Synthesis Of Acridinium Ester As A Substrate For Measurement Of PON Arylesterase Activity

The tumor is one of a killer for people life, so it is very important to discover andtreat early. But the tumor is appeared likely misdiagnose and misseddiagnosis, becausethe early symptom of tumor isn’t obviously. When the tumor happen before four toeight weeks, PON activity is clearly increase or reduce in serum or body fluid.Therefore, PON determined is one of the means of diagnosis for tumor in humoru.There are many ways to assay the PON activity, but a few methods aren’tdetermined the PON in sample of body fluid, because of interference factor andsensitivity. UV is used to assay the PON activity. The UV is a briefness method,however, its’ background noise is bigger. And the orgaism is a extremely complexsystem, other compositions may have light absorption in extracting solution, neither thesensitivity nor the singleness aren’t high-priced. But the chemiluminescence （CL） hasno use for light source, when the PON activity assayed is with no need for separatingthe chaff interferent that have absorbent of UV, because of little background noise andinterference, high response rate. We gained three compounds. It was found that thehydrolysis of9-（4-chlorophenyloxycarbonyl）-10-methylacridinium triflate （CPOCMA）could be catalyzed by recombinant human Paraoxonase1（PON1）.CPOCMA makes up the luminophore and the leaving group. Thechemiluminescence of CPOCMA is greared to the minute flash by means of researchingthe function of CPOCMA, the whole process are completed in20s. The trigger mayinfluence the luminous intensity of CPOCMA. If the triggers are0.1%H₂O₂,0.025mol/L HNO3and1.0mol/L NaOH,1.0×10^-3mol/L CTAB, the luminous intensity ofCPOCMA will be the strongest. When the substrate is stored at2℃and25℃, thestability of the reagent will be variation in the wake of the pH （6,7,8）. If the pH isincrescent, the stability will be bad.We studied the the specificity of the substrate of PON1and the method of the CL,and finded that acridinium esters are well-known highly chemiluminescent compounds.The concentration of the phenylacetate and the diazinon that are the substrate of PON1by UV is105times than the concentration of CPOCMA. The quantity of CPOCMA is afew, and the CPOCMA is cheap. In addition, in a buffer pH of physiology the assaybased on CPOCMA is determined. The apparent Kmvalue of a serum sample for thesubstrate was determined as85nmol/L, close to the Kmvalue （83nmol/L） of rHuPON1. The interferences by other esterase such as acetylcholinerase and lipases wereinvestigated. The NaCl and CaCl2as PON activity enhancers were able to improve thespecific signal respectively. The rHuPON1in presence of CaCl2showed at least7.8times selectivity over acetylcholinerase and lipases. So the activators of PON improvethe specificity and the sensitivity of the substrate.By comparing with the UV methods based on phenyl acetate and diazinonrespectively, the proposed chemiluminescent method was validated with30serumsamples. It seems that for the most of the serum samples the result measured by thechemiluminescent method was consistent with the results obtained by the UV methodsbased on phenyl acetate or diazinon. The several exceptive cases might result from PONpolymorphism. So it proposed that the chemiluminescent method should be reliable.