caa a22 4500 397494599 CHVBK 20180308164518.0 cr unu---uuuuu 161202e199512 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a125001 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a125001 Some Characteristics of the Fluorescence Lifetime of Reduced Pyridine Nucleotides in Isolated Mitochondria, Isolated Hepatocytes, and Perfused Rat Liver In Situ [Elektronische Daten] [Masayoshi Wakita, Goro Nishimura, Mamoru Tamura] By extensively examining the experimental conditions for time-resolved spectrophotome-try of non-transparent light scattering systems, we demonstrated the feasibility of quantitative analysis of both the fluorescence 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 systematically for measurements. The fluorescence decay was analyzed by the maximum likelihood method with a 4-component decay model. The lifetime of NADH 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), and it was characterized as a protein-bound form. The lifetime was not affected by either aerobic or anaerobic conditions nor by the energy state, though the intensity changed markedly. The decay curves of isolated hepatocytes under normal aerobic conditions were the same as those of isolated mitochondria, though cytosolic NADH and NADPH were superimposed. Under the conditions of "unphysiological” acidosis, the mean lifetime became about 1.5 times longer than that under normal conditions. With perfused liver, the relative contributions of cytosolic NADH and NADPH were determined by infusing lactate and tert-butylhydroperoxide. Cytosolic NADH did not contribute to the overall fluorescence of pyridine nucleotides. In contrast, about 70% of the total fluorescence intensity was due to cytosolic NADPH, but its decay parameters were essentially the same as those of mitochondrial NADH. No free form of either NADH or NADPH was detected in the cytosolic and mitochondrial spaces. We concluded that the changes in fluorescence intensity observed under the various conditions can be simply explained by a change in the amount of reduced pyridine nucleotides in tissues, rather than by changes in the microscopic environment. The wide applicability of time-resolved fluorescence photometry to in vivo studies is well documented. © Oxford University Press Regular Papers nationallicence fluorescence decay analysis nationallicence in vivo fluorometry nationallicence mitochondrial energy states nationallicence pyridine nucleotides nationallicence Wakita Masayoshi Biophysics Laboratory, Research Institute for Electronic Science, Hokkaido University, Kita-ku, Sapporo 060 aut Nishimura Goro Biophysics Laboratory, Research Institute for Electronic Science, Hokkaido University, Kita-ku, Sapporo 060 aut Tamura Mamoru Biophysics Laboratory, Research Institute for Electronic Science, Hokkaido University, Kita-ku, Sapporo 060 aut The Journal of Biochemistry Oxford University Press 118/6(1995-12), 1151-1160 0021-924X 118:6<1151 1995 118 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a125001 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a125001 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Wakita Masayoshi Biophysics Laboratory, Research Institute for Electronic Science, Hokkaido University, Kita-ku, Sapporo 060 aut NATIONALLICENCE 700 1- Nishimura Goro Biophysics Laboratory, Research Institute for Electronic Science, Hokkaido University, Kita-ku, Sapporo 060 aut NATIONALLICENCE 700 1- Tamura Mamoru Biophysics Laboratory, Research Institute for Electronic Science, Hokkaido University, Kita-ku, Sapporo 060 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 118/6(1995-12), 1151-1160 0021-924X 118:6<1151 1995 118 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford