Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA247809

Local Sample ID:	32-week db/m-3
Subject ID:	SU002564
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	db/m; db/db; db/db+retinol; db/db+retinoic acid; db/db+AAV9-RDH10; RDH10-cKO; RDH10fl/fl
Age Or Age Range:	32-week; 36-week; 21-week
Gender:	Male

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Subject:

Subject ID:	SU002564
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	db/m; db/db; db/db+retinol; db/db+retinoic acid; db/db+AAV9-RDH10; RDH10-cKO; RDH10fl/fl
Age Or Age Range:	32-week; 36-week; 21-week
Gender:	Male

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
32-week db/m-3	SA247809	FL031166	db/m+	Genotype
32-week db/m-3	SA247809	FL031166	no	Treatment

Collection:

Collection ID:	CO002557
Collection Summary:	Mice were fasted for 12 hours before sampling and testing. All animal experiments were approved by the Animal Care and Ethics Committee of Zhongshan School of Medicine, Sun Yat-sen University, and followed the National Institutes of Health Guidelines on the Care and Use of Animals (the protocol number is SYSU-IACUC-2019-B027)
Sample Type:	Cardiac tissue

Treatment:

Treatment ID:	TR002576
Treatment Summary:	RDH10-cKO mice: RDH10-cKO mice, which contain both RDH10fl/fl and MYH6-iCre, were bred by RDH10fl/fl mice and MYH6-iCre mice and were injected tamoxifen intraperitoneally (50 mg/kg, T2859, Sigma -Aldrich, St. Louis, MO) for 5 consecutive days from 5 weeks of age. Age-matched male RDH10fl/fl mice that also received TMX injections served as normal controls for RDH10-cKO mice. Animal treatments: Mice in the Rol treatment group received Rol gavage (800 IU/each, 17772, Sigma-Aldrich, St. Louis, MO) every two days from 8 weeks of age. Mice in the atRA treatment group received atRA intraperitoneal injection (5 mg/kg body weight, R2625, Sigma-Aldrich, St. Louis, MO) daily from 8 weeks of age. Gene therapy: A recombinant AAV9 vector carrying the mouse RDH10 sequence (AAV9-RDH10, DZ-AAV-Rdh10-OE, Dongze, Hanbio Inc, Shanghai, China) was used to overexpress RDH10. 0.8*10^11 vg/per animal of AA9-RDH10 was transferred into T2DM mice, respectively, by tail vein injection at the age of 16 weeks.

Treatment ID:

TR002576

Treatment Summary:

RDH10-cKO mice: RDH10-cKO mice, which contain both RDH10fl/fl and MYH6-iCre, were bred by RDH10fl/fl mice and MYH6-iCre mice and were injected tamoxifen intraperitoneally (50 mg/kg, T2859, Sigma -Aldrich, St. Louis, MO) for 5 consecutive days from 5 weeks of age. Age-matched male RDH10fl/fl mice that also received TMX injections served as normal controls for RDH10-cKO mice. Animal treatments: Mice in the Rol treatment group received Rol gavage (800 IU/each, 17772, Sigma-Aldrich, St. Louis, MO) every two days from 8 weeks of age. Mice in the atRA treatment group received atRA intraperitoneal injection (5 mg/kg body weight, R2625, Sigma-Aldrich, St. Louis, MO) daily from 8 weeks of age. Gene therapy: A recombinant AAV9 vector carrying the mouse RDH10 sequence (AAV9-RDH10, DZ-AAV-Rdh10-OE, Dongze, Hanbio Inc, Shanghai, China) was used to overexpress RDH10. 0.8*10^11 vg/per animal of AA9-RDH10 was transferred into T2DM mice, respectively, by tail vein injection at the age of 16 weeks.

