SOME CHARACTERISTICS OF THE FLUORESCENCE LIFETIME OF REDUCED PYRIDINE-NUCLEOTIDES IN ISOLATED-MITOCHONDRIA, ISOLATED HEPATOCYTES, AND PERFUSED-RAT-LIVER IN-SITU

By extensively examining the experimental conditions for time-resolved spectrophotometry of non-transparent light scattering systems, we dem onstrated the feasibility of quantitative analysis of both the fluores cence lifetime and intensity of reduced pyridine nucleotides in living tissues, suspensions of isolated liver mitochondria, and hepatocytes, as well as hemoglobin-free perfused rat liver being used systematical ly for measurements, The fluorescence decay was analyzed by the maximu m likelihood method with a 4-component decay model, The lifetime of NA DH observed in mitochondria (mean: 2.8+/-0.2 ns) was much longer than that of the free form in an aqueous solution (mean: 0.43+/-0.01 ns), a nd it was characterized as a protein-bound form, The lifetime was not affected by either aerobic or anaerobic conditions nor by the energy s tate, though the intensity changed markedly, The decay curves of isola ted hepatocytes under normal aerobic conditions were the same as those of isolated mitochondria, though cytosolic NADH and NADPH were superi mposed, Under the conditions of ''unphysiological'' acidosis, the mean lifetime became about 1.5 times longer than that under normal conditi ons, With perfused liver, the relative contributions of cytosolic NADH and NADPH mere determined by infusing lactate and tert-butylhydropero xide. Cytosolic NADH did not contribute to the overall fluorescence of pyridine nucleotides, In contrast, about 70% of the total fluorescenc e intensity was due to cytosolic NADPH, but its decay parameters were essentially the same as those of mitochondrial NADH, No free form of e ither NADH or NADPH was detected in the cytosolic and mitochondrial sp aces, We concluded that the changes in fluorescence intensity observed under the various conditions can be simply explained by a change in t he amount of reduced pyridine nucleotides in tissues, rather than by c hanges in the microscopic environment, The wide applicability of time- resolved fluorescence photometry to in vivo studies is well documented .