Establishment Probability of Anastrepha grandis and Zeugodacus cucurbitae (Diptera: Tephritidae) in Brazilian Semiarid Based on Thermal Requirements.

The quarantine pests, Anastrepha grandis (Macquart) and Zeugodacus cucurbitae (Coquillett), are economically important for the fruit fly-free area in Brazilian semiarid, an area in which they are not yet present. Therefore, the objective of this study was to assess the probability of establishment of A. grandis and Z. cucurbitae based on the estimate number of generations in different climatic regions of the Brazilian semiarid. For scenarios of future projections, it was estimated the number of generations with increase of 1°C (low radiative forcing scenario) and 4°C (high radiative forcing scenario) in the air temperature. Finally, we also estimate the quarantine period to eliminate the invading population of cucurbit fruit flies in Brazilian semiarid. For this, the average historical air temperature of 32 semiarid municipalities was used and the biology data of fruit flies (thermal threshold of development and thermal constant) were used. The fruit flies are able to present several generations per year in Brazilian semiarid. Anastrepha grandis can present from 7.99 (Sergipe) to 9.66 (Piauí) generations. The melon fly Z. cucurbitae may present from 31.25 (Sergipe) to 40.66 (Piauí) generations. The estimation of species multiplication is accentuated in any season, with greater amplitude in spring and summer. The municipalities of Piauí, Ceará, and Rio Grande do Norte presented the highest estimates of fruit fly generations. The increase of air temperature in a future scenario may favor the quarantine pests, A. grandis and Z. cucurbitae, in Brazilian semiarid. In conclusion, the species A. grandis and Z. cucurbitae can be established in Brazilian semiarid, with particular concern for the fruit fly-free area.

Converting the Distinct Heparins Sourced from Bovine or Porcine Mucosa into a Single Anticoagulant Drug.

Unfractionated heparin (UFH) and their low-molecular-weight derivatives are sourced almost exclusively from porcine mucosa (HPI); however, a worldwide introduction of UFH from bovine mucosa (HBI) has been recommended to reinforce the currently unsteady supply chain of heparin products. Although HBI has different chemical composition and about half of the anticoagulant potency of HPI (∼100 and ∼180 international unit [IU]/mg, respectively), they have been employed as interchangeable UFHs in some countries since the 1990s. However, their use as a single drug provoked several bleeding incidents in Brazil, which precipitated the publication of the first monographs exclusive for HBI and HPI by the Brazilian Pharmacopoeia. Nevertheless, we succeed in producing with high-resolution anion-exchange chromatography a novel HBI derivative with anticoagulant potency (200 IU/mg), disaccharide composition (enriched in N,6-disulfated α-glucosamine) and safety profile (bleeding and heparin-induced thrombocytopaenia potentials and protamine neutralization) similar to those seen in the gold standard HPI. Therefore, we show that it is possible to equalize the composition and pharmacological characteristics of these distinct UFHs by employing an easily implementable improvement in the HBI manufacturing.

Chemical and pharmacological aspects of neutralization of heparins from different animal sources by protamine.

Essentials Bovine (HBI) and porcine (HPI) heparins differ in structure and anticoagulant activity. Protamine-neutralization was evaluated on a variety of physical-chemical methods. HBI requires more protamine than HPI to fully neutralize its anticoagulant activity. Protamine preferentially removes higher-sulfated chains of HBI while HPI is evenly precipitated. SUMMARY: Background Protamine neutralization is an essential step for the safe use and inactivation of the unfractionated heparin (UFH) that is widely employed in surgical and non-surgical procedures involving extracorporeal circulation. Objective To compare protamine neutralization of different pharmaceutical-grade UFHs prepared from porcine or bovine intestine (HPI and HBI, respectively). HBI has approximately half the anticoagulant potency of HPI, mostly as consequence of its fraction enriched with N-sulfated α-glucosamine disaccharides. Methods Protamine neutralization of HPI and HBI was evaluated with in vitro, ex vivo and in vivo assays. We also performed in-depth assessments of the complexation of protamine with these distinct UFHs by using nuclear magnetic resonance and mass spectroscopy. Results HPI and HBI interact similarly with protamine on a mass/mass basis; however, HBI requires more protamine than HPI to have its anticoagulant activity fully neutralized, because of its lower potency, which entails the use of higher doses. Nuclear magnetic resonance spectra revealed that HPI precipitates homogeneously with protamine. On the other hand, the low-sulfated fraction of HBI, enriched with N-sulfated α-glucosamine, precipitates at higher concentrations of protamine than the fraction more like HPI, with a preponderance of N,6-disulfated α-glucosamine disaccharides. Finally, mass spectroscopy spectra showed that some of the different peptide components of protamine interact preferentially with the heparins, irrespective of their animal origin. Conclusion Our results have important medical implications, indicating that protamine neutralization of HBI, determined exclusively by point-of-care coagulation assessments, must fail because of its lower-sulfated fraction with reduced anticoagulant activity that could remain in the circulation after the neutralization procedure.

