#METABOLOMICS WORKBENCH michaelsa93_20170427_113316 DATATRACK_ID:898 STUDY_ID:ST000600 ANALYSIS_ID:AN000919 PROJECT_ID:PR000437 VERSION 1 CREATED_ON May 8, 2017, 11:11 am #PROJECT PR:PROJECT_TITLE NEFA Profile Response to Triphenyl Phosphate Exposure PR:PROJECT_SUMMARY This study aims to identify changes in non-esterified fatty acid (NEFAs) in the PR:PROJECT_SUMMARY plasma with triphenyl phosphate (TPP) exposure. UC Davis type 2 diabetes PR:PROJECT_SUMMARY mellitus (UCD-T2DM) rats were treated with TPP or not treated. Each group was PR:PROJECT_SUMMARY analyzed for non-esterified fatty acid (NEFA) changes to investigate alterations PR:PROJECT_SUMMARY in NEFAs due to TPP exposure. Targeted analysis of NEFA in rat plasma samples PR:PROJECT_SUMMARY was performed by the Newman lab. PR:INSTITUTE University of California, Davis PR:DEPARTMENT Department of Environmental Toxicology PR:LAST_NAME La Merrill PR:FIRST_NAME Michele PR:ADDRESS 1 Shields Ave., Davis, CA 95616 PR:EMAIL mlamerrill@ucdavis.edu PR:PHONE (530) 754-7254 #STUDY ST:STUDY_TITLE NEFA Profile Response to Triphenyl Phosphate Exposure ST:STUDY_SUMMARY This study aims to identify changes in non-esterified fatty acid (NEFAs) in the ST:STUDY_SUMMARY plasma with triphenyl phosphate (TPP) exposure. UC Davis type 2 diabetes ST:STUDY_SUMMARY mellitus (UCD-T2DM) rats were treated with TPP or not treated. Each group was ST:STUDY_SUMMARY analyzed for non-esterified fatty acid (NEFA) changes to investigate alterations ST:STUDY_SUMMARY in NEFAs due to TPP exposure. Targeted analysis of NEFA in rat plasma samples ST:STUDY_SUMMARY was performed by the Newman lab. ST:INSTITUTE U.S.D.A. Western Human Nutrition Research Center, University of California, ST:INSTITUTE Davis ST:DEPARTMENT Nutrition ST:LAST_NAME Newman ST:FIRST_NAME John ST:ADDRESS 430 W. Health Sciences Dr., Davis, CA 95616 ST:EMAIL john.newman@ars.usda.gov ST:PHONE +1-530-752-1009 ST:STUDY_COMMENTS The samples included a high degree of hemolysis exhibited in the plasma. One ST:STUDY_COMMENTS sample was lost during processing (Group E- Subject 78). Two samples were ST:STUDY_COMMENTS outliers for multiple analytes and were not included in the final data (E-117 & ST:STUDY_COMMENTS T-28). Of the samples reported in this data set, there were no missing values. #SUBJECT SU:SUBJECT_TYPE Animal SU:SUBJECT_SPECIES Rattus norvegicus SU:TAXONOMY_ID 10116 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS 5 LM-15 Treatment:E (Vehicle control) SUBJECT_SAMPLE_FACTORS 7 LM-12 Treatment:E (Vehicle control) SUBJECT_SAMPLE_FACTORS 39 LM-06 Treatment:E (Vehicle control) SUBJECT_SAMPLE_FACTORS 40 LM-31 Treatment:E (Vehicle control) SUBJECT_SAMPLE_FACTORS 46 LM-09 Treatment:E (Vehicle control) SUBJECT_SAMPLE_FACTORS 