Hydroxyproline assay

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R.K.B
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Hydroxyproline assay

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

cfish
cfish's picture
Hydroxyproline assay:

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

R.K.B
R.K.B's picture
Thanks for posting the link.

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

ABCD09
ABCD09's picture
R.K.B wrote:Thanks for

R.K.B wrote:

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

Could you please post or forward me the micro assay for hydroxyproline? This would be helpful for my assay.

Thanks.

R.K.B
R.K.B's picture
Hello ABCD09,

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)

Ramjee Pallela
Ramjee Pallela's picture
HI All....

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.

Ramjee Pallela
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(No subject)
R.K.B
R.K.B's picture
Ramjee Pallela wrote:

Ramjee Pallela wrote:

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]

Harvard_Med_Student
Harvard_Med_Student's picture
Hi Rajeev,

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

R.K.B
R.K.B's picture
Hello 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

Harvard_Med_Student
Harvard_Med_Student's picture
Hi 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!

R.K.B
R.K.B's picture
hello kapil

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

fatmaelzahraa
fatmaelzahraa's picture
Dear All,

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?????

missjai81
missjai81's picture
Hey,

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

Ti
Ti's picture
Hi,

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

R.K.B
R.K.B's picture
missjai81 wrote:Hey,

missjai81 wrote:

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,

Were you able to trouble shoot sample precipitation problem,

Rajeev

R.K.B
R.K.B's picture
Ti wrote:Hi,

Ti wrote:

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

Hello,

I haven't tried to analyze Hydroxyproline from cell culture system.
I think it is possible in cell culture system to estimate hydroxyproline. What exactly you are trying to do in your experiment?
Rajeev

Ti
Ti's picture
I am going to culture the

I am going to culture the cell lines in a monolayer form. Then extract the cells to see whether the cells produce collagen or not.

harp
harp's picture
Hi Ti and Rajeev,

Hi Ti and Rajeev,

Ti, since collagen is secreted by the cells (chondrocytes or fibroblasts) you should look for the collagen in the supernatant. 

Have you succeeded in measuring collagen in your cell line cultures?

I am growing chondrocytes on gel scaffolds made from polysugars and 
I am going to try this protocol to quantify the collagen in my scaffolds.
Any reccomendations from your experience?

Thanks.

Ramjee Pallela
Ramjee Pallela's picture
Dear Rajeev,

Dear Rajeev,
I have compared four to five formulas and ultimately finalized the formula, which exactly matched with the ones provoded by you. Anyways, long back I have completed my collagen quantification from marine sponge tissues by using the same formula. thanks for the detailed touch ups. take care

erikreinertsen
erikreinertsen's picture
 I have a question about the

 I have a question about the dry step. I'm performing a hydroxyproline assay on cellular supernatant. Do I need to dry the sample using an oven (and is it really 1000 degrees C?), or can I just use NaOH pellets? I'd prefer the less time-consuming method. Thanks.

R.K.B
R.K.B's picture
Hello Erikreinertsen,

Hello Erikreinertsen,

If you are still doing the hydroxyproline assay from the cellular supernatant,

- I don't think you need to dry the sample after treating with HCl or NaOH.

-The original protocl calls for treatment with HCl because, I was releasing the cross-link from collagen molecule inorder to analyze the hydroxyproline.

- If give tell exact nature of your experiment (ex: cell type, what is the end goal ), it will help to give you correct answer.

sorry for late reply
Rajeev

erikreinertsen
erikreinertsen's picture
 I am culturing fibroblasts

 I am culturing fibroblasts and keratinocytes in a fibrin-polymer scaffold, and removing supernatant (with replacement) at regular time points. I am then running assays on the supernatant to determine protein expression as a function of time and scaffold composition.

I feel like I should let the sample dry because the collagen content will be extremely dilute as it is. What do you think?

Thanks for your reply.

R.K.B
R.K.B's picture
You are right,

You are right,

I think it is good idea to dry HCl treated sample for following reasons:

- It will concentrate the treated collagen sample in small volume.

- It will also help to remove the HCl (if you are using the HCl to digest collagen protein)

Rajeev

semsem
semsem's picture
Hi Rajeev,

Hi Rajeev,
I'm working on tretment of lung fibrosis in rats. i need to measure hydroxyproline in lung homogenate. but i dried the lung using a filtration paper and freezed it rapidly in liquid nitrogen and then kept it at -80 C.  can i measure HP content of my lung homogenate after freezing? On the way i didn't add HCL before freezing.

                                           Thanks,
                                             SemSem

R.K.B
R.K.B's picture
Hello SemSem,

Hello SemSem,

I think you can measure HP content from frozen lung homogenate.  In that past, I have used frozen tissue samples to measure HP content (not the lung tissue).  Plus you are measuring the HP content which is building blocks of collagen protein and they are stable and you should be able digest the collagen protein with HCl from frozen tissue sample.

Good luck
Rajeev

strejo
strejo's picture
Hello,

Hello,

I had more luck preparing my samples (mouse spleen, heart, and quadracept) than in making the basic standards from hydroxyproline. I ordered poly-l-hydroxyproline, but it simply wouldn't go into solution.

Earlier someone posted that l-4-hydroxyproline was used, Sigma number P5607. However, the Sigma web site shows that product number as being "l-proline." Is that the right one?

Thank you very much.

Sonya

erikreinertsen
erikreinertsen's picture
A 96-well plate is extremely

A 96-well plate is extremely small; because most cell lines in small quantities don't produce a ton of collagen in vitro, I am looking into a drying step to concentrate collagen concentration (which is what most people do but I am looking at a slightly optimized version of this protocol: Reddy, G., Enwemeka, C., “A Simplified Method for the Analysis of Hydroxyproline in Biological Tissues.” Clinical Biochemistry 1996; Vol. 29, No.3 225-229. I use Nalgene 1.5 mL screwtop tubes so I can safely autoclave the contents. I'd recommend saving the supernatant, trypsinizing and spinning cells down, and running the assay on all of that combined.

Intracellular collagen will be a significant component - be sure your protocol involves lysing. If you are doing 3D culture systems, the collagen could also bind to the scaffolds (like in my case where I am using polymers and biomimetics).

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minor-latin">

MMA
MMA's picture
Hi,

Hi,
I'm going to be looking at fibrosis in lungs - and have a couple of questions - it was mentioned above that the hyp can be expressed as ug hyp / wt of tissue. I assume that's a 'dry' weight? or can you measure wet wt at time of tissue collection and use that in the calculation? If it's a dry weight and I'm freezing the tissue for future analysis, should I dry, then freeze...or freeze, then thaw, dry and weigh at time of running the assay?

On the freezing issue, I have some lung tissue that was frozen at -20 and has been stored for a few years (it was not flash frozen in liquid nitrogen). Does anyone know if the assay will still be accurate on this tissue?

thanks in advance for any suggestions.
Mandy

semsem
semsem's picture
Hi MMA: 

Hi MMA: 

I'm semsem how asked Rajeev about measuring HP in lung homogenates. 

From my readings, the wet weight was using in more than one paper (this is the method i'll use), but also u can use the dry weight. The wet weight can be measured at time of tissue collection or immediatly after thawing of tissues.

If u'll use the dry weight (used in few papers):

Simply, lung samples should be freezed firstly by snap freezing in liquid nitrogen and then stored at -80 C, then after thawing put it in regular dry oven at 80 C (in some papers for 16 hr). Then the dry weight will be available now to measure.

For ur 2nd question:

I'm sorry, i think ur tissue samples are not suitable now for ur assay as u didn't freeze it in liquid nitrogen. Also, u freeze it in -20 C and i think this temperature is not suitable for storring lung tissues for few years.

