#METABOLOMICS WORKBENCH MIDelgadoDolset_20210317_041208 DATATRACK_ID:2532 STUDY_ID:ST001733 ANALYSIS_ID:AN002822 PROJECT_ID:PR001109 VERSION 1 CREATED_ON March 25, 2021, 11:06 am #PROJECT PR:PROJECT_TITLE LC-MS Nasal Polyp analysis PR:PROJECT_SUMMARY Analysis of human samples from patients with nasal polyps with and without PR:PROJECT_SUMMARY allergy. PR:INSTITUTE CEMBIO PR:LAST_NAME Delgado Dolset PR:FIRST_NAME María Isabel PR:ADDRESS Urb. Montepríncipe s/n, Ctra. Boadilla del Monte km 5.3, Madrid, Madrid, 28668, PR:ADDRESS Spain PR:EMAIL maria.delgadodolset@beca.ceu.es PR:PHONE +34 913724700 4665 #STUDY ST:STUDY_TITLE Understanding systemic and local inflammation induced by nasal polyposis: role ST:STUDY_TITLE of the allergic phenotype (part-I) ST:STUDY_SUMMARY Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by persistent ST:STUDY_SUMMARY symptoms associated to the development of nasal polyps. To this day, the ST:STUDY_SUMMARY molecular mechanisms involved are still not well defined. However, it has been ST:STUDY_SUMMARY suggested that a sustained inflammation as allergy is involved in its onset. In ST:STUDY_SUMMARY this pilot study, we aimed to look into the effect of the allergic status of the ST:STUDY_SUMMARY patient and in their underlying mechanisms. To achieve this, we recruited 22 ST:STUDY_SUMMARY patients with CRSwNP and classified them in non-allergic and allergic using ST:STUDY_SUMMARY ImmunoCAP ISAC molecular diagnosis. Plasma samples were analyzed using liquid ST:STUDY_SUMMARY chromatography coupled to mass spectrometry (LC-MS). Subsequently, the ST:STUDY_SUMMARY identified changed metabolites from plasma that were commercially available were ST:STUDY_SUMMARY then analyzed by targeted analysis in some nasal polyps. Additionally, nasal ST:STUDY_SUMMARY polyp and mucosa tissue samples were examined for eosinophils and neutrophils. ST:STUDY_SUMMARY We found that 9 out of the 22 patients were sensitized to some aeroallergens ST:STUDY_SUMMARY (named as allergic). The other 13 patients had no sensitizations (non-allergic). ST:STUDY_SUMMARY Regarding metabolomics, we found that bilirubin, cortisol, ST:STUDY_SUMMARY lysophosphatidylcholines (LPCs) 16:0, 18:0 and 20:4 and lysophosphatidylinositol ST:STUDY_SUMMARY (LPI) 20:4, metabolites that are usually related to a sustained allergic ST:STUDY_SUMMARY inflammation, were unexpectedly increased in the plasma of non-allergic patients ST:STUDY_SUMMARY with CRSwNP compared to allergic patients with CRSwNP. LPC 16:0, LPC 18:0 and ST:STUDY_SUMMARY LPI 20:4 metabolites followed the same trend in the nasal polyp as they did in ST:STUDY_SUMMARY plasma. Comparison of nasal polyps with nasal mucosa tissue showed a significant ST:STUDY_SUMMARY increase in eosinophils (p < 0.001) and neutrophils (p < 0.01) in allergic ST:STUDY_SUMMARY patients with CRSwNP. There were also more eosinophils in the polyps of ST:STUDY_SUMMARY non-allergic patients with CRSwNP than in their nasal mucosa (p <0.01). The ST:STUDY_SUMMARY polyps from non-allergic patients with CRSwNP had less eosinophils than the ST:STUDY_SUMMARY polyps of allergic patients with CRSwNP (p < 0.05). Our data suggests that there ST:STUDY_SUMMARY is a systemic inflammatory response associated