dr.cris - 22-8-2006 at 09:25
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 mycrolitres) and triplicated test samples (homogenate 10 mycrolitres, 50mg wet tissue/ 1ml NaCl)
were placed in high temperature polypropylene tubes of 2ml capacity. Later 50 mycrolitres 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 mycrolitres of Chloramine-T was added and mixed with the hydrolyzate and oxidation was allowed to proceed for 25 minutes at room temperature.
500 mycrolitres 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
Edited title spelling. Chemoleo.
[Edited on 22-10-2006 by chemoleo]
methyl_ethyl - 26-8-2006 at 17:42
Hello,
It's almost bedtime, but I thought I would at least let you know I could possibly help you.
First of all, how do you know you have failed. What sort of problems have you been encountering?
Although I have dealt with this assay on a slightly larger scale ~2mg of collagen suspended in HOAc, I have had much success with utilizing an acid
hydrolysis (HCL) over night at 110C followed by the addition of NaOH to adjust the pH into the working range of the assay.
I have used a direct quantitative dye binding assay that utilizes sirius red for the determination of collagen in a 96well plate format, although if
Histological staining of your cardiac tissue is what you are seeking I am sure you could apply the same methodology. The Irish Company Biocolor
offers the sirius red assay i speak of.
It's been a while since I have performed this so I might have to consult my notes.
Do you have access to HPLC? Most Amino Acid Analysis procedures work fine for HYP.
I'll get back to you when I am not so sleepy 
regards,
m_e
[Edited on 27-8-06 by methyl_ethyl]
methyl_ethyl - 30-8-2006 at 18:00
hello dr.cris,
I am not sure exactly what types of collagen occur in rat myocardial tissue, however it seems that you only perform a salt extraction on the tissue.
Are you sure that your collagen of interest is soluble in netural salt solutions? If so what concentrations of salt, in what buffer have you been
using? If you are not sure that your collagen is only neutral salt soluble, then an acetic acid extraction could prove beneficial. And if you are
interested in non-soluble collagens, there are some other avenues you could explore to increase solubility into your solvent system. Recently the
reduction of disulphide bridges has been noted to move previously insoluble collagen to the soluble phase.
From what I have gathered from your post, the best, most direct route with the highest probability of success in determining collagen content from
your target tissue would be the Sirius red dye binding assay that I mentioned. HYP quantitation is an indirect method of collagen quantitation that
relies heavily on the complete extraction, and hydrolysis of collagen from your starting material. The Sirius Red procedure allows for extraction of
collagen that is salt and acid soluble, and provides a well established procedure to do so.
One more thing I just thought of, your Chloramine-T should exist as free-flowing granular crystals. If the crystals are clumped, seem overly
hydrated, or if you have to "dig" into the container to break up the crystals then your assay will not behave as expected. These aforementioned
conditions almost always result in a precipitate that does not clear after addition of perchloric acid and subsequent addition of p-DAB.
The final product after p-DAB addition should be clear and complete with no cloudiness, or precipiate noted. If so the problem is more than likely
with your Chloramine-T.
much_love,
methyl_ethyl
Hydroxyproline problem
amina_Amy27 - 23-11-2011 at 21:30
Hello everyone
I am a PhD student and i have to quatify collagen for my PhD work . I have been using
A simplified method for analysis of Hydroxiproline in Biological tissues.
Reddy GK, Enwemeka CS.
Clinical Biochemistry 1996;29:225-9.
and i am unable to obtain results
The protocol i follow is
1.10mg of dry tissue was homogenised in 1mL distilled water .
2. To 25microL of this homogenate i added 25 microL of 2N NaOH and hydrolysed it by autoclave at 120 degrees for 30 min
3. Added 450microL of chloramine T ( 0.127gm Chloramine T dissolved in 2ml 50% n-propanol and reconstituted to 10mL with acetate citrate buffer)
4. Incubated at room temperature for 25 min
5. prepared fresh PDAB ( 1.51 gm pDMAB added 2:1 npropanl : perchloric acid(60%) upto 10mL)
6. Incubated at 65degrees for 30 min. Read absorbance at 550nm
7. For standard curve, 1mg/ml stock solution of Hydroxyproline prepared in distilled water.
8. prepared standard of concentrations 2-20microgm/ml form stock in distilled water.
9. to 25microL of each standard added 50microL of 2N NaOH and hydrolysed by autoclaving at 120degrees for 30 min. Rest procedure similar
Problem:
1. After incubaton for 30 min with pDMAB, the colour developed is yellow instead of Red.
Please help in this regard. I have to submit my work next month and this standarization is left.
Thank You in advance