Statistical Analysis of Fiber Area in Human Skeletal Muscle

Transcription

1 TECHNICAL NOTES Fiber Area in Skeletal Muscle 415 Statistical Analysis of Fiber Area in Human Skeletal Muscle Michael R.M. McGuigan, William J. Kraemer, Michael R. Deschenes, Scott E. Gordon, Takashi Kitaura, Timothy P. Scheett, Matthew J. Sharman, Robert S. Staron Catalog Data McGuigan, M.R.M., Kraemer, W.J., Deschenes, M.R., Gordon, S.E., Kitaura, T., Scheett, T.P., Sharman, M.J., and Staron, R.S. (2002). Statistical analysis of fiber area in human skeletal muscle. Can. J. Appl. Physiol. 27(4): Canadian Society for Exercise Physiology. Key words: fiber type, sample size, cross-sectional area, biopsy Mots-cles: Abstract/Résumé Previous research has indicated that 50 fiber measurements per individual for type I and II fibers would be sufficient to characterize the fiber areas. This study replicated the work of McCall et al. (1998) using the three major fiber types (I, IIA, and IIB) and sampling larger populations of fibers. Random blocks of fibers were also examined to investigate how well they correlated with the overall mean average fiber area. Using random blocks of 50 fibers provided an accurate reflection of the type IIB fibers (r = ) but not for the type I (r = ) or IIA fibers (r = ). Type I fibers were consistently reflected by a random block of 150 fibers (r = ) while type IIA fibers required random blocks of 200 fibers (r = ), which appeared to provide an accurate reflection of the cross- M.R.M. McGuigan is with the Department of Exercise and Sport Science, University of Wisconsin-La Crosse, La Crosse, WI; W.J. Kraemer, T.P. Scheett, and M.J. Sharman are with the Department of Kinesiology, University of Connecticut, Storrs, CT; M.R. Deschenes is with the Department of Kinesiology, The College of William and Mary, Williamsburg, VA; S.E. Gordon is with the Human Performance Laboratory, East Carolina University, Greenville, NC; T. Kitaura is with the University of Kanazawa, Kanazawa, Japan; and R.S. Staron is with the Department of Biomedical Sciences, Ohio University, Athens, OH. 415

2 416 McGuigan, Kraemer, Deschenes, et al. sectional area. These results indicate that for a needle biopsy different numbers of fibers are needed depending on the fiber type to accurately characterize the mean fiber population. D après certaines études, il semble que l analyse d un échantillon de 50 fibres musculaires suffit pour identifier la proportion des types de fibre musculaire I et II. Cette étude reprend la démarche de McCall et coll. (1998) qui ont analysé les trois principaux types de fibre (I, IIA et IIB), mais utilise un plus gros échantillon de fibres musculaires. Un échantillon aléatoire de groupes de fibres est analysé puis mis en corrélation avec la moyenne globale des surfaces de fibre musculaire. La corrélation avec les fibres de type IIB est excellente (r = 0,96 0,98), mais pas pour les fibres de type I (r = 0,85 0,94) et les IIA (r = 0,80 0,91). Pour une bonne estimation de la surface de coupe des fibres de type I et de type IIA, il faut un échantillon aléatoire de 150 fibres (r = 0,95 0,98) et de 200 fibres (r = 0,94 0,98), respectivement. Ainsi, pour une bonne caractérisation des type de fibres musculaires prélevées par biopsie, il faut prendre en compte le type de fibres musculaire pour établir la juste taille des échantillons de fibres musculaires. Introduction Although fiber area is routinely measured in human muscle biopsies, there is no consensus on the number of fibers that must be measured to obtain reliable and valid results. Fiber area data is often used to measure morphological changes in skeletal muscle in response to different training stimuli. It is therefore important to obtain mean values based on sufficient measurements of different populations of fibers to provide an accurate reflection of the fiber size. A recent study by McCall et al. (1998) indicated that mean values calculated from at least 50 fibers for each type (I and II) were representative of an individuals muscle biopsy. As the authors did not differentiate between the major fast fiber subtypes (eg. IIA and IIB), the appropriate sample size for characterizing specific fiber subtypes could not be determined from the results. Alway et al. (1989) showed that at least 200 fibers of type I and II fibers need to be measured in elite bodybuilders to accurately determine fiber area in this population. Lexell and Taylor (1989a) showed that to obtain a good estimate of the mean fiber cross-sectional area for a whole muscle, the number of biopsies has a much greater influence on the sampling error than the number of fibers measured in each biopsy. These authors showed that when the fiber cross-sectional area in three or more biopsies is measured, it is sufficient to measure only 25 fibers in each biopsy. If less than three biopsies are taken, there is no worthwhile reduction in sampling error when more than 100 fibers are measured. Most studies in exercise science have used the vastus lateralis muscle for biopsy sampling. The majority of studies have based their fiber area measurements on lower numbers of fibers, with one study using measurements from as few as 10 fibers (Tesch et al., 1984). For the purposes of training studies it is important to get an accurate reflection of changes in fiber area of the different subtypes. Therefore, it is essential to know the number of fibers for the different subtypes that need to be measured to accurately determine fiber area. It was our aim to replicate the findings of McCall et al. (1998) using larger numbers of fibers and including the major fiber types in human limb musculature. Therefore, the purpose of the present investigation was to determine the sample

