Determination of penicillin in pharmaceutical formulations by flow injection analysis using an optimised immobilised penicillinase reactor and iodometric detection

An automated assay for the determination of penicillin in formulations suitable for use in pharmaceutical quality control is presented. The method is based on the classical iodometric penicillin assay which is incorporated in a flow injection analysis (FIA) system. The required hydrolysis is performed on-line by using an immobilised penicillinase reactor. Packed-bed and single-bead-string enzyme reactors are compared. It turns out that a packed-bed penicillinase reactor (10 cm x 1.5 mm i.d.) provides complete hydrolysis within short residence time, while only little back-pressure is generated. This enzyme reactor is stable for at least 9 months. Enzymatic hydrolysis of the beta-lactam ring results in the formation of the corresponding penicilloic acid, which consumes iodine. The iodine consumption is determined colorimetrically by measuring the decrease of the absorbance of the blue coloured iodine/starch complex. The optimum reactor length and flow rate for the colourimetrical detection reaction are determined. The optimised method is applied to the assay of penicillin in formulations and the results are compared with the "true" results obtained with a reference method: a mercurimetric titration. The reliability of the flow injection method is evaluated quantitatively by determining the maximum total error (MTE). The reliability is shown to be highest when measuring at a 0.3-mM level. Eight formulations including capsules, tablets and injectables containing penicillin G, amoxicillin or flucloxacillin are assayed. The MTE does not exceed the 6% level and the most probable MTE is between 1.5 and 3.5%.     

Epimerization and Hydrolysis of Dalvastatin,a New Hydroxymethylglutaryl Coenzyme A (HMG-CoA) Reductase Inhibitor

In aqueous solutions, dalvastatin (1) undergoes epimerization as well as hydrolysis. The transformation of the drug was studied as a function of pH at 25°C in aqueous solutions containing 20% acetonitrile. At all pH values, first-order plots for the conversion are biphasic, indicating rapid equilibration of 1 with its epimer (2) and slower hydrolysis of 1 to the corresponding -hydroxy acid (3). Apparent first-order rate constants for the biexponential equation are given as a function of pH. The alkyl–oxygen cleavage of the lactone ring results in the epimerization of 1 to 2, whereas the acyl–oxygen cleavage results in the hydrolysis of 1 to 3. The epimerization is an S_N1 reaction reaching an equilibrium of [l] _eq/[2] _eq = 1.27. The epimerization rate is increased with an increase in the water content of the solvent. The hydrolysis of 1 to 3 is acid and base catalyzed. The hydrolysis is reversible in acidic media and irreversible in neutral and basic media. At pH values greater than 9, the hydrolysis reaction proceeds more rapidly than the epimerization.     

Accumulation of Succinimide in a Recombinant Monoclonal Antibody in Mildly Acidic Buffers Under Elevated Temperatures

Purpose The purpose of this paper was to identify the location of a succinimide and determine the rate of its formation and hydrolysis in a recombinant human monoclonal IgG2 antibody aged in mildly acidic buffers at elevated temperatures. Materials and Methods Cation exchange (CEX) HPLC separated multiple Main Peaks and high levels (up to 50%) of basic variants, the identification of which was an analytical challenge and required several complementary techniques. The relative abundance of the CEX basic variants was used to quantify the percentage of succinimide and to study the rates of its formation and hydrolysis. Results Mass decrease by approximately 18 Da for intact antibodies from the CEX basic fractions suggested succinimide formation from aspartic acid as the major modification. Reversed-phase HPLC/MS of the reduced and trypsin-digested samples detected an isoaspartate 30 (isoD30) in the light chain peptide A25-R37. Direct evidence that isoD30 was from succinimide was obtained by performing succinimide hydrolysis in followed by tryptic digestion in . Conclusions Succinimide formation increased as pH became more acidic, whereas its hydrolysis was faster as pH became neutral and alkaline. Succinimide hydrolysis in a denatured sample was estimated to have completed in less than 2 h, but approximately three days for a similar pH but without denaturant. These observations suggest that protein conformation affects succinimide hydrolysis.     

Biochemical and molecular analysis of carboxylesterase-mediated hydrolysis of cocaine and heroin

Background and purpose:

Carboxylesterases (CEs) metabolize a wide range of xenobiotic substrates including heroin, cocaine, meperidine and the anticancer agent CPT-11. In this study, we have purified to homogeneity human liver and intestinal CEs and compared their ability with hydrolyse heroin, cocaine and CPT-11.

