Assessing Exposome Effects on Pregnancy through Urine Metabolomics of a Portuguese (Estarreja) Cohort.

This nuclear magnetic resonance metabolomics study compared the influence of two different central Portugal exposomes, one of which comprised an important source of pollutants (the Estarreja Chemical Complex, ECC), on the urinary metabolic trajectory of a cohort of healthy pregnant women (total n = 107). An exposome-independent description of pregnancy metabolism was found to comprise a set of 18 metabolites reflecting expected changes in branched-chain amino acid catabolism and hormone and lipid metabolisms. In addition, a set of small changes in some metabolites was suggested to be exposome-dependent and characteristic of pregnant subjects from the Estarreja region. These results suggested that the Estarreja exposome may impact to a very low extent pregnancy metabolism, inducing slight changes in amino acid metabolism (alanine, glycine, and 3-hydroxyisobutyrate, possibly involved in valine metabolism), tricarboxylic acid (TCA) cycle (cis-aconitate), diet, or gut microflora (furoylglycine) as well as allantoin, 2-hydroxyisobutyrate, and an unassigned resonance at δ 8.45. Furthermore, the urine of Estarreja subjects was found to generally contain higher levels of 4-hydroxyphenylacetate and lower levels of citrate. However, out of the above metabolites, only glycine and citrate seemed to correlate with the proximity to the ECC, with slightly relative higher levels of these compounds found for subjects living closer to the ECC. This suggested possible small effects of local pollutants on energy metabolism, with the remaining exposome-dependent metabolite changes most probably originating from other aspects of the local exposome such as diet and lifestyle. Despite the limitation of this study regarding the unavailability of objective environmental parameters for the period under study, our results confirm the usefulness of metabolomics of human urine to gauge exposome effects on human health and, particularly, during pregnancy.

Dietary and metabolomic determinants of relapse in ulcerative colitis patients: A pilot prospective cohort study.

AIM: To identify demographic, clinical, metabolomic, and lifestyle related predictors of relapse in adult ulcerative colitis (UC) patients. METHODS: In this prospective pilot study, UC patients in clinical remission were recruited and followed-up at 12 mo to assess a clinical relapse, or not. At baseline information on demographic and clinical parameters was collected. Serum and urine samples were collected for analysis of metabolomic assays using a combined direct infusion/liquid chromatography tandem mass spectrometry and nuclear magnetic resolution spectroscopy. Stool samples were also collected to measure fecal calprotectin (FCP). Dietary assessment was performed using a validated self-administered food frequency questionnaire. RESULTS: Twenty patients were included (mean age: 42.7 ± 14.8 years, females: 55%). Seven patients (35%) experienced a clinical relapse during the follow-up period. While 6 patients (66.7%) with normal body weight developed a clinical relapse, 1 UC patient (9.1%) who was overweight/obese relapsed during the follow-up (P = 0.02). At baseline, poultry intake was significantly higher in patients who were still in remission during follow-up (0.9 oz vs 0.2 oz, P = 0.002). Five patients (71.4%) with FCP > 150 µg/g and 2 patients (15.4%) with normal FCP (≤ 150 µg/g) at baseline relapsed during the follow-up (P = 0.02). Interestingly, baseline urinary and serum metabolomic profiling of UC patients with or without clinical relapse within 12 mo showed a significant difference. The most important metabolites that were responsible for this discrimination were trans-aconitate, cystine and acetamide in urine, and 3-hydroxybutyrate, acetoacetate and acetone in serum. CONCLUSION: A combination of baseline dietary intake, fecal calprotectin, and metabolomic factors are associated with risk of UC clinical relapse within 12 mo.

Metabonomic analysis of potential biomarkers and drug targets involved in diabetic nephropathy mice.

Diabetic nephropathy (DN) is one of the lethal manifestations of diabetic systemic microvascular disease. Elucidation of characteristic metabolic alterations during diabetic progression is critical to understand its pathogenesis and identify potential biomarkers and drug targets involved in the disease. In this study, (1)H nuclear magnetic resonance ((1)H NMR)-based metabonomics with correlative analysis was performed to study the characteristic metabolites, as well as the related pathways in urine and kidney samples of db/db diabetic mice, compared with age-matched wildtype mice. The time trajectory plot of db/db mice revealed alterations, in an age-dependent manner, in urinary metabolic profiles along with progression of renal damage and dysfunction. Age-dependent and correlated metabolite analysis identified that cis-aconitate and allantoin could serve as biomarkers for the diagnosis of DN. Further correlative analysis revealed that the enzymes dimethylarginine dimethylaminohydrolase (DDAH), guanosine triphosphate cyclohydrolase I (GTPCH I), and 3-hydroxy-3-methylglutaryl-CoA lyase (HMG-CoA lyase) were involved in dimethylamine metabolism, ketogenesis and GTP metabolism pathways, respectively, and could be potential therapeutic targets for DN. Our results highlight that metabonomic analysis can be used as a tool to identify potential biomarkers and novel therapeutic targets to gain a better understanding of the mechanisms underlying the initiation and progression of diseases.

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.

Urinary loss of tricarboxylic acid cycle intermediates as revealed by metabolomics studies: an underlying mechanism to reduce lipid accretion by whey protein ingestion?

