#METABOLOMICS WORKBENCH Jinquan_20220730_180727 DATATRACK_ID:3373 STUDY_ID:ST002298 ANALYSIS_ID:AN003754 PROJECT_ID:PR001472 VERSION 1 CREATED_ON September 19, 2022, 7:44 am #PROJECT PR:PROJECT_TITLE NAD(P) deficiency plays an important role in the restraint-stress-induced PR:PROJECT_TITLE depression in the rat model PR:PROJECT_SUMMARY The metabolic dysfunction or irreversible metabolic changes from stress may PR:PROJECT_SUMMARY cause body vulnerability, potentially leading to the onset of psychiatric and PR:PROJECT_SUMMARY non-psychiatric illnesses. Nevertheless, little is known about the biochemical PR:PROJECT_SUMMARY events that cause depression due to stress. Our study employed open field test, PR:PROJECT_SUMMARY plasma adrenocorticotropic hormone (ACTH) and corticosterone determination, PR:PROJECT_SUMMARY serum biochemical analysis, quantitative PCR, immunoblotting, enzyme activity PR:PROJECT_SUMMARY assay, and NMR-based metabolomics to analyze and identify the biochemical PR:PROJECT_SUMMARY variations of body fluids (serum and urine) and tissues (brain, kidney, liver, PR:PROJECT_SUMMARY lung, and spleen) in an acute restraint stress-induced rat model of depression. PR:PROJECT_SUMMARY Our data suggested that the post-stress effects on biochemical alterations PR:PROJECT_SUMMARY involved different biochemical pathways, including regulating the NAD(P) pool, PR:PROJECT_SUMMARY glucose homeostasis, biosynthesis and degradation of heme, and uric acid PR:PROJECT_SUMMARY production and metabolism. The urinary excretion of nicotinate and nicotinamide PR:PROJECT_SUMMARY N-oxide increased significantly. Thus, we conclude that the depletion of NAD(P) PR:PROJECT_SUMMARY precursors may occur in response to restraint stress. Our results show a close PR:PROJECT_SUMMARY association between NAD(P) deficiency and post-stress metabolic dysfunction, PR:PROJECT_SUMMARY which would provide a ground for developing recovery-promoting micronutrients in PR:PROJECT_SUMMARY treating depression. PR:INSTITUTE Anhui Science and Technology University PR:LAST_NAME Li PR:FIRST_NAME Jinquan PR:ADDRESS No. 9, Donghua Road, Fengyang, Anhui Province, 233100, China PR:EMAIL lijinquan@ahstu.edu.cn PR:PHONE 86 133 2875 1890 #STUDY ST:STUDY_TITLE NAD(P) deficiency plays an important role in the restraint-stress-induced ST:STUDY_TITLE depression in the rat model ST:STUDY_SUMMARY The metabolic dysfunction or irreversible metabolic changes from stress may ST:STUDY_SUMMARY cause body vulnerability, potentially leading to the onset of psychiatric and ST:STUDY_SUMMARY non-psychiatric illnesses. Nevertheless, little is known about the biochemical ST:STUDY_SUMMARY events that cause depression due to stress. Our study employed open field test, ST:STUDY_SUMMARY plasma adrenocorticotropic hormone (ACTH) and corticosterone determination, ST:STUDY_SUMMARY serum biochemical analysis, quantitative PCR, immunoblotting, enzyme activity ST:STUDY_SUMMARY assay, and NMR-based metabolomics to analyze and identify the biochemical ST:STUDY_SUMMARY variations of body fluids (serum and urine) and tissues (brain, kidney, liver, ST:STUDY_SUMMARY lung, and spleen) in an acute restraint stress-induced rat model of depression. ST:STUDY_SUMMARY Our data suggested that the post-stress effects on biochemical alterations ST:STUDY_SUMMARY involved different biochemical pathways, including regulating the NAD(P) pool, ST:STUDY_SUMMARY glucose homeostasis, biosynthesis and degradation of heme, and uric acid ST:STUDY_SUMMARY production and metabolism. The urinary excretion of nicotinate and nicotinamide ST:STUDY_SUMMARY N-oxide increased significantly. Thus, we conclude that the depletion of NAD(P) ST:STUDY_SUMMARY precursors may occur in response to restraint stress. Our results show a close ST:STUDY_SUMMARY association between NAD(P) deficiency and post-stress metabolic dysfunction, ST:STUDY_SUMMARY which would provide a ground for developing recovery-promoting micronutrients in ST:STUDY_SUMMARY treating depression. ST:INSTITUTE Anhui Science and Technology University ST:LAST_NAME Li ST:FIRST_NAME Jinquan ST:ADDRESS No. 9, Donghua Road, Fengyang, Anhui Province, 233100, China ST:EMAIL lijinquan@ahstu.edu.cn ST:PHONE 86 133 2875 1890 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Rattus norvegicus SU:TAXONOMY_ID 10116 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - serum_44001 Experimental factor:non-stress RAW_FILE_NAME=44001 SUBJECT_SAMPLE_FACTORS - serum_44002 Experimental factor:non-stress RAW_FILE_NAME=44002 SUBJECT_SAMPLE_FACTORS - serum_44003 Experimental factor:non-stress RAW_FILE_NAME=44003 SUBJECT_SAMPLE_FACTORS - serum_44004 Experimental factor:non-stress RAW_FILE_NAME=44004 SUBJECT_SAMPLE_FACTORS - serum_44005 Experimental factor:non-stress RAW_FILE_NAME=44005 SUBJECT_SAMPLE_FACTORS - serum_44006 Experimental factor:non-stress RAW_FILE_NAME=44006 SUBJECT_SAMPLE_FACTORS - serum_44007 Experimental factor:48 h post-stress RAW_FILE_NAME=44007 SUBJECT_SAMPLE_FACTORS - serum_44008 Experimental factor:48 h post-stress RAW_FILE_NAME=44008 SUBJECT_SAMPLE_FACTORS - serum_44009 Experimental factor:48 h post-stress RAW_FILE_NAME=44009 SUBJECT_SAMPLE_FACTORS - serum_44010 Experimental factor:48 h post-stress RAW_FILE_NAME=44010 SUBJECT_SAMPLE_FACTORS - serum_44011 Experimental factor:48 h post-stress RAW_FILE_NAME=44011 SUBJECT_SAMPLE_FACTORS - serum_44012 Experimental factor:48 h post-stress RAW_FILE_NAME=44012 SUBJECT_SAMPLE_FACTORS - serum_44013 Experimental factor:48 h post-stress RAW_FILE_NAME=44013 SUBJECT_SAMPLE_FACTORS - urine_44302 Experimental factor:non-stress RAW_FILE_NAME=44302 SUBJECT_SAMPLE_FACTORS - urine_44303 Experimental factor:non-stress RAW_FILE_NAME=44303 SUBJECT_SAMPLE_FACTORS - urine_44304 Experimental factor:non-stress RAW_FILE_NAME=44304 SUBJECT_SAMPLE_FACTORS - urine_44305 Experimental factor:non-stress RAW_FILE_NAME=44305 SUBJECT_SAMPLE_FACTORS - urine_44306 Experimental factor:non-stress RAW_FILE_NAME=44306 SUBJECT_SAMPLE_FACTORS - urine_44307 Experimental factor:non-stress RAW_FILE_NAME=44307 SUBJECT_SAMPLE_FACTORS - urine_44308 Experimental factor:24 h post-stress RAW_FILE_NAME=44308 SUBJECT_SAMPLE_FACTORS - urine_44309 Experimental factor:24 h post-stress RAW_FILE_NAME=44309 SUBJECT_SAMPLE_FACTORS - urine_44310 Experimental factor:24 h post-stress RAW_FILE_NAME=44310 SUBJECT_SAMPLE_FACTORS - urine_44311 Experimental factor:24 h post-stress RAW_FILE_NAME=44311 SUBJECT_SAMPLE_FACTORS - urine_44312 Experimental factor:24 h post-stress RAW_FILE_NAME=44312 SUBJECT_SAMPLE_FACTORS - urine_44313 Experimental factor:24 h post-stress RAW_FILE_NAME=44313 SUBJECT_SAMPLE_FACTORS - urine_44314 Experimental factor:24 h post-stress RAW_FILE_NAME=44314 SUBJECT_SAMPLE_FACTORS - urine_44315 Experimental factor:48 h post-stress RAW_FILE_NAME=44315 SUBJECT_SAMPLE_FACTORS - urine_44316 Experimental factor:48 h post-stress RAW_FILE_NAME=44316 SUBJECT_SAMPLE_FACTORS - urine_44317 Experimental factor:48 h post-stress RAW_FILE_NAME=44317 SUBJECT_SAMPLE_FACTORS - urine_44318 Experimental factor:48 h post-stress RAW_FILE_NAME=44318 SUBJECT_SAMPLE_FACTORS - urine_44319 Experimental factor:48 h post-stress RAW_FILE_NAME=44319 SUBJECT_SAMPLE_FACTORS - urine_44320 Experimental factor:48 h post-stress RAW_FILE_NAME=44320 SUBJECT_SAMPLE_FACTORS - urine_44321 Experimental factor:48 h post-stress RAW_FILE_NAME=44321 SUBJECT_SAMPLE_FACTORS - liver_44044 Experimental factor:non-stress RAW_FILE_NAME=44044 SUBJECT_SAMPLE_FACTORS - liver_44049 Experimental factor:non-stress RAW_FILE_NAME=44049 SUBJECT_SAMPLE_FACTORS - liver_44054 Experimental factor:non-stress RAW_FILE_NAME=44054 SUBJECT_SAMPLE_FACTORS - liver_44059 Experimental factor:non-stress RAW_FILE_NAME=44059 SUBJECT_SAMPLE_FACTORS - liver_44064 Experimental factor:non-stress RAW_FILE_NAME=44064 SUBJECT_SAMPLE_FACTORS - liver_44069 Experimental factor:non-stress RAW_FILE_NAME=44069 SUBJECT_SAMPLE_FACTORS - liver_44074 Experimental factor:48 h post-stress RAW_FILE_NAME=44074 SUBJECT_SAMPLE_FACTORS - liver_44079 Experimental factor:48 h post-stress RAW_FILE_NAME=44079 SUBJECT_SAMPLE_FACTORS - liver_44084 Experimental factor:48 h post-stress RAW_FILE_NAME=44084 SUBJECT_SAMPLE_FACTORS - liver_44089 Experimental factor:48 h post-stress RAW_FILE_NAME=44089 SUBJECT_SAMPLE_FACTORS - liver_44094 Experimental factor:48 h post-stress RAW_FILE_NAME=44094 SUBJECT_SAMPLE_FACTORS - liver_44099 Experimental factor:48 h post-stress RAW_FILE_NAME=44099 SUBJECT_SAMPLE_FACTORS - liver_44104 Experimental factor:48 h post-stress RAW_FILE_NAME=44104 SUBJECT_SAMPLE_FACTORS - kidney_44045 Experimental factor:non-stress RAW_FILE_NAME=44045 SUBJECT_SAMPLE_FACTORS - kidney_44050 Experimental factor:non-stress RAW_FILE_NAME=44050 SUBJECT_SAMPLE_FACTORS - kidney_44055 Experimental factor:non-stress RAW_FILE_NAME=44055 SUBJECT_SAMPLE_FACTORS - kidney_44060 Experimental factor:non-stress RAW_FILE_NAME=44060 SUBJECT_SAMPLE_FACTORS - kidney_44065 Experimental factor:non-stress RAW_FILE_NAME=44065 SUBJECT_SAMPLE_FACTORS - kidney_44070 Experimental factor:non-stress RAW_FILE_NAME=44070 SUBJECT_SAMPLE_FACTORS - kidney_44075 Experimental factor:48 h post-stress RAW_FILE_NAME=44075 SUBJECT_SAMPLE_FACTORS - kidney_44080 Experimental factor:48 h post-stress RAW_FILE_NAME=44080 SUBJECT_SAMPLE_FACTORS - kidney_44085 Experimental factor:48 h post-stress RAW_FILE_NAME=44085 SUBJECT_SAMPLE_FACTORS - kidney_44090 Experimental factor:48 h post-stress RAW_FILE_NAME=44090 SUBJECT_SAMPLE_FACTORS - kidney_44095 Experimental factor:48 h post-stress RAW_FILE_NAME=44095 SUBJECT_SAMPLE_FACTORS - kidney_44100 Experimental factor:48 h post-stress RAW_FILE_NAME=44100 SUBJECT_SAMPLE_FACTORS - kidney_44105 Experimental factor:48 h post-stress RAW_FILE_NAME=44105 SUBJECT_SAMPLE_FACTORS - spleen_44046 Experimental factor:non-stress RAW_FILE_NAME=44046 SUBJECT_SAMPLE_FACTORS - spleen_44051 Experimental factor:non-stress RAW_FILE_NAME=44051 SUBJECT_SAMPLE_FACTORS - spleen_44056 Experimental factor:non-stress RAW_FILE_NAME=44056 SUBJECT_SAMPLE_FACTORS - spleen_44061 Experimental factor:non-stress RAW_FILE_NAME=44061 SUBJECT_SAMPLE_FACTORS - spleen_44066 Experimental factor:non-stress RAW_FILE_NAME=44066 SUBJECT_SAMPLE_FACTORS - spleen_44071 Experimental factor:non-stress RAW_FILE_NAME=44071 SUBJECT_SAMPLE_FACTORS - spleen_44076 Experimental factor:48 h post-stress RAW_FILE_NAME=44076 SUBJECT_SAMPLE_FACTORS - spleen_44081 Experimental factor:48 h post-stress RAW_FILE_NAME=44081 SUBJECT_SAMPLE_FACTORS - spleen_44086 Experimental factor:48 h post-stress RAW_FILE_NAME=44086 SUBJECT_SAMPLE_FACTORS - spleen_44091 Experimental factor:48 h post-stress RAW_FILE_NAME=44091 SUBJECT_SAMPLE_FACTORS - spleen_44096 Experimental factor:48 h post-stress RAW_FILE_NAME=44096 SUBJECT_SAMPLE_FACTORS - spleen_44101 Experimental factor:48 h post-stress RAW_FILE_NAME=44101 SUBJECT_SAMPLE_FACTORS - spleen_44106 Experimental factor:48 h post-stress RAW_FILE_NAME=44106 SUBJECT_SAMPLE_FACTORS - lung_44047 Experimental factor:non-stress RAW_FILE_NAME=44047 SUBJECT_SAMPLE_FACTORS - lung_44052 Experimental factor:non-stress RAW_FILE_NAME=44052 SUBJECT_SAMPLE_FACTORS - lung_44057 Experimental factor:non-stress RAW_FILE_NAME=44057 SUBJECT_SAMPLE_FACTORS - lung_44062 Experimental factor:non-stress RAW_FILE_NAME=44062 SUBJECT_SAMPLE_FACTORS - lung_44067 Experimental factor:non-stress RAW_FILE_NAME=44067 SUBJECT_SAMPLE_FACTORS - lung_44072 Experimental factor:non-stress RAW_FILE_NAME=44072 SUBJECT_SAMPLE_FACTORS - lung_44077 Experimental factor:48 h post-stress RAW_FILE_NAME=44077 SUBJECT_SAMPLE_FACTORS - lung_44082 Experimental factor:48 h post-stress RAW_FILE_NAME=44082 SUBJECT_SAMPLE_FACTORS - lung_44087 Experimental factor:48 h post-stress RAW_FILE_NAME=44087 SUBJECT_SAMPLE_FACTORS - lung_44092 Experimental factor:48 h post-stress RAW_FILE_NAME=44092 SUBJECT_SAMPLE_FACTORS - lung_44097 Experimental factor:48 h post-stress RAW_FILE_NAME=44097 SUBJECT_SAMPLE_FACTORS - lung_44102 Experimental factor:48 h post-stress RAW_FILE_NAME=44102 SUBJECT_SAMPLE_FACTORS - lung_44107 Experimental factor:48 h post-stress RAW_FILE_NAME=44107 SUBJECT_SAMPLE_FACTORS - brain_44048 Experimental factor:non-stress RAW_FILE_NAME=44048 SUBJECT_SAMPLE_FACTORS - brain_44053 Experimental factor:non-stress RAW_FILE_NAME=44053 SUBJECT_SAMPLE_FACTORS - brain_44058 Experimental factor:non-stress RAW_FILE_NAME=44058 SUBJECT_SAMPLE_FACTORS - brain_44063 Experimental factor:non-stress RAW_FILE_NAME=44063 SUBJECT_SAMPLE_FACTORS - brain_44068 Experimental factor:non-stress RAW_FILE_NAME=44068 SUBJECT_SAMPLE_FACTORS - brain_44073 Experimental factor:non-stress RAW_FILE_NAME=44073 SUBJECT_SAMPLE_FACTORS - brain_44078 Experimental factor:48 h post-stress RAW_FILE_NAME=44078 SUBJECT_SAMPLE_FACTORS - brain_44083 Experimental factor:48 h post-stress RAW_FILE_NAME=44083 SUBJECT_SAMPLE_FACTORS - brain_44088 Experimental factor:48 h post-stress RAW_FILE_NAME=44088 SUBJECT_SAMPLE_FACTORS - brain_44093 Experimental factor:48 h post-stress RAW_FILE_NAME=44093 SUBJECT_SAMPLE_FACTORS - brain_44098 Experimental factor:48 h post-stress RAW_FILE_NAME=44098 SUBJECT_SAMPLE_FACTORS - brain_44103 Experimental factor:48 h post-stress RAW_FILE_NAME=44103 SUBJECT_SAMPLE_FACTORS - brain_44108 Experimental factor:48 h post-stress RAW_FILE_NAME=44108 #COLLECTION CO:COLLECTION_SUMMARY Individual urine samples were collected in ice-cooled vessels containing 1% CO:COLLECTION_SUMMARY sodium azide (0.1 ml) for 2 h using a metabolic cage at 0, 24, and 48 h CO:COLLECTION_SUMMARY post-stress, respectively, and immediately frozen at -80°C. Animals were CO:COLLECTION_SUMMARY sacrificed by exsanguination under isoflurane anesthesia at 48 h post-stress. CO:COLLECTION_SUMMARY The blood sample was divided into two aliquots, one serum for biochemical CO:COLLECTION_SUMMARY analysis and the other heparinized plasma for NMR spectroscopic analysis. After CO:COLLECTION_SUMMARY weighing, brain, kidney, liver, lung, and spleen tissue were excised in CO:COLLECTION_SUMMARY duplicate: one being fixed in 10% formalin for histopathological examination, CO:COLLECTION_SUMMARY the other immediately snap-frozen in liquid nitrogen for tissue extraction. CO:COLLECTION_SUMMARY These samples were stored at -80°C until used. CO:SAMPLE_TYPE serum, urine, brain, kidney, liver, lung, spleen #TREATMENT TR:TREATMENT_SUMMARY According to the National Institutes of Health Guide for the Care and Use of TR:TREATMENT_SUMMARY Laboratory Animals, all animals involved in this study were cared for, and TR:TREATMENT_SUMMARY protocols were reviewed and approved by the Anhui Laboratory Animal Care TR:TREATMENT_SUMMARY Committee. The specific pathogen-free (SPF) seven-week-old male Sprague Dawley TR:TREATMENT_SUMMARY (SD) rats (weight 233 ± 5 g) were purchased from Beijing Vital River Laboratory