Intact organ spectrophotometry and single-photon counting

Abstract

In toxicology, it is of interest not only to assess enzyme levels and capacities for potential fluxes, but it is also useful to develop methods for determining actual concentrations and fluxes in the intact cell and organ. To this end, several noninvasive techniques have been developed over the years. Our interest has been largely in photometric techniques. Transmission spectrophotometry through solid organs permits monitoring of the cytochromes of the mitochondrial respiratory chain and cytochrome P-450 as well as other pigments of biological interest. Furthermore, the steady state level of catalase Compound I in liver provides information on rates of H2O2 production. These are in the nM to microM concentration range. More recently, the monitoring of photoemission from intact organs has been useful in toxicological problems. The major photoemissive species, singlet molecular oxygen and excited carbonyls, can now be monitored with good signal/noise ratio. Redox cycling of quinones and the generation of photoemissive species were studied in menadione metabolism. Inhibition of phase II led to a significant increase in the steady state level of singlet oxygen, as did the inhibition of two-electron reduction by using the inhibitor dicoumarol for DT diaphorase. Conversely, the induction of DT diaphorase by pretreatment with BHA protected by decreasing the level of reactive oxygen species.