Unformatted text preview: Current Molecular Medicine 2007, 7, 397-416 397 Clinical Laboratory Testing in Human Medicine Based on the
Detection of Glycoconjugates
Benjamin L. Schulz#,1 , Wouter Laroy#,2 , $ and Nico Callewaert*,#,2,3
1GlycoINIT and Institute of Microbiology, Swiss Federal
Paulistrasse 10, CH-8093 Zürich, Switzerland
2Unit for Molecular Glycobiology, Department
Technologiepark 927, B-9052 Ghent, Belgium for Institute Molecular of Technology, Biomedical Wolfgang- Research, VIB, 3Unit for Molecular Glycobiology, Department for Biochemistry, Physiology and Microbiology, Ghent
University, K.L.-Ledeganckstraat 35, B-9000 Ghent, Belgium
Abstract: The purpose of this review is to provide a concise overview of developments over the last 15
years in the field of laboratory tests in human medicine that are based on the detection of alterations
in the glycan part of glycoconjugates. We show how glycosylation-based diagnostic testing is
widespread in the current clinical practice, in different formats. To provide the necessary focus in this
extremely broad field, we have only included assays that are either in actual clinical use or that are
under active development towards clinical use, with some bias towards assays that were recently
developed. The fields included are: cancer, infectious disease, genetic defects of glycoconjugate
biosynthesis and catabolism, auto-immunity, drug abuse and liver disease. n
rib To conclude this review, we provide a viewpoint on the future of the glyco-diagnostics field in terms of
novel technologies, especially with regard to the discovery and clinical implementation of biomarkers
that are based on pathologically altered endogenous glycotopes. t
ot GLYCOCONJUGATE-BASED TESTING IN
CURRENT CLINICAL PRACTICE OR
ADVANCED RESEARCH STAGE
Diagnostic and Prognostic Tests for Malignant
Introduction Changes in glycosylation have long been
associated with cancers. Similar to all but a few
biochemical markers for cancer, while none of these
glycosylation-based markers is 100% accurate, they
are still clinically very useful, as will be pointed out
below. Secreted and surface glycoproteins of a
broad variety of adenocarcinomas commonly express
Tn, T, sialyl-Tn glycotopes and increased levels of
the different Lewis blood group glycotopes  (see
Fig. 2 for structures of these glycotopes). Glycobiological cancer diagnosis and prognosis relies on
lectin or antibody-based glycotope
performed either on tumor biopsy material using
histochemistry, or in biofluids using solid-phase or insolution immunoassays. The detection of cancerassociated glycotopes is currently the most widely
studied subfield of glycomics-based diagnosis. The
activity in this field over the last 10 years has mainly
been in trying to establish the utility of these N *Address correspondence to this author at the Unit for Molecular
Glycobiology, Department for Molecular Biomedical Research, VIB,
Technologiepark 927, B-9052 Ghent, Belgium; Tel: +32 9 331 3620; Fax:
+32 9 331 3609; E-mail: [email protected]
$Current address: Pronota NV, VIB Bio-incubator, Technologiepark 4, B9052 Ghent, Belgium
#All these authors have contributed equally.
1566-5240/07 $50.00+.00 markers in a very broad range of carcinoma types,
for a variety of clinical purposes. A second focus has
been the elucidation of the genetic and biochemical
mechanisms behind these glycobiological alterations.
And, third, a range of studies has aimed at
mechanistically contribute to metastasis phenotype.
The latter hypothesis arose from prognostic studies
in which several of the glycobiological alterations
were found to be clearly predictive of a worse patient
outcome. In the sections that follow, we have only included
those tumor antigens with relatively well-established
validity for diagnostic purposes (i.e. in current clinical
use or well on the way to that status). Many more
might be therapeutically relevant as tumor-specific
antigens or might have been described in the
literature, but they have either not proven to yield
more accurate diagnostic information than the more
established carbohydrate-dependent tumor markers,
or are still in a very preliminary stage of validation.
Current Clinical Use, Mainly Detected Via
Histology on Tumor Tissue
Probably the best-known family of tumor antigens
of carbohydrate nature are based on the T-antigen
(Galβ-1,3-GalNAc- α -Ser/Thr) . This structure is also
known as the Thomsen-Friedenreich antigen. The
detection of the T-antigen is performed using the
lectin PNA (peanut agglutinin) or with specific
monoclonal antibodies. PNA-based detection has
© 2007 Bentham Science Publishers Ltd. 398 Current Molecular Medicine, 2007, Vol. 7, No. 4 Schulz et al. n
tio Glycolipids u
rib Polysaccharides Glycosylation
Glycoproteins N O O-glycans N-glycans N Fig. (1). Overview of the diagnostic testing procedures discussed in this review.
Glycoconjugate-based testing is widespread in the current clinical practice, and several assays are in advanced stages of
clinical development. the advantage that it is more protein-carrier
independent than the monoclonal antibodies. An
enzyme-linked PNA assay has also been developed
to measure the T-antigen in serum in the context of
cervical carcinoma (utility in other adenocarcinomas
has not been reported) . The detection of the Tantigen is currently most often used in histological
diagnosis of colorectal carcinoma , where it has a
very good specificity (i.e. ratio of number of true
positives to the number of all positive test results) as
the normal colonic mucosa is T-antigen negative.
The marker has been assessed in a large range of
other carcinomas as well, with varying degrees of clinical utility concluded (down to none in lung
cancer, where the T antigen is expressed on
terminally differentiated pneumocytes and is thus a
differentiation antigen) .
A related tumor-associated glycotope is known as
Tn (GalNAc-α -Ser/Thr). The Tn antigen itself is
detected using monoclonal antibodies, whereas the
lectin from Helix pomatia (HPA) recognizes a broad
array of GalNAc-modified glycoconjugates on cancer
cells (and some GlcNAc binding is also very likely
). Remarkably, HPA lectin histochemistry results Clinical Laboratory Testing in Human Medicine Based depend very much on the technical details of the
method [7, 8]. Given the low affinity of lectins for
their glycotopes, and the fact that lectins, unlike
antibodies, are a diverse family of proteins with
different folds, care should always be taken to not
modifications (such as conjugation to peroxidase for
direct detection). To allow detection of Tn in serum, a
heterogenous immuno-lectin sandwich assay was
glycoproteins on an anti-Tn surface, followed by
glycoprotein detection using biotinylated Vicia villosa
isolectin B4-streptavidin. The assay appeared to
have good analytical characteristics and very good
specificity for the indiscriminate detection of a range
of adenocarcinomas (test negative in all healthy
controls and in patients
diseases). However, we are not aware of it being
used in routine clinical practice.
Increased levels of this glycotope are good
adenocarcinomas of the breast, the colon and the
stomach, the skin , the ovary, oesophagus,
thyroid and prostate . Human adenocarcinoma
cell lines that are HPA positive metastasize in
immunodeficient mice models, while their HPA
negative counterparts generally do not [12, 13]. In in
vitro studies with breast cancer-derived cell lines, the
level of Tn expression correlated with the capability
of the cell lines to invade through Matrigel, but the
Tn glycotope itself appeared not to be directly
mechanistically involved in this phenotype, as the
glycotope blocking with HPA lectin did not affect the
invasivity of the cell lines . In vitro, cell lines have
been derived from a common progenitor colon
carcinoma line, which are either Tn+ or Tn-. Tn
positivity was associated with a loss of core 1 β-1,3galactosyltransferase activity . It is unsure
whether this is also the mechanism behind increased
Tn abundance during natural tumor progression. Current Molecular Medicine, 2007, Vol. 7, No. 4 the metastatic competence of adenocarcinomas.
This would explain why Tn/HPA positivity correlates
closely with increased metastasis, while it seems not
to be directly involved in this behaviour. In the case
of colon carcinoma, a very nice study  used
quantitative T/sia-T and Tn/Sia-Tn histology in a
comparison of paired primary tumor and metastatic
tissues. The metastases had a decrease in the Tn
and T structures versus the primary tumour and a
reciprocal increase in Sia-Tn and Sia-T. The authors
went on to show that the sialylated glycotopes were
important in weakening the adhesion of the
metastasizing cells to basement membranes, which
is compatible with the finding on β1-integrin
modfication. It appears that, at least in colon
carcinoma, deregulation of gene expression leads to
some cells acquiring a hyper-α -2,6-sialylation
phenotype , which helps these cells to detach
from the tissue and at the same time increases the
likelihood for these cells to re-attach at endothelial
sites elsewhere in the body. Of course, metastasis
formation is a multifactorial process which might
follow different paths in different patients, but
changes in cancer cell glycosylation certainly is an
important contributing factor. In breast carcinoma,
the detection of Sia-Tn correlates with other
predictors of poor prognosis (high nuclear grade,
aneuploidy and high S-phase fraction of epithelial
cells) , and Sia-Tn is already detectable in a
significant fraction of in situ carcinomas, indicating
that it is often expressed early on in the process of
cancer formation. N The α -2,6-sialylated derivative of Tn (Sia-Tn) is
also a cancer-associated glycotope that is used in
clinical histology, and its increased abundance on
cancer cells is often correlated with an increased
activity of the ST6 GalNAc 1 sialyltransferase. When
the latter enzyme is overexpressed in epithelial cell
lines, the cells dramatically change in morphology
and migration properties, in a phenotype that is
reminiscent of epithelial-to-mesenchymal transition
(EMT). An important pathway in this phenotype is the
modification of the β1- integrin subunit with Oglycans that are sialylated by the over-expressed
enzyme . This appears to significantly modify
integrin-initiated signaling, and might be associated
with cancer progression. In this sense, and taking
into account that the Tn epitope is the substrate for
the ST6 GalNAc 1 sialyltransferase, one would be
induced to think that Tn/HPA positivity is merely a
pre-condition for Sia-Tn formation, which then would
be the functionally important glycotope in increasing n
ot Sia-Tn Glycotope 399 Le Blood Group Determinants
The glycan structures that determine the Le blood
group have been implicated in many cancers as risk
factors for a bad prognosis [20, 21], but it seems
very difficult to draw general conclusions for different
cancer types: different cancers seem to disturb the
flux through the complex network of O-glycosylation
biosynthesis reactions in quite different ways. For
example, in pancreas cancer, Sia-Lea detection is
used clinically for diagnosis, whereas in prostate
cancer, it is Sia-Lex and Ley which are the only
blood-group related antigens that are minimal or
absent in benign epithelial cells and that are more
highly expressed in malignant tissue. It is likely that
the expression pattern of the O-glycosylation genes
in the benign tissue (which can be very different from
tissue to tissue) greatly influences the effect that upor downregulation of some of these genes has on
the O-glycosylation phenotype of the cancer. And of
course, the 'ground state' of O-glycosylation
determines whether a specific glycotope is practically
useful or not in detecting a change in that tissue: it is
much easier to detect the appearance of a normally
absent glycotope than to reliably measure -for
example- a 25% increase in the abundance of a
glycotope. As tissues differ in their 'ground state' Oglycosylation, the type of glycotopes that are useful
to detect alterations in these tissues are obviously
also different. If one general conclusion can be
drawn, it would be that if a tumor displays the altered 400 Current Molecular Medicine, 2007, Vol. 7, No. 4 glycosylation which is cancer-specific for that tissue,
then the clinical course of the cancer is most often
significantly worse than for tumors in that tissue that
do not diplay the glycosylation change. Whether all
of these different glycosylation changes contribute
causally to this more agressive behaviour or rather
merely reflect a more advanced tumor stage has not
been firmly established in all cases.
In colon carcinoma, Sia-Lex detection is predictive
for disease recurrence, whereas Sia-Lea detection is
not [22, 23]. In primary lung cancer, a high level of
Sia-Lex predicts short survival time of the patient
Sia-Lex In gastric cancer,
expression is an
independent risk factor for liver metastasis  and
Sia-Lea expression is a predictor for worse outcome
A monoclonal antibody (designated as FH6) is
available which specifically recognizes dimeric SiaLe x glycotopes. The detection of such a dimeric
glycotope would indicate a high density of Sia-Lex
and thus be particularly suitable for use in cancer
diagnostics based on Sia-Lex. Indeed, in gastric
carcinoma patients it was found that detection of this
complex glycotope was significantly associated with
venous invasion of the tumour and with a more
advanced histological classification . Moreover,
patients with a high level of this glycotope had a
shorter survival time than patients with none or low
levels. In patients with advanced stage gastric
carcinoma, dimeric Sia-Lex was the only independent
prognostic factor for patient outcome in this study. Schulz et al. increased adhesion to umbilical vein endothelial
cells. This phenotype was correlated with an
increased abundance of the Sia-Lex glycotope,
which is a ligand for the endothelial E-selectin .
In hepatocellular carcinoma and in colon carcinoma
, Mgat5 mRNA is strongly upregulated.
Markers in Current Clinical Use, Mainly Detected
Via Immuno-Assay on Body Fluids
CA 19-9 Glycotope Detection in Gastro-Intestinal
The epitope of the monoclonal antibody CA 19-9
encompasses the Sia-Lea glycotope. The detection
of this epitope in serum is currently part of routine
diagnostic testing for pancreatic cancer [34, 35], for
cholangiocarcinoma (bile duct cancer), and for
colorectal [36-38] and gastric cancer  (and the
marker has sporadically been studied for a large
range of other cancers). Approximately 80% of all
pancreatic cancer cases are positive for CA19-9, with
a false positive rate of approx. 20% in benign
pancreatic and hepatic conditions. Postoperatively
high CA19-9 or re-appearing CA19-9 upon therapy
are useful predictors of therapy failure and poor
survival . Remarkably, the CA19-9 marker, in
combination with CEA, can usefully detect even
imaging-occult cholangiocarcinoma on a background
of primary sclerosing cholangitis (the most important
risk factor for cholangiocarcinoma development) .
