ABSTRACT
The monohydroxyeicosanoid 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), which is derived from oxygenation of arachidonic acid by 12-lipoxygenase, is one of the major metabolites in platelets. In a recent study, we have showed that this eicosanoid stimulated basal sickle-red-cell-endothelial-cell adhesion. To understand the pathophysiologic significance of 12-HETE, we measured the levels of this eicosanoid in plasma and urine from children with sickle cell disease. We found that as compared with controls, plasma 12-HETE levels are increased in patients with sickle-cell disease in the steady state, and are increased further during vaso-occlusive crises. Urinary 12-HETE levels were also increased during the steady state. We also assessed plasma levels of soluble P-selectin (another potential marker for platelet activation), and found changes in the levels of this marker similar to those seen with plasma 12-HETE. In additional studies, we found that 12-HETE enhanced hypoxia-induced sickle-red-cell-endothelial adherence, and that this effect was mediated by potentiation of agonist-induced upregulation of the expression of the mRNA for vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells. Because 12-HETE appears to enhance both basal and agonist-induced sickle-red-cell adhesion, this metabolite could potentially play a role in the pathogenesis of the vaso-occlusive crisis (VOC) in sickle-cell disease.
Subject(s)
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/physiology , Anemia, Sickle Cell/physiopathology , Arterial Occlusive Diseases/physiopathology , 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/blood , 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/urine , Adolescent , Adult , Anemia, Sickle Cell/blood , Anemia, Sickle Cell/urine , Animals , Arterial Occlusive Diseases/blood , Arterial Occlusive Diseases/urine , Cattle , Cell Adhesion , Child , Child, Preschool , Endothelium, Vascular/pathology , Gene Expression Regulation/physiology , Humans , Middle Aged , P-Selectin/blood , RNA, Messenger/genetics , Vascular Cell Adhesion Molecule-1/geneticsABSTRACT
Analysis of the catechol-O-methyltransferase (COMT) enzyme in human RBC lysates from 15 samples exhibiting inherited variations in level of activity and thermal stability was performed. Electrophoretic blotting and immune fixation was carried out following sodium dodecyl sulfate polyacrylamide gel electrophoresis or isoelectric focusing of lysate protein. These techniques did not reveal a major structural alteration of the protein that could account for the observed variation in enzyme activity or thermal stability. Future studies utilizing molecular genetic techniques should make it possible to determine the basis for inherited variations in human RBC COMT activity and thermal stability.
