Formation and disposition of diethylphosphoryl-obidoxime, a potent anticholinesterase that is hydrolyzed by human paraoxonase (PON1).

The potential of pyridinium-4-aldoximes, such as obidoxime, to reactivate diethylphosphorylated acetylcholinesterases is not fully exploited due to the inevitable formation of phosphoryloximes (POX) with high anticholinesterase activity. Mono(diethylphosphoryl) obidoxime (DEP-obidoxime) was isolated for the first time showing remarkable stability under physiological conditions (half-life 13.5min; pH 7.1; 37 degrees C). The half-life was considerably extended to 20h at 0 degrees C, which facilitated the preparation and allowed isolation by HPLC. The structure was confirmed by mass spectrometry and the degradation pattern. DEP-obidoxime decomposed by an elimination reaction forming the intermediate nitrile that hydrolyzed mainly into the pyridone and cyanide. The intermediates were prepared and confirmed by mass spectroscopy. DEP-Obidoxime was an extremely potent inhibitor of human acetylcholinesterase approaching a second-order rate constant of 10(9)M(-1)min(-1) (pH 7.4; 37 degrees C). The nitrile and the pyridone were still good reactivators. In the presence of human plasma DEP-obidoxime was hydrolyzed into parent obidoxime. Calcium-dependence and sensitivity towards chelators, substitution pattern by other divalent cations and protein-modifying agents all pointed to human paraoxonase (hPON1) as the responsible protein with POX-hydrolase activity. Subjects, probably belonging to the homozygous (192)arginine subtype, were virtually devoid of POX-hydrolase activity while a highly purified hPON1 of the homozygous (192)glutamine subtype exhibited particularly high POX-hydrolase activity. Two parathion-poisoned patients with high and low POX-hydrolase activity responded well and poorly, respectively, to obidoxime treatment although the former patient had higher plasma paraoxon levels than the poor responder. Hence, the POX-hydrolase associated PON1 subtype may be another contributor that modulates pyridinium-4-aldoxime effectiveness.

Effect of human plasma on the reactivation of sarin-inhibited human erythrocyte acetylcholinesterase.

The reactivation of organophosphate-inhibited acetylcholinesterase (AChE) by oximes inevitably results in the formation of highly reactive phosphoryloximes (POX), which are able to re-inhibit the enzyme. In this study, the dependence of POX formation on AChE concentration was investigated with sarin-inhibited human erythrocyte AChE (EryAChE). A marked dependence was found with obidoxime but not with the experimental oxime HI 6, suggesting great differences in the decomposition rates of the respective POXs. At a physiological erythrocyte content the reactivation of EryAChE was markedly affected by POX with obidoxime and pralidoxime (2-PAM) but not with the newer oximes HI 6 and HLö 7. Addition of extensively dialysed, sarin-treated human plasma reduced the reactivation by obidoxime and 2-PAM even more. Obidoxime and 2-PAM were superior to HI 6 and HLö 7 in reactivating butyrylcholinesterase (BChE). This effect was pronounced in diluted plasma, but was obscured in concentrated plasma, probably because of re-inhibition by the generated POX. Addition of native erythrocytes to sarin-treated plasma resulted in marked inhibition of EryAChE in the presence of obidoxime, suggesting a higher affinity of the POX for EryAChE. The results indicate that obidoxime and 2-PAM may reactivate sarin-inhibited AChE insufficiently due to re-inhibition by the POX formed. In addition, the re-inhibition of Ery-AChE may be aggravated by the POX that is produced during BChE reactivation. These reactions must be regarded as therapeutically detrimental and disqualify those oximes which are capable of forming stable POX by reactivation of BChE.

The phosphoryl oxime-destroying activity of human plasma.

The potential of obidoxime and other pyridinium-4-aldoximes to reactivate dimethyl- and diethylphosphorylated cholinesterases is markedly restricted by the inevitable formation of rather stable phosphoryl oximes (POXs) with high anticholinesterase activity. This effect is hardly seen with very dilute enzyme preparations, but becomes significant at physiological enzyme concentrations. Human plasma with the butyrylcholinesterase irreversibly blocked by soman was able to stimulate obidoxime-induced reactivation of concentrated erythrocyte acetylcholinesterase (Ery-AChE) to the same extent as was observed with a dilute preparation, suggesting phosphoryl oxime-destroying capacity. The inactivating factor, which was tentatively termed POX-hydrolase, had (1) a molecular weight of >100 kDa; (2) required Ca2+ , which could not be substituted by Zn2+ or Mg2+; and (3) lost its catalytic activity reversibly in the presence of ethylenediamine-tetraacetic acid (EDTA). The enzyme activity varied widely (20-fold) among different subjects and did not follow the activity pattern of human serum paraoxonase (PON1). Rabbit plasma with its particularly high paraoxonase content showed only weak POX-hydrolase activity. These data suggest POX-hydrolase to be a different entity. POX-hydrolase was most active with the putative phosphoryl-obidoxime from paraoxon-ethyl, less with the product from paraoxon-methyl and least with that from diisopropylfluorophosphate. The analogue TMB-4 reacted similarly to obidoxime. The putative phosphonyl oximes arising by the reaction of obidoxime with nerve agents were apparently not cleaved. The variation in POX-hydrolase activity may additionally contribute to the variable response to oxime therapy in patients with organophosphate insecticide poisoning.

Improved determination of acetylcholinesterase activity in human whole blood.

Determination of erythrocyte acetylcholinesterase (AChE) activity is the appropriate tool for the diagnosis of organophosphate exposure and intoxication. The original colorimetric Ellman procedure is disturbed by a high hemoglobin absorption at 412 nm. In our modified method the wavelength was changed to 436 nm. This reduced the indicator absorption to 80% and the hemoglobin absorption to 25%. The signal-to-noise ratio was further enhanced by reduction of pH and substrate concentration, thus making it possible to measure 3% residual activity. AChE activity was determined in whole blood samples in the presence of the selective butyrylcholinesterase inhibitor ethopropazine. Dilution of blood samples (1:100) stops secondary reactions in the presence of inhibitor (organophosphate) and reactivator (oxime). Normalization of the AChE activity to the hemoglobin content, determined as cyanmethemoglobin, prevented dilution errors. This modified approach provides a simple way for sensitive and precise determination of AChE activity in whole blood in the presence of organophosphates even with low-tech equipment.