Determination of fibrosis from cryostat sections using high performance liquid chromatography: skeletal muscle.
Analysis of hydroxyproline (collagen) and pyridinoline (collagen cross-links) in biopsies prepared for routine histological evaluation with OCT compound was performed. Frozen sections (250 microm-thick) were cut from cardiac muscle, diaphragm, liver, and soleus muscle from the rat. After removal of OCT compound by rinsing, the samples were dried, weighed and hydrolyzed in 6 N HCl. A portion of the hydrolysate was analyzed for hydroxyproline using high performance liquid chromatography with collagen type I as the standard. Collagen concentrations ranged from 6.6 microg/mg dry weight (liver) to 74.7 microg/mg dry weight (diaphragm). From the remainder of the hydrolyzate, pyridinoline cross-links of collagen were separated and analyzed similarly by high performance liquid chromatography. The concentration of pyridinoline ranged from 2.6 ng/mg dry weight (liver) to 35.6 ng/mg dry weight (diaphragm). These techniques were adequate to analyze both collagen and pyridinoline (i.e. collagen cross-links) in small biopsy samples (< 1 mg dry weight) routinely used in clinical pathology. The method proved useful in the quantitation of focal fibrosis in a partially denervated rat soleus. Denervation was confirmed using fast myosin immunohistochemistry which revealed large areas of small myofibres containing fast myosin. Collagen concentration increased by five-fold and collagen cross-links by more than 7-fold consistent with fibrotic changes known to occur with denervation.
Miller
G R
GR
Department of Physiology, West Virginia University, Morgantown 26506, USA.
Smith
C A
CA
Stauber
W T
WT
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000378:metabolism; D005260:Female; D005353:Fibronectins / Q000032:analysis; D005355:Fibrosis / Q000378:metabolism / Q000473:pathology; D005629:Frozen Sections; D006909:Hydroxyproline / Q000032:analysis; D007150:Immunohistochemistry; D009121:Muscle Denervation; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000294:innervation / Q000473:pathology*; D009218:Myosins / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley
D000596:Amino Acids; D005353:Fibronectins; C015484:pyridinoline; D003094:Collagen; D009218:Myosins; D006909:Hydroxyproline