Peripheral artery disease and oxidative stress in patients in a program for preventing complications of diabetes mellitus and dyslipidemia / Enfermedad arterial periférica y estrés oxidativo en pacientes del programa para la prevención de complicaciones de diabetes mellitus y dislipidemias

Abstract Introduction: Peripheral artery disease (PAD) is mainly caused by atherosclerosis but also involves hyperglycemia and dyslipidemia, which trigger oxidative stress and lead to vascular damage. Objectives: To determine the prevalence of PAD in patients with type 2 diabetes mellitus (DM2) and/or prediabetes and/or dyslipidemia, to identify some risk factors and to establish whether urinary levels of 8-isoprostane-f2α (an oxidative stress marker) are elevated in patients with PAD. Design: A cross-sectional, nonprobabilistic, convenience sampling study Materials and methods: The sample included 146 patients with DM2 and/or prediabetes and/ or dyslipidemias from the Universidad Nacional de Colombia. Risk factors, symptoms related to PAD, ankle-brachial index measurement and biochemical variables (HbA1c%, fasting blood glucose, lipid profile, creatinine and albuminuria) were recorded. Urine levels of 8-isoprostane-f2α were determined by ELISA. The 8-iso-PGF2α/creatine concentration were analyzed using the statistical package R. Risk factors were compared using ANOVA/ Kruskal-Wallis. ROC curves were generated to analyze the discriminant power of the biomarkers. The joint analysis of laboratory results and risk factors was performed using multivariate logistic regressions. Results: PAD was identified in 10 diabetic patients. Risk factors were smoking, dyslipidemia, poor metabolic control, overweight or obesity. There was no evidence of increased urinary 8-isoprostane-f2α in these subjects. Conclusions: A low prevalence of PAD was found in subjects with DM2. There was no evidence of increased 8-isoprostane-f2α measured by ELISA in patients with PAD. The extension of the study with different markers of oxidative stress and the use of other techniques is recommended (Acta Med Colomb 2019; 44. DOI: https://doi.org/10.36104/amc.2019.1257).

Resumen Introducción: la enfermedad arterial periférica (EAP) es causada principalmente por aterosclerosis e intervienen la hiperglucemia y dislipidemia que desencadenan estrés oxidativo y daño vascular. Objetivo: determinar la prevalencia de EAP en pacientes con diabetes mellitus tipo 2 (DM2) y/o prediabetes y/o dislipidemias, así como algunos factores de riesgo; también, establecer si los niveles urinarios de 8-isoprostano-f2α (marcador de estrés oxidativo) están elevados en pacientes con EAP. Diseño: estudio de tipo transversal, no probabilístico, de conveniencia. Material y métodos: la muestra comprendió 146 pacientes con DM2 y/o prediabetes y/o dislipidemias de la Universidad Nacional de Colombia. Se registraron factores de riesgo, síntomas relacionados con EAP, medida índice tobillo-brazo y variables bioquímicas (HbA1c%, glucemia basal, perfil lipídico, creatinina y albuminuria). Se determinaron niveles en orina de 8-isoprostano-f2α por ELISA. Los resultados de la concentración de 8-iso-PGF2α/creatinuria se analizaron mediante el paquete estadístico R. La comparación de factores de riesgo se analizó mediante ANOVA/Kruskal-Wallis. Se generaron curvas ROC para analizar el poder discriminante del biomarcador. El análisis conjunto de resultados de laboratorios y de factores de riesgo se realizó mediante regresiones logísticas multivariantes. Resultados: se evidenció prevalencia de EAP en 10 pacientes diabéticos. Como factores de riesgo se encontraron: fumar, dislipidemia, mal control metabólico, sobrepeso u obesidad. No se evidenció aumento del 8-isoprostano-f2α urinario en estos sujetos. Conclusiones: se encontró baja prevalencia de EAP en los sujetos con DM2. No se evidenció aumento del 8-isoprostano-f2α medido por ELISA en pacientes con EAP. Se recomienda ampliar el estudio con diferentes marcadores de estrés oxidativo y uso de otras técnicas. (Acta Med Colomb 2019; 44. DOI: https://doi.org/10.36104/amc.2019.1257).

