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ABSTRACT: A simple, rapid and sensitive method has been developed and validated for the determination of cocaine and its main metabolites (benzoylecgonine and cocaethylene) in human hair. The method involved solid-phase extraction with an Oasis HLB extraction cartridge and subsequent analysis by GC/MS. The limit of detection was 0.01 ng mg(-1) for cocaine, 0.04 for benzoylecgonine and 0.03 for cocaethylene. The method validation included linearity (with a correlation coefficient >0.99 over the range 0.2-50 ng mg(-1) ), intra- and inter-day precision (always lower than 12%) and accuracy (mean relative error always below 17%) to meet the bioanalytical acceptance criteria. The procedure was further applied to 40 hair samples from self-reported cocaine users arrested by the police who provided a positive urine-analysis for cocaine, and was demonstrated to be suitable for its application in forensic toxicology. New approaches were raised to detect false-negative results that allow a better interpretation of hair testing results. Copyright © 2012 John Wiley & Sons, Ltd.
Journal of Applied Toxicology 03/2012; · 2.48 Impact Factor
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47th Congress of the European Societies of Toxicology (EUROTOX), París, Francia; 01/2011
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ABSTRACT: For biological monitoring of heavy metal exposure in occupational toxicology, usually whole blood and urine samples are the most widely used and accepted matrix to assess internal xenobiotic exposure. Hair samples and saliva are also of interest in occupational and environmental health surveys but procedures for the determination of metals in saliva and hair are very scarce and to our knowledge there is no validation of a method to quantify Cr, Cd, Mn, Ni and Pb in four different human biological materials (whole blood, urine, saliva and axilary hair) by electrothermal atomization atomic absorption spectrometry (ETAAS). In the present study, quantification methods for the determination of Cr, Cd, Mn, Ni and Pb in whole blood, urine, saliva and axilary hair were validated according to the EU common standards. Pyrolisis and atomization temperatures have been determined. The main parameters evaluated were: detection and quantification limits, linearity range, repeatability, reproducibility, recovery and uncertainty. Accuracy of the methods was tested with the whole blood, urine and hair certified reference materials and recoveries of the spiked samples were acceptable ranged from 96.3 to 107.8%.
Analytica chimica acta 02/2010; 659(1-2):60-7. · 4.31 Impact Factor
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ABSTRACT: To determine the incidence of the detection of abuse drugs in severe trauma patients
Prospective observational study conducted from July 2004 to January 2006.
Neurotrauma intensive care unit of a reference tertiary university hospital.
Trauma patients who require admission to ICU during the study period.
Determination of alcohol in blood and of toxics (cocaine, cannabis, amphetamines and other substances) in urine.
Toxicological analysis was performed in 196 of the 288 severe trauma patients admitted during the study period. The most frequently detected cause of the trauma was traffic accident (69%). The most frequently detected substance was cannabis (22.4%), followed by alcohol (17.3%) and cocaine (12.8%). Cannabis was detected in 26.1% of under-45-yr-old patients versus 9.3% of older patients (p < 0.05), and cocaine in 16.3% vs. 0% in over-45-yr-olds (p < 0.001). Some substance of abuse was detected in 45% of under-45-yr-olds versus 23% of older patients (p < 0.05).
The high proportion of positive results to toxic substances in severely traumatized patients suggests that the epidemiological environment for these patients is of great concern. These data may be of interest for the design of future prevention campaign.
Medicina Intensiva 07/2008; 32(5):222-6. · 1.07 Impact Factor
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ABSTRACT: Paraoxonase-1 (PON1) is known to play an important role in the individual susceptibility to environmental chemicals, particularly
pesticides. The major results of our studies on biochemical and clinical end-points of workers long-term exposed to pesticides
in a large intensive agriculture area from Southeast Spain are presented herein and compared with several other epidemiologic
studies performed in different scenarios. In addition of being an individual marker of susceptibility, PON1 can be also considered
a biological indicator of exposure to pesticides, since workers spraying these agents (chiefly OPs) showed decreased enzyme
levels. Besides, long-term exposure to pesticides appears to indirectly elicit higher levels of PON1, which might be regarded
as enzyme induction. On the other hand, carriers of the PON1 192R allele showed lower levels of erythrocyte cholinesterase
and catalase, but a higher glutathione reductase activity. Regarding clinical outcomes, workers with the PON1 R allele had
less risk of reporting a previous episode of pesticide poisoning as well as a lower risk of pesticide-related symptomatology.