This finding was recently validated and screening of
primary sclerosing cholangitis patients with CA19-9
and CEA was found useful . CA19-9 is thus one
biomarkers. Because its use is widespread, there is
also quite some information on non-cancer
pathologies that can cause false positive CA19-9
test results. As with most cancer biomarkers, these
pathologies often are benign alterations of the target
tissue (such as pancreatitis , gallstone-induced
bile duct inflammation , or acute hepatitis ),
but other diseases can also cause false positives in
clinically distinct settings (splenic cysts [45-47],
endometriosis , or pulmonary alveolar proteinosis
[50, 51]). Also, in the rare cases where these
cancers occur at young age, the marker has to be
used with utmost caution . The levels of CA19-9
are also influenced by the Lewis secretor phenotype
of the individual under investigation [53, 54], and
negative CA19-9 test results can be obtained in
secretor negative pancreatic cancer patients. This
rather broad understanding of the factors that can
adversely influence CA19-9 test results is telltale of a
mature biomarker, which has stood the reality test of
clinical implementation. u
ot α -1,2-Fucosylated Le Glycotopes The mAb MIA-15-5 recognizes the H/Le y/Le b
glycotopes, which have a Fuc-α -1,2-Gal determinant
in common. Patients with MIA-15-5 positive primary
lung cancers have a 3-to 6-fold reduced chance on
5y survival than those with MIA-15-5 negative tumors
. Most carcinomas express LeY glycotopes on
their plasma membrane glycoproteins, including
epidermal growth factor receptors. As was recently
demonstrated, anti-LeY antibodies can potently
interfere with EGF-R signaling and this might be a
novel target for tumor therapy . N Increased L-PHA Binding (β-1,6-GlcNAc Branching)
N-acetylglucosaminyltransferase V is the enzyme
that catalyzes β-1,6-GlcNAc branching of N-glycans
in the mammalian Golgi apparatus. The coding
Mgat5 gene is under control of Ras signaling, which
is very commonly upregulated in tumor cells. Glycans
which are modified with β-1,6-GlcNAc branches can
be detected by the lectin L-PHA (leukoagglutinating
phytohemagglutinin). Increased levels of L-PHA
binding are especially well documented in human
breast and colon carcinoma, and these increased
levels are associated with progressively advanced
stages of the diseases . In an in vitro study,
over-expression of GnT V in colon carcinoma cells
leads to decreased fibronectin adhesion, but n
tio On the mechanistic side, a recent study has
shown that the β3GalT5 β-1,3-galactosyltransferase
is important in biasing the biosynthesis of O-glycans
in pancreas cancer cells towards Sia-Lea -capped
type 1 chain O-glycans, as its silencing by antisense
RNA resulted in poly-N-acetyllactosamine chain
elongation and capping by Sia-Le x structures . Clinical Laboratory Testing in Human Medicine Based CA125 in Ovarian Carcinoma
The protein backbone of the CA125 antigen is
encoded by the Muc16 gene [56, 57]. Detection of
the CA125 glycoform of MUC16 is commonly used in
the diagnosis of epithelial ovarian cancer , using
the OC125 monoclonal antibody . As with most
cancer markers, false positives can occur in patients
with non-malignant disorders of the target tissue or
of other tissues [60, 61]. The O- and N-glycosylation
of CA125 has recently been comprehensively
characterised  (although we still do not know
which of the observed glycan structures form part of
the CA125 epitope), potentially opening the way to
yet more specific measurement, and to an
understanding of its role in ovarian cancer
Established Non-Invasive Diagnostic Utility
Next to mucinous O-glycosylation and Nglycosylation, glycolipids are also known to be
altered in malignant transformation. Especially in
brain tumors, ganglioside fingerprinting has met with
some success in classifying different tumor types
. Several gangliosides are or have been the
focus for development of tumor-targeting therapies
. In a non-invasive
glycolipids have a more limited role
glycoproteins, as they are mainly cell-associated. In
those cases where cellular material is readily
sampled, detection of altered glycolipids can be
useful, as in the case of endometrial carcinoma .
Some studies have also quantified gangliosides in
serum of cancer patients, and found ganglioside
levels to be upregulated in cancer patients as
compared to healthy patients and those patients
with benign tumors . With the development of
improved glycolipid analytical technology, we might
as yet see more studies appearing that explore the
diagnostic potential of biofluid glycolipid composition. Current Molecular Medicine, 2007, Vol. 7, No. 4 tumor marker protein. Glycans add a layer of
structural diversity to the underlying molecule
(especially protein), the quality and quantity of which
can be sensitive to the physiology of the cell
producing the molecule, often independent of the
mere abundance of this molecule. Glycosylation is a
very frequent post-translational modification of
secreted proteins (i.e. those proteins that are the
most likely ones to be found in the easily accessible
patient biofluids), in contrast to other posttranslational modifications that can be modulated by
Highly Fucosylated α -Fetoprotein (AFP) Glycoforms
for the Detection of Hepatocellular Carcinoma
The best-established example of the utility of
measuring glycoform ratios of tumor marker proteins
rather than the protein levels per se, is to be found
in the diagnosis of hepatocellular carcinoma in
patients with underlying liver cirrhosis. Clinically, an
α -fetoprotein level in serum of >20 ng/ml detects
patients with hepatocellular carcinoma with a useful
sensitivity (>60%; sensitivity is defined as the number
of afflicted individuals scoring positive on the test to
the total number of afflicted individuals in the study
group). However, the large majority of HCC arises on
a background of liver cirrhosis, and AFP levels are
often strongly increased in cirrhosis. Thus, between
20 and 500 ng/ml, there is a 'grey zone' for HCC
detection, which severly impairs the utility of AFP as
an early marker. Early observations using L ens
culinaris lectin crossed affinoimmunophoresis on AFP
demonstrated that a highly core-α -1,6-fucosylated
glycoform of AFP (designated as AFP-L3) was
carcinoma than in 'benign' liver disease, including
liver cirrhosis [67-74]. The AFP 'fucosylation index'
(normalizing the highly fucosylated forms to the
amount of AFP) is known as AFP-L3% and kits were
developed to perform this analysis [75, 76]. The cutoff value for HCC detection that is reported varies
somewhat between the studies, but appears to lie
between 10 and 15%. Soon, immunoblotting
replaced the second dimension gel in the lectin
crossed affinoimmunophoresis procedure , a
development which afforded improved sensitivity
(down to about 1 ng/ml per detected band). Using
this improved sensitivity, it was found that even when
AFP levels were below the 20 ng/ml cutoff, AFP-L3%
was still diagnostic in many HCC cases (especially
those with small tumors, i.e. <20 mm diameter). The
sensitivity of the assay for small tumors is about
35%, with a specificity of >90%. In longitudinal
studies of patients with cirrhosis, AFP-L3% has a
lead time of 9-12 months in HCC detection over
currently used imaging techniques . This offers a
time window for resection of HCC in a small (often still
localized) stage. AFP-L3% positivity of small HCC is a
risk factor for recurrence of the disease upon
treatment, so especially in these cases, waiting until
the tumor becomes discernable by imaging
techniques may prove lethal for the patient. Such a Glycoform Detection of Known Tumor Marker
Proteins or Other Proteins Produced by the
Several currently used serological tumor markers
are glycoproteins that can also be produced at a
certain level by non-cancerous tissues, and this
often limits their clinical utility. Quite often potential
pre-cancerous lesions are discovered through
imaging or clinical examination screens (of the
breast, the prostate gland, the liver,...) of relatively
large population groups, based on their sex and
age, and based on known risk factors (such as
chronic viral hepatitis, smoking habit,...). Those
patients in which suspected lesions are found could
benefit the most from high-frequency highly specific
non-invasive tumor marker testing (to yield early
warning of conversion to cancer). Recent work shows
that measuring tumor-specific glycoforms of these
tumor marker proteins can help in making a clearer
distinction between benign hyperplasia and cancer
than the measurement of the total amount of the n
ot N 401 402 Current Molecular Medicine, 2007, Vol. 7, No. 4 strategy of repeated AFP-L3% screening of cirrhosis
patients obviously presumes that good assays to
pre-symptomatically detect liver cirrhosis are
available, and also here, glycomics-based testing
provides a solution (see below). Post-operative AFPL3% positivity is the most significant independent
factor for predicting a decreased survival chance for
the patient . The AFP-L3% assay has recently
been automated  in a heterogenous liquid-phase
lectin-antibody sandwich assay, by making use of
LCA and 2 anti-AFP antibodies. The assay cycle is
now less than 4 minutes, with almost perfect
correlation of the data with the previous lectin
affinoelectrophoresis technique. This technological
feat proves that, given sufficiently convincing clinical
utility and given favorable market forces, glycoformbased cancer diagnostics are realistic and can be
indistinguishable from routine clinical assays.
Along similar lines, but less well-established, it has
been found that the sialylation level of AFP is lower
in HCC than in benign liver disease. Specifically, the
proportion of monosialylated AFP can be measured
upon isoelectric focusing of serum proteins, followed
by anti-AFP western blotting. This assay has a
reported accuracy of 89% for distinguishing HCC
from liver cirrhosis in cases with nondiagnostic total
AFP (<500 ng/ml) , but this has not (yet) been
validated by different investigators. Schulz et al. normal prostate cells synthesize sialylated and less
fucosylated structures. More recently, similar
differences have been shown for urinary PSA in a
comparison of malignant versus benign tumors .
Another recent study determined that the level of α 2,3-sialylation of PSA N-glycans is significantly
increased in prostate cancer vs. benign prostate
hyperplasia, both in seminal fluid (where it was
detected through direct glycan structural analysis)
and in serum (determined through increased binding
to the Maackia amurensis-derived lectin (MAA) ).
A determination of the clinical utility of these
glycosylation differences awaits larger-scale clinical
experimentation and the development of a simple,
standardized assay method, along the development
path that has been succesful for AFP-L3% in HCC.
Detection Antigen) N Prostate-Specific Antigen (PSA) Glycoforms Prostate cancer is one of the leading causes of
cancer-related deaths worldwide. However, it can be
treated effectively if caught in its early stages. To do
so, markers for early diagnosis are needed.
Currently, rectal examination and prostate specific
antigen (PSA) test are widely used for this purpose.
PSA, a serine protease of the kallikrein gene family
, is secreted almost exclusively by epithelial
prostate cells. In cases where disruption of the
prostate basement membrane occurs, elevated
serum PSA levels are observed. However, increased
PSA levels can also be the consequence of more
benign prostate disorders, and this yields many false
positive results in risk-population screening (positive
predictive value is about 30%) . However, recent
studies indicate that the type of N-glycosylation of
PSA is indicative for the type of prostate disease
[86-90]. Prostate cancer cells produce neutral, nonsialylated and highly fucosylated N-glycans, whereas n
tio The carcinoembryonic antigen is the longest
studied [91, 92] and still one of the most frequently
used glycosylation-dependent serum tumor markers
(originally for colon carcinoma, but now in a very wide
spectrum of carcinomas). In the current clinical CEAmeasurement practice, antibodies are used of which
the binding to CEA is carbohydrate-independent [93,
94], despite the massive glycosylation of the CEA
protein (40-65% carbohydrate). However, these CEA
measurements suffer from rather low specificity,
especially in patients with non-malignant diseases of
the intestine, the pancreas, the liver and the lung.
Therefore, the main indication for the use of the CEA
test is in the follow-up of cancer patients posttreatment: succesful therapy is hallmarked by a drop
of the CEA levels to reference values, while
remaining CEA most often indicates that the tumor
has not been completely removed or has
metastasized. The re-appearance of CEA indicates
relapse or outgrowth of previously covert micrometastases. The present CEA tests are useless for
wide at-risk population screening because of their
high false positive rates. Therefore, attempts have
been made to make use of cancer-associated CEA
carbohydrate structural alterations (presence of Siaα 2,6-Gal- β1,4-GlcNAc glycotopes on cancer patients
CEA and not on the cross-reactive molecules from
non-cancerous patients) [95-97] to improve the
specificity for cancer . However, these assays
await further validation to determine their clinical
ot Another study has shown
gammaglutamyltransferase (an enzyme of which the activity
is elevated in active liver disease) is differentially
glycosylated in hepatocellular carcinoma versus liver
cirrhosis, with useful clinical properties of the ensuing
assay . This difference was detected through
separation of GGT isoforms over boronate affinity
columns. The boronate groups interact with vicinal
cis-hydroxyl groups, found mainly on proteins in their
sugar moieties. An elucidation of the glycanstructural underpinning of this observation is not
available. Glycoform T-Antigen on MUC1 (in Histology)
The BW835 monoclonal antibody binds the
Thomsen-Friedenreich antigen on a motif within the
MUC1 repeat region . Thus, quantitative
measurement of this epitope in patient material
would elegantly make use of the combined
knowledge that MUC1 is frequently upregulated in
human cancers, and that Thomsen-Friedenreich
antigen is also upregulated in (some of) these
conditions. This strategy has been tested in gastric
carcinoma , and it was found that the BW835
immunoreactivity correlated with the presence of Clinical Laboratory Testing in Human Medicine Based lymph node metastases and was a marker of
unfavorable prognosis (although the latter analysis
was univariate). In colorectal carcinoma, BW835
staining was significantly correlated with more
advanced tumor stage . The antibody binds to
about 75% of oesophageal squamous cell
carcinomas , whereas antibodies raised against
the T-glycotope alone detected only 40% of these
Altered N-Glycan Branching of Urinary Fibronectin in
In bladder cancer patients, the lectin binding
behaviour of the common extracellular matrix protein
fibronectin, shed in urine from the bladder
epithelium, has been shown to be dramatically
altered . The lectin binding pattern suggested
Nacetylglucosaminyltransferases that are involved in
determining the branching pattern of N-glycans, and
these were subsequently assayed in patient tissues.
N-acetylglucosaminyltransferase III was especially
strongly upregulated, explaining the 5-fold reduction
in ConA binding of the fibronectin (ConA does not
bind N-glycans with a bisecting GlcNAc-modified
trimannosyl core structure). Proper clinical validation
of this finding has not yet been reported. Current Molecular Medicine, 2007, Vol. 7, No. 4 403 Diagnostic Tests to Identify the Agent of Infectious
Periodic Acid Schiff staining in histology for
fungus detection is based on the presence in the
yeast/fungal cell wall of copious quantities of
polysaccharides. Combined with the morphology of
the PAS-stained entity, this can yield conclusive
evidence of fungal infection  (in human
aspergillosis are the most frequent).
Candida albicans serotyping is based on
agglutination assays  or flow cytometry 
using specific antibodies against the yeast’s cell wall
mannan epitopes, and such a serotyping strategy is
competitive with other clinical yeast classification
assays that are based mainly on differential
carbohydrate utilitzation . n
tio Aspergillus fumigatus is the most frequent
causative agent of human aspergillosis, and its
determinants that are immunogenic. The detection of
these carbohydrate epitopes via immunoassay has a
role in the diagnosis and monitoring of aspergillosis
patients . u
ot N Fig. (2). Some of the glycotope structures measured in the discussed diagnostic tests. 404 Current Molecular Medicine, 2007, Vol. 7, No. 4 Bacteriology
The bacterial cell wall contains a range of
capsular polysaccharide-based bacterial typing using
specific antisera is still a mainstay in clinical
bacteriology, although these assays are gradually
being replaced by nucleic-acid detection tests (PCRbased) that genotype the genes that biosynthesize
the strain-specific components of the cell wall
polysaccharide biosynthesis machinery (especially
the O-antigen polymerases and the genes that are
postulated to be the O-antigen lipid precursor
flippases). However, with the advent of protein microarray technology for the fast detection of specific
pathogens (both clinically and in bio-defense
settings), the use of anti-cell wall lectins and
serotyping antisera might enjoy a revival as specific
capture reagents for the bacteria .