Resolving pathways of interaction of mipafox and a sarin analog with human acetylcholinesterase by kinetics, mass spectrometry and molecular modeling approaches.

The hydroxyl oxygen of the catalytic triad serine in the active center of serine hydrolase acetylcholinesterase (AChE) attacks organophosphorus compounds (OPs) at the phosphorus atom to displace the primary leaving group and to form a covalent bond. Inhibited AChE can be reactivated by cleavage of the Ser-phosphorus bond either spontaneously or through a reaction with nucleophilic agents, such as oximes. At the same time, the inhibited AChE adduct can lose part of the molecule by progressive dealkylation over time in a process called aging. Reactivation of the aged enzyme has not yet been demonstrated. Here, our goal was to study oxime reactivation and aging reactions of human AChE inhibited by mipafox or a sarin analog (Flu-MPs, fluorescent methylphosphonate). Progressive reactivation was observed after Flu-MPs inhibition using oxime 2-PAM. However, no reactivation was observed after mipafox inhibition with 2-PAM or the more potent oximes used. A peptide fingerprinted mass spectrometry (MS) method, which clearly distinguished the peptide with the active serine (active center peptide, ACP) of the human AChE adducted with OPs, was developed by MALDI-TOF and MALDI-TOF/TOF. The ACP was detected with a diethyl-phosphorylated adduct after paraoxon inhibition, and with an isopropylmethyl-phosphonylated and a methyl-phosphonylated adduct after Flu-MPs inhibition and subsequent aging. Nevertheless, nonaged nonreactivated complexes were seen after mipafox inhibition and incubation with oximes, where MS data showed an ACP with an NN diisopropyl phosphoryl adduct. The kinetic experiments showed no reactivation of activity. The computational molecular model analysis of the mipafox-inhibited hAChE plots of energy versus distance between the atoms separated by dealkylation showed a high energy demand, thus little aging probability. However, with Flu-MPs and DFP, where aging was observed in our MS data and in previously published crystal structures, the energy demand calculated in modeling was lower and, consequently, aging appeared as a more likely reaction. We document here direct evidence for a phosphorylated hAChE refractory to oxime reactivation, although we observed no aging.

Silencing of PNPLA6, the neuropathy target esterase (NTE) codifying gene, alters neurodifferentiation of human embryonal carcinoma stem cells (NT2).

Neuropathy target esterase (NTE) is a protein involved in the development of a polyneuropathy caused by exposure to certain organophosphorus compounds. In vivo and in vitro studies have also associated NTE with embryonic development since NTE null mice embryos are non-viable, and silencing the NTE-codifying gene (Pnpla6) in mouse embryonic stem cells strongly alters the differentiation of vascular and nervous systems. In this paper, human embryonal carcinoma stem cells human-derived NTera2/D1 (hNT2) are used as an in vitro neurodifferentiation model to determine whether PNPLA6 silencing is able to alter the differentiation process. In control cultures, PNPLA6 mRNA levels increased in parallel with other neuroectodermal markers during neurodifferentiation. PNPLA6 silencing with specific interference RNA reached a 97% decrease in gene expression 3days after transfection and with a maximum reduction in NTE enzymatic activity (50%), observed on day 4. Silencing PNPLA6 showed an 80% decrease in quantifiable neuronal cells after 13days in vitro (DIV) compared to controls and absence of different neuronal markers after 66DIV. Microarray data analysis of the PNPLA6-silenced cells showed alterations in several developmental processes, mainly neurogenesis and epithelium tube morphogenesis. PNPLA6 silencing also led to a reduction in electrical activity and an altered neuronal phenotype. This work is the first proof supporting the hypothesis that NTE plays a role in human early neurodevelopment using a human cell differentiation model.

Separating esterase targets of organophosphorus compounds in the brain by preparative chromatography.

Low level exposure to organophosphorus esters (OPs) may cause long-term neurological effects and affect specific cognition domains in experimental animals and humans. Action on known targets cannot explain most of these effects by. Soluble carboxylesterases (EC 3.1.1.1) of chicken brain have been kinetically discriminated using paraoxon, mipafox and phenylmethyl sulfonylfluoride as inhibitors and phenyl valerate as a substrate. Three different enzymatic components were discriminated and called Eα, Eß and Eγ. In this work, a fractionation procedure with various steps was developed using protein native separation methods by preparative HPLC. Gel permeation chromatography followed by ion exchange chromatography allowed enriched fractions with different kinetic behaviors. The soluble chicken brain fraction was fractionated, while total esterase activity, proteins and enzymatic components Eα, Eß and Eγ were monitored in each subfraction. After the analysis, 13 fractions were pooled and conserved. Preincubation of the soluble chicken brain fraction of with the organophosphorus mipafox gave rise to a major change in the ion exchange chromatography profile, but not in the molecular exchanged chromatography profile, which suggest that mipafox permanently modifies the ionic properties of numerous proteins.

Stereospecific hydrolysis of a phosphoramidate as a model to understand the role of biotransformation in the neurotoxicity of chiral organophosphorus compounds.

Calcium-dependent and EDTA-resistant hydrolyses of R and S isomers of O-hexyl O-2,5-dicholorophenyl phosphoramidate (HDCP) were observed in serum and subcellular fractions of liver, kidney and brain from hen, rat and rabbit. In serum, the Ca(2+)-dependent hydrolysis was much higher in rabbit than in other species. Liver showed a higher activity than kidney and brain. The S-HDCP isomer was hydrolysed to a higher extent than the other isomer. The fact that this stereospecificity favours the S-isomer is more clearly observed in rabbit serum, and in rat and rabbit liver particulate fractions. In such tissues and species, the EDTA-resistant hydrolysis was not stereospecific. Soluble fractions of rat brain and of hen liver, kidney and brain, showed a lower total activity but with a higher proportion of EDTA-resistant activity and a higher hydrolysis of the R-HDCP isomer. The Ca(2+)-dependent stereoselective biodegradation of S-HDCP is dominant in the most active tissues in rabbit and rat. It can therefore be concluded that S-HDCP would be biodegraded faster than R-HDCP. Furthermore, R-HDCP is the isomer that will remain at a higher proportion to be available for interaction with the target of neurotoxicity.

Comparison of chromaffin cells from several animal sources for their use as an in vitro model to study the mechanism of organophosphorous toxicity.

It had been observed that the chromaffin cells of bovine adrenal medulla contain high levels of neuropathy target esterase (NTE), the esterase whose inhibition and aging is associated with induction of the organophosphorous induced delayed neuropathy. In this study, total esterase and NTE activities, and their inhibition kinetics by OPs are characterized in adrenal medulla of several species in order to find the best source for chromaffin cells. Total esterase activity in membrane fraction of bovine, equine, porcine, ovine and caprine were 6100+/-840, 4200+/-270, 5000+/-120, 28800+/-3000, and 10800+/-2400mU/gtissue, respectively (mean+/-S.D., n=3-4). NTE represented around 70%, 24%, 58%, 10% and 24% of the total esterases in the same tissues, respectively. It was deduced that NTE represents between 69% and 89% of the "B-activity" (activity resistant to 40microM paraoxon) in the membrane fraction of all species. The mipafox I(50) calculated for 30-min inhibition of NTE at 37 degrees Celsius ranged between 7.4 and 12microM. These values are in the range of that for brain NTE in hen (the usual model for testing OP delayed neurotoxicity). Considering that bovine adrenal medulla contains high NTE activity, that it represents a high proportion of total activity, it is easier to dissect than adrenal medulla from equine, caprine or ovine, and is more readily available than species cited previously, and that its inhibitory properties are similar to the classical hen brain model, it is deduced that bovine adrenal medulla is the most appropriate source of chromaffin cells to study OP toxicity, with porcine as the second alternative. The kinetic properties of chromaffin cell cultures from bovine and porcine were in accordance with their properties in homogenate and subcellular fractions, and they displayed an appropriate stability and viability of the primary culture to be used in in vitro toxicological studies for both mechanistic and testing purposes.

