Cyanide intoxication: protection with chlorpromazine.

Protection against cyanide intoxication in mice can be enhanced by the administration of chlorpromazine, providing it is given with sodium thiosulfate, or the sodium thiosulfate-sodium nitrite antidotal combination. Protency ratios which were derived from the LD50 values were compared in groups of mice premedicated with chlorpromazine (10 mg/kg) and/or sodium thiosulfate (1 g/kg) and/or sodium nitrite (100 mg/kg). These results indicate that the administration of chlorpromazine alone provides no protection against the lethal effects of cyanide. Chlorpromazine also does not enhance the protective effect of sodium nitrite; however, it strikingly potentiates the effectiveness of sodium thiosulfate either alone or in combination with sodium nitrite.     

Cyanide intoxication in sheep; therapeutics

Various cyanide antidotes were evaluated by comparing the effects of delay in time of therapy following oral administration of sodium cyanide in sheep. Successful therapy of lethal doses of sodium cyanide could be accomplished with the more potent antidotes for up to 30 minutes following administration of sodium cyanide. Either 660 mg/kg sodium thiosulfate or 1 mg/kg p-aminopropriophenone were effective antidotes for moderate lethal doses (7.6 mg/kg) of sodium cyanide. The conventional low dosage nitrite/thiosulfate (6.7 mg/kg and 67 mg/kg) was much less effective. Larger doses (15.2 mg/kg) of sodium cyanide were effectively antagonized by either 660 mg/kg sodium thiosulfate alone or in combination with 1.5 mg/kg p-aminopropriophenone or 22 mg/kg sodium nitrite. At high cyanide dosage, p-aminopropriophenone alone was less effective than sodium thiosulfate alone. Sodium thiosulfate at high dosage appears to be the antidote of choice. This more closely satisfies the requirements of high efficacy and low toxicity for an antidote. Sodium nitrate or other antidotes may be used in conjunction with sodium thiosulfate, but their use is not necessary for high efficacy.     

Cyanide intoxication: protection with cobaltous chloride

Protection against the lethal effects of cyanide can be elicited by administration of cobaltous chloride, either alone or in combination with sodium nitrite and/or sodium thiosulfate. Potency ratios derived from the LD50 values were compared in groups of mice premedicated with cobaltous chloride and/or sodium thiosulfate and/or sodium nitrite. Under the conditions of our experiment cobaltous chloride alone is slightly more effective than sodium nitrite; when it is combined with sodium nitrite, an additive effect is obtained. When cobaltous chloride is administered in combination with sodium thiosulfate, a dramatic antagonism of the lethal effects of potassium cyanide is observed. The synergistic antidotal effect of cobaltous chloride may be related to the physiological disposition of the cobaltous ion and its ability to chelate both cyanide and thiocyanate ions.     

The efficacy of alpha-ketoglutaric acid in the antagonism of cyanide intoxication

It has been reported that compounds containing carbonyl groups can readily react with cyanide. Pyruvic acid, an alpha-ketocarboxylic acid, has been shown to antagonize the lethal effects of cyanide. It is suggested that its mechanism of action rests in its ability to react with or "bind" cyanide. In this study, alpha-ketoglutaric acid, also an alpha-ketocarboxylic acid, was evaluated for its ability to counteract the lethal effects of cyanide. alpha-Ketoglutaric acid increased the LD50 value of cyanide (6.7 mg/kg) by a factor of five, a value statistically equivalent to that ascertained in mice pretreated with sodium thiosulfate and sodium nitrite. The combination of alpha-ketoglutaric acid and sodium thiosulfate increased the LD50 value of cyanide to 101 mg/kg. Addition of sodium nitrite to the alpha-ketoglutaric acid/sodium thiosulfate regimen increased the LD50 value of cyanide to 119 mg/kg. Unlike sodium nitrite, no induction of methemoglobin formation was observed with alpha-ketoglutaric acid pretreatment. It is apparent from these studies that the administration of alpha-ketoglutaric acid in conjunction with sodium thiosulfate resulted in fewer animal deaths than sodium nitrite and sodium thiosulfate without the dangerous formation of methemoglobin.     

The in vivo effects of cyanide and its antidotes on rat brain cytochrome oxidase activity.

