Evaluación de aspectos clínicos relacionados con la amputación de miembros inferiores en personas que viven con diabetes mellitus tipo 2 en México / Evaluation of clinical aspects related to lower limb amputation among individuals living with type 2 diabetes mellitus in Mexico

RESUMEN Introducción: La diabetes continúa siendo una de las principales causas de discapacidad y muerte en la población mundial. Alrededor del 25% de las personas con diabetes desarrollarán una úlcera en alguno de sus miembros pélvicos inferiores. Objetivo: El presente estudio evalúa los aspectos clínicos relacionados con la amputación del miembro inferior pélvico en una cohorte de pacientes con diabetes mellitus. Métodos: Estudio retrospectivo, transversal, realizado en colaboración entre el Instituto Mexicano del Seguro Social y la Facultad de Farmacia de la Universidad Autónoma del Estado de Morelos, implicó una revisión de expedientes de pacientes con diabetes mellitus tipo 2 en el Hospital General Regional "Ignacio García Téllez". Se seleccionaron 100 expedientes clínicos y Electrónicos basados en criterios de inclusión, que incluían edad mayor de 18 años, afiliación en el sitio del estudio, evolución de la diabetes de al menos 10 años, tratamiento farmacológico para la diabetes y diagnóstico de pie diabético con curación completa o amputación como resultado. Los análisis estadísticos se realizaron mediante STATA y se obtuvo aprobación ética. Resultados: Los pacientes con un control glucémico óptimo cuantificando sus niveles de glucosa en ayunas (<130 mg/dl) así como sus valores de hemoglobina glicosilada (< 7%) tuvieron una menor frecuencia de amputaciones (p˂0,001; Chi2) en comparación con aquellos pacientes sin un control glucémico adecuado. Conclusión: Se encontró que ser hombre, valores de hemoglobina glucosilada superiores al 7% y valores promedio de glucosa en ayunas superiores a 130 mg/L aumentan la probabilidad de presentar una amputación de extremidad inferior.

ABSTRACT Introduction: Diabetes continues to be a leading cause of disability and death in the world's population. About 25% of people with diabetes will develop an ulcer in one of their lower pelvic limbs. Objective: The present study evaluates the clinical aspects related to lower pelvic limb amputation in a cohort of patients with diabetes mellitus. Lazarte Echegaray Hospital during the period 2017-2020. Methods: Retrospective, cross-sectional study, conducted in collaboration between the Mexican Institute of Social Security and the School of Pharmacy of the Autonomous University of Morelos State, involved a review of records of patients with type 2 diabetes mellitus at the Regional General Hospital "Ignacio García Téllez". One hundred clinical and electronic records were selected based on inclusion criteria, which included age over 18 years, affiliation at the study site, diabetes evolution of at least 10 years, pharmacological treatment for diabetes and diagnosis of diabetic foot with complete healing or amputation as an outcome. Statistical analyses were performed using STATA and ethical approval was obtained. Results: Patients with optimal glycemic control by quantifying their fasting glucose levels (<130 mg/dl) as well as their glycated hemoglobin values (< 7%) had a lower frequency of amputations (p˂0.001; Chi2) compared to those patients without adequate glycemic control. Conclusion: Being male, glycosylated hemoglobin values greater than 7% and mean fasting glucose values greater than 130 mg/L were found to increase the likelihood of having a lower extremity amputation.

Cu2+-dependent hydrolysis of O-hexyl 2,5-dichlorophenyl phosphoramidate by reptile sera.

This study shows the EDTA-resistant, Ca2+ and Cu2+-dependent hydrolysis of O-hexyl 2,5-dichlorophenyl phosphoramidate (HDCP) compound in reptiles sera determined by spectrophotometry UV/Vis and chiral chromatography. Samples of ten reptile species were incubated with aliquot of 100 or 400 µM HDCP in presence of 100 or 300 µM Cu2+, or 2.5 mM Ca2+ or 5 mM EDTA at 37 °C for 30-60 min. The results shown an activator effect of Cu2+ on HDCP hydrolysis in freshwater turtles sera (Trachemys scripta, Chelydra serpentina and Macrochelys temminckii) because the levels of 2,5-dichlorophenol (DCP; product hydrolysis) were similar (∼37 µM DCP) to chicken serum (positive control group). The marine turtles (Chelonia mydas and Eretmochelys imbricata) and crocodiles (Crocodylusacutus and Crocodylus moreletii) showed ∼50% less HDCPase activity (13-17 µM DCP) compared to the HDCPase activity of the freshwater turtle species. Terrestrial reptile species (snakes and lizards) showed around 25% of activity (7-13 µM DCP) with both copper concentrations. These Cu2+-dependent hydrolysis were stereospecific to R(+)-HDCP (p˂0.05) in the three freshwater turtle species that showed similar hydrolysis to the chicken serum. However, the Ca2+ did not show a significant activating effect on the HDCPase activity (1-8 µM DCP) in any reptile serum. Their hydrolysis levels were very similar to those of EDTA-resistant activity. The present study demonstrates a Cu2+-dependent A-esterase (HDCPase) activity in turtles and points serum albumin as the cuproprotein responsible for this activity, reinforcing its N-terminal sequence (DAEH) as a catalytic center.

