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Kunz 1994 Anal Biochem

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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. Analyt. Biochem. 216: 322-327.

Β» PMID: 8179187

Kunz WS, Kuznetsov AV, Winkler K, Gellerich FN, Neuhof S, Neumann HW (1994) Analyt. Biochem.

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 Ξ±-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 Ξ±-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.


β€’ O2k-Network Lab: DE_Magdeburg_Gellerich FN


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Organism: Human  Tissue;cell: Skeletal Muscle"Skeletal Muscle" is not in the list (Heart, Skeletal muscle, Nervous system, Liver, Kidney, Lung;gill, Islet cell;pancreas;thymus, Endothelial;epithelial;mesothelial cell, Blood cells, Fat, ...) of allowed values for the "Tissue and cell" property.  Preparation: Permeabilized Cell or Tissue; Homogenate"Permeabilized Cell or Tissue; Homogenate" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property. 

Regulation: Respiration; OXPHOS; ETS Capacity"Respiration; OXPHOS; ETS Capacity" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property. 


HRR: Oxygraph-2k 

Spectrophotometry; Spectrofluorimetry