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MB Sample ID: SA181885

Local Sample ID:	HFD-PB2
Subject ID:	SU002013
Subject Type:	Mammal
Subject Species:	Macaca fascicularis
Taxonomy ID:	9541
Age Or Age Range:	16.3 - 18.8 yo
Weight Or Weight Range:	5.73 - 10.56 kg
Gender:	Male
Animal Animal Supplier:	Guangzhou Huazhen Biosciences Co., Ltd, Guangzhou
Animal Housing:	Guangzhou Huazhen Biosciences Co., Ltd, Guangzhou
Animal Light Cycle:	12-h/12-h light-dark cycle
Animal Feed:	normal food (19.26 % protein, 5% fat, no sugar, and no cholesterol), or high-fat and high-sugar (HFHS) food (≥ 30% sugar, ≥ 15% fat, ≥ 10.5% protein, and ≥ 0.5% cholesterol)
Animal Inclusion Criteria:	spontaneous diabetes mellitus and HFHS food-fed

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Subject:

Subject ID:	SU002013
Subject Type:	Mammal
Subject Species:	Macaca fascicularis
Taxonomy ID:	9541
Age Or Age Range:	16.3 - 18.8 yo
Weight Or Weight Range:	5.73 - 10.56 kg
Gender:	Male
Animal Animal Supplier:	Guangzhou Huazhen Biosciences Co., Ltd, Guangzhou
Animal Housing:	Guangzhou Huazhen Biosciences Co., Ltd, Guangzhou
Animal Light Cycle:	12-h/12-h light-dark cycle
Animal Feed:	normal food (19.26 % protein, 5% fat, no sugar, and no cholesterol), or high-fat and high-sugar (HFHS) food (≥ 30% sugar, ≥ 15% fat, ≥ 10.5% protein, and ≥ 0.5% cholesterol)
Animal Inclusion Criteria:	spontaneous diabetes mellitus and HFHS food-fed

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
HFD-PB2	SA181885	FL022291	HFHS	Condition
HFD-PB2	SA181885	FL022291	PB	Tissue

Collection:

Collection ID:	CO002006
Collection Summary:	All macaques were sacrificed at about 18 years old, and the liver, PB, and HPVB samples were collected. Both blood samples were centrifuged at 3,000 rpm for 10 min at 4°C immediately to separate sera. All samples were frozen in liquid nitrogen and stored at -80°C until use. The study protocol received prior approval from the Institutional Animal Care and Use Committee of Huazhen Biosciences.
Sample Type:	Blood (serum) and Liver

Treatment:

Treatment ID:	TR002025
Treatment Summary:	Male Macaca fascicularis of about 18 years old were bought from Huazhen Biosciences (Guangzhou, China). All macaques were born from 16th August 2001 to 11th February 2004 (Supplemental Table S1) and housed in the animal rooms maintained at 16 ~ 26 °C and 40% ~ 70% room humidity on a 12-h/12-h light-dark cycle. All macaques of both NC and sDM groups were fed with the normal food (19.26 % protein, 5% fat, no sugar, and no cholesterol), whereas the HFHS monkeys were grown up on the same diet until switching to the high-fat and high-sugar (HFHS) food (≥ 30% sugar, ≥ 15% fat, ≥ 10.5% protein, and ≥ 0.5% cholesterol) on 1st March 2019 (Fig. 1a). After one year of food change, blood and urine samples were collected.

Treatment ID:

TR002025

Treatment Summary:

Male Macaca fascicularis of about 18 years old were bought from Huazhen Biosciences (Guangzhou, China). All macaques were born from 16th August 2001 to 11th February 2004 (Supplemental Table S1) and housed in the animal rooms maintained at 16 ~ 26 °C and 40% ~ 70% room humidity on a 12-h/12-h light-dark cycle. All macaques of both NC and sDM groups were fed with the normal food (19.26 % protein, 5% fat, no sugar, and no cholesterol), whereas the HFHS monkeys were grown up on the same diet until switching to the high-fat and high-sugar (HFHS) food (≥ 30% sugar, ≥ 15% fat, ≥ 10.5% protein, and ≥ 0.5% cholesterol) on 1st March 2019 (Fig. 1a). After one year of food change, blood and urine samples were collected.