3 Fiber Area in Skeletal Muscle 417 size required for each of the major fiber types (I, IIA and IIB) needed to give an accurate measurement of individual fiber area. We were also interested in examining the effect of taking random blocks of fibers and how they would relate to the overall mean fiber area of each of the subtypes. SUBJECTS Methods All 26 subjects were men and were cleared with a physical examination by a physician before the start of the study. None of the subjects had any medical or endocrine disorders that would confound or limit his ability to participate fully in the investigation. Each subject was a member of the U.S. Army and was classified as physically active, having been involved with standard military physical training programs at least three times per week for at least two years before the start of the study. MUSCLE BIOPSIES Muscle biopsies were obtained from the superficial portion of vastus lateralis muscle of the dominant thigh by utilizing the percutaneous needle biopsy technique of Bergstrom (1962) as modified by Evans et al. (1982). The muscle samples used for this investigation were from Kraemer et al. (1995). Data from repeated biopsies (randomly performed) demonstrated no significant intrabiopsy variations for fiber type distributions. Muscle tissue samples were oriented in tragancanth gum, frozen in isopentane cooled to 159 ºC with liquid N 2, and stored at 120 ºC until analyzed. Serial cross sections (12 m thick) were cut on a cryostat (American Optical, Buffalo, NY) at 20 ºC for histochemical analyses. Pre- and post-training samples were histochemically analyzed in the same assay to avoid interassay variances. Histochemical analyses used for fiber typing consisted of assaying for myofibrillar adenosinetriphosphatase (ATPase) activity after pre-incubations at ph 4.3, 4.6, and 10.3, as well as standard NADH histochemistry (Halkjaer-Kristensen et al., 1979). Muscle fiber types were divided into three groups (types I, IIA, and IIB) based on the stability of their ATPase activity (Brooke et al., 1970; Staron et al., 1983). Although hybrid fiber types were delineated (IC, IIC, and IIAB), they made up too small a percentage of the total biopsy samples to be included in the analysis (Staron et al., 1992). Therefore, it was essential to concentrate only on the major fiber types (I, IIA, and IIB). MUSCLE FIBER AREA Muscle fiber areas were determined on sections assayed for nicotinamide-adenine dinucleotide tetrazolium reductase activity to avoid any possible shrinkage due to the alcohol used in the ATPase histochemical assay. The perimeter of all intact fibers of each muscle fiber type was measured. Cross sections were projected at a constant magnification with a Zeiss microscope (standard drawing tube) onto a digitizing tablet with a self-contained computer running the appropriate morphometric programs (Kraemer et al., 1995). This was interfaced with a mainframe

4 418 McGuigan, Kraemer, Deschenes, et al. computer (VAX 11/780, Digital Equipment, Maynard, MA) system for data storage and analysis. Fiber areas were determined by tracing the perimeter of each fiber on the digitizing tablet and calculating the area with a Zeiss Interactive Digital Analysis System (ZIDAS; Zeiss, Thornwood, NY). Data from repeat biopsies (randomly performed) demonstrated insignificant intrabiopsy variations in fiber type distributions (Kraemer et al., 1995). STATISTICAL ANALYSIS The fiber sample size required for accurately determining the mean fiber areas was determined using sequential estimation analysis as previously described (Clarkson et al. 1980; McCall et al., 1998). The sequential estimation analysis involved calculating the rolling cumulative mean for each additional block of five fiber measurements for each individual. Sequential blocks of fiber measurements were drawn from each individual s total pool of measurements, beginning with the first one. Each individual s total pool of measurements were made randomly. Cumulative means were calculated from individual block means for each additional block of five fiber measurements. Only subjects from which 500 type I (n = 26), type IIA (n = 12) and 100 type IIB (n = 13) fibers could be measured were included in the analysis. A correlation matrix was constructed between the individual means for each block of five measurements as described by McCall et al. (1998). In addition, mean fiber areas were determined for a randomly selected sample of 20, 50, 75, and 100 fibers for each subject and each fiber subtype. Finally, mean fiber areas were determined for randomly selected samples of 150 and 200 fibers for the type I and IIA fiber subtypes. Statistical significance was chosen as p 0.05.<a>Results Table 1 shows the rolling cumulative means, standard deviations and interblock correlations for the different fiber area subtypes. Sequential estimation analysis for the fiber areas indicated that most individual and group means and standard deviations leveled off by 150 fibers per subject for the type I fibers (r = 0.95, p <.05) (Table 1). For the IIA fibers the means and standard deviations had stabilized after 200 fibers had been measured (r = 0.94, p <.05), whereas for the IIB fibers the stablization occurred after 50 fibers had been measured (r = 0.96, p <.05). Table 2 shows the means, standard deviations and correlation coefficients for the random blocks with the mean number of fibers for each fiber area subtype. Taking a random block of 20 fibers resulted in a variable correlation with the mean number of fibers (r = ). There were also differences in the subtypes when 50 fibers were measured, either using the first 50 fibers, final 50 fibers or a random block of 50 fibers. The correlation coefficients for the type I fibers ranged from r = , type IIA from r = , and IIB from r = For the blocks of 75 fibers the correlation coefficients for the type I fibers ranged from r = , type IIA from r = and IIB from r = Using random blocks of 150 fibers for the type I fibers ranged from r = , and random blocks of 200 fibers for the type IIA fibers ranged from r = Discussion The results of this study indicate that different numbers of fibers need to be counted depending on the fiber type that is being measured. McCall et al. (1998) showed

5 Fiber Area in Skeletal Muscle 419 Table 1 Cumulative Means, Standard Deviations, and Interblock Correlations for Fiber Cross-Sectional Areas Type I Type IIA Type IIB Mean SD r Mean SD r Mean SD r Cumulative number of fibers per subject Note: Data was obtained from an average of 853 ± 180 type I fibers, 501 ± 210 IIa fibers and 171 ± 79 IIb fibers.

6 420 McGuigan, Kraemer, Deschenes, et al. Table 2 Means and Standard Deviations Fiber Cross-Sectional Areas for Random Blocks of Fibers Type I Type IIA Type IIB Mean SD r Mean SD r Mean SD r First Final Random First Final Random First Final Random First Final Random First Final Random First Final Random

