Development of a Novel Targeted Metabolomic LC-QqQ-MS Method in Allergic Inflammation

Abstract

The transition from mild to severe allergic phenotypes is still poorly understood and there is an urgent need of incorporating new therapies, accompanied by personalized diagnosis approaches. This work presents the development of a novel targeted metabolomic methodology for the analysis of 36 metabolites related to allergic inflammation, including mostly sphingolipids, lysophospholipids, amino acids, and those of energy metabolism previously identified in non-targeted studies. The methodology consisted of two complementary chromatography methods, HILIC and reversed-phase. These were developed using liquid chromatography, coupled to triple quadrupole mass spectrometry (LC-QqQ-MS) in dynamic multiple reaction monitoring (dMRM) acquisition mode and were validated using ICH guidelines. Serum samples from two clinical models of allergic asthma patients were used for method application, which were as follows: (1) corticosteroid-controlled (ICS, n = 6) versus uncontrolled (UC, n = 4) patients, and immunotherapy-controlled (IT, n = 23) versus biologicals-controlled (BIO, n = 12) patients. The results showed significant differences mainly in lysophospholipids using univariate analyses in both models. Multivariate analysis for model 1 was able to distinguish both groups, while for model 2, the results showed the correct classification of all BIO samples within their group. Thus, this methodology can be of great importance for further understanding the role of these metabolites in allergic diseases as potential biomarkers for disease severity and for predicting patient treatment response.

Severe Allergy as a Chronic Inflammatory Condition From a Systems Biology Perspective

“…They are usually a product of the action of phospholipase (PLA) enzymes, such as PLA1 and/or PLA2, which act on cell membrane glycerophospholipids releasing a FA and a LPGL [110]. There are multiple studies suggesting LPGL involvement in asthma, allergy, COPD, obesity, diabetes and other diseases [102,125,[140][141][142][143][144]. LPCs, LPEs and LPIs have been found increased in the most severe phenotypes of allergic/eosinophilic asthma [125,145], AR [19], and nasal polyps with concomitant allergy [144] compared with either nonallergic or mild allergic patients.…”

L-Phenylalanine is a metabolic checkpoint of human Th2 cells

“…Phenylalanine concentration was analysed in Cohort D using an adaptation of the previously published method 101 . In brief, serum samples were prepared in a randomized order and measured in batches using dynamic molecular reaction monitoring on a liquid chromatography system (1260 Infinity II, Agilent Technologies, Waldbronn, Germany) coupled to a triple quadrupole mass spectrometer with electrospray ionization Agilent Jet Stream source, 6470 Agilent Technologies.…”

“…Then, serum samples were vortex-mixed for 20s, placed on ice for 20min and centrifuged at 16000g for 20min at 4°C. Then, 70µL of the supernatant was transferred into an LC vial and mixed with 50µL of ISTD mix] 101 and 440µL of the initial conditions of the mobile phases (95% B + 5% A). QC and blank samples, prepared as described above, were also measured through the run.…”

Microbiome Therapeutics for Food Allergy

“…Emerging evidence supports the connection between sphingolipids, a class of lipids with a long-chain sphingoid base, to food allergy pathogenesis [ 102 , 103 , 104 ]. Sphingolipids may be ingested, produced endogenously, and, notably, synthesized by gut microbiota, such as Bacteroidetes [ 105 ].…”