Sample Preparation:

Sampleprep ID:	SP002019
Sampleprep Summary:	The hydrophilic and hydrophobic compounds were extracted using methanol/water and MTBE/methanol/water solvent systems, respectively. Samples were first thawed on ice. To extract hydrophilic metabolites from the tissue samples, 1 ml of methanol/water (7:3, v/v) was added to 50 mg of the liver, and homogenized with steel balls for 3 min at 30 Hz, followed by 1 min of a vortex. The homogenate was then centrifuged at 12,000 rpm for 10 min at 4°C to collect the supernatant. Hydrophobic compounds were extracted from another 50 mg using a slightly modified protocol. Homogenization was done with 1 ml of MTBE/methanol (10:3, v/v) and 100 µl of water was mixed with the homogenate to extract before centrifugation. For the sera of PB and HPVB, 3 volumes (v/v) of methanol and a mixture of MTBE and methanol (10:3, v/v) were whirled with the serum samples for 3 min, followed by centrifugation at 12,000 rpm for 10 min at 4°C. All collected supernatants were dried and store at -80°C until LC-MS/MS analysis. Internal standards were dissolved in the solvents before extraction.

Sampleprep ID:

SP002019

Sampleprep Summary:

The hydrophilic and hydrophobic compounds were extracted using methanol/water and MTBE/methanol/water solvent systems, respectively. Samples were first thawed on ice. To extract hydrophilic metabolites from the tissue samples, 1 ml of methanol/water (7:3, v/v) was added to 50 mg of the liver, and homogenized with steel balls for 3 min at 30 Hz, followed by 1 min of a vortex. The homogenate was then centrifuged at 12,000 rpm for 10 min at 4°C to collect the supernatant. Hydrophobic compounds were extracted from another 50 mg using a slightly modified protocol. Homogenization was done with 1 ml of MTBE/methanol (10:3, v/v) and 100 µl of water was mixed with the homogenate to extract before centrifugation. For the sera of PB and HPVB, 3 volumes (v/v) of methanol and a mixture of MTBE and methanol (10:3, v/v) were whirled with the serum samples for 3 min, followed by centrifugation at 12,000 rpm for 10 min at 4°C. All collected supernatants were dried and store at -80°C until LC-MS/MS analysis. Internal standards were dissolved in the solvents before extraction.

Combined analysis:

Analysis ID	AN003145	AN003146	AN003147	AN003148
Analysis type	MS	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	Reversed phase	Reversed phase
Chromatography system	Shim-pack UFLC SHIMADZU CBM A system	Shim-pack UFLC SHIMADZU CBM A system	Shim-pack UFLC SHIMADZU CBM A system	Shim-pack UFLC SHIMADZU CBM A system
Column	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
MS Type	EI	EI	EI	EI
MS instrument type	Triple quadrupole	Triple quadrupole	Triple quadrupole	Triple quadrupole
MS instrument name	ABI Sciex 6500 QTrap	ABI Sciex 6500 QTrap	ABI Sciex 6500 QTrap	ABI Sciex 6500 QTrap
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	Peak area	Peak area	Peak area	Peak area

Chromatography:

