Hello to all,

Is there any one has experience with Non-HPLC method of Hydroxyproline assay using DMAB in 96 well plate ?

I am planning to run this assay in few days from tendons. Is there any critical suggestions and trouble shoots with this assay.

Thanks in advance for help
Rajeev

Hydroxyproline assay:

Title: Modified assay for determination of hydroxyproline in a tissue hydrolyzate.

Abstract:
A modified assay for the determination of hydroxyproline in tissue is presented. The modifications greatly reduce the time required for analysis of excised tissue as first introduced by Stegemann and Stalder [1]. These modifications include a change in the technique for tissue hydrolysis and a change in the preparation of the hydroxyproline oxidizing agent. The analysis utilizes the standard addition technique, eliminating the need for correction of matrix effects between the specimen and standard. This paper attempts to give a complete detailed description of the assay such that the procedure may be repeated without requiring additional reference material.

Publication Link:
http://www.brl.uiuc.edu/Publications/1980/Edwards-ClinChimActa-161-1980.pdf

Thanks for posting the link.
I got helpful hints from this paper in addition to other papers. Sorry for the delayed replay.
Finally i was able to develop micro assay (with modification) for hydroxy proline from mouse tendons.

Rajeev

Hello ABCD09,

sorry for the delayed replay. I am posting the micro method for hydroxy proline quantification. I adapted this method to measure collagen level from mouse tendons.

You may not be able see the table in its original format. If you want i can attach the protocol to your e-mail.

If you have any questions, please let me know.

Rajeev


Hydroxy proline assay: Quantitative measurement of hydroxy proline


Pre-treatment of tendons (digestion of tendons)

-Transfer the tendons from -800 C to high temperature resistant screw cap plastic tubes (VWR cat # 16466-048 or 16466-064).

- Add 200ul of 6N HCl (this should cover the tendons in the tube), put it in the glass beaker and keep thermometer in the beaker.

-Transfer the beaker in to the regular dry oven and incubate at least 16hr at 1000 C,
check the temperature in side the oven chamber rather than outside.

-After complete digestion, speed vac the sample to remove HCl. Remove HCl by re-dissolving the dried tendons many times. This process can be monitored by having few drops of color indicator in the sample.

-Finally check the pH for the neutral and dissolve the neutralized tendons in 100ul of
double distilled water.



Required reagents

1. Citric acid
From sigma

2. Sodium acetate trihydrate
From Sigma

3. L-4-hydroxyproline
Sigma # P5607

4. Ethylene glycol
Fluka cat no. 64719

5. Chloramine T
VWR cat no. VW3506-0

6. p-dimethylamino benzaldehyde (DMBA)

Sigma cat no. P7626






Preparation of solutions

1) 10X sodium acetate trihydrate buffer-for 1lit (can be stored for several months at 40C)

- 50gm of citric acid monohydrate
-120 gm of sodium acetate trihydrate
- 34gm of sodium hydroxide
- 12ml of acetic acid, final pH should be 6
Prepare 800ml of this solution and adjust the pH 6 and finally make up to 1lit


2) 20% DMBA solution (always prepare fresh solution)

-20gm of DMBA in 100ml of ethylene glycol or 2gm in 10ml of ethylene glycol
Add DMBA powder in to ethylene glycol solution and warm it at 60 degree for few minutes to dissolve the DMBA and finally make up the volume

3) Chloramine-T solution (for 10ml) (can be stored for few weeks in dark bottles at cold)

-0.141 gm of chloramines-T
-2ml double distilled water
-3ml of ethylene glycol
-5ml of 10X buffer (sodium acetate trihydrate buffer)
Filter this solution before use

4) Perchloric acid solution (stable for several hrs but not more than 2 days)

-2.7ml of 70% PCA
-7.3 ml of double distilled water
Filter this solution before use

5) Hydrolyzed collagen solution

Hydrolyze the collagen (pure colTM from INMAED) several tubes at 1mg/ml concentration (follow the tendon digestion protocol)

