Journal of Analytical Toxicology, Vol. 15, March/April 1991 NeurotoxicEsterase(NTE) Assay: OptimizedConditions Based on Detergent-Induced Shifts in the Phenol/ 4-Aminoantipyrine Chromophore Spectrum* U.S. Kayyali, T.B. Moore**, J.C. Randall, and R.J. Richardsont Neurotoxicology Research Laboratory, Toxicology Program, Department of Environmental & Industrial Health, School of Public Health, The University of Michigan, Ann Arbor, M148109 Abstract An assay for neurotoxic esterase (neuropathy target asterase, NTE) was developed by Johnson (1,2) to assess the delayed neurotoxic potential of organophosphorus compounds. NTE activity is calculated from the rate of phenyl valerate hydrolysis resistant to paraoxon and sensitive to mipafox inhibition under specified conditions of inhibitor concentrations, pH, temperature, and incubation times with inhibitors and substrate. The amount of phenol produced is measured colorimetrically after its oxidative coupling with 4- aminoantipyrine to yield 4-N-(1,4-benzoquinoneimine)- antipyrine, a chromophore with a wavelength of maximum absorbance (Lm)510 nm and corresponding molar absorptivity (molar extinction coefficient, ~) equal to 13,900 M-lcm-L The assay was improved and simplified later by Johnson (3) without any change in the ~-mor ~, even though the chromophore solvent was altered by adding the detergent, sodium dodecyl sulfate (SDS). The present work demonstrates that when the NTE assay is performed according to the improved procedure, with a final [SDS] of 3.0 mg/mL, the ~.rn of the chromophore in the assay mixture is shifted from 510 to 490 nm. The same shift in the chromophore ~rn is observed when phenol standards are coupled with 4-aminoantipyrine in solutions containing 3.0 mg/mL SDS. A systematic investigation of the dependence of the ~-mof the chromophore on [SDS] in the assay mixture revealed that the spectral shift increases rapidly at an [SDS] greater than the apparent critical micelle concentration (CMC; estimated to be 0.53 mg/mL under these conditions) and begins to plateau at [SDS] greater than 10 mg/mL. Furthermore, the sensitivity of the assay was shown to be dependent upon [SDS], reaching a peak ~-value of 16,470 M-~cm -~ at a Lm of 486 nm when the final [SDS] was 9.5 mg/mL; it is therefore recommended that these optimized values of [SDS], Lm, and ~"be adopted in the NTE assay. Finally, because there are versions of assays for other enzymes or metabolites (e.g., acetylcholinesterase, butyrylcholinesterase, glucose, triglycerides, and uric acid) that rely on the same or a related chromophore as used in the NTE assay (4-8), the potential for detergent-induced shifts in the chromophore spectrum should be taken into account when optimizing these assays in the presence of detergents. 9Presented in part at the 2Bth annual meeting of '~heSociety of Tc, xlcology, Atlanta. GA, February 27 to March 3. 1989 "'Present address: California 13epartment of Food and Agnoulture, Medical Toxicology Branch, Saotamento, CA 95814 t Author to whom all correspondence should be sent Mmhng address: Or R J Richardson. Neurotoxlcology Research Laboratory. 1420 Washington Heights, The Umverslty of Michigan, Ann Arbor, M148109-2029 Introduction Neurotoxic esterase (neuropathy target esterase, NTE) is the putative target for organophosphorus compounds (OPs) that cause organophosphorus-induced delayed neuropathy (OPIDN). This neuropathy is believed to be initiated by a concerted two- step reaction involving inhibition of NTE followed by cleavage of a secondary leaving group from the enzyme-bound inhibitor. The second step of initiation is known as aging and appears to be an obligatory element in the pathogenesis of OPIDN. Because OPs are used in a wide variety of products including pesticides, hydraulic fluids, plasticizers, and fuel additives, there are many opportunities for humans and other nontarget organ- isms to become exposed. Fortunately, the risk of OPIDN can be minimized, because the neuropathic potential of OPs can be prcdicted reliably by measuring thc inhibition of NTE by com- pounds that are capable of aging (9,10). Johnson (I.2) developed a differential assay for NTE that measures estcratic activity resistant to diethyl 4-nitrophenyl- phosphate (paraoxon) and sensitive to N.N'-di-2-propylphos- phorodiamidotquoridate (mipafox). Paraoxon is an OP inhibitor of esterases other than NTE and does not cause OPIDN. Mipafk)x is an OP inhibitor of esterases including NTE and causes OPIDN. The sttbstrate used in the assay is phenyl valerate, which is hydrolyzed by esterases to liberate phenol. The amount of phenol produced is determined colorimetrically after its oxidative coupling to 4-aminoantipyrine in alkaline potassium ferricyanide to yield 4-N-( 1,4-benzoquinoneimine)- antipyrine, a chromophore with a wavelength of maximum ab- sorbance (X.n,)of 510 nm and corresponding molar absorptivity (molar extinction coefficient, e) of 13,900 M-Icm -l (2,11 ). In the original assay procedure, the enzymatic reaction was stopped by adding perchloric acid; in a later, widely adopted modification of the assay designed to improve and simplify the procedure, per- chloric acid was replaced with the detergent sodium dodecyl- sulfate (SDS). without modifying kn, and ~: (3). Recent reports (12,13) indicate that when the NTE assay is performed according to the modified method, the ~na of the chromophore is shifted to a shorter wavelength and the sensi- tivity of the assay is enhanced because of an increase in the E of the chromophore. The present study was carried out in order to study the role of SDS in the altered absorbance of the chro- mophore, and to use this information to optimize the NTE assay. 86 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission