| Diabetes is a metabolic disease caused by different pathogenesis and etiology,which is caused by absolute or relative deficiency of insulin in the body.resulting in metabolic disorders of sugar,fat and protein,and severe hyperglycemia as the main clinical manifestation.Although modern medical technology has made great progress in the diagnosis and treatment of diabetes,diabetes and its complications are still the most common diseases in the world after cardiovascular and cerebrovascular diseases and tumors.With the improvement of national living standards,the incidence of diabetes has also shown an upward trend.Epidemiological surveys show that the number of diabetic patients worldwide was 425 million in 2017 and is expected to exceed 629 million by 2045.In China alone,there are 114 million adult diabetes patients aged 20-79,ranking first in the world.Cardiovascular and cerebrovascular diseases caused by diabetes is the main cause of death.Therefore,it is very important for the early diagnosis,course monitoring and interventional treatment of diabetes.The significant increased glucose concentration in diabetic patients causes proteins in the body to undergo a chemical reaction with glucose called non-enzymatic glycation without the involvement of glycosyltransferases.This reaction is the initial factor that causes diabetes and related complications.Human serum albumin(HSA)is a high-abundance protein in plasma.It is mainly responsible for binding and transporting various drugs,and has a profound impact on the pharmacokinetic characteristics and efficacy of various drugs.In diabetic patients,hyperglycemia leads to a higher rate of non-enzymatic glycation of high-abundance HSA in plasma than other proteins.Higher levels of non-enzymatic glycation modification will not only lead to HSA structure and function changes,but also have serious impact on the body's normal physiological activities.The different distribution of multiple non-enzymatic glycation sites on HSA in their three-dimensional structure makes them different in the degree of non-enzymatic glycation at different glucose concentrations.The non-enzymatic glycation modification of sites located in the main drug binding regions will significantly affect the ability of this regions to bind drugs,thereby affecting the pharmacokinetic properties and efficacy of the drug.The incidence of cardiovascular and cerebrovascular disease in diabetic patients is much higher than that in normal people,so the probability of taking anticoagulant drugs is greater than that of non-diabetic patients.Warfarin and heparin are two commonly used anticoagulants in clinical anticoagulation therapy.If used incorrectly,it will cause side effects such as severe bleeding.Therefore,relevant indicators must be strictly tested.In order to understand the effect of HSA glycation on the binding,transportation,and efficacy changes of these two anticoagulants in diabetic patients,it is necessary to start with early HSA glycation products.Studies on the qualitative,quantitative and functional changes of glycation sites on HSA can help us to understand the role and significance of HSA glycation in clinical anticoagulation therapy.This paper used liquid chromatography coupled with mass spectrometry to qualitatively and quantitatively analyze HSA non-enzymatic glycation sites in plasma samples from healthy subjects and diabetic patients.The effects of HSA glycation on the binding,pharmacokinetic and pharmacodynamic properties of anticoagulants(warfarin and heparin)were investigated in vitro and in vivo.The effects of glycation at specific sites on the entire HSA drug binding function were studied by computer simulation combined with amino acid mutation technology.In this paper,the glycation sites of HSA were identified more comprehensively,and an accurate and high-throughput method for the quantification of glycation sites was established by combining the isotope labeling relative and absolute quantification(iTRAQ)technique with the three-stage mass spectrometry.Subsequently,the effect of HSA glycation on the efficacy of warfarin in diabetes was demonstrated in vitro and in vivo.The research results of this paper are helpful to deepen our understanding of the pathogenesis of diabetes and its complications,and provide a strong theoretical basis for early diagnosis and guidance of clinical diabetes medication.The main achievements of this paper are as follows:1.Qualitative comparison of non-enzymatic glycation sites of HSA in the plasma of healthy and diabetic subjectsThe glycation sites of HSA in the healthy and diabetic groups were qualitatively analyzed using high-efficiency separation and high resolution of nano liquid chromatography-mass spectrometry(nLC-MSn).A total of 49 HSA non-enzymatic glycation sites were identified in this experiment,and 7 of them were identified for the first time.Healthy and diabetic groups identified 40 and 47 glycation sites,respectively.Compared with the healthy group,there are more non-enzymatic glycation sites in the diabetic group,which is necessarily related to the hyperglycemia of the diabetic patients.By exploring the distribution of HSA non-enzymatic glycation sites on its three-dimensional structure,it was found that most of the sites were distributed on the surface of the three-dimensional structure of HSA and were close to basic amino acids.The non-enzymatic glycation sites identified in this paper are more comprehensive than the sites reported so far,which enriching the report of existing non-enzymatic glycation sites in the body,and expanding our further understanding of the development of diabetes and its complications.2.Quantitative comparison of glycation sites of HSA in the plasma of healthy and diabetic subjectsThe glycation sites of HSA in the healthy and diabetic groups were quantitatively compared using the high-throughput iTRAQ labeling technique,combined with the high resolution and high accuracy of nLC-MS3.In this part,iTRAQ labeling technology,borate affinity chromatography and three-stage mass spectrometry were combined for the first time to establish a new method for the quantification of non-enzymatic glycated peptides.A total of 21 glycation sites were quantified,and 19 of them were significantly different between the diabetic group and the healthy group(p<0.05).Seven sites were located in the Subdomains IIA and III A,the main drug binding regions of HSA.Therefore,it is speculated that the abnormal non-enzymatic glycation of these sites in the drug-binding region will cause changes in HIS A drug-binding function.Among them,K199 glycated peptide showed the most significant degree of glycation(p<0.01).After further verification with the highly sensitive nLC-MS/MS-multiple reaction monitoring(MRM),it was found that the glycation degree of K199 was consistent with the change trend in iTRAQ-nLC-MS3,and there was still a very significant difference(p<0.0001),indicating that K199 was more sensitive to changes in plasma glucose concentration than other sites.Therefore,the glycated K199 peptide can be used as a candidate early warning marker for the diagnosis of clinical diabetes.More importantly,K199 is not only located in the drug binding region Subdomains IIA,but also close to Subdomains IIIA.Therefore,it is speculated that the glycation of K199 has an important impact on drug transport and efficacy in patients with diabetes,which provides further research direction for the study on the change of drug transport function of diabetes HSA.3.Effect of non-enzymatic glycation of K199 on the binding of HSA to anticoagulantsK199,the most significant site in the quantitative results,was selected as the main research object.The effect of non-enzymatic glycation of K199 on HSA combined with anticoagulants warfarin and heparin was studied using a docking technique.The results showed that K199 glycation increased the binding of warfarin by nearly two times,but had no effect on heparin binding.It is considered that in addition to K199,other glycation sites located at or near the the HSA drug binding region will also affect the binding of warfarin.We combined the simulation docking technology with nLC-MS/MS-MRM quantitative technology and compared the contribution of glycation sites at or near the drug-binding region to warfarin bindining.The non-enzymatic glycation of K199 was found to be the main cause of the change in warfarin binding.PCR mutation technology was used to further verify the effect of non-enzymatic glycation of K199 on warfarin binding.The results showed that there was no significant effect in the binding of warfarin with and without glycation of HSA(K199M).indicating that the binding of warfarin with glycation at sites other than K199 on HSA(K199M)had little effect.In contrast.site K199 contributed the most to the increased binding affinity to warfarin during diabetes.This chapter demonstrates the role of K199 non-enzymatic glycation in HSA binding to different anticoagulants.and provides theoretical guidance for the study of the effect of non-enzymatic glycation on the distribution of anticoagulant drugs in patients with diabetes4.Effect of HSA non-enzymatic glycation on anticoagulants binding and transportThe effect of non-enzymatic glycation of HSA on the binding of warfarin and enoxaparin sodium was investigated in vitro and in vivo using ultrafiltration combined with LC-MS/MS-MRM technology.An analytical method for the analysis of heparin polysaccharides in plasma based on ultrafiltration and 2-aminoacridone(AMAC)labeling technology was established for the first time.Fresh plasma of diabetes and healthy subjects was incubated with the anticoagulant drugs warfarin and enoxaparin sodium in vitro,and the free drug concentration was determined.Free warfarin concentrations in diabetic patients were significantly reduced relative to the healthy group(p<0.005).while there was no significant difference for free enoxaparin sodium(p=0.17).This shows that non-enzymatic glycated HSA has a stronger binding effect on warfarin in diabetes.but has no effect on enoxaparin binding.In order to verify the above results in vivo.we chose a rat model of diabetes for drug administration.The plasma concentrations of free warfarin and enoxaparin sodium at each time point were determined to compare the pharmacokinetic characteristics of the two anticoagulants in normal and diabetic rats.It was found that the free drug concentration curve of warfarin in diabetic rats was different from that of normal rats.and the free warfarin concentration was significantly lower than that of the normal group after 8 hours of administration.In contrast.enoxaparin sodium did not differ significantly between the two groups.It was further confirmed that the combination of glycated albumin with warfarin increased in diabetic patients.which led to a decrease in the amount of free warfarin.Since only free drugs play a pharmacological role in the process of drug efficacy.therefore the binding affinity of HSA on drugs will determine the level of free drug and affect its efficacy.Considering the high incidence of cardiovascular and cerebrovascular diseases in diabetes,the anticoagulant effect of warfarin in clinical diabetic patients may also be affected.The results of this experimental study will provide a certain reference for the selection of clinical anticoagulant drugs for patients combine with diabetes and cardiovascular and cerebrovascular diseases,and the change of the efficacy of warfarin drug treatment is estimated.The analysis method of plasma enoxaparin sodium established in this experiment will provide a valuable reference method for analyzing polysaccharide drugs in animal disease models or clinical samples.5.Preliminary clinical retrospective study on the efficacy of anticoagulant drugs in patients with diabetesTo assess the effect of non-enzymatic glycation on the efficacy of anticoagulants in patients with diabetes,we conducted a preliminary clinical retrospective study involving 53 subjects.Comparing diabetic and non-diabetic patients taking warfarin and heparin,it was found that both the the international normalized ratio(INR)24 h of diabetic patients after taking warfarin and the INR difference before and after taking warfarin was significantly smaller than that of non-diabetics(p<0.05),the difference between INR 24 h and INR differnence after taking heparin was not significantly different from that of non-diabetic patients(p>0.05).After taking warfarin,the INR value of diabetic patients is reduced,which indicates that the plasma concentration of free warfarin in diabetic patients is reduced,thereby reducing the drug efficacy,which makes diabetic patients more likely to clot.In addition,diabetes is inherent a hypercoagulable state,so warfarin administration in diabetes requires constant adjustment and close attention to INR values.Combined with the fact that warfarin has different pharmacokinetic behaviors for diabetic and non-diabetic rats,extra caution must be used in the clinical use of warfarin as an anticoagulant in patients with diabetes.In terms of drug equivalence for diabetic and non-diabetic patients,heparin is safer and more effective than warfarin.This experiment evaluated the safety and risk of anticoagulant drugs used in patients with diabetes,and provided a reference value for the guidance of clinical anticoagulant drugs. |
PDF Full Text Request