47 LM-08 Treatment:E (Vehicle control) SUBJECT_SAMPLE_FACTORS 54 LM-32 Treatment:E (Vehicle control) SUBJECT_SAMPLE_FACTORS 56 LM-13 Treatment:E (Vehicle control) SUBJECT_SAMPLE_FACTORS 80 LM-01 Treatment:E (Vehicle control) SUBJECT_SAMPLE_FACTORS 109 LM-22 Treatment:E (Vehicle control) SUBJECT_SAMPLE_FACTORS 111 LM-10 Treatment:E (Vehicle control) SUBJECT_SAMPLE_FACTORS 119 LM-24 Treatment:E (Vehicle control) SUBJECT_SAMPLE_FACTORS 135 LM-14 Treatment:E (Vehicle control) SUBJECT_SAMPLE_FACTORS 136 LM-16 Treatment:E (Vehicle control) SUBJECT_SAMPLE_FACTORS 15 LM-20 Treatment:T (Treated with TPhP) SUBJECT_SAMPLE_FACTORS 16 LM-05 Treatment:T (Treated with TPhP) SUBJECT_SAMPLE_FACTORS 22 LM-27 Treatment:T (Treated with TPhP) SUBJECT_SAMPLE_FACTORS 23 LM-02 Treatment:T (Treated with TPhP) SUBJECT_SAMPLE_FACTORS 32 LM-28 Treatment:T (Treated with TPhP) SUBJECT_SAMPLE_FACTORS 61 LM-26 Treatment:T (Treated with TPhP) SUBJECT_SAMPLE_FACTORS 63 LM-17 Treatment:T (Treated with TPhP) SUBJECT_SAMPLE_FACTORS 71 LM-30 Treatment:T (Treated with TPhP) SUBJECT_SAMPLE_FACTORS 72 LM-21 Treatment:T (Treated with TPhP) SUBJECT_SAMPLE_FACTORS 94 LM-19 Treatment:T (Treated with TPhP) SUBJECT_SAMPLE_FACTORS 96 LM-03 Treatment:T (Treated with TPhP) SUBJECT_SAMPLE_FACTORS 101 LM-25 Treatment:T (Treated with TPhP) SUBJECT_SAMPLE_FACTORS 102 LM-11 Treatment:T (Treated with TPhP) SUBJECT_SAMPLE_FACTORS 127 LM-07 Treatment:T (Treated with TPhP) SUBJECT_SAMPLE_FACTORS 128 LM-29 Treatment:T (Treated with TPhP) #COLLECTION CO:COLLECTION_SUMMARY "Prior to sacrifice, rats were fasted between 8 and 12 h,their body weights CO:COLLECTION_SUMMARY recorded, and blood was collected from the tail as above prior to euthanasia. CO:COLLECTION_SUMMARY Rats were anesthetized with sodium pentobarbital and euthanized with a 1 mL/kg CO:COLLECTION_SUMMARY intracardiac injection of saturated potassium chloride. Once cardiac movement CO:COLLECTION_SUMMARY had stopped for 30 s the rat was decapitated and the hypothalamus, liver, CO:COLLECTION_SUMMARY pancreas, heart, mesenteric adipose tissue, quadriceps, kidney, gonadal adipose CO:COLLECTION_SUMMARY tissue, inguinal adipose tissue, and brown adipose tissue were collected. All CO:COLLECTION_SUMMARY tissues were removed in the order listed above, wet weighed, and snap frozen in CO:COLLECTION_SUMMARY liquid nitrogen" CO:COLLECTION_PROTOCOL_FILENAME Green_et_al_2017-TPP_Exposure_Accelerat_T2DM_Rats.pdf CO:SAMPLE_TYPE Blood CO:BLOOD_SERUM_OR_PLASMA Plasma #TREATMENT TR:TREATMENT_SUMMARY Adult non-pregnant female UCD-T2DM rats (n = 16; 3 months old) were paired with TR:TREATMENT_SUMMARY males (n = 10; 3–4 months old) for a 24 h period at which point males were TR:TREATMENT_SUMMARY