2.4. Determination of hydroxyproline levels
On the day 21 after surgery, a piece of skin from the healed
wound area was collected and analyzed for hydroxyproline
content. As a major part of collagen, hydroxyproline has an
essential role in collagen stability. The collagen is the major
component of extracellular tissue, which gives support and
strength. The hydroxyproline contents were estimated using a
method described by others [28]. Briefly,tissues were dried in a hot
air oven at 60–70 8C to constant weight and were hydrolyzed in 6 N
HCl at 130 8C for 4 h in sealed tubes. The hydrolysate was
neutralized to pH 7.0 and was subjected to chloramine-T oxidation
for 20 min. The reaction was terminated by addition of 0.4 M
perchloric acid and color was developed with the help of Ehrlich
reagent at 60 8C and measured at 557 nm using UV–visible
spectrophotometer (CamSpec M330, Cambridge CB2 4BG, UK).