Exposure to low doses of pesticides which are metabolically activated in the liver seems to elicit subtle and early biochemical
changes of hepatotoxicity. It is concluded that epidemiological studies addressing health or biochemical outcomes of workers
occupationally exposed to pesticides should determine PON1 genotypes and phenotypes (activities), as these biomarkers may
help in identifying those individuals at increased risk of developing pesticide toxicity or who are showing early effects
after pesticide exposure
12/2007: pages 221-237;
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ABSTRACT: The effect of several metal ions and calcium on purified paraoxonases (PON1 and PON3) from rat liver was studied. PON1 and PON3 were also inhibited by EDTA and both enzyme activities were restored by the addition of free calcium. The reactivation by calcium was a time-dependent effect for PON1; however, this was not the case for PON3. We also studied the response of PON1 and PON3 to several inhibitors: Co, Cu, Mn, Hg and p-hydroxymercurybenzoate (pOHMB), and determined the type of inhibition and the inhibition constants. Among all the compounds tested, mercurials (Hg and pOHMB) were the most potent inhibitors of PON1. For PON3 mercurials and copper showed the highest inhibitory potency. Purified PON3 also showed different inhibition patterns as compared to PON1. A comparison of PON1 and PON3 shows qualitative and quantitative differences in the sensitivity against the inhibitors tested, showing major differences in the case of cobalt, copper and pOHMB, which may be related to structural differences of both PONs. These results increase our knowledge of the biochemical properties of PON1 and PON3 and may help in the understanding of their physiological role as a potential detoxification mechanism against environmental metal ions.
Chemico-Biological Interactions 05/2007; 167(1):63-70. · 2.46 Impact Factor
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ABSTRACT: Studies on the localization of paraoxonases (PON's) are of interest because of its involvement in both the detoxication of activated organophosphorus pesticides and in the prevention of peroxidative damage to phospholipids and cholesteryl-esters in LDL and HDL particles and cell membranes during the atherogenic process. In the present study, we have investigated the cellular localization of PON1 by immunohistochemistry in different rat tissues. The protein was mainly detected in the endothelial lining of every tissue studied (liver, kidney, lung and brain). Besides, it was found in hepatocytes from the centrolobular region of the liver, in the glomeruli and basal pole of the proximal convoluted tubule of the kidney, in cells from bronchiolar epithelium and type I pneumocytes of the lung, and in leptomeningeal cells, ependymal cells and ventricular side of choroid plexus cells of the brain. However, neurons and glia lacked immunostaining. After 3-methylcholanthrene induction an increase in the intensity of immunostaining was observed in the same areas, as well as an additional staining in midzonal hepatocytes. On the basis of the tissue distribution observed for PON1, it is proposed that this enzyme might have a function related to the inactivation of oxidative stress by-products (either at a cellular level or blood-vessel wall) and other environmental chemicals. At present it has not yet been established whether the paraoxonase detected in the various tissues is truly a product of the PON1 gene or could represent products of the PON2 or PON3 genes.
Chemico-Biological Interactions 09/2001; 137(2):123-37. · 2.46 Impact Factor
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ABSTRACT: The existence of two or more enzyme forms with paraoxonase activity has been reported in sheep, rabbit, human and rat serum and recently in mouse and rat liver. In this study we describe the presence of two peaks with paraoxonase activity (M1 and M2) after non-specific affinity chromatography of rat liver microsomes on Cibacron Blue 3GA. The first peak (M1) was obtained during the washing of the column and coeluted with albumin. The second active peak (M2) was eluted with 1 M NaCl. The characterization of each peak was determined by SDS/PAGE electrophoresis and Western-blotting. A comparison of both active fractions on the basis of kinetic parameters, heat inactivation and pH stability, calcium requirement and inhibition by EDTA and several metals was performed. Our results support the fact that two proteins capable of hydrolyzing paraoxon are present in rat liver microsomes. Furthermore, during the purification to homogeneity of rat liver paraoxonase we have performed a study of its hydrolytic ability against three different substrates: paraoxon, phenylacetate and phenyl thioacetate (Paraoxonase (PON), Arylesterase (ArE), Phenyl thioacetate esterase (PTase)). The elution profile in different chromatographic steps, as well as the activity ratios from the crude extract throughout the purification process, heat inactivation and effect of inhibitors were used as identity criteria for the three hydrolytic activities. Our results show evidence for the hydrolysis of paraoxon and phenylacetate by the same protein from rat liver (paraoxonase).
