A comparison of maximal bioenergetic enzyme activities obtained with commonly used homogenization techniques.
Homogenization of tissue for analysis of bioenergetic enzyme activities is a common practice in studies examining metabolic properties of skeletal muscle adaptation to disease, aging, inactivity or exercise. While numerous homogenization techniques are in use today, limited information exists concerning the efficacy of specific homogenization protocols. Therefore, the purpose of this study was to compare the efficacy of four commonly used approaches to homogenizing skeletal muscle for analysis of bioenergetic enzyme activity. The maximal enzyme activity (Vmax) of citrate synthase (CS) and lactate dehydrogenase (LDH) were measured from homogenous muscle samples (N = 48 per homogenization technique) and used as indicators to determine which protocol had the highest efficacy. The homogenization techniques were: (1) glass-on-glass pestle; (2) a combination of a mechanical blender and a teflon pestle (Potter-Elvehjem); (3) a combination of the mechanical blender and a biological detergent; and (4) the combined use of a mechanical blender and a sonicator. The glass-on-glass pestle homogenization protocol produced significantly higher (P < 0.05) enzyme activities compared to all other protocols for both enzymes. Of the four protocols examined, the data demonstrate that the glass-on-glass pestle homogenization protocol is the technique of choice for studying bioenergetic enzyme activity in skeletal muscle.
Grace
M
M
Department of Exercise and Sport Sciences and Physiology, University of Florida, Gainesville, USA.
Fletcher
L
L
Powers
S K
SK
Hughes
M
M
Coombes
J
J
D000222:Adaptation, Physiological; D000375:Aging; D000704:Analysis of Variance; D000818:Animals; D002417:Cattle; D002950:Citrate (si)-Synthase / Q000032:analysis*; D003902:Detergents; D004194:Disease; D004734:Energy Metabolism; D005898:Glass; D006651:Histocytochemistry / Q000295:instrumentation / Q000379:methods; D007770:L-Lactate Dehydrogenase / Q000032:analysis*; D008931:Mitochondria, Muscle / Q000201:enzymology; D018485:Muscle Fibers, Skeletal / Q000201:enzymology; D018482:Muscle, Skeletal / Q000201:enzymology*; D005082:Physical Exertion; D011138:Polytetrafluoroethylene; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D014465:Ultrasonics
D003902:Detergents; D011138:Polytetrafluoroethylene; D007770:L-Lactate Dehydrogenase; D002950:Citrate (si)-Synthase