Department of Neurological Surgery, Division of Surgical Sciences, Vanderbilt University Medical Center, Nashville, TN, USA.
Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA; Department of Pathobiological Sciences, School of Veterinary Medicine, Louisina State University and Agricultural and Mechanical College, Baton Rouge, LA, USA.
Blood Cells Mol Dis. 2023 Jan;98:102699. doi: 10.1016/j.bcmd.2022.102699. Epub 2022 Aug 15.
Elevated levels of circulating cell-free hemoglobin (CFH) are an integral feature of several clinical conditions including sickle cell anemia, sepsis, hemodialysis and cardiopulmonary bypass. Oxidized (Fe, ferric) hemoglobin contributes to the pathophysiology of these disease states and is therefore widely studied in experimental models, many of which use commercially sourced CFH. In this study, we treated human endothelial cells with commercially sourced ferric hemoglobin and observed the appearance of dense cytoplasmic aggregates (CAgg) over time. These CAgg were intensely autofluorescent, altered intracellular structures (such as mitochondria), formed in multiple cell types and with different media composition, and formed regardless of the presence or absence of cells. An in-depth chemical analysis of these CAgg revealed that they contain inorganic components and are not pure hemoglobin. To oxidize freshly isolated hemoglobin without addition of an oxidizing agent, we developed a novel method to convert ferrous CFH to ferric CFH using ultraviolet light without the need for additional redox agents. Unlike commercial ferric hemoglobin, treatment of cells with the fresh ferric hemoglobin did not lead to CAgg formation. These studies suggest that commercially sourced CFH may contain stabilizers and additives which contribute to CAgg formation.
循环无细胞血红蛋白 (CFH) 水平升高是几种临床情况的一个固有特征，包括镰状细胞贫血、败血症、血液透析和心肺旁路。氧化（Fe、三价铁）血红蛋白促成了这些疾病状态的病理生理学，因此在实验模型中得到了广泛研究，其中许多模型使用商业来源的 CFH。在这项研究中，我们用商业来源的三价铁血红蛋白处理人内皮细胞，随着时间的推移观察到密集的细胞质聚集物 (CAgg) 的出现。这些 CAgg 具有强烈的自发荧光，改变了细胞内结构（如线粒体），在多种细胞类型和不同的培养基组成中形成，并且无论是否存在细胞都会形成。对这些 CAgg 的深入化学分析表明，它们含有无机成分，而不是纯血红蛋白。为了在不添加氧化剂的情况下氧化刚分离的血红蛋白，我们开发了一种新方法，使用紫外线将亚铁 CFH 转化为三价 CFH，而无需额外的氧化还原试剂。与商业三价血红蛋白不同，用新鲜的三价血红蛋白处理细胞不会导致 CAgg 的形成。这些研究表明，商业来源的 CFH 可能含有稳定剂和添加剂，这些稳定剂和添加剂促成了 CAgg 的形成。
