Ammonia Metabolic Pathway And Mechanism Of Tissue Specific Free Ammonia And Amino Acids

Objective Ammonia is an intermediate form of nitrogen nutrients metabolized by human and exists as free ammonia and bound ammonia.The amount of free ammonia depends on the pH value in the blood.Under normal physiological conditions,the concentration of free ammonia in the body of healthy people is kept at a low level(<50uM),which will not cause damage to tissues,especially in brain.In contrast,bound ammonia is mainly involved in the formation of large molecules such as amino acids and nucleic acids.Clinically,patients with gastrointestinal surgery will undergo different levels of fasting,which can cause nutrition metabolic adaptations as well as catabolism enhancement,causing the ammonia metabolism disorder in the body,which will directly influence the postoperative recovery of patients.Patients with severe trauma and infection often develop a disorder called free ammonia metabolism disorder.Due to the disorder of free ammonia metabolism,hepatic encephalopathy can occur,and the latter is more common in patients after portal-body venous shunt,but the specific mechanism is not yet clear.Therefore,the influence of ammonia metabolism imbalance on postoperative patients has been given much attention over the years[2],but the specific mechanism and role of the link are not clear.In particular,there are not many methods for studying ammonia metabolism in the tissue.The development of effective prevention and treatment strategies in clinical settings brings great challenges.Our research group has long studied the pathways and mechanisms of tissue-specific ammonia metabolism.Looking at the physiological characteristics of the microenvironment between neural retina and RPE(Retina Pigment Epithelium cells)/choroidal tissues,we have established an innovative system into studying ammonia metabolism in tissue,which is beneficial to the in-depth study of the mechanism of hepatic encephalopathy and other diseases of ammonia metabolism disorders.Methods In this study,we use Gas Chromatography-Mass Spectrometry(Gas Chromatography-Mass Spectrometry,GC-MS)with Stable Isotope labeling(Stable Isotope-based metabolic analyze)test labeled ammonium and amino acids in different tissues on mice model in vivo and in vitro.In vivo,we used stable isotope 15N labeled NH4CL to do intraperitoneal injection,at 0 minutes,5 minutes,15 minutes,30 minutes,60 minutes and collected mouse liver,retina,retina pigment epithelial(RPE)/Choroid,brain,and plasma which were then analyzed using GC-MS to examine the dynamic changes of each metabolites.In the in vitro experiment,retina and RPE/Choroid cultured in KRB medium with different tracers(15N-amine-Glutamine and 15N-amide-Glutamine,15N-Aspartate,15N-Alanine,13C-Alanine)After incubating for 2 hours in a 5%CO2 incubator at 37℃,tissue and culture medium were collected was run with the GC-MS.Results:1.Metabolic characteristics of free ammonia in different tissues(1)Characteristics of ammonia metabolism in retinal and brain.15N can label the following metabolites:Glutamine,Urea,Asparagine,5-Oxoproline,Glutamate,Aspartate,GABA,Alanine.This suggests that the basic ammonia metabolic pathways of the two nerve tissues are similar.However,they remove ammonia at different rates:The accumulation of ammonia metabolites in brain peaks at 60 minutes,in retinal reached its peak at 15 minutes.(2)Characteristics of ammonia metabolism in liver.The liver metabolized ammonia differently from the nerve tissues:There are more than 70%of glutamine was labeled at five minutes.Urea,Glutamate,Alanine,Aspartate,Hydroxy-proline,and GABA labeled at 5 min.That means liver cleared ammonia faster than brain and the retina.2.Metabolic characteristics of amino acid metabolism pathways in retina and RPE/choroid.To explore the metabolic pathways and mechanisms of Glutamine,Aspartate and Alanine in retina and RPE/Choroid.Since their remarkable changes in previous study.1)To explore the characteristics of Glutamine metabolism pathway.Since Glutamine has two nitrogenous groups We first labeled 15N-amine-Glutamine,Approximately 80%of Glutamine was labeled in both retina and RPE/Choroid.The amount of labeled Glutamate and Aspartate in the retina increased 2 times compared with the control group,while other metabolites were not labeled.In RPE/Choroid,the increase was more significant at 4 and 8 times respectively.In addition,there was a nearly 3-fold increase in labeled Alanine and a nearly 2-fold increase in labeled BCAAs(Leucine,Valine,Isoleucine),Serine,and Glycine.The above results indicate that the 15 N-amine-Glutamine metabolism pathway of RPE/Choroid is more vigorous and complex than that of retina.2)Further,15N-amide-Glutamine shows the concentration of Glutamine increased by 75 times in the retina,and the labeled Asparagine increased about 2 times.In RPE/Choroid,the amount of labeled Glutamine increased about 60 times,while showing no change in Asparagine.The above results indicate that the 15N-amide-Glutamine metabolism pathway is recycling to synthesize Glutamine and Asparagine and Asparagine cannot be synthesized in RPE/Choroid.3)To explore the characteristics of Aspartate ammonia metabolism pathway.The 15N-Aspartate shows in retina that there is a 5-fold increase in the amount of labeled Asparagine as well as a 6-fold increase in Glutamine and a 3-fold increase in Glutamate.In RPE/Choroid,labeled Glutamine and Glutamate increased by 3 and 3.5 times respectively.In addition,the labeled Alanine,Serine,and Glycine increased more than 2 times,but Asparagine remained unlabeled and unchanged.These results suggest that Aspartate is the primary material for the synthesis of Glutamate,Glutamine,and Asparagine in retina.However,Aspartate is more commonly used for the synthesis of Alanine,Serine,and Glycine in RPE/Choroid.4)To explore the characteristics of the Alanine ammonia metabolism pathway.The retina only have the labeled 15N-Alanine,no other labeled metabolites shows there.In RPE/Choroid,the labeled Alanine increased nearly 40-fold.In addition,Aspartate,Glutamate and Glycine increased more than 2-fold,Leucine and Serine nearly increased 2-fold.The ratio of Glutamate/α-KG in retina was 30 times higher than that in RPE/Choroid.This is potentially the reason why retina could not synthesize new amino acids by using the nitrogen-containing groups of Alanine but RPE/Choroid can.3.Characteristics of metabolic pathway of major amino acids after inhibit Pyruvate transporter(MPC)Alanine produces Pyruvate,which is the hub of sugar and amino acid and fat metabolism.Pyruvate is required to enter the mitochondrial oxidative phosphorylation through MPC.We have reported that Aspartate builds up after the MPC is blocked.To study the sources of Aspartate,we labeled the 2 times most likely sources of amino acids,Glutamine and Alanine.(1)The characteristics of the ammonia metabolic pathway of Glutamine after the inhibition of MPC.The results of labeled 15N-amine-Glutamine shows that,Pyruvate increased 2.5 times,α-KG decreased by about 50 times,unlabeled Aspartate increased by 4.5 times and labeled Aspartate increased by 20 times in the retina tissue.Pyruvate in RPE/Choroid increased by 2.5 times,α-KG decreased by about 30 times,unlabeled Aspartate increased by 4 times,and labeled Aspartate increased by 20 times.This means that when MPC was blocked,the amine group of Glutamine was the most important source of Aspartate in retina and RPE/Choroid(2)The characteristics of the ammonia metabolic pathway of Alanine after the inhibition of MPC.The results of labeled 15N-Alanine shows that Pyruvate increased by nearly 2 times,α-KG decreased by about 30 times,and unlabeled Aspartate increased by nearly 6 times,but labeled Aspartate increased slightly(<2 times)in the retina.Pyruvate in RPE/Choroid increased by 2.5 times,α-KG decreased by 30 times,unlabeled Aspartate increased by 3 times,and labeled Aspartate decreased by nearly 2 time.It indicates that,Alanine is not an important nitrogen source of Aspartate in both retina and RPE/Choroid.However,when MPC is inhibit,the retina may use Alanine to synthesize Aspartate.However,RPE/Choroid does not have this pathway.To test this hypothesis,we performed a marker study on Alanine carbon metabolism.