Dilution Effects on the Measurement of Blood Cholinesterase Inhibition by Carbamates

Abstract

UNDER “steady state” conditions in the absence of its normal substrate (acetylcholine), or when the substrate concentration can be neglected, the inhibition of acetyl-cholinesterase (ChE) by insecticidal or related carbamates, such as 1-naphthyl N-methylcarbamate, o-isopropoxy-phenyl N-methylcarbamate (IPMC) and eserine, can be defined by the equation The symbols v′ and v are the rates of substrate hydrolysis by the inhibited and uninhibited enzyme respectively, i is the concentration of carbamate, K₁ the effective bimolecular carbamylation rate constant, and K₂ the effective unimolecular rate constant for the hydrolytic regeneration of free enzyme^1,2. It followed from (1) that if v′₀/v₀ represents the steady state inhibition obtaining in vivo or as a result of the pre-incubation of tissue and inhibitor in vitro, and v′_z/v_z that estimated after diluting the tissue z times and allowing a new steady state to be achieved When estimating human blood ChE inhibition as an index of accidental or occupational exposure to insecticidal carbamates it is therefore important to keep z minimal or to carry out the assay so rapidly that the change towards the new steady state can be neglected. Fortunately, the values of K₂ for typical insecticidal carbamates^2,3 are such that, if assays can be completed within a few minutes of sample dilution, the changes before and during the measurement are relatively small. A simple radiometric method for estimating blood ChE under field conditions has been described⁴ which enables an assay to be made within 2 min of dilution and involves diluting a 20 µl. blood sample a nominal eleven times. When this method was applied to samples of undiluted human blood pre-incubated with IPMC or eserine to simulate conditions in vivo, it was found that the new steady state effects of subsequent dilution were invariably less than those indicated by equation (2). The steady state inhibition obtaining at the instant of dilution was estimated by extrapolating the time-inhibition curves obtained after dilution. The results obtained in experiments with IPMC and eserine at 25° C are summarized in Table 1. In these experiments the actual dilution was ten times, because of a small volume of aqueous inhibitor that was present in the whole blood. The observed steady state inhibition by eserine has been corrected for the slow enzymatic decomposition of the inhibitor itself which was expected at the relatively high enzyme concentrations used². This decomposition tends to exaggerate and extend the dilution effect so that reactivation of the diluted enzyme appeared to continue almost to completion after 18 h. Because of its lower carbamylation rate, no correction was required for the steady state inhibition by IPMC, and this did not change significantly over a period of 15 h. The discrepancy between the observed and calculated effects of dilution illustrated in Table 1 may be explained as a partition phenomenon resulting in a lowering of the concentration of free inhibitor in the aqueous phase as follows.