Chromatography ID:	CH002327
Chromatography Summary:	The analysis of hydrophilic metabolites used 0.1% formic acid (in water) and acetonitrile with 0.1% formic acid as mobile phases, with a gradient (V/V) as following: 95:5 at 0 min, 10:90 at 11.0 min, 10:90 at 12.0 min, 95:5 at 12.1 min, and 95:5 at 14.0 min.
Instrument Name:	Shim-pack UFLC SHIMADZU CBM A system
Column Name:	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
Flow Gradient:	95:5 at 0 min, 10:90 at 11.0 min, 10:90 at 12.0 min, 95:5 at 12.1 min, and 95:5 at 14.0 min.
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH002328
Chromatography Summary:	The two mobile phases for hydrophobic compounds were 0.04% acetic acid and acetonitrile with 0.04% acetic acid, and the gradient (V/V) program was 95:5 at 0 min, 5:95 at 11.0 min, 5:95 at 12.0 min, 95:5 at 12.1 min and 95:5 at 14.0 min.
Instrument Name:	Shim-pack UFLC SHIMADZU CBM A system
Column Name:	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
Flow Gradient:	95:5 at 0 min, 5:95 at 11.0 min, 5:95 at 12.0 min, 95:5 at 12.1 min and 95:5 at 14.0 min.
Solvent A:	100% water; 0.04% acetic acid
Solvent B:	100% acetonitrile; 0.04% acetic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002925
Analysis ID:	AN003145
Instrument Name:	ABI Sciex 6500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	EI
MS Comments:	LIT and triple quadrupole (QQQ) scans were acquired in both positive and negative ion modes under the control of Analyst 1.6.3 software (Sciex, Washington, USA). The ion spray voltage (IS) of the ESI source was 5500 and -4500 V for positive and negative modes, respectively. Source temperature was set at 500 C, ion source gas I (GSI), gas II (GSII), curtain gas (CUR) at 55, 60, and 25.0 psi, respectively, and collision gas (CAD) high. Instrument tuning and mass calibration in QQQ and LIT modes were performed with 10 and 100 μmol/L polypropylene glycol solutions, respectively. Specific sets of multiple reaction monitoring (MRM) transitions of various periods of retention time were monitored according to an in-house library of metabolites.
Ion Mode:	POSITIVE

MS ID:	MS002926
Analysis ID:	AN003146
Instrument Name:	ABI Sciex 6500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	EI
MS Comments:	LIT and triple quadrupole (QQQ) scans were acquired in both positive and negative ion modes under the control of Analyst 1.6.3 software (Sciex, Washington, USA). The ion spray voltage (IS) of the ESI source was 5500 and -4500 V for positive and negative modes, respectively. Source temperature was set at 500 C, ion source gas I (GSI), gas II (GSII), curtain gas (CUR) at 55, 60, and 25.0 psi, respectively, and collision gas (CAD) high. Instrument tuning and mass calibration in QQQ and LIT modes were performed with 10 and 100 μmol/L polypropylene glycol solutions, respectively. Specific sets of multiple reaction monitoring (MRM) transitions of various periods of retention time were monitored according to an in-house library of metabolites.
Ion Mode:	NEGATIVE

MS ID:	MS002927
Analysis ID:	AN003147
Instrument Name:	ABI Sciex 6500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	EI
MS Comments:	LIT and triple quadrupole (QQQ) scans were acquired in both positive and negative ion modes under the control of Analyst 1.6.3 software (Sciex, Washington, USA). The ion spray voltage (IS) of the ESI source was 5500 and -4500 V for positive and negative modes, respectively. Source temperature was set at 500 C, ion source gas I (GSI), gas II (GSII), curtain gas (CUR) at 55, 60, and 25.0 psi, respectively, and collision gas (CAD) high. Instrument tuning and mass calibration in QQQ and LIT modes were performed with 10 and 100 μmol/L polypropylene glycol solutions, respectively. Specific sets of multiple reaction monitoring (MRM) transitions of various periods of retention time were monitored according to an in-house library of metabolites.
Ion Mode:	POSITIVE

MS ID:	MS002928
Analysis ID:	AN003148
Instrument Name:	ABI Sciex 6500 QTrap
Instrument Type:	Triple quadrupole
MS Type:	EI
MS Comments:	LIT and triple quadrupole (QQQ) scans were acquired in both positive and negative ion modes under the control of Analyst 1.6.3 software (Sciex, Washington, USA). The ion spray voltage (IS) of the ESI source was 5500 and -4500 V for positive and negative modes, respectively. Source temperature was set at 500 C, ion source gas I (GSI), gas II (GSII), curtain gas (CUR) at 55, 60, and 25.0 psi, respectively, and collision gas (CAD) high. Instrument tuning and mass calibration in QQQ and LIT modes were performed with 10 and 100 μmol/L polypropylene glycol solutions, respectively. Specific sets of multiple reaction monitoring (MRM) transitions of various periods of retention time were monitored according to an in-house library of metabolites.
Ion Mode:	NEGATIVE