Measurement of fluorescence changes of NAD(P)H and of fluorescent flavoproteins in saponin-skinned human skeletal muscle fibers.
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 alpha-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 alpha-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.
Kunz
W S
WS
Institut für Biochemie, Medizinische Akademie Magdeburg, Germany.
Kuznetsov
A V
AV
Winkler
K
K
Gellerich
F N
FN
Neuhof
S
S
Neumann
H W
HW
D000244:Adenosine Diphosphate / Q000378:metabolism; D005260:Female; D005420:Flavoproteins / Q000378:metabolism*; D005453:Fluorescence; D005470:Fluorometry; D006801:Humans; D008875:Middle Aged; D008931:Mitochondria, Muscle / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism*; D009132:Muscles / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010085:Oxidative Phosphorylation; D012503:Saponins; D013050:Spectrometry, Fluorescence
D005420:Flavoproteins; D009124:Muscle Proteins; D012503:Saponins; D009249:NADP; D000244:Adenosine Diphosphate