Very long reply about EDTA RE: [Histonet] rat myelofibrosis questions
gcallis <@t> montana.edu
Fri Nov 3 14:05:05 CST 2006
Barry and fellow Histonetters,
This will be a long reply on EDTA decalcification - delete if not
interested. Barry, you were correct in your thinking, and not confused. I
feel much of the confusion that arises about EDTA decalcification comes
from several things. One is not only adjusting the pH upwards but actually
changing the EDTA molecule in chemical terms to make decalcification with
this molecule work.
1. It helps to understand how EDTA chelation functions to decalcify bone
or chelate calcium.
EDTA binds to calcium as a function of pH (Harris DE, Quantitative
Chemical Analysis, Ch: EDTA Titrations, p271-289, 1982 and the
molecule is made up of tetracarboxylic acid (COOH) and 2 amine (NH) groups
that are fully protonated at an acidic, LOW pH so that decalcification
cannot occur. As one progressively raises the pH (using sodium hydroxide)
the protons dissociate from these groups, known as unprotonation, and
decalcification become possible. Above pH 10, these groups
are completely unprotonated and this form of EDTA binds calcium the
strongest. This also makes decalcification proceed at a
faster rate. Unfortunately, but this high pH can be damaging to
alkaline sensitive protein linkages.
Consequently, if you try to use EDTA at pH 3, decalcification is
not occuring in fact, it only begins to
decalcify very slowly around pH 4. As you continue to raise the
pH to 7, decalcification is faster than at pH 4, and at pH 8, the EDTA is
even more efficient. However pH 8 is approaching too alkaline for
those sensitive protein linkages. This was discussed in JOH, March
1998 Review of Decalcification, Callis and Sterchi.
EDTA formulations and EDTA with sodium salts ARE more soluble as the number
of sodiums increase. Disodium salt of EDTA is less soluble than tetrasodium
salt of EDTA but it is important to note that their pH's are different too.
2. EDTA powders (please note plural here) used for decalcification can be
several EDTA formulations
a. EDTA (edetic acid) has formula weight of 292, is soluble in
water at approx 10%, a concentration that is useful
for decalcification. To get this EDTA into solution at this high
concentration, heat and adding sodium hydroxide is used which also
contributes to helps "unprotonating" the molecule. Dissolved in water,
this EDTA probably has acidic pH around 4 or so before adding the sodium
hydroxide (your memory served you well! )
b. EDTA disodium has a formula weight 372. A 5% solution has pH 4
to 5 at RT. Once again is soluble in water up to 10% concentration,
but people can experience difficulty making up solution until sodium
hydroxide is added which adjusts the pH to 7 or so.
c. EDTA tetrasodium salt has a formula weight of 380 and is
highly soluble in water (14% of more) with a pH of 9.5 -
12. This molecule of EDTA is fully unprotonated, but the high pH
is not good for the sensitive protein linkages. It also will
decalcify faster at this high pH, but adjusting the pH DOWN with
acetic acid to pH 7.4 or a pH needed for enzyme histochemical work is
advisable. We have actually decalcified at pH 7.6, which is the pH of our
TRIS buffered saline, and people have good immunostaining
results. I have seen pH 7.4 as being ideal rather than pushing it to 7.6.
When doing enzyme histochemical staining, we stay within a specific, narrow
pH range needed for the enzyme. That pH could be even lower than 7.
In 1997, Diane Sterchi and I embarked on a decalcification
publication. She did all the bone decalcification testing including using
10% EDTA to see the effects of these different pH's (3.2, 7, 10.3) for
speed and success with EDTA decalcification. I guess you could call it
testing the function of pH of EDTA for calcium binding, and her results
were consistent with that dependence on pH. All bones were the same size,
from the same animal and she used FAXITRON X-ray to determine
endpoint. The results were:
EDTA pH 3.2 - the bone never decalcified!
EDTA, pH 7.0 - bone decalcified in 40 days
EDTA, pH 10.3 - bone decalcified in 16 days
This was an eye opener and set standards here for using EDTA
decalcification. Now we pay attention to which EDTA is used, it's
solubility, original pH. Alll this is found in a Merck Index or some
chemical catalog (Aldrich). We then proceed with either using sodium
hydroxide to unprotonate the molecule and adjust to a higher pH (7 - 7.4)
for faster decalcification without damaging alkaline sensitive protein
linkages. OR our favorite, highly soluble tetrasodium EDTA at 14% to put
more molecules into solution and available for chelating the calcium with
simple pH adjustment with glacial acetic acid. We never used hydrochloric
acid for this purpose and followed a Webb Jee publication method using the
acetic acid, a decision to follow a bone expert's good results.
I have never tried to put EDTA disodium into solution at 14% concentration,
but it may work by adding the sodium hydroxide pellets. It sometimes is a
guessing game when publications do not tell you the exact formula weight
nor nomenclature of EDTA, but I guess one could assume it is usually
disodium or the Edetic acid (EDTA alone). So much for exact scientific
I have to thank my physical chemist spouse for lectures on unprotonating
the EDTA molecule, complete with charts from a textbook, and no tuition -
his fee was h'ors deours (mispelled that one!) dinner, and clean clothes
and house for a lifetime.
Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717
12:04 PM 11/2/2006, you wrote:
>I am a little confused so should put my thoughts down.
>EDTA has four possible chelation sites for chelating calcium and other ions.
>If you start with EDTA powder and adjust the pH upwards you will first get
>mono, then di, then tri and finally tetrasodium EDTA.
>EDTA powder by itself if not very soluble until the pH is adjusted. Most
>recipes start with the disodium salt.
>I forget the actual pHs but memory is that they are around
>4.6, 6.5, 8.5 and 11 respectively (forgive me if incorrect but I am not
>at my office computer).
>Whenever you adjust the pH you alter the EDTA salts that are produced.
>So that at pH 7.0 or so you have a micture of disodium and trisodium.
More information about the Histonet