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MB Sample ID: SA033558

Local Sample ID:OHSUserum_PO6927_3
Subject ID:SU000631
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Species Group:Human

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Subject:

Subject ID:SU000631
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Species Group:Human

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
OHSUserum_PO6927_3SA033558FL007454serumSample_Type
OHSUserum_PO6927_3SA033558FL007454caseFactor

Collection:

Collection ID:CO000625
Collection Summary:Matched DBS and serum samples were simultaneously collected in 2000–2001 from 10 overnight fasted male participants having an average age of 75.5 ± 6.15 years. The institutional review boards at the participating institutions approved the protocol and the study participants provided informed consent. Case participants included 5 diabetic men with high BMIs, TGs and glucose levels, and low amounts of HDL. The 5 control participants had normal metabolic profiles (normal BMIs, TGs, HDL and glucose levels).
Sample Type:Blood
Storage Conditions:The DBS were stored at room temperature in the dark until analysis, while the serum was stored at –80°C.

Treatment:

Treatment ID:TR000645
Treatment Summary:Samples collected in 2000–2001 were stored until June 2015.

Sample Preparation:

Sampleprep ID:SP000638
Sampleprep Summary:For the serum samples, 10 mL of whole blood was drawn and centrifuged so the serum could be extracted. For the DBS samples, 500 μL of blood was collected and 125 μL was blotted in four areas on a Whatman FTA Classic Card. Polar metabolites and lipid extracts were derived from the same sample type (DBS or serum) using a modified Folch extraction in June 2015 and analyzed with MS shortly after. The DBS punch from each patient was transferred into to a 2.0 mL tube where 50 μL of water and then 1200 μL of –20°C 2:1 chloroform/methanol were added. Each sample was vortexed for 30 s then transferred into a shaker at 22°C for 60 min at 600 rpm. The samples were vortexed again for 30 s and then 250 μL of water was added to induce a phase separation. The sample was gently inverted several times, placed at room temperature for 5 min and then centrifuged at 10,000 g for 5 min at 4°C and put on ice to maintain the clear phase separation. Finally, 400 μL of the top polar layer was removed, dried in a Speedvac, and stored at –80°C for analysis of polar metabolites, while 700 μL of the bottom nonpolar layer was removed, dried in a Speedvac, and stored at –20°C in 250 μL of 2:1 chloroform/methanol for lipid analyses. Serum lipids were extracted using a similar procedure except 25 μL of serum was used and then 600 μL of –20°C 2:1 chloroform/methanol was added. After vortexing and shaking, 125 μL of water was added to induce a phase separation and 200 μL of the top polar layer and 350 μL of the bottom nonpolar lipid layer were removed and stored as outlined above. Prior to analysis, the total lipid extracts were dried down and then reconstituted in 70 μL and 100 μL of MeOH for the DBS and serum samples, respectively. To generate pooled case and control samples for LC/MS/MS and LC/IMS-MS analyses, 5 μL aliquots from each reconstituted DBS and serum sample were removed and combined.

Combined analysis:

Analysis ID AN000929 AN000930 AN000931
Analysis type MS MS MS
Chromatography type Reversed phase Reversed phase GC
Chromatography system Waters NanoAcquity Waters NanoAcquity Agilent 7890A
Column Waters Acquity HSS T3 (150 x 1mm,1.8um) Waters Acquity HSS T3 (150 x 1mm,1.8um) Agilent HP5-MS (30m × 0.25mm, 0.25 um)
MS Type ESI ESI EI
MS instrument type Orbitrap Orbitrap Single quadrupole
MS instrument name Thermo Velos Pro Orbitrap Thermo Velos Pro Orbitrap Agilent 7890A
Ion Mode POSITIVE NEGATIVE POSITIVE
Units Peak Height Peak Peak Area

Chromatography:

Chromatography ID:CH000663
Instrument Name:Waters NanoAcquity
Column Name:Waters Acquity HSS T3 (150 x 1mm,1.8um)
Chromatography Type:Reversed phase
  
Chromatography ID:CH000664
Instrument Name:Agilent 7890A
Column Name:Agilent HP5-MS (30m × 0.25mm, 0.25 um)
Chromatography Type:GC

MS:

MS ID:MS000825
Analysis ID:AN000929
Instrument Name:Thermo Velos Pro Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:POSITIVE
  
MS ID:MS000826
Analysis ID:AN000930
Instrument Name:Thermo Velos Pro Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:NEGATIVE
  
MS ID:MS000827
Analysis ID:AN000931
Instrument Name:Agilent 7890A
Instrument Type:Single quadrupole
MS Type:EI
Ion Mode:POSITIVE
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