Measurement of Fluorescence Changes of NAD(P)H and of Fluorescent Flavoproteins in Saponin-Skinned Human Skeletal Muscle Fibers

• Published in: Analytical biochemistry 1994-02, Vol.216 (2), p.322-327
• Main Authors: Kunz, W.S., Kuznetsov, A.V., Winkler, K., Gellerich, F.N., Neuhof, S., Neumann, H.W.
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - metabolism; Biological and medical sciences; Cell physiology; Female; Flavoproteins - metabolism; Fluorescence; Fluorometry; Fundamental and applied biological sciences. Psychology; Humans; Middle Aged; Mitochondria, Muscle - metabolism; Molecular and cellular biology; Muscle contraction; Muscle Proteins - metabolism; Muscles - chemistry; Muscles - drug effects; Muscles - metabolism; NADP - metabolism; Oxidation-Reduction; Oxidative Phosphorylation; Saponins; Spectrometry, Fluorescence
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1994.1048

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.