#METABOLOMICS WORKBENCH yfulcher_20160413_075611 DATATRACK_ID:581 STUDY_ID:ST000406 ANALYSIS_ID:AN000646 PROJECT_ID:PR000318 VERSION 1 CREATED_ON May 31, 2016, 4:27 pm #PROJECT PR:PROJECT_TITLE Biomarker Identification of EBC samples PR:PROJECT_TYPE Data processing and multivariate statistical analysis of NMR spectra PR:PROJECT_SUMMARY Multivariate Statistics of NMR Spectra of Exhaled Breath Condensate of Early PR:PROJECT_SUMMARY Allergic Asthma in Cats PR:INSTITUTE University of Missouri-Columbia PR:DEPARTMENT Biochemistry Department, Department of Veterinary Medicine and Surgery PR:LABORATORY Van Doren Lab PR:LAST_NAME Van Doren PR:FIRST_NAME Steven PR:ADDRESS 117 Schweitzer Hall, Biochemistry Department, University of Missouri-Columbia, PR:ADDRESS Columbia, MO 65211 PR:EMAIL VanDorenS@missouri.edu PR:PHONE 5738846405 PR:FUNDING_SOURCE Morris Animal Foundation grants D10FE-803A and D14FE-507 and University of PR:FUNDING_SOURCE Missouri Research Council grant URC-13-040 #STUDY ST:STUDY_TITLE Noninvasive Recognition and Biomarkers of Early Allergic Asthma in Cats using ST:STUDY_TITLE Multivariate Statistics of NMR Spectra of Exhaled Breath Condensate ST:STUDY_TYPE Statistical Analysis of NMR spectra of EBC samples ST:STUDY_SUMMARY Asthma is prevalent in children and cats, and needs means of noninvasive ST:STUDY_SUMMARY diagnosis. We sought to distinguish noninvasively the differences in 53 cats ST:STUDY_SUMMARY before and soon after induction of allergic asthma, using NMR spectra of exhaled ST:STUDY_SUMMARY breath condensate (EBC). Statistical pattern recognition was improved by ST:STUDY_SUMMARY preprocessing the spectra with glog transformation. Classification of the 106 ST:STUDY_SUMMARY preprocessed spectra by principal component analysis, partial least squares with ST:STUDY_SUMMARY discriminant analysis (PLS-DA), and multi-level PLS-DA appears to be impaired by ST:STUDY_SUMMARY variances unrelated to eosinophilic asthma. By subtracting out confounding ST:STUDY_SUMMARY variances, orthogonal signal correction (OSC) PLS-DA greatly improved the ST:STUDY_SUMMARY separation of the healthy and early asthmatic states, attaining 94% specificity ST:STUDY_SUMMARY and 71% sensitivity in predictions. OSC-PLS-DA results highlight the elevation ST:STUDY_SUMMARY of acetone in two-thirds of the cats with early asthma. Asthma also decreased at ST:STUDY_SUMMARY least a dozen compounds, especially carboxylic acids such as short chain fatty ST:STUDY_SUMMARY acids and amino acids. These trends suggest that a majority of the cats with ST:STUDY_SUMMARY allergic asthma underwent alteration of metabolic fluxes through pyruvate and ST:STUDY_SUMMARY acetyl-CoA to promote ketosis. The noninvasive detection of early experimental ST:STUDY_SUMMARY asthma, its biomarkers in EBC, and metabolic rerouting invite further ST:STUDY_SUMMARY investigation of the diagnostic potential in humans. ST:INSTITUTE University of Missouri-Columbia ST:DEPARTMENT Department of Biochemistry, Department of Veterinary Medicine and Surgery ST:LABORATORY Van Doren Lab & Reinero Lab ST:LAST_NAME Van Doren ST:FIRST_NAME Steven ST:ADDRESS 117 Schweitzer Hall, Biochemistry Department, University of Missouri-Columbia, ST:ADDRESS Columbia, MO 65211 ST:EMAIL VanDorenS@missouri.edu ST:PHONE 5738846405 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 53 #SUBJECT SU:SUBJECT_TYPE Animal SU:SUBJECT_SPECIES Felis catus SU:TAXONOMY_ID 9685 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - Vixen-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Twix-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Tux-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Titan-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Tacoma-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Sully-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Snickers-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Skittles-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Sedona-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Sable-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Rio-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Randall-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Paris-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Morgan-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Metro-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Mcfly-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Maxima-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Marti-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Lancer-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Kitcat-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Fox-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Focux-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