Daño a células endoteliales en cultivo inducido por el isoprostano 8-iso-PGF2a / Damage to cultured endothelial cells induced by isoprostane 8-iso PGF2

INTRODUCCIÓN: el endotelio vascular posee un papel esencial en los procesos asociados a la enfermedad cardiovascular. Existe estrecha relación entre el desbalance redox de estas células y la aparición y evolución de estas enfermedades. Entre los marcadores de daño oxidativo a los lípidos de membranas se encuentra el isoprostano 8-iso-PGF2a, que aumenta en estos pacientes. OBJETIVO: evaluar el efecto del isoprostano 8-iso-PGF2a sobre células endoteliales en cultivo y la protección con la proteína de estrés térmico a-cristalina. MÉTODOS: se cultivaron células endoteliales de la línea H5V y se evaluó el efecto del isoprostano 8-iso-PGF2a y del análogo del tromboxano A2, U46619, sobre la supervivencia celular. Se evaluó el efecto protector de la proteína de estrés térmico a-cristalina a través de la incubación de los cultivos con 1 mg/ml de la proteína previo a la inducción del daño con los compuestos en estudio. RESULTADOS: la supervivencia celular disminuyó proporcional al aumento de la concentración del isoprostano y del U46619. La a-cristalina aumentó la supervivencia celular en un 20 % al preincubar los cultivos sometidos al efecto de ambos compuestos. CONCLUSIONES: el isoprostano 8-iso-PGF2a, además, de ser un marcador de daño oxidativo puede ser considerado un inductor directo de daño a las células del endotelio vascular, efecto mediado a través, de la generación de tromboxano A2 o la activación de su receptor. La proteína de estrés térmico a-cristalina, añadida de forma exógena, puede considerarse un protector endotelial.

INTRODUCTION: the vascular endothelium plays an essential role in processes associated with cardiovascular disease. There is a close relationship between redox imbalance in these cells and the appearance and evolution of such diseases. Increased isoprostane 8-iso PGF2 is among the markers of oxidative damage to membrane lipids in these patients. OBJECTIVE: evaluate the effect of isoprostane 8-iso PGF2 on cultured endothelial cells and the protection provided by -crystallin heat-shock stress protein. METHODS: endothelial cells from line H5V were cultured to evaluate the effect of isoprostane 8-iso PGF2 and thromboxane A2 analog U46619 on cell survival. An evaluation was conducted of the protective effect of -crystallin heat-shock stress protein by incubation of the cultures with 1 mg/ml of the protein prior to damage induction with the study compounds. RESULTS: cell survival decreased as isoprostane and U46619 concentration increased. -Crystallin increased cell survival by 20% upon preincubation of the cultures subjected to both compounds. CONCLUSIONS: besides being an oxidative damage marker, isoprostane 8-iso PGF2 may be considered a direct inducer of damage to vascular endothelial cells. This effect is mediated by the generation of thromboxane A2 or the activation of its receptor. Added exogenously, -crystallin heat-shock stress protein may be considered to be an endothelial protector.

Cysteinyl leukotriene and 8-isoprostane in exhaled breath condensate of asthmatic children / 国际儿科学杂志

Objective To analyze the change of cysteinyl leukotriene ( Cys-LTs) levels and 8-Isopros-tane (8-iso-PG) levels in the exhaled breath condensate (EBC) of asthmatic children from acute exacerbation to clinical remission, and investigate the role of the detection of Cys-LTs and 8-iso-PG in EBC in its severity and pathogenesis , and explore the relationship between the Cys-LTs and 8-iso-PG through measuring Cys-LTs levels and 8-iso-PG levels in the EBC of asthmatic children. Methods The outpatient or inpatient asthmatic children of the pediatrics and a group of healthy children were studied. All subjects′ EBC were collected by the R-Tube produced by American Respiratory Research. The concentration of Cys-LTs and 8-iso-PG in EBC were measured by enzyme-linked immunosorbent assay (ELISA), and compared among children in asthmatic exacerbation, asthmatic remission, and healthy condition. The relevance of their change would be explored at the same time. Results (1) Cys-LTs levels in EBC were higher in asthma exacerbation, compared to healthy controls (P0. 05 ) . ( 2 ) 8-iso-PG levels was higher in asthmatic exacerbation compared to asth-matic remission ( P<0. 05 ) . Moreover, the 8-iso-PG levels were significantly higher in asthmatic remission than in healthy controls (P<0. 05). (3) Through the relevance analysis of the Cys-LTs and 8-iso-PG levels in EBC among the three groups, Cys-LTs levels in EBC of asthmatic exacerbation significantly were correlated with 8-iso-PG levels (n1 =35, r1 =0. 61, P<0. 05), while there was no significant correlation between 8-iso-PG levels and Cys-LTs levels in asthmatic remission. Conclusion The increase of 8-iso-PG levels in EBC of bronchial asthmatic patients correlates with the disease and its control. Therefore, 8-iso-PG can be an objective indicator for asthmatic diagnosis and healing efficacy. Cy-LTs levels increase in the EBC of bronchial asthmatic according to disease severity. The two levels correlate during asthmatic exacerbation, indicating that a link be-tween airway oxidative stress and inflammation among asthmatics.

