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MB Sample ID: SA021828

Local Sample ID:sample38
Subject ID:SU000455
Subject Type:Animal
Subject Species:Homo sapiens
Taxonomy ID:9606
Species Group:Human

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Subject:

Subject ID:SU000455
Subject Type:Animal
Subject Species:Homo sapiens
Taxonomy ID:9606
Species Group:Human

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
sample38SA021828FL005331NDtreatment

Collection:

Collection ID:CO000449
Collection Summary:At 5:00 AM after an overnight fast, baseline blood samples were collected from study participants. Plasma samples were stored at 80°C until analysis.
Sample Type:Blood. Plasma was isolated for MS analysis.

Treatment:

Treatment ID:TR000469
Treatment Summary:Participants were admitted to the Clinical Research Unit at St Mary’s Hospital (Rochester, Minnesota) the evening before the study and spent overnight in the Clinical Research Unit. The participants were given a standard meal on the evening of the admission after which they fasted overnight. Participants with T1D were treated with insulin as per their usual individual programs. At 5:00 AM after an overnight fast, baseline blood samples were collected from study participants. Plasma samples were stored at 80°C until analysis.

Sample Preparation:

Sampleprep ID:SP000462
Sampleprep Summary:Plasma quality-control samples used in the study were prepared from pooled plasma spiked with a selection of metabolites to mimic elevated levels of metabolites during I− (insulin withdrawn) condition. Plasma was spiked with a standard mixture (3:1 ratio of plasma to spiking solution) containing 100 μg/mL niacin, hypoxanthine, leucine, isoleucine, phenylalanine, tryptophan, citric acid, glucose, hippuric acid, and taurocholic acid dissolved in 1:1 acetonitrile/water. All plasma samples (200 μL) were thawed on ice at 4°C followed by deproteinization with methanol (1:4 ratio of plasma to methanol) and vortexed for 10 s, followed by incubation at −20°C for 2 h. The samples were then centrifuged at 15,871g for 30 min at 4°C. The supernatants were lyophilized (Savant, Holbrook, NY) and stored at −20°C prior to analysis. The samples were reconstituted in 50% H2O/acetonitrile and passed through a Microcon YM3 filter (Millipore Corporation). The supernatants were transferred to analytical vials, stored in the autosampler at 4°C, and analyzed within 48 h of reconstitution in buffer.

Combined analysis:

Analysis ID AN000684
Analysis type MS
Chromatography type Reversed phase
Chromatography system Cohesive TX2 liquid chromatography system
Column Ascentis C18 (150 x 2.1mm,2.7um)
MS Type ESI
MS instrument type Triple quadrupole
MS instrument name ABI Sciex API 5000 QQQ
Ion Mode NEGATIVE
Units micromolar

Chromatography:

Chromatography ID:CH000496
Chromatography Summary:Concentration and isotopic enrichment of palmitic acid from extracted plasmas were simultaneously measured against an extracted concentration standard curve as well as an enrichment standard curve on the Applied Biosystem API5000 mass spectrometer-MS (Foster City, CA) coupled with a Cohesive TX2 liquid chromatography system-LC (Franklin, MA). This system provides two LCs connected in parallel to the mass spectrometer, allowing twice the number of samples to be analyzed by using tandem sample injection. The LCs were controlled by Aria software (Cohesive Technology). Palmitic and heptadecanoic acids were separated on the LCs using an Ascentis C18, 2.7 μm, 2.1 × 150 mm column using two buffers. Buffer A was 80% acetonitrile, 0.5 mM ammonium acetate; buffer B was 99% acetonitrile, 1% 0.5 mM ammonium acetate. The flow rate was 0.4 ml/min, and the gradient conditions were as follows: 0–8 min isocratic at 55% B, 8–8.5 min 55⇒95% B, 8.5–10 min isocratic at 95% B, 10–10.5 min 95⇒55% B, and 10.5–12 min isocratic at 55% B. One tenth of the volume of each concentration standard and each plasma sample were resuspended in 400 μl of buffer A prior to injecting 10 μl onto the LC/MS. The chromographic separation of linoleic acid, palmitic acid, oleic acid, eladic acid, and heptadecanoic acid (internal standard) are depicted in Fig. 1A. Although palmitic and oleic acid peaks overlap, they are distinguishable by MS, whereas eladic acid, which is indistinguishable from oleic acid by MS, is easily separated by chromatography. Fig. 1B depicts the M+2 peaks for palmitate and heptadecanoate and the M+16 peak for palmitate of a plasma sample from a volunteer receiving a [U-13C]palmitate infusion. Palmitate elutes at 6.2 min and heptadecanoate acid at 7.1 min; however, in Fig. 1B the retention times are shown as 1.2 and 2.9 min because of the 3 min delay set in the LC method before diverting the flow to the MS for acquisition. The MS was set to acquire for only 5 min, allowing MS data acquisition from the second LC to begin while the first LC finishes its 12 min gradient. The MS was equipped with a turbo ion spray interface with the heater set at 60°C, spray voltage at 5500 V, sheath gas at 50, sweep gas at 40, and transfer capillary at 270°C. Palmitate, [13C16]palmitate and heptadecanoate were selectively monitored at m/z 257, m/z 271 in negative mode. Palmitate was monitored as [M+2−H]− and [M+16−H]−. Heptadecanoic was also monitor as [M+2−H]− . Therefore m/z 271 was either [13C16]palmitate or heptadecanoate, depending on where it eluted in the LC gradient.
Instrument Name:Cohesive TX2 liquid chromatography system
Column Name:Ascentis C18 (150 x 2.1mm,2.7um)
Flow Gradient: 0–8 min isocratic at 55% B, 8–8.5 min 55⇒95% B, 8.5–10 min isocratic at 95% B, 10–10.5 min 95⇒55% B, and 10.5–12 min isocratic at 55% B.
Flow Rate:0.4 ml/min
Solvent A:80% acetonitrile/20% water; 0.5 mM ammonium acetate
Solvent B:99% acetonitrile/1% water; 0.5 mM ammonium acetate
Chromatography Type:Reversed phase

MS:

MS ID:MS000610
Analysis ID:AN000684
Instrument Name:ABI Sciex API 5000 QQQ
Instrument Type:Triple quadrupole
MS Type:ESI
Ion Mode:NEGATIVE
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