TR:TREATMENT_SUMMARY Animal Technology Co., Ltd and used in this study. The environmental conditions TR:TREATMENT_SUMMARY were set at 21-26°C with a relative humidity of 50 ± 10% and a 12/12 h TR:TREATMENT_SUMMARY light/dark cycle. Food and tap water were provided ad libitum, and body weights TR:TREATMENT_SUMMARY were recorded daily. After one week of acclimatization, rats were randomly TR:TREATMENT_SUMMARY assigned to the groups of non-stressed control (n = 6) or the stressed (n = 7). TR:TREATMENT_SUMMARY For restraint stress, rats were individually placed in a ventilated plastic tube TR:TREATMENT_SUMMARY restrainer for 120 minutes, using a previously modified method. According to the TR:TREATMENT_SUMMARY general protocol, control rats were left undistributed in a home cage and TR:TREATMENT_SUMMARY allowed to contact each other without food and water. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples of plasma (255 μl) were mixed with 255 μl of phosphate D2O buffer SP:SAMPLEPREP_SUMMARY solution (NaH2PO4 and K2HPO4, 60 mM, pH 7.4). After centrifugation at 10000 × g SP:SAMPLEPREP_SUMMARY at 4°C for 10 min to remove the precipitates, the supernatants were transferred SP:SAMPLEPREP_SUMMARY to 5 mm NMR tubes and analyzed by NMR. Samples of urine (455 μl) were mixed SP:SAMPLEPREP_SUMMARY with 55 μl of D2O buffer solution (NaH2PO4 and K2HPO4, 1.5 M, including 0.1% SP:SAMPLEPREP_SUMMARY TSP (sodium 3-(trimethylsilyl) propionate-2,2,3,3-d4), pH 7.4) to minimize any SP:SAMPLEPREP_SUMMARY gross variation in the pH of the urine samples. The mixture was left to stand SP:SAMPLEPREP_SUMMARY for 10 min and centrifuged at 10000 × g at 4°C for 10 min to remove the SP:SAMPLEPREP_SUMMARY precipitates. The supernatants were transferred to 5 mm NMR tubes and analyzed SP:SAMPLEPREP_SUMMARY by NMR. The polar metabolites in the rat tissue were extracted according to the SP:SAMPLEPREP_SUMMARY protocol established in our previous work. In brief, pre-weighed brain, kidney, SP:SAMPLEPREP_SUMMARY liver, lung, or spleen samples (100 mg) were homogenized in 400 μl of CH3OH and SP:SAMPLEPREP_SUMMARY 85 μl of H2O at 4°C. The homogenates were transferred into a 2.5-ml tube, SP:SAMPLEPREP_SUMMARY combined with 400 μl of CHCl3 and 200 μl of H2O, and then kept in a vortex for SP:SAMPLEPREP_SUMMARY 60 s. After 10-min partitioning on ice, the samples were centrifuged for 5 min SP:SAMPLEPREP_SUMMARY (10000 × g, 4°C). The upper supernatants were transferred into 1.5-ml tubes SP:SAMPLEPREP_SUMMARY and lyophilized to remove CH3OH and H2O. The extracts were reconstituted in 0.5 SP:SAMPLEPREP_SUMMARY ml D2O containing 1 mM TSP, then transferred into 5-mm NMR tubes and analyzed by SP:SAMPLEPREP_SUMMARY NMR spectroscopy. #ANALYSIS AN:ANALYSIS_TYPE NMR #NMR NM:INSTRUMENT_NAME Varian 500 MHz spectrometer/Bruker-AV600 spectrometer NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 500 MHz/600 MHz NM:NMR_RESULTS_FILE ST002298_AN003754_Results.txt UNITS:Peak area #END