Biochemical verification of quantitative histochemical analysis of succinate dehydrogenase activity in skeletal muscle fibres

Abstract

The purpose of this investigation was to examine critically the validity of a computerized quantitative microphotometric histochemical technique for the determination of succinate dehydrogenase (SDH) activity in skeletal muscle fibres. Sections from the anterior costal diaphragm were removed from Fischer-344 rats (n = 12) and assayed histochemically to determine SDH activity. The SDH activity in individual muscle fibres was computed using a computerized microphotometric histochemical technique which involves measurement of the optical density of deposited diformazan derived from nitroblue tetrazolium within the fibres. To validate the histochemical technique, whole muscle SDH activities were calculated from the histochemical procedure and were compared to SDH activities determined from whole muscle homogenates via a standard quantitative biochemical assay. The mean within-day variability of the computerized microphotometric histochemical technique of determining SDH activity was 6% (range = 0.5-10.9%) for an area containing approximately 50 fibres and 6.1% (range = 1.05-14.9%) for an individual muscle fibre. Similarly, the mean between-day variability of the microphotometric histochemical technique of determining SDH activity was 5.9% (range = 2.6-13.9%) for an area containing approximately 50 fibres and 6.6% (range = 2.2-13.9%) for an individual muscle fibre. The inter-class correlation coefficient between biochemically determined SDH activity and histochemically determined SDH activity was r = 0.83 (p < 0.05). Collectively, these data demonstrate that the quantitative histochemical technique of Blanco et al. (1988) is both valid and reliable in the determination of SDH activity in skeletal muscle fibres.

Effect of training on the activity of five muscle enzymes studied on elite cross‐country skiers

“…To obtain reliable histochemical data (sampling error ≈6%) from repeated biopsy samples, a minimum of 150 muscle fibres are required ( Blomstrand & Ekblom 1982, Lexell et al . 1985 , Powers et al . 1993 ).…”

27-hydroxycholesterol and the expression of three estrogen-sensitive proteins in MCF7 cells

“…In active cells, tetrazolium salts are reduced in vivo to formazan by the superoxide ion (31); therefore, NBT is widely used as an indicator of mitochondrial metabolism (11,32). Exposing MCF7 cells to either E 2 or 27OHC, we observed an increased production of superoxide radical in comparison to the non-treated, control cells.…”

Time-dependent increase of succinate dehydrogenase activity in low-frequency stimulated rabbit muscle: a comparison between microphotometric and biochemical methods

“…Contrary to commonly used single point ("end point") measurements (Martin et al 1986;Blanco et al 1988;Powers et al 1993;Nakae and Stoward 1995;Sieck et al 1995), kinetic microphotometric determination of local enzyme activities in tissue sections is based on the evaluation of maximum reaction rates (Nolte and Pette 1972;Wimmer 1979, 1980;Lomax et al 1989;Old and Johnson 1989;Pette and Reichmann 1989;Nakae and Stoward 1995). As has been outlined elsewhere (Leeuw and Pette 1994), measurements of reaction rates by integrating microdensitometry within the same section largely exclude artefacts due to distributional error and non-uniform illumination (Goldstein 1981).…”

“…It is obvious, however, that this procedure, which has also been applied by other investigators (e.g. Blanco et al 1988;Kugler et al 1988;Old and Johnson 1989;Powers et al 1993), does not represent an ideal solution, as might be achieved by individually correcting each fibre for its nothing-dehydrogenase reaction. In the present study, determination of nothing-dehydrogenase reaction in 200 fibres yielded a mean value with a relatively high coefficient of variation (±40%).…”