietary restrictions. We examined the effect of strawberry consumption on ferric reducing ability (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (DPPH-test) of native and non-urate plasma in healthy subjects on their usual diet. Eleven subjects consumed strawberries (500 g daily) for 9 days. Fasting and 3-h postprandial plasma and 24-h urine collection were obtained before, during and after strawberry course for FRAP, DPPH-test and polyphenols determination. Fifteen subjects served as a control in respect to plasma antioxidant activity changes and effect of 300 mg of oral ascorbate. First, 5th and 9th strawberry dose increased 3-h postprandial DPPH-test by 17.4, 17.6 and 12.6%, and FRAP by 15.5, 25.6 and 21.4% in comparison to fasting values in non-urate plasma (p<0.05). In native plasma only a trend was observed to higher postprandial values for both tests. Strawberries increased urinary urolithin A and 4-hydroxyhippuric acid whereas plasma polyphenols were stable. No changes of FRAP and DPPH-test were noted in controls and after ascorbate intake. Strawberries transiently increased non-urate plasma antioxidant activity but this cannot be attributed to direct antioxidant effect of polyphenols and ascorbate.

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.

Deregulated tyrosine-phenylalanine metabolism in pulmonary tuberculosis patients.

Metabolic profiling of biofluids from tuberculosis (TB) patients would help us in understanding the disease pathophysiology and may also be useful for the development of novel diagnostics and host-directed therapy. In this pilot study we have compared the urine metabolic profiles of two groups of subjects having similar TB symptoms and categorized as active TB (ATB, n = 21) and non-TB (NTB, n = 21) based on GeneXpert test results. Silylation, gas chromatography mass spectrometry, and standard chemometric methods were employed to identify the important molecules and deregulated metabolic pathways. Eleven active TB patients were followed up on longitudinally for comparative urine metabolic profiling with healthy controls (n = 11). A set of 42 features qualified to have a variable importance parameter score of > 1.5 of a partial least-squares discriminate analysis model and fold change of > 1.5 at p value < 0.05 between ATB and NTB. Using these variables, a receiver operating characteristics curve was plotted and the area under the curve was calculated to be 0.85 (95% CI: 0.72-0.96). Several of these variables that represent norepinephrine, gentisic acid, 4-hydroxybenzoic acid, hydroquinone, and 4-hydroxyhippuric acid are part of the tyrosine-phenylalanine metabolic pathway. In the longitudinal study we observed a treatment-dependent trend in the urine metabolome of follow-up samples, and subjects declared as clinically cured showed similar metabolic profile as those of asymptomatic healthy subjects. The deregulated tyrosine-phenylalanine axis reveals a potential target for diagnostics and intervention in TB.

Addition of Strawberries to the Usual Diet Decreases Resting Chemiluminescence of Fasting Blood in Healthy Subjects—Possible Health-Promoting Effect of These Fruits Consumption

Objective: Regular strawberry consumption augmented plasma antioxidant activity and decreased lipid peroxidation suggests preventive potential of these fruits against oxidative stress-dependent disorders. Blood phagocytes are important source of oxidants that may contribute to systemic oxidative stress. We examined the effect of strawberry consumption on the luminol enhanced whole blood chemiluminescence (LBCL) reflecting oxidants generation by circulating phagocytes in healthy subjects.Methods: Thirty-one healthy subjects (being on their usual diet) consumed 500 g of strawberry pulp daily (between 11.00–14.00) for 30 days (1st strawberry course) and after 10 day wash-out the cycle was repeated (2nd strawberry course). Fasting blood and spot morning urine samples were collected before and after each strawberry course for measuring resting and agonist (fMLP)-induced LBCL, various phenolics and plasma antioxidant activity. Twenty subjects served as a control in respect to LBCL changes over the study period.Results: Strawberry consumption decreased median resting LBCL and this effect was more evident after the 1st course (by 38.2%, p < 0.05) than after the the 2nd one (18.7%), while fMLP-induced LBCL was constant. No changes in LBCL were noted in controls. Strawberries increased fasting plasma levels of caffeic acid and homovanillic acid as well as urolithin A and 4-hydroxyhippuric acid in spot urine. Plasma antioxidant activity and the number of circulating phagocytes did not change over the study period. Resting LBCL correlated positively with the number of circulating polymorphonuclear leukocytes at all occasions and negative correlation with plasma 4-hydroxyhippuric acid was noted especially after the first strawberry course (r = −0.46, p < 0.05).Conclusions: The decrease in resting LBCL suggests that regular strawberry consumption may suppress baseline formation of oxidants by circulating phagocytes. This may decrease the risk of systemic imbalance between oxidants and anti-oxidants and be one of mechanisms of health-promoting effect of these fruits consumption.