Computational study to find the goat serum albumin (GSA) binding site as A-esterase.

A-esterases are a classical term applied to enzymatic activity of the proteins by a mechanism not involving intermediate covalent phosphorylation, but requiring a divalent cation cofactor. Recently, a copper-dependent A-esterase activity has been identified in goat serum albumin (GSA) on the organophosphorus insecticide trichloronate. This hydrolysis was identified ex vivo with spectrophotometry and chromatography techniques. Albumin mechanism of action and catalytic site as Cu2+-dependent A-esterase are still unknown. Therefore, to know the copper bind to albumin is relevant. N-terminal sequence has been reported as the high affinity site for this cation, due to the histidine in position 3. The aim of this work in silico is to explore how occurs this metallic binding and active the esterase catalytic function. The GSA crystallized structure (PDB: 5ORI) was chosen for molecular docking and dynamics. A site-directed docking, for N-terminal site and a blind docking was done with trichloronate as ligand. Root-mean-square deviation and frequency plot was calculated to find the most frequent predicted structure and visualize the amino acids involved in binding site. The affinity energy in the blind docking (-5.80 kcal/mol) is almost twice lower than site-directed docking (-3.81 kcal/mol) and N-terminal amino acids do not appear in the most repeated structure binding site, suggesting that the protein has a site with higher affinity to the trichloronate ligand. His145 could be involved in the binding site as has been reported in previous studies.

The Neurotoxic Effect of Ochratoxin-A on the Hippocampal Neurogenic Niche of Adult Mouse Brain.

Ochratoxin A (OTA) is a common secondary metabolite of Aspergillus ochraceus, A. carbonarius, and Penicillium verrucosum. This mycotoxin is largely present as a contaminant in several cereal crops and human foodstuffs, including grapes, corn, nuts, and figs, among others. Preclinical studies have reported the involvement of OTA in metabolic, physiologic, and immunologic disturbances as well as in carcinogenesis. More recently, it has also been suggested that OTA may impair hippocampal neurogenesis in vivo and that this might be associated with learning and memory deficits. Furthermore, aside from its widely proven toxicity in tissues other than the brain, there is reason to believe that OTA contributes to neurodegenerative disorders. Thus, in this present in vivo study, we investigated this possibility by intraperitoneally (i.p.) administering 3.5 mg OTA/kg body weight to adult male mice to assess whether chronic exposure to this mycotoxin negatively affects cell viability in the dentate gyrus of the hippocampus. Immunohistochemistry assays showed that doses of 3.5 mg/kg caused a significant and dose-dependent reduction in repetitive cell division and branching (from 12% to 62%). Moreover, the number of countable astrocytes (p < 0.001), young neurons (p < 0.001), and mature neurons (p < 0.001) negatively correlated with the number of i.p. OTA injections administered (one, two, three, or six repeated doses). Our results show that OTA induced adverse effects in the hippocampus cells of adult mice brain tissue when administered in cumulative doses.

The acute systemic toxicity of thallium in rats produces oxidative stress: attenuation by metallothionein and Prussian blue.

Thallium (TI) is one of the most toxic heavy metals. Human exposure to Tl occurs through contaminated drinking water and from there to food, a threat to health. Recently, environmental contamination by Tl has been reported in several countries, urging the need for studies to determine the impact of endogenous and exogenous mechanisms preventing thallium toxicity. The cytoprotective effect of metallothionein (MT), a protein with high capacity to chelate metals, at two doses (100 and 600 µg/rat), was tested. Prussian blue (PB) (50 mg/kg) was administered alone or in combination with MT. A dose of Tl (16mg/kg) was injected i.p. to Wistar rats. Antidotes were administered twice daily, starting 24h after Tl injection, for 4 days. Tl concentrations diminished in most organs (p < 0.05) by effect of PB, alone or in combination with MT, whereas MT alone decreased Tl concentrations in testis, spleen, lung and liver. Likewise, brain thallium also diminished (p < 0.05) by effect of PB and MT alone or in combination in most of the regions analyzed (p < 0.05). The greatest diminution of Tl was achieved when the antidotes were combined. Plasma markers of renal damage increased after Tl administration, while PB and MT, either alone or in combination, prevented the raise of those markers. Only MT increased the levels of reduced glutathione (GSH) in the kidney. Finally, increased Nrf2 was observed in liver and kidney, after treatment with MT alone or in combination with PB. Results showed that MT alone or in combination with PB is cytoprotective after thallium exposure.

