Kunz 1994 Anal Biochem
|Kunz WS, Kuznetsov AV, Winkler K, Gellerich FN, Neuhof S, Neumann HW (1994) Measurement of fluorescence changes of NAD(P)H and fluorescent flavoproteins in saponin-skinned fibers. Anal Biochem 216:322-7.|
Abstract: Saponin-skinned human muscle fibers from M. vastus lateralis were immobilized in a quartz capillary to detect the fluorescence changes of NAD(P)H and of fluorescent flavoproteins. To get sufficient intense fluorescence signals from a small amount of muscle tissue the NAD(P)H fluorescence was excited by means of an HeCd laser at 325 nm and the flavoprotein fluorescence by an argon-ion laser at 454 nm or by the second wavelength of a HeCd laser at 442 nm. Using this experimental setup the fluorescence spectra of NAD(P)H, of α-lipoamide dehydrogenase and of electron-transfer flavoprotein were detected in saponin-skinned human muscle fibers. These fibers behaved identically to isolated mitochondria: (i) The addition of substrates caused an increase in reduction of mitochondrial NAD+, (ii) the addition of ADP caused its reoxidation, and (iii) the addition of respiratory chain inhibitors led to an almost complete reduction of NAD+. It was observed that the redox state of the NAD(P) system and of the α-lipoamide dehydrogenase reached after addition of 1 mM ADP correlates with the rate of active state respiration with NAD-dependent substrates. Therefore, this fluorimetric method is suitable to compare the mitochondrial oxidation capacities of NAD-dependent substrates in less then 5 mg wet weight muscle tissue. Moreover, the maximal changes in fluorescence of NAD(P)H and flavoproteins correlate with the amount of mitochondrial marker enzymes per milligram muscle tissue. Using this method a myopathy caused by a diminished content of mitochondria per milligram muscle tissue was observed.
Labels: MiParea: Respiration
Organism: Human Tissue;cell: Skeletal muscle Preparation: Permeabilized tissue
Coupling state: OXPHOS