Chemico-Biological Interactions 06/1999; 119-120:263-75. · 2.46 Impact Factor
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ABSTRACT: The paraoxonase/arylesterase phenotype was measured in a Spanish population as previous studies have reported that the polymorphic variation in serum paraoxonase activity may affect the metabolism of organophosphates in individuals at risk of chronic intoxication. The prevalence of congenital deficiency in serum cholinesterase was also established in order to ascertain whether individuals with a congenital defect would be at a higher risk against a potential organophosphate exposure. We consider it useful to incorporate these two biomarkers into the health programme of agricultural workers with the purpose of monitoring workers who spray organophosphate pesticides, as they provide reliable indications of early-stage effects related to biochemical alterations that might precede overt clinical pictures.
Chemico-Biological Interactions 06/1999; 119-120:201-9. · 2.46 Impact Factor
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ABSTRACT: A 32-y-old woman was admitted to Granada University Hospital for attempted suicide by ingestion of an ant-killer containing 10% sodium arsenate and 5% pyrethrins. Neither gastrointestinal distress nor hepatic, renal, or neurologic disturbances were clinically observed. However, the presence of toxic levels of arsenic (14 mg/L) was confirmed by atomic absorption spectrophotometry in a sample of urine taken about 12 h after poisoning. An uneventful clinical course was observed, and the patient was discharged after 6 days upon her request. Long-term follow-up was unavailable. From a Medline search over the years 1985-1998 only one similar report also dealing with sodium arsenate was found. Different pathogenic hypotheses are discussed in the light of the clinical data.
Veterinary and human toxicology 01/1999; 40(6):344-5.
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ABSTRACT: CASE REPORT: An 18-year-old female who accidentally ingested strychnine developed chemical pancreatitis in addition to the classical clinical picture of strychnine poisoning. Many drugs or chemicals have been reported to be associated with pancreatitis; however, this paper provides us with the first evidence that acute pancreatitis may follow strychnine poisoning. The patient survived despite the development of seizures, lactic acidosis, rhabdomyolysis, and pulmonary infiltrates. Toxicology testing confirmed the presence of strychnine in blood (2.17 mg/L), gastric aspirate, and urine. Attention is drawn to the fact that survival can follow the ingestion of large doses of strychnine providing there is no delay in diagnosis and treatment. The pathophysiologic mechanism of chemical pancreatitis is discussed.
Journal of toxicology. Clinical toxicology 02/1998; 36(1-2):67-71.
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ABSTRACT: The subcellular localization and different biochemical properties of a human hepatic microsomal enzyme that hydrolyses paraoxon (paraoxonase, PON1) were studied and compared to the paraoxon hydrolase activity found in human plasma as well as in rat liver and plasma. Having evaluated the influence of the postmortem interval by a parallel experiment performed in rats, we conclude that the paraoxonase activity was preferentially localized in the microsomal fraction. The enzyme reaction was optimized according to temperature, pH, buffer, ionic strength, substrate concentration, and enzyme protein concentration. The characterization of human liver paraoxonase included the study of optimum pH, pH stability, heat inactivation assays, and kinetic parameters (K(m) and Vmax). In addition, the enzyme activity showed an absolute requirement for exogenous calcium. The activity was lost after incubation with EDTA and partially restored by the addition of calcium; however, other metals assayed were not able to activate the human liver enzyme as did calcium. Our results support the possible identity between human plasma and liver paraoxonases. In spite of the technical difficulties of this study and the possible interference of the postmortem changes in the results, this article represents the first systematic approach to the characterization of human liver paraoxonase.