removed. This was defined as gestational day zero (G0) if a sperm plug was TR:TREATMENT_SUMMARY observed or if the female rats gained at least 30 g of body weight over the next TR:TREATMENT_SUMMARY 7 days. The day of birth was designated postnatal day zero (P0). Pregnant dams TR:TREATMENT_SUMMARY were randomly assigned to an exposure group (n = 8 per group), and received TR:TREATMENT_SUMMARY daily oral TPhP or ethanol vehicle exposure from G8 through weaning (P21) as TR:TREATMENT_SUMMARY described in Section 2.2 below. Gestational length and litter size were recorded TR:TREATMENT_SUMMARY on P0 and the sex of pups was determined and recorded on P4. Body weights of all TR:TREATMENT_SUMMARY pups in each litter were obtained periodically from P4–21. On P4 the litters TR:TREATMENT_SUMMARY were culled to 8 pups ensuring up to 4males and 2 females in each litter by TR:TREATMENT_SUMMARY random selection (Fig. 1A & B). This was done to ensure consistent exposure of TR:TREATMENT_SUMMARY pups between litters [13,23]. The time ittakes to develop T2DM is accelerated TR:TREATMENT_SUMMARY among UCD-T2DM rats with higher body weights on P21. Hence at weaning the TR:TREATMENT_SUMMARY largest pups were housed in same sex littermate groups of two females and up to TR:TREATMENT_SUMMARY four males as available (Fig. 1A & B). Urine was collected from the dams using TR:TREATMENT_SUMMARY an adapted plastic wrap method outlined by Kurien [24], 60 mins after final TR:TREATMENT_SUMMARY dose. Dams were placed in clean cages without bedding for at least 20 min then TR:TREATMENT_SUMMARY using a pipette up to 500 L of urine was collected in ethanol rinsed glass vials TR:TREATMENT_SUMMARY and placed on ice. At weaning all dams and remaining weanlings were sacrificed TR:TREATMENT_SUMMARY (90–330 min post-exposure) by CO2 asphyxiation and rapid decapitation. Two TR:TREATMENT_SUMMARY male rats weighing between 350–400 g on P61, from the TPhP group and the TR:TREATMENT_SUMMARY vehicle group were weight-matched across treatments for the diabetes study to TR:TREATMENT_SUMMARY eliminate confounding effects of body mass on the association between TPhP and TR:TREATMENT_SUMMARY T2DM onset (Fig. 1B). This weight range was selected because male UCD-T2DM rats TR:TREATMENT_SUMMARY that are between 350 and 400 g at 8 weeks of age develop T2DM at approximately TR:TREATMENT_SUMMARY 23 weeks of age [18]. Weight-matched rats were followed until 26 weeks or until TR:TREATMENT_SUMMARY they developed T2DM, which was defined as two consecutive weekly non-fasting TR:TREATMENT_SUMMARY glucose measurements of ≥200 mg/dL [18] in accordance with the American TR:TREATMENT_SUMMARY Diabetes Association (ADA) guideline of diagnosing diabetes with a