Experimental approach:

The hydrolysis of heroin and cocaine by recombinant human CEs was evaluated and the kinetic parameters determined. In addition, microsomal samples prepared from these tissues were subjected to chromatographic separation, and substrate hydrolysis and amounts of different CEs were determined.

Key results:

In contrast to previous reports, cocaine was not hydrolysed by the human liver CE, hCE1 (CES1), either as highly active recombinant protein or as CEs isolated from human liver or intestinal extracts. These results correlated well with computer-assisted molecular modelling studies that suggested that hydrolysis of cocaine by hCE1 (CES1), would be unlikely to occur. However, cocaine, heroin and CPT-11 were all substrates for the intestinal CE, hiCE (CES2), as determined using both the recombinant protein and the tissue fractions. Again, these data were in agreement with the modelling results.

Conclusions and implications:

These results indicate that the human liver CE is unlikely to play a role in the metabolism of cocaine and that hydrolysis of this substrate by this class of enzymes is via the human intestinal protein hiCE (CES2). In addition, because no enzyme inhibition is observed at high cocaine concentrations, potentially this route of hydrolysis is important in individuals who overdose on this agent.     

A Mechanistic and Kinetic Study of the E-Ring Hydrolysis and Lactonization of a Novel Phosphoryloxymethyl Prodrug of Camptothecin

Purpose. This study was done to determine the E-ring hydrolysis and lactonization mechanism of a water-soluble 20-phosphoryloxymethyl (POM) prodrug of camptothecin (P-CPT). Specifically, the role of the phosphate group in facilitating E-ring hydrolysis was examined. Methods. Resolution between the lactone and carboxylate forms of P-CPT and camptothecin (CPT) was achieved with a RPHPLC assay using UV-visible detection. E-ring P-CPT hydrolysis and lactonization kinetics were followed using 20 mM acetate or phosphate buffer ( = 0.15 NaCl) over the pH range of 4 to 8 at 25.0°C. A kinetic solvent isotope effect (KSIE) study was used to further probe the mechanism of E-ring hydrolysis. Results. The hydrolysis and lactonization reactions followed pseudo-first-order kinetics in the approach to equilibrium. The equilibrium ratio of the open and closed forms of P-CPT was dependent on pH, with the closed form dominant at low pH and the open form dominant at high pH. Buffer concentration changes had little to no effect on the rate of P-CPT E-ring hydrolysis. The KSIE study provided an overall isotope effect of 2.47 and a proton inventory KSIE consistent with an intramolecular general base catalysis. Conclusions. P-CPT has a pH-dependent equilibrium between the lactone and carboxylate forms similar but not identical to that of CPT. The results suggest a hydrolysis reaction mechanism that involves a single site hydrogen exchange facilitated intramolecularly by the dianionic phosphate moiety of P-CPT via either general base catalysis of the lactone ring attack by water or breakdown of the tetrahedral intermediate.     

Amine Prodrugs Which Utilize Hydroxy Amide Lactonization. II. A Potential Esterase-Sensitive Amide Prodrug

In an effort to develop esterase-sensitive pro-prodrugs for amines, an amide derivative of 3-(2-acetoxy-4,6-dimethylphenyl)-3,3- dimethylpropionic acid (4-methoxyaniline amide (8) was synthesized and its stability investigated. This esterifled hydroxy amide was found under all conditions to degrade via a two-step process initiated by acetyl ester hydrolysis generating the hydroxy amide intermediate 9a. The lactonization of this intermediate 9a in the second step resulted in the formation of 4-methoxyaniline (10) and 4,4,5,7-tetramethyl-3,4-dihydrocoumarin (la). The pro-prodrug 8 was observed to possess the following half-lives at 37°C under various conditions: 4030 min in phosphate buffer (50 mM, µ = 0.15) fixed to pH 7.4, 11.9 min in the same buffer containing a porcine liver esterase, 53.7 min in plasma, and 475 min in plasma containing diisopropylfluorophosphate. These results suggest that in a biological milieu the ester hydrolysis will occur by the enzymic hydrolysis rather than the chemical hydrolysis and that the enzymic hydrolysis of 8 in plasma is due, in part, to the action of serine-dependent esterases.     

