#METABOLOMICS WORKBENCH MIDelgadoDolset_20210317_070437_mwtab.txt DATATRACK_ID:2533 STUDY_ID:ST001734 ANALYSIS_ID:AN002823 PROJECT_ID:000000 VERSION 1 CREATED_ON March 25, 2021, 11:12 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-II) 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-04 group:non-allergic RAW_FILE_NAME=POL04b.d POL04c.d POL04d.d POL04e.d SUBJECT_SAMPLE_FACTORS - POL-06 group:non-allergic RAW_FILE_NAME=POL06b.d POL06c.d POL06d.d POL06e.d SUBJECT_SAMPLE_FACTORS - POL-14 group:non-allergic RAW_FILE_NAME=POL14b.d POL14c.d POL14d.d POL14e.d SUBJECT_SAMPLE_FACTORS - POL-15 group:allergic RAW_FILE_NAME=POL15b.d POL15c.d POL15d.d POL15e.d SUBJECT_SAMPLE_FACTORS - POL-19 group:allergic RAW_FILE_NAME=POL19b.d POL19c.d POL19d.d POL19e.d SUBJECT_SAMPLE_FACTORS - POL-36 group:allergic RAW_FILE_NAME=POL36b.d POL36c.d POL36d.d POL36e.d #COLLECTION CO:COLLECTION_SUMMARY During endoscopic surgical procedures to remove the polyp, 5 mm biopsies of CO:COLLECTION_SUMMARY nasal polyp were obtained and kept in RNA later. CO:SAMPLE_TYPE Nasal Polyp tissue CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Nasal polyp samples were kept in RNA later and stored at -80ºC until TR:TREATMENT_SUMMARY preparation #SAMPLEPREP SP:SAMPLEPREP_SUMMARY RNAlater solvent was removed by washing the tissue 3 times with PBS 1X. Then, SP:SAMPLEPREP_SUMMARY the polyp was frozen in liquid nitrogen for 30 seconds. The frozen sample was SP:SAMPLEPREP_SUMMARY put in cryoPREPTMCP02 (Covaris, MA, United States) plastic bags and submerged SP:SAMPLEPREP_SUMMARY again for 30 seconds in liquid nitrogen. Once the plastic bag was inside the SP:SAMPLEPREP_SUMMARY automated cryoPREPTMCP02, two consecutive impact forces of levels 2 and 4 out of SP:SAMPLEPREP_SUMMARY 6 were applied. The resultant powder was gathered and weighted. Then, 100 µL of SP:SAMPLEPREP_SUMMARY cold methanol:ethanol (1:1, v/v) and 0.5 µL of internal standard (LPC 18:1-d7; SP:SAMPLEPREP_SUMMARY 0.01mM) were added per each 10 mg of tissue for metabolite extraction and SP:SAMPLEPREP_SUMMARY protein precipitation. Samples were then vortex-mixed and homogenized using SP:SAMPLEPREP_SUMMARY Tissue-Lyser LT homogenizer (Qiagen, Germany) for 5 min at 50 Hz, 3 times. SP:SAMPLEPREP_SUMMARY Supernatant containing the metabolites was separated from the pellet by SP:SAMPLEPREP_SUMMARY centrifugation (2,000 rcf for 10 min at 4 °C). Then, an aliquot of 70 µL was SP:SAMPLEPREP_SUMMARY transferred to an LC vial and diluted with 490 µL of mobile phase (5% water: SP:SAMPLEPREP_SUMMARY 95% acetonitrile; both with 7.5 mM ammonium acetate and 0.1% acetic acid). All SP:SAMPLEPREP_SUMMARY samples were randomized before metabolite extraction and for the corresponding SP:SAMPLEPREP_SUMMARY analytical run. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY We used HPLC system (1260 Infinity, Agilent Technologies). Metabolites were CH:CHROMATOGRAPHY_SUMMARY separated on a Kinetex hydrophilic interaction liquid chromatography (HILIC) CH:CHROMATOGRAPHY_SUMMARY silica column (150 mm × 2.1 mm, particle size 100Å, Phenomenex, USA) CH:CHROMATOGRAPHY_SUMMARY maintained at 25 ºC. The mobile phases consisted of A) water, and B) CH:CHROMATOGRAPHY_SUMMARY acetonitrile, both with 7.5 mM ammonium acetate and 0.1% acetic acid, with a CH:CHROMATOGRAPHY_SUMMARY final pH of 4.0 in the aqueous phase. The flow rate was 0.5 mL/min with an CH:CHROMATOGRAPHY_SUMMARY injection volume of 5 µl. Gradient started with 5% of A for 2 min, then CH:CHROMATOGRAPHY_SUMMARY increased up to 50% until 12 min, and back to initial conditions until 22 min. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent 1260 CH:COLUMN_NAME Phenomenex Kinetex HILIC 100A (50 x 2.1 mm, 2.6um) CH:FLOW_RATE 0.5 mL/min CH:COLUMN_TEMPERATURE 25ºC CH:SOLVENT_A 7.5 mM ammonium acetate + 0.1% acetic acid on water (pH=4) CH:SOLVENT_B 7.5 mM ammonium acetate + 0.1% acetic acid on acetonitrile #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME CEMBIO #MS MS:INSTRUMENT_NAME Agilent 6470 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The MS conditions were: 5500 V of capillary voltage in positive ESI mode, a MS:MS_COMMENTS nebulizer gas flow rate of 11.0 L/min; a source temperature of 250 °C; and a MS:MS_COMMENTS source pressure of 60 psi. The sample tray temperature was maintained at 4 °C. MS:MS_COMMENTS Each transition was optimized adjusting the fragmentor and collision energy MS:MS_COMMENTS voltages. Data were acquired in MassHunter Workstation B.05.00 (Agilent MS:MS_COMMENTS Technologies), and re-processed using MassHunter QQQ Quantitative Analysis MS:MS_COMMENTS B.08.00 (Agilent) where peak areas were integrated. Concentration of metabolites MS:MS_COMMENTS were calculated using calibration curves with the standard addition method. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ug/mg MS_METABOLITE_DATA_START Samples POL-04 POL-06 POL-14 POL-15 POL-19 POL-36 Factors group:non-allergic group:non-allergic group:non-allergic group:allergic group:allergic group:allergic Bilirubin 8.57E-05 7.69E-04 1.40E-05 4.76E-06 8.54E-04 1.93E-03 LPI 20 4 7.42E-02 9.49E-02 4.04E-02 4.36E-02 3.76E-02 5.44E-02 LPC 18 0 1.11E-01 9.26E-02 4.39E-02 6.11E-02 6.30E-02 8.51E-02 LPC 16 0 2.45E-01 1.89E-01 1.19E-01 1.64E-01 1.60E-01 2.11E-01 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Bilirubin LPI 20 4 LPC 18 0 LPC 16 0 METABOLITES_END #END