Patterson C E, Rhoades R A, Garcia J G
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
J Appl Physiol (1985). 1992 Mar;72(3):865-73. doi: 10.1152/jappl.1992.72.3.865.
Determination of protein transfer across the endothelial barrier or the entire alveolar capillary membrane is critical for investigation of mechanisms leading to pulmonary edema. The purpose of this study was to evaluate Evans blue dye for determination of protein clearance across cultured bovine pulmonary artery endothelial cell monolayers and as a quantitative marker for albumin leakage to the air spaces in isolated perfused rat lungs. Evans blue dye bound tightly to albumin (EBA) as determined by lack of transfer through dialysis membranes and specific elution with albumin from a molecular exclusion column. EBA was equivalent to 125I-labeled albumin for calculation of albumin clearance rates (Calb) across intact and challenged monolayers [Calb (+ vehicle) = 0.12 microliters/min; Calb (+10 nM alpha-thrombin) = 0.47 microliters/min; Calb (+5 mg/ml trypsin) = 1.29 microliters/min]. Transfer of EBA was linear with time in both the endothelial cell monolayer model and the perfused lung. EBA was a sensitive marker for early edema in the perfused lung (before detectable weight gain) as well as for severe edema in the oxidant-injured lung (marked EBA accumulation in lavage fluid) and was a more specific marker for protein transfer than lavage fluid protein. EBA transfer is a convenient, reproducible, and accurate means to assess alterations in vascular permeability.
测定蛋白质穿过内皮屏障或整个肺泡毛细血管膜的情况对于研究导致肺水肿的机制至关重要。本研究的目的是评估伊文思蓝染料用于测定蛋白质通过培养的牛肺动脉内皮细胞单层的清除率，以及作为分离的灌注大鼠肺中白蛋白漏入气腔的定量标志物。通过透析膜无转移以及从分子排阻柱用白蛋白特异性洗脱确定，伊文思蓝染料与白蛋白紧密结合（EBA）。在计算完整和受刺激单层的白蛋白清除率（Calb）时，EBA等同于125I标记的白蛋白[Calb（+赋形剂）=0.12微升/分钟；Calb（+10 nMα-凝血酶）=0.47微升/分钟；Calb（+5毫克/毫升胰蛋白酶）=1.29微升/分钟]。在内皮细胞单层模型和灌注肺中，EBA的转移随时间呈线性。EBA是灌注肺早期水肿（在可检测到体重增加之前）以及氧化损伤肺中严重水肿（灌洗液中有明显的EBA积累）的敏感标志物，并且是比灌洗液蛋白更特异的蛋白质转移标志物。EBA转移是评估血管通透性改变的一种方便、可重复且准确的方法。