                                                                                                  GOOD LUCK 
                                                                                                    SEMSEM

semsem
semsem's picture
Hello Rajeev,

Hello Rajeev,

Thanks alot for ur reply. but i've now some questions:

1- I did homogenate from my lung tissues by using PBS (phosphate buffer saline), and then centrifuged the samples at 14.000Xg for 10 min. The supernatants were collected to measure HP. Did this way affect my results. Or i should homogenate my samples in HCL.  By the way, should i dry my supernatant befor adding HCL or what.?

2- I put my supernatants (0.5 ml) in pyrex screw tightly capped tubes and added 1ml of 6M HCL then put it in regular dry oven for 16 hr on 110 C (as i read in papers). But my samles were evaporated  and just few burned tissues were in the bottom of the tube. what can i do. How can i make my samples not evaporated? and can i reduce the time and elevate temperature (for example 120 C for 8 hr, readed in some papers)? Also can i use autoclave to by pass this step? I'm really so depreesed as this step is an obstcle to me.  

3- I don't have a speed vaccum. Can i neutrilize the samples without using the speed vaccum to dry the HCL? Also,when i'm not using speed vaccum,i read in some papers that i can use filtration membrane to filtrate the samples after neutralization by NaOH, Is it true?

                                                                   Thanks, 
                                                                   semsem

BRL
BRL's picture
 Hello all 

 Hello all 

I am new to lab and i am experiencing problem with estimation of hydroxyproline from tissue. 
The standards i prepare about 2-20 microgm/ml in citrate acetate buffer or in distilled water are not developing the red-purple colour instead its yellow. Can someone help me here. Point out my mistake in procedure. 

i am following this. 

1.Prepared 1mg/ml Hyp stock in citrate acetate buffer
2. prepared varius standard concentration by diluting with distilled water 
3. 1ml of standard added 1 ml chloramine T , incubation 25 min 
4. 1ml PDMAB , incubate at 65 degrees for 20-25 min 
5. The colour developed is yellow only . 
please advice if autoclaving sample at 120degrees to hydrolyse it is necessary ? 

Thanks in advance. 
Please Help 

fatma_elzahraa
fatma_elzahraa's picture
Hi...

Hi...
I didn't get if the problem in the std measurement or in the sample.....
If it is in std, plz check ur chemicals expiry dates..... and I remembered that chloramine T should be freshly prepared....
So, Try it again after preparing fresh reagents.... then tell us what is happened to re-think about the problem again....
For the sample, I hydrolyzed my samples in sealed ampoules to reduce oxygen reaction with the sample constituents.... also, I hydrolyzed them in autoclave for 20 min.... then try to take the clear part to work with....

Waiting for ur news.... :)

BRL
BRL's picture
 Hi Fatma 

 Hi Fatma 
Thank you for ur reply 
I tried again and the colour developed is still yellow. 

I prepared (2-20)microgm/ml of standard. To 500microL of each standard added 50uL NaOH and hydrolysed it by autoclaving at 120degrees for 20 min .
Can you please help me with the volume of standard i should take for hydrolysing . 

Please suggest 

fatma_elzahraa
fatma_elzahraa's picture
Dear BRL

Dear BRL
I think u shouldn't autoclave ur std...... try it without autoclaving.....
also, I autoclaved my samples with HCl not NaOH...... try this too..... put an equivolume of ur std and conc HCl in a sealed tube or ampoule then autoclave it.....

Try this and tell me about ur progress....

God be with u.....

