Sensitivity of Griess Reagent - How could they do it!? (Oct/21/2008 )

Hello there!

I will soon enough work with polymer films that release nitric oxide (NO). I haven't started the experience yet. But I do have already issues and questions about this Griess Assay, mostly about the sensitivity of the assay.

according to the degradation equation of NO, it means that if the griess assay can only detect more than 3uMole of NO (by detecting more than 3uMole of Nitrite)? the thing is that I have been reading a lot and I found 2 papers that could present NO release kinetic graphs lower than 1umole of NO while using the Griess assay.. O_o how did they do it? is that possible?

I am asking because i will probably need an assay which can detect less than 3uMole of NO...but I don't think i can buy the chemilumeniscence machine (sievers NO analyzer)...is there any kit that can measure lower than 1uMole of NO? (most of the kit a found can only measure between 1uMole to 100uMole)

thanks a lot for any suggestions.

Evelyne

Ho-wook Jun and al., Nitric oxide producing polyurethane, biomacromolecule, 2004
Kelly A. Mowery and al., Preparation and characterization of hydrophobic polymeric films that are thromboresistant via nitric oxide release, Biomaterials, 1999

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Dear Evelyne,

The Griess assay will only be sensitive at 3uM Nitrite and above. The Sievers machine you mentioned is what we have and can go down to 50nM Nitrite. I have 20 years experience in NO detection in various cellular based systems. The Griess reaction is quick, easy and cheap but relatively insensitive. The Sievers machine is expensive, requires regular maintenance and a level of technical competence.
If you can only afford to use the Griess reaction then why don't you reduce the Nitrate to Nitrite using a Nitrate reduction step. Then you will be measuring TOTAL NITRIC OXIDE(NO)
REMEMBER that NO oxidises to Nitrite and Nitrate. Combining the 2 together may get your total Nitrite to levels well above this 3uM level.

Group papers: Moncada et al: Nature, Vol.320, No. 6061, pp. 454-456, 3rd April 1986.


Moncada et al: Nature, Vol. 327, No.6122, pp. 524-526, 11th June 1987.


Hope this is useful.

Rhombus

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ahh. thanks, I completely forgot about the nitrate. but I dont get you when you say TOTAL NO...according to the degradation reaction, I thought that one mole of NO gives 1 mole of Nitrate and 1mole of Nitrite, is that right?
If we should reduce nitrate to have the total of NO, how come a few griess assay kit do not proceed this reduction step (they only perform the nitrite measurement)? does that mean we always get a wrong NO measurement through only Nitrite measurement?

thanks again. ^^

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Dear Evelyne,

The reference below may help. In our hands NO is oxidised in cell systems to NO2- and NO3-. The relative amounts vary. In animal cells such as Rat, Mice, Cow and Pig, Griess will pick up approximately 60%-80% NO2-. The rest will be Nitrate. However with Human cells, the ratio is completely reversed. 80% of the NO is oxidised to NO3-, 20% to NO2-.

I hope this is useful again

Rhombus.



Proc Natl Acad Sci U S A > v.90(17); Sep 1, 1993
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PubMed articles by:
Ignarro, L.
Fukuto, J.
Griscavage, J.
Byrns, R. Proc Natl Acad Sci U S A. 1993 September 1; 90(17): 8103–8107. PMCID: PMC47296

Copyright notice
Oxidation of nitric oxide in aqueous solution to nitrite but not nitrate: comparison with enzymatically formed nitric oxide from L-arginine.
L J Ignarro, J M Fukuto, J M Griscavage, N E Rogers, and R E Byrns
Department of Pharmacology, University of California, School of Medicine, Los Angeles 90024.

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ok ^^ thanks a lot for this precious information.

ill be back to you in a few month (or next week ... ) to tell you if everything works.

evelyne

P.s. i am still curious though on how these people i mentioned could detect so small amount of NO with the griess assay (0-1uMole)...

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Some other tips I forgot to mention. Make sure that the media that you are using is Nitrite/Nitrate free. We use DMEM liquid media as it has no Nitrate in it. RPMI on the other hand has mM amounts of Nitrate, which would swamp any small amounts of NO oxidised to NO3-. If you are using water then use the best quality water, again with low amounts of NO2-/NO3-.

Because the Griess is an end point, colour change assay, using water/media alone will nearly always give you a background colour. In my hands, making up all the ingredients myself, rather than using a pre-made kit, the standard curve which you get at the lower range (0-3uM) is too unreliable. I was always taught to use the middle part of the standard curve in order to reduce standard errors.

If I was reviewing a paper that had data showing levels of below 3uM then I would ask to see their standard curves from the spectrophotometer.

I hope your research is successful.

Regards

Rhombus

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Keep going! any advice from you is so much interesting and more efficient than looking all over the website. thanks a lot. I already tried by the way to contact the researchers that show surprising very low NO release...but no answer yet...

so you do advice me to make my own "griess assay" rather than use a kit from a company...?

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Dear Evelyne,

I use my own ingredients because once you have purchased the Sulphanilamide, N-1- napthylethylene diamine dihydrochloride and Sodium Nitrite, it is CHEAPER than buying kits......Kits remember have use by dates. The chemicals can be kept for years to make up as many solutions as you need. Again you have to be diligent in making up the stocks. However you will know straight away if you have made a mistake....no colour or dilution errors will show up in the standard curve.

Interestingly I have just googled Griess reaction and the Promega site, which sells one of the commercially available kits states "the lowest sensitivity for our kit is 2.5uM"........"standards made up in ultra pure dionised distilled water"

Nice to converse with someone who thinks about their experiments before performing them.....saves time and money

Kindest regards

Rhombus

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well even if this Griess assay seems to be very easy to perform, it gives me a hard time, as there are many adaptations of this assay (as you already know, I am looking for a detection range from 0 to 5umole)...I've been looking for a nice modified griess assay protocols that could help me to reduce the detection range from 100umole down to 10umole (while increasing the detection limit of course), but these papers were not available online.

i keep looking on internet...but i think ill start with a very cheap kit, just to perform the protocol...when the real experiment is going to start, hopefully i will have found a protocol adapted to my NO releasing levels.

wish me more than luck and thank you!

p.s. ho by the way, is there specific details that i must pay attention to if i want to do the nitrate reduction step before doing the griess assay? I read somewhere that the compounds used for this reduction step can interfere with the further diazotiziation reaction...again, there are so many different protocols for the nitrate reduction step...

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