{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000620","ANALYSIS_ID":"AN000952","VERSION":"1","CREATED_ON":"June 15, 2017, 2:42 pm"},

"PROJECT":{"PROJECT_TITLE":"DBA2J Mice Aqueous Humor and Trabecular Meshwork Sphingolipids","PROJECT_SUMMARY":"Identified the sphingolipid and ceramide species and their quantitative differences between normotensive and hypertensive intraocular pressure states in DBA/2J mouse aqueous humor (AH) and trabecular meshwork (TM).","INSTITUTE":"University of Miami","LAST_NAME":"Bhattacharya","FIRST_NAME":"Sanjoy","ADDRESS":"1638 NW 10th Avenue, Miami, Florida, 33136, USA","EMAIL":"sbhattacharya@med.miami.edu","PHONE":"3054824103"},

"STUDY":{"STUDY_TITLE":"Sphingolipid Analysis of hyper and normotensive DBA2J mice aqueous humor and trabecular meshwork","STUDY_SUMMARY":"To determine the differential profiles of sphingomyelin, sphingoid base, sphingoid base-1-phosphate, and ceramide and their quantitative differences between trabecular meshwork (TM) and aqueous humor (AH) derived from normotensive and hypertensive intraocular pressure states of DBA/2J mice.","INSTITUTE":"University of Miami","LAST_NAME":"Bhattacharya","FIRST_NAME":"Sanjoy","ADDRESS":"McKnight Vision Research Building, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 1638 NW 10th Avenue, Room 706A, Miami, Florida 33136","EMAIL":"SBhattacharya@med.miami.edu","PHONE":"305-482-4103"},

"SUBJECT":{"SUBJECT_TYPE":"Mouse","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"3M_FAH",
"Factors":{"Phenotype":"Normotensive"},
"Additional sample data":{"Tissue Location":"AH","Age (months)":"3","Sex":"F"}
},
{
"Subject ID":"-",
"Sample ID":"3M_FTM",
"Factors":{"Phenotype":"Normotensive"},
"Additional sample data":{"Tissue Location":"TM","Age (months)":"3","Sex":"F"}
},
{
"Subject ID":"-",
"Sample ID":"3M_MAH",
"Factors":{"Phenotype":"Normotensive"},
"Additional sample data":{"Tissue Location":"AH","Age (months)":"3","Sex":"M"}
},
{
"Subject ID":"-",
"Sample ID":"3M_MTM",
"Factors":{"Phenotype":"Normotensive"},
"Additional sample data":{"Tissue Location":"TM","Age (months)":"3","Sex":"M"}
},
{
"Subject ID":"-",
"Sample ID":"7.5M_FAH",
"Factors":{"Phenotype":"Normotensive"},
"Additional sample data":{"Tissue Location":"AH","Age (months)":"7.5","Sex":"F"}
},
{
"Subject ID":"-",
"Sample ID":"7.5M_FTM",
"Factors":{"Phenotype":"Normotensive"},
"Additional sample data":{"Tissue Location":"TM","Age (months)":"7.5","Sex":"F"}
},
{
"Subject ID":"-",
"Sample ID":"7.5M_MAH",
"Factors":{"Phenotype":"Normotensive"},
"Additional sample data":{"Tissue Location":"AH","Age (months)":"7.5","Sex":"M"}
},
{
"Subject ID":"-",
"Sample ID":"7.5M_MTM",
"Factors":{"Phenotype":"Normotensive"},
"Additional sample data":{"Tissue Location":"TM","Age (months)":"7.5","Sex":"M"}
},
{
"Subject ID":"-",
"Sample ID":"8-8.5M_FAH",
"Factors":{"Phenotype":"Hypertensive"},
"Additional sample data":{"Tissue Location":"AH","Age (months)":"8-8.5","Sex":"F"}
},
{
"Subject ID":"-",
"Sample ID":"8-8.5M_FTM",
"Factors":{"Phenotype":"Hypertensive"},
"Additional sample data":{"Tissue Location":"TM","Age (months)":"8-8.5","Sex":"F"}
},
{
"Subject ID":"-",
"Sample ID":"8-8.5M_MAH",
"Factors":{"Phenotype":"Hypertensive"},
"Additional sample data":{"Tissue Location":"AH","Age (months)":"8-8.5","Sex":"M"}
},
{
"Subject ID":"-",
"Sample ID":"8-8.5M_MTM",
"Factors":{"Phenotype":"Hypertensive"},
"Additional sample data":{"Tissue Location":"TM","Age (months)":"8-8.5","Sex":"M"}
},
{
"Subject ID":"-",
"Sample ID":"10M_FAH",
"Factors":{"Phenotype":"Hypertensive"},
"Additional sample data":{"Tissue Location":"AH","Age (months)":"10","Sex":"F"}
},
{
"Subject ID":"-",
"Sample ID":"10M_FTM",
"Factors":{"Phenotype":"Hypertensive"},
"Additional sample data":{"Tissue Location":"TM","Age (months)":"10","Sex":"F"}
},
{
"Subject ID":"-",
"Sample ID":"10M_MAH",
"Factors":{"Phenotype":"Hypertensive"},
"Additional sample data":{"Tissue Location":"AH","Age (months)":"10","Sex":"M"}
},
{
"Subject ID":"-",
"Sample ID":"10M_MTM",
"Factors":{"Phenotype":"Hypertensive"},
"Additional sample data":{"Tissue Location":"TM","Age (months)":"10","Sex":"M"}
},
{
"Subject ID":"-",
"Sample ID":"12M_FAH",
"Factors":{"Phenotype":"Hypertensive"},
"Additional sample data":{"Tissue Location":"AH","Age (months)":"12","Sex":"F"}
},
{
"Subject ID":"-",
"Sample ID":"12M_FTM",
"Factors":{"Phenotype":"Hypertensive"},
"Additional sample data":{"Tissue Location":"TM","Age (months)":"12","Sex":"F"}
},
{
"Subject ID":"-",
"Sample ID":"12M_MAH",
"Factors":{"Phenotype":"Hypertensive"},
"Additional sample data":{"Tissue Location":"AH","Age (months)":"12","Sex":"M"}
},
{
"Subject ID":"-",
"Sample ID":"12M_MTM",
"Factors":{"Phenotype":"Hypertensive"},
"Additional sample data":{"Tissue Location":"TH","Age (months)":"12","Sex":"M"}
}
],
"COLLECTION":{"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_SUMMARY":"No treatment was applied"},

"SAMPLEPREP":{"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)."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"None (Direct infusion)","INSTRUMENT_NAME":"None","COLUMN_NAME":"none"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Thermo Quantiva Access Max","INSTRUMENT_TYPE":"Triple quadrupole","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED"},

"MS_METABOLITE_DATA":{
"Units":"Peak Area",

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}

}