Journal of Biochemical and Molecular Toxicology 02/1998; 12(1):61-9. · 1.38 Impact Factor
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ABSTRACT: Inhibition of paraoxon hydrolase (paraoxonase) activity by 'in vitro' exposure to EDTA, Mg2+, Co2+, Ba2+, La3+, Zn2+, Cu2+, Hg2+, p-hydroxymercuribenzoate (p-OH-MB) and phenyl mercuric acetate (PMA) was investigated in human liver microsomes. Enzyme activity was totally inhibited by 1 mM EDTA in a time-dependent manner, in contrast to previous data obtained in rat liver where an EDTA-resistant fraction was detected. The possible influence of postmortem changes in these results was checked in a parallel experiment using rat livers with different postmortem intervals. From our results the existence in human liver of an EDTA-resistant fraction cannot be discarded. Ba, La and PMA showed immediate inhibition. By contrast the other compounds tested were time-dependent inhibitors. Ba and Zn showed the highest IC50 values. Cu and mercurials (Hg, p-OH-MB, PMA) were the most potent inhibitors of human liver paraoxonase. Kinetic analysis (Lineweaver-Burk and Dixon plots) indicated that different inhibitors exhibit different inhibition patterns: competitive (EDTA, Ba, La, Cu, p-OH-MB and PMA), non competitive (Zn) and mixed (Hg). The pretreatment of sample with dithiothreitol (DTT) protects against the inhibitory effect of mercurials. Furthermore after inhibition by mercurials the activity was restored by DTT. These results confirmed the essential role of the -SH groups to maintain the catalytic activity of paraoxonase and suggest the existence of two types of -SH groups that could differ in their localization.
Chemico-Biological Interactions 09/1997; 105(3):169-79. · 2.46 Impact Factor
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ABSTRACT: Paraoxonase (paraoxon hydrolase), an enzyme that hydrolyses paraoxon (O,O-diethyl O-p-nitrophenyl phosphate), is located in mammals primarily in the serum and liver. Although considerable information is available regarding serum paraoxonase, little is known about the hepatic form of this enzyme. The present work represents the first study on the purification of rat liver paraoxonase. This enzyme has been purified 415-fold to apparent homogeneity with a final specific activity of 1370 units/mg using a protocol consisting of five steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA and anion exchange on Mono Q HR 5/5. The presence of Ca2+ and Triton X-100 in the buffers throughout the purification procedure was essential for maintaining enzyme activity. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent Mr of 45 000. N-terminal and internal amino acid sequences were determined and compared with those of paraoxonases from human and rabbit serum and mouse liver, showing a high similarity. The pH profile showed optimum activity at pH 8.5. The pH stability and heat inactivation of the enzyme were also studied. The Km for liver paraoxonase was 1.69 mM.
Biochemical Journal 03/1997; 321 ( Pt 3):595-601. · 4.90 Impact Factor
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ABSTRACT: The effects of three different enzyme-inducing agents (phenobarbital, 3-methylcholanthrene and rifampicin) on plasma and liver microsomal fraction paraoxonase and arylesterase were studied in rats. Although phenobarbital and 3-methylcholanthrene each increased the esterase activities in microsomal fraction, only 3-methylcholanthrene was capable to increase them in plasma. By contrast, the administration of rifampicin decreased both enzyme activities in liver and plasma. The results indicate that at least there exists two esterase activities in rat liver microsomes which hydrolyse both paraoxon and phenylacetate, but only one of them is released into the blood.
Environmental Toxicology and Pharmacology 02/1997; 3(1):83-6. · 1.47 Impact Factor
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ABSTRACT: 1 This study was conducted with the aim of evaluating the impact on health produced by the use of different types of pesticides in greenhouses. It is based on the need to practice and develop biological monitoring techniques to assess exposure and predict health risk in workers occupationally exposed to pesticides. 2 Two groups of greenhouse workers with either high or low exposure to a combination of pesticides was taken in Almería, a Spanish province where cultures under plastic are very extended. 3 One hundred and five sprayers were interviewed to collect information about symptoms and signs related to past exposures. Each pesticide sprayer was examined by a physician, and a blood sample was drawn for plasma and red blood cell cholinesterases, complete blood count, and liver and renal function tests. 4 Exposure of workers to a combination of pesticides resulted in 37% of the workers showing toxic signs and symptoms. The main toxic effect observed were a high incidence of spontaneous abortion, depression, and certain neurologic disorders like headache, tremor and paraesthesia. 5 The major analytical change was a decrease of the mean corpuscular haemoglobin concentration in 38% of the cases. However, no significant decrease in both serum and erythrocyte cholinesterase activities was observed. 6 The sprayers were not usually aware of the potential hazards of pesticides and did not try their best to maintain personal hygiene.