Bovine chromaffin cell cultures as model to study organophosporus neurotoxicity.

Based on the high level of phenyl valerate esterase activities, and in particular of neuropathy target esterase (NTE) found in bovine adrenal medulla, chromaffin cells culture have been proposed as an alternative model for the study of organophosphorus neurotoxicity. Organophosphorus-induced polyneuropathy is a syndrome related to the inhibition and further modification by organophosphorus compounds of NTE (a protein that displays phenyl valerate esterase activity resistant to mipafox and sensitive to paraoxon). Total phenyl valerate esterase activities found in homogenate, particulate and soluble fractions of bovine adrenal medulla were 5200+/-35, 5000+/-280 and 1700+/-260 mU/g tissue, respectively. Cultured chromaffin cells displayed a total hydrolysing activity of 41+/-5 mU/10(6) cells. Homogenates of bovine adrenal medulla displayed only about 6% of activity sensitive to paraoxon. Most of the phenyl valerate esterase activity inhibited by mipafox (a neuropathy inducing compound) was found in particulate fraction. Cultured chromaffin cells displayed kinetics of inhibition by mipafox similar to the kinetics displayed by homogenates of bovine adrenal medulla. We conclude that NTE could be assayed in this system by only using one inhibitor (mipafox) instead of two (paraoxon and mipafox). Also, the proposal is supported of using chromaffin cells as in vitro model for the study of the role of NTE and related esterases in organophosphorus-induced polyneuropathy.

Results of the cemented SR trapeziometacarpal prosthesis in the treatment of thumb carpometacarpal osteoarthritis.

PURPOSE: The purpose of this study was to analyze our experience in the treatment of trapeziometacarpal (TMC) osteoarthritis with a cemented surface replacement arthroplasty. METHODS: We did a retrospective study of 19 patients with 20 hands operated on with this technique, with a follow-up evaluation of 33 months. We analyzed the preoperative stage, the postoperative clinical results, measured the radiographic changes found at the end of the study, and correlated all these data with a statistical study. For statistical analysis chi(2) test and Student t-test were used. The level of significance was set at a p value of.05 or less. RESULTS: With an average follow-up period of 33 months (range, 24-45 mo) the following rates were obtained: 55% loosenings and 15% ankylosis owing to periprosthetic calcifications (70% of failures of the technique). Four patients (20%) needed a revision surgery (a salvage procedure with a dynamic ligament reconstruction arthroplasty). In the subjective functional evaluation only 8 cases of 19 patients (40%) maintained an excellent/good result at the end of the study. CONCLUSIONS: In our group of patients the cemented surface replacement TMC prosthesis was not an adequate surgical option for the treatment of TMC osteoarthritis. Further studies are necessary to clarify the role of this surgical technique in this pathology.

Distribution of serum paraoxon hydrolyzing activity in a large Spanish population using a routine automized method in clinical laboratory.

This work was performed to adapt the manual laboratory method of measuring serum paraoxonase activity using a routine automatized method in the clinical laboratory and to study the distribution of paraoxonase activity in a large population from Alcoy, a region of Spain. The serum samples for the study were obtained from extractions of blood from 2891 individuals, distributed by sex and age groups, in a routine check in a primary care facility of Alcoy. Paraoxonase activity was assayed by measuring the release of p-nitrophenol according to a previously published method adapted to an automatized analyzer. The mean paraoxonase activity recorder was 70.2 +/- 16.5 IU/L. Paraoxonase activity in children (both males and females) was significantly lower (p < 0.0005) than in older individuals. Paraoxonase activity detected in males and females older than 56 was slightly lower than that detected in younger individuals, although in this case the difference was not statistically significant. The paraoxonase activity shows higher mean values in females than in males (p < 0.0005). Human paraoxonase activity shows a unimodal distribution pattern in the studied population, which is in contrast with other studies showing bimodal distribution.