random plasma TR:TREATMENT_SUMMARY glucose of 200 mg/dL or higher [19]. The remaining rats were not weight-matched TR:TREATMENT_SUMMARY and followed for the 3.5 month obesity study (Fig. 1A). TR:TREATMENT_PROTOCOL_FILENAME Green_et_al_2017-TPP_Exposure_Accelerat_T2DM_Rats.pdf #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Plasma non-esterified fatty acids (NEFAs) were isolated as previously described SP:SAMPLEPREP_SUMMARY by Smedes (1). and Gladine et al. (2). Specifically, plasma aliquots (100 mL) SP:SAMPLEPREP_SUMMARY were enriched with 5 mL 0.2 mg/ml butylated hydroxytoluene/EDTA in 1:1 SP:SAMPLEPREP_SUMMARY methanol:- water, and a suite of extraction surrogates, which included SP:SAMPLEPREP_SUMMARY deuterated-tri-palmitoyl glycerol (d31-16:0-TG; CDN Isotopes, Pointe-Claire, SP:SAMPLEPREP_SUMMARY Quebec, Canada), deuterated distearoylphosphotidylcholine (d35-18:0-PC; Avanti SP:SAMPLEPREP_SUMMARY Polar Lipids, Alabaster, Alabama), dodeca-(9E)-enoyl cholesterylesters SP:SAMPLEPREP_SUMMARY (22:1n9-CE; NuChek Prep, Elysian MN) and dodecatrienoic acid (22:3n3; NuChek SP:SAMPLEPREP_SUMMARY Prep). Lipids were then extracted with 10:8:11 cylcohexane: 2- propanol:ammonium SP:SAMPLEPREP_SUMMARY acetate. Briefly, enriched samples were mixed with cyclopropane/2-propanol, SP:SAMPLEPREP_SUMMARY phases were split with ammonium acetate, the organic phase was isolated and the SP:SAMPLEPREP_SUMMARY aqueous phase was re-extracted with cyclohexane. The combined organic total SP:SAMPLEPREP_SUMMARY lipid extract was reduced to dryness and reconstituted in 200 µL of 1:1 SP:SAMPLEPREP_SUMMARY methanol/toluene and the total lipid extract was used to quantify plasma fatty SP:SAMPLEPREP_SUMMARY acids as methyl esters by gas chromatography-mass spectrometry (GC-MS). It was SP:SAMPLEPREP_SUMMARY derivitized by adding 45 μL 2M (trimethylsilyl) diazomethane in hexanes and SP:SAMPLEPREP_SUMMARY spiked with 15:1n5 free acid to track methylation efficiency. Next, it was SP:SAMPLEPREP_SUMMARY brought to a final volume of 200 mL with 90:10 methanol/toluene (v/v) and left SP:SAMPLEPREP_SUMMARY at room temperature for 30 min, before being brought to dryness. The remaining SP:SAMPLEPREP_SUMMARY fatty acid methyl esters (FAMEs) were re-constituted in 300 mL Hexane plus 10 uL SP:SAMPLEPREP_SUMMARY of 44 mM tricosanoate methyl ester (23:0; NuChek Prep), vortexed, and 100 uL was SP:SAMPLEPREP_SUMMARY transferred to a GC-MS Vial for analysis. SP:SAMPLEPREP_PROTOCOL_FILENAME NEFA_Plasma_Newman_Data_Report.docx #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 6890N CH:COLUMN_NAME Agilent DB-225 (30m x 0.25mm x 0.5um) CH:SAMPLE_INJECTION 1 µl #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent MS:INSTRUMENT_TYPE