The in vivo effects of sodium cyanide and its antidotes, sodium nitrite, sodium thiosulfate and 4-dimethylaminophenol (DMAP), as well as the alpha-adrenergic blocking agent phentolamine, on rat brain cytochrome oxidase were studied. The course of inhibition was time-dependent and a peak of 40% was attained between 15 and 20 min after the s.c. injection of 1.3 LD50 (12 mg/kg) of cyanide. Pronounced dose-dependence was observed in the inhibition of the enzyme, at this relatively low, but lethal dose. Further observation was impossible because of rapidly lethal effects of cyanide. In animals artificially ventilated with room air, observation was possible up to 60 min. However, maximum inhibition was also 40%. When antidotes were applied 30 min after 20 mg/kg of cyanide, marked reactivation of cytochrome oxidase activity was observed with all antidotes (particularly with thiosulfate) except for phentolamine which had no effect. Prevention of methemoglobin forming with toluidine blue did not affect the reactivating ability of nitrite or DMAP, thus suggesting more complex protective mechanisms then simple methemoglobin formation. The high efficacy of thiosulfate may be attributed to its rhodanese catalyzed, direct binding to free blood cyanide, leading thus to its dissociation from cytochrome oxidase. The theory that cytochrome oxidase inhibition is a basic mechanism of cyanide toxicity could not be disproved.     

Protection against cyanide-induced convulsions with alpha-ketoglutarate

Protection against convulsions induced by cyanide was observed after treatment with alpha-ketoglutarate, either alone or in combination with sodium thiosulfate, a classical antagonist for cyanide intoxication. However, sodium thiosulfate alone did not protect against cyanide (30 mg/kg)-induced convulsions. gamma-Aminobutyric acid (GABA) levels in brain were decreased by 31% in KCN-treated mice exhibiting convulsions. The combined administration of alpha-ketoglutarate and sodium thiosulfate completely abolished the decrease of GABA levels induced by cyanide. Furthermore, sodium thiosulfate alone also completely abolished the decrease of GABA levels. These results suggest that the depletion of brain GABA levels may not directly contribute to the development of convulsions induced by cyanide. On the other hand, cyanide increased calcium levels by 32% in brain crude mitochondrial fractions in mice with convulsions. The increased calcium levels were completely abolished by the combined administration of alpha-ketoglutarate and sodium thiosulfate, but not affected by sodium thiosulfate alone. These findings support the hypothesis proposed by Johnson et al. (Toxicol. Appl. Pharmacol., 84 (1986) 464) and Robinson et al. (Toxicology, 35 (1985) 59) that calcium may play an important role in mediating cyanide neurotoxicity.     

Antagonism of cyanide intoxication with sodium pyruvate

Cyanide intoxication in mice can be effectively antagonized by sodium pyruvate, particularly if it is administered in combination with the antidotes, sodium nitrite and sodium thiosulfate. Potency ratios derived from the LD50 data were compared in groups of mice treated with sodium nitrite, sodium thiosulfate, and sodium pyruvate either alone or in various combinations. These results indicate that the administration of sodium pyruvate alone does provide minimal, but statistically significant, protection against the lethal effects of cyanide. Sodium pyruvate does not enhance the effect of sodium nitrite; however, it does potentiate the antidotal effect of sodium thiosulfate. The sodium thiosulfate and sodium pyruvate combination is not as effective as the sodium nitrite and sodium thiosulfate combination, but the addition of sodium pyruvate to the sodium nitrite-sodium thiosulfate combination further enhances the antidotal effect. No further enhancement is observed when sodium nitrite, sodium thiosulfate, and sodium pyruvate are combined with oxygen.     

Effects of various known and potential cyanide antagonists and a glutathione depletor on acute toxicity of cyanide in mice