[Costs of a low payment maintenance treatment with methadone]. / Estudio de costes de un tratamiento de mantenimiento con metadona de bajo nivel de prestaciones.

BACKGROUND: Little is known about the cost of methadone maintenance treatments in opioid-dependent individuals. PATIENTS AND METHODS: Calculation of cost was achieved by applying an activity-based costing and management method from Conselleria de Sanitat of Catalonia, Spain (1997). RESULTS: The cost of each methadone dose was 76 ptas. First medical visits cost 4,517 ptas. Urine tests cost 760 ptas. Transportation amounted to 18,895 ptas. CONCLUSION: Such a costing method is applicable and reveals the low cost of this methadone maintenance treatment in opioid-dependent subjects.

Dichlorophenyl phosphoramidates as substrates for avian and mammalian liver phosphotriesterases: activity levels, calcium dependence and stereospecificity.

The present study shows the existence of both Ca2+-dependent and EDTA-resistant hydrolysing activities against HDCP and paraoxon in the particulate and soluble fractions of hen, rat and rabbit liver. HDCP was more extensively hydrolysed than paraoxon in both subcellular fractions and each of three individuals of the three animal species under study in spite of wide interindividual variations. However the ratio of HDCP versus paraoxon hydrolysing activity (HDCPase/paraoxonase), although within the same order of magnitude, cannot be considered as constant as it ranges one- to seven-fold between individuals of the same species. Also there is no constant ratio of Ca2+-dependent/EDTA-resistant activities. Rabbit liver showed the highest rates of Ca2+-dependent hydrolysis for both organophosphorus compounds whereas the hen paraoxonase activity was not inhibited by EDTA. The stereospecific hydrolysis of HDCP was mostly a Ca2+-dependent one, the S-HDCP isomer being hydrolysed faster than the R-HDCP one. The suggestion is made that HDCP could be conveniently used to measure PTE activity in the liver.

NTE soluble isoforms: new perspectives for targets of neuropathy inducers and promoters.

Neural carboxylesterases can be discriminated by differential inhibition assays with organophosphorus compounds (OPs), paraoxon (O,O'-diethyl p-nitrophenyl phosphate) and mipafox (N,N'-diisopropyl phosphorodiamidofluoridate) being the ones used to discriminate esterases that should be either irrelevant or candidates as targets of the mechanism of induction of the organophosphorus-induced delayed polyneuropathy (OPIDP). The brain membrane-bound phenyl valerate esterase (PVase) defined by Dr Johnson in 1969 as neuropathy target esterase (NTE) and recently cloned by Dr Glynn and coworkers is termed here as particulate NTE due to its association to the membrane particulate fraction. It is considered as the target of OPIDP and is the activity measured in standard NTE assays and toxicity tests. Following the same operational criteria in the soluble fraction of sciatic nerve a paraoxon-resistant but mipafox-sensitive PVase activity was described and termed as S-NTE, with an apparent lower sensitivity to some inhibitors than particulate NTE. Two isoforms (S-NTE1 and S-NTE2) were subsequently separated by gel filtration chromatography. In a partly purified S-NTE2 preparation polypeptides were identified in western blots by labelling with S9B [1-(saligenin cyclic phospho)-9-biotinyldiaminononane], the same biotinylated OP used to label and isolate particulate NTE, but not with anti-particulate NTE antibodies. From sequential inhibition protocols, inhibitor washing-out and time course inhibition studies it is deduced that reversibility of inhibition is a new factor introducing a higher complexity in the identification of the esterases that could be candidates as targets of the mechanisms of induction and/or promotion of neuropathy. We have evidences that in sciatic nerve soluble fraction a high proportion (about 70%) of the activity that is inhibited by paraoxon in the usual concurrent assay is quickly reactivated after removing paraoxon and it is permanently inhibited by mipafox. Under this improved sequential paraoxon/mipafox inhibition procedure S-NTE represents about 50% of total PVases while in the usual concurrent assay it was only apparently about 1-2%. Moreover with such criteria, S-NTE2 isoform(s) represents about 97-99% of total S-NTE, and S-NTE1 is only a marginal amount probably resulting of a partial solubilization from particulate NTE. Fixed time inhibiton curves with variable mipafox concentration failed to discriminate more than one component. However kinetic behaviour of the time progressive inhibition cannot be explained by a simple model with a single exponential mathematical component, indicating that either the possibility of more than one component or a more complex mechanistic model should be considered. Consequently both particulate NTE and S-NTE assay protocols and their role in induction and promotion of neuropathies will need to be reviewed. Data published by Drs Lotti, Moretto and coworkers suggest that particulate NTE cannot be the target of promotion of axonopathies. The proposal that S-NTE2 could be such a target is suggestive and under collaborative biochemical and toxicological studies.

