[Histonet] Re: chondroitin sulfate & hyaluronic acid (Long)

[John Kiernan]

A proper answer to Elizabeth's question depends on
the detailed requirements of the investigation: just
what questions do you need to answer? Does it really
matter to be absolutely specific about which of
the chondroitin sulphates? Must you be certain that
the staining of hyaluronic acid is not being
mimicked by another weakly acidic macromolecule?
If rigorous specificity is needed, the following
remarks will not be helpful. If it is sufficient to
stain sulphated carbohydrates differently from
big carbohydrates that are carboxylic acids, this
can be done quite easily with dyes and other simple
reagents. Several controls are always needed. They
are all logically obvious, but also easily missed
for reasons of expediency, impatience etc etc.

Nearly all staining with dyes involves dye ions
or molecules being attracted to and then binding
to much larger molecules such as proteins, nucleic
acids or macromolecular carbohydrates.  

The chondroitin sulphates are salts of a strong
acid (sulphuric). As such, their macromolecular
anions are always negatively charged, even at
very low pH. Any cationic dye used at pH 1 will
probably stain only the chondroitin sulphates in 
a section of cartilage. (I say probably because
I don't know if cartilage also contains heparan
or keratan sulphates, which are similar 
extracellular glycosaminoglycans of connective
tissues.)  Alcian blue at pH 1 is often used
for this job.

Hyaluronic acid is a weak acid, so the pH of a
solution of a cationic dye needs to be 2.5 or
higher for staining to occur. Alcian blue is
better than other basic dyes for this application
because it does not stain nucleic acids. (The
reason is out there, in the Literature.) At
pH 2.5, however, alcian blue stains the
sulphated carbohydrates as well as the weaker
acids such as hyaluronic. To stain the carboxyl
items (like hyaluronate) selectively you must 
first remove the sulphate groups from chondroitin 
etc.

Sulphate removal is a two-step process, and you
will need to consult a histochemistry textbook
for technical details. No unusual chemicals or
apparatus are needed, but the procedures can
remove sections from slides, so be sure to have
plenty of sections. You may need to experiment
with different ways of sticking the sections
on the slides.

The first step is METHYLATION. This permanently
removes sulphate groups and reversibly blocks
carboxyl groups. After methylation nothing should
be stainable with any cationic dye at any pH.

The second step is SAPONIFICATION. This restores
methylated carboxyl groups but it cannot restore
removed sulphate groups. Saponification is also 
a potent remover of sections from slides.

After methylation and saponification, all staining
of extracellular material by alcian blue is due to 
carboxylate anions of hyaluronic acid and/or similar 
macromolecules. The stainability of chondroitin
sulphates cannot be restored by saponification.

As an additional check, you can incubate some
control sections in a solution of hyaluronidase,
an enzyme that catalyses the hydrolysis of the
huge hyaluronate polyanion into small, water-soluble 
fragments. If the enzyme treatment prevents the
staining, it was probably hyaluronic acid. A
control for the enzyme digestion is important,
because staining may be prevented by digestion in
water or a buffer. If this happens, the stainable
material may or may not have been hyaluronic acid. 

There are chondroitinases, which might be exploited 
in the histochemical analysis of chondroitin 
sulphates. I don't think they have been investigated
in the context  of dye-based carbohydrate
histochemistry. Please let me know if I've been
missing some important developments in this field.
-- 
-------------------------
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
   kiernan <@t> uwo.ca
   http://publish.uwo.ca/~jkiernan/
____________________________________________
Elizabeth Chlipala <lizchlipala <@t> premierhistology.com>
asked:
> Is it possible to stain specifically for chondrotin sulfate or hyaluronic acid in the cartilage of decalcified knee
> joints (guinea pig).  I know that Alcian Blue ph 2.5 will stain for hyaluronic acid.  I do have a previous post
> from John Kiernan that explains a pretreatment process for the Alcian Blue pH 2.5 to make it specific for
> hyaluronic acid. But I was wondering if anyone had any other suggestions and what I could do for staining
> of chondrotin sulfate.