F2α-isoprostane, Na+-K+ ATPase and membrane fluidity of placental syncytiotrophoblast cell in preeclamptic women with vitamin E supplementation.

Background: The aim of our study was to analyze F2α-isoprostane level, Na+-K+ ATPase activity and placental syncytiotrophoblast cell membrane fluidity in preeclamptic women who received vitamin E supplementation. Methods: The study was conducted between September 2003 and February 2005 at Budi Kemuliaan Maternity Hospital, Central Jakarta. Samples were 6 preeclamptic women with vitamin E supplementation, 6 preeclamptic women without vitamin E supplementation and 6 normal pregnant women. The dose of vitamin E was 200 mg daily. F2α-isoprostane was measured with ELISA reader at λ of 450 nm. Cell membrane fluidity was measured by comparing the molar ratio of total cholesterol and cell membrane phospholipid concentration. The cholesterol was measured by Modular C800 using Roche reagent. Phospholipid was measured by Shimadzu RF5301PC spectrofluorometer (excitation 267 nm, emission 307 nm). Na+-K+ ATPase activity was inhibited by ouabain. Pi production was measured with Fiske and Subbarow method using spectrophotometer at λ of 660 nm. Data was analyzed using F test with one-way ANOVA. Results: Vitamin E supplementation in preeclamptic women decreased the oxidative stress, indicated by significantly lower level of F2α-isoprostane compared to those without vitamin E (26.72 ± 11.21 vs 41.85 ± 7.09 ng/mL, respectively, p = 0.017). Membrane fluidity in syncytiotrophoblast cell of preeclampsia with vitamin E group was maintained at 0.39 ± 0.08 while in those without vitamin E was 0.53 ± 0.14 (p = 0.04). Na+-K+ ATPase activity in syncytiotrophoblast cell membrane was not affected by vitamin E (p = 0.915). Conclusion: Vitamin E supplementation in preeclamptic women decreases F2α-isoprostane level and maintains cell membrane fluidity of syncytiotrophoblast cells; however, it does not increase Na+-K+ ATPase enzyme activity.

Exhaled breath condensate analysis in chronic obstructive pulmonary disease.

The increasing focus on airway inflammation in the pathogenesis of chronic obstructive pulmonary disease (COPD) has led to development and evolution of tools to measure it. Direct assessment of airway inflammation requires invasive procedures, and hence, has obvious limitations. Non-invasive methods to sample airway secretions and fluids offer exciting prospects. Analysis of exhaled breath condensate (EBC) is rapidly emerging as a novel non-invasive approach for sampling airway epithelial lining fluid and offers a convenient tool to provide biomarkers of inflammation. It has definite advantages that make it an attractive and a feasible option. It is a source of mediators and molecules that are the causes or consequences of the inflammatory process. Measurement of such markers is increasingly being explored for studying airway inflammation qualitatively and quantitatively in research studies and for potential clinical applications. These biomarkers also have the potential to develop into powerful research tools in COPD for identifying various pathways of pathogenesis of COPD that may ultimately provide specific targets for therapeutic intervention. The EBC analysis is still an evolving noninvasive method for monitoring of inflammation and oxidative stress in the airways. The limited number of studies available on EBC analysis in COPD have provided useful information although definite clinical uses are yet to be defined. Evolving technologies of genomics, proteomics, and metabonomics may provide deeper and newer insights into the molecular mechanisms underlying the pathogenesis of COPD.