GC-TOF MS:MS_TYPE EI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Concentration (µM) MS_METABOLITE_DATA_START Samples LM-15 LM-12 LM-06 LM-31 LM-09 LM-08 LM-32 LM-13 LM-01 LM-22 LM-10 LM-24 LM-14 LM-16 LM-20 LM-05 LM-27 LM-02 LM-28 LM-26 LM-17 LM-30 LM-21 LM-19 LM-03 LM-25 LM-11 LM-07 LM-29 Factors Treatment:E (Vehicle control) Treatment:E (Vehicle control) Treatment:E (Vehicle control) Treatment:E (Vehicle control) Treatment:E (Vehicle control) Treatment:E (Vehicle control) Treatment:E (Vehicle control) Treatment:E (Vehicle control) Treatment:E (Vehicle control) Treatment:E (Vehicle control) Treatment:E (Vehicle control) Treatment:E (Vehicle control) Treatment:E (Vehicle control) Treatment:E (Vehicle control) Treatment:T (Treated with TPhP) Treatment:T (Treated with TPhP) Treatment:T (Treated with TPhP) Treatment:T (Treated with TPhP) Treatment:T (Treated with TPhP) Treatment:T (Treated with TPhP) Treatment:T (Treated with TPhP) Treatment:T (Treated with TPhP) Treatment:T (Treated with TPhP) Treatment:T (Treated with TPhP) Treatment:T (Treated with TPhP) Treatment:T (Treated with TPhP) Treatment:T (Treated with TPhP) Treatment:T (Treated with TPhP) Treatment:T (Treated with TPhP) C15:0 1.50 1.45 1.86 1.82 1.65 1.91 2.26 1.63 2.29 1.77 1.63 1.44 1.61 1.87 1.70 1.90 1.92 1.66 1.45 1.14 1.93 2.00 2.61 3.01 2.54 1.39 2.05 1.97 1.67 C16:0 190.35 148.95 181.04 234.79 157.23 173.86 223.34 175.37 233.52 180.43 143.62 109.36 186.66 226.29 168.11 251.50 285.25 208.24 223.51 150.40 204.02 191.91 275.19 219.41 297.42 146.55 284.11 187.27 179.40 C17:0 1.20 1.18 1.21 1.24 1.21 1.60 1.48 1.15 1.65 1.46 1.28 1.30 1.28 1.39 1.50 1.45 1.51 1.33 1.21 0.95 1.31 1.68 1.82 1.63 2.07 1.17 1.62 1.43 1.23 C18:0 37.08 32.91 31.84 41.70 38.06 48.41 42.61 34.60 39.49 41.81 34.79 30.31 35.08 41.76 36.63 43.31 45.29 34.70 35.10 29.73 35.58 46.70 48.84 40.81 53.60 37.61 47.44 35.88 36.28 C20:0 0.53 0.76 0.44 0.60 0.65 0.59 1.08 0.55 0.60 0.64 0.75 0.82 0.64 0.59 0.71 0.44 0.70 0.68 0.71 0.44 0.60 0.75 0.60 0.85 0.62 0.60 0.62 0.64 0.75 C16:1n7t 3.40 4.76 4.12 6.15 4.28 3.50 6.28 3.37 6.33 4.48 3.82 2.78 4.22 5.19 4.45 5.10 5.28 4.34 5.52 2.97 4.72 4.49 6.52 7.03 5.38 3.65 4.57 4.26 4.20 C16:1n7 47.61 37.51 45.02 40.35 46.98 30.40 42.40 24.16 63.45 41.54 28.57 16.45 57.38 61.54 43.71 57.32 52.51 55.39 109.35 48.93 49.42 42.25 73.04 36.57 85.25 41.17 89.02 36.54 32.97 C18:1n9 127.95 110.70 119.47 160.86 116.08 105.17 143.06 114.21 153.46 128.16 95.85 76.48 127.68 153.12 127.63 160.04 193.84 138.05 144.32 108.31 130.17 127.26 176.79 140.24 194.74 108.79 187.51 123.60 127.57 C18:1n7 22.75 20.05 21.48 22.01 21.56 18.99 27.62 20.15 23.13 22.27 17.72 11.66 23.35 28.87 24.69 33.07 35.85 23.84 22.78 20.07 23.70 24.00 