Peripheral nerve soluble esterases are spontaneously reactivated after inhibition by paraoxon: implications for a new definition of neuropathy target esterase.

Soluble extracts of chicken peripheral nerve contain detectable amounts of phenyl valerate esterase (PVase) activity (about 2000 nmol/min per g of fresh tissue). More than 95% of this activity is inhibited in assays where substrate has been added to a preincubated mixture of tissue with the non-neuropathic organophosphorus compound (OP) paraoxon (O,O'-diethyl p-nitrophenyl phosphate): residual activity includes soluble neuropathy target esterase (S-NTE) which, by definition, is considered resistant to long-term progressive (covalent) inhibition by paraoxon. However we have previously shown that paraoxon strongly interacts with S-NTE so interfering with its sensitivity to other inhibitors. We now show that, surprisingly, removal of paraoxon by ultrafiltration ('P' tissue) in order to avoid such an interference results in the reappearance of about 65% of total original soluble PVase activity which is inhibited in the presence of this OP. Although a purely reversible non-progressive inhibition might be suspected, kinetic analysis data show a time-progressive inhibition which suggests that such PVase(s) covalently bind paraoxon. Also a time-dependent recovery due to spontaneous reactivation of the PVase activity was observed after dilution of the inhibitor. Gel filtration chromatography of 'P' tissue in Sephacryl S-300 shows that the reactivated activity is associated with proteins of about 100-kDa mass which include S-NTE and an, as yet, unknown number of other PVases. The implications of these findings in the definition of NTE in a target tissue for the so-called organophosphorus-induced delayed polyneuropathy (OPIDP) are discussed.

The role of phosphotriesterases in the detoxication of organophosphorus compounds.

The enzymes that hydrolyze organophosphorus compounds are called phosphotriesterases. The presence of phosphotriesterases has been described in a variety of tissues. The physiological role of these enzymes is not known, although a clear correlation exists between the levels of phosphotriesterases and susceptibility of the species to the toxic effects of organophosphorus compounds. Thus, mammals that possess high levels of phosphotriesterases in serum and liver are more tolerant to the toxic effects of these compounds than birds and insects - these being species considered lacking of phosphotriesterases. Because most of these enzymes are not well characterized, they are usually differentiated according to their different patterns of response to activators and/ or inhibitors. Phosphotriesterases usually depend of divalent cations and therefore EDTA usually inhibits them. A peculiar EDTA-resistant phosphotriesterase has been described in serum albumin. The biotechnological and therapeutical applications of phosphotriesterases are currently subject to study.

EDTA-resistant and sensitive phosphotriesterase activities associated with albumin and lipoproteins in rabbit serum.

Phosphotriesterase (PTE) activities in mammalian serum are typically found in the lipoprotein fraction. This PTE requires Ca++ for activity and is consequently inactivated by ethylenediaminetetraacetic acid (EDTA). There is also a little known PTE in mammal serum that is resistant to EDTA inactivation. In this work, the PTE activities for the substrates O-hexyl O-2, 5-dichlorophenyl phosphoramidate (HDCP) and O,O'-diethyl p-nitrophenyl phosphate were purified from rabbit serum by ultracentrifugation, molecular exclusion, and anion exchange chromotography. Rabbit serum produced two PTE activities. One was sensitive and the other was resistant to EDTA inhibition. The EDTA-resistant HDCP hydrolyzing activity and paraoxonase activities of rabbit serum were purified to homogeneity. These activities copurified and were associated to albumin. This EDTA-resistant activity exhibited no stereoselectivity in the hydrolysis of HDCP. The EDTA-sensitive activity was isolated in the lipoprotein fraction and stereoselectively hydrolyzed the S-HDCP over the R-HDCP. Other differences between the EDTA-sensitive paraoxonase and HDCP hydrolyzing activity were discovered in response to p-nitrophenylbutkyrate, 5,5-dithio-bis(2-nitrobenzoic acid), caprylic acid, sodium ions, and ammonium ions. This work demonstrates the existence of two well differentiated PTE activities in rabbit serum. One is sensitive to EDTA, stereoselective, and found in the lipoprotein fraction, and the other is resistant to EDTA inhibition and nonstereospecific.