Effects of N-Acetylcysteine on Nicotinamide Dinucleotide Phosphate Oxidase Activation and Antioxidant Status in Heart, Lung, Liver and Kidney in Streptozotocin-Induced Diabetic Rats

PURPOSE: Hyperglycemia increases reactive oxygen species (ROS) and the resulting oxidative stress plays a key role in the pathogenesis of diabetic complications. Nicotinamide dinucleotide phosphate (NADPH) oxidase is one of the major sources of ROS production in diabetes. We, therefore, examined the possibility that NADPH oxidase activation is increased in various tissues, and that the antioxidant N-acetylcysteine (NAC) may have tissue specific effects on NADPH oxidase and tissue antioxidant status in diabetes. MATERIALS AND METHODS: Control (C) and streptozotocin-induced diabetic (D) rats were treated either with NAC (1.5 g/kg/day) orally or placebo for 4 weeks. The plasma, heart, lung, liver, kidney were harvested immediately and stored for biochemical or immunoblot analysis. RESULTS: levels of free 15-F2t-isoprostane were increased in plasma, heart, lung, liver and kidney tissues in diabetic rats, accompanied with significantly increased membrane translocation of the NADPH oxidase subunit p67phox in all tissues and increased expression of the membrane-bound subunit p22phox in heart, lung and kidney. The tissue antioxidant activity in lung, liver and kidney was decreased in diabetic rats, while it was increased in heart tissue. NAC reduced the expression of p22phox and p67phox, suppressed p67phox membrane translocation, and reduced free 15-F(2t)-isoprostane levels in all tissues. NAC increased antioxidant activity in liver and lung, but did not significantly affect antioxidant activity in heart and kidney. CONCLUSION: The current study shows that NAC inhibits NADPH oxidase activation in diabetes and attenuates tissue oxidative damage in all organs, even though its effects on antioxidant activity are tissue specific.

Efecto del ibuprofeno y ácido acetilsalicílico sobre el deterioro cognitivo, poder antioxidante total e isoprostanos en suero / The effect of ibuprofen and acetylsalicylic acid on cognitive impairment, total antioxidant power and isoprostane serum

Introducción: Existen estudios controvertidos sobre la prevención de la enfermedad de Alzheimer y el uso de antiinflamatorios no esteroideos. El objetivo fue evaluar el efecto del ibuprofeno y ácido acetilsalicílico sobre el deterioro cognitivo, poder antioxidante total (PAT) e isoprostanos (8-iso-PGF2á) séricos. Material y métodos: Entre abril de 2004 y febrero de 2006, a 18 mujeres mayores de 55 años de edad se les realizó escrutinio con la Prueba Mínima del Estado Mental de Folstein (MMSE); Prueba Corta para la Evaluación de la Memoria y la Atención, Syndrome Kurtz Test (SKT) y Escala de Depresión Geriátrica de Yasevage. Fueron asignadas aleatoriamente para recibir 400 mg/día de ibuprofeno (n=9) o 500 mg/día de ácido acetilsalicílico (n=9) durante un año. En la visita basal, seis meses y al año se determinó PAT y 8-iso-PGF2á séricos. Resultados: A un año de intervención, en cinco mujeres (55.6%) el MMSE aumentó cuatro puntos con ácido acetilsalicílico comparado con tres (33.3%) de ibuprofeno (p=0.028). El PAT aumentó (p=0.01) y disminuyeron los 8-iso-PGF2á (p=0.01) en ambos grupos en comparación con los valores basales. Conclusiones: Ambos medicamentos mejoraron el estado cognitivo y el perfil oxidativo en la población estudiada.