Whey protein intake is associated with the modulation of energy metabolism and altered body composition both in human subjects and in animals, but the underlying mechanisms are not yet elucidated. We fed obesity-prone C57BL/6J mice high-fat diets with either casein (HF casein) or whey (HF whey) for 6 weeks. At equal energy intake and apparent fat and nitrogen digestibility, mice fed HF whey stored less energy as lipids, evident both as lower white adipose tissue mass and as reduced liver lipids, compared with HF-casein-fed mice. Explorative analyses of 48 h urine, both by (1)H NMR and LC-MS metabolomic platforms, demonstrated higher urinary excretion of tricarboxylic acid (TCA) cycle intermediates citric acid and succinic acid (identified by both platforms), and cis-aconitic acid and isocitric acid (identified by LC-MS platform) in the HF whey, relative to in the HF-casein-fed mice. Targeted LC-MS analyses revealed higher citric acid and cis-aconitic acid concentrations in fed state plasma, but not in liver of HF-whey-fed mice. We propose that enhanced urinary loss of TCA cycle metabolites drain available substrates for anabolic processes, such as lipogenesis, thereby leading to reduced lipid accretion in HF-whey-fed compared to HF-casein-fed mice.

Analysis of urinary metabolic signatures of early hepatocellular carcinoma recurrence after surgical removal using gas chromatography-mass spectrometry.

The objective of present study was to offer insights into the metabolic responses of hepatocellular carcinoma (HCC) to surgical resection and the metabolic signatures latent in early HCC recurrence (one year after operation). Urinary metabolic profiling employing gas chromatography time-of-flight mass spectrometry (GC-TOF MS) was utilized to investigate the complex physiopathologic regulations in HCC after operational intervention. It was revealed that an intricate series of metabolic regulations including energy metabolism, amino acid metabolism, nucleoside metabolism, tricarboxylic acid (TCA) cycle, gut floral metabolism, etc., principally leading to the direction of biomass synthesis, could be observed after tumor surgical removal. Moreover, metabolic differences between recurrent and nonrecurrent patients had emerged 7 days after initial operation. The metabolic signatures of HCC recurrence principally comprised notable up-regulations of lactate excretion, succinate production, purine and pyrimidine nucleosides turnover, glycine, serine and threonine metabolism, aromatic amino acid turnover, cysteine and methionine metabolism, and glyoxylate metabolism, similar to metabolic behaviors of HCC burden. Sixteen metabolites were found to be significantly increased in the recurrent patients compared with those in nonrecurrent patients and healthy controls. Five metabolites (ethanolamine, lactic acid, acotinic acid, phenylalanine and ribose) were further defined; they were favorable to the prediction of early recurrence.

Preliminary determination of a molecular basis of chronic fatigue syndrome.

Chronic fatigue syndrome (CFS/ME) is a debilitating fatigue illness that has an unknown etiology. We studied 20 chronic fatigue syndrome (CFS) patients, who complied with the Oxford and American CDC definitions, and 45 non-CFS subjects. Participants completed questionnaires, were clinically examined, and had first morning urine specimens collected, which were screened by gas chromatography-mass spectrometry for changes in metabolite excretion. Multivariate analysis of the urinary metabolite profiles differed significantly in the CFS patients compared to the non-CFS patients (P < 0.004). The CFS patients had increases in aminohydroxy-N-methylpyrrolidine (P < 0.00003, referred to as chronic fatigue symptom urinary marker 1, or CFSUM1), tyrosine (P < 0.02), beta-alanine (P < 0.02), aconitic acid (P < 0.05), and succinic acid (P < 0.05) and reductions in an unidentified urinary metabolite, CFSUM2 (P < 0.0007), alanine (P < 0.005), and glutamic acid (P < 0.02). CFSUM1, beta-alanine, and CFSUM2 were found by discriminant function analysis to be the first, second, and third most important metabolites, respectively for discriminating between CFS and non-CFS subjects. The abundances of CFSUM1 and beta-alanine were positively correlated with symptom incidence (P < 0.01 and P < 0.001, respectively), symptom severity, core CFS symptoms, and SCL-90-R somatization (P < 0.00001), suggesting a molecular basis for CFS.

Organic acids or urine in multiple sclerosis.

The urinary excretion of organic acids was examined in 509 cases with multiple sclerosis and in 50 age- and sex-matched controls. The concentrations of the acids were related to creatinine. No differences were found for compounds such as glycolic acid, 2-methyl-3-hydroxybutyric acid, 2-ethylhydracrylic acid, 4-hydroxyphenylacetic acid, suberic acid and many other acids. However, the mean excretion of 2-hydroxy-2-methyl-3-butenoic acid was increased two-fold in the MS group. 2 MS cases had a very high excretion of 3-methylglutaconic acid, and another 6 cases had moderate elevations, which were fairly constant over a time period of several months. Moderate elevations were also noted in 2 healthy controls. 1 MS case had a very high excretion of 3-hydroxyisovaleric acid. 7 MS cases, and none in the control group, had elevated excretion of adipic acid. Differences were also noted for lactic acid, succinic acid, aconitic acid and 3-methyladipic acid. An oral dose of deuterium-labelled acetate was given to one of the patients with high excretion of 3-methylglutaconic acid. Deuterium was incorporated into this metabolite. 3-methylglutaconic acid, 2-hydroxy-2-methyl-3-butenoic acid, 3-hydroxy-isovaleric acid and 3-methyladipic acid are all potential isoprenoid metabolites. A possible defect in the pathway of isoprenoid biosynthesis is discussed.

Urinary organic acid profiles of Reye's syndrome patients.

The status of mitochondrial functioning in Reye's syndrome was assessed by comparing organic acid profiles from nine pathological urines with those from normal urines. It was found that Reye's syndrome urines have a normal content of succinic, oxaloacetic, aconitic, and citric acids suggesting that the enzymes of the acid cycle are functional. Elevated pyruvate and depressed alpha-ketoglutarate levels were observed. Abnormal urinary constituents detected were salicylic and adipic acids. Presence of the latter indicates that the enzymes of fatty acid beta-oxidation are functional.