Rapid spectrophotometric determination of oxygen consumption using hemoglobin, in vitro: Light scatter correction and expanded dynamic range

The method of using absorbance in conjunction with hemoglobin (Hb) to monit or rapid changes in oxygen consumption in vitro was improved by using a non linear calibration technique and multiwavelength spectroscopy. The O-2 depe ndence of Hb absorbance was effectively linearized using the current techni que (R-2 = 0.990 +/- 0.0021 n = 3), and extended the dynamic range of [O-2] determinations by 1.6-fold over previous approaches. The association/disso ciation rates of O-2 and Hb were evaluated using the current approach and w ere not significant on the 100-ms time domain. A method was also developed for compensating for large amplitude light scattering changes in turbid med ia using multiwavelength analysis. Both the nonlinear calibration curve and light scattering corrections were validated in isolated porcine heart mito chondrial preparations. (C) 2000 Academic Press.