Best regards

BRL
BRL's picture
 Dear Fatma  and everyone

 Dear Fatma  and everyone else

I have been following this protocol 
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

If i hydrolyse samples in HCL i have to neutralise them also . Will do and tell you . 
Thank you for the help so far. Hopefully i succeeed inshaAllah 

fatma_elzahraa
fatma_elzahraa's picture
Yeah... I read the methodolgy

Yeah... I read the methodolgy in the paper u sent...
the pink color was developed with me in std solutions.... std was prepapred in dil HCl.... but this is n't the real problem....
just try acidic hydrolysis.... and tell me about the color.....
but plz... check of ur chemicals....
Once, I had a problem in color developed in another assay.... finally, i discovered that one of my chemicals was expired or its color in its package was changed.... when i relaced it with another one, the assay worked fine....
we will try here together.... till isA we will get the clue to develop the pink color....
Best regards

BRL
BRL's picture
 Hi Fatma , 

 Hi Fatma , 

I did try preparing standards in 0.001N HCl and it definitely developed colour.
Also i found that my CHloramine T isnt working fine. Its hydrated and not free flowing powder anymore. So may be that was a problem. I changed my protocol to Nueman 1950 and it developed pink colour with a good standard curve with slight modification. 
Today am trying with the tissue sample. Hope to succeed . 
Will share with you the protocol then. 

Thanks for the encouragement :) 

fatma_elzahraa
fatma_elzahraa's picture
 That's very good news....

 That's very good news....
really, I tried Newman assay too, and it worked with std solutions.... but I couldn't recommend that from the start as there may be some type of small problems in the assay u used before....
generally, I am happy to hear that....
just tell me about ur samples after hydrolysis with HCl....
u can try it by the two methods of hydrolysis (one by HCl and the other by NaOH) and see the difference....
I hope it gave u good results with samples too.... as really, it didn't work with me that way and I had to use HPLC method.... but I didn't want to tell that too.... hope ur problem solved in spectrophotometric assay and not going to use HPLC at all.....

Nice again to hear that... and waiating for ur other good news..... :)

SKP
SKP's picture
 Dear Fatma and BRL

 Dear Fatma and BRL
 I am estimating hydroxyproline in urine by Neuman metod. I have a problem in estimating hydroxyproline. I took all the care about temperature, vigorous shaking.  But-the urine samples are producing an orange red hue, whereas the serial standards  treated similarly for plotting standard curve are producing  pink colour. Is any interfering substance present in the urine hindering the pink colour formation? Should i change my method to chloramin T  .Please give your valuable suggestions  to overcome this problem. 

BRL
BRL's picture
 Dear SKP 

 Dear SKP 

If you followed all protocol properly and your standards are giving the colour pinkinsh then there is some problem with your sample processing. Did you hydrolyse the samples before starting? If yes then try out hydrolysis with 2.5N NaOH followed by autoclave for 30 min. 
Tell me about it if any success.

SKP
SKP's picture
 Dear BRL Thank u so much for

 Dear BRL Thank u so much for your prompt reply
I am estimating HYP in urine samples with out any pretreatment.After your suggetion i tried hydrolysis with 2.5N NaoH and autoclave but still the colours not pink.Please suggest me what should i do.is there any important steps to observe in hydrolysis (inomplete hydrolysis ?) 

fatma_elzahraa
fatma_elzahraa's picture
Hey SKP.... I ahd the same

Hey SKP.... I ahd the same problem in urine samples.... so I measured HYP in serum samples .... I hydrolysed the samples with 6N HCl then derivatized by a reagent called dabsyl chloride and estimating HYP using HPLC ...... as spectrophotometric method didn't work in urine samples... if u have the instrument and the reagents, tell me to send u the procedure I followed..... God be with us all.... :)

SKP
SKP's picture
 Hello Fatma

 Hello Fatma
Thank u so much for ur reply .we dont have HPLC so i cant adapt that method.I wrote a letter to auther " Ashuma sachdeva" paper published in indian journal.waiting for their reply.
Can u predict what facters hinder the colour formation in urine.is it NPNsubstances? In what form HYP present in urine. still not getting idia

