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MB Sample ID: SA032765

Local Sample ID:LAM-05
Subject ID:SU000620
Subject Type:Animal
Subject Species:Mus musculus
Taxonomy ID:10090
Species Group:Mammal

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Subject ID:SU000620
Subject Type:Animal
Subject Species:Mus musculus
Taxonomy ID:10090
Species Group:Mammal


Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
LAM-05SA032765FL007287T (treated with TPhP)Treatment


Collection ID:CO000614
Collection Summary:Prior to sacrifice, rats were fasted between 8 and12 h,theirbody weights recorded, and blood was collected from the tail as above prior to euthanasia. Rats were anesthetized with sodium pentobarbital and euthanized with a 1 mL/kg intracardiac injection of saturated potassium chloride. Once cardiac movement had stopped for 30 s the rat was decapitated and the hypothalamus, liver, pancreas, heart, mesenteric adipose tissue, quadriceps, kidney, gonadal adipose tissue, inguinal adipose tissue, and brown adipose tissue were collected. All tissues were removed in the order listed above, wet weighed, and snap frozen in liquid nitrogen
Collection Protocol Filename:Green_et_al_2017-TPP_Exposure_Accelerat_T2DM_Rats.pdf
Sample Type:Tissue


Treatment ID:TR000634
Treatment Summary:Adult non-pregnant female UCD-T2DM rats (n = 16; 3 months old) were paired with males (n = 10; 3–4 months old) for a 24 h period at which point males were removed. This was defined as gestational day zero (G0) if a sperm plug was observed or if the female rats gained at least 30 g of body weight over the next 7 days. The day of birth was designated postnatal day zero (P0). Pregnant dams were randomly assigned to an exposure group (n = 8 per group), and received daily oral TPhP or ethanol vehicle exposure from G8 through weaning (P21) as described in Section 2.2 below. Gestational length and litter size were recorded on P0 and the sex of pups was determined and recorded on P4. Body weights of all pups in each litter were obtained periodically from P4–21. On P4 the litters were culled to 8 pups ensuring up to 4males and 2 females in each litter by random selection (Fig. 1A & B). This was done to ensure consistent exposure of pups between litters [13,23]. The time ittakes to develop T2DM is accelerated among UCD-T2DM rats with higher body weights on P21. Hence at weaning the largest pups were housed in same sex littermate groups of two females and up to four males as available (Fig. 1A & B). Urine was collected from the dams using an adapted plastic wrap method outlined by Kurien [24], 60 mins after final dose. Dams were placed in clean cages without bedding for at least 20 min then using a pipette up to 500 L of urine was collected in ethanol rinsed glass vials and placed on ice. At weaning all dams and remaining weanlings were sacrificed (90–330 min post-exposure) by CO2 asphyxiation and rapid decapitation. Two male rats weighing between 350–400 g on P61, from the TPhP group and the vehicle group were weight-matched across treatments for the diabetes study to eliminate confounding effects of body mass on the association between TPhP and T2DM onset (Fig. 1B). This weight range was selected because male UCD-T2DM rats that are between 350 and 400 g at 8 weeks of age develop T2DM at approximately 23 weeks of age [18]. Weight-matched rats were followed until 26 weeks or until they developed T2DM, which was defined as two consecutive weekly non-fasting glucose measurements of ≥200 mg/dL [18] in accordance with the American Diabetes Association (ADA) guideline of diagnosing diabetes with a random plasma glucose of 200 mg/dL or higher [19]. The remaining rats were not weight-matched and followed for the 3.5 month obesity study (Fig. 1A).
Treatment Protocol Filename:Green_et_al_2017-TPP_Exposure_Accelerat_T2DM_Rats.pdf

Sample Preparation:

Sampleprep ID:SP000627
Sampleprep Summary:Oxylipins and endocannabinoids were isolated using a Waters Ostro Sample Preparation Plate (Milford, MA). Hypothalamus samples were pulverized and aliquoted (~20-25mg) were added to 2mL polypropylene tubes and spiked with a 5 µL anti-oxidant solution (0.2 mg/ml solution BHT/EDTA in 1:1 MeOH:water) and 10 μL 1000nM analytical deuterated surrogates. A total of 100 µL methanol was added to the sample and vrtexed 90 sec. Next, 500 µL D.I. water and 1000 µL ethyl acetate was added and the tube was vortexed 3 minutes, before being centrifuged at 15,000g for 10 min at room temp. The supernate was then transferred into a clean 2 mL autosampler vial. The extraction with ethyl acetate was repeated and the eluent was dried by speed vacuum for 35 min at the medium BP setting. Once dry, samples were re-constituted with the internal standard 1-cyclohexyl ureido, 3-dodecanoic acid (CUDA) and 1-Phenyl 3-Hexadecanoic Acid Urea (PHAU) at 100 nM (50:50 MeOH:CAN), vortexed 1 min, transferred to a spin filter (0.1 µm, Millipore, Billerica, MA), centrifuged for 3 min at 6ºC at <4500g (rcf), before being transferred to 2 mL LC-MS amber vials. Extracts were stored at -20ºC until analysis by UPLC-MS/MS. The internal standard was used to quantify the recovery of surrogate standards.
Sampleprep Protocol Filename:Hypothal_Newman_Data_Report.docx

Combined analysis:

Analysis ID AN000915
Analysis type MS
Chromatography type Reversed phase
Chromatography system Waters Acquity
Column Waters Acquity BEH C18 (150 x 2.1mm,1.7um)
MS instrument type Triple quadrupole
MS instrument name ABI Sciex API 4000 QTrap
Units Concentration pmol/g


Chromatography ID:CH000652
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH C18 (150 x 2.1mm,1.7um)
Chromatography Type:Reversed phase


MS ID:MS000814
Analysis ID:AN000915
Instrument Name:ABI Sciex API 4000 QTrap
Instrument Type:Triple quadrupole