to CRSwNP in the absence of ST:STUDY_SUMMARY allergy, which could be accountable for the nasal polyp development. Allergic ST:STUDY_SUMMARY patients with CRSwNP presented a higher number of eosinophils located in nasal ST:STUDY_SUMMARY polyps suggesting that eosinophilia might be connected to the development of ST:STUDY_SUMMARY nasal polyps in these patients. ST:INSTITUTE CEMBIO ST:LAST_NAME Delgado Dolset ST:FIRST_NAME María Isabel ST:ADDRESS Urb. Montepríncipe s/n, Ctra. Boadilla del Monte km 5.3, Madrid, Madrid, 28668, ST:ADDRESS Spain ST:EMAIL maria.delgadodolset@beca.ceu.es ST:PHONE +34 913724700 4665 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 22 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #FACTORS #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 - POL_11 group:allergic RAW_FILE_NAME=POL_11.d SUBJECT_SAMPLE_FACTORS - POL_12 group:allergic RAW_FILE_NAME=POL_12.d SUBJECT_SAMPLE_FACTORS - POL_14 group:non-allergic RAW_FILE_NAME=POL_14.d SUBJECT_SAMPLE_FACTORS - POL_15 group:allergic RAW_FILE_NAME=POL_15.d SUBJECT_SAMPLE_FACTORS - POL_19 group:allergic RAW_FILE_NAME=POL_19.d SUBJECT_SAMPLE_FACTORS - POL_2 group:non-allergic RAW_FILE_NAME=POL_2.d SUBJECT_SAMPLE_FACTORS - POL_20 group:non-allergic RAW_FILE_NAME=POL_20.d SUBJECT_SAMPLE_FACTORS - POL_21 group:allergic RAW_FILE_NAME=POL_21.d SUBJECT_SAMPLE_FACTORS - POL_22 group:non-allergic RAW_FILE_NAME=POL_22.d SUBJECT_SAMPLE_FACTORS - POL_23 group:non-allergic RAW_FILE_NAME=POL_23.d SUBJECT_SAMPLE_FACTORS - POL_24 group:allergic RAW_FILE_NAME=POL_24.d SUBJECT_SAMPLE_FACTORS - POL_25 group:allergic RAW_FILE_NAME=POL_25.d SUBJECT_SAMPLE_FACTORS - POL_28 group:allergic RAW_FILE_NAME=POL_28.d SUBJECT_SAMPLE_FACTORS - POL_29 group:non-allergic RAW_FILE_NAME=POL_29.d SUBJECT_SAMPLE_FACTORS - POL_31 group:non-allergic RAW_FILE_NAME=POL_31.d SUBJECT_SAMPLE_FACTORS - POL_4 group:non-allergic RAW_FILE_NAME=POL_4.d SUBJECT_SAMPLE_FACTORS - POL_5 group:non-allergic RAW_FILE_NAME=POL_5.d SUBJECT_SAMPLE_FACTORS - POL_6 group:non-allergic RAW_FILE_NAME=POL_6.d SUBJECT_SAMPLE_FACTORS - POL_8 group:non-allergic RAW_FILE_NAME=POL_8.d #COLLECTION CO:COLLECTION_SUMMARY We obtained 20 ml of heparinized blood from 19 out of the 22 patients. We used a CO:COLLECTION_SUMMARY Ficoll-Paque (GE Healthcare™) density gradient centrifugation to obtain CO:COLLECTION_SUMMARY plasma. Plasma samples were stored at -80ºC. CO:SAMPLE_TYPE Blood (plasma) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Plasma from heparinized blood was collected using a Ficoll-Paque (GE TR:TREATMENT_SUMMARY Healthcare™) density gradient centrifugation. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Plasma proteins were removed by adding 300 µL of cold (-20 ℃) SP:SAMPLEPREP_SUMMARY methanol:ethanol (1:1) to 100 µL of sample. Samples were then vortex-mixed and SP:SAMPLEPREP_SUMMARY stored on ice for 5 min. Supernatant containing the metabolites was separated SP:SAMPLEPREP_SUMMARY from the pellet by centrifugation (16,000 × g for 20 min at 4 ℃), then put SP:SAMPLEPREP_SUMMARY into a LC vial for analysis. Quality control sample (QC) was prepared by pooling SP:SAMPLEPREP_SUMMARY equal volumes of plasma from each sample. QC followed the same procedure applied SP:SAMPLEPREP_SUMMARY for the experimental samples and was analysed throughout the run to provide a SP:SAMPLEPREP_SUMMARY measurement of system stability, performance and reproducibility. All samples SP:SAMPLEPREP_SUMMARY were randomised before metabolite extraction and for the corresponding SP:SAMPLEPREP_SUMMARY analytical run. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Compound separation was performed on an Agilent HPLC system (1200 series, CH:CHROMATOGRAPHY_SUMMARY Agilent Technologies, Waldbronn, Germany) equipped with a degasser, two binary CH:CHROMATOGRAPHY_SUMMARY pumps, and a thermostated auto sampler. A volume of 10 μL of sample were CH:CHROMATOGRAPHY_SUMMARY injected into a Discovery HS C18 column (2.1 mm × 150 mm, 3.0 μm; Supelco, CH:CHROMATOGRAPHY_SUMMARY Sigma Aldrich, Germany), with a guard column Discovery® HS C18 (2 cm × 2.1 mm, CH:CHROMATOGRAPHY_SUMMARY 3 μm; Supelco, Sigma Aldrich, Germany), both maintained at 40 ℃. The flow CH:CHROMATOGRAPHY_SUMMARY rate was set at 0.6 mL/min. The elution gradient involved a mobile phase CH:CHROMATOGRAPHY_SUMMARY consisting of: (A) 0.1% formic acid (FA) in water, and (B) 0.1% FA in CH:CHROMATOGRAPHY_SUMMARY acetonitrile. Initial conditions were set at 25% phase B, which increased to 95% CH:CHROMATOGRAPHY_SUMMARY phase B in 35 min; then, it was re-equilibrated for 1 min and finally held for 9 CH:CHROMATOGRAPHY_SUMMARY min in the initial conditions. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1200 CH:COLUMN_NAME Discovery HS C18 column (2.1 mm × 150 mm, 3.0 μm) CH:FLOW_RATE 0.6 mL/min CH:COLUMN_TEMPERATURE 40ºC CH:SOLVENT_A 0.1% formic acid in water CH:SOLVENT_B 0.1% FA in 100% acetonitrile #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME CEMBIO #MS MS:INSTRUMENT_NAME Agilent 6520 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The capillary voltage was set at 3,500 V. The drying gas flow rate was 10.5 MS:MS_COMMENTS L/min at 330 ℃ and gas nebulizer at 52 psi; fragmentor voltage was 175 V; MS:MS_COMMENTS skimmer and octopole radio frequency voltage (OCT RF Vpp) were set to 65 and 750 MS:MS_COMMENTS V. Data were collected in the centroid mode at a scan rate of 1.2 spectra per MS:MS_COMMENTS second. Mass spectrometry detection was performed in full scan from 100 to 1200 MS:MS_COMMENTS m/z. The reference m/z ions were purine (121.0508) and HP-0921 (922.0097). These MS:MS_COMMENTS masses were continuously infused into the system to allow constant mass MS:MS_COMMENTS correction. Samples were analysed in separate runs. Acquired data were cleaned MS:MS_COMMENTS of background noises and unrelated ions using MassHunter Profinder (B.06.00, MS:MS_COMMENTS Agilent Technologies) software. “Molecular feature extraction” and “Find MS:MS_COMMENTS by ion” algorithms were applied to reduce the size and complexity of data, and MS:MS_COMMENTS to improve the reliability in finding the features. 1654 chemical signals were MS:MS_COMMENTS obtained. Then, data was filtered, and only those features detected in >50% in MS:MS_COMMENTS QCs and with a Relative Standard Deviation (RSD) <30% in Qcs were kept, MS:MS_COMMENTS resulting in 535 signals. MS:MS_RESULTS_FILE ST001733_AN002822_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END