Cu2+-dependent stereoselective hydrolysis of a chiral organophosphonothioate insecticide for domestic mammals' sera and its albumins.

Acute toxicity of organophosphate (OPs) pesticides is a public health problem. The adverse effects are associated with the inhibition and aging of nervous system B-esterases such as acetyl cholinesterase (AChE) and neuropathic target esterase (NTE). Treatment based on A-esterases such as mammal serum paraoxonase-1 has been suggested. This ex vivo study shows the Cu2+-dependent hydrolysis of trichloronate (TCN), a racemic organophosphonothioate insecticide, in human and domestic mammal serum (dog, goat, pig, sheep and cow). Ca2+-dependent (2.5 mM) or EDTA-resistant (5 mM) activity (1-6%) was not significant (p>0.05) in all samples, except goat serum and its albumin, which showed higher levels of TCN hydrolysis (38-58%) than other mammals with 100 and 300 µM copper sulfate at physiological conditions for 60 min. Goat serum albumin (GSA) showed significant (p˂0.05) stereoselective hydrolysis (+)-TCN ˃ (-)-TCN (45% versus 33%). This suggests that GSA is the protein responsible for Cu2+-dependent TCNase activity in goat serum. This is the first report on Cu2+-dependent A-esterase activity in mammalian tissues. This goat serum cuproprotein could be considered as an alternative in future biotechnological applications including enantiomeric synthesis, bioremediation and antidotal treatment of organophosphonothioate pesticide poisoning.

Antioxidant Effect of Hydroxytyrosol, Hydroxytyrosol Acetate and Nitrohydroxytyrosol in a Rat MPP+ Model of Parkinson's Disease.

3,4-Dihydroxyphenyl ethanol, known as hydroxytyrosol (HTy), is a phenylpropanoid found in diverse vegetable species. Several studies have demonstrated that HTy is a potent antioxidant. Thus, our study is aimed to evaluate the antioxidant effect of HTy and its derivatives, hydroxytyrosol acetate (HTyA) and nitrohydroxytyrosol (HTyN), in a model of oxidative stress induced by 1-methyl-4-phenylpyridinium (MPP+) in rats. Rats were administered intravenously (i.v.) in the tail with 1 mL saline solution or polyphenol compound (1.5 mg/kg) 5 min before intrastriatal infusion of 10 µg MPP+/8 µL. We found that rats injured with MPP+, pretreatment with HTy, HTyA or HTyN significantly decreased ipsilateral turns. This result was consistent with a significant preservation of striatal dopamine levels and decreased lipid fluorescence products (LFP), a marker of oxidative stress. Brain GSH/GSSG ratio, from rats pretreated with HTy or HTyN showed a significant preservation of that marker, decreased as a consequence of MPP+-induced oxidative damage. These results show an antioxidant effect of HTy, HTyA and HTyN in the MPP+ model of Parkinson's disease in the rat.

DAEH N-terminal sequence of avian serum albumins as catalytic center of Cu (II)-dependent organophosphorus hydrolyzing A-esterase activity.

O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) induces delayed neuropathy. The R (+)-HDCP inhibits and caused the so call "aging reaction" on inhibited-NTE. This enantiomer is not hydrolyzed by Ca(II)-dependent A-esterases in mammal tissues but is hydrolyzed by Cu(II)-dependent chicken serum albumin (CSA). With the aim of identifying HDCP hydrolysis by other vertebrate albumins, we incubated albumin with 400 µM racemic HDCP in the presence of 100 µM copper sulfate. HDCPase activity was assessed by measurement of HDCP with chiral chromatography. Human, sheep, dog, pig, lamprey or cobra serum albumin did not show a significant activity (~10%). Rabbit and bovine albumins hydrolyzed both enantiomers of HDCP (25% and 50% respectively). Turkey serum albumin had more HDCPase activity (~80 µM remaining) than the chicken albumin (~150 µM remaining). No animal albumins other than chicken showed stereoselective hydrolysis. Preincubation of chicken albumin with 1 mM the histidine modifying agents, 100 µM N-bromosuccinimide (NBS) and Zn(II), inhibited its Cu(II)-dependent R (+)-HDCPase activity, where as other mM amino acids modifiers had no inhibitory effects. . These results confirm that the stereoselective hydrolysis of (+)-HDCP is a specific A-esterase catalytic property of chicken albumin. The higher HDCPase activity by turkey albumin suggests the amino-terminal sequence of avian albumins (DAEHK) is the active center of this Cu(II)-dependent A-esterase activity.