Sample Preparation:

Sampleprep ID:	SP002570
Sampleprep Summary:	Sample preparation Homogenized the heart tissue in 200μL of cold NaCl solution (0.9%) for 5 seconds, add 100μL retinol-D4 (IR-23012, IsoSciences, Ambler, PA, USA) as internal standard for Rol and atRA, and retinyl acetate (46958, Sigma-Aldrich, St. Louis, MO) as internal standard for retinyl esters, then homogenize for another 5 seconds. For Rol and at RA, 1mL formic acid n-hexane solution (1%) was added to the homogenizing to facilitate phase separation by a 5-minutes vortex and 5-min centrifugation (16,200 g). The supernatants were collected in a new tube and blown dry with nitrogen followed by re-dilution with 100μL 70% methanol. For retinyl esters, 24 μL 0.5 M NaOH solution was added to tissue homogenate, followed by 1 mL n-hexane. The mixture was votexed 5 min and centrifuged at 16,200 g for 5 min. The supernatants were collected in a new tube and blown dry with nitrogen followed by re-dilution with 100μL 100% methanol. The processes above should be protected from the light.

Sampleprep ID:

SP002570

Sampleprep Summary:

Sample preparation Homogenized the heart tissue in 200μL of cold NaCl solution (0.9%) for 5 seconds, add 100μL retinol-D4 (IR-23012, IsoSciences, Ambler, PA, USA) as internal standard for Rol and atRA, and retinyl acetate (46958, Sigma-Aldrich, St. Louis, MO) as internal standard for retinyl esters, then homogenize for another 5 seconds. For Rol and at RA, 1mL formic acid n-hexane solution (1%) was added to the homogenizing to facilitate phase separation by a 5-minutes vortex and 5-min centrifugation (16,200 g). The supernatants were collected in a new tube and blown dry with nitrogen followed by re-dilution with 100μL 70% methanol. For retinyl esters, 24 μL 0.5 M NaOH solution was added to tissue homogenate, followed by 1 mL n-hexane. The mixture was votexed 5 min and centrifuged at 16,200 g for 5 min. The supernatants were collected in a new tube and blown dry with nitrogen followed by re-dilution with 100μL 100% methanol. The processes above should be protected from the light.

Combined analysis:

Analysis ID	AN004041
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Sciex Triple Quad TM 4500MD MS
Column	Phenomenex Kinetex C18 (50 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	QTRAP
MS instrument name	ABI Sciex 5500 QTrap
Ion Mode	POSITIVE
Units	ng/ml

Chromatography:

Chromatography ID:	CH002989
Instrument Name:	Sciex Triple Quad TM 4500MD MS
Column Name:	Phenomenex Kinetex C18 (50 x 2.1mm,1.7um)
Column Temperature:	25
Flow Gradient:	initial conditions 75% B; from 0 to 3.5min linear increase to 95% B; between 3.5 and 4.5min 95% B was retained; at 4.6 min back to initial conditions with 75% B; finally, 75% B was held from 4.6 to 5.9min
Flow Rate:	0.2ml/min
Solvent A:	40% acetonitrile/30% methanol/30% water; 0.1 % formic acid
Solvent B:	55% acetonitrile/30% methanol/15% water; 0.1 % formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003788
Analysis ID:	AN004041
Instrument Name:	ABI Sciex 5500 QTrap
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	The MS conditions were as follows: electrospray ionization (ESI) under positive mode; nebulizer gas: nitrogen; curtain gas,30 psi; ion spray voltage, 4000V; temperature, 400ºC; gas 1 and gas 2, 35 and 40 psi, respectively; collision gas, 10 psi. The parameters of the mass spectrometer were optimized, and the multiple reaction monitoring (MRM) transitions of retinol, all-trans-retinoic acid, as well as D4-retinol were chosen as 269.2>93.1, 273.1>94.0, and 301.2>123.1, respectively. The MRM transitions for retinyl esters were chosen as 329.3>269.3 for retinyl acetate, 524.4>268.1 for retinyl palmitate (16:0), 552.5>268.2 for retinyl stearate (18:0), 522.4>268, retinyl palmitoleate (16:1), and retinyl oleate (18:1). The quantification was performed using the calibration curve.
Ion Mode:	POSITIVE