Mechanism Of High Unconjugated Bilirubinemia Induced By Albumin Homocysteinylation

Background and Aims: Hyperbilirubinemia refers to the increase of serum bilirubin level caused by various reasons.Among them,neonatal hyperbilirubinemia is one of the most common diseases in neonatal clinic,and its incidence and hospitalization rate rank first among neonatal hospitalization diseases.Severe hyperbilirubinemia causes pathological damage to multiple organs and tissues,especially bilirubin encephalopathy(Kernicterus),which HSA a very high mortality and disability rate.In the past,the level of serum total bilirubin(TB)was used as a key index to evaluate the toxicity of hyperbilirubinemia.However,with the progress of researches on hyperbilirubinemia,scholars agree that the level of serum TB is affected by the state of the body and the internal environment,and HSA limited value in predicting the toxic damage of hyperbilirubinemia to the body.Unconjugated bilirubin(UCB)does not bind to glucuronic acid and mainly binds to albumin in the form of a complex in circulation.It is insoluble in water,fat-soluble.It can be aggregated,bound,and passed through a cell biofilm,which is the key to tissue and organ damage.At present,the clinical prevention and treatment of hyperbilirubinemia commonly used treatment schemes mainly include phototherapy,blood exchange,touch drug treatment and so on.Each method HSA a certain indication,but there are still some limitations and safety problems.Therefore,exploring the mechanism of elevated serum UCB level in hyperbilirubinemia can provide a new strategy and direction for clinical prevention and treatment of hyperbilirubinemia and its complications.The metabolic process of bilirubin in human body includes source and production,binding and transportation,uptake and transformation of hepatocytes,and excretion.Among them,UCB is mainly transported in circulation in the form of albumin binding complex,which maintains the dynamic balance of serum UCB level.When albumin changes in mass and/or affinity,it can lead to the decrease of the binding capacity of UCB to albumin,which is one of the important mechanisms of hyper-UCB.Whether there a change in the quality and/or affinity of serum albumin in hyperbilirubinemia and the mechanism HSA not yet been reported.By consulting the literature,we know that homocysteine(Hcy)and its derivatives can react with ?-amino group of protein lysine residue to form lysine residue(protein N-hypercysteine)complex,resulting in protein Hcy,As a result,the spatial folding and conformation of the modified protein were changed,and the molecular structure and physical and chemical properties of the protein were changed.However,it HSA been confirmed that the level of serum Hcy is increased in hyperbilirubinemia,and it is related to the toxic injury of hyperbilirubinemia.Therefore,this study mainly explores whether there is an increase in serum Hcy level in hyperbilirubinemia,albumin cyclization occurs at the amino acid residue site of modified albumin,and the spatial folding and conformation of albumin are changed.Competitive inhibition of UCB binding to albumin leads to hyper-UCB.Research methods:(1)The hyper-UCB model was established by subcutaneous injection of phenylhydrazine hydrochloride into neonatal 7-day-old Wistar rats.The concentrations of serum bilirubin,albumin and Hcy were measured by automatic biochemical analyzer.The level of albumin homocysteinylation was detected by Western Blot with K-Hcy antibody.The binding capacity of serum albumin and bilirubin was detected by HBABA dye binding method.We also explored the effect of anti-Hcy(folic acid)on the level of albumin homocysteinylation and UCB concentration,and the binding capacity of serum albumin and bilirubin.(2)L-homocysteine thiolactone(HTL)was incubated with human serum albumin(HSA)in vitro and the modification effect of Hcy on HSA was verified by Western Blot with K-Hcy antibody.The specific amino acid residues of Hcy modified HSA were identified and analyzed by liquid chromatography-tandem mass spectrometry(LC-MS/MS),and the binding capacity of Hcy modified HSA to bilirubin was determined by oxidase method.