Measurement of Fluorescence Changes of NAD(P)H and of Fluorescent Flavoproteins in Saponin-Skinned Human Skeletal Muscle Fibers
✍ Scribed by W.S. Kunz; A.V. Kuznetsov; K. Winkler; F.N. Gellerich; S. Neuhof; H.W. Neumann
- Publisher
- Elsevier Science
- Year
- 1994
- Tongue
- English
- Weight
- 493 KB
- Volume
- 216
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Saponin-skinned human muscle fibers from (M). vastus lateralis were immobilized in a quartz capillary to detect the fluorescence changes of (\mathrm{NAD}(\mathrm{P}) \mathrm{H}) and of fluorescent flavoproteins. To get sufficient intense fluorescence signals from a small amount of muscle tissue the (\mathrm{NAD}(\mathrm{P}) \mathrm{H}) fluorescence was excited by means of an (\mathrm{HeCd}) laser at (325 \mathrm{~nm}) and the flavoprotein fluorescence by an argon-ion laser at (454 \mathrm{~nm}) or by the second wavelength of a HeCd laser at (442 \mathrm{~nm}). Using this experimental setup the fluorescence spectra of (\mathrm{NAD}(\mathrm{P}) \mathrm{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 (\mathrm{NAD}^{+}), (ii) the addition of ADP caused its reoxidation, and (iii) the addition of respiratory chain inhibitors led to an almost complete reduction of (\mathrm{NAD}^{+}). It was observed that the redox state of the (\mathrm{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 \mathrm{mg}) wet weight muscle tissue. Moreover, the maximal changes in fluorescence of (\mathrm{NAD}(\mathrm{P}) \mathrm{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. 1984 Academic Press, Inc.