{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001734","ANALYSIS_ID":"AN002823","VERSION":"1","CREATED_ON":"March 25, 2021, 11:12 am"},

"PROJECT":{"PROJECT_TITLE":"LC-MS Nasal Polyp analysis","PROJECT_SUMMARY":"Analysis of human samples from patients with nasal polyps with and without allergy.","INSTITUTE":"CEMBIO","LAST_NAME":"Delgado Dolset","FIRST_NAME":"María Isabel","ADDRESS":"Urb. Montepríncipe s/n, Ctra. Boadilla del Monte km 5.3, Madrid, Madrid, 28668, Spain","EMAIL":"maria.delgadodolset@beca.ceu.es","PHONE":"+34 913724700 4665"},

"STUDY":{"STUDY_TITLE":"Understanding systemic and local inflammation induced by nasal polyposis: role of the allergic phenotype (part-II)","STUDY_SUMMARY":"Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by persistent symptoms associated to the development of nasal polyps. To this day, the molecular mechanisms involved are still not well defined. However, it has been suggested that a sustained inflammation as allergy is involved in its onset. In this pilot study, we aimed to look into the effect of the allergic status of the patient and in their underlying mechanisms. To achieve this, we recruited 22 patients with CRSwNP and classified them in non-allergic and allergic using ImmunoCAP ISAC molecular diagnosis. Plasma samples were analyzed using liquid chromatography coupled to mass spectrometry (LC-MS). Subsequently, the identified changed metabolites from plasma that were commercially available were then analyzed by targeted analysis in some nasal polyps. Additionally, nasal polyp and mucosa tissue samples were examined for eosinophils and neutrophils. We found that 9 out of the 22 patients were sensitized to some aeroallergens (named as allergic). The other 13 patients had no sensitizations (non-allergic). Regarding metabolomics, we found that bilirubin, cortisol, lysophosphatidylcholines (LPCs) 16:0, 18:0 and 20:4 and lysophosphatidylinositol (LPI) 20:4, metabolites that are usually related to a sustained allergic inflammation, were unexpectedly increased in the plasma of non-allergic patients with CRSwNP compared to allergic patients with CRSwNP. LPC 16:0, LPC 18:0 and LPI 20:4 metabolites followed the same trend in the nasal polyp as they did in plasma. Comparison of nasal polyps with nasal mucosa tissue showed a significant increase in eosinophils (p < 0.001) and neutrophils (p < 0.01) in allergic patients with CRSwNP. There were also more eosinophils in the polyps of non-allergic patients with CRSwNP than in their nasal mucosa (p <0.01). The polyps from non-allergic patients with CRSwNP had less eosinophils than the polyps of allergic patients with CRSwNP (p < 0.05). Our data suggests that there is a systemic inflammatory response associated to CRSwNP in the absence of allergy, which could be accountable for the nasal polyp development. Allergic patients with CRSwNP presented a higher number of eosinophils located in nasal polyps suggesting that eosinophilia might be connected to the development of nasal polyps in these patients.","INSTITUTE":"CEMBIO","LAST_NAME":"Delgado Dolset","FIRST_NAME":"María Isabel","ADDRESS":"Urb. Montepríncipe s/n, Ctra. Boadilla del Monte km 5.3, Madrid, Madrid, 28668, Spain","EMAIL":"maria.delgadodolset@beca.ceu.es","PHONE":"+34 913724700 4665","NUM_GROUPS":"2","TOTAL_SUBJECTS":"22"},

