Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA008100

Local Sample ID:	4C
Subject ID:	SU000168
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

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Subject:

Subject ID:	SU000168
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Species Group:	Human

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
4C	SA008100	FL001967	High Insulin	Sample Group

Collection:

Collection ID:	CO000154
Collection Summary:	Percutaneous muscle biopsy specimens of the vastus lateralis (?300 mg) were performed at 180 and 480 minutes under local anesthesia (2% lidocaine). Muscle samples were blotted, were dissected free of fat and connective tissue, and then were frozen. Biopsy specimens were collected from the opposite legs on a single visit. Indirect calorimetry was performed at 11:00 am for 20 minutes using a ventilated hood (Parvo Medics). The final 10 minutes of steady-state oxygen consumption (Vo2) and carbon dioxide production (Vco2) determined the respiratory exchange ratio (RER = Vco2/Vo2). Urine was collected throughout the clamp and analyzed for urea content at a core laboratory.
Sample Type:	Muscle

Treatment:

Treatment ID:	TR000173
Treatment Summary:	Low Insulin \| High Insulin \| Low Ins+ Saline \| Low Ins +EAA \| High Insulin+Saline \| High Insulin + EAA

Sample Preparation:

Sampleprep ID:	SP000168
Sampleprep Summary:	MMP and muscle fractions were isolated from frozen samples using differential centrifugation (18, 19). Biopsy samples were homogenized with protease and phosphatase inhibitors (Halt; Thermo Fisher Scientific) and centrifuged to pellet myofibrillar (MYO) proteins. The supernatant was centrifuged to pellet mitochondrial (MITO) proteins, and the final supernatant was deproteinated with cold ethanol (1:9, v/v) and then centrifuged to pellet sarcoplasmic (SARC) proteins. Aliquots from MMP, MYO, SARC, and MITO were acid hydrolyzed, and free amino acids were purified using cation exchange columns and then were dried. Plasma phenylalanine enrichment was determined using gas chromatography (GC) and mass spectrometry (MS) as described previously (19). Samples were derivatized to a heptafluorobutyryl isobutyl ester and identified with a Micromass Quattro Micro triple quadrupole GC-MS system (Waters) operating under negative ion chemical ionization using isobutane as the reactant gas. Selected ion monitoring of m/z 399.2 and 403.2 M + 2 and M + 6 fragments of phenylalanine and the l-[ring-13C6]phenylalanine, respectively, was performed.

Sampleprep ID:

SP000168

Sampleprep Summary:

MMP and muscle fractions were isolated from frozen samples using differential centrifugation (18, 19). Biopsy samples were homogenized with protease and phosphatase inhibitors (Halt; Thermo Fisher Scientific) and centrifuged to pellet myofibrillar (MYO) proteins. The supernatant was centrifuged to pellet mitochondrial (MITO) proteins, and the final supernatant was deproteinated with cold ethanol (1:9, v/v) and then centrifuged to pellet sarcoplasmic (SARC) proteins. Aliquots from MMP, MYO, SARC, and MITO were acid hydrolyzed, and free amino acids were purified using cation exchange columns and then were dried.
Plasma phenylalanine enrichment was determined using gas chromatography (GC) and mass spectrometry (MS) as described previously (19). Samples were derivatized to a heptafluorobutyryl isobutyl ester and identified with a Micromass Quattro Micro triple quadrupole GC-MS system (Waters) operating under negative ion chemical ionization using isobutane as the reactant gas. Selected ion monitoring of m/z 399.2 and 403.2 M + 2 and M + 6 fragments of phenylalanine and the l-[ring-13C6]phenylalanine, respectively, was performed.

Combined analysis:

Analysis ID	AN000236
Analysis type	MS
Chromatography type
Chromatography system
Column
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	ABI Sciex API 5000 QQQ
Ion Mode	POSITIVE
Units	uM

Chromatography:

Chromatography ID:	CH000165

Chromatography ID:

CH000165

MS:

MS ID:	MS000187
Analysis ID:	AN000236
Instrument Name:	ABI Sciex API 5000 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Dried amino acids were prepared as isobutyl ester derivatives, and the mass was determined by an API 5000 triple quadrupole mass spectrometer with a TurboIonSpray source (Applied Biosystems).
Ion Mode:	POSITIVE