Evaluation of Resuscitation Fluids for Maintaining Human Endothelial Cell Antioxidant Status In Vitro

With hemorrhage a major cause of death in trauma patients, infusing resuscitation fluids is still considered an important treatment strategy. The endothelial cell lining all blood vessels is the first cell type exposed to these intravenous solutions and at the highest concentrations. Crystalloid resuscitation fluids (RF) such as Ringer's solution, normal saline (NS), lactated Ringer's (LR) and colloids such as 5% albumin (Buminate 5%, Baxter Healthcare, Westlake Village, CA) and Hextend (BioTime, Berkeley, CA) have shown similar hemodynamic responses in animal hemorrhage models. However, choosing the most efficacious resuscitation fluid might be more rationally based on maintenance of endothelial antioxidant status, which may be adversely affected by restrictions of cellular metabolites that occur with fluids intended mainly to maintain intravascular volume. In this study, human umbilical vein endothelial cells (HUVEC) were exposed to various resuscitation fluids, and total antioxidant status was assessed. Pooled gender‐mixed (HUVEC) were cultivated on 75‐cm 2 culture flasks in Medium 131, and endothelial supplements. Stock cultures were cultivated at 37°C in a humidified atmosphere of 3% oxygen and 5% CO 2 with medium changes every 2 days until confluent. Prior to an experiment, cells were subcultivated with Trypsin/EDTA onto Costar® 6‐well or 96‐well multiplates at 5000 cells/cm 2 and used when confluent. Ringer's, LR, NS, 5% Alb, Hextend, and also 10% human plasma (George King Bio‐Medical, Overland, KA), were buffered with 10 mM Hepes (pH 7.3) and made 0.025% in phenol red to monitor pH. Replicate cultures in multiplates were exposed to each fluid for 3 hrs sealed in a humidified box at 37°C. ATP levels in the cells were measured at the end of exposure utilizing the luciferase reaction from the CellTiter‐Glo Luminescent Cell Viability Assay. The number of cells per well was monitored with the PicoGreen DNA assay. Reactive Oxygen Species (ROS) Generation was estimated with CellROX® Deep Red Reagent (Life Technologies, Grand Island, NY). Incubation with NS resulted in about a 20% reduction in cellular DNA compared with control cells, however ATP levels were generally maintained but reduced 40% in albumin treated cells compared with controls grown in complete medium. Morphologically it appeared that apoptosis occurred in cells treated with Hextend. A marked elevation in ROS was observed in albumin treated cells compared to the other groups, whereas ROS production in other cells was about 50% of controls. Taken together these data suggest that RF appear to affect mitochondria in endothelial cells as they regulate apoptosis and ATP production and they are the principal site of ROS production. Support or Funding Information This study was funded by the US Army Medical Research and Materiel Command