Biochemical assays in lung homogenates: Artifacts caused by trapped blood after perfusion

Abstract

The amounts of trapped whole blood in well-perfused homogenates of rat lung (from rat lungs weighing approximately 1–1.5 g wet weight) were quantitatively determined by a method which utilizes the difference in hemoglobin (Hb) absorbance at 415 nm after reduction of HbO 2 to deoxyHb by dithionite. By adding known amounts of whole blood to unfiltered crude lung homogenates, we constructed standard curves which allowed us to determine residual blood contents of perfused homogenates. Homogenates contained a mean of 20 ± 2.4 (SE) μ l of blood/lung. These data were used to calculate the effects that contaminating whole blood might make upon measured values of various biochemical parameters in lung cytosols and homogenates. Depending on the specific parameter evaluated, blood contamination can contribute a major (e.g., catalase, β - N -acetylglucosaminidase and protein), a trivial (e.g., lysozyme, DNA) or an intermediate (e.g., superoxide dismutase, glutathione peroxidase) fraction of the total amount of a given component present in lung homogenate or cytosol. In studies in which the overall magnitudes of measured lung biochemical changes have been ascribed mechanistic significance (such as in lung damage, repair, protective, or adaptative processes) or in which lung enzyme specific activities are being expressed, the effects of blood contamination must be taken into account.