{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002438","ANALYSIS_ID":"AN003972","VERSION":"1","CREATED_ON":"January 12, 2023, 3:00 pm"},

"PROJECT":{"PROJECT_TITLE":"Ozone alters glycosphingolipid metabolism and exacerbates characteristics of asthma in mice","PROJECT_SUMMARY":"Asthma is a common chronic respiratory disease exacerbated by multiple environmental factors, including exposure to air pollutants such as ozone. Acute ozone exposure has previously been implicated in airway inflammation, airway hyperreactivity, and other characteristics of asthma. Altered sphingolipid metabolism following ozone exposure may contribute to the molecular mechanisms underlying these previously reported effects. This study aimed to identify changes in metabolomic profiles and characteristics of asthma in allergen-sensitized mice following ozone exposure to provide insights regarding mechanisms of ozone-induced exacerbations in asthma. Adult male and female BALB/c mice were sensitized intranasally to house dust mite allergen (HDM) on days 1, 3, and 5 followed by HDM challenge on days 12-14. Mice were subsequently exposed to ozone following each HDM challenge for 6 hr/day. Bronchoalveolar lavage, plasma, whole lung lobes, and microdissected lung airways were collected from 8 female and 8 male mice for metabolomics analysis. 6 female and 6 male mice underwent methacholine challenge using a forced oscillation technique to assess pulmonary function. HDM-sensitized male mice exposed to ozone displayed synergistically increased airway hyperreactivity as well as increased airway inflammation and eosinophilia relative to control mice. Effects in male mice were significantly more severe than the effects observed in females. Both HDM-sensitized male and female mice exposed to ozone displayed significant decreases in multiple classes of sphingolipids in microdissected airways. However, glycosphingolipids were significantly increased in females and to a lesser extent in males. These results potentially implicate glycosphingolipids in protecting against severe outcomes of ozone exposure that coincide with exacerbation of allergic asthma.","INSTITUTE":"University of California Davis","LAST_NAME":"Stevens","FIRST_NAME":"Nathanial","ADDRESS":"451 Health Sciences Drive, Davis, CA, 95616, USA","EMAIL":"ncstevens@ucdavis.edu","PHONE":"8282844315","FUNDING_SOURCE":"R21 ES030276, T32 ES007059, T32 HL007013, NIH U19 AG023122"},

"STUDY":{"STUDY_TITLE":"Ozone alters glycosphingolipid metabolism and exacerbates characteristics of asthma in mice","STUDY_SUMMARY":"Asthma is a common chronic respiratory disease exacerbated by multiple environmental factors, including exposure to air pollutants such as ozone. Acute ozone exposure has previously been implicated in airway inflammation, airway hyperreactivity, and other characteristics of asthma. Altered sphingolipid metabolism following ozone exposure may contribute to the molecular mechanisms underlying these previously reported effects. This study aimed to identify changes in metabolomic profiles and characteristics of asthma in allergen-sensitized mice following ozone exposure to provide insights regarding mechanisms of ozone-induced exacerbations in asthma. Adult male and female BALB/c mice were sensitized intranasally to house dust mite allergen (HDM) on days 1, 3, and 5 followed by HDM challenge on days 12-14. Mice were subsequently exposed to ozone following each HDM challenge for 6 hr/day. Bronchoalveolar lavage, plasma, whole lung lobes, and microdissected lung airways were collected from 8 female and 8 male mice for metabolomics analysis. 6 female and 6 male mice underwent methacholine challenge using a forced oscillation technique to assess pulmonary function. HDM-sensitized male mice exposed to ozone displayed synergistically increased airway hyperreactivity as well as increased airway inflammation and eosinophilia relative to control mice. Effects in male mice were significantly more severe than the effects observed in females. Both HDM-sensitized male and female mice exposed to ozone displayed significant decreases in multiple classes of sphingolipids in microdissected airways. However, glycosphingolipids were significantly increased in females and to a lesser extent in males. These results potentially implicate glycosphingolipids in protecting against severe outcomes of ozone exposure that coincide with exacerbation of allergic asthma.","INSTITUTE":"University of California, Davis","LAST_NAME":"Stevens","FIRST_NAME":"Nathanial","ADDRESS":"451 Health Sciences Drive","EMAIL":"ncstevens@ucdavis.edu","PHONE":"8282844315"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","GENOTYPE_STRAIN":"BALB/c","AGE_OR_AGE_RANGE":"8-10 wks.","GENDER":"Male and female","ANIMAL_ANIMAL_SUPPLIER":"Envigo","ANIMAL_LIGHT_CYCLE":"12/12 light/dark"},
"SUBJECT_SAMPLE_FACTORS":[
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"Factors":{"Factor":"HO3_F"},
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{
"Subject ID":"-",
"Sample ID":"mx626289_OzAW_BR1",
"Factors":{"Factor":"BR"},
"Additional sample data":{"RAW_FILE_NAME":"mx626289_OzAW_BR1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"mx626289_OzAW_BR2",
"Factors":{"Factor":"BR"},
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{
"Subject ID":"-",
"Sample ID":"mx626289_OzAW_BR3",
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"Subject ID":"-",
"Sample ID":"mx626289_OzAW_BR4",
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{
"Subject ID":"-",
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{
"Subject ID":"-",
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{
"Subject ID":"-",
"Sample ID":"mx626289_OzAW_MB01",
"Factors":{"Factor":"MB"},
"Additional sample data":{"RAW_FILE_NAME":"mx626289_OzAW_MB01.raw"}
},
{
"Subject ID":"-",
"Sample ID":"mx626289_OzAW_MB02",
"Factors":{"Factor":"MB"},
"Additional sample data":{"RAW_FILE_NAME":"mx626289_OzAW_MB02.raw"}
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{
"Subject ID":"-",
"Sample ID":"mx626289_OzAW_MB03",
"Factors":{"Factor":"MB"},
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{
"Subject ID":"-",
"Sample ID":"mx626289_OzAW_MB04",
"Factors":{"Factor":"MB"},
"Additional sample data":{"RAW_FILE_NAME":"mx626289_OzAW_MB04.raw"}
},
{
"Subject ID":"-",
"Sample ID":"mx626289_OzAW_MB05",
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{
"Subject ID":"-",
"Sample ID":"mx626289_OzAW_MB06",
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{
"Subject ID":"-",
"Sample ID":"mx626289_OzAW_MB07",
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"Subject ID":"-",
"Sample ID":"mx626289_OzAW_P1",
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"Sample ID":"mx626289_OzAW_P2",
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{
"Subject ID":"-",
"Sample ID":"mx626289_OzAW_P3",
"Factors":{"Factor":"P"},
"Additional sample data":{"RAW_FILE_NAME":"mx626289_OzAW_P3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"mx626289_OzAW_P4",
"Factors":{"Factor":"P"},
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},
{
"Subject ID":"-",
"Sample ID":"mx626289_OzAW_P5",
"Factors":{"Factor":"P"},
"Additional sample data":{"RAW_FILE_NAME":"mx626289_OzAW_P5.raw"}
},
{
"Subject ID":"-",
"Sample ID":"mx626289_OzAW_P6",
"Factors":{"Factor":"P"},
"Additional sample data":{"RAW_FILE_NAME":"mx626289_OzAW_P6.raw"}
},
{
"Subject ID":"-",
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],
"COLLECTION":{"COLLECTION_SUMMARY":"Right lung lobes were microdissected following a previous protocol (Plopper et al. 1991; Stevens et al. 2021). Briefly, 3 right lobes from each mouse collected during necropsy were blunt dissected on ice by removal of the surrounding lung parenchyma from the airways. The dissected lung airways were promptly transferred and stored at -80 degrees C until preparation for metabolomics analysis.","SAMPLE_TYPE":"Lung airways"},

"TREATMENT":{"TREATMENT_SUMMARY":"Mice underwent intranasal instillation with either house-dust mite dissolved in PBS or vehicle on days 1,3, and 5. Mice were challenged with HDM or vehicle on days 12-14 and subsequently exposed to either filtered air or ozone (0.5ppm, 6hr./day) after each challenge."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Extraction of Mammalian Tissue Samples: Liver 1. References: Fiehn O, Kind T (2006) Metabolite profiling in blood plasma. In: Metabolomics: Methods and Protocols. Weckwerth W (ed.), Humana Press, Totowa NJ (in press) 2.Starting material: Liver sample: weigh 4mg per sample into 2mL Eppendorf tubes. 3. Equipment: Centrifuge (Eppendorf 5415 D) Calibrated pipettes 1-200μl and 100-1000μl Eppendorf tubes 2mL, clear (Cat. No. 022363204) Centrifuge tubes 50mL, polypropylene Eppendorff Tabletop Centrifuge (Proteomics core Lab.) ThermoElectron Neslab RTE 740 cooling bath at –20°C MiniVortexer (VWR) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Speed vacuum concentration system (Labconco Centrivap cold trap) Turex mini homogenizer 4. Chemicals Acetonitrile, LCMS grade (JT Baker; Cat. No.9829-02) Isopropanol, HPLC grade (JT Baker; Cat. No. 9095-02) Methanol Acetone Crushed ice 18 MΩ pure water (Millipore) Nitrogen line with pipette tip pH paper 5-10 (EMD Chem. Inc.) 5. Procedure Preparation of extraction mix and material before experiment: Switch on bath to pre-cool at –20°C (±2°C validity temperature range) Check pH of acetonitrile and isopropanol (pH7) using wetted pH paper Make the extraction solution by mixing acetonitrile, isopropanol and water in proportions 3 : 3 : 2 De-gas the extraction solution for 5 min with nitrogen. Make sure that the nitrogen line was flushed out of air before using it for degassing the extraction solvent solution Sample Preparation Weigh 4mg tissue sample in to a 2mL Eppendorf tube. Add 1mL extraction solvent to the tissue sample and homogenize for 45 seconds ensuring that sample resembles a powder. In between samples, clean the homogenizer in solutions of methanol, acetone, water, and the extraction solvent in the order listed. Vortex samples for 10 seconds, then 5 minutes on 4°C shaker. Centrifuge the samples for 2 minutes at 14,000 rcf. Aliquot 500µL supernatant for analysis, and 500µL for a backup. Store backup aliquots in the -20°C freezer. Evaporate one 500µl analysis aliquot in the Labconco Centrivap cold trap concentrator to complete dryness (typically overnight). The dried aliquot is then re-suspended with 500μl 50% acetonitrile (degassed as given) Centrifuge for 2 minutes at 14,000 rcf using the centrifuge Eppendorf 5415. Remove supernatant to a new Eppendorf tube. Evaporate the supernatant to dryness in the the Labconco Centrivap cold trap concentrator. Submit to derivatization. The residue should contain membrane lipids because these are supposedly not soluble enough to be found in the 50% acetonitrile solution. Therefore, this ‘membrane residue’ is now taken for membrane lipidomic fingerprinting using the nanomate LTQ ion trap mass spectrometer. Likely, a good solvent to redissolve the membrane lipids is e.g. 75% isopropanol (degassed as given above). If the ‘analysis’ aliquot is to be used for semi lipophilic compounds such as tyrosine pathway intermediates (incl. dopamine, serotonine etc, i.e. polar aromatic compounds), then these are supposedly to be found together with the ‘GCTOF’ aliquot. We can assume that this mixture is still too complex for Agilent chipLCMS. Therefore, in order to develop and validate target analysis for such aromatic compounds, we should use some sort of Solid Phase purification. We re-suspend the dried ‘GCTOF’ aliquot in 300 l water (degassed as before) to take out sugars, aliphatic amino acids, hydroxyl acids and similar logP compounds. The residue should contain our target aromatics .We could also try to adjust pH by using low concentration acetate or phosphate buffer. The residue could then be taken up in 50% acetonitrile and used for GCTOF and Agilent chipMS experiments. The other aliquot should be checked how much of our target compounds would actually be found in the ‘sugar’ fraction. 6. Problems To prevent contamination disposable material is used. Control pH from extraction mix. 7. Quality assurance For each sequence of sample extractions, perform one blank negative control extraction by applying the total procedure (i.e. all materials and plastic ware) without biological sample. 8. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Waters Acquity CSH C18 (100 x 2.1mm, 1.7um)"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive HF hybrid Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"The ion source conditions were set as follows: spray voltage, -3.0 kV; sheath gas flow rate, 60 arbitrary units; aux gas flow rate, 25 arbitrary units; sweep gas flow rate, 2 arbitrary units; capillary temp, 300 °C; S-lens RF level, 50; Aux gas heater temp, 370 °C. The following acquisition parameters were used for MS1 analysis: resolution, 60000, AGC target, 1e6; Maximum IT, 100 ms; scan range 60-900 m/z; spectrum data type, centroid. Data dependent MS/MS parameters: resolution, 15000; AGC target, 1e5; maximum IT, 50 ms; loop count, 4; TopN, 4; isolation window, 1.0 m/z; fixed first mass, 70.0 m/z; (N)CE/ stepped nce, 20, 30, 40; spectrum data type, centroid; minimum AGC target, 8e3; intensity threshold, 1.6e5; exclude isotopes, on; dynamic exclusion, 3.0 s. To increase the total number of MS/MS spectra, five runs with iterative MS/MS exclusions were performed using the R package “IE-Omics”18 for both positive and negative electrospray conditions."},

"MS_METABOLITE_DATA":{
"Units":"Relative abundance",

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