Discussion

In this study，anaphylaxis models were successfully established in guinea pigs，which expressed typical clinical symptoms of anaphylaxis and high IgE level in serum after challenge with allergen on the 14th day of sensitization.Serum metabolic profiling was performed by GC-MSanalysis.The GC-MS method was validated by measuring repeatability，and good repeatability(RSD＜10%)was observed for six tested metabolites tentatively identified in serum samples.From metabolic profiling，metabolic differences between the control group and the model groups were characterized by multivariate pattern-recognition.Variables with significant differences between the control group and the model groups were observed(Tables 3 and 4).The metabolites predominantly responsible for the differences(p＜0.05)might range from amino acids，simple hydroxyacids，and fatty acids to sugars，sugar alcohols，and polyols.In addition，metabolic change of these metabolites can be putatively interpreted in a functional way.

Glucose is a major source of energy for living organisms.Fumaric acid is an intermediate in the tricarboxylic acid cycle(TCA).The lower levels of both in the model groups might be a result of high energy expenditure in the anaphylaxis reaction，which indicates energy metabolism is a significant aspect of anaphylaxis.As we observed，the anaphylaxis occurring in the model groups is IgE-dependent；IgE may induce smoothmuscle contraction and increase vascular contractility［22］.Anaphylaxis is an acute multisystem syndrome.It may affect multiple systems and organs including the respiratory system，cardiovascular system，and smooth muscle［22］.These reactions occurring during anaphylaxis lead to sudden energy expenditure.As a result，there might be a sharp increase of glucose consumption［23］with acceleration of energy metabolism and increased consumption of intermediate metabolites.

In contrast，higher levels of n-pentadecanoic acid and(R)-3-hydroxybutyric acid were observed in the model groups.Usually，when blood glucose and adenosine triphosphate(ATP)are low，the organism will burn fat to produce energy.As a result，the fat was degraded to free fatty acids，for example n-pentadecanoic acid.The fatty acids are then oxidized by theβ-oxidation pathway to form acetyl CoA，which leads to the formation of ketone bodies［24］.(R)-3Hydroxybutyric acid(beta-hydroxybutyrate)is one of these ketone bodies，and is an important source of energy for tissues such as the brain，heart，and skeletal muscle［24，25］.(https://www.daowen.com)

A rapid decrease of glucose and fumaric acid with release of a large amount of n-pentadecanoic acid and synthesis of a large amount of(R)-3-hydroxybutyric acid are suggestive of depletion of cellular energy stores in anaphylaxis.This could partly explain why irreversible anaphylaxis shock and death developed so rapidly in model groups after challenge.

myo-Inositol is a carbocyclic polyol which is the structural basis for several secondary messengers.Phosphorylated myo-inositol derivatives have essential second messenger functions in many immunocytes.Antigen/IgE-mediated mast cell activation can be markedly enhanced by generation of inositol 1，4，5-trisphosphate(IP3)，which also has an important function in T cell activation［26，27］.SHIP-1(Src homology region 2 domaincontaining inositol 5＇-phosphatase)negatively regulates cytokine immunoreceptors and amplifies signaling pathways in different cell types，including T cells，B cells，mast cells，basophils，and neutrophils［28，29］.It has been reported recently that SHIP-1 is critically involved in the adaptive Th2 immune response to aeroallergens［29］.The increased level of inositol detected in the model groups indicates inositol is involved in signal transduction during the allergic response.The last tentatively identified metabolite，L-arabitol，is a sugar alcohol metabolite involved in the pentose and glucuronate interconversions pathway.The detailed mechanism of involvement of L-arabitol in anaphylaxis needs further study.

In summary，this is the first report of comprehensive metabolic profiling analysis in anaphylaxis.Samples from a control group and model groups were successfully classified by GC-MS-based metabolomics analysis，and nine variables accounting for the classification were observed.Among the nine variables，glucose，fumaric acid，L-arabitol，(R)-3-hydroxybutyric acid，and myo-inositol closely associate anaphylactic reaction with the mechanisms of energy metabolism and signal transduction.These results suggest that GC-MS-based metabolic profiling analysis might be an effective tool for investigating metabolic regulation of anaphylaxis，and anaphylaxis could be diagnosed at the level of a small molecule in serum.