To compare the protective potencies of a large number of known and potential cyanide antagonists in one stock of mice, groups (N = 10) of male CF-1 Swiss-Webster mice were given a single maximal or near-maximal intraperitoneal injection of each substance. Ethyl maleate, a glutathione (GSH) depletor and potential enhancer of cyanide toxicity, was given to other groups. Thirty min later, the mice were given subcutaneous injections of graded doses of KCN. In untreated control mice, the 24-hr median lethal dose (LD50) of KCN was 11 mg/kg of body weight (potency ratio, PR = 1.0). In comparison, protective effects of traditional antagonists thiosulfate and nitrite produced PR values of 1.48 and 2.95, respectively. Tetrathionate, sulfate, dithionite, methionine, hydroxocobalamin, ascorbate, pyridoxal phosphate, alpha-ketoglutarate, alpha-ketobutyrate, GSH, GSH disulfide (GSSG) and selenite were similar in efficacy to thiosulfate (P less than 0.05; PR values 1.35-1.59). Cysteine, diethyldithiocarbamate (DEDC), and cobaltous chloride were more effective than thiosulfate (PR values 1.68, 1.69, and 1.85, respectively). Phentolamine and dicobalt EDTA were ineffective, whereas papaverine enhanced toxicity (PR 0.72). Agents with significant PR values (greater than or equal to 1.14) but which were less effective than thiosulfate included sulfite, dimercaptosuccinic acid, pyruvate, citrate, alpha-ketovalerate, naloxone, and corn oil. Ethyl maleate in corn oil markedly enhanced KCN lethality (PR 0.57 compared to corn oil alone), and caused prolonged illness in several mice. Vitamin E in corn oil had no effect. Dual mixtures of thiosulfate with other selected substances were also tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of chlorpromazine on cyanide intoxication

Previous reports from our laboratory indicated that prophylactic protection against cyanide intoxication in mice can be enhanced by administration of chlorpromazine when it is given with sodium thiosulfate. The mechanism of potentiation of sodium thiosulfate by chlorpromazine was studied alone and in combination with sodium nitrite. Although chlorpromazine was found to induce a hypothermic response, the mechanism of enhancement of the antagonism of cyanide by chlorpromazine does not correlate with the hypothermia produced. Various other possible mechanisms were investigated, such as rate of methemoglobin formation, enzymatic activity of rhodanese and cytochrome oxidase, and alpha-adrenergic blockade. The alpha-adrenergic blocking properties of chlorpromazine may provide a basis for its antidotal effect, since this protective effect can be reversed with an alpha-agonist, methoxamine.     

Protection by sodium thiosulfate and thiourea against lethal toxicity of cis-diamminedichloroplatinum (II) in bacteria and mice

The protective effect of sodium thiosulfate and thiourea on the lethal toxicity of the antitumor drug, cis-diamminedichloroplatinum (II) (cis-DDP), was investigated in bacteria and mice. Initially, the agents capable of antagonizing bactericidal activity of cis-DDP were screened using WP2 uvra, a strain of E. coli sensitive to this drug. Of the ten sulfur-containing compounds tested, thiourea and sodium thiosulfate exhibited potent protecting effects against cis-DDP cytotoxicity in bacteria. Propylthiouracil and methimazole showed intermediate levels of such protection, but the other 6 compounds had little or no protective effects. Thiourea and sodium thiosulfate were then subjected to the acute lethal toxicity test in mice to assess their protective activity in vivo. We found that cis-DDP i.v. lethality against mice can be blocked almost completely by excess amounts of thiourea or sodium thiosulfate. Thiourea protected against cis-DDP toxicity with a narrow range among the effective doses, while sodium thiosulfate was protective with a remarkably wide range of effective doses. The effectiveness of sodium thiosulfate was also indicated in experiments in which the LD50 dose of cis-DDP (16 mg/kg) i.p. increased over the level of greater than 200 mg/kg with concomitant administration of sodium thiosulfate i.p.     

Antagonism of cyanide poisoning by dihydroxyacetone

Dihydroxyacetone (DHA) effectively antagonized the lethal effect of cyanide in mice and rabbits, particularly if administered in combination with thiosulfate. Oral DHA (2 and 4 g/kg) given to mice 10 min before injection (i.p.) of cyanide increased the LD50 values of cyanide from 5.7 mg/kg to 12 and 17.6 mg/kg, respectively. DHA prevented cyanide-induced lethality most effectively, if given orally 10-15 min before injection of cyanide. A combination of pretreatment with oral DHA (4 g/kg) and post-treatment with sodium thiosulfate (1 g/kg) increased the LD50 of cyanide by a factor of 9.9. Furthermore, DHA given intravenously to rabbits 5 min after subcutaneous injection of cyanide increased the LD50 of cyanide from 6 mg/kg to more than 11 mg/kg, while thiosulfate (1 g/kg) given intravenously 5 min after cyanide injection increased the LD50 of cyanide only to 8.5 mg/kg. DHA also prevented the convulsions that occurred after cyanide intoxication.     

Nitroprusside intoxication: protection of alpha-ketoglutarate and thiosulphate.

Protection against the lethal effects of sodium nitroprusside (SNP) was observed in mice after treatment with alpha-ketoglutarate (AKG), either alone or in combination with sodium thiosulphate (STS). The LD50 of SNP was 12.0 (11.0-13.0) mg/kg in mice. Ip injection of AFG (500 mg/kg twice in 20 min) increased the LD50 1.7-fold in mice. STS (1 g/kg, ip) alone increased the LD50 5.5-fold. Furthermore, combined administration of AKG and STS increased the LD50 6.9-fold. SNP elicited increased cyanide levels in blood of mice in a dose-dependent manner. SNP (10 mg/kg, sc) administration gave rise to blood cyanide levels of 73.2 +/- 3.0 microM, 30 min after treatment. Ip injection of AKG significantly decreased blood cyanide levels by 30% in mice 30 min after treatment with 10 mg SNP/kg. A single injection of STS (1 g/kg) or a combination of AKG and STS reduced in blood cyanide levels by 88 or 98%, respectively, in mice after treatment with 10 mg SNP/kg. In addition, the increase in blood cyanide levels induced by injection of 50 mg SNP/kg was markedly inhibited by a combination of AKG and STS or (to a lesser extent) by STS alone. These results suggest that the combined administration of AKG and STS, by preventing the increase in blood cyanide levels induced by SNP, may afford protection against the toxic effects of SNP.     

Effects of oxygen on the antagonism of cyanide intoxication: cytochrome oxidase, in vitro

Since oxygen was reported to be an effective cyanide antagonist in vivo, particularly in the presence of the classic antidotal combination of sodium nitrite and sodium thiosulfate, in vitro studies were initiated in an attempt to investigate the mechanism of oxygen-mediated cyanide antagonism. The effect of oxygen on cyanide-inhibited cytochrome oxidase with and without cyanide antagonist(s) was investigated in a purified membraneous enzyme system prepared from rat liver mitochondria. Cyanide produced a concentration dependent inhibition of cytochrome oxidase, and 100% oxygen did not alter the inhibition produced by KCN either in the presence or absence of sodium thiosulfate. However, the addition of sodium thiosulfate and rhodanese to the assay reactivated the cyanide-inhibited cytochrome oxidase. Kinetic analysis indicated rhodanese competes with cytochrome oxidase for cyanide, and oxygen had no effect on this coupled reaction. In conclusion, the in vivo antidotal properties of oxygen cannot be attributed to oxygen-mediated reactivation of cyanide-inhibited cytochrome oxidase or an oxygen-mediated acceleration of rhodanese detoxification.     

Acute, sublethal cyanide poisoning in mice is ameliorated by nitrite alone: complications arising from concomitant administration of nitrite and thiosulfate as an antidotal combination

Sodium nitrite alone is shown to ameliorate sublethal cyanide toxicity in mice when given from ～1 h before until 20 min after the toxic dose as demonstrated by the recovery of righting ability. An optimum dose (12 mg/kg) was determined to significantly relieve cyanide toxicity (5.0 mg/kg) when administered to mice intraperitoneally. Nitrite so administered was shown to rapidly produce NO in the bloodsteam as judged by the dose-dependent appearance of EPR signals attributable to nitrosylhemoglobin and methemoglobin. It is argued that antagonism of cyanide inhibition of cytochrome c oxidase by NO is the crucial antidotal activity rather than the methemoglobin-forming action of nitrite. Concomitant addition of sodium thiosulfate to nitrite-treated blood resulted in the detection of sulfidomethemoblobin by EPR spectroscopy. Sulfide is a product of thiosulfate hydrolysis and, like cyanide, is known to be a potent inhibitor of cytochrome c oxidase, the effects of the two inhibitors being essentially additive under standard assay conditions rather than dominated by either one. The findings afford a plausible explanation for an observed detrimental effect in mice associated with the use of the standard nitrite-thiosulfate combination therapy at sublethal levels of cyanide intoxication.     