"SUBJECT":{"SUBJECT_TYPE":"Human","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"POL-04",
"Factors":{"group":"non-allergic"},
"Additional sample data":{"RAW_FILE_NAME":"POL04b.d POL04c.d POL04d.d POL04e.d"}
},
{
"Subject ID":"-",
"Sample ID":"POL-06",
"Factors":{"group":"non-allergic"},
"Additional sample data":{"RAW_FILE_NAME":"POL06b.d POL06c.d POL06d.d POL06e.d"}
},
{
"Subject ID":"-",
"Sample ID":"POL-14",
"Factors":{"group":"non-allergic"},
"Additional sample data":{"RAW_FILE_NAME":"POL14b.d POL14c.d POL14d.d POL14e.d"}
},
{
"Subject ID":"-",
"Sample ID":"POL-15",
"Factors":{"group":"allergic"},
"Additional sample data":{"RAW_FILE_NAME":"POL15b.d POL15c.d POL15d.d POL15e.d"}
},
{
"Subject ID":"-",
"Sample ID":"POL-19",
"Factors":{"group":"allergic"},
"Additional sample data":{"RAW_FILE_NAME":"POL19b.d POL19c.d POL19d.d POL19e.d"}
},
{
"Subject ID":"-",
"Sample ID":"POL-36",
"Factors":{"group":"allergic"},
"Additional sample data":{"RAW_FILE_NAME":"POL36b.d POL36c.d POL36d.d POL36e.d"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"During endoscopic surgical procedures to remove the polyp, 5 mm biopsies of nasal polyp were obtained and kept in RNA later.","SAMPLE_TYPE":"Nasal Polyp tissue","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"Nasal polyp samples were kept in RNA later and stored at -80ºC until preparation"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"RNAlater solvent was removed by washing the tissue 3 times with PBS 1X. Then, the polyp was frozen in liquid nitrogen for 30 seconds. The frozen sample was put in cryoPREPTMCP02 (Covaris, MA, United States) plastic bags and submerged again for 30 seconds in liquid nitrogen. Once the plastic bag was inside the automated cryoPREPTMCP02, two consecutive impact forces of levels 2 and 4 out of 6 were applied. The resultant powder was gathered and weighted. Then, 100 µL of cold methanol:ethanol (1:1, v/v) and 0.5 µL of internal standard (LPC 18:1-d7; 0.01mM) were added per each 10 mg of tissue for metabolite extraction and protein precipitation. Samples were then vortex-mixed and homogenized using Tissue-Lyser LT homogenizer (Qiagen, Germany) for 5 min at 50 Hz, 3 times. Supernatant containing the metabolites was separated from the pellet by centrifugation (2,000 rcf for 10 min at 4 °C). Then, an aliquot of 70 µL was transferred to an LC vial and diluted with 490 µL of mobile phase (5% water: 95% acetonitrile; both with 7.5 mM ammonium acetate and 0.1% acetic acid). All samples were randomized before metabolite extraction and for the corresponding analytical run."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"We used HPLC system (1260 Infinity, Agilent Technologies). Metabolites were separated on a Kinetex hydrophilic interaction liquid chromatography (HILIC) silica column (150 mm × 2.1 mm, particle size 100Å, Phenomenex, USA) maintained at 25 ºC. The mobile phases consisted of A) water, and B) acetonitrile, both with 7.5 mM ammonium acetate and 0.1% acetic acid, with a final pH of 4.0 in the aqueous phase. The flow rate was 0.5 mL/min with an injection volume of 5 µl. Gradient started with 5% of A for 2 min, then increased up to 50% until 12 min, and back to initial conditions until 22 min.","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Agilent 1260","COLUMN_NAME":"Phenomenex Kinetex HILIC 100A (50 x 2.1 mm, 2.6um)","FLOW_RATE":"0.5 mL/min","COLUMN_TEMPERATURE":"25ºC","SOLVENT_A":"7.5 mM ammonium acetate + 0.1% acetic acid on water (pH=4)","SOLVENT_B":"7.5 mM ammonium acetate + 0.1% acetic acid on acetonitrile"},

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

"MS":{"INSTRUMENT_NAME":"Agilent 6470 QQQ","INSTRUMENT_TYPE":"Triple quadrupole","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"The MS conditions were: 5500 V of capillary voltage in positive ESI mode, a nebulizer gas flow rate of 11.0 L/min; a source temperature of 250 °C; and a source pressure of 60 psi. The sample tray temperature was maintained at 4 °C. Each transition was optimized adjusting the fragmentor and collision energy voltages. Data were acquired in MassHunter Workstation B.05.00 (Agilent Technologies), and re-processed using MassHunter QQQ Quantitative Analysis B.08.00 (Agilent) where peak areas were integrated. Concentration of metabolites were calculated using calibration curves with the standard addition method."},

"MS_METABOLITE_DATA":{
"Units":"ug/mg",

"Data":[{"Metabolite":"Bilirubin","POL-04":"8.57E-05","POL-06":"7.69E-04","POL-14":"1.40E-05","POL-15":"4.76E-06","POL-19":"8.54E-04","POL-36":"1.93E-03"},{"Metabolite":"LPI 20 4","POL-04":"7.42E-02","POL-06":"9.49E-02","POL-14":"4.04E-02","POL-15":"4.36E-02","POL-19":"3.76E-02","POL-36":"5.44E-02"},{"Metabolite":"LPC 18 0","POL-04":"1.11E-01","POL-06":"9.26E-02","POL-14":"4.39E-02","POL-15":"6.11E-02","POL-19":"6.30E-02","POL-36":"8.51E-02"},{"Metabolite":"LPC 16 0","POL-04":"2.45E-01","POL-06":"1.89E-01","POL-14":"1.19E-01","POL-15":"1.64E-01","POL-19":"1.60E-01","POL-36":"2.11E-01"}],

"Metabolites":[{"metabolite_name":"Bilirubin"},{"metabolite_name":"LPI 20 4"},{"metabolite_name":"LPC 18 0"},{"metabolite_name":"LPC 16 0"}]
}

}