Or use hydroxy proline amino acid solution as standard for this assay







Running the experiment:



hydrolyzed
collagen or (hydroxy proline amino acid) control 400ng 800ng 1200ng 1600ng 2000ng

volume

0 l
Calculate the volumes based on the concentration of the stock solution


1x buffer
40 l
Calculate based on the volume of the standard (max. volume should be
40ul)


Chloramine-T
solution
Add 20 l to all the tubes and incubate at room temperature for 15 minutes


Perchloric acid
solution
Add 20 l to all the tubes and mix it

DMBA solution
Finally add 20 l of DMBA solution, heat the solution at 60 degree for 20 min

(Final color of the solution should be pink for the presence of hydroxy proline).

cool the tubes and transfer the solution in to 96 well plate and read it at 560nm




Interpretation of the result from the assay:

1. Collagen content calculations from hydroxy proline assay:

g of hydroxy proline/ml X dilution factor (if any) X conversion factor (7.5) = g of

Collagen.



2. To calculate the molarity of collagen:

g of collagen/ 300 (M.Wt of the collagen)




References:

Darwin J. Prockop and Sidney udenfriend, Analytical biochemistry, 1, 228-239 (1960)

C.A.Edwards and W.D.O Brien, JR. Clinica chimica Acta, 104. 161-167 (1980)

Hermann Stegemann and Karlheinz Stalder, Clinica Chimica Acta, 18, 267-273 (1967)

HI All....
thank you for the discussion with valid points. I have followed Jamall et al protocol for the hydroxyproline estimation. My problem is to calculate the collagen content from the calculated Hyp concentration(g/ml or g/mg dry or wet tissue or so.....). As RKB suggested one calculation method for collagen content(g of collagen)...is it giving the collagen per ml of the specified sluntion(where g/ml of Hyp taken) or per tissue wt....??? Please make me clear and kindly suggest me which formula could be the best to calculate the collagen content if i have hyp concentration(concn./ml or concn./g tissu wt)???
thank u.

[quote=Ramjee Pallela][/quote] Hello Ramjee Pallela. There are many ways you can normalize the amount of collagen in a given tissue. 1. You can express hydroxyproline (HYP) concentration as- ug of HYP/ ug of total protein or ug of HYP / mg of total protein or ug of HYP / wt of given tissue (if you can weigh the tissue) 2. Once you caluclate the HYP concentration, you can caluclate the collagen concentration using the given formula (ug of HYP x 7.5 = ug of collagen). Later you can express the ug collagen / mg of total protein or ug collagen / tissue weight. If you can measure the weight of the tissue, you don't have take trouble to measure the total protein concentration. Total protein con. measurement by calorimetric assay is questionable , because hydroxy proline (major a.a in collagen) gives different color for nin hydrin and it is difficult to differentiate the color from hydroxy proline from rest of the amino acids in a given acid hydrolyzed tissue. Alternatively you can measure the total protein using amino acid analyzer from hydrolyzed tissue. The bottom line is, ug of collagen / weight of given tissue is acceptable (as long as you can measure the weight of the tissue). Hope this make sense. Rajeev [b][/b][b][u][/u]

Hi Rajeev,

Thanks for this protocol, it's very helpful (I'm starting a similar protocol for a porcine skin model). I had a few questions, though:

1- I'm just worried about how to best remove the HCl after hydrolyzing the tissues. You suggest to speed vac the HCl; but isn't it possible that once we speed-vac, and remove the supernatant that we're assuming is all HCl, that the supernatant we're removing actually has some hydroxyproline dissolved in it? (and so won't we be skewing the data, by removing any dissolved hydroxyproline that's in the HCl?)

2- Also, you say that after speed-vacing and removing the HCl to
"Remove HCl by re-dissolving the dried tendons many times" ... what do you mean by that; just dissolve the tissue, speed vac again, and remove the supernatant? Do you have to take this into account when doing the final HP calculations then, since you just dissolved the HP in the original tissue?

3- Instead of trying to remove the HCl by speed-vaccing, I read in a paper somewhere that I could just add NaOH to neutralize ... if I did this, wouldn't this mess up the HP concentrations (since from my high school gen chem I remember that acid + base forms salt plus WATER, which I'm assuming would then dissolve the HP... I read the NaCl salt that's formed isn't a big deal since it doesn't interfere with the chromaphore reaction)? How would I take this extra dilution by water into account for the final HP concentrations?

Sorry for all the annoying/specific q's, and thanks for any help in advance!
>Kapil

Hello Kapil,

Thanks for your interesting questions.

Sorry for the delayed replay. I was swamped in my work for a while, needless to explain the pressure.

Answer to your 1st question: As we all know, speed vac or centrifugal evaporator works by reducing the pressure under vacuum so does the boiling point of a given solvent. The centrifugal force generated during this process will evaporate the solvent from top, thus it prevent loss or solute by solvent bumping (in other words, by violent boiling). During centrifugal evaporation of HCl from our sample, we all assume that there will be a small loss of hydroxy proline (HYP) but this will be a similar error if we treat all our samples in the same way. It is known that the loss of HYP on drying is ≤ 0.1% at 110 C and I am sure centrifugal evaporator with no heat (assuming we are not using heating option in the instrument) will not reach this temp.

In summary, you have to dry the HCl in the absence of heat in centrifugal evaporator. If you are critical about loss of HYP during drying, you can have a positive control (dissolve known amount of HYP amino acid in HCl) and treat it exactly like your samples and find out % loss. At the end of the experiment, use the % loss of HYP amino acid from the positive sample to correct the concentration of HYP amino acid in your test samples.

Answer to your 2nd question: I sorry for the confusion in my protocol. Remove HCl by re-dissolving the dried tendons many times means re-dissolve the dried tendon in double distilled water. More clearly- speed vac to remove acid - re-dissolve dried tendon extracts in water - speed vac again to remove water - re-dissolve in water. This is a repeating step. After couple of cycle, dissolve the sample in water, add pH indicator and titrate with 0.1M sodium hydroxide, in this way you will be safe from salt formation through strong acid + base reaction.

Answer to your 3rd question: As I mentioned in the above answer, I wouldnt add base to the HCl solution at the first step rather, I would dilute the acid in water (by doing couple of speed vac dry) and then add base to neutralize it. You can find out empirically how many drops of sodium hydroxide you have to use to get the neutral pH in the extract (you dont have to use pH indicator). You are also right about NaCl salt that it is not going interfere with chromophore reaction.

Hope my comments helps.

Rajeev

Hi Rajeev, Thanks a lot- your comments are very helpful. I just 2 clarifications: 1- After I hydrolyze my tissues (skin), they seem pretty well diluted in the HCl to me - will speed-vacing actually remove the acid and result in solid precipitate left?? I'm just worried speed-vacing will just remove everything, and I won't be left with any solid precipitate to actually run the HP assay on! 2- Do you remember how long you had to speed-vac (and at how many rotations per minute) to remove the HCl? Thanks so much for all the help!

hello kapil

Yes, speed vac will remove the acid and you will have either thin layer of coated ppt along the surface of the tube or you will have a kind of powder like ppt at the bottom.

As long as your speed vac instrument is working properly, you will not loose any of your sample. Even if you don't see any ppt after 1st cycle of speed vac (it depends on quantity of your starting material) , add d.d.water and rotate the tube so that water touches every centimeter of inner surface of the tube.

The time required to remove acid is again depends on the volume of acid. For 200ul acid it will take at least 1 to 2hrs speed vac (in the absence of heat) to remove acid or it might less depending on the efficiency of your speed vac.

Good luck
Rajeev

Dear All,
Really I need to perform working HPLC method for determination of hydroxyproline in urine...... really I found too much papers and I am so confused in choosing the best one using UV-Vis detector.....
Anyone have a good advice?????

Hey,

I am having problems with the assay. After the reaction with the chloramine T and aldehyde, my samples precipitate out to were there is no longer a color change. I dont know what is going on? Has any one had this problem before? Are my reagents bad? Any help you can give me will be great!!

Crazy Lab Jai

Hi,
I am going to do this HA assay. But from the protocol people use tissue. How about if I am going to use cells? Or more specific cell line in culture? Should I centrifuge the cells and take out the pellet and digest it or take out the supernatant and digest it?

Thank you