Cholinesterase reactivation in organophosphorus poisoned patients depends on the plasma concentrations of the oxime pralidoxime methylsulphate and of the organophosphate.

We measured in nine patients, poisoned by organophosphorus agents (ethyl parathion, ethyl and methyl parathion, dimethoate, or bromophos), erythrocyte and serum cholinesterase activities, and plasma concentrations of the organophosphorus agent. These patients were treated with pralidoxime methylsulphate (Contrathion), administered as a bolus injection of 4.42 mg.kg-1 followed by a continuous infusion of 2.14 mg.kg-1/h, a dose regimen calculated to obtain the presumed "therapeutic" plasma level of 4 mg.l-1, or by a multiple of this infusion rate. Oxime plasma concentrations were also measured. The organophosphorus agent was still detectable in some patients after several days or weeks. In the patients with ethyl and methyl several days or weeks. In the patients with ethyl and methyl parathion poisoning, enzyme reactivation could be obtained in some at oxime concentrations as low as 2.88 mg.l-1; in others, however, oxime concentrations as high as 14.6 mg.l-1 remained without effect. The therapeutic effect of the oxime seemed to depend on the plasma concentrations of ethyl and methyl parathion, enzyme reactivation being absent as long as these concentrations remained above 30 micrograms.l-1. The bromophos poisoning was rather mild, cholinesterases were moderately inhibited and increased under oxime therapy. The omethoate inhibited enzyme could not be reactivated.

Phosphonylation of purified human, canine and porcine cholinesterase by soman. Stereoselective aspects.

Cholinesterases (EC 3.1.1.8, acylcholine acylhydrolase) from the sera of man, dog and pig were purified 400-600-fold using a combination of ion-exchange and affinity chromatography. In a first approach, phosphonylation by soman was studied by using the half-resolved epimers C(+)P(+/-)-soman and C(-)P(+/-)-soman. The degradation of soman at the nanomolar level was followed in time by determining the remaining soman by capillary gas chromatography with NP detection. In the three sera investigated the P-(-)-epimer phosphonylates at a higher rate than its corresponding P(+)-counterpart and the stereoselectivity is greater for the C(+)-epimers than for the C(-)-epimers. Individual soman isomers were isolated from C(+)- and C(-)-epimers and quantified by gas chromatography. Second-order rate constants were determined for the phosphonylation of purified cholinesterase by isolated soman isomers. The C(+)P(-)-isomer has the highest phosphonylation rate for the three species; the other toxic isomer, C(-)P(-), has a five to ten-fold lower rate. The overall stereoselectivity is more marked in human cholinesterase than in canine. Porcine serum cholinesterase is phosphonylated by the P(-)-isomers at a slightly higher rate than the human enzyme.

High doses of soman protect against organophosphorus-induced delayed polyneuropathy but tabun does not.

Organophosphorus-induced delayed polyneuropathy (OPIDP) is thought to result from organophosphorylation of neuropathy target esterase (NTE; formerly known as neurotoxic esterase), followed by an "aging" of the phosphorylated NTE. Protection against OPIDP should thus be achieved by production of an inhibited but "nonaging" NTE. Inhibited NTE produced in vitro by interaction with any of the four resolved isomers of soman aged negligibly (M. K. Johnson, D. J. Read, and H. P. Benschop, 1985a, Biochem. Pharmacol., 34, 1945-1951). Therefore both unresolved soman and the most inhibitory isomer (C(-)P(+)) were tested in adult hens for effects on NTE and for ability to produce OPIDP. With improved prophylaxis and therapy of acute intoxication, birds survived greater than 100 X LD50 of unresolved soman and did not develop OPIDP. One day after dosing, about half of brain and spinal cord NTE was in an unmodified (unaged) inhibited form; at this time eight survivors were challenged with a neuropathic dose of diisopropyl phosphorofluoridate (DFP). No neuropathy developed in four out of eight birds and mild to moderate signs were seen in the other four. Nine challenge control birds receiving DFP after solvent all developed severe neuropathy. Partial protection was seen in three out of three birds dosed prior to DFP challenge with sufficient C(-)P(+) isomer of soman (1.2 mg/kg sc) to convert about half of the spinal cord NTE to unaged inhibited form. Protection was not related to cholinergic shock. Two birds which survived out of eight pretreated with tabun (12 mg/kg sc) had about as much NTE inhibited as after soman administration but it was all in the modified (aged) inhibited form; these birds were not protected against DFP-induced neuropathy. A limited histopathologic examination showed that typical neurodegenerative lesions were seen only in birds with clear clinical neuropathy.

Stereoselective phosphonylation of human serum proteins by soman.

Phosphonylation has been reported as part of the degradation of soman in human serum. The concentration of phosphonylation sites can be quantified by comparing the degradation in serum, preincubated with soman (all sites occupied), with the degradation in serum not preincubated. The mean value of 73 nM of phosphonylation sites is in agreement with the concentration of active sites of butyrylcholinesterase (EC 3.1.1.8.), which is known to be phosphonylated by soman. Hence, it is concluded that butyrylcholinesterase accounts for all the phosphonylation sites present in human serum. The stereoselectivity of the reaction was investigated by using epimeric pairs of soman, in casu C(+)P(+/-)- and C(-)P(+/-)-soman. In a first approach enzymatic hydrolysis was blocked and the ratios of phosphonylation rate constants, C(+)P(+)/C(+)P(-) and C(-)P(+)/C(-)P(-), were determined to be 0.15 and 0.31, respectively. In a second approach, in untreated serum, the bimolecular phosphonylation rate constants of C(+)P(-)- and C(-)P(-)-soman were determined, neglecting their small hydrolysis rate and taking advantage of the fast enzymatically catalysed disappearance of their respective P(+)-epimeric counterparts. Values for C(+)P(-)- and C(-)P(-)-soman are 3.6 X 10(7) and 0.6 X 10(7) M-1.min-1, respectively. Using a combination of both approaches, a relative ranking of phosphonylation rates of the four isomers was found to be C(+)P(-) much greater than C(+)P(+) approximately equal to C(-)P(-) greater than C(-)P(+).