Cyanide Antagonism with Carrier Erythrocytes and Organic Thiosulfonates

Previous studies reported that resealed erythrocytes containingrhodanese (CRBC) and Na₂S₂O₃ rapidly metabolize cyanide to theless toxic thiocyanate both in vitro and in vivo. This provideda new conceptual approach to prevent and treat cyanide intoxication.Although the rhodanese-containing carrier cells with thiosulfateas the sulfur donor were efficacious, this approach has potentialdisadvantages, as thiosulfate has limited penetration of cellmembrane and product inhibition of rhodanese can occur due toinorganic sulfite accumulation. In order to circumvent substratelimitation and product inhibition by sodium thiosulfate, organicthiosulfonates were explored. These thiosulfonates have higherlipid solubility than thiosulfate and therefore can replenishthe depleted sulfur donor, as they can readily penetrate cellmembranes. Also, product inhibition of rhodanese is less aptto occur. This change in sulfur donors should greatly enhancecyanide detoxication, replenish the sulfur donor, and minimizeproduct inhibition of rhodanese. Present studies demonstratethe enhanced efficacy of exogenous organic thiosulfonates oversodium thiosulfate in the CRBC antidotal system to detoxifythe lethal effects of cyanide either alone or in combinationswith exogenously administered NaNO₂. Murine carrier erythrocytescontaining purified bovine liver rhodanese were administeredintravenously into male Balb/C mice. Subsequently, butanethiosulfonate(BTS) or Na₂S₂O₃ (ip), and NaNO₂ (sc) were co-administered priorto KCN (sc). Potency ratios, derived from the LD₅₀ values, werecompared in groups of mice treated with CRBC-Na₂S₂O₃ or CRBC-BTSeither alone or in combination with NaNO₂. The CRBC-BTS antidotalsystem shows strikingly enhanced protective effect over thatof the CRBC-thiosulfate system either alone or in combinationwith sodium nitrate.     

Morphine stimulates cholinergic neuronal activity in the rat hippocampus

The effects of morphine on cholinergic neuronal activity in the rat hippocampus were examined by measuring the rate of high-affinity, Na-dependent [³H]-choline uptake (HANDCU) into synaptosomes isolated from hippocampus. Morphine (6 mg/kg, i.p.) significantly stimulated HANDCU into hippocampal synaptosomes relative to control, and pretreatment with naloxone (1 mg/kg, i.p.) antagonized this effect. At a dose of 12 mg/kg, morphine did not alter the choline transport system. Dextrorphan, the inactive stereoisomer of the opiate agonist levorphanol, had no effect on HANDCU. Interestingly, higher doses of naloxone (15mg/kg; 30mg/kg, i.p.) produced significant increases in HANDCU, and the effects of naloxone (30 mg/kg) and morphine (6 mg/kg) on HANDCU appeared to be additive.     

In vivo studies on rhodanese encapsulation in mouse carrier erythrocytes

Resealed erythrocytes containing sodium thiosulfate and rhodanese (CRBC) are being employed as a new approach in the antagonism of cyanide intoxication. In earlier in vitro studies, the behavior of red blood cells containing rhodanese and sodium thiosulfate was investigated with regard to their properties and their capability of metabolizing cyanide to thiocyanate. The present studies are concerned with the properties of these rhodanese-containing carrier erythrocytes in the intact animal. These carrier erythrocytes were administered intravenously and the survival of the encapsulated enzyme was compared with the administration (iv) of free exogenous enzyme. Also, the amount of leakage of the encapsulated rhodanese from the red blood cell was determined. The survival of the carrier red blood cell. prepared by hypotonic dialysis, was found to be characterized by a biphasic curve. There was an initial rapid loss of approximately 40 to 50% of the carrier cells with a t1/2 = 2.5 hr. Subsequently the remaining resealed annealed carrier erythrocytes persisted in the vascular system with a t1/2 = 8.5 days. When free exogenous rhodanese was administered directly into the vascular system, it was rapidly eliminated with a t1/2 = 53 min. Red blood cells containing sodium thiosulfate and rhodanese apparently are effective in vivo in the biotransformation of cyanide. In animals pretreated with encapsulated rhodanese and sodium thiosulfate, blood cyanide concentrations are appreciably decreased with a concomitant increase in thiocyanate ion, a metabolite of cyanide. When erythrocytes, which contained no rhodanese or sodium thiosulfate, were subjected to hypotonic dialysis, cyanide was not metabolized to any appreciable extent. Furthermore, carrier erythrocytes containing rhodanese and sodium thiosulfate were found to increase the protection against the lethal effects of cyanide by approximately twofold. The ability of these carrier erythrocytes alone to metabolize cyanide and to antagonize the lethal effects of cyanide reflects the potential of this new antidotal approach in the antagonism of chemical toxicants.     