Hydrolysis of chiral organophosphorus compounds by phosphotriesterases and mammalian paraoxonase-1.

Some organophosphorus compounds (OPs), which are used in the manufacturing of insecticides and nerve agents, are racemic mixtures with at least one chiral center with a phosphorus atom. Acute exposure of humans to these mixtures induces the covalent modification of acetylcholinesterase (AChE) and neuropathy target esterase (NTE) and causes a cholinergic syndrome or organophosphate-induced delayed polyneuropathy syndrome (OPIDP). These irreversible neurological effects are due to the stereoselective interaction of the racemic OPs with these B-esterases (AChE and NTE) and such interactions have been studied in vivo, ex vivo and in vitro, using stereoselective hydrolysis by A-esterases or phosphotriesterases (PTEs) and the PTE from Pseudomonas diminuta, and paraoxonase-1 (PON1) from mammalian serum. PON1 has a limited hydrolytic potential of the racemic OPs, while the bacterial PTE exhibits a significant catalytic activity on the less toxic isomers P(+) of the nerve agents. Avian serum albumin also shows a hydrolyzing capacity of chiral OPs with oxo and thio forms. There are ongoing environmental and bioremediation efforts to design and produce recombinants as bio-scavengers of OPs.

Protection induced by estradiol benzoate in the MPP+ rat model of Parkinson's disease is associated with the regulation of the inflammatory cytokine profile in the nigro striatum.

Previously, we have demonstrated that ß-estradiol-3-benzoate (EB) has a protective effect on the neurodegenerative experimental model of Parkinson's disease. The protective effect is through the induction of the expression of paraoxonase-2 (PON2) in the striatum. PON2 has proven to have antioxidant and anti-inflammatory activity, this protein has a beneficial effect in MPP+ model in rats decreasing the lipid peroxidation and the oxidative stress. Furthermore, the molecular effect and the pathway by which EB induces protection were not further pursued. This study shows the regulation by EB of the anti-inflammatory effect through the modulation of cytokines, antioxidant enzymes and PON2 in the rat striatum. Rats were gonadectomized and 30 days after were randomly assigned into four experimental groups; only vehicles (Control group); EB treatment (EB group); MPP+ injury (M group); EB plus MPP+ injured (EB/M group). EB treatment consisted of 100 µg of the drug administered every 48 h for 11 days. Results showed that EB (group EB/M) treatment decrease significantly (40%) the number of ipsilateral turns respect to the M group and prevents significantly the dopamine (DA) decreased induced by MPP+ (~75%). This results are correlate with a significant decrease in the level of lipid peroxidation (60%) of the EB/M group respect to the M group. The EB treatment showed protection against neurotoxicity induced with MPP+, this could be due to EB capacity to prevent the increase in the expression level of proinflammatory cytokines TNF-α, IL-1 and IL-6 induced by MPP+. While, TGF-ß1 and TGF-ß3 expression was reduced in the rats treated only with MPP+, in the rats of EB/M group the expression of both cytokines was increased. EB protective effect against MPP+ neurotoxicity is related to antioxidant effect of PON2, pro-inflammatory cytokines and GSHR but not to SOD2, catalase, GPX1 or GPX4.

Hydroxytyrosol inhibits MAO isoforms and prevents neurotoxicity inducible by MPP+ invivo.

Parkinson's disease is considered to be due to an increase in the catabolism of dopamine by the action of monoamine oxidase (MAO) enzymes which leads to an increase in reactive oxygen species (ROS) and loss of dopaminergic neurons. Here, in a model of neurotoxicity inducible by 1-methyl-4-phenylpyridinium (MPP+), we tested the effect of hydroxytyrosol (HTy), a potent antioxidant, on generation of ROS. Five minutes after a single intravenous administration of 1.5 mg/Kg of Hty, Wistar rats received an intrastriatal micro-injection of 10 micrograms of MPP+ while control animals received saline solution. Six days later, all animals were treated with apomorphine (1 mg/Kg), subcutaneously and ipsilateral rotations were assessed within an hour. Then, the rats were sacrificed, striatal tissues were removed and their catecholamines and MAO-A and B activities were quantitated. Pretreatment with HTy significantly diminished the number of ipsilateral rotations. This recovery correlated with significant preservation of striatal dopamine and significant inhibition of of the MAO activity. These results are consistent with the inhibitory effect of HTy on the MAO isoforms and form a basis for the neuroprotective mechanism of this phenylpropanoid in MPP+ induced Parkinson's disease.