INTRODUCTION: There are controversial studies on the prevention of Alzheimer's disease with nonsteroidal antiinflammatory drugs (NSAIDs). The objective of this study was to evaluate the effect of ibuprofen and acetylsalicylic acid on cognitive impairment, serum total antioxidant power (TAP) and isoprostane (8-iso-PGF2alpha). METHODS: From April 2004 to February 2006, a Folstein mini-mental state (MMSE), Syndrome Kurtz Test (SKT) and a geriatric depression scale (Yasevage) were applied to eighteen, 55-56 years old eligible women. All women (n= 18) with normal cognitive state were randomized to ibuprofen 400 mg per day (n= 9) and acetylsalicylic acid 500 mg per day (n= 9) for one year. Serum TAP and 8-iso-PGF2alpha were performed at baseline, after six months and one year of treatment. RESULTS: After one year of treatment with acetylsalicylic acid five women (55.6%) raised their score 4 points in MMSE compared with 3 points increased (33.3%) showed by the ibuprofen group. TAP increased (p=0.01) and 8-iso-PGF2alpha reduced (p=0.01) in both groups compared with baseline. CONCLUSIONS: Both drugs improved the cognitive state andoxidative status of our population.

Exposição aguda ao material particulado total em suspensão proveniente de diferentes fontes e suas repercussões nas respostas inflamatórias, sistêmica e local, em ratos / Acute exposure to total suspended particles from different sources and their impacts on both, systemic and local inflammatory responses, in rats

A poluição do ar está associada ao aumento da morbimortalidade pulmonar e cardiovascular. O objetivo desse estudo foi o de avaliar e comparar a resposta inflamatória à exposição a três diferentes tipos de particulado total em suspensão (fonte automativa, industrial e da queimada da cana de açúcar), às baixas concentrações e de forma aguda; comparando-as ao grupo controle (black carbon). Os resultados mostraram uma maior inflamação pulmonar induzida pela fonte automotiva, uma maior oxidação cardíaca induzida pela biomassa de queima de cana de açúcar, e uma mais intensa alteração na contagem de células sanguíneas nas partículas industriais. Concluímos que o aerossol ambiente pode induzir inflamação subclínica a baixas concentrações, e que a composição do particulado afetou a magnitude e o tipo de resposta.

Air pollution is associated with increased pulmonary and cardiovascular morbidity and mortality. The objective of this study is to assess and to compare inflammatory responses of acute exposures to total suspended particles generated from three different sources (automotive, burning cane sugar and industrial) at lower concentrations, comparing to the control group (carbon black). The results showed a great pulmonary inflammation induced by automotive source, a strong cardiac oxidation induced by sugar cane burning source, and an intense alteration in the blood cells count induced by the industrial source. We concluded that the environmental aerosol can induce sub clinic inflammation at lower concentrations and that the composition of total suspended particles affects magnitude and kind of responses.

Oxidatvive Stress in Rat Model of Preeclampsia and Clinical Correlates

There are growing evidences suggesting a pivotal role of oxidative stress in the pathophysiology of preeclampsia. We investigated oxidative stress in the rat model of preeclampsia, and in clinical cases. Pregnant female rats were injected intraperitoneally with deoxycorticosterone acetate (DOCA) and given 0.9% saline as drinking water during their pregnancy. We assessed plasma F2-isoprostane (8-iso-PGF2alpha) and malondialdehyde (MDA) in a rat model, and the same markers in the plasma of maternal blood and fetal cord blood in pregnant women with preclampsia. Blood samples from the umbilical arteries and veins were collected separately. The concentrations of MDA were increased in the preeclampsia groups of animal and humans, compared with the control group; it was significantly increased in the umbilical artery and vein of the preeclampsia group. The concentrations of F2-isoprostane were elevated in the preeclampsia groups of animal and humans, compared with the control group, and the increase in F2-isoprostane concentration was prominent in the umbilical vein than umbilical artery of the preeclampsia group. Therefore, it appears that the placenta has an important role in the pathophysiology of preeclampsia, and the F2-isoprostanes of the umbilical vein may serve as a relatively reliable marker for ischemic/hypoxic injury to the fetus during the perinatal period.