Expo-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Echo-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Eagle-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Dino-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Diablo-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Dexter-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Deb-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Cudberry-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Civic-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Capri-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Camry-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Butterfinger-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Boxer-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Boo-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Biff-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Aspen-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Amigo-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Altima-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Aerio-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Accent-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Anthony-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Bonnie-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Cleopatra-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Clyde-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Fry-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Hansolo-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Jill-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Juliet-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Katniss-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Mickey-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Romeo-H Type:Healthy SUBJECT_SAMPLE_FACTORS - Vixen-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Twix-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Tux-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Titan-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Tacoma-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Sully-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Snickers-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Skittles-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Sedona-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Sable-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Rio-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Randall-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Paris-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Morgen-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Metro-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Mcfly-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Maxima-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Marti-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Lancer-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Kitkat-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Fox-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Focus-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Expo-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Echo-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Eagle-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Dino-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Diablo-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Dexter-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Deb-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Cudberry-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Civic-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Capri-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Camry-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Butterfinger-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Boxer-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Boo-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Biff-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Aspen-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Amigo-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Altima-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Aerio-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Accent-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Anthony-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Bonnie-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Cleopatra-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Clyde-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Fry-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Hansolo-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Jill-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Juliet-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Katniss-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Mickey-A Type:Asthmatic SUBJECT_SAMPLE_FACTORS - Romeo-A Type:Asthmatic #COLLECTION CO:COLLECTION_SUMMARY 1D 1H NMR spectra were collected using W5-WATERGATE water suppression CO:COLLECTION_SUMMARY supplemented by presaturation of the water resonance at minimal power. CO:COLLECTION_PROTOCOL_FILENAME protocol.pdf CO:SAMPLE_TYPE EBC (Exhaled breath condensate) and BALF (bronchoalveolar lavage fluid) #TREATMENT TR:TREATMENT_SUMMARY The care of the 53 cats in the study followed the NIH Guide for the Care and Use TR:TREATMENT_SUMMARY of Laboratory Animals. The University of Missouri Animal Care and Use Committee TR:TREATMENT_SUMMARY approved the study design (ACUC protocols #6912 and 7891). In order to induce TR:TREATMENT_SUMMARY allergic asthma, on day 0, cats <1 year of age were administered 12 µg of TR:TREATMENT_SUMMARY Bermuda grass allergen (BGA) in 10 mg of alum and 100 ng Bordetella pertussis TR:TREATMENT_SUMMARY toxin (both subcutaneously). On day 14, intranasal BGA (75 µg of BGA in 200 µL TR:TREATMENT_SUMMARY of phosphate-buffered saline) was given; and on day 21, 12 µg of BGA in 10 mg TR:TREATMENT_SUMMARY alum was injected subcutaneously. On day 28, the formation of wheals on an TR:TREATMENT_SUMMARY intradermal test confirmed sensitization to BGA. For the next 2 weeks, intensive TR:TREATMENT_SUMMARY aerosol challenges were administered to all cats with BGA (500 µg of BGA in 4 TR:TREATMENT_SUMMARY mL of phosphate-buffered saline) over 10 min in awake and unrestrained cats in a TR:TREATMENT_SUMMARY sealed plastic chamber. A nebulizer (Acorn nebulizer, model 646, Devilbis Health TR:TREATMENT_SUMMARY Care, Somerset, PA) delivered an aerosol of the BGA solution at an air flow rate TR:TREATMENT_SUMMARY of 9.3 l /min. An air compressor (Easy Air 15, Precision Medical, Inc., TR:TREATMENT_SUMMARY Northampton, PA) supplied the compressed air flow at a pressure of 2.93 kg/cm2. TR:TREATMENT_SUMMARY At week 6, samples were collected 24 h after challenge with BGA aerosol (see TR:TREATMENT_SUMMARY below). TR:TREATMENT_COMPOUND Bermuda grass allergen (BGA) #SAMPLEPREP SP:SAMPLEPREP_SUMMARY In all cats, Exhaled breath condensate (EBC)and Bronchoalveolar lavage fluid SP:SAMPLEPREP_SUMMARY (BALF) and were collected before (day 0) and after (week 6) sensitization to SP:SAMPLEPREP_SUMMARY BGA. The EBC was collected non-invasively just prior to BALF collection by SP:SAMPLEPREP_SUMMARY placing cats in a 25 L plexiglass chamber for 20-30 min according to a SP:SAMPLEPREP_SUMMARY previously published design with modifications [29]. BALF was collected in a SP:SAMPLEPREP_SUMMARY blind fashion under anesthesia using an 8 French red rubber catheter passed SP:SAMPLEPREP_SUMMARY through an endotracheal tube according to a previously described protocol [30]. SP:SAMPLEPREP_SUMMARY The percentage of eosinophils in each BALF modified Wright’s stained cytospin SP:SAMPLEPREP_SUMMARY was determined by counting 200 nucleated cells, with an asthmatic phenotype SP:SAMPLEPREP_SUMMARY defined as >17% eosinophils.The EBC and the supernatant of centrifuged BALF SP:SAMPLEPREP_SUMMARY remaining were promptly stored at -80 °C until further analysis. NMR spectra of SP:SAMPLEPREP_SUMMARY the EBC were measured for use in training and evaluating statistical approaches SP:SAMPLEPREP_SUMMARY and biomarkers. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE - CH:INSTRUMENT_NAME - CH:COLUMN_NAME - #ANALYSIS AN:ANALYSIS_TYPE NMR AN:LABORATORY_NAME Van Doren Lab AN:SOFTWARE_VERSION Topspin 3.1 #NMR NM:INSTRUMENT_NAME Bruker Avance III NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 800 MHz NM:BINNED_INCREMENT 0.02 NM:BINNED_DATA_EXCLUDED_RANGE 4.5-5.38 #NMR_BINNED_DATA NMR_BINNED_DATA:UNITS Peak area NMR_BINNED_DATA_START Bin range(ppm) Vixen-H Twix-H Tux-H Titan-H Tacoma-H Sully-H Snickers-H Skittles-H Sedona-H Sable-H Rio-H Randall-H Paris-H Morgan-H Metro-H Mcfly-H Maxima-H Marti-H Lancer-H Kitcat-H Fox-H Focux-H Expo-H Echo-H Eagle-H Dino-H Diablo-H Dexter-H Deb-H Cudberry-H Civic-H Capri-H Camry-H Butterfinger-H Boxer-H Boo-H Biff-H Aspen-H Amigo-H Altima-H Aerio-H Accent-H Anthony-H Bonnie-H Cleopatra-H Clyde-H Fry-H Hansolo-H Jill-H Juliet-H Katniss-H Mickey-H Romeo-H Vixen-A Twix-A Tux-A Titan-A Tacoma-A Sully-A Snickers-A Skittles-A Sedona-A Sable-A Rio-A Randall-A Paris-A Morgen-A Metro-A Mcfly-A Maxima-A Marti-A Lancer-A Kitkat-A Fox-A Focus-A Expo-A Echo-A Eagle-A Dino-A Diablo-A Dexter-A Deb-A Cudberry-A Civic-A Capri-A Camry-A Butterfinger-A Boxer-A Boo-A Biff-A Aspen-A Amigo-A Altima-A Aerio-A Accent-A Anthony-A Bonnie-A Cleopatra-A Clyde-A Fry-A Hansolo-A Jill-A Juliet-A Katniss-A Mickey-A Romeo-A NMR_BINNED_DATA_END #END