Study On Changes Of Metabolites Of L-Arginine In Rats With Acute Pancreatitis Based On UPLC-TOF-MS/MS Technology

Background:Acute Pancreatitis Syndrome（AP） is a common clinical Acute abdomen characterized by rapid onset,multiple complications and high mortality.In order to study the pathophysiology,cell biology,pathogenesis and treatment of AP,many invasive and non-invasive animal models of AP were established.However,the common disadvantage of them is that the model is not stable and has poor reversibility,and the model is easy to be contaminated by bacteria,so it is not suitable for secondary infection of AP.Recent studies have shown that AP rat models can be established by intraperitoneal injection of excessive L-arginine（L-arg）,and the method is simple,inexpensive and reproducible,it is an ideal animal model for studying acute pancreatitis and control measures.However,the detailed mechanism of L-arg-induced AP remains unclear.In particular,the metabolism of L-arg absorbed by intraperitoneal injection is unclear.There are two ways of liquid absorption in abdominal cavity:entering liver through portal vein,then entering systemic circulation after metabolism in liver,and entering systemic circulation directly through peritoneum.In this study,ultra-High-performance liquid chromatography ionization tandem mass spectrometry（UPLC-MS/MS）was used to detect the metabolites in blood and liver of rats after intraperitoneal injection of L-arg.Objective:To investigate the changes of metabolites in serum and liver tissue during intraperitoneal injection of L-arg in AP model.Methods:48 Male SD rats were randomly divided into normal Saline Control Group（NS Group,n=24） and L-arg Experimental Group（L-arg Group,N=24）.In L-arg group,Rats were injected with 20% L-arg twice intraperitoneally,one hour interval,2.5g/kg body weight per dose,and AP model was made.In NS Group,NS made the control group model in the same way as 12.5ml/kg body weight per dose.Rats in NS group and L-arg group were divided into 4 subgroups（N=6 per group） at the time points of 3h,6h,12h and 24h after intraperitoneal injection of L-arg and saline.The rats in each subgroup were killed at the corresponding time point.Before death,the serum amylase,Lipase,Serum calcium and L-arg metabolites were measured,and the pathological changes of pancreas and liver were observed The changes of L-arg metabolites in blood and liver were analyzed by UPLC-MS/MS and metabolize pilot software.Results:1.Serum amylase and lipase levels:There was no significant difference at each time point in NS group（P>0.05）.The levels of serum amylase and Lipase in L-arg group increased at 3h,peaked at 12h and decreased at 24h,but were still higher than those in the control group,the difference was statistically significant（P<0.01）.2.Serum calcium level:There was no significant difference in NS group at each time point（P>0.05）.At the same sampling time point,the serum calcium level in L-arg group was significantly lower than that in NS group（P<0.01）.3.Histopathological change:The pancreatic tissue in NS group was intact.In L-arg group,the structure of pancreatic lobules was disordered,the lobules were hyperemic and edematous with a lot of inflammatory cells infiltration.Liver tissue cells in NS group were intact,hepatic sinusoids in L-arg group were hyperemic,hepatocyte ballooning,punctate necrosis,a large number of inflammatory cells infiltrating,cells swelling,and the structure of Hepatic lobules was incomplete,even a large number of liver cell necrosis and liver lobular bleeding.The pathological injury scores of pancreas and liver were significantly different（P<0.01）.4.L-arg metabolites:compared with NS group,10 metabolites were detected during AP development in L-arg group.（1）In blood:L-arg and its eight metabolites were detected.L-arg and its five metabolites were detected at 3h,which were M1（NH⁺desaturated product of L-arg）,M2（N₂H₂⁺methylated product of L-arg）,M3（proline）,M4（glutamic acid）and M5（citrulline）.L-arg and eight metabolites were detected at 6h,which were M1,M2,M3（proline）,M4（glutamic acid）,M5（citrulline）,M6（the product of losing NH⁺ and generating ketone）,M7（the product of losing CH₂N₂⁺ and generating ketone）,M8（the product of losing CH₂N₂⁺ and generating ketone）.At 12h and 24h,compared with the NS group,the L-arg Group did not show a new peak.（2）five metabolites of L-arg were detected in the liver.Five metabolites of L-arg were detected at 3h,including M3（proline）,M5（glutamic acid）,M6,M9（product of losing CH₂N₂）,M10（product of losing NH and NH⁺,demethylating and forming carboxylic acid）,and three kinds of metabolites of L-arg were detected at 6h,they were:M3（proline）,M5（citrulline）,M6;At 12h and 24h,compared with NS group,no new peak was detected in L-arg group.Conclusion:1.AP Rat model was successfully induced by intraperitoneal injection of L-arg.2.After intraperitoneal injection of L-arg,10 kinds of L-arg metabolites were detected in rats,including 8 kinds in serum and 5 kinds in liver.Among them,3 kinds of metabolites（proline,glutamate and citrulline）were identified.