Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA125583

Local Sample ID:	13202411
Subject ID:	SU001564
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	18-45
Gender:	Male and female
Human Nutrition:	Low glycemic load and high glycemic load diet
Human Inclusion Criteria:	We recruited non-smoking, healthy individuals between the ages of 18-45 years from the Greater Seattle area.
Human Exclusion Criteria:	Exclusion criteria consisted of impaired fasting glucose (fasting blood glucose ≥5.6 mmol/L), any physician-diagnosed condition requiring a restricted diet, food allergies, regular use of hormones or anti-inflammatory medication, current pregnancy or lactation or plans to become pregnant, or heavy use of alcohol (>2 drinks/d)

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Sample Preparation:

Sampleprep ID:	SP001572
Sampleprep Summary:	Lipidomics Sample Preparation and Mass Spectrometry Frozen plasma samples were thawed at room temperature (25 °C) for 30 min, vortexed, and 25 µL plasma was transferred to a borosilicate glass culture tube (16 x 100 mm). Next, 0.475 mL water, 1.45 mL 1:0.45 methanol:dichloromethane, and 25 µL isotope-labeled internal standards mixture were added to the tube. The Lipidyzer isotope labeled internal standards mixture consisted of 54 isotopes from 13 lipid classes (Sciex, Framingham, MA). The mixture was vortexed for 5 sec and incubated at room temperature for 30 min. Next, another 0.5 mL water and 0.45 mL dichloromethane were added to the tube, followed by gentle vortexing for 5 sec, and centrifugation at 2500 g at 15 °C for 10 min. The bottom organic layer was transferred to a new tube and 0.9 mL of dichloromethane was added to the original tube for a second extraction. The combined extracts were concentrated under nitrogen and reconstituted in 0.25 mL of the mobile phase (10 mM ammonium acetate in 50:50 methanol:dichloromethane). Quantitative lipidomics was performed with the Sciex Lipidyzer platform consisting of Shimadzu Nexera X2 LC-30AD pumps, a Shimadzu Nexera X2 SIL-30AC autosampler, and a Sciex QTRAP® 5500 mass spectrometer equipped with SelexION® for differential mobility spectrometry (DMS). 1-propanol was used as the chemical modifier for the DMS. Samples were introduced to the mass spectrometer by flow injection at 8 µL/min. Each sample was injected twice, once with the DMS on [phosphatidylcholines (PC); phosphatidylethanolamines (PE); lysophosphatidylcholines (LPC); lysophosphatidylethanolamines (LPE); sphingomyelins (SM)], and once with the DMS off ([cholesterol esters (CE); ceramides (CER); diacylglycerols (DAG); dihydroceramides (DCER)/ free fatty acids(FFA); hexosylceramides (HCER); lactosylceramides (LCER); triacylglycerols (TAG)]. The lipid molecular species were measured using multiple reaction monitoring and positive/negative polarity switching. Positive ion mode detected lipid classes SM, DAG, CE, CER, DCER, HCER, DCER, and TAG, and negative ion mode detected lipid classes LPE, LPC, PC, PE, and FFA. A total of 1070 lipids and fatty acids were targeted in the analysis. Data was acquired and processed using Analyst 1.6.3 and Lipidomics Workflow Manager 1.0.5.0.

Sampleprep ID:

SP001572

Sampleprep Summary:

Lipidomics Sample Preparation and Mass Spectrometry Frozen plasma samples were thawed at room temperature (25 °C) for 30 min, vortexed, and 25 µL plasma was transferred to a borosilicate glass culture tube (16 x 100 mm). Next, 0.475 mL water, 1.45 mL 1:0.45 methanol:dichloromethane, and 25 µL isotope-labeled internal standards mixture were added to the tube. The Lipidyzer isotope labeled internal standards mixture consisted of 54 isotopes from 13 lipid classes (Sciex, Framingham, MA). The mixture was vortexed for 5 sec and incubated at room temperature for 30 min. Next, another 0.5 mL water and 0.45 mL dichloromethane were added to the tube, followed by gentle vortexing for 5 sec, and centrifugation at 2500 g at 15 °C for 10 min. The bottom organic layer was transferred to a new tube and 0.9 mL of dichloromethane was added to the original tube for a second extraction. The combined extracts were concentrated under nitrogen and reconstituted in 0.25 mL of the mobile phase (10 mM ammonium acetate in 50:50 methanol:dichloromethane). Quantitative lipidomics was performed with the Sciex Lipidyzer platform consisting of Shimadzu Nexera X2 LC-30AD pumps, a Shimadzu Nexera X2 SIL-30AC autosampler, and a Sciex QTRAP® 5500 mass spectrometer equipped with SelexION® for differential mobility spectrometry (DMS). 1-propanol was used as the chemical modifier for the DMS. Samples were introduced to the mass spectrometer by flow injection at 8 µL/min. Each sample was injected twice, once with the DMS on [phosphatidylcholines (PC); phosphatidylethanolamines (PE); lysophosphatidylcholines (LPC); lysophosphatidylethanolamines (LPE); sphingomyelins (SM)], and once with the DMS off ([cholesterol esters (CE); ceramides (CER); diacylglycerols (DAG); dihydroceramides (DCER)/ free fatty acids(FFA); hexosylceramides (HCER); lactosylceramides (LCER); triacylglycerols (TAG)]. The lipid molecular species were measured using multiple reaction monitoring and positive/negative polarity switching. Positive ion mode detected lipid classes SM, DAG, CE, CER, DCER, HCER, DCER, and TAG, and negative ion mode detected lipid classes LPE, LPC, PC, PE, and FFA. A total of 1070 lipids and fatty acids were targeted in the analysis. Data was acquired and processed using Analyst 1.6.3 and Lipidomics Workflow Manager 1.0.5.0.