The rapid hydrolysis of chlordiazepoxide to demoxepam may affect the outcome of chronic osmotic minipump studies

Background

In chronic studies, the classical benzodiazepine chlordiazepoxide (CDP) is often the preferred drug because, unlike other benzodiazepines, it is soluble in water. However, rapid CDP hydrolysis in solution has been described. This would diminish plasma levels in chronic minipump studies and introduce the corelease of active compounds.

Methods

Therefore, the present study aimed to explore the putative hydrolysis of CDP in aqueous solution over time and to identify the hydrolysis products. Moreover, we aimed to characterize the hydrolysis products for their in vitro (³H-flunitrazepam binding and oocyte electrophysiology) and in vivo (stress-induced hyperthermia paradigm) GABA_A receptor potency.

Results

CDP in solution hydrolyzed to the ketone structure demoxepam which was confirmed using mass spectrometry. The hydrolysis was concentration dependent (first-order kinetics) and temperature dependent. CDP exerted greater potency compared to demoxepam in vitro (increased activity at GABA_A receptors containing α₁ subunits) and in vivo (stress-induced hyperthermia), although ³H-flunitrazepam binding was comparable.

Conclusions

The classical benzodiazepine CDP is rapidly hydrolyzed in solution to the active compound demoxepam which possesses a reduced activity at the GABA_A receptor. Chronic studies that use CDP in aqueous solution should thus be interpreted with caution. It is therefore important to consider drug stability in chronic minipump applications.     

The Relationship of Diastereomer Hydrolysis Kinetics to Shelf-Life Predictions for Cefuroxime Axetil

Cefuroxime axetil, an ester prodrug of cefuroxime, is comprised of a 50:50 mixture of diastereomers A and B. The first-order hydrolysis kinetics of cefuroxime axetil were investigated as a function of pH, temperature, buffers, and ionic strength. Chromatographically identified hydrolysis products were cefuroxime, ²-cefuroxime axetil, and ,-sulfoxides. Buffer catalysis was observed in acetate and phosphate buffers. No significant kinetic effect was observed for ionic strength in the range µ = 0.1-1.0. The pH–rate profiles for hydrolysis of cefuroxime axetil isomeric mixture were obtained at 45, 35, and 25°C. The equation defining the cefuroxime axetil hydrolysis rate constant as a function of pH was k _obs = k _H(a _H) + k _s + k _OH(K _w/a _H), exhibiting maximal stability in the pH range 3.5 to 5.5. The predicted profile at 5°C was in excellent agreement with experimental data in the pH range 3.6 to 5.5. In the pH range 1 to 9, the maximum difference observed for individual hydrolysis constants of isomers was 27%. Shelf-life estimates based on the hydrolysis rate constants for cefuroxime axetil as an isomeric mixture were shown to be equivalent to those based on individual hydrolysis rate constants for isomers A and B.     

Investigation of the hydrolysis of ginsenosides by high performance liquid chromatography-electrospray ionization mass spectrometry

The hydrolysis of ginsenoside standards and the crude extracts of ginseng has been investigated at different pH values (2.4-11.2) using high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS). The experimental results indicated that the pH value of aqueous solutions is an important factor in changing the composition of ginsenosides. For (20 S)-protopanaxadiol ginsenosides, ginsenosides with a large mass hydrolyzed to form hydrolysates (20 S)-Rg3 and (20 R)-Rg3 at pH 4.3. There were more hydrolyzed products observed at pH 3.3: (20 S)-F2, C-25,26 hydrated ginsenoside "C-Y1" and "C-Y2" (MW=802 Da) accompanied with (20 S)-Rg3, (20 R)-Rg3. At pH 2.4, only (20 R)-Rg3, (20 S)-F2, a small quantity of (20 S)-Rg3 and three C-25,26 hydrated ginsenosides were obtained. For (20 S)-protopanaxatriol Re, no hydrolysates were observed at pH 4.3; it was hydrolyzed at pH 3.3 to form hydrolysates (20 S)-Rg2, (20 R)-Rg2 and hydrated C-25,26 (MW=802 Da) and at pH 2.4 only C-25,26 hydrated ginsenosides "C-Y1" and "C-Y2" (MW=802 Da) were left in the solution. Similar hydrolysis reactions could be also observed for the crude extracts of ginseng. It showed that HPLC/ESI-MS is a fast and convenient method to study the hydrolysis of ginseng.     

