Integrating untargeted metabolomics, genetically informed causal inference, and pathway enrichment to define the obesity metabolome

ABSTRACTBackgroundObesity and its associated diseases are major health problems characterized by extensive metabolic disturbances. Understanding the causal connections between these phenotypes and variation in metabolite levels can uncover relevant biology and inform novel intervention strategies. Recent studies have combined metabolite profiling with genetic instrumental variable (IV) analyses to infer the direction of causality between metabolites and obesity, but often omitted a large portion of untargeted profiling data consisting of unknown, unidentified metabolite signals.MethodsWe expanded upon previous research by identifying body mass index (BMI)-associated metabolites in multiple untargeted metabolomics datasets, and then performing bidirectional IV analysis to classify these metabolites based on their inferred causal relationships with BMI. Meta-analysis and pathway analysis of both known and unknown metabolites across datasets were enabled by our recently developed bioinformatics suite, PAIRUP-MS.ResultsWe identified 10 known metabolites that are more likely to be the causes (e.g. alpha-hydroxybutyrate) or effects (e.g. valine) of BMI, or may have more complex bidirectional cause-effect relationships with BMI (e.g. glycine). Importantly, we also identified about 5 times more unknown than known metabolites in each of these three categories. Pathway analysis incorporating both known and unknown metabolites prioritized 40 enriched (p < 0.05) metabolite sets for the cause versus effect groups, providing further support that these two metabolite groups are linked to obesity via distinct biological mechanisms.ConclusionsThese findings demonstrate the potential utility of our approach to uncover causal connections with obesity from untargeted metabolomics datasets. Combining genetically informed causal inference with the ability to map unknown metabolites across datasets provides a path to jointly analyze many untargeted datasets with obesity or other phenotypes. This approach, applied to larger datasets with genotype and untargeted metabolite data, should generate sufficient power for robust discovery and replication of causal biological connections between metabolites and various human diseases.

Responses of Metabolites in Soybean Shoot Apices to Changing Atmospheric Carbon Dioxide Concentrations

Soybean seedlings were grown in controlled environment chambers with CO2partial pressures of 38 (ambient) and 72 (elevated) Pa. Five or six shoot apices were harvested from individual 21- to 24-day-old plants. Metabolites were analyzed by gas chromatography and, out of 21 compounds, only sucrose and fructose increased in response to CO2enrichment. One unidentified metabolite, Unk-21.03 decreased up to 80% in soybean apices in response to elevated CO2. Levels of Unk-21.03 decreased progressively when atmospheric CO2partial pressures were increased from 26 to 100 Pa. Reciprocal transfer experiments showed that Unk-21.03, and sucrose in soybean apices were altered slowly over several days to changes in atmospheric CO2partial pressures. The mass spectrum of Unk-21.03 indicated that this compound likely contained both an amino and carboxyl group and was structurally related to serine and aspartate. Our findings suggested that CO2enrichment altered a small number of specific metabolites in soybean apices. This could be an important step in understanding how plant growth and development are affected by carbon dioxide enrichment.

Identification of Urinary Biomarkers of Colon Inflammation in IL10-/-Mice Using Short-Column LCMS Metabolomics

The interleukin-10-deficient (IL10-/-) mouse develops colon inflammation in response to normal intestinal microflora and has been used as a model of Crohn's disease. Short-Column LCMS metabolite profiling of urine from IL10-/-and wild-type (WT) mice was used, in two independent experiments, to identify mass spectral ions differing in intensity between these two genotypes. Three differential metabolites were identified as xanthurenic acid and as the glucuronides of xanthurenic acid and of α-CEHC (2,5,7,8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman). The significance of several differential metabolites as potential biomarkers of colon inflammation was evaluated in an experiment which compared metabolite concentrations in IL10-/-and WT mice housed, either under conventional conditions and dosed with intestinal microflora, or maintained under specific pathogen-free (SPF) conditions. Concentrations of xanthurenic acid, α-CEHC glucuronide, and an unidentified metabolitem/z495-/497+were associated with the degree of inflammation in IL10-/-mice and may prove useful as biomarkers of colon inflammation.