SKP
SKP's picture
 Hello Fatma

 Hello Fatma
Thank u so much for ur reply .we dont have HPLC so i cant adapt that method.I wrote a letter to auther " Ashuma sachdeva" paper published in indian journal.waiting for their reply.
Can u predict what facters hinder the colour formation in urine.is it NPNsubstances? In what form HYP present in urine. still not getting idia

fatma_elzahraa
fatma_elzahraa's picture
hi SKP

hi SKP
logically, hydrolysis was made to release HYP from any peptides and also break down all other interefering compounds..... so, it is weird to get such color with both of us..... but really, iam so interested in knowing the reason as I searched and asked alot and nobody guided me.....
we can search together and i will look it up again..... as I need to know and really need to help u too... :)
if anyone of us get anything, just inform the other.... God be with u.... :) 

drblzn
drblzn's picture
 Hi, just came by this post

 Hi, just came by this post when I was looking for the hydroxyproline content of alpha 1 and 2 chains of bovine collagen. Anyway, we do the hydroxyproline assay regularly in my lab and I think you need to add 3M perchloric acid and incubate for approx. 5 mins after the Chloramine T and before the p-DABA step. If you need any more help, you can email me at eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%64%72%62%6c%7a%6e%40%67%6d%61%69%6c%2e%63%6f%6d%22%3e%64%72%62%6c%7a%6e%40%67%6d%61%69%6c%2e%63%6f%6d%3c%2f%61%3e%27%29%3b')).

littlelingxin
littlelingxin's picture
 Hi guys, firstly thank you

 Hi guys, firstly thank you all for the answers. I was struggling with the technique recently and your answers gave great hints to solve my problem. so I simply came to contribute a bit more. 
Please be very careful about the chloramine-T. I think this reagent got a limited shelf life. It's supposed to be free-flowing powder, but when moistured, it sticks to each other and form something like a brick. If your assay is not working even if with standard and your chloramine-T is not free-flowing powder, highly suspect this reagent. It might have expired.
I checked some suppliers' website. chloramine-T got a shelf life of 24 months. http://www.grainger.com/Grainger/SPECTRUM-ChloramineT-6PLA1
Pls be very careful about this, because I did order some new chloramine-T, but the reagent is not free-flowing powder when it came!!! and the assay is also not working with new chloramine-T even with standard!!! I checked the website of the supplier. they did not have shelf life information for this reagent.

deedee24
deedee24's picture
Hi,

Hi,

I am having huge problem with the assay. I can't use perchloric acid in the lab. So, my only choice is to use Leach method. I tried 10 times but still can't get standard curve. There is no pink color. Only yellow to amber. Briefly, hydroxyproline std conc 10 ug/ml, 20 ug/ml, 30 ug/ml and blank 0.0001 N HCl. One ml of each std / blank is reacted with 1 ml of 0.05 M CuSO4 and 1 ml of 2.5 N NaOH and tube is vortexed after each addition. Then, test tubes are placed in 40 C water bath and 1 ml of 6% H2O2 is added while tubes in water bath. Tubes are incubated in 40 C water bath for 10 minutes. Cool, then add 3 N H2SO4 and 5% DMAB in N-propanol. Heat in 70 C water bath for 16 minutes. I also tried with 30% DMAB. No pink color formation. only yellow to amber.

Any suggestion what could go wrong? Thank you so much. I need to hydrolyze my tissue but I have to get standard curve and assay to work.

kevin liu
kevin liu's picture
Hi Semsem

Hi Semsem

I have a similar problem as yours, have you found out the way to fix it up?

AKG
AKG's picture
Dear Rajeev,

Dear Rajeev,
I went through your protocol but unable to see the table in its original format.
As color after rection should be pink but I found it yellow-green
Can you attach the protocol to my e-mail. 
eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%6b%69%73%68%6f%72%65%61%6d%69%74%65%73%68%40%67%6d%61%69%6c%2e%63%6f%6d%22%3e%6b%69%73%68%6f%72%65%61%6d%69%74%65%73%68%40%67%6d%61%69%6c%2e%63%6f%6d%3c%2f%61%3e%27%29%3b'))

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