Human & Experimental Toxicology 01/1997; 15(12):957-63. · 1.77 Impact Factor
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ABSTRACT: A 41-year-old man was found dead in a hotel room. He was previously diagnosed with depression. Multiple containers of medication and paraphenalia were found at the scene. Autopsy findings included fully developed rigor mortis and pulmonary edema with hemorrhage. Toxicologic analysis of different body fluids was performed and the following drugs were identified in the blood (mg/L): moclobemide (59.76), clomipramine (1.69), tramadol (10.89), diazepam (2.08), nordiazepam (0.82) and caffeine (9.64). A fatal serotonin syndrome was presumably developed as a result of moclobemide-clomipramine interaction as has been recently reported. Tramadol could have a synergistic effect on that syndrome. The forensic pathologists ruled that the cause of death was multiple drug intoxication resulting in serotonin syndrome and that the manner of death was suicide. However, an accidental death from drug abuse could be an alternative diagnosis.
Journal of Forensic Sciences 02/1995; 40(1):128-30. · 1.23 Impact Factor
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ABSTRACT: The properties of a rat hepatic microsomal enzyme that hydrolyses O,O-diethyl-p-nitrophenylphosphate (paraoxon) were studied and compared to the paraoxon hydrolase activity found in rat plasma. The pH stability for both enzyme activities was optimum between pH 6.0 and 9.0. An overall analysis of the data showed that the microsomal fraction was less resistant to the effect of the pH than plasma. The kinetic constants for heat inactivation evaluated for paraoxonase in rat plasma and liver microsomal fraction indicate that paraoxonase tends to inactivate faster in rat liver microsomes than in rat plasma. The apparent activation energies of the heat inactivation process were 77.7 and 61.1 kcal/mol for rat plasma and microsomal fraction, respectively. Enzyme activity was lost after both dialysis and incubation with EDTA and partially restored by the addition of calcium. In rat plasma samples the requirement for calcium was absolute (essential activator) while in the microsomal fraction the reaction may occur, to a minimum extent, in the absence of the activator (non-essential activator). Calcium restored 85% activity when added immediately after EDTA; restored activity decreased when the time interval between addition of EDTA and calcium was increased. Other metals were not able to restore activity previously inhibited by EDTA or dialysis. The response to several inhibitors (EDTA, Mn, Co, Zn, Ba, Mg, Cu, La, Hg and p-hydroxy-mercuribenzoate) of rat plasma and microsomal fraction was studied, determining the type of inhibition and the inhibition constants. Plasma enzyme was always more resistant than liver sample to the effect of the inhibitors and showed different types of inhibition than the liver microsomal fraction. In general we found more differences than analogies between the rat plasma and liver enzyme which suggests the presence of two enzymes or two different forms of the same enzyme. Furthermore the existence of an EDTA-resistant fraction in rat liver microsomes suggests that more than one enzyme capable of hydrolysing paraoxon is present in the microsomal fraction of rat liver.
Biochemical Pharmacology 11/1994; 48(8):1559-68. · 4.70 Impact Factor
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ABSTRACT: 1. A 27-year-old, previously healthy, worker accidentally drank a solution containing 1,3-dichloropropene. 2. He developed gastrointestinal distress, adult respiratory distress syndrome, haematological and hepatorenal functional impairment and died after 40 h. 3. Damage to the pancreas was also thought to have been caused by the chemical as part of a multiorgan disorder.
Human & Experimental Toxicology 06/1994; 13(5):303-6. · 1.77 Impact Factor
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ABSTRACT: A method for the partial purification of rat liver paraoxonase is presented. The method consists of the following steps: preparation of microsomes, solubilization with Triton X-100, adsorption on hydroxylapatite and chromatography on DEAE-52 cellulose. A partially purified preparation of rat liver paraoxonase has been obtained, showing a specific activity of 422 mU/mg with a yield of about 22% and a purification factor of 77-fold.
Chemico-Biological Interactions 07/1993; 87(1-3):69-75. · 2.46 Impact Factor
