Development and application of novel ELISA-based analytical tools for assessing nitroxidative distress biomarkers in ischemic stroke: implications for improved diagnosis and clinical management.

Ischemic cerebrovascular accident (iCVA) is a public health issue, whose subjacent events involve the development of nitroxidative distress. Identifying biomarkers that assist in the diagnosis of this disease has clinically relevant implications. The aim of this study was to develop an analytic tool for measuring nitroxidative distress biomarkers, intended for application in clinical practice to enhance patient healthcare. Three enzyme linked immunosorbent assays (ELISA) were developed, with different detection objectives. One of them, in a sandwich format, quantifies the amount of fibrinogen in human plasma, an important glycoprotein involved in the blood coagulation process, contributing to thrombus formation and thereby participating in the mechanism of ischemic stroke. Another ELISA, also in a sandwich format, detects the presence of nitrotyrosine residues in fibrinogen from human plasma, a nitroxidative posttranslational modification resulting from the attack of peroxynitrite by-products on tyrosine residues present in proteins. The third one, in inhibition format, determines human plasma nitrotyrosine total content and was used to analyze human plasma samples from control and iCVA patients. Those two groups of plasma samples were analyzed using inhibition ELISA, revealing statistically significant differences in their nitrotyrosine content and molar ratios of nitrotyrosine to fibrinogen, which were higher in the iCVA group. This study provides evidence that nitroxidative distress occurs in ischemic stroke, as indicated by the detection of the biomarker nitrotyrosine. This finding supports other studies that also identified nitrotyrosine in ischemic stroke, through several different methods. This specific ELISA method is applicable for the rapid analysis of clinical samples, making it a potential clinical tool for assessing iCVA patients.

Evaluación de parámetros inflamatorios, trombóticos y variantes genéticas en pacientes con ataque cerebrovascular isquémico que se asisten en un hospital de Uruguay

Introducción: El ataque cerebrovascular es la segunda causa de muerte en adultos en el mundo occidental y una de las principales causas de discapacidad permanente, aumentando su frecuencia con la edad, el 85 % es de tipo isquémico. Objetivos: Analizar parámetros trombofílicos, hipofibrinolíticos y genéticos en pacientes con ataque cerebrovascular isquémico y evaluar la posible asociación de estos con factores de riesgo cardiovascular. Metodología: Se utilizó un cuestionario para evaluar la presencia de factores de riesgo cardiovascular en 114 pacientes incluidos en el estudio con diagnóstico de ataque cerebrovascular isquémico. Proteína C y antitrombina fueron determinados mediante métodos cromogénicos, resistencia a la proteína C activada e inhibidor lúpico mediante métodos coagulométricos y proteína S libre, inhibidor del activador del plasminógeno-1, homocisteína y lipoproteína (a) por métodos inmunoquímicos. Fibrinógeno fue determinado por coagulometría y proteína C reactiva por inmunoturbidimetría, ambos contra un grupo control. Las variantes genéticas factor V Leiden, protrombina G20210A, rs1205 (gen PCR), rs1800779 (gen NOS3) y rs2257073 (gen ASB10) fueron analizadas mediante real-time PCR, comparando los últimos tres con una población de referencia. La alteración de las frecuencias de las variables fue determinada por análisis estadístico chi-cuadrado. Resultados: Tres de los cuatro pacientes jóvenes estudiados presentaron indicadores de trombofilia. El resto de los parámetros alterados fueron homocisteína 30.1% (22.4-39.1), lipoproteína (a) 32.1% (24.1-41.4), inhibidor del activador del plasminógeno-1 36.0% (27.8-45.1), fibrinógeno 12.3% (7.5-19.6) y proteína C reactiva 78.1% (69.6-84.7). Se encontró asociación (p < 0.05) entre ciertos factores de riesgo cardiovascular y los parámetros evaluados como hipertensión/proteína C reactiva, dislipemia/lipoproteína (a), arritmia/lipoproteína (a) y arritmia/fibrinógeno. Para pacientes con ataque cerebrovascular isquémico solo la variante rs1205 mostró una frecuencia más alta del alelo T. Conclusiones: Este estudio revela la importancia de analizar la trombofilia en pacientes jóvenes, especialmente en aquellos sin factores de riesgo cardiovascular, así como el rol de la hipofibrinolisis, inflamación y algunas variantes genéticas en el desarrollo de ataque cerebro vascular isquémico.

Introduction: Stroke is the second cause of death in adults in the Western world and one of the main causes of permanent disability, increasing in frequency with age; 85% are ischemic. Objectives: To analyze thrombophilic, hypofibrinolytic, inflammatory, and genetic parameters in patients with ischemic stroke and evaluate possible associations with vascular risk factors. Methodology: Questionnaires were used to evaluate vascular risk factors in 114 patients included in the study with ischemic stroke diagnosis. Protein C and Antithrombin were determined by chromogenic assays, Activated Protein C Resistance and Lupus Anticoagulant were determined with by coagulometry and Free Protein S, Plasminogen activator inhibitor-1, Homocysteine and Lipoprotein (a) by immunochemistry. Fibrinogen was assayed by coagulometry and C-reactive protein by immunoturbidimetry, both against a control group. Factor V Leiden, Prothrombin G20210A, rs1205 (CRP gene), rs1800779 (NOS3 gene) and rs2257073 (ASB10 gene) genetic variants were analyzed by Real-Time PCR, comparing the last three with a reference population. Alteration frequencies of the variables were determined by chi-square statistical analysis. Results: Three out of four of the young patients studied presented indicators of thrombophilia. The rest of the altered parameters were Homocysteine 30.1% (22.4-39.1), Lipoprotein (a) 32.1% (24.1-41.4), Plasminogen activator inhibitor-1 36.0% (27.8-45.1), Fibrinogen 12.3% (7.5-19.6) and C-reactive protein 78.1% (69.6-84.7). Associations were found (p<0.05) between certain vascular risk factors and parameters evaluated, namely hypertension/C-reactive protein, dyslipidemia/lipoprotein (a), arrhythmia/lipoprotein (a) and arrhythmia/fibrinogen. For ischemic stroke patients only the genetic variant rs1205 showed higher frequency of the T allele. Conclusions: This study reveals the importance of analyzing thrombophilia in young patients, especially those without vascular risk factors, as well as the role of hypofibrinolysis, inflammation and some genetic variants in the development of ischemic stroke.