Stereoselective hydrolysis of soman in human plasma and serum.

The contribution of various human serum and plasma fractions to the total hydrolysis rate constants of the four isomers of soman is studied. Spontaneous hydrolysis (as measured in buffer) occurs at a faster rate for the C(+)P(+)- and C(-)P(-)-isomers. A stereoselectively catalyzed hydrolysis of soman occurs in serum fractions IV and V (albumin). In fraction V the C(+)P(+)- and C(-)P(-)-isomers are hydrolyzed at a faster rate than their respective epimers, while in fraction IV-1 a stereoselective effect towards C(+)P(+)-soman is found. All the forementioned contributions, however, are negligible in comparison with the stereoselective enzymatic hydrolysis of the P(+)-isomers. The latter reaction is characterized by a significant lowering of the activation energy as compared with the spontaneous hydrolysis of the P(+)-isomers. Such a lowering in activation energy is not found for the hydrolysis of the P(-)-isomers in whole serum or plasma; hence it can be concluded that a phosphorylphosphatase hydrolyzes the P(+)-isomers in a stereoselective way, the P(-)-isomers either not being affected by this (these) enzyme(s) or the mechanism of catalysis being fundamentally different. This conclusion is in agreement with the observations on the influence of Hg2+ on the hydrolysis of soman in serum; the hydrolysis of the P(+)-isomers is significantly inhibited by 1 mM of Hg2+ while the P(-)-hydrolysis is unaffected by this concentration of Hg2+. The action of some potential inhibitors on this phosphorylphosphatase activity was studied. Iodoacetate did not inhibit nor did Ba2+, Sr2+, Co2+ or Mn2+ show a significant effect on the hydrolysis of the P(+)-isomers. On the other hand the hydrolytic activity in serum was nearly completely inhibited by EDTA but restored upon addition of Ca2+. These findings suggest that this enzymatic activity can be classified as an arylesterase (paraoxonase). Finally, the influence of pH on the hydrolytic activity shows a different pattern for C(+)P(+)- and C(-)P(+)-soman, which may suggest that more than one enzyme is involved in the degradation of soman.

In vitro detoxification of soman in human plasma.

The degradation of soman in human plasma and at a physiological pH has been studied at the high nanomolar level. From a comparison between degradation of racemic soman in preincubated and unpreincubated plasma the concentration of phosphonylation sites was estimated at some 120 nM. The contribution of phosphonylation to the degradation of soman is eliminated by preincubation of the plasma with racemic soman. The rate constants of the total hydrolysis (enzymatic and nonenzymatic) were calculated from the semi-log plot of the degradation of C(+)- or C(-)-soman in preincubated plasma/Tris (50% v/v). The nonenzymatic part of the hydrolysis was estimated from experiments in plasma ultrafiltrate (100,000 NMWL)/Tris (50% v/v). The nonenzymatic contribution to the hydrolysis of the P(+)-isomers is negligible compared to the enzymatic hydrolysis.

In vitro degradation of the four isomers of soman in human serum.

Starting from racemic soman (1,2,2-trimethylpropyl methylphosphonofluoridate), the degradation of its four stereoisomers in human serum (25 degrees, pH 8.8), has been studied at the nM level. Phosphylation of serum proteins is eliminated by preincubation of the serum with soman. A capillary gas chromatographic method with nitrogen-phosphorous detection allows the separation of the diastereoisomers. The total hydrolysis (enzymatic and non-enzymatic) rate constants of the isomers can then be resolved indirectly on the basis of the important rate difference between P(+) and P(-) isomers. The enzymatic hydrolysis rate constants are obtained by subtracting, for each isomer, the spontaneous (non-enzymatic) rate constant from the total hydrolysis rate constant. The non-enzymatic part of the hydrolysis is obtained from experiments in serum ultrafiltrate (30,000 NMWL). Enzymatic hydrolysis of C(+) P(+) soman occurs so rapidly that only a lower limit of the rate constant can be given. The other enzymatic rate constants are 0.016 min-1 for C(+)P(-), 0.74 min-1 for C(-)P(+) and 0.028 min-1 for C(-)P(-).

Delayed neuropathy by the organophosphorus nerve agents soman and tabun.

The organophosphorus nerve agents soman and tabun were tested in the hen at doses 120-150 times higher than their acute LD50, as it was assumed that these doses would produce delayed neuropathy. The animals were protected against the acute lethal effect of these agents by pretreatment with atropine, physostigmine, diazepam, and the oxime HI-6 or obidoxime. The surviving animals were followed for 30 days and the occurrence of delayed neuropathy was clinically diagnosed. Soman produced severe delayed neuropathy at a dose of 1.5 mg/kg, a dose which produced acute lethality in five animals out of six. Tabun elicited very mild neuropathic symptoms in one animal out of two at a dose of 6 mg/kg given on 2 consecutive days. Delayed neuropathy was not seen in the hens that survived the acute toxicity of a single dose of tabun , 12 mg/kg (three out of six) or 15 mg/kg (two out of six).