Very long reply about EDTA RE: [Histonet] rat myelofibrosis questions

Gayle Callis gcallis <@t>
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.

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