34.01 26.20 35.14 21.30 31.08 22.31 25.99 C20:1n9 1.98 2.17 1.42 3.38 4.73 4.73 2.45 1.30 5.39 2.11 1.38 2.47 0.66 2.34 3.24 1.69 3.88 0.87 3.62 1.16 2.73 2.51 3.33 1.42 2.73 2.56 1.83 3.49 3.53 C18:2n6 175.93 179.02 177.13 129.24 186.20 200.85 165.25 156.95 240.84 240.32 178.53 160.90 208.57 228.93 211.89 208.64 241.62 192.29 189.82 172.17 185.32 205.74 254.08 186.98 268.81 166.33 267.41 206.10 196.70 C18:3n6 1.94 1.73 1.95 2.80 2.46 1.91 2.91 1.53 1.59 3.04 1.81 1.76 2.17 2.15 2.56 2.59 3.41 1.47 2.47 1.80 2.42 2.30 3.04 2.98 1.91 1.89 3.34 2.73 2.73 C20:2n6 3.19 0.97 3.68 2.71 3.40 4.43 2.88 2.67 5.53 4.93 3.50 2.74 2.60 4.50 4.59 4.26 4.68 4.01 3.38 1.96 2.75 4.14 4.55 2.56 3.72 3.30 4.68 5.32 4.46 C20:3n6 4.87 3.52 4.99 7.18 4.77 4.44 7.93 6.41 4.49 4.25 3.54 2.14 4.74 4.67 5.14 7.83 9.41 5.99 3.46 1.65 6.38 4.64 6.50 5.63 7.85 3.84 4.77 5.88 4.25 C20:4n6 61.35 61.37 64.68 61.33 73.10 94.01 58.33 61.86 70.91 74.25 63.09 54.09 62.87 72.92 64.43 75.08 67.07 70.74 57.99 39.63 63.13 73.02 79.43 58.33 92.76 70.21 84.45 66.08 61.67 C22:2n6 0.13 1.13 0.49 0.76 0.78 0.37 0.17 0.39 0.46 1.13 0.49 0.41 0.29 0.41 0.49 0.70 0.12 0.78 0.26 0.64 0.31 0.48 0.62 0.13 0.54 0.20 0.68 0.83 0.35 C22:4n6 5.47 4.68 5.58 6.89 5.74 5.97 6.54 6.40 5.50 5.73 6.14 4.26 4.29 6.84 6.60 8.22 9.14 7.01 3.87 3.49 6.20 6.18 25.43 5.50 12.36 6.15 7.36 5.86 6.28 C22:5n6 2.85 1.89 2.68 3.99 2.52 2.71 3.22 2.35 1.56 2.54 2.61 2.22 2.50 2.50 2.07 3.74 3.82 2.63 2.45 1.37 1.82 2.52 14.45 2.50 3.10 2.46 3.72 2.88 1.56 C18:3n3 14.94 13.03 15.38 9.44 15.94 16.05 14.46 11.39 22.41 20.09 14.85 12.20 20.25 21.57 16.66 19.66 20.06 17.59 20.17 13.55 18.30 16.83 25.14 15.51 25.57 13.54 27.84 16.71 13.98 C20:4n3 0.61 0.74 0.46 0.73 0.59 0.59 0.75 0.54 0.48 0.46 0.57 0.33 0.72 0.53 0.62 1.00 0.83 0.71 0.65 0.42 0.69 0.50 0.52 0.49 0.50 0.49 0.51 0.75 0.37 C20:5n3 1.76 1.95 1.48 1.62 1.52 1.58 1.75 1.40 1.88 2.27 1.50 1.47 1.13 2.08 2.23 2.38 2.45 1.96 1.46 1.21 1.33 1.65 2.19 1.37 2.04 1.48 3.03 3.59 1.51 C22:5n3 5.65 3.66 6.69 6.06 4.04 5.39 5.34 5.12 6.03 5.02 4.33 2.80 3.77 5.04 6.06 6.65 10.24 7.41 4.14 2.15 4.34 5.13 9.39 4.83 5.94 3.86 6.07 6.49 5.86 C22:6n3 39.46 36.38 46.05 43.24 44.32 60.94 43.42 45.55 20.30 45.13 43.54 29.39 41.29 46.53 42.45 56.92 54.35 21.85 32.98 35.70 47.66 45.44 54.69 37.93 26.53 42.84 54.34 45.13 38.13 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID C15:0 985 C16:0 10465 C17:0 5281 C18:0 5282745 C20:0 C16:1n7t 5282761 C16:1n7 142770 C18:1n9 5280450 C18:1n7 5280644 C20:1n9 5280933 C18:2n6 6439848 C18:3n6 444899 C20:2n6 5497181 C20:3n6 6441454 C20:4n6 5280934 C22:2n6 C22:4n6 5312539 C22:5n6 446284 C18:3n3 5282850 C20:4n3 445580 C20:5n3 13849 C22:5n3 5312508 C22:6n3 5312513 METABOLITES_END #END