11. M U C O P O L Y S A C C H A R I D E S IN T I S S U E C U L T U R E
359
chromatically with toluidine blue, to make a negligible contribution to
tissue metachromasia, and considers the latter to be due entirely to
sulphated mucopolysaccharide such as chondroitin sulphate, the
interaction of which with dye has been studied by Pal and Schubert
(1961). Miiller (1959), in a comparison of toluidine blue metachromasia
with Alcian blue and periodic acid-Schiff staining procedures, concluded that metachromasia is essentially a property of sulphated
mucopolysaccharide, but the various methods do not give comparable
results. The use of enzymes in conjunction with such staining procedures
has been described by Fullmer (1960). A review by Curran (1961)
on the histology of mucopolysaccharides, points out a number of
possible fallacies in the identification of these substances by enzymic
methods. Herring and Kent (1958) separated the mucoproteins from
ox tracheal cartilage and showed that a polysaccharide that contained
neutral sugars was responsible for the periodic acid-Schiff reaction.
The reaction of polysaccharides to this procedure is dependent upon
the presence of free hydroxyl groups, and complete esterification with
either sulphate or acetate precludes a positive reaction. Thus in general
this procedure is not satisfactory for the location of the acid mucopolysaccharides. Metachromatic staining and affinity for Alcian blue,
however, are not restricted to acid mucopolysaccharides, but are shown
by non-sulphated sialoglycoproteins, since digestion with sialidase
eliminates these properties (Spicer and Warren, 1960). The periodic
acid-Schiff procedure also stains sialoglycoproteins, since the intensity
of the stain is reduced after treatment with sialidase (Montreuil,
Defretin, Clay and Carnes, 1959).
D. A N A L Y S I S OF M U C O P O L Y S A C C H A R I D E S
A detailed survey of chemical procedures for the analysis of mucopolysaccharides is outside the scope of this chapter. Nevertheless, the
authors feel that any work on mucopolysaccharide metabolism in
Tissue Culture is so dependent upon efficient fractionation and analytical techniques, that a critical review of available methods applicable
to the micro-scale of tissue culture is necessary.
At present no methods are available for the direct determination
of mucopolysaccharides in tissues, whilst estimations based on analysis
for components in unfractionated tissue hydrolysates are, at best,
unreliable and generally valueless.
E. E X T R A C T I O N OF M U C O P O L Y S A C C H A R I D E S
Since mucopolysaccharides exist in combination with protein, the
first problem in analysis is the removal of the mucopolysaccharides
359
chromatically with toluidine blue, to make a negligible contribution to
tissue metachromasia, and considers the latter to be due entirely to
sulphated mucopolysaccharide such as chondroitin sulphate, the
interaction of which with dye has been studied by Pal and Schubert
(1961). Miiller (1959), in a comparison of toluidine blue metachromasia
with Alcian blue and periodic acid-Schiff staining procedures, concluded that metachromasia is essentially a property of sulphated
mucopolysaccharide, but the various methods do not give comparable
results. The use of enzymes in conjunction with such staining procedures
has been described by Fullmer (1960). A review by Curran (1961)
on the histology of mucopolysaccharides, points out a number of
possible fallacies in the identification of these substances by enzymic
methods. Herring and Kent (1958) separated the mucoproteins from
ox tracheal cartilage and showed that a polysaccharide that contained
neutral sugars was responsible for the periodic acid-Schiff reaction.
The reaction of polysaccharides to this procedure is dependent upon
the presence of free hydroxyl groups, and complete esterification with
either sulphate or acetate precludes a positive reaction. Thus in general
this procedure is not satisfactory for the location of the acid mucopolysaccharides. Metachromatic staining and affinity for Alcian blue,
however, are not restricted to acid mucopolysaccharides, but are shown
by non-sulphated sialoglycoproteins, since digestion with sialidase
eliminates these properties (Spicer and Warren, 1960). The periodic
acid-Schiff procedure also stains sialoglycoproteins, since the intensity
of the stain is reduced after treatment with sialidase (Montreuil,
Defretin, Clay and Carnes, 1959).
D. A N A L Y S I S OF M U C O P O L Y S A C C H A R I D E S
A detailed survey of chemical procedures for the analysis of mucopolysaccharides is outside the scope of this chapter. Nevertheless, the
authors feel that any work on mucopolysaccharide metabolism in
Tissue Culture is so dependent upon efficient fractionation and analytical techniques, that a critical review of available methods applicable
to the micro-scale of tissue culture is necessary.
At present no methods are available for the direct determination
of mucopolysaccharides in tissues, whilst estimations based on analysis
for components in unfractionated tissue hydrolysates are, at best,
unreliable and generally valueless.
E. E X T R A C T I O N OF M U C O P O L Y S A C C H A R I D E S
Since mucopolysaccharides exist in combination with protein, the
first problem in analysis is the removal of the mucopolysaccharides