1H NMR-based metabolic fingerprinting of urine metabolites after consumption of lingonberries (Vaccinium vitis-idaea) with a high-fat meal

The use of NMR metabolomics in clinical trials is growing; however, reports of postprandial experiments in humans are scarce. The present study investigated whether consumption of lingonberries as a supplement to an oil-rich meal modifies the postprandial fingerprints of human urine. Urine samples were analysed by 1H NMR, and untargeted multivariate analysis was applied to the data for comprehensive fingerprinting. A clear separation of postprandial lingonberry meal samples was revealed. To evaluate statistical differences, a targeted approach was applied for the informative spectral areas. Significantly (p<0.05) increased levels of polyphenol metabolites, hippuric acid and 4-hydroxyhippuric acid, and decreased creatinine and dimethylamine levels were the major explanations for the grouping of the postprandial samples after the different meals. Thus, inclusion of polyphenol-rich lingonberry powder in a rapeseed oil-rich meal modifies the metabolic profile of urine which may be used to reveal both consumption of berries and health-promoting changes in the common metabolism.

SPE–NMR metabolite sub-profiling of urine

NMR-based metabolite profiling of urine is a fast and reproducible method for detection of numerous metabolites with diverse chemical properties. However, signal overlap in the (1)H NMR profiles of human urine may hamper quantification and identification of metabolites. Therefore, a new method has been developed using automated solid-phase extraction (SPE) combined with NMR metabolite profiling. SPE-NMR of urine resulted in three fractions with complementary and reproducible sub-profiles. The sub-profile from the wash fraction (100 % water) contained polar metabolites; that from the first eluted fraction (10 % methanol-90 % water) semi-polar metabolites; and that from the second eluted fraction (100 % methanol) aromatic metabolites. The method was validated by analysis of urine samples collected from a crossover human nutritional intervention trial in which healthy volunteers consumed capsules containing a polyphenol-rich mixture of red wine and grape juice extract (WGM), the same polyphenol mixture dissolved in a soy drink (WGM_Soy), or a placebo (PLA), over a period of five days. Consumption of WGM clearly increased urinary excretion of 4-hydroxyhippuric acid, hippuric acid, 3-hydroxyphenylacetic acid, homovanillic acid, and 3-(3-hydroxyphenyl)-3-hydroxypropionic acid. However, there was no difference between the excreted amounts of these metabolites after consumption of WGM or WGM_Soy, indicating that the soy drink is a suitable carrier for WGM polyphenols. Interestingly, WGM_Soy induced a significant increase in excretion of cis-aconitate compared with WGM and PLA, suggesting a higher demand on the tricarboxylic acid cycle. In conclusion, SPE-NMR metabolite sub-profiling is a reliable and improved method for quantification and identification of metabolites in urine to discover dietary effects and markers of phytochemical exposure.

The metabolic fate of red wine and grape juice polyphenols in humans assessed by metabolomics

The metabolic impact of polyphenol-rich red wine and grape juice consumption in humans was studied using a metabolomics approach. Fifty-eight men and women participated in a placebo-controlled, double-crossover study in which they consumed during a period of 4 wk, either a polyphenol-rich 2:1 dry mix of red wine and red grape juice extracts (MIX) or only a grape juice extract (GJX). Twenty-four-hour urine samples were collected after each intervention. (1)H NMR spectroscopy was applied for global metabolite profiling, while GC-MS was used for focused profiling of urinary phenolic acids. Urine metabolic profiles after intake of both polyphenol-rich extracts were significantly differentiated from placebo using multilevel partial least squares discriminant analysis. A significant 35% increase in hippuric acid excretion (p<0.001) in urine was measured after the MIX consumption as) or only a red grape juice dry extract (GJX). 24-h urine samples were collected after each intervention. 1H-NMR spectroscopy was applied for global metabolite profiling, while gas chromatography-mass spectrometry (GC-MS) was used for focused profiling of urinary phenolic acids. Urine metabolic profiles after intake of both polyphenol-rich extracts were significantly differentiated from placebo using multilevel partial least squares discriminant analysis (ML-PLS-DA). A significant 35% increase in hippuric acid excretion (p<0.001) in urine was measured after the MIX consumption compared with placebo, whereas no change was found after GJX consumption. GC-MS-based metabolomics of urine allowed identification of 18 different phenolic acids, which were significantly elevated following intake of either extract. Syringic acid, 3- and 4-hydroxyhippuric acid and 4-hydroxymandelic acid were the strongest urinary markers for both extracts. MIX and GJX consumption had a slightly different effect on the excreted phenolic acid profile and on endogenous metabolite excretion, possibly reflecting their different polyphenol composition.