Enzyme concentration as an important factor in the in vitro testing of the stereospecificity of the enzymatic hydrolysis of organophosphorus compounds.

A report is made of important differences in the Ca(2+)-dependent hydrolysis of the chiral phosphoramidate O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) when recorded using different quantities of hen liver microsomes. In a colorimetric microassay using the microsomes from 5mg tissue in the presence of HDCP stereoisomers and 2.5mM calcium, the R-HDCP isomer was hydrolysed at a rate similar to or slightly faster than S-HDCP isomer (14% v. 11%), while the S-HDCP stereoisomer was hydrolysed faster than R-HDCP (17% v. 25% and 21% v. 43%) when HDCP isomers hydrolysis was assayed in the presence of the microsomes from 10 or 20mg, respectively. This stereospecific hydrolysis was verified assaying racemic HDCP and quantities of liver microsomes from 10 to 80mg of tissue, using a chiral chromatographic method; thus, the increase in the ratio of remaining R-HDCP/S-HDCP was dependent on the amount of liver microsomes (range one- to threefold). This study demonstrates that the concentration of the subcellular fraction in in vitro assays is a critical factor to be taken into account in securing a more realistic approximation to the stereospecific enzymatic processes occurring in biological systems. Our data concerning the hydrolysis of HDCP by liver microsomes at high enzyme concentrations afford a better fit to the in vivo toxicological response with HDCP than assays performed with the most commonly used highly diluted preparations.

Chicken serum albumin hydrolyzes dichlorophenyl phosphoramidates by a mechanism based on transient phosphorylation.

The hydrolyzing activities of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) and p-nitrophenyl butyrate (p-NPB) in chicken serum had been found to copurify in the same protein, identified as albumin. The hydrolyzing activities of both chicken serum and commercial serum albumins from different species were inhibited in a dose-dependent manner by short chain fatty acids. On simultaneous incubation of chicken serum with HDCP and p-NPB, a competitive interaction was detected between the two substrates. This behavior suggests that both are hydrolyzed in the same albumin active site. When chicken serum was preincubated with one of the substrates, and the latter were withdrawn by large dilution, the hydrolyzing activities with both substrates were found to be reduced. This reduction was in turn dependent upon the time of preincubation with the first substrate. These results suggest that HDCP and p-NPB are hydrolyzed by the same albumin active site, via a mechanism based on transient phosphorylation/acylation of the active site. The proposed hydrolysis mechanism would account for the hydrolytic kinetics of both substrates.

Phosphotriesterase activity identified in purified serum albumins.

The phosphotriesterase in chicken serum that hydrolyses O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) was purified in three chromatographic steps. The activity copurified to apparent homogeneity with albumin monitoring by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS/ PAGE) and by SDS-capillary electrophoresis in the purified fractions. Commercial chicken serum albumin was further purified and the phosphotriesterase activity remained associated with albumin. Capillary electrophoresis established a molecular weight of 59 +/- 4 kDa for both purified proteins (chicken serum and commercial chicken serum albumin). The purified samples were assayed for hydrolytic activity against several carboxylesters, organophosphates and phosphoramidates. From carboxylesters, only p-nitrophenylbutyrate (p-NPB) hydrolysing activity was found to copurify with the phosphotriesterase. The purified human, chicken, rabbit and bovine serum albumins and recombinant human serum albumin obtained from commercial sources hydrolysed HDCP and p-NPB. Serum albumin also hydrolysed O-butyl O-2,5-dichlorophenyl phosphoramidate, O-ethyl O-2,5-dichlorophenyl phosphoramidate and O-2,5-dichlorophenyl ethylphosphonoamidate but not other organophosphates and phosphoramidates.