In settings where rapid and simple testing for
specific pathogens is required or cost-efficient,
immunological assays, often detecting pathogenspecific carbohydrate antigens, are still very
competitive, and we discuss a number of cases that
were reported during the review period (many more
were commercially developed into point-of-care tests
and are not necessarily formally reported in the peerreviewed literature). Schulz et al. patients. The latter might be very valuable, especially
in low-resource medical settings: urine is generally
sterile and its testing does not require BSL3
containment laboratories . When 3 serological
tests (including anti-PGL-1 and anti-LAM) are
combined, the sensitivity for M. tuberculosis can be
as high as 85% even in acid fast smear negative/
culture negative patients, with about 10-15% false
positives in a healthy control population .
development (the whole field was reviewed recently:
). A remarkable case is the use of keyhole
limpet hemocyanin (KLH) in the diagnosis of acute
schistosomiasis: this protein, derived from a
completely different organism than the parasite
(Fucα 1,2Fucα 1,3)GlcNAc β1-] and variants thereof
[119, 120] in common with the surface of
schistosomula. Thus, KLH can be used as standard
antigen in ELISA assays to detect the active stage
of schistosomiasis with high accuracy . It is
even so that the shared glycotope between KLH
and Schistosoma is the relevant pathogenetic factor
in the induction of hepatic granulomas by S.
mansoni eggs that are trapped in the liver (this is the
main pathology of the disease) . Schistosoma
mansoni also sheds antigens of glycoconjugate
nature that are filtered into the urine and can be
detected there using specific monoclonal antibodies,
with an efficiency which approaches diagnosis using
serum . u
ot Rapid immunoassays for the detection of
streptococcal infection from throat swabs, based on
detection of the Group A streptococcal carbohydrate
diagnostics in sensitivity (and, obviously, speed)
 and can be formulated in point-of-care
Bacterial endocarditis caused by gram-positive
bacterial infection can be detected using an ELISA
for lipid S (a glycolipid which is related to lipoteichoic
acid). This assay has sensitivity and specificity of
close to 90%, is rapid, and complements the results
from traditional culture microbiology and from
endocardial imaging . N A field that is still under active development is the
detection of mycobacterial infection using blood or
urine as the matrix. Several of these assays use
antibodies that specifically recognize
mycobacterial glycolipids (amongst others: phenolic
glycolipid I ; lipoarabinomannan (commercial
name of the assay: MycoDot TM ). For M. leprae
infection, the anti-PGL-I IgM ELISA has been widely
used for the testing of high-risk groups in countries
where leprosy is endemic, in an effort to detect
subclinical infections. However, the assay has a low
predictive value in this setting of early leprosy
detection . A more recent study demonstrated
that the levels of PGL-I and anti-PGL-I IgM in the
serum of leprosy patients correlates with the bacterial
load in the patients . For M. tuberculosis,
available with serum as the matrix, and this antigen
can also be detected in the urine of tuberculosis n
tio In the case of leishmaniasis (Leishmania
donovani ), surface expression of 9-O-acetylated
sialic acid glycotopes on infected hematopoietic cells
and erythrocytes is elevated (produced
promastigotes of the parasite) . Patients have
increased titers of anti-9-O-acetylsialic acid IgM
antibodies in their serum, which forms the basis for a
novel diagnostic test with good clinical characteristics
[125, 126]. The standard antigen in this ELISA test
is bovine submaxillary mucin, which contains the
same 9-O-acetylsialic acid glycotope as Leishmania.
The parasite also appears to shed low-molecular
weight glycans that are filtered by the kidney into the
urine, where they can be detected using antiLeishmania glycoconjugate antibodies .
In infections caused by Trichinella spiralis
(trichinellosis), the diagnostic antigen in use contains
terminal tyvelose residues (3,6-dideoxy-D-arabinose).
The tyvelose-GalNAc determinant was chemically
synthesized and coupled to BSA to serve as
standard antigen in an ELISA with 100% sensitivity
neurocysticercosis can be quite reliably diagnosed by
measuring Lens culinaris-reactive parasite proteins
via ELISA or immunoblot [129, 130]. The diagnostic Clinical Laboratory Testing in Human Medicine Based C-antigens for Echinococcal infections in humans are
also of glycoconjugate nature [131, 132], though the
detailed structure of these remains unknown.
Airway and Salivary Mucus Glycosylation as
Predisposing Factors for Infectious Disease
Glycoconjugates are abundant at mucosal
surfaces, which are often the initial sites of infection.
Changes in mucosal glycosylation as a consequence
of an underlying disease are under investigation in
mucosa that are accessible for clinical diagnostic
sampling, as risk factors for infectious disease in
compromised patients, especially in cystic fibrosis
Cystic fibrosis (CF) is caused by lack of function of
cystic fibrosis transmembrane conductance regulator
protein (CFTR) , which results in increased
viscosity , and reduced clearance [134, 135], of
pulmonary mucus. At present, CF diagnosis is
specifically and accurately performed with genetic
screening . Nevertheless, the disease burden
generally comes in bouts, and there are currently no
biochemical predictors for when a new bout is
upcoming in a patient.
The severity of airway infection in CF lung disease
and in chronic bronchitis is correlated with high
sialylation of airway mucus glycoconjugates .
Moreover, increased sulfation of respiratory mucins in
CF was observed relative to other respiratory
diseases including chronic bronchitis  and
asthma . It is still a matter of debate whether
these glycosylation changes are a consequence of
altered conditions in the mucin-secreting cells'
secretory pathway due to the primary CFTR defect,
or whether secondary effects such as chronic airway
inflammation and infection lead to these mucin
glycosylation changes that have been observed in
vivo. Some in vitro analyses
glycoconjugates in CF have reported increased
sulfation [140-143], which would be in support of the
first hypothesis, whereas others have failed to detect
Methodological differences in these studies are a
likely cause of this discrepancy.
Whatever may be the underlying cause, the
correlation of mucin sialylation with infection severity
 suggests that prognosis of the progression of
CF lung disease may be possible by measuring
changes in airway mucus glycosylation. Such
prognostic biochemical marker would complement
the currently available prognostic analyses such as
forced expiratory volume in 1 second (FEV1) and
high-resolution computer aided tomography (HRCT)
scanning [146, 147]. Accurate biochemical markers
that provide this prognostic information would be
clinically useful in CF management, as they could
trigger prophylactic antibiotic treatment to stem the
severe airway infection that often accompanies an
active CF period. Current Molecular Medicine, 2007, Vol. 7, No. 4 (MG1) and soluble monomeric MUC7 (MG2) .
These mucins are heavily glycosylated with different
glycans: MUC7 contains sTn, Le x, sLex and binds LSelectin  and various streptococci through
these epitopes . On the other hand, MUC5B
contains blood group epitopes (A, B, H/O, Lea, Lex,
Le b, Le y), sialylated epitopes and many sulfated
structures [151-153]. As a consequence
glycosylation of MUC5B is heavily dependent on the
subject's blood group status: MUC5B carries blood
group antigens corresponding to the subject's blood
groups in secretors, but increased sialyl-Le a in nonsecretors . Using lectins, salivary mucous
glycosylation differences have been noted between
individuals with high vs. low susceptibility to dental
caries . It would be interesting to correlate the
lectin data with direct structural analysis of salivary
mucin glycosylation, to perhaps find out which
glycotopes confer increased resistance to the dental
caries pathogens (especially Streptococcus mutans). u
rib Mucins are also a major component of saliva, and
are present in two main fractions, gel-forming MUC5B n
tio Diagnostic Tests for Inherited Glycoconjugate
Anabolic and Catabolic Defects: Congenital
Disorders of Glycosylation and Lysosomal
Storage Diseases t
ot N 405 Lysosomal Storage Diseases The lysosomal storage diseases are a family of
rare genetic diseases of which the causative
mutation(s) result in a defective lysosomal catabolism
of cellular constituents. This defective catabolism
causes these cellular constituents to accumulate in
the lysosomal compartment of the cell, and the family
of diseases owes its name to this lysosomal ‘storage’
phenotype. There are several ways in which storage
diseases can be diagnosed. The profiling of
glycoconjugates in patient urine plays an important
role in newborn screening for lysosomal storage
diseases and in the diagnostic workup of identified
cases, as most of these defects are in the
catabolism of glycoconjugates. As the lysosomal
load becomes too large, cells die and release the
storage product in the interstitial medium, and at
least some of this storage product is filtered out in
the kidney and is excreted in urine. The analysis is
oligosaccharides [156-158]. For specific subcategories, especially the mucopolysaccharidosis
group of diseases (caused by defects in the
catabolism of glycosaminoglycans), Fluorophore
Assisted Carbohydrate Electrophoresis (FACE) on
electrospray mass spectrometry  can be used.
The diagnosis of a lysosomal storage disorder is
complemented by histology on patient cells
(morphology and general chemical reactivity of the
storage product). Quantitative assaying of the
suspected enzymatic activity (or activities) is also a
mainstay for these diagnoses, nowadays often
accompanied by genetic analysis of the locus or loci
of interest for mutations (such as for Tay-Sachs
disease in known risk populations). 406 Current Molecular Medicine, 2007, Vol. 7, No. 4 Congenital Disorders of Glycosylation
Congenital Disorders of Glycosylation (CDGs) are
a rapidly growing chapter in pediatrics, after the first
description of such disease [161, 162].
The original diagnostic test is probably still the
best: iso-electric focusing of serum proteins, followed
by immunodetection of the transferrin isoforms .
Transferrin is a major iron transporting glycoprotein
synthesized by the liver. The two N-glycans present
can have two to four antennae, each of which can
be sialylated or not. Differential sialylation (either
because entire N-glycan chains or lost or because
the structure of the N-glycans contains fewer sialic
acid residues) results in several transferrrin isoforms
with different isoelectric points that can be separated
using iso-electric focusing.
CDG type I is defined as those CDGs with defects
that affect the biosynthesis of the N-glycan precursor
Glc3Man 9GlcNAc2PPDol, or the transfer of the
Glc3Man 9GlcNAc2 moiety to N-glycosylation sites on
proteins. These defects generally cause a lower
efficiency of N-glycosylation site occupancy, which is
detected in transferrin IEF as increased abundance
of the disialo-and asialo-isoforms of transferrin. On
the other hand, defects in the processing of the
precursor glycan after transfer to the protein are
classified as CDG type II. The underlying functions
that are affected are very diverse: they can be in
glycosidases, glycosyltransferases, sugar-nucleotide
transporters, and also in components that are
involved in secretory pathway homeostasis. Defects
in these biosynthetic steps towards fully processed
complex-type N-glycans most often result in a lower
degree of sialylation of glycoproteins (because of
incomplete processing towards the galactosylated
glycans that are the substrate of sialyltransferases,
because of altered branching, or because of defects
in the sialylation process itself). Thus, type II CDGs
have a transferrin IEF pattern that also tends to
have increased abundance of the monosialylated
and trisialylated isoforms, or more generally, their
transferrin IEF is ‘not normal and not type I’. The
transferrin IEF is a rapid, cheap, quite reliable assay
for the biosynthetic pathway that assembles the bulk
of serum N-glycans. It is suitable for screening
purposes, and this is its current role in the clinic.
Nevertheless, the assay’s utility only stretches as far
as its molecular basis allows: it will not detect defects
in O-glycan biosynthesis, and also not in the
biosynthesis of N-glycan modifications that are not
present on transferrin (for example brain-specific
modifications). It also does not detect defects in noncharged substituents on transferrin glycans (such as
fucosylation and bisecting GlcNAc). For fine-typing
the N-glycan structural differences in type II CDGs,
one can either resort to direct serum protein N-glycan
analysis using mass spectrometry, HPLC or capillary
electrophoresis, or to mass spectrometrical analysis
of transferrin using MALDI or ESI technology
[164,165]. Immunoaffinity-LC-ESI  is now
routinely used in many centers in the US for neonate
screening for CDG. Schulz et al. A range of congenital muscular dystrophies
disease and Fukuyama muscular dystrophy) are
caused by defects in O-mannosylation . MEB is
caused by a defect in the gene coding for POMGnTI
O-mannosylation-N-acetylglucosaminyltransferase I). An enzymatic assay that should be
useful in screening for this disorder amongst
muscular dystrophy patients has recently been
Further, Ehler Danlos syndrome is caused by
glycosylation defects in a small proteoglycan .
Hereditary multiple exostoses are probably caused
by defects in heparan
biosynthetic glycosyltransferases [170, 171].
Although technically not a 'congenital' disorder in
the true sense of the word, paroxysmal nocturnal
hemoglobinuria (PNH)  somehow belongs to the
same category of genetic disorders in glycosylation
biosynthesis. This is a rare hemolytic anemia
characterized by the increased sensitivity of cells of
the myeloid lineage to complement. This leads to
periodic intravascular hemolysis and hemoglobinuria,
accompanied with an increased risk on thrombosis.
The disease is caused by acquired mutations in the
PIG-A gene in haematogenic progenitor cells (stem
cells), followed by periodical clonal expansion of
and thus, PIG-A deficient cells are devoid of or have
a strongly reduced abundance of GPI-anchored cell
surface proteins (amongst others: those that protect
cells against complement lysis: CD55, CD59 and
C8BP). This forms the basis of the current diagnostic
methods for the disease . In one method, flow
cytometry is used on blood cells to detect GPIanchored proteins (CD55 and CD59), whereas in a
second method, fluorescently labeled, non-toxic
mutants of bacterial GPI-binding toxins (aerolysine or
mutants of Clostridium septicum U-toxin ) are
used for blood cell staining and flow cytometry. u
ot N n
tio Diagnostic Tests for Drugs of Abuse and Illicit
Carbohydrate Deficient Transferrin (CDT) for the
Detection of Alcohol Abuse
Alcohol is considered the major drug of abuse
worldwide, with important social and economical
consequences. Therefore, there is a need for the
diagnosis and follow-up of alcohol abuse. Since the
discovery that alcohol abuse leads to increased
levels of under-sialylated transferrin isoforms as
compared to subjects with moderate or no alcohol
use, carbohydrate-deficient transferrin (CDT) has
become the standard marker to evaluate these
patients [175-177]. The procedure is the same as for
transferrin IEF in the diagnosis of Congenital
Disorders of Glycosylation (see above), although the
phenotype due to alcohol abuse is usually less
severe than the one observed in CDG patients.
However, overlaps do exist and there are cases Clinical Laboratory Testing in Human Medicine Based known of heterozygous carriers of 'severe' CDG-I
causing mutations that score positive on the CDT
test for alcoholism.