Differential receptor occupancy requirements for muscarinic cholinergic stimulation of inositol lipid hydrolysis in brain and in neuroblastomas

The potency with which carbamoylcholine enhances phosphoinositide (PPI) hydrolysis in different brain regions (neostriatum, cerebral cortex, and hippocampus) and in two neuroblastomas (the murine N1E-115 and human SK-N-SH) differs by 10- to 20-fold. To determine whether the presence of a muscarinic receptor (mAChR) reserve might account for these differences, we have examined the effect of propylbenzilylcholine mustard (PrBCM) on mAChR number and on agonist-stimulated PPI hydrolysis. In the cerebral cortex, in hippocampus, and in N1E-115 cells, PrBCM treatment resulted in a loss of the PPI response, as measured by the release of [3H]inositol phosphates, that was equal to or greater than the reduction in receptor number, as determined by the loss of either [3H]quinuclidinylbenzilate- or [3H]N-methylscopolamine-binding sites. From dose response curves for carbamoylcholine, it was determined that alkylation of mAChRs resulted in a reduction in the maximum release of inositol phosphates but had no effect on agonist potency. The KA values for carbamoylcholine obtained following receptor inactivation were similar to those for the EC50 (120-316 microM). In contrast, in both the neostriatum and SK-N-SH cells, PrBCM treatment resulted in a greater loss of mAChR number than of stimulated inositol phosphate release, and dose response curves for carbamoylcholine were shifted to higher agonist concentrations. The KA values (34-65 microM) were 2- to 9-fold higher than the comparable EC50 values. Moreover, in both tissues the PPI response elicited by partial agonists was more susceptible to receptor alkylation than that elicited by carbamoylcholine. The two groups of tissues also differ in their sensitivity to pirenzepine, which is a markedly weaker antagonist of stimulated PPI hydrolysis in SK-N-SH cells and neostriatum (Ki 160-250 nM), than in the cerebral cortex, hippocampus, and N1E-115 cells (Ki 10-20 nM). These results suggest: 1) that a population of "spare" receptors exists for mAChR-mediated inositol lipid hydrolysis in some neuronal tissues, 2) that both M1 and M2 mAChRs may be coupled to PPI turnover, and 3) that M2 mAChRs appear to be more efficiently coupled to phosphoinositide hydrolysis than their M1 counterparts.     

Purification and Partial Characterization of an Indomethacin Hydrolyzing Enzyme from Pig Liver

Purpose. Indomethacin is well known to be metabolized via O-demethylation and N-deacylation. In this paper we found an enzyme involved in the hydrolysis of amide-linkage of indomethacin and partially characterized it as well as its substrate specificity. Methods. An indomethacin hydrolyzing enzyme was purified to homogeneity from pig liver microsomes using columns of Q-Sepharose, Red-Sepharose and Blue-Sepharose. The enzyme activity was assayed by measuring of -chlorobenzoic acid liberated from indomethacin by HPLC. Results. The purified enzyme effectively hydrolyzed the amide linkage in indomethacin but not those in -naphthylacetate and -nitrophenylacetate, which are typical substrates for carboxylesterase. The subunit molecular mass of the enzyme was 65 kDa according SDS-polyacrylamide gel electrophoresis. The Michaelis constant (Km) and maximum velocity (Vmax) values for indomethacin were 67.8 µM and 9.02 nmol/min/mg protein, respectively. The amino acid sequence analysis of the enzyme after cyanogen bromide cleavage showed high homology with a mouse carboxylesterase isozyme designated as ES-male. The activity of indomethacin hydrolysis was relatively high in the pig, rabbit and human liver homogenate, but not in those from rat and mouse. On the other hand, purified human liver carboxylesterases pl 5.3 and 4.5, and pig liver carboxylesterases have no catalytic activity for indomethacin. Conclusions. These results indicate that the hydrolysis of amide-linkage of indomethacin in humans would be associated with an enzyme similar to the indomethacin hydrolyzing enzyme from pig liver microsomes described here.     

The hydrolysis of acetanilide and some of its derivatives by enzymes in the microsomal and soluble fraction prepared from livers of various species

Summary The velocity of hydrolysis of some acetanilide derivatives by guinea pig liver microsomes was found to be proportional to the microsome content of the suspension.Several extractions of the homogenate prepared from the livers of various species were needed for extracting most of the activity located in the soluble and the microsomal fraction.Acetanilide, p-acetaminophenol, phenacetin, and a ring-substituted phenacetin derivative (compound IX) were used as substrates in measuring the hydrolytic activity in the microsomal and soluble fraction from liver. The ratio between these activities was found to vary widely with the substrate and the species (rabbit, guinea pig, dog, and cat) used. The soluble fractions of all species hydrolyzed acetanilide more rapidly than the microsomes, and the microsomes were more active than the soluble fractions in hydrolyzing the ring-substituted phenacetin derivative.In a study of the hydrolysis of acetanilide and 12 derivatives by the microsomes prepared from the livers of rabbits, dogs, and guinea pigs the replacement of the N-acetyl residue in phenacetin by -dimethylamino--hydroxybutyric acid was found to decrease the rate of hydrolysis. Substitution of an acetyl, propionyl, or butyryl residue in m-position to the amino group of phenacetin increased the rate of hydrolysis.N-methyl phenacetin was hydrolyzed more slowly than phenacetin by the soluble fractions prepared from guinea pig's, rabbit's dog's and cat's liver and by the microsomes except those from guinea pigs. The substitution of an N-methyl group in -dimethylamino--hydroxybutyric acid phenetidide produced a compound which was found not to be hydrolyzed by microsomes or soluble fraction from the livers of the 4 species—or so slowly that it could not be detected in our experiments.     

Serum-catalyzed hydrolysis of metronidazole amino acid esters

Glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), and lysine (Lys) esters of metronidazole were synthesized using dicyclohexylcarbodiimide (DCC) coupling or a mixed-anhydride route, using tert-butyloxycarbonyl (tert-Boc) amino acids. Human serum-catalyzed hydrolysis of these esters at 37 degrees C give half-lives varying from 4.5 min for the Phe ester to 96 h for the Ile ester. Also determined was the pH-rate profile for hydrolysis in aqueous buffers at 25 degrees C. A linear relationship was observed between the logarithmic value of the hydrolysis rate constant in serum and that of the OH- -catalyzed hydrolysis of cationic esters. This finding may indicate that the esters studied are "equally" poor substrates for binding to the enzymes in serum and, thus, the difference observed in the serum-catalyzed hydrolysis rate is solely derived from the chemical lability of an ester bond. Interestingly, the extent of chemical activation observed in the buffer system appears to be amplified in the serum-catalyzed hydrolysis.     

Raising the ambient potassium ion concentration enhances carbachol stimulated phosphoinositide hydrolysis in rat brain hippocampal and cerebral cortical miniprisms

Summary The influence of the ambient potassium ion concentration ([K⁺]_ upon agonist stimulated hydrolysis of phosphoinositides (PI) has been studied in isolated miniprisms of rat hippocampus and cerebral cortex. When the external [K⁺] was raised from 6 to 18 mmol/l, there was little or no increase in the hydrolysis of PI in the absence of agonist, however, carbachol (100 mol/l) stimulated hydrolysis was greatly enhanced in both brain regions studied. Thus, carbachol stimulated the hydrolysis of PI to 146% and 386% of control levels at potassium concentrations of 5.8 and 18.2 mmol/l, respectively, in the rat hippocampus. A similar enhancement of muscarine (100 mol/l) stimulation was observed in cortical miniprisms with 18 mmol/l [K⁺]. A further enhancement was seen at higher ambient [K⁺], although basal hydrolysis of PI was then also increased. The carbachol-stimulated hydrolysis of PI found at both 6 and raised [K⁺] was prevented by atropine (1 and 10 mol/l) and tetraethylammonium (20 mmol/l), but not by 10 mmol/l Mg²⁺. Pirenzepine (50 nmol/l) also reduced this response. The ions Cs⁺ and Rb⁺ (but not Li⁺ or Tris⁺) produced a similar enhancement of the carbachol stimulation to that found with K⁺. At a buffer [K⁺] of 6 mmol/l, noradrenaline (100 mol/l) produced a 2-fold increase in the hydrolysis of PI whereas 5-hydroxytryptamine (100 mol/l) and histamine (500 mol/l) had little or no effect. However, histamine and 5-hydroxytryptamine did stimulate the hydrolysis of PI when [K⁺] was increased. Miniprism ATP content was not changed by a rise in [K⁺] to 18 mmol/l. The significance of these results is discussed in terms of the postsynaptic cellular events following cholinergic stimulation.     

