[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.

Partial left ventriculectomy (Batista procedure) in the treatment of dilated cardiomyopathy: Makati Medical Center Philippine experience.

In the Philippines patients with end-stage heart disease refractory to conventional medical and surgical treatment do not have alternative choices. More than 99% of the population cannot afford cardiac transplantation. Partial left ventriculectomy (PLV) is a surgical procedure that improves cardiac function and refractory congestive heart failure (CHF). Between October 1997 and February 1998 eight patients had PLV at the Makati Medical Center, Philippines. All patients had end-stage dilated cardiomyopathy. Six patients had an idiopathic etiology, one was ischemic and one valvular. Seven of eight operations were done with the heart beating and all had transesophageal echo monitoring. An average of a 2-cm reduction in the left ventricle diameter was achieved and ejection fraction improved in all cases. There were no operative deaths. There were three late deaths. Two patients died of refractory CHF and ventricular arrhythmias and one patient died of massive cerebral hemorrhage with coumadin therapy. The five survivors are all doing well with no CHF. Follow-up two-dimensional echo shows stable left ventricular (LV) size and improved ejection fraction. Our initial experience shows that PLV, at least in the short-term, has beneficial effects in the treatment of end-stage dilated cardiomyopathy and might become an alternative to cardiac transplantation.

Discrimination of carboxylesterases of chicken neural tissue by inhibition with a neuropathic, non-neuropathic organophosphorus compounds and neuropathy promoter.

Carboxylesterases are enzymes present in neural and other tissues that are sensitive to organophosphorus compounds. The esterase activity in particulate forms, resistant to paraoxon and sensitive to mipafox have been implicated in the initiation of organophosphorus-induced delayed polyneuropathy (OPIDP) and is called neuropathy target esterase (P-NTE). Certain esterases inhibitors such as phenylmethylsulfonyl fluoride (PMSF), can also irreversibly inhibit P-NTE and by this mechanism PMSF 'protects' from further effect of neuropathic OPs. However, if PMSF is dosed after a low non-neuropathic dose of a neuropathic OP, its neurotoxicity is 'promoted', causing severe neuropathy. The molecular target of promotion has not yet been identified and it has been shown that it is unlikely to be the P-NTE. In order to discriminate the different esterases, we used non-neuropathic (paraoxon), and neuropathic organophosphorus compounds (mipafox, DFP) and a neuropathy promoter (PMSF). They were used alone or in concurrent inhibition to study particulate and soluble fractions of brain, spinal cord and sciatic nerve of chicken. From the experimental data, a matrix was constructed and equations deduced to estimate the proportions of the different potential activity fractions that can be discriminated by their sensitivity to the tested inhibitors. It was deduced that only combinations of up to three inhibitors can be used for the analysis with consistent results. In all tissues, inside the paraoxon sensitive activity, most of the activity was sensitive either to mipafox, to PMSF or both. In all fractions, except brain soluble fractions, within the paraoxon resistant activity, a mipafox sensitive component was detected that is operationally considered NTE (P-NTE and S-NTE in particulate and soluble fractions, respectively). Most of this activity was also sensitive to PMSF, and this should be considered the target of organophosphorus inducing neuropathy and of PMSF protective effect. Either in brain and spinal cord, a significant amount of the activity resistant to 40 microM paraoxon and 250 microM mipafox (usually called 'C' activity) is sensitive to PMSF. It could be a good candidate to contain the target of the promotion effect of PMSF as well as the S-NTE activity that is also PMSF sensitive.

Reversible inhibition can profoundly mislead studies on progressive inhibition of enzymes: the interaction of paraoxon with soluble neuropathy target esterase.

Neuropathy target esterase (NTE) is suggested to be the molecular target for the initiation of the organophosphorus induced delayed polyneuropathy (OPIDP). O,O'-diethyl p-nitrophenyl phosphate (paraoxon) was the non-neurotoxic OP of choice for the standard assay of NTE to block the non-relevant esterases (phenylvalerate hydrolases) because it was supposed not to inhibit the enzymic activity of the target protein while N,N'-diisopropyl phosphorodiamidofluoridate (mipafox) is the neuropathic OP used to inhibit (and so to detect) NTE activity. A soluble form of NTE (S-NTE) had previously been described in peripheral nerve which showed a different inhibitor response from that of the particulate NTE (P-NTE). The use of a sequential type of inhibition protocol revealed the presence of an activity component within S-NTE which was extremely sensitive to different esterase inhibitors. Such a soluble activity component remained hidden under the usual concurrent inhibition procedure with paraoxon and was about one order of magnitude more sensitive than P-NTE to the inhibitors studied in the present article. Our results suggest that paraoxon could produce a strong reversible effect on S-NTE when the concurrent procedure is used so that it interferes with its inhibition by both neuropathy inducers and promoters. As a result S-NTE seems to be much more sensitive, than previously believed, to several esterase inhibitors involved in either the genesis of delayed polyneuropathy and/or axonopathy promotion.

Separation of two forms of neuropathy target esterase in the soluble fraction of the hen sciatic nerve.

Neuropathy target esterase (NTE) activity is operatively defined in this paper as the phenyl valerate esterase activity resistant to 40 microM paraoxon but sensitive to 250 microM mipafox. Molecular exclusion column chromatography with Sephacryl S-300 of the soluble (S) fraction from chick sciatic nerve demonstrated two NTE activity peaks. The first eluted with the front, thus indicating a mol. wt. of over 700 kDa (peak Vo), while the second peak eluted with kd = 0.36, suggesting a mol. wt. of about 100 kDa. The curve of total phenyl valerate (PVase) activity inhibition with paraoxon (0.19-200 microM) shows that at a concentration of 40 microM the esterases highly sensitive to paraoxon are inhibited in the Vo and 100-kDa peaks. The NTE activity in these two peaks in turn represented 31% and 44% of the 40 microM paraoxon resistant activity, respectively. The mipafox inhibition curves (1.0-250 microM) revealed different sensitivities to mipafox, with I50 values (t = 30 min) of approximately 1.47 and 63 microM, for Vo and 100-kDa peaks respectively. Mipafox sensitivity of the Vo and 100-kDa peaks correlates with the two components, that had been deduced from the kinetic properties of the S-fraction.

Organophosphorus inhibition and heat inactivation kinetics of particulate and soluble forms of peripheral nerve neuropathy target esterase.

Neuropathy target esterase (NTE) is the proposed target site for the mechanism of initiation of the so-called organophosphorus-induced delayed polyneuropathy (OPIDP). NTE is operationally defined in this article as the phenylvalerate esterase activity which is resistant to inhibition by 40 microM paraoxon and sensitive to 250 microM mipafox. Soluble (S-NTE) and particulate (P-NTE) forms of NTE had first been identified in hen sciatic nerve [E. Vilanova, J. Barril, V. Carrera, and M. C. Pellín (1990). J. Neurochem., 55, 1258-1265]. P-NTE and S-NTE showed different sensitivities to the inhibition by several organophosphorus compounds over a range of inhibitor concentrations for a 30 or 120 minute fixed inhibition time at 37 degrees C. S-NTE was less sensitive to the inhibition by O,O'-diisopropyl phosphorofluoridate (DFP), hexyl 2,5-dichlorophenyl phosphoramidate (H-DCP), and mipafox than P-NTE and brain NTE, while the opposite was true for O,S-dimethyl phosphoroamidothioate (methamidophos). For each of the four inhibitors assayed, S-NTE showed two components of different sensitivity according to the inhibition curves fitted with exponential models. However, the inhibition of P-NTE by mipafox, DFP, and HDCP did not show the presence of a considerable proportion of a second component. The kinetics of heat inactivation showed that P-NTE inactivated faster and to a greater extent than S-NTE. It is concluded that (1) sciatic nerve S-NTE is more different from brain NTE than P-NTE; (2) P-NTE and S-NTE have different sensitivities to the inhibition by the studied organophosphorous compounds; (3) the inhibition curves suggest that S-NTE has two different enzymatic components while these are not so evident for P-NTE.

Cloning and sequencing of the Lactococcus lactis subsp. lactis dnaK gene using a PCR-based approach.

The coding region for the dnaK gene from Lactococcus lactis subsp. lactis LM0230 was isolated and sequenced. An internal 789-bp fragment was amplified by the polymerase chain reaction (PCR) using a pair of degenerate oligodeoxyribonucleotide primers designed on the basis of amino acid (aa) sequences conserved in a number of DnaK. This PCR product was cloned, sequenced and used as a Southern hybridization probe to locate the flanking regions of the gene. The sequence of this central region from dnaK was also used to design two sets of inverse PCR primers to amplify, separately, the upstream and downstream regions. The inverse PCR products were then cloned and partially sequenced. The complete nucleotide sequence was obtained from overlapping cloned fragments of the gene and found to consist of a single 1824-bp open reading frame coding for a 602-aa protein. Alignment of the deduced aa sequence with those of other bacterial DnaK showed a high degree of homology and is most similar to the Bacillus megaterium DnaK.

Non-calcium dependent activity hydrolysing organophosphorus compounds in hen plasma.

O-Hexyl O-2,5, dichlorophenyl phosphoramidate (HDCP) is a chiral compound that induces delayed neuropathy in hens. The chicken has very low activity of Ca-dependent organophosphorus-hydrolases (OP-hydrolases) such as paraoxonase. HDCP is degraded at a similar rate in rat and hen plasma (16 and 21 nmol/min/microliters plasma, respectively) when measured by the loss of its anti-cholinesterase potency (Díaz-Alejo et al., 1990). The time course of the HDCP hydrolysis was not significantly affected by the following treatments: (a) 0.5-1 mM Ca2+ or 1-10 mM EDTA added at 30 min before starting the reaction at 37 degrees C; (b) preincubation with a carboxylesterase inhibitor 100 microM diisopropyl phosphorosfluoridated (DFP) for 60 min at 37 degrees C; (c) preincubation with 100 microM HDCP for 60 min at 37 degrees C; and (d) the presence of 50 microM DCP. However, the hydrolysis of HDCP was slightly modified by the other product of its hydrolysis. There is no contribution to the HDCP hydrolysis by covalent binding to carboxylesterase proteins. The course of the hydrolysis of HDCP was similar when measured by either the loss of anti-cholinesterase potency or the DCP liberated. HDCP is hydrolysed by an OP-hydrolase which is not Ca-dependent and is present in hen in contrast to the best known OP-hydrolases which are Ca-dependent and are undetectable in birds.

Biochemical properties and possible toxicological significance of various forms of NTE.

NTE (neuropathy target esterase) is considered to be the target for organophosphorus-induced delayed polyneuropathy and is operationally measured by radiolabelling or by determining its esteratic activity as the paraoxon-resistant mipafox-sensitive phosphorylable site(s). From electrophoresis and density gradient centrifugation using radiolabelling techniques, several phosphorylable sites have been described in hen brain that are paraoxon-resistant mipafox-sensitive; however, only the majority electrophoresis band (155 kDa) shows properties related with the aging reaction. Kinetic criteria have also suggested two components of brain NTE (NTEA and NTEB). Most brain NTE is recovered in the particulate microsomal fraction and only about 1% in soluble fraction. In sciatic nerve about 50%/50% activity is recovered as soluble (S-NTE) or particulate (P-NTE) forms. A similar distribution were observed in hen, cat, rat and young chick. The fixed time inhibition curves show that P-NTE is more sensitive to mipafox, DFP and hexyl-DCP than S-NTE, while the reverse is true for methamidophos. P-NTE fits properly to one sensitive component while S-NTE fits better to two sensitive component models, except in the case of methamidophos. In vivo, significant differences in the inhibition of P- and S-NTE by mipafox were found only when using low non-neuropathic dosing. The possible significance of different NTE forms are discussed.

Properties of partly preinhibited hen brain neuropathy target esterase.

NTE inhibitors cause different toxicological consequences (protection, induction or potentiation/promotion of neuropathy) depending on the order of dosing. These effects might be explained in terms of several phosphorylable sites with 'allosteric irreversible' behaviour. Brain neuropathy target esterase (NTE) has been preinhibited with phenylmethylsulphonyl fluoride (PMSF) (0, 5, 10, 15, 30 and 60 microM) or with diisopropylphoshoro fluoridate (DFP) (0, 0.2, 0.5, and 1 microM) at 37 degrees C for 30 min. After washing by centrifugation, tissues were then reinhibited with a range of PMSF (0 to 80 microM) or DFP (0 to 1 microM) concentrations. The slopes of the inhibition curves (log % activity vs. concentration) of pretreated tissues were identical to those of the non-pretreated tissues, with non-distinguishable I50 values. It is concluded that allosteric effects are not likely to be involved in membrane-bound NTE of hen brain.

Local application of neuropathic organophosphorus compounds to hen sciatic nerve: inhibition of neuropathy target esterase and peripheral neurological impairments.