Tetrasialylated transferrin is the most predominant
glycoform in healthy controls (70-80% of total
transferrin). The analysis of serum from alcoholic
patients typically reveals elevated levels of di- and
asialo forms that are formed due to impaired
glycosylation: one or two entire N-glycan chains are
missing . The percentage of these two
glycoforms found in serum is used to demonstrate
alcohol abuse (definitions of abuse vary, but
>50g/day for more than 5 days is about the average
of the definitions). As transferrin has a half-life of ten
to fifteen days, a change of this value will be seen
when a patient entered a withdrawal treatment, or
when a patient returns to alcohol abuse while under
treatment. Although CDT is currently the best
available marker to detect recent alcohol abuse in at
risk populations, the positive predictive value that
can be attained in the general population is less
than 50% (when compared to self-declared alcohol
consumption as 'gold standard', which is certainly not
perfect) . It is the higher prevalence of alcohol
abuse in risk groups that gives the CDT test a
sufficient positive predictive value to be useful. Liver
disease appears to only interfere substantially with
CDT measurements in a very severe stage
(including transferrin) is observed. Current Molecular Medicine, 2007, Vol. 7, No. 4 polymorphisms often yield inaccurate results in
immunoassay-based CDT tests, and IEF, CE or
HPLC-based  CDT testing is recommended to
confirm immunoassay results in critical cases .
Recombinant EPO Detection
Erythropoietin (EPO) is a glycosylated hormone
produced by the kidney in human adults. It induces
increased red blood cell mass, haemoglobin
concentration and aerobic power. For those reasons,
the protein is used therapeutically in the treatment of
certain forms of anemia . As the administration
of this hormone and its analogues also leads to a
substantial improvement of exercise performance, it
has been abused as a doping agent by endurance
athletes . Both the International Olympic
Committee (IOC) and the World Anti-Doping Agency
(WADA) have banned the use of recombinant
human EPO (rhEPO). Currently, the use of rhEPO is
tested using a direct identification method based on
isoelectric focusing and double blotting of the EPO
present in urine . As in the case of transferrin
(described above), differences in the degree and
type of sialylation of EPO between endogenous
EPO and exogenously administered rhEPO lead to
diffent isoelectric points. Human EPO has 3 N- and
one O-glycosylation site [193, 194]. Glycosylation is
pharmacokinetic properties [194,
commonly, rhEPO is produced in Chinese hamster
ovary cells (CHO) or human kidney cells. In both
cases, glycosylation is different from human
endogenous EPO [193, 196]. n
ot CDT measurements can currently be performed
using a choice of commercially available and
homebrew test formats, which all differ somewhat
from each other in their analytical properties .
This does not make it easier to compare the data
from one study to another, but most studies seem to
result in a sensitivity of 40-70% for a specificity of
>80% (reviewed in ). Because of the higher test
volume than in the setting of CDG screening,
classical (rather laborious) IEF is not much used
anymore, although it remains the gold standard. The
commercial CDTect test uses anion exchange microcolumns to separate CDT from non-CDT isoforms,
and quantifies transferrin in both fractions using antitransferrin enzyme-linked immunoassay . The
%CDT-TIA (turbidimetric immunoassay) and its 2nd
generation %CDT use rate nephelometry to quantify
total transferrin and CDT upon anion exchange
separation. Capillary electrophoresis with UV
absorbance detection of total serum proteins can be
used to determine CDT: although all serum proteins
are detected, immunosubtraction experiments were
used to pinpoint the transferrin isoforms amongst the
observed peaks. The assay is available commercially
with excellent analytical characteristics (CeoFix CDT)
[183-185], and some laboratories have developed a
homebrew variant [186, 187]. As for IEF-based CDT
tests, transferrin genetic variations that result in a
more anionic or cationic transferrin protein molecule
shift all transferrin glycoform peaks in the IEF pattern
transferrin N 407 There are some disadvantages related to the
analysis of urinary EPO. The major drawback is the
quick disappearance of measurable rhEPO levels
soon after administration. After about four days [197,
198], levels have dropped back to baseline. In
contrast, the athlete retains the benefits associated
to its use for a longer time. Moreover, conditions like
proteinurea influence urinary EPO levels  and
endurance sports influence renal functions .
Therefore, alternative tests are under investigation,
which analyze total serum EPO in combination with
reticulocytes, heamoglobulin and soluble transferrin
receptor concentrations .
Diagnostic Tests in Auto-Immune Disorders and
Other Chronic Inflammatory States
Reactivity with Glycoconjugate (Cross) Anti- S. Cerevisiae Mannan Antibodies (ASCAs) in
A subgroup of Crohn's disease patients develops
an adaptive immune response against S. cerevisiae
mannan (detected as Anti- S. cerevisiae Antibodies
or ASCAs) [202, 203]. When used in a clinical
context, the ASCA assay has a high specificity in the
important differential diagnosis from ulcerative colitis
. It was recently found that low-expressing 408 Current Molecular Medicine, 2007, Vol. 7, No. 4 alleles of Mannose Binding Lectin (MBL) are very
significantly more frequent in ASCA-positive than in
ASCA-negative Crohn's disease patients .
ASCA-positive patients also had a significantly lower
MBL protein level than ASCA-negative patients. This
would indicate a mechanism in which the increased
intestinal permeability in Crohn's disease causes an
increased S. cerevisiae burden, which would
normally be handled by the MBL-mediated branch of
the alternative complement activation pathway. This
would not be the case in MBL-deficient patients, who
hence develop an anti- S. cerevisiae humoral and
cellular immune response.
Anti-Ganglioside Antibodies in Several Neuropathies
A large body of data (about 500 studies, curated
bibliography available upon request) is available in
the literature on disorders of the Guillain-Barre
syndrome family and the role that anti-ganglioside
antibodies play in the pathogenesis of these
diseases. These syndromes are strongly associated
with previous infection with different pathogens, but
most notably with Campylobacter jejuni and related
species. Conclusive evidence has recently been
Oligosaccharide in the cell wall of these pathogens
contains an epitope which induces antibodies that
are cross-reactive with gangliosides that are
prevalent in the myelin sheath of peripheral nerves
. These antibodies block
conduction and may attract phagocytic cells to attack
the antibody-decorated Schwann cells. Schulz et al. cannot be established by a single test, but is
accomplished by a combination of laboratory test
results and clinical scoring systems. Now that new
therapies are starting to make an impact, more
biomarkers are needed to follow-up the condition of
these patients (as objective measures of response to
these expensive therapies that are not devoid of
serious side-effects such as emergence of
tuberculosis [217, 218]).
One of the possible bases for such a therapymonitoring marker is the well-established observation
that the N-glycans of immunoglobulin-G molecules
are hypo-galactosylated in RA-patients [219-221].
However, a lower galactosylation degree of IgGs was
later also observed in other inflammatory diseases
like lupus, tuberculosis, Crohn’s disease, systemic
vasculitis and in some cancers  (possibly
reflecting inflammatory aspects of the disease),
whereas an increase in galactosylation is associated
with pregnancy . Because of this lack of
specificity, this marker is not in widespread clinical
use for diagnostic purposes. With the advent of
DMARDs, however, there is currently more need for
non-invasive markers to assess chronic rheumatoid
disease activity, progression rate and response to
therapy. We believe that IgG galactosylation status
could play a much more useful role here than for the
differential diagnosis purposes for which it was
originally conceived. We recently completed a study
to explore the utility of this marker in a number of
these clinical contexts [Laroy and Callewaert,
manuscript submitted]. The technologies used are
immunoglobulins, followed by N-glycan preparation
and fluorescent labeling and profiling of these
glycans using HPLC or CE. The entire procedure can
now be performed with clinically relevant throughput. u
ot Because of the pathophysiological role of
ganglioside-crossreactive antibodies in the Guillain
Barre syndrome and related disorders such as Fisher
Syndrome and Bickerstaff's brainstem encephalitis,
the laboratory detection of such antibodies (using
immunoassays with the gangliosides as antigens)
aids in the diagnosis of the disease [207-210]. N Under-Galactosylation of Serum IgG in a Range of
Chronic Inflammatory Disorders Rheumatoid arthritis (RA) is a major cause of
disability, morbidity and mortality [211, 212]. It is a
chronic, systemic inflammatory autoimmune disease
with as the primary target the synovium. It affects
almost 1% of the adults worldwide and is three times
more common in women, in whom it also has an
earlier onset. Although the exact mechanisms
behind the disease are yet unknown,
understanding of cytokine networks responsible for
the ongoing inflammatory response and of the
pathophysiology of the disease have led to several
therapies that modifiy the disease process.
Methotrexate is currently the most prescribed of
(DMARDs), and new therapies directly target the
proinflammatory cytokines TNFα and IL1. An early
diagnosis is critical for successful treatment, as a
delay of treatment of only three months results in
substantially more bone damage after 5 years . At present, however, a definite diagnosis n
tio Recently, it was indicated that differences in IgG
galactosylation may be due to increased sialylation
of the β-1,4-galactosyltransferase
rheumatoid arthritis, which inhibits its enzymatic
activity . However, this finding was based on
serum β-1,4-galactosyltransferase isoform profiling
and should be confirmed on plasma cell β-1,4galacytosyltransferase.
Cholinesterase Glycoforms in the Diagnosis of
One laboratory has reported that an increased
level of non-concanavalin A (ConA)-binding isoforms
of acetylcholinesterase in lumbar cerobrospinal fluid
is diagnostic for Alzheimer's disease and was not
detected in other illnesses that cause dementia
. This increase in non-ConA binding was also
found in a mouse model of AD (APP Tg2576
transgenic mice) . In a more recent study, the
level of non-ConA binding CSF acetylcholinesterase
was found to positively correlate with the duration of
Alzheimer's disease, but to be not suitable for earlier
diagnosis of AD than is currently clinically possible. Clinical Laboratory Testing in Human Medicine Based These findings are very interesting, but there are
as yet no reports available from other laboratories to
validate this assay. In our own studies involving CSF
glycomics (unpublished), we have found substantial
differences in glycosylation profiles with samples from
different clinics. This most likely reflects different
(centrifugation, storage,...) possibly compounded by
contamination of these samples. CSF sampling and
handling requires substantially more skill and is
unfortunately much less standardized in the medical
practice than blood serum sampling and handling.
Diagnostic Tests to Monitor Status of the Liver
Chronic liver pathology is a complex syndrome
with different stages. As a result of the exposure to
hepatotoxic agents (alcohol, viruses), liver tissue
undergoes inflammatory necrosis. As a reaction,
activated hepatic stellate cells in this tissue will
secrete extracellular matrix components. Due to the
chronic character of the necrosis, this process (which
is a component of normal healing) overshoots,
leading to excessive deposition of extracellular matrix
and a disturbance in liver architecture. This is called
fibrosis and different stages are histologically
distinguishable. The most severe form of fibrosis is
liver cirrhosis and is characterized by regenerative
nodules: clusters of replicating hepatocytes in an
abnormal architecture. This chronic regenerative
activity is a major risk factor for the development of
hepatocellular carcinoma. Up till now, the clinical
assessment of a patient's liver condition is achieved
by the histological examination of liver tissue. The
invasive percutanous biopsy which is needed to
obtain this tissue involves serious discomfort to the
patient, and it is a costly procedure (estimated at
1500 USD). Moreover, the procedure is not entirely
risk-free, especially in patients with rather advanced
liver disease. All disadvantages taken together, liver
biopsy is not a suitable procedure for regular followup of patients who have been diagnosed with a
chronic liver disorder, and non-invasive alternatives
are needed to this end. Current Molecular Medicine, 2007, Vol. 7, No. 4 liver cirrhosis strongly pre-disposes patients for the
development of liver cirrhosis (about 40-fold
increased risk), another important question is: does
the patient have the chronic regenerative activity
associated with liver cirrhosis ? This entails
detection of the regenerative activity before clinical
signs of cirrhosis appear, to be able to put these
patients on an intensified screening regime for HCC.
answering these questions, based on the fact that
the major fraction of serum glycoprotein is
synthesized by hepatocytes, while the liver also
resorbs a large amount of serum glycoproteins.
Therefore, changes in the whole serum protein
glycosylation profile reflect the liver condition.
A very basic approach
monosaccharide composition measurement of serum
proteins , with conclusions that fucose and
GlcNAc are elevated when normalized per 3 mol
mannose (3 mannose residues are the common
element of every abundant serum N-glycan) in
severe liver disease. The increase in GlcNAc was
interpreted as an increased degree of branching, but
taking the result of recent studies  on the
structures of serum protein N-glycans in liver disease
into account, one has to revise this interpretation:
the extra GlcNAc in the early studies most likely
ensues from an increase in 'bisecting' GlcNAc rather
than from an increase in branching of the N-glycans. A typical setting in which such repetitive
assessment is needed, is in determining whether a
patient responds to therapy with a stabilisation or
improvement of the fibrosis (therapies are either antiviral as in the case of hepatitis C infection, or antifibrotic (the latter still experimental)).
Changes in a number of serological parameters
(most notably hyaluronic acid concentration, α 2macroglobulin
activities, bilirubin concentration, etc.) correlate
somewhat with the stage of liver fibrosis. These
changes have been mathematically combined in a
number of regression models to obtain scores that
better answer the clinical questions at hand than the
single parameters. One important question is: is
there 'clinically significant' fibrosis in the patient's liver
or not (important in making treatment decisions)? As n
ot N 409 The quantitation of Aleuria aurantia lectin-binding
(i.e. fucosylated) serum cholinesterase isoforms,
relative to the total amount of serum cholinesterase,
can distinguish compensated liver cirrhosis (Child's
stage A) from chronic active hepatitis with an
accuracy of 70% . Along similar lines, the
fucosylation of α 1-acid glycoprotein was found to be
higher in patients with liver cirrhosis than in patients
with non-cirrhotic liver disease , and the
diagnostic accuracy of the lectin immunoassay that
was developed for this purpose was very similar to
that of hyaluronic acid (about 75-80%), although the
cirrhosis patients in this study were not stratified
according to clinical severity. As mentioned above,
the core-fucosylation of α -fetoprotein is higher in
patients with hepatocellular carcinoma than in those
with non-HCC liver disease, and this is useful
diagnostically. However, AFP levels and AFP
fucosylation are also increased in conditions
associated with massive hepatic regeneration, such
as acute hepatitis. As a consequence, the AFP-L3
assay should be used with caution and probably
only in patients with chronic liver disease (and not in
the acute phase of the disease). Curiously, AFP
fucosylation does not correlate with the fucosylation
of other, abundant serum glycoproteins (tested were
transferrin and α -1-antitrypsin) , which probably
hyperfucosylated in HCC, whereas hyperfucosylation
of the abundant proteins can be used diagnostically
for liver cirrhosis (which normally precedes HCC in
95% of the cases). 410 Current Molecular Medicine, 2007, Vol. 7, No. 4 Schulz et al. Some studies have explored imaging of the
biodistribution of technetium 99m-labeled, galactosesubstituted human serum albumin to measure the
activity of the liver asialoglycoprotein-mediated
clearance (and thereby of the functional reserve of
the liver for this function), and one generally finds
good correlations with other such assays of liver
In our own work , we have developed a
profiling tool for the total serum protein N-glycome.
This technology is very rapid and robust and strikes
a good balance between a low technical complexity
(as is desirable for clinical implementation) on the
one hand and yielding sufficient analyte structural
information on the other hand (as is desirable to link
the diagnostic proflie changes to known aspects of
disease pathology). This glycomics diagnostic
technology was explored for the diagnosis of liver
cirrhosis, and we found that about 75-80% of
compensated liver cirrhosis cases can be detected
by measuring changes in the serum protein N-glycan
profile, with very high specificity. We also found
evidence to show that the serum protein N-glycan
profile contains information that can be used in
monitoring the progression of liver fibrosis from early
stages onwards. Some of the structural alterations
that were detected are compatible with the lectinbased studies that have
fucosylation of serum proteins in liver cirrhosis, but
the best diagnostic parameter that was derived from
the serum N-glycome profiles was not dependent on
fucosylation, but rather on an increased abundance
of glycans that are substituted with a bisecting
GlcNAc residue and a decreased abundance of the
triantennary non-fucosylated structure. Based on
lectins, it would be very difficult to find and reliably
measure this combination of glycan structural
alterations. stringency of the clinical laboratory in terms of
requirements, and not in the least: cost pressure.
Therefore, a realistic perspective on the booming
diagnostic 'omics' efforts must be that the
technologies used for discovery of novel markers will
generally not be the ones that will be used for the
final clinical implementation of these markers. DNA
arrays with tens of thousands of features will hardly
be used in the routine clinical lab, shotgun
proteomics measurements also not, and highresolution mass spectrometrical
profiling neither. The goal of the discovery stage with
high-complexity, comprehensive profiling technology
must be to identify as small a set of analytes as
possible that, when measured together, contain as
much of the diagnostic information contained in the
entire dataset as possible. Then, focused test
methodology needs to be devised to measure this
limited set of analytes in a format that allows
thorough validation and easy implementation in a
moderate complexity clinical laboratory environment.
In practice, such tests are almost invariably based on
specific binding with the analytes of interest, be they
nucleic acids (hybridization), proteins (antibodies) or
glycans (lectins). In rare cases, robust separation
technology and direct non-analyte specific detection
can be clinically implemented. This is mainly the case
when the diagnostic information is contained in the
major components of a mixture. This is the case for
serum protein electrophoresis, for total serum protein
N-glycome profiling, and for capillary electrophoresisbased Carbohydrate Deficient Transferrin detection,
all of which have diagnostic utility. u
ot N All of these findings are currently being validated
in larger studies of different designs, to fully assess
the cllinical utility of these novel assays. As the
assays are based on analytes which have not been
used before in a clinical context, we are investing
significant efforts at present in the analysis of factors
that may influence the serum protein N-glycome, in
order to establish sound guidelines for physicians
and clinical laboratories that will perform the assay
(patient pre-conditioning, choice of serum or plasma
as the matrix, way of preparing the biofluid, potential
influence of different storage conditions etc.). GLYCOCONJUGATE BIOMARKER DISCOVERY/TESTING TECHNOLOGIES
FUTURE OF GLYCOCONJUGATE-BASED
Development Reality of Clinical
Assay All 'omics' technologies that move out of the
academic research laboratory soon encounter the n
tio Glycoconjugate Mass Spectrometry
Since the introduction of soft
techniques, mass spectrometry (MS) has increased
in importance and success as a carbohydrate
analytical technique . The benefits of
carbohydrate analysis using MS include remarkable
sensitivity (sub-picomolar to femtomolar),
possibility for a large degree of automation, high
analysis speed (minutes to seconds per analysis
depending on the specific MS technique), the ability
oligosaccharides with MS detection, and most
importantly: more or less unbiased detection of a
wide variety of molecular species in the same
characterisation by MS relies heavily on biological
rules determined previously or in combination with
other methods, such as NMR or glycosidase
digestion. For instance, oligosaccharide linkage
anomericity is very difficult to determine by MS alone.
However, once these general rules have been
outlined for a given biological system, MS provides a
very useful compromise between speed and extent
of characterisation. Because it lends itself to robust
medium- to high-throughput analyses, MS shows
great promise as a discovery tool in diagnostic
glycomics. Clinical Laboratory Testing in Human Medicine Based Essentially all modern mass spectrometers can be
used successfully for oligosaccharide analysis and
characterisation . These different techniques
have various advantages. For instance, MALDI
analysis is typically faster, while on-line LC and GC
allow separation and detection of isomeric
oligosaccharides, and ion trap instruments typically
allow easier structural analysis through ion
fragmentation. MS-based approaches have certainly
shown their merit for sensitive and detailed
characterisation of carbohydrates, but the robust
performance required for routine clinical application is
often lacking. Therefore, the challenge is now for
instrument manufacturers, researchers, and clinicians
to develop MS as a robust and cost-effective
technique for general clinical application. The recent
success of clinical applications of MS (particularly
ESI-MS/MS) for inherited metabolic
screening (including amino acid, acylcarnitine, steroid
and lysosomal enzymes analysis) (reviewed in 
and ) bodes well for the future.
Glycodiagnostic markers must not only be well
characterised, but also relatively or absolutely
quantifiable. Standard MS techniques provide only
limited quantitation, in terms of total ion current
measurements, which can
between measurements. A rapidly expanding
technique for relative quantitation of analyte
abundances from different samples is labelling with
stable isotope-containing reagents . The central
premise of quantitation through stable isotope
coding is to label analytes from different samples
with the same chemical derivative, but containing
different amounts of heavy isotope atoms (typically
deuterium, 13C or 15N). The samples are then mixed,
and detected in a single MS experiment. Peptides
are detected as doublets with molecular weights
differing by the difference in mass of the heavy and
unlabelled reagents, and quantiation is performed by
comparing the relative intensities of peaks from each
sample. The most commonly used reactive groups
on proteinaceous analytes are cysteine residues, the
peptide amino and carboxy termini, and lysine ε amino groups . It is conceivable that
oligosaccharides could be similarly derivatised with
heavy and light reagents (for example through
reducing terminal reductive amination) for relative or
absolute quantitation in MS. This would enable more
diagnosis and prognosis based
biomarkers. Current Molecular Medicine, 2007, Vol. 7, No. 4 been developed to provide equivalents to peptide
mass fingerprinting  of peptides, or SEQUEST
pattern matching of peptide fragmentation. After
glycan characterisation, the large amounts of
processed information also require consistent
(including carbohydrate structure, disease state and
analytical methodology). Such databases are now
available: GlycoSuite (http://www.glycosuite.com/)
 and SweetDB (http://www.glycosciences.de/)
. Central data repositories enable the
accumulation of many researchers’ work, and
become acutely necessary as the amount of data
generated increases. The value of genomic and
proteomic databases is apparent, and the same will
become true of glycomic databases, enabling
efficient data mining and critical assessment of the
usefulness and validity of potential glycodiagnostics. MS analytical techniques are a very useful tool in
glycome characterisation by enabling the collection
of vast amounts of data describing the structures of
large numbers of glycans in a single experiment. In
these circumstances, structural and statistic data
analysis is often much more time consuming and
difficult than the wet-lab data collection itself. These
large data sets necessitate efficient bioinformatic
tools to assist data handling and processing. Tools
such as the glycan-fragment mass-fingerprinting tool
GlycosidIQ  or glyco-search-ms  have n
Glycoprotein Enrichment Technologies Selective A consistent problem in (glyco)proteomic analyses
is the orders of magnitude difference in protein
abundances in a single sample. This causes
difficulties because analytical techniques have
inherently limited dynamic detection ranges, and also
because more abundant components will tend to
mask lower abundant components. A solution to
these problems is to prefractionate proteins in a
sample before analysis . Similar approaches are
also possible for glycomic and glycoproteomic
analysis. For instance, affinity chromatography
enrichment of glycoproteins for glycosylation or
proteomic analysis  has been performed with
lectins [246, 247] and boronate , while
hydrazide chemistry allows the covalent capture of
glycopeptides . These various approaches
allow a focused investigation of glycosylation and
glycosylated proteins and can improve the chances
to detect also the low-abundance glycoproteins
which often contain the most useful diagnostic
information (as is the case for tumor marker
glycoproteins in early tumor stages). However, it must
be said that virtually all of these enrichment
methodologies are, to a different extent, selective for
certain glycan structures. u
ot N 411 Capillary Array Analyzers as a Platform for
Analyzers that use an array of 96 parallel
capillaries for capillary electrophoresis were the
workhorse platforms in the sequencing of the human
and many other genomes. At present, such
instruments are available from several manufacturers
at scales of 1 up to 384 capillaries, thus fitting the
most diverse needs of genetic analysis laboratories.
For DNA-analysis, these analyzers are equipped with
4-or 5-colour fluorescence detection, most often
excited by an argon laser at 488 nm, but also from
red diode lasers or from very bright green or blue
light emitting diodes. The technology is now also
being implemented in small, less versatile, cheaper, 412 Current Molecular Medicine, 2007, Vol. 7, No. 4 dedicated analyzers that are more suitable for the
clinical laboratory, to cope with the increasing
number of DNA-based diagnostic tests, especially in
such high-throughput fields as food and clinical
microbiology [250, 251].
We implemented carbohydrate analysis on the
existing DNA-analyzers to benefit from
technological developments in the higher-volume
DNA-diagnostics market. Virtually no modification is
needed to the buffer systems, capillary dynamic
coatings and separation polymers that were
optimized for DNA separation to obtain state-of-theart resolution for 8-amino-1,3,6-pyrenetrisulfonic acid
labeled glycans on the most widely used DNAanalysis systems (; Laroy et al. manuscript in
preparation). Indeed, DNA fragment analysis and
glycan analysis can effectively be performed in
parallel, if needed even in the same electrophoresis
run. This makes for very cost-effective glycan
analysis in molecular biology laboratories, for which
glycan analysis is often an infrequent need which
would not justify the major expenses needed for
dedicated glycan analytical equipment.
We are currently also starting to explore capillary
array DNA-analysers for different glycomics purposes
besides fluorophore-labeled glycan analysis. Schulz et al. field of glycosylation-based diagnostics would greatly
benefit from a set of lectins with simple and strict
binding specificity and high binding affinity to
common, small substructures of human O-and Nglycans. However, as most of these human
glycotopes are 'self' to the species in general use for
monoclonal antibody generation (mouse, rat),
classical immunisation strategies of these species
generally fail in yielding these desired antibodies.
One recent spin-off of the generation of
glycosyltransferase knock-outs in mice has been that
these mice can be used for the generation of
antibodies against the glycotope which is missing as
a consequence of the biosynthetic defect .
However, with many glycosyltransferase activities
being encoded by multigene families, it is unlikely
that this will become a generic strategy for the
production of better glycotope-binding proteins of
desired specificity. What remains is the everexpanding range of in vitro panning strategies of
huge libraries of sequence-varied protein scaffolds to
select those few sequence variants that bind the
glycotope of interest (and not other, structurally
related ones in the mixture in which the glycotope
has to be specifically detected to be of diagnostic
utility). This can be achieved by repetitive positive
and negative selection rounds, followed by more
directed in vitro affinity maturation of the few clones
with promising characteristics. In the hands of careful
and skilled specialists, these strategies can be
succesful in rapidly generating a binding protein with
the desired characteristics. Nevertheless, these
techniques currently seem under-used for the
generation of glycoconjugate-recognizing proteins.
The most important problem is that most display
technologies are geared towards monomeric binding
proteins, whereas high-affinity in carbohydrate
recognition (at least in nature) is generally achieved
through avidity effects based on oligomeric binding
proteins. Even in phage display experiments of
monomeric carbohydrate-recognizing modules, a
strong selection pressure towards mutants that
oligomerise the modules instead of mutants with
higher intrinsic affinity of the binding site has been
observed . This causes problems in then going
from the phage-displayed protein to recombinant
protein production, as the protein might not
oligomerize as it did in the phage context, unless the
adaptation to oligomerisation is very strong. A very
recent study has indeed built upon that logic in the
case of generating single-chain antibodies against
the T-antigen that are not protein-dependent, by
keeping the linker between the phage protein and
the single chain scaffold very short (0 or 1 amino
acids). This yielded oligomeric single chains which
were also oligomerizing when expressed in E. coli
out of the phage context . The future will tell
whether this strategy is of general applicability in
generating glycan-binding proteins with the desired
ot Upon detection
differences amongst the many samples that need to
be analyzed in clinical discovery programs, the
analytes of interest can then be identified using
focused, low-throughput CE-MS, which is now
coming of age as a powerful analytical technique
. Development of Carbohydrate-Binding Proteins
with Higher Specificity and Binding Affinity N Much
glyco(proteo)mics research depends on complex
analytical methodologies for the profiling of
glycoconjugate mixtures. Many of these methods rely
on multi-dimensional separations and sophisticated
mass spectrometry techniques, as described above.
However, once a glycosylation-based biomarker is
discovered using these technologies, a clinically
useful assay then has to be developed which can be
performed in an economical way in the high qualitystandard analytical environment of a routine clinical
laboratory. Most successful such assays rely on
specific binding proteins to either capture the
molecules that contain the biomarker information, to
detect the glycotope(s) of interest on these
molecules, or both. Most of the currently available
lectins have complex specificities, which can cause
difficulties in developing an assay for the
glycosylation-based marker that was found in the
discovery phase of a project. Moreover, the binding
affinity generally is orders of magnitude lower than
one typically can count on for antibodies (micromolar
instead of nanomolar), which can severely affect the
robustness of lectin-based assays. Therefore, the n
tio We have recently explored the Yeast Surface
Display system for the purpose of expression cloning Clinical Laboratory Testing in Human Medicine Based of glycan-binding proteins from complex DNA libraries
(Ryckaert, S., Callewaert, N. et al. manuscript
submitted). The salient features of this system are
cell-surface wide expression of thousands of
molecules consisting of a fusion between the protein
of interest and a yeast cell-wall associated protein,
very much mimicking the cell surface of a lectinexpressing cell. By using multivalent
molecules or high-density glycan surfaces as the
selection agents, fast and strong enrichment of
those yeast cells that express the lectin fusion
proteins with desired specificity and high affinity can
be achieved. We are now exploring the utility of this
system for true carbohydrate binding-site affinity
maturation. Current Molecular Medicine, 2007, Vol. 7, No. 4
The field of glycoconjugate-based diagnostics
has a long tradition and has provided a multitude of
very useful disease markers that are now part of the
routine testing procedures in pathogen identification
and classification, in cancer diagnosis and
monitoring, in liver disease assessment, in the
tracking of drug abuse,...) With the advent of better
analytical technologies for glycoconjugates, the field
is undergoing a revival: old markers are being
structurally characterized to gain the knowledge
required to improve their diagnostic performance;
new markers and testing procedures are being
discovered; and technologies geared towards the
clinical laboratory are being devised. Structural and
quantitative aspects of glycoconjugates often reflect
the identity and the condition of the cell that
produces them, and we have been, are, and will be
tapping into this information resource to diagnose,
predict the course of and the response to therapy of
a broad range of human diseases. 
 Hanisch, F. G. and Baldus, S. E. (1997) Histol. Histopathol., 12 ,
Reddi, A. L., Sankaranarayanan, K., Arulraj, H. S., Devaraj, N. and
Devaraj, H. (2000) Cancer Lett., 149 , 207-211.
Cao, Y., Karsten, U. R., Liebrich, W., Haensch, W., Springer, G. F.
and Schlag, P. M. (1995) Cancer, 76 , 1700-1708.
Toma, V., Sata, T., Vogt, P., Komminoth, P., Heitz, P. U. and Roth,
J. (1999) Cancer, 85 , 2151-2159.
Brooks, S. A. and Carter, T. M. (2001) Acta Histochem., 103 , 37-51.
Brooks, S. A., Lymboura, M., Schumacher, U. and Leathem, A. J.
(1996) J. Histochem. Cytochem., 44 , 519-524.
Brooks, S. A. and Wilkinson, D. (2003) A cta Histochem., 105 , 205212.
Osinaga, E., Babino, A., Grosclaude, J., Cairoli, E., Batthyany, C.,
Bianchi, S., Signorelli, S., Varangot, M., Muse, I. and Roseto, A.
(1996) Int. J. Oncol., 8, 401-406.
Thies, A., Moll, I., Berger, J. and Schumacher, U. (2001) Br. J.
Cancer, 84 , 819-823.
Brooks, S. A. (2000) Histol. Histopathol., 15 , 143-158.
Mitchell, B. S. and Schumacher, U. (1999) Histol. Histopathol., 14 ,
Schumacher, U. and Adam, E. (1997) Histochem. J., 29 , 677-684.
Brooks, S. A. and Hall, D. M. S. (2002) Clin. Exp. Metastasis, 19 ,
Brockhausen, I., Yang, J., Dickinson, N., Ogata, S. and Itzkowitz,
S. H. (1998) Glycoconjug. J., 15 , 595-603.
Clement, M., Rocher, J., Loirand, G. and Le Pendu, J. (2004) J. Cell
Sci., 117 , 5059-5069.
Bresalier, R. S., Ho, S. B., Schoeppner, H. L., Kim, Y. S.,
Sleisenger, M. H., Brodt, P. and Byrd, J. C. (1996)
Gastroenterology, 110 , 1354-1367.
Vierbuchen, M. J., Fruechtnicht, W., Brackrock, S., Krause, K. T.
and Zienkiewicz, T. J. (1995) Cancer, 76 , 727-735.
Cho, S. H., Sahin, A., Hortobagyi, G. N., Hittelman, W. N. and
Dhingra, K. (1994) Cancer Res., 54 , 6302-6305.
Le Pendu, J., Marionneau, S., Cailleau-Thomas, A., Rocher, J., Le
Moullac-Vaidye, B. and Clement, M. (2001) A pmis, 109 , 9-31.
Thurin, M. and Kieber-Emmons, T. (2002) Hybrid. Hybridomics, 21 ,
Nakamori, S., Kameyama, M., Imaoka, S., Furukawa, H., Ishikawa,
O., Sasaki, Y., Kabuto, T., Iwanaga, T., Matsushita, Y. and Irimura,
T. (1993) Cancer Res., 53 , 3632-3637.
Nakamori, S., Kameyama, M., Imaoka, S., Furukawa, H., Ishikawa,
O., Sasaki, Y., Izumi, Y. and Irimura, T. (1997) Dis. Colon Rectum,
40 , 420-431.
Ogawa, J., Inoue, H. and Koide, S. (1997) Cancer, 79 , 1678-1685.
Tatsumi, M., Watanabe, A., Sawada, H., Yamada, Y., Shino, Y. and
Nakano, H. (1998) C lin. Exp. Metastasis, 16 , 743-750.
Nakamori, S., Furukawa, H., Hiratsuka, M., Iwanaga, T., Imaoka, S.,
Ishikawa, O., Kabuto, T., Sasaki, Y., Kameyama, M., Ishiguro, S.
and Irimura, T. (1997) J. Clin. Oncol., 15 , 816-825.
Amado, M., Carneiro, F., Seixas, M., Clausen, H. and SobrinhoSimoes, M. (1998) Gastroenterology, 114 , 462-470.
Miyake, M., Taki, T., Hitomi, S. and Hakomori, S. (1992) N. Engl. J.
Med., 327 , 14-18.
Klinger, M., Farhan, H., Just, H., Drobny, H., Himmler, G., Loibner,
H., Mudde, G. C., Freissmuth, M. and Sexl, V. (2004) Cancer Res.,
64 , 1087-1093.
Dennis, J. W., Granovsky, M. and Warren, C. E. (1999) B iochim.
Biophys. Acta, 1473 , 21-34.
Fernandes, B., Sagman, U., Auger, M., Demetrio, M. and Dennis, J.
W. (1991) Cancer Res., 51 , 718-723.
Murata, K., Miyoshi, E., Ihara, S., Noura, S., Kameyama, M.,
Ishikawa, O., Doki, Y., Yamada, T., Ohigashi, H., Sasaki, Y.,
Higashiyama, M., Tarui, T., Takada, Y., Kannagi, R., Taniguchi, N.
and Imaoka, S. (2004) Oncology, 66 , 492-501.
Petretti, T., Kemmner, W., Schulze, B. and Schlag, P. M. (2000)
Gut, 46 , 359-366.
Cappelli, G., Paladini, S. and D'Agata, A. (1999) Tumori, 85 , S19S21.
Sawabu, N., Watanabe, H., Yamaguchi, Y., Ohtsubo, K. and Motoo,
Y. (2004) Pancreas, 28 , 263-267.
Shimono, R., Mori, M., Akazawa, K., Adachi, Y. and Sgimachi, K.
(1994) Am. J. Gastroenterol., 89 , 101-105.
Alvarez, J. A., Marin, J., Jover, J. M., Fernandez, R., Fradejas, J.
and Moreno, M. (1995) Dis. Colon Rectum, 38 , 535-542.
Reiter, W., Stieber, P., Reuter, C., Nagel, D., Lau-Werner, U. and
Lamerz, R. (2000) Anticancer Res., 20 , 5195-5198.
Haglund, C., Roberts, P. J., Jalanko, H. and Kuusela, P. (1992)
Scand. J. Gastroenterol., 27 , 169-174.
Sperti, C., Pasquali, C., Catalini, S., Cappellazzo, F., Bonadimani,
B., Behboo, R. and Pedrazzoli, S. (1993) J. Surg. Oncol., 52 , 137141.
Ramage, J. K., Donaghy, A., Farrant, J. M., Iorns, R. and Williams,
R. (1995) Gastroenterology, 108 , 865-869.
Siqueira, E., Schoen, R. E., Silverman, W., Martini, J., Rabinovitz,
M., Weissfeld, J. L., Abu Elmaagd, K., Madariaga, J. R. and
Slivka, A. (2002) Gastrointest. Endosc., 56 , 40-47.
Yoshida, E. M., Scudamore, C. H., Erb, S. R., Owen, D. A. and
Silver, H. K. (1995) Can. J. Surg., 38 , 83-86.
Adachi, Y., Iso, Y., Moriyama, M., Kasai, T. and Hashimoto, H.
(1998) Hepato-Gastroenterol., 45 , 77-80.
Fabris, C., Falleti, E., Pirisi, M., Soardo, G., Toniutto, P., Vitulli, D.,
Bortolotti, N., Gonano, F. and Bartoli, E. (1995) Clin. Chim. Acta,
243 , 25-33.
Decker, D., Bollmann, R., Hirner, A. and Stratmann, H. (1998)
Zentralbl Chir., 123 , 855-857. N 
 ACKNOWLEDGEMENTS We thank Markus Aebi for providing us with the
necessary time to write this review. Research in the
author's labs is funded by the Swiss Federal Institute
of Technology (GlycoINIT project) and a Marie Curie
Excellence grant to N.C. N.C. holds an honorary
fellowship of the Fund for Scientific Research
Flanders. W.L. is a postdoctoral fellow of the IWTFlanders. Karl Rumbold is acknowledged for
proofreading the manuscript.
A complete manually
(including >1500 relevant references) is available
upon request from the authors. Readers are
encouraged to provide us with crucial references that
we might have missed, and we regret that space
limitations have forced us to omit a lot of interesting
work.  
 Brockhausen, I. (2003) in: Glycobiology and Medicine, Advances
in Experimental Medicine and Biology, 535 , 163-188.  n
 413 414 Current Molecular Medicine, 2007, Vol. 7, No. 4
  
 Hammarstrom, S., Engvall, E., Johansson, B. G., Svensson, S.,
Sundblad, G. and Goldstein, I. J. (1975) Proc. Natl. Acad. Sci.
USA, 72 , 1528-1532.
Kuroki, M., Arakawa, F., Haruno, M., Murakami, M., Wakisaka, M.,
Higuchi, H., Oikawa, S., Nakazato, H. and Matsuoka, Y. (1992)
Hybridoma, 11 , 391-407.
Yamashita, K., Totani, K., Kuroki, M., Matsuoka, Y., Ueda, I. and
Kobata, A. (1987) Cancer Res., 47 , 3451-3459.
Yamashita, K., Totani, K., Iwaki, Y., Kuroki, M., Matsuoka, Y.,
Endo, T. and Kobata, A. (1989) J. Biol. Chem., 264 , 17873-17881.
Fukushima, K., Ohkura, T., Kanai, M., Kuroki, M., Matsuoka, Y.,
Kobata, A. and Yamashita, K. (1995) G lycobiology, 5, 105-115.
Yamashita, K., Fukushima, K., Sakiyama, T., Murata, F., Kuroki, M.
and Matsuoka, Y. (1995) Cancer Res., 55 , 1675-1679.
Baldus, S. E., Hanisch, F. G., Monaca, E., Karsten, U. R., Zirbes, T.
K., Thiele, J. and Dienes, H. P. (1999) Histol. Histopathol., 14 ,
Baldus, S. E., Zirbes, T., Glossmann, J., Fromm, S., Hanisch, F. G.,
Monig, S. P., Schroder, W., Schneider, P. M., Flucke, U., Karsten,
U., Thiele, J., Holscher, A. H. and Dienes, H. P. (2001) Oncology,
61 , 147-155.
Baldus, S. E., Zirbes, T. K., Hanisch, F. G., Kunze, D., Shafizadeh,
S. T., Nolden, S., Monig, S. P., Schneider, P. M., Karsten, U.,
Thiele, J., Holscher, A. H. and Dienes, H. P. (2000) Cancer, 88 ,
Flucke, U., Zirbes, T. K., Schroder, W., Monig, S. P., Koch, V.,
Schmitz, K., Thiele, J., Dienes, H. P., Holscher, A. H. and Baldus,
S. E. (2001) Anticancer Res., 21 , 2189-2193.
Guo, J. M., Zhang, X. Y., Chen, H. L., Wang, G. M. and Zhang, Y. K.
(2001) J. Cancer Res. Clin. Oncol., 127 , 512-519.
Hayden, R. T., Qian, X., Procop, G. W., Roberts, G. D. and Lloyd, R.
V. (2002) Diagn. Mol. Pathol., 11 , 119-126.
Shinoda, T., Kaufman, L. and Padhye, A. A. (1981) J. Clin.
Microbiol., 13 , 513-518.
Mercure, S., Senechal, S., Auger, P., Lemay, G. and Montplaisir, S.
(1996) J. Clin. Microbiol., 34 , 2106-2112.
Heelan, J. S., Sotomayor, E., Coon, K. and D'Arezzo, J. B. (1998)
J. Clin. Microbiol., 36 , 1443-1445.
Patterson, T. F., Miniter, P., Patterson, J. E., Rappeport, J. M. and
Andriole, V. T. (1995) J. Infect. Dis., 171 , 1553-1558.
Ertl, P. and Mikkelsen, S. R. (2001) Anal. Chem., 73 , 4241-4248.
Harbeck, R. J., Teague, J., Crossen, G. R., Maul, D. M. and
Childers, P. L. (1993) J. Clin. Microbiol., 31 , 839-844.
Connaughton, M., Lang, S., Tebbs, S. E., Littler, W. A., Lambert, P.
A. and Elliott, T. S. J. (2001) J. Infect., 42 , 140-144.
Agis, F., Schlich, P., Cartel, J. L., Guidi, C. and Bach, M. A. (1988)
Int. J. Lepr. Other Mycobact. Dis., 56 , 527-536.
Del Prete, R., Picca, V., Mosca, A., D'Alagni, M. and Miragliotta, G.
(1998) Int. J. Tuberc. Lung Dis., 2, 160-163.
Chanteau, S., Glaziou, P., Plichart, C., Luquiaud, P., Plichart, R.,
Faucher, J. F. and Cartel, J. L. (1993) Int. J. Lepr. Other Mycobact.
Dis., 61 , 533-541.
Cho, S. N., Cellona, R. V., Villahermosa, L. G., Fajardo, T. T.,
Balagon, M. V. F., Abalos, R. M., Tan, E. V., Walsh, G. P., Kim, J.
D. and Brennan, P. J. (2001) Clin. Diagn. Lab. Immunol., 8, 138142.
Hamasur, B., Bruchfeld, J., Haile, M., Pawlowski, A., Bjorvan, B.,
Kallenius, G. and Svenson, S. B. (2001) J. Microbiol. Methods, 45 ,
Okuda, Y., Maekura, R., Hirotani, A., Kitada, S., Yoshimura, K.,
Hiraga, T., Yamamoto, Y., Itou, M., Ogura, T. and Ogihara, T. (2004)
J. Clin. Microbiol., 42 , 1136-1141.
Nyame, A. K., Kawar, Z. S. and Cummings, R. D. (2004) Arch.
Biochem. Biophys., 426 , 182-200.
Nyame, A. K., Leppanen, A. M., Bogitsh, B. J. and Cummings, R.
D. (2000) Exp. Parasitol., 96 , 202-212.
van Remoortere, A., Vermeer, H. J., van Roon, A. M., Langermans,
J. A., Thomas, A. W., Wilson, R. A., van die, I., van den Eijnden,
D. H., Agoston, K., Kerekgyarto, J., Vliegenthart, J. F., Kamerling,
J. P., van dam, G. J., Hokke, C. H. and Deelder, A. M. (2003) Exp.
Parasitol., 105 , 219-225.
Alvesbrito, C. F., Simpson, A. J. G., Bahiaoliveira, L. M. G.,
Rabello, A. L. T., Rocha, R. S., Lambertucci, J. R., Gazzinelli, G.,
Katz, N. and Correaoliveira, R. (1992) Trans. Roy. Soc. Trop. Med.
Hyg., 86 , 53-56.
de Vijver, K. K. V., Hokke, C. H., van Remoortere, A., Jacobs, W.,
Deelder, A. M. and Van Marck, E. A. (2004) Int. J. Parasitol., 34 ,
Shaker, Z. A., Kaddah, M. A., Hanallah, S. B. and El-Khodary, M. I.