O-hexyl O-2,5-dichlorophenyl phosphoramidate as a substrate for domestic and sea bird serum A-esterases: Hydrolysis levels, Cu2+- and Zn2+-dependence and stereoselectivity.

O-Hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) induces delayed neuropathy in hens. It has been used as a tool to identify new A-esterase activities in animal tissues. This study shows the EDTA-resistant, Cu2+- and Zn2+-dependent hydrolysis of racemic HDCP in domestic and sea bird serum using UV/Vis spectrophotometry and chiral chromatography. The results clearly show a significant (p ˂ 0.05) Cu2+- and Zn2+-dependent HDCP hydrolysis in the serum of all bird species versus EDTA, except for the Zn2+-dependent HDCPase activity from Yucatecan quail serum. The ratio of Cu2+/Zn2+ hydrolysis varied between 1 and 7 (intraspecies) and 15.6 (interspecies). EDTA affected the Cu2+- and Zn2+-dependent HDCPase activity in the range of 37-95% and 40-50%, respectively. HDCP hydrolysis activated by Cu2+ was significantly (p ˂ 0.05) stereoselective (R-(+)-HDCP ˃ S-(-)-HDCP) in chicken and sea bird serum. Its R-(+)-HDCP/S-(-)-HDCP ratios were 6.8 and 1.6-2.8, respectively. EDTA-resistant and zinc-dependent HDCP hydrolysis were not stereospecific in all bird sera tested. The present ex vivo study reinforces the idea that bird sera have HDCPase activity that is sensitive to divalent metals, resistant to EDTA and possibly associated with the protein albumin.

Copper(II)-dependent hydrolysis of trichloronate by turkey serum albumin.

Trichloronate is a racemic organophosphonothioate insecticide that induced delayed neuropathic in hens and human. The avian are species with great susceptibility to organophosphorus poisoning due to their low levels of A-esterases. However, a significant copper-dependent A-esterase activity has been identified in chicken and turkey serum. This study aims at characterizing the trichloronate enantiomers hydrolysis by serum and albumin from chicken (CSA) and turkey (TSA) in the presence of copper by chiral chromatography. A significant Cu2+-dependent hydrolysis for both trichloronate enantiomers (38%) was observed in turkey serum and TSA (p < 0.05). The hydrolysis is stereoselective for (-)-trichloronate (p < 0.05). TSA incubation (200 µg) showed residual values of 56 µM and 20 µM of (+)-trichloronate and (-)-trichloronate, respectively; while the chicken serum and CSA presented a slight hydrolysis (1-7%) of both enantiomers. This copper-dependent hydrolysis and stereoselectivity of trichloronate by TSA was proportional to the incubation time. The increase of TSA in the assay (200-3000 µg) in the presence of 100 µM copper did not significantly increase the levels of hydrolysis and stereoselectivity, an opposite effect was observed for turkey serum (100-200 mL), which totally inhibited this copper-dependent activity of both isomers. The present study evidences an A-esterase activity of TSA on a thio form OP compound, which is stereoselective and activated by copper.

Copper-dependent hydrolysis of trichloronate by turkey serum studied with use of new analytical procedure based on application of chiral chromatography and UV/Vis spectrophotometry.

Trichloronate is a racemic organophosphate, which has been used for the manufacture of insecticides. This compound induces delayed neuropathy in hen and humans. This study shows the Cu2+-dependent hydrolysis of trichloronate by turkey serum using UV/Vis spectrophotometry and chiral chromatography. The CHIRALCEL OD column and mobile phase of heptane allowed a resolution of 1.15 of its two enantiomers, while the liquid-liquid extraction showed a recovery of 95-98%. The optimum linear response was of 50 to 800 µM with a detection and quantification limit of 0.6 and 2 µM for (+)-trichloronate, and 0.7 and 2.3 µM for (-)-trichloronate. The levels of Cu2+-dependent hydrolysis (µM remaining concentration) quantified for 60 min at 37 °C and pH 7.4 were statistically higher (p ˂ 0.05) for (-)-trichloronate (65%) than (+)-trichloronate (32%). This stereoselective hydrolysis was confirmed by UV/Vis spectrophotometry using 2,4,5­trichlorophenol as standard, each of the enantiomers (93-95% purity) collected by HPLC, as well as aminoantipyrine and ferricyanide reagents to yield a colored product. This method exhibited an optimal linearity (r > 0.99) and a higher Cu2+-dependent hydrolysis (p < 0.05) to (-)-trichloronate (47%) than its corresponding (+)-form (31%). This results shows the Cu2+-dependent stereoselective hydrolysis of a racemic OP in its thio form (P = S) by an A-esterase of the turkey serum through the development of a colorimetric method and optimization of an existing chiral chromatographic method.

