Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA034156

Local Sample ID:	NAH16L1
Subject ID:	SU000639
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

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Sample Preparation:

Sampleprep ID:	SP000646
Sampleprep Summary:	Aqueous humor samples were subjected to extraction of lipids using suitable and minimal modification of Bligh and Dyer method. The lower organic phase containing the extracted lipids was isolated and solvent dried with a Speed-Vac (Model 7810014; Labconco, Kansas City, MO). Samples were subsequently flushed with argon gas to prevent oxidation. Proteins recovered from the corresponding upper aqueous phase were quantified using the Bradford method. A subset of protein samples were also subjected to densitometric quantification using bovine serum albumin (BSA) as a standard (amino acid quantified) after electrophoretic separation on a PHAST (GE Healthcare Bio-Sciences AB, Sweden) gel system. We repeated protein estimations using an amino acid analyzer after overnight digestion in hydrochloric acid following previously published protocols. The protein amounts determined using amino acid analyzer were utilized in normalization of lipids per amount of proteins. In order to determine and ensure extraction efficiency, ovine wool cholesterol (molecular mass 386.7; catalog no. 700000; Avanti Polar Lipids, Albaster, AL) was premixed with AH prior to extraction. All extractions and subsequent handling were made using glass vials to avoid contaminating impurities. The quantification lipid standards (all procured from Avanti Polar Lipids, Albaster, AL) we used were ditridecanoyl-sn-glycero-3-phosphocholine (molecular mass 649.89, catalog no. 850340) for PCs, 1,2-dioleoyl-snglycero- 3-phospho-L-serine (molecular mass 810.03, catalog no. 840035) for PSs, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (molecular mass 744.04, catalog no. 850725) for PEs and 1,2-dioleoyl-sn-glycero-3-phospho-(10-myo-inositol) (molecular mass 880.15, catalog no. 850149) for PIs.

Sampleprep ID:

SP000646

Sampleprep Summary:

Aqueous humor samples were subjected to extraction of lipids using suitable and minimal modification of Bligh and Dyer method. The lower organic phase containing the extracted lipids was isolated and solvent dried with a Speed-Vac (Model 7810014; Labconco, Kansas City, MO). Samples were subsequently flushed with argon gas to prevent oxidation. Proteins recovered from the corresponding upper aqueous phase were quantified using the Bradford method. A subset of protein samples were also subjected to densitometric quantification using bovine serum albumin (BSA) as a standard (amino acid quantified) after electrophoretic separation on a PHAST (GE Healthcare Bio-Sciences AB, Sweden) gel system. We repeated protein estimations using an amino acid analyzer after overnight digestion in hydrochloric acid following previously published protocols. The protein amounts determined using amino acid analyzer were utilized in normalization of lipids per amount of proteins. In order to determine and ensure extraction efficiency, ovine wool cholesterol (molecular mass 386.7; catalog no. 700000; Avanti Polar Lipids, Albaster, AL) was premixed with AH prior to extraction. All extractions and subsequent handling were made using glass vials to avoid contaminating impurities. The quantification lipid standards (all procured from Avanti Polar Lipids, Albaster, AL) we used were ditridecanoyl-sn-glycero-3-phosphocholine (molecular mass 649.89, catalog no. 850340) for PCs, 1,2-dioleoyl-snglycero- 3-phospho-L-serine (molecular mass 810.03, catalog no. 840035) for PSs, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (molecular mass 744.04, catalog no. 850725) for PEs and 1,2-dioleoyl-sn-glycero-3-phospho-(10-myo-inositol) (molecular mass 880.15, catalog no. 850149) for PIs.