Establishment Of A Detection Method For BHMT And Preliminary Clinical Study As A Novel Serum Marker For Acute Liver Injury

Acute liver injury is one of the leading causes of death in current clinical studiesand a main obstacle to drug development. Among the many reported indicators ofliver damage, only traditional alanine aminotransferase (ALT), aspartateaminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase(GGT) and bilirubin (BIL) are commonly used clinically. However, the sensitivity andliver-specificity of these traditional indicators are unsatisfactory, so betterclinically-used indicators are also in great demand. Finding an earlier, moreliver-specific and sensitive indicators of acute liver injury was the objective of thisresearch project. In addition, immunological detection instrument for clinicallaboratories has come into use in recent years and has been improved greatly inautomation, miniaturization and high-throughput. Immunological approaches aremore specific than the biochemical enzymatic method because the interference of theisozyme can be prevented. Conventional liver function test is almost entirely based onthe biochemical enzymatic method, so the development of a detection method ofliver functions based on immunological methods is what this paper attempted toachieve.With the help of systems biology technology and massive parallel sequencing(MPSS) databases of large-scale high-throughput genetic analysis on human andmouse tissues of NIH, we obtained the expression profiles of mRNA of these tissues.According to the expression data of mRNA in livers and other tissues, we chose133liver-specific expression genes. Through the NCBI EST expression database,107ofthese genes were identified before some of them were ruled out based on whethertheir expression protein belonged to one family. Through the model of acute liverinjury caused by overdose of paracetamol (APAP), mouse serum was analyzed byproteomic technology, such as protein profiling, protein chip, and Western blot.Ultimately,20liver-specific genes were used as serum markers of liver injury in mice.In this study, one of the above proteins-BHMT (betaine homocysteineS-methyltransferase)-was selected. A detection method of BHMT in human serum was established and a preliminary clinical study carried out.BHMT is the methyltransferase containing Zn2+, involved in SAH(S-adenosylhomocysteine) and homocysteine metabolic regulation. BHMT is highlyliver-specific by the standards for screening liver-specific genes:1)the targetexpression level in the liver should be five times higher than that of any non-liverorgan or two-fold higher than those of all non-liver organs combined.2) targets wereexcluded if expression levels were not higher than10tpm (transcripts per million).The conventional liver function markers (ALT, AST, ALP, GGT) fall short of thisstandard. Moreover, BHMT is quite abundant in the liver, representing about0.6-1.6%of liver total protein. Therefore, BHMT is likely to possess important features of newserum markers of acute liver injury.In our laboratory, recombinant human BHMT protein expressed in Escherichiacoli BL21were previously prepared. Purified protein was used as an immunogen toobtain mouse anti-human BHMT monoclonal antibody4D1and rabbit anti-humanBHMT polyclonal antibody2-4-2.On this basis, our study conducted a more in-depth exploration and achieved thefollowing results:1. High purity recombinant human BHMT expressed ineukaryocytes was obtained. The plasmid pGEX-4T1-BHMTcontaining full-lengthsequence of human BHMT and kept in our laboratory as a template amplified thefragment of BHMT coding before being inserted into the pCMV-Flag vector andtransfected into293T cells so that the eukaryotic expression of recombinant humanBHMT was achieved. Using agarose affinity media (Ni), the expressed protein waspurified and used as a standard substance for the next ELISA.2. ELISA doubleantibody sandwich method was established in detecting BHMT of human serum.Mouse anti-human BHMT monoclonal antibody4D1, rabbit anti-human BHMTpolyclonal antibody2-4-2and depurated recombinant human BHMT expressed ineukaryocytes were prepared for standard ELISA double antibody sandwich method.By orthogonal test, the best experimental system was selected. The protocol of ELISAwas based on the best experimental system. Detection ranged from0.82ng/ml to106.6ng/ml, able to reach two orders of magnitude. The linear correlation coefficientof the detection fitting curve was up to0.999, suggesting better accuracy.3. Clinicalresearch of BHMT. The established ELISA method was used in the detection of theBHMT in clinical serum samples to explore the relationship between BHMT and acute liver injury. The results were as follows:(1) The medical reference range ofBHMT in normal human serum was established.(2) The correlation between BHMTand ALT was investigated.48samples from suspected acute liver injury in patientshospitalized with ALT>40U/L were detected (clinical ALT medical reference rangein the serum was0to40U/L).The statistically analysis indicating a good positivecorrelation between BHMT and ALT.(3) BHMT for the diagnosis of acute liverinjury was evaluated.126cases of clinical samples were detected including normaland acute suspicious cases of liver injury. There were two diagnostic methods ofBHMT as acute liver injury indicators that were respectively analyzed by statisticalKappa coefficient test with the current clinical standard diagnostic method. Theresults prompted that two diagnostic methods and the standard method have a highconsistency.(4) In addition,126cases of clinical samples mentioned above underwentROC analysis. The obtained Cut-off value of BHMT as a diagnostic index of acuteliver injure was65.92ng/ml, with a sensitivity of0.94, specificity of0.94, accuracyof0.94, and Youden index of0.89. These results indicated that the index BHMT wasof a high diagnostic value.(5) The specificity of BHMT in reflecting liver injury wasdetermined. BHMT also exists in the kidney, so a comparison of the content ofBHMT was made between the serum of renal damage patient and the normal humanserum.Through the statistical group of rank-sum test, P>0.05, indicating that therewas no significant difference in the BHMT content between the serum of patients withrenal injury and the normal human serum, and that BHMT may not be closely relatedto kidney injury,while may be more specfic to liver injure.(6) The sensitivity ofBHMT in reflecting liver damage was explored. BHMT in the serum of five differentcases of acute liver injury with time trends were detected, using the above standardELISA method, and five trend charts were drawn. The changes in trends suggest thatBHMT seems to be more sensitive than ALT in the process of liver damage. However,the sample size was not enough, for this was nothing more than preliminaryspeculation. Further clinical validation is necessary.In conclusion, BHMT has a better performance in terms of specificity andsensitivity for the diagnosis of acute liver injury, can be used as a novel marker ofacute liver injury, and deserve more in-depth study.