[Cris]

Hello everybody

I am a medical student in Peru.  At present, I have been developing my thesis in ventricular remodeling; therefore I need to quantify “collagen content” in rat heart, as fibrosis index. I have tried to quantify “collagen content by hydroxyproline determination” several times in my laboratory, but I failed.

I have been following Reddy’s technique:

A simplified method for analysis of Hydroxiproline in Biological tissues.
Reddy GK, Enwemeka CS.
Clinical Biochemistry 1996;29:225-9.

My procedure:   
   
Preparation of myocardial samples:
The rats were weighed, anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal), and underwent median thoracotomy for heart removal. The left ventricle was separated and weighed. Later, the myocardial samples were “dehydrated” in pure alcohol for 24 hours (is this correct?). Finally, 50mg of wet tissue sample was manually homogenized (manualmente en un mortero) in 1mL NaCl.
   
Quantification of hydroxyproline
Duplicated Hyp standard (0, 2.0, 4.0, 6.0, 8.0, 10.0?l) and triplicated test samples (homogenate 10?l, 50mg wet tissue/ 1ml NaCl) were placed in high temperature polypropylene tubes of 2ml capacity. Later 50?l of 2N sodium hydroxide was added to each tube with Hyp standard and test samples and mixed at room temperature for 20 minutes.
Samples were hydrolyzed (alkali hydrolysis in 2N NaOH) by fire (estufa) at 120°C for 20min.

50?l of Chloramine-T was added and mixed with the hydrolyzate and oxidation was allowed to proceed for 25 minutes at room temperature.

500?l of 1M Ehrlich’s reagent was added to each sample and the samples were mixed and incubated at 65°C for 20 minutes.
Finally the absorbance of samples was read at 550nm using a spectrophotometer.

1. Is it necessary to dehydrate the sample for a later analysis?

2. Is alkaline hydrolysis (2N NaOH) the better way to liberate Hyp from samples?

3. Did the method use autoclave to hydrolyzate? I have been use a fire (estufa)?
Is this correct?
 
4. How can I estimate Hydroxyproline content in rat heart (mg/g)?


Does anybody know how I can measure collagen content in rat heart?

Has anyone done a Sirius red stain for collagen?  If so, could I please have your protocol? 

Help me please I am desperate, because I must present my thesis next October.   

Thank you in advance.
Cristian Aguilar
dr.cris85@yahoo.com
Universidad Nacional de Trujillo

[GSR]

hi,

you might get a good response from biochemistry forum.

cheers

GSR