Applicability of the Kubelka-Munk theory for the evaluation of reflectancespectra demonstrated for haemoglobin-free perfused heart tissue

Reflectance spectrometry is a useful tool for studying in vivo kinetic chan ges in the oxygen saturation of haemoglobin and myoglobin as well as the re dox state of cytochromes. A method is given which allows the quantification of tissue reflectance spectra using multicomponent analysis. This method u tilizes the Kubelka-Munk theory for modelling the measured tissue spectra. To test this approach, reflectance spectra of a haemoglobin-free perfused g uinea pig heart were measured by a fast scanning spectrophotometer (100 spe ctra/s, spectral resolution 1.0 nm) and evaluated using the component absor bance spectra measured separately. A relative mean spectral residual error of 0.15% was achieved by least-squares fitting. Using statistical error pro pagation, oxygenation of myoglobin is obtained within a relative precision of 1%, and the redox state of cytochromes aa(3) and c are determined simult aneously within a margin of 3%; the results for the redox-state of cytochro me b, however, are less precise. Special component error functions are pres ented to provide a reliability measure for the concentration prediction usi ng this multicomponent assay. The consistency of the theory and the compone nt absorptivity data is tested by regressing the actual concentrations obta ined for each of the redox pair components during the various states of tis sue oxygenation. A method is described for the recognition and reduction of systematic errors.