(1998) Int. J. Parasitol., 28 , 1893-1901.
Bandyopadhyay, S., Chatterjee, M., Sundar, S. and Mandal, C.
(2003) Glycoconjug. J., 20 , 531-536.
Chatterjee, M., Sharma, V., Mandal, C., Sundar, S. and Sen, S.
(1998) Glycoconjug. J., 15 , 1141-1147.
Bandyopadhyay, S., Chatterjee, M., Pal, S., Waller, R. F., Sundar,
S., McConville, M. J. and Mandal, C. (2004) Diagn. Microbiol.
Infect. Dis., 50 , 15-24.
Sarkari, B., Chance, M. and Hommel, M. (2002) Acta Trop., 82 , 339348.
Bruschi, F., Moretti, A., Wassom, D. and Fioretti, D. P. (2001)
Parasite-J. Soc. Fr. Parasitol., 8, S141-S143.
Restrepo, B. I., Obregon-Henao, A., Mesa, M., Gil, D. L., Ortiz, B. L.,
Mejia, J. S., Villota, G. E., Sanzon, F. and Teale, J. M. (2000) Int. J.
Parasit., 30 , 689-696.
Prabhakaran, V., Rajshekhar, V., Murrell, K. D. and Oommen, A.
(2004) Trans. Roy. Soc. Trop. Med. Hyg., 98 , 478-484.
Sato, C. and Furuya, K. (1994) Jpn. J. Med. Sci. Biol., 47 , 65-71.
Sato, C., Kawase, S. and Yano, S. (1999) Jpn. J. Infect. Dis., 52 ,
  
ot   Galizia, G., Lieto, E., Ferraraccio, F., Castellano, P., De Vita, F.,
Orditura, M., Romano, C. and Pignatelli, C. (2003) Dig. Surg., 20 ,
Ishibashi, R., Sakai, T., Yamashita, Y., Maekawa, T., Hideshima,
T. and Shirakusa, T. (1999) Int. Surg., 84 , 151-154.
Harada, T., Kubota, T. and Aso, T. (2002) Fertil. Steril., 78 , 733-739.
Hirakata, Y., Kobayashi, J., Sugama, Y. and Kitamura, S. (1995)
Eur. Respir. J., 8, 689-696.
Holtzman, R. N. N., Heymann, A. D., Bordone, F., Marinoni, G.,
Barillari, P. and Wahl, S. J. (2001) Arch. Pathol. Lab. Med., 125 ,
Angel, C. A., Pratt, C. B., Rao, B. N., Schell, M. J., Parham, D. M.,
Lobe, T. E. and Fleming, I. D. (1992) Cancer, 69 , 1487-1491.
Ichihara, T., Sakamoto, J., Nakao, A., Furukawa, K., Watanabe, T.,
Suzuki, N., Horisawa, M., Nagura, H., Lloyd, K. O. and Takagi, H.
(1993) Cancer, 71 , 71-81.
Nishihara, S., Narimatsu, H., Iwasaki, H., Yazawa, S., Akamatsu,
S. ando, T., Seno, T. and Narimatsu, I. (1994) J. Biol. Chem., 269 ,
Mare, L. and Trinchera, M. (2004) Eur. J. Biochem., 271 , 186-194.
Yin, B. W., Dnistrian, A. and Lloyd, K. O. (2002) Int. J. Cancer, 98 ,
Yin, B. W. and Lloyd, K. O. (2001) J. Biol. Chem., 276 , 2737127375.
Maggino, T. and Gadducci, A. (2000) Eur. J. Gynaecol. Oncol., 21 ,
Bast, R. C., Feeney, M., Lazarus, H., Nadler, L. M., Colvin, R. B.
and Knapp, R. C. (1981) J. Clin. Invest., 68 , 1331-1337.
Buamah, P. (2000) J. Surg. Oncol., 75 , 264-265.
D'Aloia, A., Faggiano, P., Aurigemma, G., Bontempi, L., Ruggeri,
G., Metra, M., Nodari, S. and Dei Cas, L. (2003) J. Am. Coll.
Cardiol., 41 , 1805-1811.
Kui Wong, N., Easton, R. L., Panico, M., Sutton-Smith, M.,
Morrison, J. C., Lattanzio, F. A., Morris, H. R., Clark, G. F., Dell, A.
and Patankar, M. S. (2003) J. Biol. Chem., 278 , 28619-28634.
Sung, C. C., Pearl, D. K., Coons, S. W., Scheithauer, B. W.,
Johnson, P. C. and Yates, A. J. (1994) Cancer, 74 , 3010-3022.
Ragupathi, G. (1996) Cancer Immunol. Immunother., 43 , 152-157.
Kobayashi, Y., Tsukazaki, K., Kubushiro, K., Sakayori, M. and
Nozawa, S. (1996) Clin. Cancer Res., 2, 749-754.
Mondal, S. and Saha, S. (2000) J. Exp. Clin. Cancer Res., 19 , 317327.
Taga, H., Hirai, H., Ishizuka, H. and Kaneda, H. (1988) Tumor Biol.,
Taketa, K. and Hirai, H. (1989) Electrophoresis, 10 , 562-567.
Du, M. Q., Hutchinson, W. L., Johnson, P. J. and Williams, R.
(1991) Cancer, 67 , 476-480.
Vanstaden, L., Bukofzer, S., Kew, M. C. and Savage, N. (1992) J.
Gastroenterol. Hepatol., 7, 260-265.
Hirai, H. and Taketa, K. (1992) J. Chromatogr., 604 , 91-94.
Taketa, K., Endo, Y., Sekiya, C., Tanikawa, K., Koji, T., Taga, H.,
Satomura, S., Matsuura, S., Kawai, T. and Hirai, H. (1993) Cancer
Res., 53 , 5419-5423.
Sato, Y., Nakata, K., Kato, Y., Shima, M., Ishii, N., Koji, T., Taketa,
K., Endo, Y. and Nagataki, S. (1993) N. Engl. J. Med., 328 , 18021806.
Shiraki, K., Takase, K., Tameda, Y., Hamada, M., Kosaka, Y. and
Nakano, T. (1995) Hepatology, 22 , 802-807.
Magne, D., Seta, N., Lebrun, D., Durand, G. and Durand, D. (1992)
Clin. Chem., 38 , 1418-1424.
Shimizu, K., Taniichi, T., Satomura, S., Matsuura, S., Taga, H. and
Taketa, K. (1993) Clin. Chim. Acta, 214 , 3-12.
Albanese, E. A., Bachl, B. L. and Mulcahy, G. M. (1995) Ann. Clin.
Lab. Sci., 25 , 158-168.
Li, D., Mallory, T. and Satomura, S. (2001) Clin. Chim. Acta, 313 ,
Okuda, K., Tanaka, M., Kanazawa, N., Nagashima, J., Satomura,
S., Kinoshita, H., Eriguchi, N., Aoyagi, S. and Kojiro, M. (1999) Int.
J. Oncol., 14 , 265-271.
Yamagata, Y., Shimizu, K., Nakamura, K., Henmi, F., Satomura, S.,
Matsuura, S. and Tanaka, M. (2003) Clin. Chim. Acta, 327 , 59-67.
Poon, T. C. W., Mok, T. S. K., Chan, A. T. C., Chan, C. M. L.,
Leong, V., Tsui, S. H. T., Leung, T. W. T., Wong, H. T. M., Ho, S. K.
W. and Johnson, P. J. (2002) Clin. Chem., 48 , 1021-1027.
Yamamoto, T., Amuro, Y., Matsuda, Y., Nakaoka, H., Shimomura,
S., Hada, T. and Higashino, K. (1991) Am. J. Gastroenterol., 86 ,
Watt, K. W., Lee, P. J., M'Timkulu, T., Chan, W. P. and Loor, R.
(1986) Proc. Natl. Acad. Sci. USA, 83 , 3166-3170.
Troyer, D. A., Mubiru, J., Leach, R. J. and Naylor, S. L. (2004) Dis.
Markers, 20 , 117-128.
Huber, P. R., Schmid, H. P., Mattarelli, G., Strittmatter, B.,
Vansteenbrugge, G. J. and Maurer, A. (1995) Prostate, 27 , 212-219.
Sumi, S., Arai, K., Kitahara, S. and Yoshida, K. (1999) J.
Chromatogr. B Biomed. Sci. Appl., 727 , 9-14.
Peracaula, R., Tabares, G., Royle, L., Harvey, D. J., Dwek, R. A.,
Rudd, P. M. and de Llorens, R. (2003) G lycobiology, 13 , 457-470.
Jankovic, M. M. and Kosanovic, M. M. (2005) Clin. Biochem., 38 ,
Basu, P. S., Majhi, R. and Batabyal, S. K. (2003) Clin. Biochem.,
36 , 373-376.
Ohyama, C., Hosono, M., Nitta, K., Oh-eda, M., Yoshikawa, K.,
Habuchi, T., Arai, Y. and Fukuda, M. (2004) G lycobiology, 14 , 671679.
Gold, P. and Freedman, S. O. (1965) J. Exp. Med., 122 , 467-481.
Thomson, D. M., Krupey, J., Freedman, S. O. and Gold, P. (1969)
Proc. Natl. Acad. Sci. USA, 64 , 161-167. Schulz et al. Clinical Laboratory Testing in Human Medicine Based
 Riordan, J. R., Rommens, J. M., Kerem, B., Alon, N., Rozmahel, R.,
Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J. L. and
al., e. (1989) S cience, 245 , 1066-1073.
Verkman, A. S., Song, Y. and Thiagarajah, J. R. (2003) Am. J.
Physiol. Cell Physiol ., 284 , C2-15
Wine, J. J. and Joo, N. S. (2004) Proc. Am. Thorac. Soc., 1, 47-53.
Lyon, E. and Miller, C. (2003) Arch. Pathol. Lab. Med., 127 , 11331139.
Davril, M., Degroote, S., Humbert, P., Galabert, C., Dumur, V.,
Lafitte, J. J., Lamblin, G. and Rousse, P. (1999) G lycobiology, 9,
Boat, T. F., Cheng, P. W., Iyer, R. N., Carlson, D. M. and Polony, I.
(1976) Arch. Biochem. Biophys., 177 , 95-104.
Chace, K. V., Flux, M. and Sachdev, G. P. (1985) B iochemistry, 24 ,
Zhang, Y., Doranz, B., Yankaskas, J. R. and Engelhardt, J. F.
(1995) J. Clin. Invest., 96 , 2997-3004.
Mohapatra, N. K., Cheng, P. W., Parker, J. C., Paradiso, A. M.,
Yankaskas, J. R., Boucher, R. C. and Boat, T. F. (1995) Pediatr.
Res., 38 , 42-48.
Mendicino, J. and Sangadala, S. (1999) Mol. Cell. Biochem., 201 ,
Cheng, P. W., Boat, T. F., Cranfill, K., Yankaskas, J. R. and
Boucher, R. C. (1989) J. Clin. Invest., 84 , 68-72.
Holmen, J. M., Karlsson, N. G., Abdullah, L. H., Randell, S. H.,
Sheehan, J. K., Hansson, G. C. and Davis, C. W. (2004) Am. J.
Physiol. Cell. Mol. Physiol., 287 , L824-834.
Schulz, B. L., Sloane, A. J., Robinson, L. J., Sebastian, L. T.,
Glanville, A. R., Song, Y., Verkman, A. S., Harry, J. L., Packer, N.
H. and Karlsson, N. G. (2005) Biochem. J., 387 , 911-919.
Tiddens, H. A. (2002) Pediatr. Pulmonol., 34 , 228-231.
Helbich, T. H., Heinz-Peer, G., Fleischmann, D., Wojnarowski, C.,
Wunderbaldinger, P., Huber, S., Eichler, I. and Herold, C. J. (1999)
AJR Am. J. Roentgenol., 173 , 81-88.
Zalewska, A., Zwierz, K., Zolkowski, K. and Gindzienski, A. (2000)
Acta Biochim. Pol., 47 , 1067-1079.
Prakobphol, A., Thomsson, K. A., Hansson, G. C., Rosen, S. D.,
Singer, M. S., Phillips, N. J., Medzihradszky, K. F., Burlingame, A.
L., Leffler, H. and Fisher, S. J. (1998) B iochemistry, 37 , 4916-4927.
Murray, P. A., Prakobphol, A., Lee, T., Hoover, C. I. and Fisher, S.
J. (1992) Infect. Immun., 60 , 31-38.
Thomsson, K. A., Prakobphol, A., Leffler, H., Reddy, M. S., Levine,
M. J., Fisher, S. J. and Hansson, G. C. (2002) G lycobiology, 12 , 114.
Schulz, B. L., Packer, N. H. and Karlsson, N. G. (2002) Anal.
Chem., 74 , 6088-6097.
Klein, A., Carnoy, C., Wieruszeski, J. M., Strecker, G., Strang, A.
M., van Halbeek, H., Roussel, P. and Lamblin, G. (1992)
Biochemistry, 31 , 6152-6165.
Thomsson, K. A., Schulz, B. L., Packer, N. H. and Karlsson, N. G.
(2005) G lycobiology, 15 , 791-804.
Seemann, R., Zimmer, S., Bizhang, M. and Kage, A. (2001) Caries
Res., 35 , 156-161.
Sewell, A. C. (1980) Eur. J. Pediatr. 134 , 183-194.
Paschke, E. and Stockler, S. (1992) Wien. Klin. Wochen., 104 ,
Meikle, P. J., Ranieri, E., Simonsen, H., Rozaklis, T., Ramsay, S.
L., Whitfield, P. D., Fuller, M., Christensen, E., Skovby, F. and
Hopwood, J. J. (2004) P ediatrics, 114 , 909-916.
Giudici, T. A., Sunico, H. and Blaskovics, M. (1996) J. Inherit.
Metab. Dis., 19 , 263-266.
Ramsay, S. L., Meikle, P. J. and Hopwood, J. J. (2003) Mol. Genet.
Metab., 78 , 193-204.
Jaeken, J., Vanderschueren-Lodeweyckx, M., Casaer, P., Snoeck,
L. and Corbeel, L. (1980) Pediatr. Res., 14 , 179.
Jaeken, J., van Eijk, H. G., van der Heul, C., Corbeel, L., Eeckels,
R. and Eggermont, E. (1984) Clin. Chim. Acta, 144 , 245-247.
Stibler, H. and Kristiansson, B. (1991) Acta Paediatr. Scand., 3238.
Wada, Y., Gu, J. G., Okamoto, N. and Inui, K. (1994) Biol. Mass
Spectrom., 23 , 108-109.
