Paraoxon aging

Recently, Noort et al developed a procedure that is based on straightforward isolation of adducted BuChE from plasma by means of affinity chromatography with a procainamide column, followed by pepsin digestion and LC/electrospray tandem MS analysis of a specific nonapeptide containing the phosphonylated active site serine-198 residue (5). This method surpasses the limitations of the fluoride-reactivation method, since it can also deal with dealkylated ( aged ) phosphonylated BuChE. The method allowed the positive analysis of several serum samples of Japanese victims of the terrorist attack in the Tokyo subway in 1995. Furthermore, the method could be applied for detection of ChE modifications induced by, e.g., diethyl paraoxon and pyridostigmine bromide, illustrating the broad scope of this approach. This new approach... 

Methyl parathion itself is not a strong cholinesterase inhibitor, but one of its metabolites, methyl paraoxon, is an active inhibitor. Methyl paraoxon inactivates cholinesterase by phosphorylation of the active site of the enzyme to form the dimethylphosphoryl enzyme. Over the following 24-48 hours there is a process, called aging, of conversion to the monomethylphosphoryl enzyme. ... 

FIGURE 69.2. Reaction scheme of acetylcholinesterase inhibition, reactivation, and protection activities. OPH, 2-PAM, and paraoxon are used in the example. Reaction (I) - cholinesterase AChE reaction with ASCh Reaction (II) - inhibition of AChE inhibition by paraoxon Reaction (III) - aging of AChE associated with OP exposure Reaction (TV) -reactivation of AChE by 2-PAM Reaction (V) - hydrolysis of paraoxon by OPH Reaction (VI) - reaction of 2-PAM oxime with ASCh and Reaction (VII) - DTNB reaction with SCh. The indicates a photometrically detectable metabolite. 

Carr. L. R., and Chambers, J. E. (1996). Kinetic analysis of in vitro inhibition, aging and reactivation of brain acetylcholinesterase from rat and channel cat fish by paraoxon and chlorpyrifo.s-oxon, Toxicol. Appl. Pharmacol. 139, 365-373,... 

FIGURE 64.1 MS/MS spectrum of the active site peptide of human AChE, covalently bound to tabun and dealkylated by aging or by acid. The identical modified peptide is derived from human BChE treated with tabun, chlorpyrifos oxon, paraoxon, or diazoxon. The A symbol indicates ions that have lost the OP and a molecule of water, converting serine to dehydroalanine. The peak height for 778.2t /2 was 2.5-fold higher than indicated it was shortened to show the less intense ions. 

A problem for CarbE as a scavenger for nerve gases is that VX and echothiophate are both 10,000-fold better inhibitors of AChE than CarbE. This is due to the anion attraction of ChE as described in the section "Active site," earlier in this chapter. OP-inhibited CarbE differ from OP-AChE in that they do not age. Instead, they spontaneously reactivate similar to a slow substrate. The half-life for spontaneous reactivahon of sarin-inhibited CarbE is 2h, whereas for soman or paraoxon, it is 20 h and for DFP, it is 40 h (Maxwell and Brecht, 2001). This agrees with results from a series of experiments with repetitive injection of 0.5x LD50 of soman in guinea pigs at different intervals, which demonstrated that soman was completely removed within 24 h and plasma CarbE (tribut5U inase) activity was fully recovered (Sterri et al., 1981). The slow reactivation of DEP from CarbE explains its low tolerance to repetitive administration. 

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