(3)According to the results of mass spectrometry,using Flag-HSA wild type gene as template and PCR site-directed mutagenesis technique,the eukaryotic expression plasmid of site-directed HSA mutant was constructed in vitro,and the transfection technique mediated by cationic liposome was used.The HSA mutant plasmid was transfected into HEK293 cells.The expression of HSA mutant plasmid was detected by Western Blot and immunofluorescence.The target protein was purified by immunoprecipitation and the binding ability of HSA mutant to bilirubin was detected by oxidase method.Research results:(1)The hyper-UCB model was successfully established by subcutaneous injection of phenylhydrazine hydrochloride into neonatal 7-day-old Wistar rats.Compared with the control group,the concentration of serum Hcy in hyperbilirubinemia group was increased.The level of albumin homocysteinylation was increased and the binding capacity of albumin to UCB was decreased.Anti-Hcy(folic acid)treatment decreased the level of albumin homocysteinylation and UCB concentration,but increased the binding capacity of albumin to UCB.(2)Western Blot results showed that the level of modified HSA increased with the HTL concentration,and lysine at position 28 and position 569 were the specific amino acid residues of Hcy modified HSA by liquid chromatography-tandem mass spectrometry(LC-MS/MS).The binding capacity of Hcy modified HSA to bilirubin was significantly decreased by oxidase method.(3)Restriction endonuclease digestion and sequencing confirmed that the eukaryotic plasmid of HSA mutant was successfully constructed,and the results of Western Blot and immunofluorescence showed that the transfection efficiency of HSA mutant plasmid in HEK293 cells was high.A large number of HSA mutants were successfully obtained by Flag-tag antibody and Immunoprecipitation,and the binding ability of HSA mutants to bilirubin was significantly decreased by oxidase assay.Conclusion:(1)There was an increase in serum Hcy and albumin cyclization levels but a decrease in the binding capacity of UCB to albumin in hyper-UCB.This suggests that hyper-UCB is associated with serum Hcy levels.(2)The level of HSA did not change in the early stage of hyperbilirubinemia,but the binding capacity of albumin to UCB decreased significantly,which indicated that the increase of serum UCB level might be related to the change of HSA quality and/or affinity.(3)Anti-Hcy(folic acid)treatment could decrease the concentration of serum UCB and the level of albumin cyclization in Wistar rats with hyperbilirubinemia,and enhance the binding ability of serum albumin to UCB.This indicates that serum Hcy can be used as a biochemical index to evaluate hyperbilirubinemia and its toxic injury,and is expected to become a new strategy and direction for clinical prevention and treatment of hyperbilirubinemia and its toxic injury.(4)The results of co-incubation of HSA and HTL in vitro showed that the level of HSA modification increased with the HTL concentration,which confirmed that HSA could undergo cyclization modification.Through LC-MS/MS identification and analysis,it was found that the specific amino acid residues were lysine at position 28 and position 569 in the molecular structure of HSA.This lays a theoretical foundation for the next step to explore the interaction between Hcy modified HSA and bilirubin.(5)The binding capacity of Hcy modified HSA to bilirubin was detected by oxidase method.The results suggested that Hcy modification can inhibit the binding of bilirubin to HSA,which could be related to the change of molecular space folding and conformation of HSA.(6)The binding ability of the site-directed HSA mutant at K28/K569 locus to bilirubin was decreased.These results suggest that Hcy modification competitively inhibits the binding of HSA to bilirubin,which may be one of the important mechanisms of hyper-UCB.In conclusion,hyper-UCB is significantly correlated with serum Hcy concentration,albumin cyclization level,HSA and bilirubin binding capacity,Hcy can modify the amino acid residue site of HSA molecule,and change the spatial folding and conformation of HSA.Competitive inhibition of bilirubin binding to HSA is one of the important mechanisms of hyperbilirubinemia.Anti-Hcy(folic acid)therapy is expected to become a new strategy and direction for clinical prevention and treatment of hyperbilirubinemia and its toxic injury.