Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA034998

Local Sample ID:	8-8.5M_MAH
Subject ID:	SU000643
Subject Type:	Mouse
Subject Species:	Mus musculus
Taxonomy ID:	10090
Species Group:	Mammal

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Subject:

Subject ID:	SU000643
Subject Type:	Mouse
Subject Species:	Mus musculus
Taxonomy ID:	10090
Species Group:	Mammal

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
8-8.5M_MAH	SA034998	FL007482	Hypertensive	Phenotype

Collection:

Collection ID:	CO000637
Collection Summary:	Breeding pairs of DBA/2J mice originally procured from The Jackson Laboratory (Bar Harbor, ME) were maintained at the animal facility/McKnight vivarium at University of Miami Miller School of Medicine. All animal protocols were reviewed and approved by Institutional Animal Care and Use Committee (IACUC) and in accordance with the ARVO statement for use of animals in ophthalmic and vision research. Mice gender and ages used are as indicated in individual experiments with an n=40 for each time point unless stated otherwise. IOP measurements were obtained using a TonoLab instrument (Colonial Medical Supplies, NH). Average IOP from two weeks of twice daily measurements were calculated. We considered ~3-7.5 month old mice as normotensive (IOP ≤15 mm of Hg) and ~8-12 month old mice as hypertensive when carefully measured IOP showed an IOP ≥18mm of Hg at every single measurement. For the normotensive group no single IOP measurement had been >17 mm of Hg and average IOP over two weeks of measurement had been ≤15 mm of Hg. At 8-9 months of age the IOP elevation in DBA/2J mice is asynchronous and thus any mice that showed an IOP <18 mm of Hg at any single point measurement was excluded from the study. Normotensive and hypertensive AH was sampled from the anterior chamber of DBA/2J mice by paracentesis via syringe (catalog no. 7635-01 Hamilton, Reno, Nevada) with a small 33 gauge removable needle (701RN, catalog no. 7803-05 Hamilton, Reno, Nevada), which is a well established procedure. The syringe and needle were rinsed with LC-MS grade water 5 times preceding and following extraction to flush out impurities. To ensure that samples are contamination free, in between the samples, the wash from a syringe was also evaluated using mass spectrometry. Prior to collection, mice were anesthetized with an injection (0.1ml) of ketamine (100mg/kg) and xylazine (9mg/kg) administered intraperitoneally. A drop of tetracaine hydrochloride ophthalmic solution, 0.5% (Bausch & Lomb, USA) was used to anesthetize the ocular surface just prior to sample collection. A total of ~1-2 μl of AH was collected per animal and used for lipid extraction. Surgical removal of TM samples after euthanasia followed established protocols.

Collection ID:

CO000637

Collection Summary:

Breeding pairs of DBA/2J mice originally procured from The Jackson Laboratory (Bar Harbor, ME) were maintained at the animal facility/McKnight vivarium at University of Miami Miller School of Medicine. All animal protocols were reviewed and approved by Institutional Animal Care and Use Committee (IACUC) and in accordance with the ARVO statement for use of animals in ophthalmic and vision research. Mice gender and ages used are as indicated in individual experiments with an n=40 for each time point unless stated otherwise. IOP measurements were obtained using a TonoLab instrument (Colonial Medical Supplies, NH). Average IOP from two weeks of twice daily measurements were calculated. We considered ~3-7.5 month old mice as normotensive (IOP ≤15 mm of Hg) and ~8-12 month old mice as hypertensive when carefully measured IOP showed an IOP ≥18mm of Hg at every single measurement. For the normotensive group no single IOP measurement had been >17 mm of Hg and average IOP over two weeks of measurement had been ≤15 mm of Hg. At 8-9 months of age the IOP elevation in DBA/2J mice is asynchronous and thus any mice that showed an IOP <18 mm of Hg at any single point measurement was excluded from the study. Normotensive and hypertensive AH was sampled from the anterior chamber of DBA/2J mice by paracentesis via syringe (catalog no. 7635-01 Hamilton, Reno, Nevada) with a small 33 gauge removable needle (701RN, catalog no. 7803-05 Hamilton, Reno, Nevada), which is a well established procedure. The syringe and needle were rinsed with LC-MS grade water 5 times preceding and following extraction to flush out impurities. To ensure that samples are contamination free, in between the samples, the wash from a syringe was also evaluated using mass spectrometry. Prior to collection, mice were anesthetized with an injection (0.1ml) of ketamine (100mg/kg) and xylazine (9mg/kg) administered intraperitoneally. A drop of tetracaine hydrochloride ophthalmic solution, 0.5% (Bausch & Lomb, USA) was used to anesthetize the ocular surface just prior to sample collection. A total of ~1-2 μl of AH was collected per animal and used for lipid extraction. Surgical removal of TM samples after euthanasia followed established protocols.

Treatment:

Treatment ID:	TR000657
Treatment Summary:	No treatment was applied

Sample Preparation:

Sampleprep ID:	SP000650
Sampleprep Summary:	AH and TM samples were subjected to lipid extraction using suitable modification of the Bligh and Dryer method. All extractions were made without addition of any extraneous ions such as lithium or acetate ions unless stated otherwise. Some confirmatory additional experiments were performed, where 1mM LiCl or 1mM ammonium acetate (for positive and negative ion mode analyses, respectively) was added to the aliquot being analyzed just before analyses. The samples were dried with a Speed-Vac and flushed with argon gas. Uniformity of extraction was ensured with an added external standard (1, 2-ditridecanoyl-snglycero-3-phosphocholine) during tissue homogenization. All extractions and subsequent handling were done using glass vials to avoid contaminating impurities. Extracted lipids were dried and resuspended in LC-MS grade Acetonitrile:Isopropanol (1:1).

Sampleprep ID:

SP000650

Sampleprep Summary:

AH and TM samples were subjected to lipid extraction using suitable modification of the Bligh and Dryer method. All extractions were made without addition of any extraneous ions such as lithium or acetate ions unless stated otherwise. Some confirmatory additional experiments were performed, where 1mM LiCl or 1mM ammonium acetate (for positive and negative ion mode analyses, respectively) was added to the aliquot being analyzed just before analyses. The samples were dried with a Speed-Vac and flushed with argon gas. Uniformity of extraction was ensured with an added external standard (1, 2-ditridecanoyl-snglycero-3-phosphocholine) during tissue homogenization. All extractions and subsequent handling were done using glass vials to avoid contaminating impurities. Extracted lipids were dried and resuspended in LC-MS grade Acetonitrile:Isopropanol (1:1).

Combined analysis:

Analysis ID	AN000952
Analysis type	MS
Chromatography type	None (Direct infusion)
Chromatography system	None
Column	none
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	Thermo Quantiva Access Max
Ion Mode	UNSPECIFIED
Units	Peak Area

Chromatography:

Chromatography ID:	CH000677
Instrument Name:	None
Column Name:	none
Chromatography Type:	None (Direct infusion)

MS:

MS ID:	MS000847
Analysis ID:	AN000952
Instrument Name:	Thermo Quantiva Access Max
Instrument Type:	Triple quadrupole
MS Type:	ESI
Ion Mode:	UNSPECIFIED