Umbilical Cord Arterial Concentrations of Isoprostane(8-iso-PGF2alpha) in Newborn Infants

PURPOSE: We measured the umbilical cord arterial concentrations of isoprostane(8-iso-PGF2alpha) and intended to decide whether the umbilical cord arterial concentrations of isoprostane could be used as a useful parameter for lipid peroxidation in newborn infants. METHODS: The isoprostane and malondialdehyde(MDA) concentrations of the umbilical cord were measured by enzyme immunoassay and TBARS(thiobarbituric acid reactive substance) assay in 33 preterm and 28 term infants, respectively. The concentrations of isoprostane and MDA were compared between preterm infants and term infants, and were analysed for association with perinatal risk factors and neonatal complications. RESULTS: Umbilical cord arterial concentrations of isoprostane were 704.7+/-635.6 pg/mL and 421.9+/-306.5 pg/mL in preterm and term infants, respectively. Umbilical cord arterial concentrations of MDA were 44.0+/-22.9 micrometer/L and 26.2+/-10.7 micrometer/L in preterm and term infants, respectively. Umbilical cord arterial concentrations of isoprostane and MDA in preterm infants were significantly higher than those in term infants(P<0.05). The umbilical cord arterial concentrations of isoprostane were significantly associated with perinatal risk factors such as fetal distress, oligohydramnios, and breech delivery in preterm infants and pregnancy-induced hypertension in term infants(P<0.05). CONCLUSION: Umbilical cord arterial concentrations of isoprostane in preterm infants were higher than those in term infants, and those are significantly associated with some perinatal risk factors.

The Effect of Repetitive Hypoxia on Production of Lipid Peroxidation in Newborn Rat Brain

PURPOSE: Among many pathophysiologic mechanisms of hypoxic-ischemic brain injury, reactive oxygen species (ROS) cause or contribute to brain damage relates to their ability to attack the fatty acid moiety of plasma and subcellular membranes. Because ROS are generated by hypoxia-ischemia especially during reperfusion period of recovery, repetitive hypoxia-reoxygenation in newborn brain may result in more severe damage than a similar single insult. It is to determine whether repetitive hypoxia-reoxygenation may produce more ROS than a similar single insult in newborn rat brain. METHODS: We compared the production of lipid peroxidation in 3 days old rat brain following normoxia, repetitive hypoxia-reoxygenation and an equal duration of sustained hypoxia-reoxygenation by measuring 8-isoprostane-F2alpha. 8-isoprostane-F2alpha is free radical catalyzed metabolites of arachidonic acid, which is produced independent of cyclooxygenase. RESULTS: Compared to a single duration hypoxia-reoxygenation, repetitive hypoxia- reoxygenation produce more ROS (8-isoprostane-F2alpha) in newborn rat brain (P < 0.005). CONCLUSION: It can be speculated that repetitive hypoxia is more detrimental than equal duration of single insult in new born rat brain. Relations between increased ROS production and brain injury following repetitive hypoxia-reoxygenation should be evaluated.

Efficacy of midtrimester amniotic fluid 8-isoprostane measurement in the prediction of severe preeclampsia / 대한산부인과학회잡지

OBJECTIVE: The objective of this study is to investigate whether the amniotic fluid 8-isoprostane levels at the time of genetic amniocentesis is a marker for severe preeclampsia. METHODS: A case-control study was conducted to compare mid-trimester concentrations of amniotic fluid 8-isoprostane in women with normal pregnancies (n=22) and in those who subsequently developed severe preeclampsia (n=22). Amniotic fluid was also obtained by amniocentesis from another women who already developed severe preeclampsia (n=22) after 20 weeks of gestation. The 8-isoprostane levels were measured by enzyme-linked immunoassay. For statistical analysis, nonparametric tests and receiver-operating characteristic curves were used where appropriate. Statistical significance was considered when probability was <0.05. RESULTS: The levels of midtrimester amniotic fluid 8-isoprostane were found to be significantly decreased in the women who subsequently developed severe preeclampsia in comparison with those who underwent normal pregnancies (P<0.05). The levels of 8-isoprostane in preeclamptic amniotic fluid were found to be significantly decreased with respect to that in midtrimester amniotic fluid (P<0.05). No relationship was found between the midtrimester amniotic fluid 8-isoprostane levels and preeclampsia with small-for- gestational-age. After the onset of severe preeclampsia, however, the amniotic fluid 8-isoprostane levels were significantly decreased in women with small-for-gestational-age. The midtrimester amniotic fluid 8-isoprostane level of 170 pg/ml had a sensitivity of 72.7% and a specificity of 63.6% in the prediction of severe preeclampsia. CONCLUSION: The midtrimester amniotic fluid 8-isoprostane levels may predict the later occurrence of severe preeclampsia. This study not only presents a new information that 8-isoprostane is detected in human amniotic fluid, but also provides a convincing evidence that a subclinical process from faulty placentation in early gestation is important for the occurrence of preeclampsia. Further studies are warranted to determine which mechanism causes such decrease in amniotic fluid 8-isoprostane in preeclampsia.

