Re-evaluation of Evans Blue dye as a marker of albumin clearance in murine models of acute lung injury

Quantifying the amount of albumin conjugated to Evans Blue dye (EBA) fluxing across organ-specific vascular barriers is a popular technique to measure endothelial monolayer integrity in rodent and murine models of human diseases. We have re-evaluated this technique with a specific focus of assessing the commonly used turbidity correction factors. These factors, originally developed and required in a spectrophotometric assay to quantify Evans Blue (EB) in human infant or dog serum, produced negative numbers when applied to murine models of acute lung injury. We next sought to determine tissue-specific correction factors for murine tissues and experimentally derived such factors, which allow estimation of the amount of EB in formamide extracts of murine tissues as positive numbers. Utilization of a best fit correction factor in a lipopolysaccharide (LPS)-induced murine model of acute lung injury resulted in significantly increased sensitivity and repeatability of the EB dye tissue extravasation assay. This factor may be of significant utility in animal models of inflammatory injury.