Dear all,
I need to measure the rate of bone resorption....
One parameter is to measure total and/or free pyridinoline and deoxypyridinoline (collagen degradation products) in serum or urine
My problem is that i could not get some old papers that the new papers take as reference....
Also, there is no available standard form of these biological compounds, as researchers made this work before have synthesized these standards with time-consuming methods...
I need help in both....
How getting those old papers, as our university database of e-journals is limited..
And how to get those standards, or how to synthesize them with a time-saving method
If anyone has another assay, please contact me...
Best Regards....
Fatma elzahraa

Hello Fatma elzahraa,

You can measure pyridinoline and deoxypyridinoline using HPLC method. This is highly sensitive method.

Unfortunately nobody is selling the these standards, you have to make it your self except pentosidine (pentosidine is available for purchase)

i tried to attach papers for above method but, the maximum size allowed is 100kb. Alternatively i can send the papers to your e-mail.



Rajeev

Thanks alot....
Please can you send me these papers....
Really I need papers about the preparation or isolation of these degradation products
my email is fatma_elzahraa_2004@yahoo.com
Best regards

for the old papers not in your library,

Try
illiad (Interlibrary Loan), you supposed to find it somewhere at your library homepage.

dear rajeev

I found only one company that can purchase these collagen degradation standards,.....
Thanks alot for your help.....

fatmaelzahraa said:

dear rajeev I found only one company that can purchase these collagen degradation standards,..... Thanks alot for your help.....

Hi Fatmaelzahraa,

Are you still interested in making collagen cross-link standard (hydroxylysyl pyridinoline) your self ?

Rajeev

sure, I am intersted in doing such a challenging procedure, as there is no one here in my faculty work in this field....
Do you have a simpler method....???
as the methods I read before are so difficult and need a large budget (here, we are paying for everything we need in research work).....
also the commercial standards are sooooo expensive....
If you have a good method, please tell me about it.....
Also, I do not have a fluorescence detector for HPLC.... but there is an available HPTLC with fluorescence detection....
so I have another idea, but I do not know if it will work well or not....
What is your opinion in applying the plasma samples on HPTLC plates RP C18 with a mobile phase used before in papers..????
Do you think it will work????
Really, I need your help.......

Hello Fatmaelzahraa,

Indeed, the collagen cross-link preparation is challenging but, you can prepare it through simple procedure.

As we all know type II collagen has more cross-links (hydroxylysyl and lysyl pyridinolines) compare to other types of collagen.

Here is the protocol to make type II collagen from bovine cartilage (you may have the protocol to make type II collagen in your book). Once you prepare type II collagen, acid hydrolyze it to release the cross-links.



- Get a fresh bovine joint from slaughter house and store it in -80 C until its use.

- One day before the experiment, thaw the joint in cold room. Scrape the hyaline cartilage (white translucent layer, image can be obtained from Google image) using single edged blade on ice.

- Chop the scraped cartilage in to small pieces with blade or it can be done quickly by using laboratory blender (use some ice cubes along with cartilage to keep it cold). Transfer the ground cartilage in to 50 ml conical tube or to a conical flask and wash with 70% ethyl alcohol. Ethyl alcohol wash can be done by stirring the cartilage in alcohol first for few minutes and then centrifuging at 10,000 rpm. Repeat this washing step at least two times.

- After washing with alcohol, wash with double distill water by mixing and centrifugation process.

-Important: measure the weight of the wet cartilage before proceed to the next step

- Transfer the cartilage in to the beaker and add 4 M Guanidine hydrochloride at 10:1 ratio to cartilage (Prepare 4M Guanidine hydrochloride in 0.05 M Tris buffer and filter it. It can be stored up to 6 months in cold). If you have a sonicator, sonicate the cartilage for 5-10 minutes in presence of 4M Gn-HCL solution in cold (sonication helps the better digestion). Digest the cartilage with Gn-HCL overnight in cold room.

- Next day, centrifuge the digested cartilage at 8000rpm for 30 minutes and remove the supernatant. To the pellets add 10-15ml of distilled water / gm of cartilage and centrifuge again for 30 minutes at 8000 rpm. Repeat this process at least 3 to 4 times to remove all the Gn-HCL solution, alternatively digested cartilage can be dialyzed against distilled water for 24 hrs with 3 to 4 changes of water in cold.


-prepare 500mM acetic acid solution and adjust the pH to 2.8 with formic acid solution for this step. Use pHed 500mM acetic acid solution at 10:1 ratio to the cartilage original wet weight. Dissolve pepsin enzyme powder in to this solution (keep the pepsin enzyme at 1:20 ratio to the wet weight of the cartilage) Transfer the cartilage pellets from the above step in to the glass beaker and add 500mM acetic acid solution with pepsin enzyme in it. Digest the cartilage with pepsin enzyme for 24 hr in cold room.