Changes in the Levels of Eicosanoids and Isoprostane (8-iso-PGF2alpha) in the Newborn Rat Brain after Hypoxic-Ischemic Injury

PURPOSE: The changes in the levels of eicosanoids and isoprostane (8-iso-PGF2alpha) were investigated in brain tissue of 7 day-old rats after hypoxic-ischemic (HI) injury. METHODS: The 7 day-old newborn rats underwent right unilateral common carotid artery ligation followed by exposure to hypoxia with 8% oxygen for 150 minutes. There after, the pups were decapitated during reoxygenation 21% period of 0, 1, 6, 24, and 72 hours and their cerebral hemisheres were dissected through sagittal plane. Ipsilateral and contralateral cerebral hemesheres to common carotid artery ligation were used to determine the water content for estimation of severity of brain edema (n=5) and to measure the levels of eicosanoid and isoprostane (n=7). The levels of 6-keto-PGF1alpha, TXB2, and PGE2 were measured by RP-HPLC (reversed-phase high-performance liquid chromatography) and the levels of isoprostane (8-iso-PGF2alpha) were measured by enzyme immunoassay. The changes of eicosanoid and isoprostane levels during reoxygenation period were observed and comparisons between ipsilateral and contralateral hemispheres were done. RESULTS: The edema of ipsilateral cerebral hemesheres to common carotid artery ligation was more severe than that of contralateral cerebral hemisheres (P<0.05). The levels of 6-keto-PGF1alpha, TXB2, and PGE2 were found to increase during the early period of reoxygenation after HI insult, peak at 1 hour, and then decrease to the control levels at 72 hour (P<0.05). But, the levels of 8-iso-PGF2alpha did not significantly increase during the period of reoxygenation. The levels of 6-keto-PGF1alpha, TXB2, and PGE2 of ipsilateral hemispheres had a tendency to be higher than those of contralateral hemispheres during the initial 6 hour reoxygenation period, but the levels of 8-iso-PGF2alpha of ipsilateral hemispheres were significantly higher than those of contralateral hemispheres during the relatively later reoxygenation period (P<0.05). CONCLUSION: Reoxygenation after hypoxic-ischemic injury increased the levels of 6-keto-PGF1alpha, TXB2, and PGE2 in 7 day-old rat brain during the early period of reoxygenation, but the levels of isoprostane (8-iso-PGF2alpha) were not significantly increased during the reoxygenation period after HI injury.

Simultaneous HPLC analysis of arachidonic acid metabolites in biological samples with simple solid phase extraction

A reversed-phase high-performance liquid chromatography (RP-HPLC) has been developed to analyze the metabolites of arachidonic acid based on the specificities of ultraviolet absorption of these various metabolites and is sensitive to the nanogram level. This procedure makes it possible to extract complex mixtures of eicosanoids efficiently with a single step and to analyze them simultaneously by RP-HPLC from biological samples using octadesylsilyl silica extraction column and PGB2 as an internal standard. The cyclooxygenase, products (prostaglandin (PG)D2, PGE1, PGE2, PGF1alpha, PGF2alpha, 6-keto-PGF1alpha, and thromboxane B2 (TXB2)) and lipid peroxidation product, isoprostanes, of arachidonic acid were monitored by one isocratic HPLC system at 195 nm wavelength. The lipoxygenase products (leukotriene(LT)B4, LTC4, LTD4, and 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, 15-HETE) were measured by another isocratic HPLC system at 280 nm for LTs and 235 nm for HETEs. This method provides a simple and reliable way to extract and assess quantitatively the final arachidonic acid metabolites.