358
N.K.KARAMANOS
GPC on Sepharose CL-6B, Sephacyl S-200 or S-500 of the GAG population isolated by ion-exchange chromatography may provide useful information on the
homogeneity of the population according to its size. The average molecular mass
can also be determined using standard GAG preparations with known Mrs. When
low amount of GAGs is available, gel-permeation HPLC coupled with refractive
index detection of eluting polymers could be used (Arvaniti et al. 1994).
5.2
Strategies to Characterize Glycosaminoglycans
Various strategies may be used to determine GAG structures. Analysis of intact
polymers may give information on charge density, polydispersity and the molecular size of the GAG chain. Information on the chain composition can be
obtained by depolymerization of GAGs to unit fragments. Modern separations
allow most of this information to be deduced from the disaccharide fragments
obtained by enzymic cleavage. Harsher hydrolytic procedures yielding monosaccharide components are rarely needed. When determining longer sequences,
larger fragments must be analyzed. Such fragments can be obtained by employing enzymes, with specific cleavage sites distributed less frequently along the
GAG chain.
The improved resolution provided by HPLC and CE permitted the accurate
analysis of crucial parameters such as the pattern of sulfation, types of uronic
acid and hexosamine (Karamanos et al. 1988, Hjerpe et al. 1980). Clinically, the
simple HPLC or CE based determination of HA in effusion has proven to be of
great value for diagnosis malignant mesotheliomas (Karamanos and Hjerpe
1997). Combining different HPLC and CE separations one could even identify a
sequence charac~eristic of an entire DS chain (Karamanos et al. 1995a, 1995b).
With chromatographic methods, flow is more or less laminar, while in CE flow is
more homogeneous, giving narrower bands/peaks and, therefore, higher resolving power. When similar detector sensitivity in terms of concentration is needed,
the narrower bands also provide improved sensitivity and consequently, better
possibilities for further elucidation of GAG structures. Both HPLC and CE can be
used to elucidate GAG structures, CE is advantageous when working with biological samples because of the trace amount of sample needed. Furthermore the low
consumption of solvents seems desirable for environmental concerns. Applications of both HPLC and CE in combination to characterize the fine chemical
structure of GAGs have been reviewed by Karamanos and Hjerpe, 1996. For further insight into principles of CE and applications to carbohydrate analysis the
books written by R. Kuhn 1993 and Z. El Rassi 1996 are recommended. Some
reviews (El Rassi and Mechref 1996, Grimshaw 1997, Karamanos and Hjerpe 1998
and Lamari and Karamanos 1999) on the structural characterization of GAGs/
PGs and the relation of GAG/PGs to disease diagnosis (Hjerpe and Karamanos
1998), using CE could be recommended.
N.K.KARAMANOS
GPC on Sepharose CL-6B, Sephacyl S-200 or S-500 of the GAG population isolated by ion-exchange chromatography may provide useful information on the
homogeneity of the population according to its size. The average molecular mass
can also be determined using standard GAG preparations with known Mrs. When
low amount of GAGs is available, gel-permeation HPLC coupled with refractive
index detection of eluting polymers could be used (Arvaniti et al. 1994).
5.2
Strategies to Characterize Glycosaminoglycans
Various strategies may be used to determine GAG structures. Analysis of intact
polymers may give information on charge density, polydispersity and the molecular size of the GAG chain. Information on the chain composition can be
obtained by depolymerization of GAGs to unit fragments. Modern separations
allow most of this information to be deduced from the disaccharide fragments
obtained by enzymic cleavage. Harsher hydrolytic procedures yielding monosaccharide components are rarely needed. When determining longer sequences,
larger fragments must be analyzed. Such fragments can be obtained by employing enzymes, with specific cleavage sites distributed less frequently along the
GAG chain.
The improved resolution provided by HPLC and CE permitted the accurate
analysis of crucial parameters such as the pattern of sulfation, types of uronic
acid and hexosamine (Karamanos et al. 1988, Hjerpe et al. 1980). Clinically, the
simple HPLC or CE based determination of HA in effusion has proven to be of
great value for diagnosis malignant mesotheliomas (Karamanos and Hjerpe
1997). Combining different HPLC and CE separations one could even identify a
sequence charac~eristic of an entire DS chain (Karamanos et al. 1995a, 1995b).
With chromatographic methods, flow is more or less laminar, while in CE flow is
more homogeneous, giving narrower bands/peaks and, therefore, higher resolving power. When similar detector sensitivity in terms of concentration is needed,
the narrower bands also provide improved sensitivity and consequently, better
possibilities for further elucidation of GAG structures. Both HPLC and CE can be
used to elucidate GAG structures, CE is advantageous when working with biological samples because of the trace amount of sample needed. Furthermore the low
consumption of solvents seems desirable for environmental concerns. Applications of both HPLC and CE in combination to characterize the fine chemical
structure of GAGs have been reviewed by Karamanos and Hjerpe, 1996. For further insight into principles of CE and applications to carbohydrate analysis the
books written by R. Kuhn 1993 and Z. El Rassi 1996 are recommended. Some
reviews (El Rassi and Mechref 1996, Grimshaw 1997, Karamanos and Hjerpe 1998
and Lamari and Karamanos 1999) on the structural characterization of GAGs/
PGs and the relation of GAG/PGs to disease diagnosis (Hjerpe and Karamanos
1998), using CE could be recommended.