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Oral Gavage Delivery of Stable Isotope Tracer for In Vivo Metabolomics.

ABSTRACT: Stable isotope-resolved metabolomics (SIRM) is a powerful tool for understanding disease. Advances in SIRM techniques have improved isotopic delivery and expanded the workflow from exclusively in vitro applications to in vivo methodologies to study systemic metabolism. Here, we report a simple, minimally-invasive and cost-effective method of tracer delivery to study SIRM in vivo in laboratory mice. Following a brief fasting period, we orally administered a solution of [U-¹³C] glucose through a blunt gavage needle without anesthesia, at a physiological dose commonly used for glucose tolerance tests (2 g/kg bodyweight). We defined isotopic enrichment in plasma and tissue at 15, 30, 120, and 240 min post-gavage. ¹³C-labeled glucose peaked in plasma around 15 min post-gavage, followed by period of metabolic decay and clearance until 4 h. We demonstrate robust enrichment of a variety of central carbon metabolites in the plasma, brain and liver of C57/BL6 mice, including amino acids, neurotransmitters, and glycolytic and tricarboxylic acid (TCA) cycle intermediates. We then applied this method to study in vivo metabolism in two distinct mouse models of diseases known to involve dysregulation of glucose metabolism: Alzheimer's disease and type II diabetes. By delivering [U-¹³C] glucose via oral gavage to the 5XFAD Alzheimer's disease model and the Lep^ob/ob type II diabetes model, we were able to resolve significant differences in multiple central carbon pathways in both model systems, thus providing evidence of the utility of this method to study diseases with metabolic components. Together, these data clearly demonstrate the efficacy and efficiency of an oral gavage delivery method, and present a clear time course for ¹³C enrichment in plasma, liver and brain of mice following oral gavage of [U-¹³C] glucose-data we hope will aid other researchers in their own ¹³C-glucose metabolomics study design.

SUBMITTER: Williams HC

PROVIDER: S-EPMC7764755 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Oral Gavage Delivery of Stable Isotope Tracer for In Vivo Metabolomics.

Williams Holden C HC Piron Margaret A MA Nation Grant K GK Walsh Adeline E AE Young Lyndsay E A LEA Sun Ramon C RC Johnson Lance A LA

Metabolites 20201208 12

Stable isotope-resolved metabolomics (SIRM) is a powerful tool for understanding disease. Advances in SIRM techniques have improved isotopic delivery and expanded the workflow from exclusively in vitro applications to in vivo methodologies to study systemic metabolism. Here, we report a simple, minimally-invasive and cost-effective method of tracer delivery to study SIRM in vivo in laboratory mice. Following a brief fasting period, we orally administered a solution of [U-<sup>13</sup>C] glucose ...[more]

PMID: 33302448

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Project description:Untargeted approaches and thus biological interpretation of metabolomics results are still hampered by the reliable assignment of the global metabolome as well as classification and (putative) identification of metabolites. In this work we present an liquid chromatography-mass spectrometry (LC-MS)-based stable isotope assisted approach that combines global metabolome and tracer based isotope labeling for improved characterization of (unknown) metabolites and their classification into tracer derived submetabolomes. To this end, wheat plants were cultivated in a customized growth chamber, which was kept at 400 ± 50 ppm 13CO2 to produce highly enriched uniformly 13C-labeled sample material. Additionally, native plants were grown in the greenhouse and treated with either 13C9-labeled phenylalanine (Phe) or 13C11-labeled tryptophan (Trp) to study their metabolism and biochemical pathways. After sample preparation, liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis and automated data evaluation, the results of the global metabolome- and tracer-labeling approaches were combined. A total of 1,729 plant metabolites were detected out of which 122 respective 58 metabolites account for the Phe- and Trp-derived submetabolomes. Besides m/z and retention time, also the total number of carbon atoms as well as those of the incorporated tracer moieties were obtained for the detected metabolite ions. With this information at hand characterization of unknown compounds was improved as the additional knowledge from the tracer approaches considerably reduced the number of plausible sum formulas and structures of the detected metabolites. Finally, the number of putative structure formulas was further reduced by isotope-assisted annotation tandem mass spectrometry (MS/MS) derived product ion spectra of the detected metabolites. A major innovation of this paper is the classification of the metabolites into submetabolomes which turned out to be valuable information for effective filtering of database hits based on characteristic structural subparts. This allows the generation of a final list of true plant metabolites, which can be characterized at different levels of specificity.

Project description:BackgroundKnowledge of the physiologic requirement for iron, the core index for the formulation of a dietary reference intake (DRI), is of great importance for the health of a pregnant woman and her fetus, and can help a mother accurately plan her iron supplementation. However, direct measurements of the physiologic requirement for iron during pregnancy are still lacking.MethodEleven women of reproductive age from Hebei Province, China, who planned to become pregnant in the near future, were enrolled between January and March 2015 and included in the final analysis. Subjects participated in a 2-week metabolic trial in which they consumed 50 mg of the stable isotope 58Fe, and were then followed for ~ 2 years. The abundance of 58Fe and the total iron concentration in the circulation were measured using Multi-collector Inductively-Coupled Plasma Mass Spectrometry and Atomic Absorption Spectroscopy, respectively. The physiologic requirement for iron during pregnancy was then calculated by the formula derived from our previously published work.ResultsThe mean physiologic requirement for iron in the 11 subjects, across their entire pregnancies, was 3.05 mg.d- 1 in total and 44.0 μg.kg- 1.d- 1 after adjustment for body mass. The physiologic requirements for iron in the first, second, and third trimesters were 2.04 mg.d- 1, 3.26 mg.d- 1, and 4.13 mg.d- 1, respectively. When adjusted for body mass, the physiologic requirements for iron in different trimesters were 32.3 μg.kg- 1.d- 1, 46.9 μg.kg- 1.d- 1, and 55.7 μg.kg- 1.d- 1, respectively.ConclusionWe preliminarily explored the physiologic requirement for iron in pregnant women. The data demonstrated that pregnant women needed about twice iron than non-pregnant women. This research may be helpful for the design of future studies and the modification of iron DRIs.Trial registrationChiCTR, ChiCTR-OCH-14004302. Registered 14 February 2014, http://www.chictr.org.cn/showproj.aspx?proj=5267.

Dataset Information

Oral Gavage Delivery of Stable Isotope Tracer for In Vivo Metabolomics.

Publications

Oral Gavage Delivery of Stable Isotope Tracer for In Vivo Metabolomics.

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