[Histonet] Determination of hydroxyproline. Help me please

[cris aguilar]

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, 10.0ml) and triplicated test
samples (homogenate 10ml, 50mg wet tissue/ 1ml NaCl) were placed in high
temperature polypropylene tubes of 2ml capacity. Later 50ml 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.



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



500ml 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

            Universidad Nacional de Trujillo