(3)The characteristics of the carbon metabolic pathway of Alanine after the inhibition of MPC.The 13C-Alanine shows 3-times increased in the retina.The amino acids changes in the Pyruvate metabolic pathway were characterized as follows:Pyruvate,Lactate,Fumarate,Aspartate and Malate increased by more than 2.5 times,the labeled Citrate and α-KG decreased by 2 times,and the Succinate decreased by about 0.2 times.In RPE/Choroid,the labeled Alanine and Pyruvate increased nearly 2-fold,consistent with the retina.However,the increase of Malate and Aspartate was smaller than that of retina.It shows that inhibition of MPC increased the content of Citrate metabolites upstream but decreased the content of Citrate metabolites downstream in retina.There is no unlabeled Citrate in the retina but the labeled Citrate content increased by 1500.In RPE/Choroid tissue,the unlabeled Citrate content was close to 6000,6 times that of the labeled Citrate.These results suggest that when MPC is inhibited,partial Alanine can enter the retina mitochondria to compensate for Pyruvate deficiency,but this pathway is not present in RPE/Choroid.ConclusionCharacteristics of free ammonia metabolism1)Brain is similar to the free ammonia metabolism pathway of retina,mainly metabolizing ammonia through the Glutamine pathway,and partly through the synthesis of Asparagine pathway.However,ammonia metabolites in brain are more likely to accumulate,which may related to ammonia poisoning in brain.2)The free ammonia metabolism pathway of the liver is the most active,mainly including the urea circulation pathway,the Glutamine pathway and part of the de’ novol Glutamate synthesis pathway.Moreover.the ability of urea cycle pathway and Glutamine pathway to metabolize free ammonia is approximately equal.3)In the free ammonia metabolism pathway,there are similarities between the retina and RPE/Choroid in that they both synthesize Glutamine from free ammonia.However,only the retina can metabolize ammonia through the Asparagine pathway and the RPE/Choroid metabolizes ammonia by synthesizing a variety of amino acids.Characteristics of amino acid metabolism pathway in retina and RPE/Choroid in microenvironment1)The amine and amide metabolic pathways of Glutamine in retina and RPE/Choroid are different and tissue specific.The amine groups in Glutamine are the main nitrogen sources of Glutamate and Aspartate,and the acyl groups are mainly Glutamine and Asparagine synthesized through recycling.The amine-Glutamine metabolic pathway of RPE/Choroid is more vigorous and complex than retina.Asparagine could not be synthesized from amide-Glutamine in RPE/Choroid.2)The ammonia metabolism pathway of Aspartate the retina and RPE/Choroid is tissue specific.Aspartate is the main source of nitrogen for the synthesis of Glutamate,Glutamine and Asparagine in retina.However,RPE/Choroid is an important source of nitrogen for Alanine,Serine and Glycine.3)The ammonia metabolism pathway of Alanine in the retina and RPE/Choroid is tissue specific.When MPC function is normal,the retina does not use the Alanine,but RPE/Choroid tissue can use Alanine to synthesize amino acids.When MPC is blocked,the Alanine pathway in the retina activated.A portion of that Alanine can directly enter the mitochondria to compensate for the lack of Pyruvate,but there is no such complementary pathway in RPE/ChoroidTo sum it up,ammonia metabolism has significant tissue specificity,and the expression of retina and retina pigment epithelial cells in ammonia metabolism is different.At the same time,retina pigment epithelial cells may support retina metabolism by activating multiple amino acid metabolism,maintaining a symbiotic balance between retina tissue and RPE/Choroid.