7 Fiber Area in Skeletal Muscle 421 that among recreational resistance-trained men, mean values calculated from 50 fibers were representative of an individual s type I and II fibers. Table 1 indicates that the different fiber subtypes varied as to where the group cumulative mean and standard deviations stabilized (p < 0.05). It would appear that type I fibers require 150 fibers, type IIA 200 fibers and type IIB 50 fibers to be measured to obtain accurate results for mean fiber area. The reason for the type IIB fiber population requiring less total fibers to be measured could perhaps be related to them being a minority population of the three major fiber types. If 50 type IIB fibers were measured in a sample, that would represent a greater percentage of the total of type IIB fibers. If one is sampling from fewer total motor units, it would be expected that the variance would be lower. Table 2 shows that there are differences in the mean fiber areas after taking random blocks of the different fiber subtypes. Taking a random block of 20 fibers did not provide an accurate reflection of the mean fiber area for any of the three subtypes (r = ). Taking random blocks of 50 fibers resulted in different degrees of accuracy depending on which fiber type was being measured. Type IIB fibers appeared to have the highest correlation with 50 fibers measured and overall mean (r = ). However the type IIA (r = ) and type I (r = ) fiber areas were not accurately reflected by 50 fibers. Type I fibers were consistently reflected by a random block of 150 fibers (r = ), while type IIA fibers required random blocks of 200 fibers (r = ) to provide an accurate reflection of the cross-sectional area. This provides further evidence that different numbers of fibers need to be measured in order to accurately determine the different fiber subtypes. The data analyzed from this retrospective analysis appears to confirm that to accurately determine fiber cross-sectional area 150 type I fibers, 200 type IIA fibers and 50 type IIB fibers are required. In summary, it would appear that the number of fibers needed to be measured to determine fiber cross-sectional area varies depending on the fiber subtype. It is clear that caution should be taken when interpreting the results of previous investigations that have taken muscle biopsies to determine changes in crosssectional area of the fibers. The results from our study, using muscle biopsy data from physically active men, would suggest that to determine type I fiber area, 150 fibers are required, 200 fibers for type IIA and 50 fibers for type IIB. As type IIB fibers represent a smaller percentage of the three major fiber types, a smaller sample size would represent a greater percentage of the total number of type IIB fibers. References Alway, S.E., Grumbt, W.H., Gonyea, W.J., and Stray-Gundersen, J. (1989). Contrasts in muscle and myofibers of elite male and female bodybuilders. J. Appl. Physiol. 67(1): Bergstrom, J. (1962). Muscle electrolytes in man. Scand. J. Clin. Lab. Invest. 68: Brooke, M.H., and Kaiser, K.K. (1970). Three myosin ATPase systems: the nature of their ph lability and sulfhydryl dependence. J. Histochem. Cytochem. 18(9):

8 422 McGuigan, Kraemer, Deschenes, et al. Clarkson, P.M., Katch, F.I., Kroll, W., Lane, R., and Kamen, G. (1980). Regional adipose cellularity and reliability of adipose cell size determination. Am. J. Clin. Nutr. 33(11): Evans, W.J., Pinney, S.D., and Young, V.R. (1982). Suction applied to a muscle biopsy maximises sample size. Med. Sci. Sports Exerc. 14: Halkjaer-Kristensen, J., and Ingemann-Hansen, T. (1979). Microphotometric analysis of NADH-tetrazolium reductase and a- glycerophosphate dehydrogenase in human quadriceps muscle. Histochem. J. 11: Kraemer, W.J., Patton, J.F., Gordon, S.E., Harman, E.A., Deschenes, M.R., Reynolds, K., Newton, R.U., Triplett, N.T., and Dziados, J.E. (1995). Compatibility of high-intensity strength and endurance training in hormonal and skeletal muscle adaptations. J. Appl. Physiol. 78: Lexell, J., and Taylor, C.C. (1989a). Variability in muscle fibre areas in whole human quadriceps muscle. How much and why? Acta. Physiol. Scand. 136: McCall, G.E., Byrnes, W.C., Dickinson, A.L. and Fleck, S.J. (1998). Sample size required for the accurate determination of fiber area and capillarity of human skeletal muscle. Can. J. Appl. Physiol. 23(6): Staron, R.S., and Hikida, R.S. (1992). Histochemical, biochemical, and ultrastructural analyses of single human muscle fibers, with special reference to the C-fiber population. J. Histochem. Cytochem. 40: Staron, R.S., Hikida, R.S., and Hagerman, F.C. (1983). Myofibrillar ATPase activity in human muscle fast-twitch subtypes. Histochem. 78: Tesch, P.A., Thorsson, A., and Kaiser, P. (1984). Muscle capillary supply and fiber type characteristics in weight and power lifters. J. Appl. Physiol. 56(1): Received March 19, 2001; accepted in final form September 6, 2001.