Anaerobic Degradation of Benzene, Toluene, Ethylbenzene, and Xylene Compounds by Dechloromonas Strain RCB

ABSTRACT Dechloromonas strain RCB has been shown to be capable of anaerobic degradation of benzene coupled to nitrate reduction. As a continuation of these studies, the metabolic versatility and hydrocarbon biodegradative capability of this organism were investigated. The results of these revealed that in addition to nitrate, strain RCB could alternatively degrade benzene both aerobically and anaerobically with perchlorate or chlorate [(per)chlorate] as a suitable electron acceptor. Furthermore, with nitrate as the electron acceptor, strain RCB could also utilize toluene, ethylbenzene, and all three isomers of xylene (ortho-, meta-, and para-) as electron donors. While toluene and ethylbenzene were completely mineralized to CO2, strain RCB did not completely mineralize para-xylene but rather transformed it to some as-yet-unidentified metabolite. Interestingly, with nitrate as the electron acceptor, strain RCB degraded benzene and toluene concurrently when the hydrocarbons were added as a mixture and almost 92 μM total hydrocarbons were oxidized within 15 days. The results of these studies emphasize the unique metabolic versatility of this organism, highlighting its potential applicability to bioremediative technologies.

Degradation of 2,4-dinitrophenol and selected nitroaromatic compounds bySphingomonassp. UG30

Sphingomonas strain UG30 mineralizes both p-nitrophenol (PNP) and pentachlorophenol (PCP). Our current studies showed that UG30 oxidatively metabolized certain other p-substituted nitrophenols, i.e., p-nitrocatechol, 2,4-dinitrophenol (2,4-DNP), and 4,6-dinitrocresol with liberation of nitrite. 2,6-DNP, o- or m-nitrophenol, picric acid, or the herbicide dinoseb were not metabolized. Studies using14C-labelled 2,4-DNP indicated that in glucose-glutamate broth cultures of UG30, greater than 90% of 103 µM 2,4-DNP was transformed to other compounds, while 8-19% of the 2,4-DNP was mineralized within 5 days. A significant portion (20-50%) of the 2,4-DNP was metabolized to highly polar metabolite(s) with one major unidentified metabolite accumulating from 5 to 25% of the initial radioactivity. The amounts of 2,4-DNP mineralized and converted to polar metabolites was affected by glutamate concentration in the medium. Nitrophenolic compounds metabolized by UG30 were also suitable substrates for the UG30 PCP-4-monooxygenase (pcpB gene expressed in Escherichia coli) which is likely central to degradation of these compounds. The wide substrate range of UG30 could render this strain useful in bioremediation of some chemically contaminated soils.Key words: bioremediation, dinitrophenol, metabolism, nitroaromatic, pentachlorophenol, Sphingomonas.

Microsomal metabolism of carbamazepine and oxcarbazepine in liver and placenta

Metabolism of both carbamazepine (CBZ) and oxcarbaze-pine (OCBZ) were catalyzed by human liver microsomes and microsomes from livers of CBZ-induced or non-induced C57BL/6 mice. Human placental microsomes metabolized only OCBZ. Mouse liver microsomes metabolized CBZ to carbamazepine-10,11-epoxide (CBZ-E), 10- hydroxy-10,11-dihydro-carbamazepine (10-OH-CBZ), 3- hydroxy-carbamazepine (3-OH-CBZ), 10,11-trans-dihydroxy-10,11-dihydro-carbamazepine (10,11-D) and to an unidentified metabolite. CBZ-pretreatment of mice increased both ethoxyresorufin O-deethylase activity in the liver and the amount of CBZ-E in microsomal incubations regardless of the age of mice. Human liver microsomes catalyzed the formation of CBZ to 9-hydroxymethyl-10-carbamoyl acridan (9-AC) in addition to CBZ-E, 3-OH-CBZ and 10-OH-CBZ. OCBZ was metabolized to its active metabolite in all incubations. An unknown metabolite was also present in some of the incubations. Human liver microsomes catalyzed only minute covalent binding of CBZ and OCBZ to DNA. Binding of OCBZ was, however, one order of magnitude greater than binding of CBZ. Human placental micro-somes from the mothers on CBZ therapy did not catalyze CBZ metabolism. The same microsomes catalyzed OCBZ metabolism to 10-OH-CBZ and to an unknown metabolite. These results indicate autoinduction in CBZ metabolism in mouse liver. Due to the higher binding of OCBZ than CBZ to DNA in vitro, further studies on the potential mutagenicity of OCBZ may be warranted.

Liquid Chromatography-EIectrochemical Detection of Furazolidone and Metabolite in Extracts of Incurred Tissues

One-day-old chicks were raised to maturity on a diet fortified with 0.0055% furazolidone. Analyses of tissue extracts by a liquid chromatographic-electrochemlcal detection screening procedure for nltro-contalning drugs disclosed, in addition to the parent drug, an unidentified metabolite in the liver and breast tissue of the mature birds sacrificed while on the fortified feed. No evidence of residues of the drug or metabolite was found In birds removed from the medicated feed 48 h prior to sacrifice. In view of the rapid In vivo and postmortem metabolism of the parent drug In liver tissue, the metabolite can serve as an alternative means of detecting furazolidone residues In chicken tissues.

Methionine transport by mycelia of Fusarium oxysporum f. sp. lycopersici

Mycelia of Fusarium oxysporum f. sp. lycopersici accumulated L-methionine by an energy-dependent process, and the energy required for uptake may be derived from either respiration or glycolysis. The pH optimum for transport was 4 and the temperature optimum was 35 °C. The apparent Km for methionine uptake was 2.5–3.3 μM and the Vmax was 0.24–0.30 nmol∙min−1∙mg dry weight−1. S-adenosylhomocysteine (Ado-Hcys) was the major metabolic product of methionine although S-adenosylmethionine (Ado-Met), homocysteine (Hcys), and an unidentified metabolite (compound X) were also detected. The failure to demonstrate efflux of accumulated methionine in the presence of the uncoupler 2,4-dinitrophenol or excess unlabeled methionine was probably due to the fact that methionine was rapidly metabolized within the cells.Acidic and basic amino acids, and those amino acids having less than a four-carbon chain, did not inhibit methionine uptake. The rate of uptake of methionine, which was greatest in log phase mycelia, decreased substantially as the cells entered the stationary phase.

Absorption, Translocation, and Metabolism of 14C-Buthidazole in Alfalfa (Medicago sativa) and Quackgrass (Agropyon repens)

The patterns of buthidazole {3-[5-(1,1-dimethylethyl)-1,3,4-thiadiazol-2-yl]-4-hydroxy-1-methyl-2 imidazolidinone} uptake, translocation, and metabolism, and their potential contribution to crop selectivity, were studied in tolerant alfalfa (Medicago sativa L. ‘Vernal’) and susceptible quackgrass [Agropyron repens (L.) Beauv.]. 14C-Buthidazole was absorbed from a 5-μl droplet by leaves of both alfalfa and quackgrass plants, but did not move basipetally to the roots or nontreated leaves in 14 days. 14C-Buthidazole was absorbed very rapidly from Hoagland's solution by the roots of both species and translocated apoplastically to the leaves. Absorption and translocation of buthidazole did not differ between species, indicating that these processes did not contribute to crop selectivity. The herbicide and five metabolites were present in alfalfa leaf extracts 6 days after treatment. One unidentified metabolite accounted for 40% of the detected radioactivity 6 days after root application. Unmetabolized 14C-buthidazole accounted for 87% of the total radioactivity in quackgrass after 6 days. Metabolites having different chromatographic properties were recovered from the two species. Differential rate and type of metabolism appeared to contribute to buthidazole selectivity between the species.