Abstract
The increase in the levels of protein carbonyls, biomarkers of oxidative stress, appears to play an important role in aging skeletal muscle. However, the exact distributions of carbonyls among various skeletal muscle microstructures still remain largely unknown, partly owing to the lack of adequate techniques to carry out these measurements. This report describes an immunohistochemical approach to determine the relative abundance of carbonyls in the intermyofibrillar mitochondria (IFM), the subsarcolemmal mitochondria (SSM), the cytoplasm, and the extracellular space of skeletal muscle. These morphological features were defined by labeling the nucleus, the Z-lines, and mitochondria. Carbonyls were detected by derivatization with dinitrophenylhydrazine followed by labeling with an Alexa 488-labeled anti-dinitrophenyl primary antibody. Alexa 488 fluorescence (green) in different fiber microstructures was used to estimate the relative abundance of carbonyls. On the basis of the samples examined, preliminary results suggest that the most dramatic age-related changes in carbonyl levels occur in the extracellular space, followed in a decreasing order by SSM, IFM, and the cytoplasm. These observations were confirmed in the soleus and semimembranosus muscles composed predominantly of type I and type II fibers, respectively. This approach could easily be extended to the investigation of carbonyl levels in other muscles (composed of mixed skeletal muscle fiber types) or other tissues in which protein carbonyls are present.
Similar content being viewed by others
References
Reid MB (2001) Med Sci Sports Exerc 33:371–376
Levine RL, Williams JA, Stadtman ER, Shacter E (1994) Methods Enzymol 233:346–357
Mecocci P, Fano G, Fulle S, MacGarvey U, Shinobu L, Polidori MC, Cherubini A, Vecchiet J, Senin U, Beal MF (1999) Free Radic Biol Med 26:303–308
Sundaram K, Panneerselvam KS (2006) Biogerontology 7:111–118
Stadtman ER (2001) Ann N Y Acad Sci 928:22–38
Cavaletto M, Ghezzi A, Burlando B, Evangelisti V, Ceratto N, Viarengo A (2002) Comp Biochem Physiol C Toxicol Pharmacol 131:447–455
England K, Cotter T (2004) Biochem Biophys Res Commun 320:123–130
van der Vlies D, Pap EH, Post JA, Celis JE, Wirtz KW (2002) Biochem J 366:825–830
Costa VM, Amorim MA, Quintanilha A, Moradas-Ferreira P (2002) Free Radic Biol Med 33:1507–1515
Hood DA (2001) J Appl Physiol 90:1137–1157
Palmer JW, Tandler B, Hoppel CL (1977) J Biol Chem 252:8731–8739
Menshikova EV, Ritov VB, Fairfull L, Ferrell RE, Kelley DE, Goodpaster BH (2006) J Gerontol A Biol Sci Med Sci 61:534–540
Allen DL, Roy RR, Edgerton VR (1999) Muscle Nerve 22:1350–1360
Ohira Y, Yoshinaga T, Ohara M, Nonaka I, Yoshioka T, Yamashita-Goto K, Shenkman BS, Kozlovskaya IB, Roy RR, Edgerton VR (1999) J Appl Physiol 87:1776–1785
Rosser BW, Dean MS, Bandman E (2002) Int J Dev Biol 46:747–754
Tseng BS, Kasper CE, Edgerton VR (1994) Cell Tissue Res 275:39–49
Fannin SW, Lesnefsky EJ, Slabe TJ, Hassan MO, Hoppel CL (1999) Arch Biochem Biophys 372:399–407
Chen JC, Warshaw JB, Sanadi DR (1972) J Cell Physiol 80:141–148
Muscari C, Frascaro M, Guarnieri C, Caldarera CM (1990) Biochim Biophys Acta 1015:200–204
Craig EE, Hood DA (1997) Am J Physiol 272:H2983–H2988
Hansford RG (1978) Biochem J 170:285–295
Manzelmann MS, Harmon HJ (1987) Mech Ageing Dev 39:281–288
Judge S, Jang YM, Smith A, Hagen T, Leeuwenburgh C (2005) FASEB J 19:419–421
Smith MA, Perry G, Richey PL, Sayre LM, Anderson VE, Beal MF, Kowall N (1996) Nature 382:120–121
Smith MA, Sayre LM, Anderson VE, Harris PL, Beal MF, Kowall N, Perry G (1998) J Histochem Cytochem 46:731–735
Ahmadzadeh H, Andreyev D, Arriaga EA, Thompson LV (2006) J Gerontol A Biol Sci Med Sci 61:1211–1218
Radak Z, Sasvari M, Nyakas C, Pucsok J, Nakamoto H, Goto S (2000) Arch Biochem Biophys 376:248–251
Radak Z, Takahashi R, Kumiyama A, Nakamoto H, Ohno H, Ookawara T, Goto S (2002) Exp Gerontol 37:1423–1430
Vlassara H, Bucala R (1996) Diabetes 45(Suppl 3):S65–S66
Avery NC, Bailey AJ (2005) Scand J Med Sci Sports 15:231–240
Bank RA, Bayliss MT, Lafeber FP, Maroudas A, Tekoppele JM (1998) Biochem J 330(1):345–351
Judge S, Jang YM, Smith A, Selman C, Phillips T, Speakman JR, Hagen T, Leeuwenburgh C (2005) Am J Physiol Regul Integr Comp Physiol 289:R1564–R1572
Suh JH, Heath SH, Hagen TM (2003) Free Radic Biol Med 35:1064–1072
Soper DS (2007) Free statistics calculators. http://www.danielsoper.com/statcalc/
Loschen G, Azzi A, Richter C, Flohe L (1974) FEBS Lett 42:68–72
Boveris A, Chance B (1973) Biochem J 134:707–716
Beckman KB, Ames BN (1998) Physiol Rev 78:547–581
Drew B, Leeuwenburgh C (2002) Ann N Y Acad Sci 959:66–81
Cadenas E, Davies KJ (2000) Free Radic Biol Med 29:222–230
Espinosa A, Leiva A, Pena M, Muller M, Debandi A, Hidalgo C, Carrasco MA, Jaimovich E (2006) J Cell Physiol 209:379–388
Jackson MJ (2005) Philos Trans R Soc Lond B Biol Sci 360:2285–2291
Acknowledgements
This work was supported by the National Institutes of Health (AG025371). E.A.A. acknowledges the support of NIH by a Career Award (1K02-AG21453). The authors also thank Janice Shoeman from the Department of Physical Medicine and Rehabilitation of the University of Minnesota for preparing the muscle cross sections.
Author information
Authors and Affiliations
Corresponding author
Additional information
An erratum to this article is available at http://dx.doi.org/10.1007/s00216-008-2389-x.
Rights and permissions
About this article
Cite this article
Feng, J., Navratil, M., Thompson, L.V. et al. Estimating relative carbonyl levels in muscle microstructures by fluorescence imaging. Anal Bioanal Chem 391, 2591–2598 (2008). https://doi.org/10.1007/s00216-008-2187-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-008-2187-5