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

doi:10.1016/j.trsl.2007.03.013

Translational Research

Volume 150, Issue 4, October 2007, Pages 253-265

https://doi.org/10.1016/j.trsl.2007.03.013 Get rights and content

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.

Section snippets

Chemicals

Tetrasodium salt of EB (tetrasodium 4-amino-6-[4-[4-(8-amino-1-hydroxy-5,7-disulfonato-naphthalen-2-yl)diazenyl-3-methyl-phenyl]-2-methyl-phenyl]diazenyl-5-hydroxy-naphthalene-1,3-disulfonate; MW 960.8), bacterial LPS (serotype O127:B8), and bovine serum albumin (BSA) (fraction V, low-endotoxin; MW 66,000) were from Sigma Chemical Co. (St. Louis, Mo). All other chemicals were analytical or cell-culture grade and were obtained from various commercial sources. Evans Blue dye conjugated to albumin

A lung-specific tissue-specific correction factor

To initially test whether a correction factor (as in Linderkamp et al¹⁸) can at all be obtained from mouse lung homogenates, we recorded absorbances at both 620 nm and 740 nm of such homogenates prepared as described in the Materials and Methods section, and we calculated the least-squares fit between the paired absorbance recordings for each sample (Fig 1). We found that a significant fit (P < 0.01) could be obtained even with a small sample size (N = 18), in contrast to the large N (147)

Discussion

These data clearly demonstrate that the widely used EB dye extravasation method to quantify barrier dysfunction can be improved in terms of repeatability and sensitivity by using tissue-specific turbidity correction factors. One clue as to why that is so may be found by comparing the commonly used serum correction factor¹⁸ with the lung-specific ones experimentally determined in this manuscript. Such a comparison shows that, although the slopes are not significantly different, the intercepts of

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Acute lung injury (ALI) remains a serious challenge in the intensive care unit. Inflammation plays a key role in the progression of ALI. Chrysin (CHR) is a natural flavonoid with anti-inflammatory functions. We investigated the anti-inflammatory effects in a mouse model of ALI induced by lipopolysaccharide (LPS), and identified the underlying mechanisms of its action. Following CHR administration, mice were challenged with LPS intratracheally for 6 h to induce ALI. Compared to mice challenged with LPS alone, the presence of CHR showed a reduction in the development of lung injuries, as confirmed by histopathological observation. Pre-treatment with CHR attenuated inflammation by reducing the production of myeloperosidase (MPO), and pro-inflammatory cytokine levels in the lung and bronchoalveolar lavage fluid (BALF). Furthermore, CHR improved lung edema by reducing the vascular permeability, as demonstrated by less evans blue staining in the lung tissue and low levels of protein in BALF. In addition, our results proved that CHR improved the antioxidant capacity by increasing the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the lung tissue. Results of western blot assays suggested that CHR suppressed the LPS-induced expression of glucose-regulated protein 78 (GRP78) and phosphorylated inositol-requiring enzyme 1α (p-IRE1α). We also found that CHR suppressed the expression of thioredoxin interaction protein (TXNIP), nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and cleaved caspase-1. In conclusion, CHR improved vascular permeability and mitigated the inflammatory response of lung tissue by suppressing the IRE1α/TXNIP/NLRP3 pathway, thereby alleviating LPS-induced ALI in the lungs of mice.
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: Supported by the NIH/NHLBI Grant P50 HL 73994 (to J.G.N.G.).

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

Section snippets

Chemicals

A lung-specific tissue-specific correction factor

Discussion

Clin Chim Acta

J Neurosci Meth

Ann Thorac Surg

Resp Physiol

J Biol Chem

J Lab Clin Med

J Lab Clin Med

Evans blue dye as a marker of albumin clearance in cultured endothelial monolayer and isolated lung

J Appl Physiol

In vivo vascular leakage assay

Clinical studies of the blood volumeI. Clinical application of a method employing the azo dye “Evans Blue” and the spectrophotometer

Clin Invest

The binding of T-1824 and structurally related diazo dyes by the plasma proteins

Am J Physiol

Equilibrium and kinetic properties of the Evans Blue-albumin system

Am J Physiol

Combination of Evans Blue with plasma protein: its significance in capillary permeability studies, blood dye disappearance curves, and its use as a protein tag

Am J Physiol

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