Cholinesterases reactivation rates

A review of the literature disclosed no long-term effects of cholinesterase reactivators (Appendix A). They are eliminated rapidly and produce a variety of short-term, reversible acute effects. These short-term effects might explain in part the slightly increased (nonsignificant) rates of admission to Army hospitals during the first 5 years after testing (Table 12). However, there was no evidence of a difference in current health status between these subjects and the other subjects. Nor was there evidence of differences in the current social functioning of these subjects, e.g., In employment, marital status, and family life. 

CHOLINESTERASE REACTIVATORS Although the phosphorylated esteratic site of AChE undergoes hydrolytic regeneration at a slow or negligible rate, nucleophilic agents, such as hydroxylamine (NH OH), hydroxamic adds (RCONH-OH), and oximes (RCH=NOH), reactivate the enzyme more rapidly than does spontaneous hydrolysis. Reactivation with prahdoxime (Figure 8-1E) occurs at a million times the rate of that with hydroxylamine. Several h/s-quaternary oximes are even more potent as reactivators for insectidde and nerve gas poisoning (e.g., HI-6, used in Europe as an antidote). 

Thus, the biochemical characteristics affecting the reactivation of cholinesterase are complex and only partially understood. Knowledge of the kinetics of the various rate-determining processes is essential to the understanding of the inhibitor-reactivation process. 

FIGURE 7.3 Comparative reactivation kinetics of soman-inhibited human butyryl-cholinesterase single mutant G117H ( ) and double mutant G117H/E197Q ( ). Note that the recovery rate of the double mutant is very fast (with reaction rates of 77,000 and 128,000 per minute for the PgCj and PgC isomers of soman, respectively), while the single mutant does not recover measurably. The insert shows that reactivation of the double mutant with soman can be treated as a first-order reaction for at least 2.5 X lO s. 

The biomolecular inhibition rate constant (ifj) describes both the affinity and the rate of cholinesterase phosphorylation and is an indicator of inhibitory potency (Kousba et al 2004 Kardos and Sultatos, 2000 Amitai et al., 1998 Carr and Chambers, 1996). A typical determination is illustrated in Fig. 10 for the in vitro inhibition of rat BuChE with chlorpyrifos-oxon. In this example, the Kj was determined by incubating BuChE with varying concentrations of ehloropyrifos-oxon (0.25-5 nAf) the maximum inhibition ranged from 10 to 90% during a 7- to 30-min incubation period (Kousba et al., 2003). The slopes obtained from this analy.sis were then analyzed by linear regression to calculate a K (Fig. lOB). Similar in vitro approaches have been used to calculate the spontaneous first-order reactivation... 

Compound.s with an oxime group in the molecule reactivate phosphyiated cholinesterases much faster than water due to the powerful nucieophiliciiy of the oxime group. In the reactivation process, reactivated enzyme and phos-phylatcd oximes are formed (Eq. 7), and the time course of reactivation follows (Eq. 8), where It is the second-order rate constant of oxime reactivation ... 

Dilution of the sample will slow down further cholinesterase inhibition and further oxime reactivation because both reactions depend on the concentrations of the inhibitor and oxime (Eqs. 4 and 8). However, spontaneous reactivation will continue irrespective of dilution (Eq. 6). Because all reactions arc temperature dependent, samples can be stored at a low temperature but not below zero if one wants to have nonhemolyzed erythrocytes. All three reactions are also pH dependent. Oximes reactivate when they are deproto-nated, and lowering the pH therefore decreases rates of oxime reactivation. The pH profiles of inhibition and spontaneous reactivation depend on the structure of the inhibitor, but for the majority of compounds these rates decrease with decreasing pH. BkHxl samples can therefore be diluted with a buffer of pH between 5 and 6. If cholinestera.se activities are measured in haemoly.sed blood, dilution with water is. suggested, and these samples may be stored below zero. 

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