Paraoxonase-1 polymorphisms and cerebral ischemic stroke: a pilot study in mexican patients.

BACKGROUND: The serum paraoxonase-1 (PON1) associated to HDL presents two common polymorphisms in the positions 192 and 55. These polymorphisms are considered determinant of the capacity of HDL to protect LDL from their oxidative modification. In this context, the PON1 genotype has been associated with cardiovascular diseases, including stroke. OBJECTIVE: To determine the allelic and genotypic frequencies of PON1 L55M and Q192R as well as the enzymatic activities of PON1 in subjects with and without atherothrombotic stroke. METHODS: There were included 28 people with atherothrombotic stroke and 29 without stroke. The genotyping was carried out by PCR-RFLP and the phenotyping by measurement of the activities of paraoxonase and arylesterase in serum. RESULTS: For the polymorphism Q192R, the allelic frequencies (Q/R) were 0.46/0.54 and 0.48/0.52 (p= 0.843) for the control group and the group with stroke, respectively. While for the polymorphism L55M, the allelic frequencies (L/M) were 0.81/0.19 for the control group, and 0.78/0.22 for the group with stroke (p= 0.610). The activity levels of paraoxonase were not significantly different between the control and stroke groups (450 vs. 348 UI/mL, p= 0.093) While the activity levels of arylesterase were significantly different between the studied groups (90 vs. 70 UI/mL, p= 0.001); however, upon adjustment by multiple linear regression, it was not longer significant. CONCLUSION: The polymorphisms Q192R and L55M, and the paraoxonase activity of PON1 are not risk factors for atherothrombotic stroke according to the results of this study.

INTRODUCCIÓN: La paraoxonasa-1 (PON1) sérica asociada a las HDL presenta dos polimorfismos comunes en las posiciones 192 y 55. Estos polimorfismos se consideran determinantes para la capacidad de las HDL de proteger a las LDL de su modificación oxidativa. En este contexto, el genotipo de PON1 se ha asociado con enfermedades cerebrovasculares, que incluyen el infarto cerebral. OBJETIVO: Determinar las frecuencias alélicas y genotípicas de PON1-L55M y PON1- Q192R, así como las actividades enzimáticas de PON1 en sujetos con y sin infarto cerebral aterotrombótico. MÉTODOS: Se incluyeron 28 personas con infarto cerebral aterotrombótico y 29 sin infarto. Las genotipificaciones se realizaron mediante PCR-RFLP y las fenotipificaciones mediante la medición de las actividades paraoxonasa y arilesterasa en suero. RESULTADOS: Para el polimorfismo Q192R, las frecuencias alélicas (Q/R) fueron 0.46/0.54 y 0.48/0.52 (p= 0.843) para el grupo control y el grupo con infarto, respectivamente. Mientras que para el polimorfismo L55M, las frecuencias alélicas (L/M) fueron 0.81/0.19 para el grupo control y 0.78/0.22 para el grupo con infarto (p= 0.610). Los niveles de actividad paraoxonasa no fueron significativamente diferentes entre los grupos control y con infarto (450 vs. 348 Ul/mL, p= 0.093). Mientras que los niveles de actividad arilesterasa fueron significativamente diferentes entre los grupos estudiados (90 vs. 70 Ul/mL, p= 0.001), sin embargo, al ajustarla por regresión lineal múltiple, dejo de ser significativa. CONCLUSIÓN: Los polimorfismos Q192R y L55M, y la actividad paraoxonasa de la PON1 no son factores de riesgo para el infarto cerebral aterotrombótico en este estudio.

