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Identification of Immunoglobulins that Recognize 3-Nitrotyrosine in Patients with Acute Lung Injury after Major Trauma

Stokes Research Institute and Department of Pediatrics and Pharmacology, Children's Hospital of Philadelphia and University of Pennsylvania School of Medicine; Pulmonary, Allergy and Critical Care Division, Department of Medicine, and Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; and Departments of Cell Biology and Cardiovascular Medicine, Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic Foundation, Cleveland, Ohio

Correspondence and requests for reprints should be addressed to Leonor Thomson, M.D., Ph.D., Stokes Research Institute, Children's Hospital of Philadelphia, 416D Abramson Center, 34th Street and Civic Center Blvd., Philadelphia, PA 19104-4318. E-mail: thomson{at}email.chop.edu. Permanent address: Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay.

Abstract

Tyrosine nitration is a nitric oxide–derived post-translational modification of proteins. Elevated levels of specific plasma proteins modified by tyrosine nitration have been detected during acute and chronic inflammatory conditions, including acute lung injury (ALI). In the present study we examined whether circulating immunoglobulins against nitrated proteins are present in the plasma of subjects with clinically documented ALI. Affinity chromatography using covalently linked 3-nitrotyrosine was employed to identify plasma proteins that bind to this unusual amino acid. Western blotting and liquid chromatography-tandem mass spectrometry of in-gel digested protein bands revealed that the major proteins eluted from the affinity column were IgM and IgG. An enzyme-linked immunosorbent assay (ELISA) based on competition of horseradish peroxidase–derivatized 3-nitrotyrosine binding to plasma with unlabeled 3-nitrotyrosine was developed and validated. Using this ELISA, the levels of immunoglobulins that recognize 3-nitrotyrosine were significantly higher in the plasma of subjects with ALI compared with both normal control subjects and subjects with major trauma who did not develop ALI (0.36± 0.14 versus 0.03 ± 0.05, and 0.25 ± 0.15; P < 0.001 and P = 0.006, respectively). These data indicate that tyrosine-nitrated proteins induce the production of specific immunoglobulins during acute phase response and inflammation.

Key Words: tyrosine nitration • oxidative stress • immunoglobulins • affinity chromatography • biological markers

CLINICAL RELEVANCE This study provides the first evidence that immunoglobulins against tyrosine-nitrated proteins are present in the plasma of subjects with acute lung injury (ALI). This finding may provide a novel biomarker to ascertain risk for the development of ALI.

 

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