RELIABILITY OF AN ELECTROPHORETIC AND IMAGE-PROCESSING ANALYSIS OF HUMAN SKELETAL-MUSCLE TAKEN FROM M-VASTUS-LATERALIS

The relative content of myosin heavy chain (MHC) isoforms IIb, IIa and I in human skeletal muscle taken from the m. vastus lateralis of 30 h ealthy male subjects was analysed using mini-gel electrophoresis. Repe ated electrophoretic gels utilizing the same methods were produced for all subjects and the determination of MHC protein bands was performed using a digital scanner and National Institutes of Health (NIH) Image software and laser densitometry. A comparison between the NIH Image p rocessing technique and laser densitometry revealed differences of 6.4 7%, 6.35% and 6.84% between these measurement techniques for MHC-IIb, -IIa and -I isoforms, respectively. The percentage technical error of measurement (TEM%) between electrophoretic gels was shown to be 19.1%; 17.8% and 14.2%, with regard to percentage of occurrence of MHC-IIb, -IIa and -I isoforms respectively. The variation in electrophoretic ge l analyses was shown to be 5.7%. 7.3% and 5.5%. with regard to the per centage of MHC-IIb. -IIa and -I isoforms respectively. Intra-class cor relations comparing NIH Image and laser densitometry produced I values in the range 0.38-0.63. Comparisons between and within gel analyses p roduced r values in the range 0.59-0.94 and 0.93-0.98, respectively. A nalyses of variance revealed no significant differences (P < 0.05) bet ween analysis techniques. between gels or within gels for the measurem ent of MHC-IIb, -IIa and -I isoforms. The inter-gel error between fibr e subgroups was moderate for the two type-II MHC populations and less fur type-I MHC; the intraindividual error in the measuring technique u sed for classifying the MHC-IIb, -IIa and -I protein bands was small. The results obtained in this investigation showed consistent tl ends w hich may reflect a false classification of the type-II MHC populations for the inter-gel and intra-individual analyses. The NIH Image softwa re and digitizing process was shown to be a valid and reliable method for distinguishing between MHC protein bands of human skeletal tissue as separated by mini-gel electrophoretic techniques.