Summary of Study ST002497

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 PR001612. The data can be accessed directly via it's Project DOI: 10.21228/M8BB1T This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

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.

Study ID	ST002497
Study Title	Postnatal hyperglycemia alters amino acid profile in retinas
Study Summary	Nutritional deprivation occurring in most preterm infants postnatally, can induce hyperglycemia, a significant and independent risk factor for suppressing physiological retinal vascularization (Phase I retinopathy of prematurity (ROP)), leading to compensatory but pathological neovascularization. Amino acid supplementation reduces retinal neovascularization in mice. Little is known about amino acid contribution to Phase I ROP. Significant changes in retinal amino acids (including most decreased L-leucine, L-isoleucine and L-valine) were found in mice modeling hyperglycemia-associated Phase I ROP, and parenteral (i.p.) L-isoleucine suppressed physiological retinal vascularization. In premature infants, severe ROP was associated with a higher mean intake of parenteral versus enteral amino acids in the first two weeks of life after adjustment for treatment group, gestational age at birth, birth weight and sex. The number of days with parenteral amino acids support independently predicted severe ROP. Further understanding and modulating amino acids may help improve nutritional intervention and prevent Phase I ROP
Institute	Boston Children's Hospital
Last Name	Fu
First Name	Zhongjie
Address	1 Blackfan Circle, Boston, MA 02114
Email	Zhongjie.Fu@childrens.harvard.edu
Phone	617-919-2534
Submit Date	2023-02-16
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2023-03-22
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001612
Project DOI:	doi: 10.21228/M8BB1T
Project Title:	Postnatal hyperglycemia alters amino acid profile in retinas (model of Phase I ROP)
Project Summary:	Nutritional deprivation occurring in most preterm infants postnatally, can induce hyperglycemia, a significant and independent risk factor for suppressing physiological retinal vascularization (Phase I retinopathy of prematurity (ROP)), leading to compensatory but pathological neovascularization. Amino acid supplementation reduces retinal neovascularization in mice. Little is known about amino acid contribution to Phase I ROP. Significant changes in retinal amino acids (including most decreased L-leucine, L-isoleucine and L-valine) were found in mice modeling hyperglycemia-associated Phase I ROP, and parenteral (i.p.) L-isoleucine suppressed physiological retinal vascularization. In premature infants, severe ROP was associated with a higher mean intake of parenteral versus enteral amino acids in the first two weeks of life after adjustment for treatment group, gestational age at birth, birth weight and sex. The number of days with parenteral amino acids support independently predicted severe ROP. Further understanding and modulating amino acids may help improve nutritional intervention and prevent Phase I ROP
Institute:	Boston Childrens Hospital
Last Name:	Fu
First Name:	Zhongjie
Address:	1 Blackfan Circle, Boston, MA 02114
Email:	Zhongjie.Fu@childrens.harvard.edu
Phone:	617-919-2534

Subject:

Subject ID:	SU002594
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

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

mb_sample_id	local_sample_id	Group
SA249794	P10N ctrl_3	control group mouse retinas
SA249795	P10N ctrl_2	control group mouse retinas
SA249796	P10N ctrl_4	control group mouse retinas
SA249797	P10N ctrl_5	control group mouse retinas
SA249798	P10N ctrl_6	control group mouse retinas
SA249799	P10N ctrl_1	control group mouse retinas
SA249788	P10HAR_3	HAR mouse retinas
SA249789	P10HAR_4	HAR mouse retinas
SA249790	P10HAR_6	HAR mouse retinas
SA249791	P10HAR_2	HAR mouse retinas
SA249792	P10HAR_5	HAR mouse retinas
SA249793	P10HAR_1	HAR mouse retinas

Showing results 1 to 12 of 12

Showing results 1 to 12 of 12

Collection:

Collection ID:	CO002587
Collection Summary:	Retinas were collected at P10 following a single incision across the sclera and immediately snap frozen in liquid nitrogen and stored at -80 ºC until sample processing 2. Samples were processed and analyzed by LC-MS/MS by the NYU Metabolomics Core Resource Laboratory, New York, NY, USA, as described previously 3,4. Briefly, samples were homogenized using a bead blaster for 10 cycles with 30 seconds on and 30 seconds off. Metabolites were extracted using 80% methanol and dried down using a speedvac. Next, samples were reconstituted in 50 µL MS-grade water and sonicated for two minutes. Samples were then spun down in a centrifuge at 10 G for 4 min and finally transferred to MS vials for analysis. Internal standards were used for correction of retention time and identification of metabolites. Six retinas were pooled as one replicate to reduce biological variability for metabolomics analysis in each group, n=6 per group (HAR vs. control).
Sample Type:	Retina

Treatment:

Treatment ID:	TR002606
Treatment Summary:	To study the metabolic alterations occurring in hyperglycemia-associated Phase I ROP, we applied quantitative metabolomics and proteomics on mouse retinas from HAR and normal control mice. C57BL/6J (Jackson Laboratory, Bar Harbor, ME) mice, of each sex, aged 10-12 weeks, were purchased, housed and bred in the institutional vivarium and maintained on a 12hour/12hour light/dark cycle with mouse chow provided ad libitum. Neonatal mice were randomly assigned to experimental groups. Induction of hyperglycemia was accomplished as previously described 1. Neonatal mice were intraperitoneally injected with 50mg/kg/day STZ consecutively from P1 to P9 using a 34-G needle (Hamilton syringe) (Fig. 1B). Vehicle control animals received equal volumes of vehicle phosphate-buffered saline (PBS, Gibco, Waltham, MA). Hyperglycemia is induced around P8 and delayed retinal vascularization is found at P10 1. Mice with weight range 4 to 5 grams were used for further metabolomics and proteomics analysis. Mouse litters were randomly assigned to HAR or control groups, both sexes were used. The cages were located at close spots to minimize the potential housing influences. All procedures were approved by our Institutional Animal Care and Use Committee and adhered to ARRIVE guidelines and the NIH Guide for the Care and Use of Laboratory Animals. With conditions tested with β=0.8 and α=0.05, at least n=6 per group will be needed for the analysis. Control was re-named as group 1 and HAR was re-named as group 2 for analysis.

Treatment ID:

TR002606

Treatment Summary:

To study the metabolic alterations occurring in hyperglycemia-associated Phase I ROP, we applied quantitative metabolomics and proteomics on mouse retinas from HAR and normal control mice. C57BL/6J (Jackson Laboratory, Bar Harbor, ME) mice, of each sex, aged 10-12 weeks, were purchased, housed and bred in the institutional vivarium and maintained on a 12hour/12hour light/dark cycle with mouse chow provided ad libitum. Neonatal mice were randomly assigned to experimental groups. Induction of hyperglycemia was accomplished as previously described 1. Neonatal mice were intraperitoneally injected with 50mg/kg/day STZ consecutively from P1 to P9 using a 34-G needle (Hamilton syringe) (Fig. 1B). Vehicle control animals received equal volumes of vehicle phosphate-buffered saline (PBS, Gibco, Waltham, MA). Hyperglycemia is induced around P8 and delayed retinal vascularization is found at P10 1. Mice with weight range 4 to 5 grams were used for further metabolomics and proteomics analysis. Mouse litters were randomly assigned to HAR or control groups, both sexes were used. The cages were located at close spots to minimize the potential housing influences. All procedures were approved by our Institutional Animal Care and Use Committee and adhered to ARRIVE guidelines and the NIH Guide for the Care and Use of Laboratory Animals. With conditions tested with β=0.8 and α=0.05, at least n=6 per group will be needed for the analysis. Control was re-named as group 1 and HAR was re-named as group 2 for analysis.

Sample Preparation:

Sampleprep ID:	SP002600
Sampleprep Summary:	Both retinas from each mouse were collected, pooled, and prepared for targeted MS-based proteomics as described previously 1. After sample preparation, LC-tandem MS analysis was performed using selected reaction monitoring (SRM) 5 on a Thermo Scientific TSQ Vantage mass spectrometer equipped with an Eksigent splitless nanoflow HPLC system. 7 µL aliquots of each sample were injected onto a 10 cm x 75 µm i.d. capillary column packed with Phenomenex Jupiter C18 reversed phase beads. The column was eluted at 150 nL/min with a 60 min linear gradient of acetonitrile in 0.1% formic acid. The SRM assays were developed and validated to monitor two peptides per protein. Each peptide was monitored in a 6-min window centered on the known elution time of the peptide 6-8. Approximately 30 protein assays are grouped into a panel of proteins that are measured in a single LC-tandem MS run.

Sampleprep ID:

SP002600

Sampleprep Summary:

Both retinas from each mouse were collected, pooled, and prepared for targeted MS-based proteomics as described previously 1. After sample preparation, LC-tandem MS analysis was performed using selected reaction monitoring (SRM) 5 on a Thermo Scientific TSQ Vantage mass spectrometer equipped with an Eksigent splitless nanoflow HPLC system. 7 µL aliquots of each sample were injected onto a 10 cm x 75 µm i.d. capillary column packed with Phenomenex Jupiter C18 reversed phase beads. The column was eluted at 150 nL/min with a 60 min linear gradient of acetonitrile in 0.1% formic acid. The SRM assays were developed and validated to monitor two peptides per protein. Each peptide was monitored in a 6-min window centered on the known elution time of the peptide 6-8. Approximately 30 protein assays are grouped into a panel of proteins that are measured in a single LC-tandem MS run.

Combined analysis:

Analysis ID	AN004101
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Ultimate 3000
Column	SeQuant ZIC-pHILIC (150 x 2.1mm, 5um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap
Ion Mode	UNSPECIFIED
Units	Absolute Intensity

Chromatography:

Chromatography ID:	CH003036
Instrument Name:	Thermo Ultimate 3000
Column Name:	SeQuant ZIC-pHILIC (150 x 2.1mm, 5um)
Column Temperature:	25
Flow Gradient:	80-20%B (0-30 min), 20-80%B (30-31 min), 80-80%B (31-42 min)
Flow Rate:	0.1mL/min
Solvent A:	10 mM ammonium carbonate in water, pH 9.0
Solvent B:	acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS003848
Analysis ID:	AN004101
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Initial data analysis (metabolite identification & quantification) was performed by the NYU metabolomics facility 3,9-11. Subsequent downstream bioinformatic analysis was performed using MetaboAnalyst R-based statistical and pathway analysis (v5.0) as described by Petrova et al. 12 using updates as described by Pang et al. 13. Differences in metabolite levels between groups were assessed using unpaired t test and considered statistically significant if P<0.05. For pathway analysis SMPDB database was reviewed, applying Fisher’s Exact Test for mapping. Only metabolites meeting P-value criteria were loaded for the analysis.
Ion Mode:	UNSPECIFIED