Summary of Study ST001130

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000755. The data can be accessed directly via it's Project DOI: 10.21228/M8367J This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST001130
Study TitleUrea cycle and 1C/serine metabolism in the prevention of oxygen induced retinopathy
Study SummaryUntargeted metabolite profiling links the urea cycle and 1C/serine metabolism in the prevention of oxygen induced retinopathy by hepatic HIF stabilization. Premature infants require oxygen supplementation to survive that is simultaneously toxic to developing tissues. We have demonstrated that hypoxia inducible factor (HIF) stabilization during hyperoxia prevents oxygen induced retinopathy (OIR) and lung disease. Here, untargeted metabolite profiling coupled to XCMS systems biology analysis finds that serine/1C and urea cycles dominate pathway enrichment graphs. MS1 peak areas and MS2 library matches reveal 50% or more increased levels of plasma and retina serine, glycine, hypotaurine, methionine, and taurine. In addition, N-acetylglutamate increased 4-fold in serum, while orotate, citrulline, arginine, aspartate, glutamine were at least 50% increased after HIF stabilization. Targeted data analysis in vivo finds that retinal serine and glycine were derived from liver. HIF-1α2lox/2lox; albumin-cre KO had reduced levels of serine and retinal glycine. Inhibition of 1C metabolism blocked rescue by HIF stabilization. The metabolic phenotype of mice protected from OIR by HIF stabilization is dependent on hepatic serine/1C metabolism and urea cycle.
Institute
Cole Eye Institute
DepartmentCleveland Clinic
Last NameSingh
First NameCharandeep
Address9500 Euclid Avenue
Emailcxs065@gmail.com
Phone(216) 444-8232
Submit Date2019-01-31
Analysis Type DetailLC-MS
Release Date2020-06-20
Release Version1
Charandeep Singh Charandeep Singh
https://dx.doi.org/10.21228/M8367J
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000755
Project DOI:doi: 10.21228/M8367J
Project Title:Plasma metabolite profiling
Project Type:MS untargeted data
Project Summary:Untargeted LC- data-dependent MS2 in positive mode to find difference in OIR model mice and FG-4592 treated mice plasma.
Institute:Cole Eye Institute
Department:Cleveland Clinic
Laboratory:Dr. Jonathan Sears lab
Last Name:Singh
First Name:Charandeep
Address:9500 Euclid Avenue, Cleveland, Select One, 44195, USA
Email:cxs065@gmail.com
Phone:(216) 444-8232

Subject:

Subject ID:SU001191
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6J

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Treatment
SA078083Pos_hyper3incluMS2Hyperoxia
SA078084Pos_hyper4incluMS2Hyperoxia
SA078085Pos_hyper6incluMS2Hyperoxia
SA078086Pos_hyper2incluMS2Hyperoxia
SA078087Pos_hyper5incluMS2Hyperoxia
SA078088Pos_hyper1incluMS2Hyperoxia
SA078089Pos_FG1incluMS2Hyperoxia+FG4592
SA078090Pos_FG2incluMS2Hyperoxia+FG4593
SA078091Pos_FG3incluMS2Hyperoxia+FG4594
SA078092Pos_FG4incluMS2Hyperoxia+FG4595
SA078093Pos_FG5incluMS2Hyperoxia+FG4596
SA078094Pos_FG6incluMS2Hyperoxia+FG4597
Showing results 1 to 12 of 12

Collection:

Collection ID:CO001185
Collection Summary:To extract the blood samples, mice were anesthetized with ketamine/xylazine mix and then blood was extracted from the heart of the animal with a 27.5 gauge needle fixed onto a 1cc heparinized syringe and added to 1.5 ml tubes containing 1.5 µl of Heparin 10,000 USP units/ml (Hospira, Lake forest, IL, USA) and stored on ice until further use. Plasma was obtained from blood samples by centrifuging samples at 1,100 x g at 4⁰C for 20 min.
Sample Type:Blood (plasma)
Storage Conditions:-80℃

Treatment:

Treatment ID:TR001206
Treatment Summary:All experimental procedures involving live animals were conducted in accordance with the guidelines of the NIH Guide for the Care and Use of Laboratory Animals and approved by the Cleveland Clinic institutional animal care and use committee (IACUC, protocol #2016-1677). Wild type C57BL/6J mice were supplied by the Jackson Laboratory (Bar Harbor, ME). OIR model was based on the previously described procedure developed by LE Smith(Smith et al, 1994) and was described in detail elsewhere(Hoppe et al, 2014b; Sears et al, 2008; Singh et al, 2018). Metabolomics experiment was performed on p10 old OIR model mice Smith LE, Wesolowski E, McLellan A, Kostyk SK, D'Amato R, Sullivan R, D'Amore PA (1994) Oxygen-induced retinopathy in the mouse. Investigative ophthalmology & visual science 35: 101-111 Sears JE, Hoppe G, Ebrahem Q, Anand-Apte B (2008) Prolyl hydroxylase inhibition during hyperoxia prevents oxygen-induced retinopathy. Proceedings of the National Academy of Sciences 105: 19898-19903 Singh C, Sharma A, Hoppe G, Song W, Bolok Y, Sears JE (2018) 3-Hydroxypyruvate Destabilizes Hypoxia Inducible Factor and Induces Angiostasis. Investigative Ophthalmology & Visual Science 59: 3440-3448

Sample Preparation:

Sampleprep ID:SP001199
Sampleprep Summary:Metabolites from plasma were extracted by adding 20 µl of plasma to 500 µl of 50% cold acetonitrile. Samples were centrifuged at 15,000 x g for 10 min and 400 µl supernatant was carefully removed into fresh tubes without disturbing the cell pellet. Supernatants were injected directly into the LC-MS

Combined analysis:

Analysis ID AN001855
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Vanquish
Column SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap
Ion Mode POSITIVE
Units Area under the curve Arbitrary units

Chromatography:

Chromatography ID:CH001342
Chromatography Summary:Chromatographic separation was performed on SeQuant ZIC-HILIC column with dimensions 150 x 2.1 mm, 3.5 µm, 100 Å (Merck, Darmstadt, Germany) attached to a precolumn SeQuant ZIC-HILIC with dimensions 20 x 2.1 mm, 3.5 µm, 100 Å (Merck, Darmstadt, Germany). The LC method used for separation of metabolites using the ZIC-HILIC column was adapted from Singh et al. al.(Singh et al, 2017). Briefly, gradient of solvent A (0.1% formic acid in water) and solvent B (0.08% formic acid in ACN) ramped from 80% B to 35% B in 23 minutes, followed by a wash step with 5% B from 25-30 min and then re-equilibration with 80% B from 25-30 min. Column oven was set to 20⁰C and a constant flow of solvents was set to 150 µl min-1. Samples were kept on 4⁰C auto-sampler throughout the measurements and 10 μl (plasma) of sample was injected. Singh C, Glaab E, Linster CL (2017) Molecular Identification of d-Ribulokinase in Budding Yeast and Mammals. The Journal of biological chemistry 292: 1005-1028
Instrument Name:Thermo Vanquish
Column Name:SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)
Column Temperature:20
Flow Gradient:ramped from 80% B to 35% B in 23 minutes, followed by a wash step with 5% B from 25-30 min and then re-equilibration with 80% B from 25-30 min.
Flow Rate:150 µl/min
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.08% formic acid
Chromatography Type:HILIC

MS:

MS ID:MS001715
Analysis ID:AN001855
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:LC-MS data from ddMS2 measurement were converted to mzML format using compound discoverer (version 2.1) (Thermo Scientific, Bellefonte, PA, USA) Data were analyzed using XCMS online tool(Forsberg et al, 2018). The metabolites were mapped onto the mouse database available on XCMS(Forsberg et al, 2018). Data was also analyzed with MSdial tool to confirm some of the metabolite annotations, using library provided with the MSdial software(Lai et al, 2017; Tsugawa et al, 2015). Forsberg EM, Huan T, Rinehart D, Benton HP, Warth B, Hilmers B, Siuzdak G (2018) Data processing, multi-omic pathway mapping, and metabolite activity analysis using XCMS Online. Nat Protoc 13: 633-651 Lai Z, Tsugawa H, Wohlgemuth G, Mehta S, Mueller M, Zheng Y, Ogiwara A, Meissen J, Showalter M, Takeuchi K, Kind T, Beal P, Arita M, Fiehn O (2017) Identifying metabolites by integrating metabolome databases with mass spectrometry cheminformatics. Nature methods 15: 53 Tsugawa H, Cajka T, Kind T, Ma Y, Higgins B, Ikeda K, Kanazawa M, VanderGheynst J, Fiehn O, Arita M (2015) MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis. Nature methods 12: 523-526
Ion Mode:POSITIVE
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