Paraoxonase-1 polymorphisms and cerebral ischemic stroke: a pilot study in mexican patients / Caracterización polimórfica de la paraoxonasa-1 en el infarto cerebral aterotrombótico de una poblacion mexicana: estudio piloto

Abstract Background: The serum paraoxonase-1 (PON1) associated to HDL presents two common polymorphisms in the positions 192 and 55. These polymorphisms are considered determinant of the capacity of HDL to protect LDL from their oxidative modification. In this context, the PON1 genotype has been associated with cardiovascular diseases, including stroke. Objective: To determine the allelic and genotypic frequencies of PON1 L55M and Q192R as well as the enzymatic activities of PON1 in subjects with and without atherothrombotic stroke. Methods: There were included 28 people with atherothrombotic stroke and 29 without stroke. The genotyping was carried out by PCR-RFLP and the phenotyping by measurement of the activities of paraoxonase and arylesterase in serum. Results: For the polymorphism Q192R, the allelic frequencies (Q/R) were 0.46/0.54 and 0.48/0.52 (p= 0.843) for the control group and the group with stroke, respectively. While for the polymorphism L55M, the allelic frequencies (L/M) were 0.81/0.19 for the control group, and 0.78/0.22 for the group with stroke (p= 0.610). The activity levels of paraoxonase were not significantly different between the control and stroke groups (450 vs. 348 UI/mL, p= 0.093) While the activity levels of arylesterase were significantly different between the studied groups (90 vs. 70 UI/mL, p= 0.001); however, upon adjustment by multiple linear regression, it was not longer significant. Conclusion: The polymorphisms Q192R and L55M, and the paraoxonase activity of PON1 are not risk factors for atherothrombotic stroke according to the results of this study.

Resumen Introducción: La paraoxonasa-1 (PON1) sérica asociada a las HDL presenta dos polimorfismos comunes en las posiciones 192 y 55. Estos polimorfismos se consideran determinantes para la capacidad de las HDL de proteger a las LDL de su modificación oxidativa. En este contexto, el genotipo de PON1 se ha asociado con enfermedades cerebrovasculares, que incluyen el infarto cerebral. Objetivo: Determinar las frecuencias alélicas y genotípicas de PON1-L55M y PON1- Q192R, así como las actividades enzimáticas de PON1 en sujetos con y sin infarto cerebral aterotrombótico. Métodos: Se incluyeron 28 personas con infarto cerebral aterotrombótico y 29 sin infarto. Las genotipificaciones se realizaron mediante PCR-RFLP y las fenotipificaciones mediante la medición de las actividades paraoxonasa y arilesterasa en suero. Resultados: Para el polimorfismo Q192R, las frecuencias alélicas (Q/R) fueron 0.46/0.54 y 0.48/0.52 (p= 0.843) para el grupo control y el grupo con infarto, respectivamente. Mientras que para el polimorfismo L55M, las frecuencias alélicas (L/M) fueron 0.81/0.19 para el grupo control y 0.78/0.22 para el grupo con infarto (p= 0.610). Los niveles de actividad paraoxonasa no fueron significativamente diferentes entre los grupos control y con infarto (450 vs. 348 Ul/mL, p= 0.093). Mientras que los niveles de actividad arilesterasa fueron significativamente diferentes entre los grupos estudiados (90 vs. 70 Ul/mL, p= 0.001), sin embargo, al ajustarla por regresión lineal múltiple, dejo de ser significativa. Conclusión: Los polimorfismos Q192R y L55M, y la actividad paraoxonasa de la PON1 no son factores de riesgo para el infarto cerebral aterotrombótico en este estudio.

Albumin, the responsible protein of the Cu2+-dependent hydrolysis of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) by chicken serum "antagonistic stereoselectivity".

O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) is a chiral analogous compound of the methamidophos insecticide that induces delayed neuropathy, and the R-(+)-HDCP enantiomer is an inhibitor of neuropathy target esterase (NTE). This enantiomer is not hydrolized by Ca2+-dependent phosphotriesterases in mammal tissues. Our group had reported R-(+)-HDCP hydrolysis in chicken serum enhanced by 30-250 µM copper in ex vivo assays, which we call "antagonistic stereoselectivity". We checked the hypothesis of the role of cupper binding proteins. Two hundred micrograms of human serum ceruloplasmine or horse kidney methallotionein in 1 mL containing 400 µM HDCP for 60 min showed no significant Cu2+-dependent hydrolysis. However under the same conditions, 10 µL of chicken serum or 10 µL of buffer containing 216 µg of chicken serum albumin (CSA) (amount of albumin content in this serum volume) with 100 µM Cu2+ showed the same stereoselectivity and similar levels to the Cu2+-dependent R-(+)-HDCP hydrolysis. About 75% of R-(+)-HDCP were hydrolyzed after 120 min in the presence of 100 µM Cu2+ (inhibited by 5 mM EDTA). No effects was observed by divalent cations Cu2+, Zn2+, Fe2+, Ca2+, Mn2+ and Mg2+. These results confirm that albumin is the protein responsible for "antagonistic stereoselectivity" observed in chicken serum.