- After pepsin enzyme digestion, cartilage solution will be viscous in nature. Centrifuge the enzyme digested cartilage for 30 minutes at 6000 rpm and remove the supernatant.
If you see any debris in the supernatant, repeat the centrifugation process to remove any undigested cartilage.

-Transfer the clear supernatant solution in to the beaker and add 5 M sodium chloride solution slowly with slow stirring (this step will initiate the precipitation of the collagen). Incubate the solution in cold room for 24hr in cold (if you want you can stir the solution slowly and this will ensure the uniform mixing of supernatant with sodium chloride solution).

- After 24hrs, centrifuge the solution at 5000 rpm for 60 minutes in cold. Remove supernatant and dissolve the pellet in to 100mM acetic acid (2:1ratio to the wet weight of the cartilage for example if you started with 100gm of cartilage, use 200ml of 100mM acetic acid solution) slowly and keep it in cold room like in previous step. After 24 hr, centrifuge the solution and dissolve the pellet in 100mM acetic acid and repeat the precipitation of collagen with sodium chloride. Repeat this step at least 2 to 3 times. If you want to make pure collagen type II, experienced people will advice to repeat this step at least 4 to 5 times to get pure collagen type II.

- Prepare 500mM sodium phosphate stock buffer for this step. Prepare 500mM of Na2HPO4 and NaH2PO4 separately and keep the Na2HPO4 and add NaH2PO4 solution to get the pH 7.2. After dissolving the final collagen precipitate in 100mM acetic acid in the above step dialyze it against 2 liter of 10mM sodium phosphate buffer pH 7.2 for 5 days. During 5 days dialysis, change the phosphate buffer 2 times per day and this is critical as this will help to remove sodium chloride and residual pepsin enzyme. Please note that, during this step you will see cloudy appearance and collagen solution and this is normal

After 5 days dialysis, centrifuge white collagen solution at 9000rpm for 40 minutes to precipitate the collagen. If you want you can wash the collagen ppt with 70% ethyl alcohol one or two times and centrifuge again at 9000rpm for 40 minutes. Finally lyophilize the collagen.

The concept in this protocol is well known and all the credit should go to every individuals who dedicated their entire life for collagen research.


For Collagen cross-link preparation:

- Acid hydrolyze the collagen in 6N hydrochloric acid in acid resistant, temperature resistant screw cap tube for 16hr at 110C in oven.

- After 16hr hydrolysis, remove the acid by speed vac and neutralize the acid with mild sodium hydroxide. After one more speed vac, dissolve the hydrolyzed collagen in double distilled water and test it for the presence of collagen cross-link.

Question is How you are going to confirm that you got the cross-link in the acid hydrolyzed collagen?

If you can tell me your C18 column specification,i can tell you solvents for HPLC run and the retention time for the collagen cross-link.

Regarding HPTLC, I am sorry i didn't worked with HPTLC.

I completely understand your situation about budget tight (we all have to deal with it one way or other way another way don't we all?).

Now with your post, I didn't get the last part ('applying the plasma sample on HPTLC'), what is your hypothesis in this?

In my understanding (i may be wrong), grant agencies may not like the idea of just measuring the cross-links (they will think it is of no use). But, if you link alteration of cross-link with a specific human abnormalities then grant agencies may say yes.


Hope this protocol is going to help you.

Rajeev

Dear Mr. Rajeev,
Really, Thanks very much for your help.. the procedure is simple ..
First.I need your personal email to contact you directly
Second. I think I can test the presence of the collagen crosslinks by fluorescence
I have a spectrofluorimeter in my lab so I can detect them.. but the real problem is how to separate them to isolate a pure pyridinoline and deoxypyridinoline??? Do you have a suggestion as the isolation procedure needs sephadex column and a fluorescence detector for HPLC to isolate them well..
Do you think that I can use cellulose column, as I read in some papers , that they use microgranular cellulose column to prepare the plasma sample. Is it efficient to isolate the two compounds of my interest???
Also, I have microcrystalline cellulose, is it equivalent in its efficiency and separation to microgranular cellulose????
Did you think about prepare these crosslinks by organic synthesis???....Do you think that it is easier than their isolation from joints or it will be more difficult????
Please, can you provide me with a reference for the procedure you sent me before.
I have C8 and C18 HPLC columns.. length : 220 mm, particle size: 5u, I.D.: 4.6 mm BrownLee columns (Perkin Elmer, USA)but my problem is that I have no fluorescence detector.. I have UV-Visible detector and electrochemical detector (and electrochemical detector has not worked yet due to many obstacles)
As a result, I thought about HPTLC (high performance thin layer chromatography).
By using RP C18 or RP C8 plates and applying the same system of HPLC on these plates. Then, if it is efficient, we can use this technique to measure the amount of pyridinoline and deoxypyridinoline in plasma or urine of osteoporotic patients.
As after developing the plate in the mobile phase, it will be scanned by certain type of HPTLC scanner to make integration for the spot on the plate in the form of peaks given with their areas and heights. i.e. it is more or less equivalent to HPLC.
Additionally, I want to make this work for my master, so I am responsible of all its expenses without any help of any agency..
Agencies may offer help if my idea (an idea to treat osteoporosis) worked well. And it is too early now to say such a thing. i.e. I made all this work by myself with help of my administrators only.
Can you tell me about the systems that I can try to apply on HPTLC..???

I have a choice to make this previous work to measure PYD and DPD or measure osteocalcin in plasma by ELISA kit what is your opinion. Which is better for me
Do you know anything about a simple method to measure hydroxyproline in plasma or in urine.(colorimetric or HPLC with UV detector.)Please inform me if you have any idea about it

Thanks a lot for your great help.
Best regards

Hello Fatma,
Here is my e-mail address: rajeev04mys@yahoo.com. I don't think you need to have pure hydroxylysyl pyridinolines (HP) and lysyl pyridinolines (LP) (deoxypyridinoline) both, only pure HP is enough for your work. The LP differ from HP by one aminoacid ( lysine amino acid), there fore LP always elute right next the HP.
Unless your thesis deals about the characterization of these two cross-links, then, you can prepare them chemically and characterize them by different method. I assume that is not your aim.

Preparing the cross-links by organic synthesis is ok but, in this method couple of steps may give you trouble. In addition, it is the question of how much time you can devote to make these cross-links. I prefer to make cross-links through the isolation of type II collagen from hyaline cartilage because, this way you will also have semi-pure or ultra pure (if you want have ultra pure collagen) collagen type II and collagen cross-links.

In my study, I am trying to understand the effect of the deficiency of Fibrillin protein on the development of extracellular matrix in a given connective tissue and collagen turn over. So, I didnt take the trouble of making cross-links by chemical method. I prepared standard cross-links through the isolation of type II collagen from hyaline cartilage. (I will e-mail you the reference for this method including other papers).

To measure collagen cross-links use RP-C-18 column, HPTLC should work. You might want to look up the research articles that describe HPTLC with RP-C18 column .

Your question; can you tell me about the systems that I can try to apply on HPLC??
Do you mean the solvent systems? I am sorry I didnt understand this question completely.

About your choice for your thesis, it depends on what is your future interest? And how much time you have?
Working with HP and LP- 1) you have to make working standard 2) you have to develop the HPLC method to study the alterations in HP and LP 3) you have to maintain HPLC system to make sure it is going to function properly all the time.

Working with osteocalcin in plasma by ELISA kit- 1) you have to do one time standardization of your ELISA experiment for your sample 2) You dont have to worry about optimizing the antibody because it is pre-determined by the company that provide the kit 3) cost for this ELISA kit is a question.
Finally you and your mentor has to decide about the project
For the method to measure hydroxyproline please refer to my post in this community with the title hydroxyproline assay
Rajeev

Dear all,

I am new in this forum. I saw this topic while browsing the website. Actually, I am facing a similar problem like Fatma.

I have some rabbit serum which I suspect, has arthritis. I would like to measure markers such as, hydroxyproline, glycosaminoglysans, osteocalcin and other bone turnover markers. My problem is, I can't get the papers describing assays of serum, which are very old.

Please advise. Thank you.

Dear
there are alot of bone markers
bone formation markers including osteocalcin (measured by ELISA) and alkaline phosphatase (colorimetric method) and if you have lectin, you can measure bone alkaline phosphatase isoform alone....
for bone resorption markers:
tartrate resistant acid phosphatase (by colorimetric method)
PYD and DPD by HPLC if you can get standards or by ELISA
Hydroxyproline in urine by HPLC or colorimetric method...
those are the major markers used....
if you need any assay of them just contact me....
best regards

Dear Fatma,

Thank you for your prompt reply.
We prefer colorimetric assays to measure bone resorption. But, we only have serum as samples. So, we were wondering whether you could provide some advise on the methods to be used. There are numerous biomarkers to detect collagen degradation. We do not know which can be detected in serum by colorimetric assays, since certain biomarkers are better measured in urine.

Thank you.

dear,
I think that tartrate resistant acid phosphatase in serum is the most suitable marker you can measure....
it can be measured by a spectrophotometric assay....
also you can measure hydroxyproline level in serum by spectrophotometric assay or by chromatographic assay....
other markers are polypeptides, i.e. they can be measured by ELISA or radioimmunoassay and there is no possible and specific spectrophotometric assay for measuring one of them...
I attach a paper including information about bone markers... I hope it may benefit you.... if it is not functioning, tell me to send it to your email instead...
I can send you the papers concerning these assays if you need them....
my email is fatma_elzahraa_2004@yahoo.com.....
Feel free to cantact me...
best regards