High-performance liquid chromatographic determination of cephacetrile.

A rapid and accurate quantitative determination of cephacetrile in finished bulk and dosage forms is reported. The high-performance liquid chromatographic method is free of interference by acetyl hydrolysis products and synthesis by-products. The assay can be performed in about 15 min, affording less than 0.7% coefficients of variation within and between days. The chromatographic results are in good agreement with the microbiological assay requested by the "Code of Federal Regulations" for certification of cephacetrile sodium.     

Menthol-β-D-Glucuronide: A Potential Prodrug for Treatment of the Irritable Bowel Syndrome

Menthol--D-glucuronide is a potential prodrug for colonic delivery of the spasmolytic agent menthol. Menthol is the primary constituent of peppermint oil, which is used to treat the irritable bowel syndrome. The chemical stability of menthol--D-glucuromde was assessed at various pHs (1.5,4.5, 6.0 and 7.4) over a 4 to 24 h period at 37°C. The prodrug was stable, i.e., there was less than 0.1% hydrolysis of the prodrug, at pHs of 4.5, 6.0 and 7.4. At pH 1.5, the prodrug was about 20% hydrolyzed over a 4 h period suggesting the need for an enteric coating to prevent premature hydrolysis in the stomach. The stability of the prodrug was also assessed in luminal contents of the laboratory rat and in human stool samples. These studies were performed at concentrations designed to assess relative velocities of hydrolysis (i.e., substrate concentrations in excess of the K_m). The prodrug was stable in luminal contents of the rat stomach, proximal small intestine, and the distal small intestine. The rate of hydrolysis of menthol--D-glucuronide was 6.26 ± 2.88 nmol min^–l mg^–l and 2.34 ± 1.22 nmol min^–l mg^–l in luminal contents of the rat cecum and colon, respectively. The hydrolysis rate of menthol--D-glucuronide was lower in human stool samples (0.52 ± 0.46 nmol min^–1 mg^–!). The prodrug had a measured log octanol/buffer partition coefficient of –1.61 suggesting it should be poorly absorbed from the lumen of the gastrointestinal tract. The data support the hypothesis that menthol--D-glucuronide is a candidate for the delivery of menthol to the large intestine under in vivo conditions.     

Pharmacokinetic profile of atenolol aspirinate

We report microwave-assisted synthetic routes, the pharmacokinetic profile along with results from ulcerogenicity and mutagenicity studies of atenolol aspirinate, and an already described derivative, in which acetyl salicylic acid (aspirin) was connected to atenolol by an ester linkage. Atenolol aspirinate was stable towards aqueous hydrolysis but rapidly hydrolyzed in plasma (t(1/2) = 7.6 min). The results showed that the rapid and complete hydrolysis generates atenolol salicylate, which assumes a conformation stabilized by two intramolecular H-bonds, avoiding its further hydrolysis to salicylic acid and atenolol.     

Human serum "A"-esterases. Hydrolysis of O,O-dimethyl-2,2-dichlorovinyl phosphate

Some characteristics of the hydrolysis of O,O-dimethyl-2,2 dichlorovinyl phosphate (DDVP) by human serum are reported and compared with the hydrolysis of O,O-diethyl-4-nitrophenyl phosphate (paraoxon) which is a substrate for Paraoxonase, a known "A"-esterase of human serum. When incubated with human serum, DDVP was losing its inhibitory power toward acetylcholinesterase (AChE). The loss of DDVP followed first order kinetics and was proportional to serum dilution. The disappearance of DDVP after incubation with human serum was not due to protein binding. Apparent Km and Vm for the hydrolysis of DDVP were 7.1 mM and 143 nmol.min-1.ml-1. The pH sensitivity, EDTA inhibitory and Ca2+ requirements of DDVP-ase were similar to those of Paraoxonase. DDVP inhibited the Paraoxonase activity and paraoxon inhibited the DDVP-ase activity. Ca2+, Ag+ and Hg2+ were better inhibitors of the Paraoxonase than the DDVP-ase. The rate of heat inactivation was also different; at 55 degrees Paraoxonase inactivated almost completely within 10 min, while DDVP-ase lost only about 10% activity over 1 hr. Consequently, DDVP-ase and Paraoxonase can be differentiated by means of heat sensitivity. The DDVP-ase was normally distributed in a population of 60 individuals, while Paraoxonase is known to show a marked polymorphism.