Relationship Between Paraoxonase-1 and Butyrylcholinesterase Activities and Nutritional Status in Mexican Children.

BACKGROUND: The enzymes butyrylcholinesterase (BuChE) and paraoxonase-1 (PON1) are the primary bioscavenging enzymes in serum and exhibit antioxidant and anti-inflammatory activities. PON1 has been associated with diseases caused by high oxidative stress, whereas BuChE appears to be involved in the pathophysiology of the metabolic syndrome and related disorders. It has been suggested that children from rural communities in Mexico may have a predisposition to develop obesity or type 2 diabetes during adolescence or adulthood. The objective of this study was to determine whether associations exist between the paraoxonase (PONase)/arylesterase (AREase) activity of PON1, its PON1-Q192R and PON1-L55M polymorphisms, and BuChE activity with the nutritional status and lipid profiles in a group of children from rural communities in Mexico. METHODS: A group of 97 boys and girls from a rural community in Mexico were assessed for body mass index, the enzymatic activities of BuChE, PONase, and AREase were measured in serum, and their lipid profiles were determined. Genetic polymorphisms of PON1-L55M and PON1-Q192R were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The children were classified into four groups: thinness, normal weight, overweight, and obese. Of the children studied, 34.4% were overweight and obese. The mean age of the participants was 9.5 years (standard deviation = 1.8). The L allele of the PON1-L55M genotype was the most frequent (83.3%), and the R allele of the PON1-Q192R genotype was the most frequent (61.8%). Overweight and obese children had higher values of BuChE, total cholesterol, triglycerides (TG), and lower high-density lipoprotein (HDL-C) values than children with thinness or normal weight (P = 0.028, P = 0.019, P = 0.004, P = 0.069 and P = 0.021, respectively). The levels of AREase and PONase and the prevalence of PON1-L55M and PON1-Q192R genotypes were similar between groups (P = 0.484 and P = 0.380, respectively). CONCLUSIONS: This study establishes a positive association of BuChE activity with nutritional status and serum TG.

ß-Estradiol-3-benzoate confers neuroprotection in Parkinson MPP+ rat model through inhibition of lipid peroxidation.

Estradiol (E2), in addition to its known hormone function, is a neuroactive steroid that has shown neuroprotective profile in several models of neurological diseases. The present study explores the antioxidant effect of ß-estradiol-3-benzoate (EB) on the neurotoxicity elicited by MPP+ in rat striatum. Male Wistar rats, that were gonadectomized 30days prior to EB, were given 100µgEB per rat every 48h for 11days and animals were infused with MPP+ via intrastriatal at day six after beginning EB treatment. EB treatment completely prevented the fall in dopamine caused by MPP+, such result was related with decreased lipid peroxidation, a marker of oxidative stress; diminished number of ipsilateral-to-lesion turns and increased signal of the dopamine-synthesizing enzyme Tyrosin Hydroxylase in substantia nigra. The protection elicited by EB was not related to Mn or Cu-Zn superoxide dismutase enzymatic activities or glutathione modulation since none of these parameters were influenced by EB at the times assayed. Whereas, increased expression of PON2 as a result of EB treatment was observed, this phenomenon could be one of the mechanism by which the steroid conferred protection to dopaminergic cells against MPP+ injury.

Copper activation of organophosporus compounds detoxication by chicken serum.

Avian species contain low levels of enzymes that hydrolyze organophosphorus compounds (OPs), and chickens are used as a model of OPs delayed neurotoxicity. For both reasons, we studied the ability of chicken tissue for OP detoxication. A significant activating effect of Cu2+ on the hydrolysis of O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) was observed in hen plasma and the microsomal fractions of the liver, brain, and mainly in hen serum, by spectrophotometric and chiral chromatography methods. The concentration of 1 mM of Cu2+ or Zn2+ showed 200% and 168% activation, respectively, in hen plasma compared with the Ca2+-dependent hydrolysis, whereas these cations had an inhibitory effect on soluble liver and brain fractions. An increase of 1.5 to 19.5 fold in HDCP hydrolyzing activity was obtained for the 30-250 µM Cu2+ range when using chicken serum instead of hen plasma. This Cu2+-dependent hydrolysis in chicken serum was stereoselective for the R-(+)-HDCP isomer, which proved the opposite to the Ca2+-dependent stereoselective hydrolysis of the S-(-)-HDCP isomer reported in rat and rabbit serum. The level of copper needed to exert this effect should be further evaluated for its suitability for potential therapeutic and biotechnological applications.