Yamashita, K., Ohkura, T., Ideo, H., Ohno, K. and Kanai, M. (1993)
J. Biochem. (Tokyo), 114 , 766-769.
Lacey, J. M., Bergen, H. R., Magera, M. J., Naylor, S. and O'Brien,
J. F. (2001) Clin. Chem., 47 , 513-518.
Muntoni, F. (2004) A cta Myol., 23 , 79-84.
Zhang, W. L., Vajsar, J., Cao, P. J., Breningstall, G., Diesen, C.,
Dobyns, W., Herrmann, R., Lehesjoki, A. E., Steinbrecher, A.,
Talim, B., Toda, T., Topaloglu, H., Voit, T. and Schachter, H. Y.
(2003) Clin. Biochem., 36 , 339-344.
Quentin, E., Gladen, A., Roden, L. and Kresse, H. (1990) Proc. Natl.
Acad. Sci. USA, 87 , 1342-1346.
Lind, T., Tufaro, F., McCormick, C., Lindahl, U. and Lidholt, K.
(1998) J. Biol. Chem., 273 , 26265-26268.
McCormick, C., Duncan, G., Goutsos, K. T. and Tufaro, F. (2000)
Proc. Natl. Acad. Sci. USA, 97 , 668-673.
Bessler, M., Schaefer, A. and Keller, P. (2001) in Transf. Med.
Rev., 15 , 255-267.
Krauss, J. S. (2003) Ann. Clin. Lab. Sci., 33 , 401-406.
Shin, D. J., Lee, J. J., Choy, H. E. and Hong, Y. J. (2004) B iochem.
Biophys. Res. Commun., 324 , 753-760.
Stibler, H., Allgulander, C., Borg, S. and Kjellin, K. G. (1978) Acta
Med. Scand., 204 , 49-56.
Stibler, H. (1991) Clin. Chem., 37 , 2029-2037.
Arndt, T. (2001) Clin. Chem., 47 , 13-27.
Bergen, H. R., Lacey, J. M., O'Brien, J. F. and Naylor, S. (2001)
Anal. Biochem., 296 , 122-129.
Alte, D., Luedemann, J., Rose, H. J. and John, U. (2004)
Alcoholism (NY), 28 , 931-940. Current Molecular Medicine, 2007, Vol. 7, No. 4
 Anttila, P., Jarvi, K., Latvala, J. and Niemela, O. (2004) A lcohol
Alcohol, 39 , 59-63.
Koch, H., Meerkerk, G. J., Zaat, J. O. M., Ham, M. F., Scholten, R.
and Assendelft, W. J. J. (2004) A lcohol Alcohol, 39 , 75-85.
Arndt, T., Kropf, J., Brandt, R., Gressner, A. M., Hackler, R., Herold,
M., Van Pelt, J., Martensson, O., Salzmann, K. and Velmans, M. H.
(1998) A lcohol Alcohol, 33 , 639-645.
Wuyts, B., Delanghe, J. R., Kasvosve, I., Wauters, A., Neels, H.
and Janssens, J. (2001) Clin. Chem., 47 , 247-255.
Lanz, C., Kuhn, M., Deiss, V. and Thormann, W. (2004)
Electrophoresis, 25 , 2309-2318.
Martello, S., Trettene, M., Cittadini, F., Bortolotti, F. B., De Giorgio,
F., Chiarotti, M. and Tagliaro, F. (2004) Forensic Sci. Int., 141 , 153157.
Iourin, O., Mattu, T. S., Mian, N., Keir, G., Winchester, B., Dwek, R.
A. and Rudd, P. M. (1996) Glycoconjug. J., 13 , 1031-1042.
Fermo, I., Germagnoli, L., Soldarini, A., Dorigatti, F. and Paroni, R.
(2004) Electrophoresis, 25 , 469-475.
Renner, F. and Kanitz, R. D. (1997) Clin. Chem., 43 , 485-490.
Helander, A., Eriksson, G., Stibler, H. and Jeppsson, J. O. (2001)
Clin. Chem., 47 , 1225-1233.
Winearls, C. G., Oliver, D. O., Pippard, M. J., Reid, C., Downing, M.
R. and Cotes, P. M. (1986) Lancet, 2, 1175-1178.
Pascual, J. A., Belalcazar, V., de Bolos, C., Gutierrez, R., Llop, E.
and Segura, J. (2004) Ther. Drug Monit., 26 , 175-179.
Lasne, F., Martin, L., Crepin, N. and de Ceaurriz, J. (2002) Anal.
Biochem., 311 , 119-126.
Takeuchi, M., Takasaki, S., Miyazaki, H., Kato, T., Hoshi, S.,
Kochibe, N. and Kobata, A. (1988) J. Biol. Chem., 263 , 3657-3663.
Takeuchi, M. and Kobata, A. (1991) G lycobiology, 1, 337-346.
Koury, M. J. (2003) Trends Biotechnol., 21 , 462-464.
Nimtz, M., Martin, W., Wray, V., Kloppel, K. D., Augustin, J. and
Conradt, H. S. (1993) Eur. J. Biochem., 213 , 39-56.
Souillard, A., Audran, M., Bressolle, F., Gareau, R., Duvallet, A.
and Chanal, J. L. (1996) Br. J. Clin. Pharmacol., 42 , 355-364.
Breidbach, A., Catlin, D. H., Green, G. A., Tregub, I., Truong, H. and
Gorzek, J. (2003) Clin. Chem., 49 , 901-907.
Nowicki, M., Kokot, F., Kokot, M., Bar, A. and Dulawa, J. (1994) Int.
Urol. Nephrol., 26 , 691-699.
Neumayr, G., Pfister, R., Hoertnagl, H., Mitterbauer, G., Getzner, W.,
Ulmer, H., Gaenzer, H. and Joannidis, M. (2003) Int. J. Sports Med.,
24 , 131-137.
Gore, C. J., Parisotto, R., Ashenden, M. J., Stray-Gundersen, J.,
Sharpe, K., Hopkins, W., Emslie, K. R., Howe, C., Trout, G. J.,
Kazlauskas, R. and Hahn, A. G. (2003) Haematologica, 88 , 333344.
Sendid, B., Colombel, J. F., Jacquinot, P. M., Faille, C., Fruit, J.,
Cortot, A., Lucidarme, D., Camus, D. and Poulain, D. (1996) Clin.
Diagn. Lab. Immunol., 3, 219-226.
Annese, V., Piepoli, A., Perri, F., Lombardi, G., Latiano, A.,
Napolitano, G., Corritore, G., Vandewalle, P., Poulain, D.,
Colombel, J. F. and Andriulli, A. (2004) Aliment Pharmacol. Ther.,
20 , 1143-1152.
Kim, J. E., Kim, K. S. and Seo, J. K. (2003) Korean J.
Gastroenterol., 42 , 297-302.
Konrad, A., Rutten, C., Flogerzi, B., Styner, M., Goke, B. and
Seibold, F. (2004) Inflamm. Bowel. Dis., 10 , 97-105.
Galassi, G., Susuki, K., Quaglino, D. and Yuki, N. (2004) Eur. J.
Neurol., 11 , 790-791.
Ang, C. W., Laman, J. D., Willison, H. J., Wagner, E. R., Endtz, H.
P., De Klerk, M. A., Tio-Gillen, A. P., Van den Braak, N., Jacobs, B.
C. and Van Doorn, P. A. (2002) Infect. Immun., 70 , 1202-1208.
Hirano, M., Kusunoki, S., Asai, H., Tonomura, Y., Morita, D. and
Ueno, S. (2003) Neurology, 60 , 1719-1720.
Nishimoto, Y., Odaka, M., Hirata, K. and Yuki, N. (2004) J.
Neuroimmunol., 148 , 200-205.
Nagashima, T., Koga, M., Odaka, M., Hirata, K. and Yuki, N. (2004)
J. Neurol. Sci., 219 , 139-145.
Pincus, T. (1995) Br. J. Rheumatol., 34 (Suppl 2), 59-73.
Young, A., Dixey, J., Kulinskaya, E., Cox, N., Davies, P., Devlin,
J., Emery, P., Gough, A., James, D., Prouse, P., Williams, P. and
Winfield, J. (2002) Ann. Rheum. Dis., 61 , 335-340.
Munro, R., Hampson, R., McEntegart, A., Thomson, E. A., Madhok,
R. and Capell, H. (1998) Ann. Rheum. Dis., 57 , 88-93.
Zeidler, H. K., Kvien, T. K., Hannonen, P., Wollheim, F. A., Forre,
O., Geidel, H., Hafstrom, I., Kaltwasser, J. P., Leirisalo-Repo, M.,
Manger, B., Laasonen, L., Markert, E. R., Prestele, H. and Kurki, P.
(1998) Br. J. Rheumatol., 37 , 874-882.
Tsakonas, E., Fitzgerald, A. A., Fitzcharles, M. A., Cividino, A.,
Thorne, J. C., M'Seffar, A., Joseph, L., Bombardier, C. and Esdaile,
J. M. (2000) J. Rheumatol., 27 , 623-629.
Smolen, J. S., Aletaha, D. and Machold, K. P. (2005) Best Pract.
Res. Clin. Rheumatol., 19 , 163-177.
Keane, J., Gershon, S., Wise, R. P., Mirabile-Levens, E.,
Kasznica, J., Schwieterman, W. D., Siegel, J. N. and Braun, M. M.
(2001) N. Engl. J. Med., 345 , 1098-1104.
Gardam, M. and Iverson, K. (2003) J. Rheumatol., 30 , 1397-1399.
Parekh, R. B., Dwek, R. A., Sutton, B. J., Fernandes, D. L., Leung,
A., Stanworth, D., Rademacher, T. W., Mizuochi, T., Taniguchi, T.,
Matsuta, K., Takeuchi, F.; Nagano, Y.; Miyamotom T.; and Kobata,
A. (1985) Nature, 316 , 452-457.
Hansler, M., Kotz, K. and Hantzschel, H. (1995) Electrophoresis,
16 , 811-812.
Martin, K., Talukder, R., Hay, F. C. and Axford, J. S. (2001) J.
Rheumatol., 28 , 1531-1536.
Kanoh, Y., Mashiko, T., Danbara, M., Takayama, Y., Ohtani, S.,
Imasaki, T., Abe, T. and Akahoshi, T. (2004) Oncology, 66 , 365370. 
 N 
ot  415 416 Current Molecular Medicine, 2007, Vol. 7, No. 4
 Alavi, A., Arden, N., Spector, T. D. and Axford, J. S. (2000) J.
Rheumatol., 27 , 1379-1385.
Alavi, A., Axford, J. S. and Pool, A. J. (2004) J. Rheumatol., 31 ,
Saez-Valero, J., Barquero, M. S., Marcos, A., McLean, C. A. and
Small, D. H. (2000) J. Neurol. Neurosurg. Psychiatry, 69 , 664-667.
Fodero, L. R., Saez-Valero, J., McLean, C. A., Martins, R. N.,
Beyreuther, K., Masters, C. L., Robertson, T. A. and Small, D. H.
(2002) J. Neurochem., 81 , 441-448.
Sherman, M. and Takayama, Y. (2004) Gastroenterol. Clin. North
Am., 33 , 671-691.
Mann, A. C., Record, C. O., Self, C. H. and Turner, G. A. (1994) Clin.
Chim. Acta, 227 , 69-78.
Callewaert, N., Van Vlierberghe, H., Van Hecke, A., Laroy, W.,
Delanghe, J. and Contreras, R. (2004) Nat. Med., 10 , 429-434.
Hada, T., Kondo, M., Yasukawa, K., Amuro, Y. and Higashino, K.
(1999) Clin. Chim. Acta, 281 , 37-46.
Ryden, I., Pahlsson, P. and Lindgren, S. (2002) Clin. Chem., 48 ,
Seta, N., Gayno, S., JezequelCuer, M., Toueg, M. L., Erlinger, S.
and Durand, G. (1997) J. Hepatol., 26 , 265-271.
Kudo, M., Todo, A., Ikekubo, K. and Hino, M. (1992) Am. J.
Gastroenterol., 87 , 865-870.
Dell, A. and Morris, H. R. (2001) S cience, 291 , 2351-2356.
Zaia, J. (2004) Mass Spectrom. Rev., 23 , 161-227.
Rashed, M. S. (2001) J. Chromatogr. B Biomed. Sci. Appli., 758 ,
Rinaldo, P., Tortorelli, S. and Matern, D. (2004) Curr. Opin. Pediatr.,
16 , 427-433.
Julka, S. and Regier, F. (2004) J. Proteome Res., 3, 350-363.
Joshi, H., Harrison, M. J., Schulz, B. L., Cooper, C. A., Packer, N.
H. and Karlsson, N. G. (2004) Proteomics, 4, 1650-1664. Schulz et al.
 Lohmann, K. K. and von der Leith, C. W. (2004) Nucleic Acids
Res., 32 , W261-266.
Cottrell, J. S. (1994) Pept. Res., 7, 115-124.
Cooper, C. A., Joshi, H. J., Harrison, M. J., Wilkins, M. R. and
Packer, N. H. (2003) N ucleic Acids Res., 31 , 511-513.
Loss, A., Bunsmann, P., Bohne, A., Loss, A., Schwarzer, E., Lang,
E. and von der Leith, C. W. (2002) Nucleic Acids Res., 30 , 405408.
Righetti, P. G., Castagna, A. and Herbert, B. (2001) Anal. Chem.,
73 , 320A-326A.
Lee, W. C. and Lee, K. H. (2004) A nal. Biochem ., 324 , 1-10.
Hortin, G. L. and Trimpe, B. L. (1990) Anal. Biochem., 188 , 271-277.
Kaji, H., Saito, H., Yamauchi, Y., Shinkawa, T., Taoka, M.,
Hirabayashi, J., Kasai, K., Takahashi, N. and Isobe, T. (2003) Nat.
Biotechnol., 21 , 667-672.
Hagglund, P., Bunkenborg, J., Elortza, F., Jensen, O. N. and
Roepstorff, P. (2004) J. Proteome Res., 3, 556-566.
Zhang, H., Li, X. J., Martin, D. B. and Aebersold, R. (2003) Nat.
Biotechnol., 21 , 660-666.
Liu, M. S. and Amirkhanian, V. D. (2003) Electrophoresis, 24 , 9395.
Callewaert, N., Contreras, R., Mitnik-Gankin, L., Carey, L.,
Matsudaira, P. and Ehrlich, D. (2004) Electrophoresis, 25 , 31283131.
Zamfir, A. and Peter-Katalinic, J. (2004) Electrophoresis, 25 , 19491963.
Lee, J., Park, S. H. and Stanley, P. (2002) Glycoconj. J., 19 , 211219.
Ravn, P., Danielczyk, A., Jensen, K. B., Kristensen, P.,
Christensen, P. A., Larsen, M., Karsten, U. and Goletz, S. (2004) J.
Mol. Biol., 343 , 985-996. t
ot N n
rib Received: November 15, 2006 Revised: December 21, 2006 Accepted: February 20, 2007 ...
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