Introdução: O AVC é a segunda causa de morte em adultos no mundo ocidental e uma das principais causas de incapacidade permanente, aumentando de frequência com a idade; 85% são isquémicos. Metas: Analisar os parâmetros trombofílicos, hipofibrinolíticos e genéticos em pacientes com acidente vascular cerebral isquêmico e avaliar a possível associação com fatores de risco cardiovascular. Metodologia: Um questionário foi utilizado para avaliar a presença de fatores de risco cardiovascular em 114 pacientes incluídos no estudo com diagnóstico de acidente vascular cerebral isquêmico. Proteína C e antitrombina foram determinadas por métodos cromogênicos, resistência à proteína C ativada e inibidor de lúpus por métodos coagulométricos e proteína S livre, inibidor do ativador do plasminogênio-1, homocisteína e lipoproteína (a) por métodos imunoquímicos. O fibrinogênio foi determinado por coagulometria e a proteína C-reativa por imunoturbidimetria, ambos contra um grupo controle. As variantes genéticas fator V Leiden, protrombina G20210A, rs1205 (gene PCR), rs1800779 (gene NOS3) e rs2257073 (gene ASB10) foram analisadas por PCR em tempo real, comparando as três últimas com uma população de referência. As frequências de alteração das variáveis ​​foram determinadas pela análise estatística qui-quadrado. Resultados: Três dos quatro pacientes jovens estudados apresentaram indicadores de trombofilia. O resto dos parâmetros alterados foram homocisteína 30,1% (22,4-39,1), lipoproteína (a) 32,1% (24,1-41,4), inibidor do ativador de plasminogênio-1 36,0% (27,8-45,1), fibrinogênio 12,3% (7,5-19,6) e proteína C reativa 78,1% (69,6-84,7). Foi encontrada associação (p<0,05) entre alguns fatores de risco cardiovascular e os parâmetros avaliados como hipertensão/proteína C reativa, dislipidemia/lipoproteína (a), arritmia/lipoproteína (a) e arritmia/fibrinogênio. Para pacientes com acidente vascular cerebral isquêmico apenas a variante rs1205 apresentou maior frequência do alelo T. Conclusões: Este estudo revela a importância de analisar a trombofilia em pacientes jovens, especialmente aqueles sem fatores de risco cardiovascular, bem como o papel da hipofibrinólise, inflamação e algumas variantes genéticas no desenvolvimento do acidente vascular cerebral isquêmico.

Identification and relative quantification of 3-nitrotyrosine residues in fibrinogen nitrated in vitro and fibrinogen from ischemic stroke patient plasma using LC-MS/MS.

Ischemic stroke is one of the leading causes of death and disability worldwide. This acute vascular event interferes with blood supply to the brain and induces a burst of free radicals such as nitric oxide and superoxide, producing peroxynitrite, a precursor of strong nitrating agents. Fibrinogen is one of the most abundant plasma proteins; it plays a role in the hemostatic system, mediating clot formation, which can be affected by nitrotyrosine formation. We hypothesized that nitration of fibrinogen by ONOOH and ONOOCO2- radical products could be one of the early events of the ischemic stroke, and protein-bound 3-nitrotyrosine could be a potential biomarker for diagnosis and/or prognosis of this condition. A targeted mass spectrometry approach was developed to analyze the nitration of fibrinogen and its association with ischemic stroke. First, a comprehensive mapping of 3-nitrotyrosine locations and their relative quantification was performed by LC-MS/MS, using in vitro nitrated fibrinogen samples. Twenty different 3-nitrotyrosine residues were identified on fibrinogen nitrated in vitro, varying with the peroxynitrite tofibrinogen molar ratio used. Nine tyrosine residues that were consistently modified at different treatment ratios were chosen to perform a targeted LC-MS/MS analysis in clinical samples. Enriched fibrinogen fractions from clinical samples from 24 ischemic stroke and 12 patients with non-inflammatory conditions were analysed with this method. Three of the nine tyrosine residues analysed (ßY452, ßY475 and γY380) showed a significant difference between the ischemic stroke and non-inflammatory disease groups. ROC curve analysis suggested an association of these residues either individually or in combination with ischemic stroke. Different tyrosine nitration patterns were also observed in fibrinogen modified in vitro and in vivo, suggesting differences in the nitration process in these situations. This is the first study showing a putative association between the nitration profile of specific tyrosine residues in human fibrinogen and ischemic stroke.