Diisopropyl phosphorofluoridate (DFP), mipafox, cresylsaligenyl phosphate, and phenylsaligenyl phosphate were applied to a 1.5-cm segment of the common trunk of the sciatic nerve in adult hens. At doses of 18-182 micrograms mipafox and 9-110 micrograms DFP, inhibition of neuropathy target esterase (NTE) for the treated segment was over 80%, whereas for the adjacent distal and proximal segments inhibition was under 40%, 15 min after application. NTE was not affected in the peripheral distal terminations arising from the common sciatic nerve (peroneal branches), contralateral sciatic nerve, brain, and spinal cord. A 24-hr study suggested a displacement of the activity-free region toward more distal segments of the nerve. All animals treated with 55 and 110 micrograms DFP or 110 micrograms mipafox lost a characteristic avian retraction reflex in the treated leg 9-15 days after dosing, suggesting peripheral neurological alterations. Only hens dosed at the maximum dose in both extremities presented alterations in motility (Grade 1 or 2 on a 0-8 scale), suggesting no significant central nervous system alterations. Electron microscopy of peroneal branches showed axon swelling and accumulation of smooth endoplasmic reticulum similar to animals dosed systemically (s.c.) with 1-2 mg/kg DFP. The branches also contained granular and electron-dense materials, as well as some intraaxonal and intramyelinic vacuolization. Clinical effects were not observed in animals protected with a 30 mg/kg (s.c.) dose of phenylmethanesulphonyl fluoride. It is concluded that the peripheral neurological effects of local dosing correlate with the specific modification of NTE in a segment of sciatic nerve and that the axon is a more likely target than the perikaryon or nerve terminal in the triggering mechanism of this axonopathy.

Soluble and particulate forms of the organophosphorus neuropathy target esterase in hen sciatic nerve.

Neuropathy target esterase (NTE) is the suggested "target" molecule involved in the initiation of organophosphorus-induced delayed polyneuropathy. Sciatic nerve NTE was separated into particulate (P-NTE) and soluble (S-NTE) fractions by ultracentrifugation at 100,000 g for 1 h in 0.32 M sucrose and compared with the corresponding brain extract. Total sciatic NTE activity was 80-100 nmol/min/g tissue from which 50-60% was recovered in the soluble supernatant fraction and the remaining 40-50% in the pellet fraction. About 90% of brain tissue activity (approximately 1,800 nmol/min/g tissue) was recovered as P-NTE. A similar distribution was obtained when more drastic centrifugation without sucrose was performed. P-NTE and S-NTE were distributed with the membrane and cytosolic markers assayed, respectively, glucose-6-phosphatase, Na+,K(+)-ATPase, 5'-nucleotidase, phospholipids, and lactate dehydrogenase. When the pH during the centrifugation was increased from 6.4 to 11, recovered P-NTE activity decreased from 1,750 to 118 nmol/min/g tissue for brain and from 31 to 12 nmol/min/g for sciatic nerve. However, S-NTE activity and total nonfractionated control activity were only slightly affected by the same pH treatment. The distribution pattern encountered may be better understood as representing two different proteins than an equilibrium between soluble and membrane-bound portions of a single protein, with P-NTE activity depending on a membrane factor from which it is separated through fractionation at high pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Sciatic nerve neuropathy target esterase. Methods of assay, proximo-distal distribution and regeneration.

Some organophosphorus compounds (OP) induce a delayed polyneuropathy (OPIDP) which is initiated by the phosphorylation of the so-called neuropathy target esterase (NTE). In this work some aspects of hen sciatic nerve NTE are studied. The assay method is reported and modifications are discussed and a combined method proposed. Proximo-distal distribution showed a significant difference from proximal (100 +/- 10%) to distal (69 +/- 9%) fragments, in accordance with reported data. The time course of in vivo regeneration after a single TOCP dose (200 mg/kg, post oral) showed some differences when compared with hen brain NTE. Sciatic nerve NTE showed a delay of 2-3 days before regeneration but then regenerated faster (74% activity at day 7) than brain NTE (50% activity at day 7). A slower rate of regeneration of distal than proximal segments has been suggested to explain higher sensitivity of distal segments [3], however in this work no significant differences were observed in the rate of regeneration when comparing proximal and distal fragments.

Low non-neuropathic tri-o-cresyl phosphate (TOCP) doses inhibit neuropathy target esterase near the neuropathic threshold in n-hexane pretreated hens.

Simultaneous intoxication with hexacarbon solvents and organophosphorus compounds has been considered a possible critical factor in some occupational neuropathies and their interactions proved to cause potentiation effects in hens [1-3]. A high degree of inhibition of neuropathy target esterase (NTE) is needed to develop organophosphorus induced polyneuropathy (OPIDP). In this work, the inhibition of NTE, BuChE and AChE by TOCP on control and n-hexane pretreated (7-15 days, 300 mg/kg per day) hens is studied. Using a single TOCP dose of 200 mg/kg, n-hexane pretreated hens showed synergistic effects, but no significant differences were observed in the inhibition of cholinesterases and NTE in brain or spinal cord. With lower TOCP dose (20 mg/kg) statistically significant differences were observed, which were not drastic but could be important because they involved an increase of inhibition up to critical threshold values (from 40-50% to 60-70% inhibition). However, no clinical effects were observed in these animals. Possible mechanisms of neurotoxic interaction are discussed.