Naloxone as a stimulus in drug discrimination learning: generalization to other opiate antagonists.

Nonopiate dependent animals were trained to discriminate the opiate antagonist naloxone (1 mg/kg) from distilled water within the conditioned taste aversion baseline of drug discrimination learning. Specifically, rats injected with naloxone prior to a saccharin-LiCl pairing, and with its vehicle prior to saccharin alone, rapidly acquired the drug discrimination, avoiding saccharin following the administration of naloxone and consuming saccharin following its vehicle after only three conditioning trials. Once the discrimination was acquired, generalization tests revealed that the opiate antagonists diprenorphine and naltrexone and the mixed opiate agonist/antagonist nalorphine completely generalized to the naloxone cue at doses of 1.8, 5.6 and 18 mg/kg, respectively. That discriminative control was established with a low dose of naloxone (i.e., 1 mg/kg) and other compounds with opiate antagonist activity generalized to the naloxone cue suggest that the stimulus effects of naloxone were likely mediated through the opiate receptor. Because each of these compounds are reported to bind to the mu receptor (with varying affinities and varying degrees of selectivity), the stimulus properties of naloxone are likely mediated at this specific receptor subtype.     

Nitrite/thiosulfate treated acute cyanide poisoning: estimated kinetics after antidote

A 34 year old, 73 kg man ingested a 1 gram potassium cyanide pellet in a suicide attempt. Within one hour, coma, apnea, metabolic acidosis, and seizures developed. Sodium nitrite and sodium thiosulfate were administered. Dramatic improvement in the clinical condition occurred by the completion of antidote infusion. Methemoglobin level was 2% immediately after nitrite administration. Serial whole blood cyanide levels were obtained, documenting a highest measured level of 15.68 mcg/mL. Estimations of toxicokinetic parameters including terminal half-life (t 1/2) (19 hours), clearance (163 mL/minute), and volume of distribution (Vd) (0.41 L/kg) were calculated. The nitrite/thiosulfate combination was clinically efficacious in this case and resulted in complete recovery.     

Enzyme therapy in cyanide poisoning: Effect of rhodanese and sulfur compounds

Crystalline bovine liver rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) was evaluated as an antidote in combination with different sulfur compounds against cyanide poisoning in mice. The prophylactic antidote effect, when the antidote was injected i.v. 1 min prior to i.p. injection of cyanide, was dependent on both the dose of the enzyme and the dose of the sulfur compound. An optimal dose of the enzyme of about 2,000 U/kg (3 mg/kg of pure enzyme) was found. This enzyme dose combined with 2 mmol/kg of sodium thiosulfate raised the LD₅₀ for potassium cyanide 7.6 times. When thiosulfate was replaced with equimolar doses of ethanethiosulfonate and propanethiosulfonate, the corresponding values were 10.3 and 9.3 times, respectively. Maximum antidote effect was obtained when the doses of ethanethiosulfonate and propanethiosulfonate were raised to 4 mmol/kg, increasing the LD₅₀ for cyanide 20.8 and 15.4 times, respectively. On the other hand, when given without rhodanese, ethanethiosulfonate and propanethiosulfonate were no better antidotes than thiosulfate.Rhodanese and a sulfur compound given therapeutically to mice when symptoms of cyanide poisoning had occurred, also had a very good antidote effect. The prophylactic antidote effect of rhodanese plus thiosulfate rapidly decreased with increasing time interval between injection of the antidote and cyanide. Thus, when rhodanese and thiosulfate were given 20 min prior to cyanide, the antidote effect was of the same order as that of thiosulfate alone. The antidote effect of the latter did not decrease significantly within the same time interval.Enzyme activity in plasma decreased rapidly after i.v. injection of rhodanese, and enzyme activity in urine was detected following injection. No appreciable inactivation occurred when the enzyme was incubated with whole blood in vitro, but a strong and rapid inhibition, about 85%, of the enzyme occurred in fresh mouse urine in vitro.