High-throughput determination of urinary hexosamines for diagnosis of mucopolysaccharidoses by capillary electrophoresis and high-performance liquid chromatography

Article abstract of DOI:10.1016/j.ab.2010.12.016

Mucopolysaccharidoses (MPS) diagnosis is often delayed and irreversible organ damage can occur, making possible therapies less effective. This highlights the importance of early and accurate diagnosis. A high-throughput procedure for the simultaneous determination of glucosamine and galactosamine produced from urinary galactosaminoglycans and glucosaminoglycans by capillary electrophoresis (CE) and HPLC has been performed and validated in subjects affected by various MPS including their mild and severe forms, Hurler and Hurler-Scheie, Hunter, Sanfilippo, Morquio, and Maroteaux-Lamy. Contrary to other analytical approaches, the present single analytical procedure, which is able to measure total abnormal amounts of urinary GAGs, high molecular mass, and related fragments, as well as specific hexosamines belonging to a group of GAGs, would be useful for possible application in their early diagnosis. After a rapid urine pretreatment, free hexosamines are generated by acidic hydrolysis, derivatized with 2-aminobenzoic acid and separated by CE/UV in ～10 min and reverse-phase (RP)-HPLC in fluorescence in ～21 min. The total content of hexosamines was found to be indicative of abnormal urinary excretion of GAGs in patients compared to the controls, and the galactosamine/glucosamine ratio was observed to be related to specific MPS syndromes in regard to both their mild and severe forms. As a consequence, important correlations between analytical response and clinical diagnosis and the severity of the disorders were observed. Furthermore, we can assume that the severity of the syndrome may be ascribed to the quantity of total GAGs, as high-molecular-mass polymers and fragments, accumulated in cells and directly excreted in the urine. Finally, due to the high-throughput nature of this approach and to the equipment commonly available in laboratories, this method is suitable for newborn screening in preventive public health programs for early detection of MPS disorders, diagnosis, and their treatment.

Full text of DOI:10.1016/j.ab.2010.12.016

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Analytical Biochemistry 411 (2011) 32–42
Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
High-throughput determination of urinary hexosamines for diagnosis
of mucopolysaccharidoses by capillary electrophoresis and high-performance liquid
chromatography
Giovanni V. Coppa a, Fabio Galeotti b, Lucia Zampini a, Francesca Maccari b, Tiziana Galeazzi a, Lucia Padelia a,
Lucia Santoro a, Orazio Gabrielli a, Nicola Volpi b,
⇑
a Pediatric Division, Department of Clinical Sciences, Polytechnic University of the Marche, Ospedali Riuniti, Presidio Salesi, Ancona, Italy
b Department of Biology, University of Modena & Reggio Emilia, Via Campi 213/D, 41100 Modena, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Mucopolysaccharidoses (MPS) diagnosis is often delayed and irreversible organ damage can occur, mak-
ing possible therapies less effective. This highlights the importance of early and accurate diagnosis. A
high-throughput procedure for the simultaneous determination of glucosamine and galactosamine pro-
duced from urinary galactosaminoglycans and glucosaminoglycans by capillary electrophoresis (CE) and
HPLC has been performed and validated in subjects affected by various MPS including their mild and
severe forms, Hurler and Hurler-Scheie, Hunter, Sanfilippo, Morquio, and Maroteaux-Lamy. Contrary to
other analytical approaches, the present single analytical procedure, which is able to measure total
abnormal amounts of urinary GAGs, high molecular mass, and related fragments, as well as specific
hexosamines belonging to a group of GAGs, would be useful for possible application in their early diag-
nosis. After a rapid urine pretreatment, free hexosamines are generated by acidic hydrolysis, derivatized
with 2-aminobenzoic acid and separated by CE/UV in ꢀ10 min and reverse-phase (RP)-HPLC in fluores-
cence in ꢀ21 min. The total content of hexosamines was found to be indicative of abnormal urinary
excretion of GAGs in patients compared to the controls, and the galactosamine/glucosamine ratio was
observed to be related to specific MPS syndromes in regard to both their mild and severe forms. As a con-
sequence, important correlations between analytical response and clinical diagnosis and the severity of
the disorders were observed. Furthermore, we can assume that the severity of the syndrome may be
ascribed to the quantity of total GAGs, as high-molecular-mass polymers and fragments, accumulated
in cells and directly excreted in the urine. Finally, due to the high-throughput nature of this approach
and to the equipment commonly available in laboratories, this method is suitable for newborn screening
in preventive public health programs for early detection of MPS disorders, diagnosis, and their treatment.
Ó 2010 Elsevier Inc. All rights reserved.
Received 27 September 2010
Received in revised form 4 December 2010
Accepted 6 December 2010
Available online 13 December 2010
Keywords:
Mucopolysaccharidoses
Hexosamines
Capillary electrophoresis
Glucosamine
Galactosamine
Glycosaminoglycans
Mucopolysaccharidoses (MPS)1 are a group of inherited lyso-
somal storage disorders characterized by a deficiency in one of the
lysosomal enzymes required to degrade glycosaminoglycans (GAGs)
[1–3]. In all MPS subtypes, partially degraded GAG(s) accumulate in
the lysosomes of affected cells and/or are eliminated in the blood
and excreted in the urine. MPS types I through IX involve deficien-
cies of one of the key 11 enzymes needed for the stepwise degrada-
tion of various GAGs, i.e., dermatan sulfate (DS), heparan sulfate
(HS), keratan sulfate (KS), chondroitin sulfate (CS), very rarely hyalu-
ronan (HA), or in combination.
MPS incidence is estimated as one out of 25,000 live births [1],
and, in general, MPS newborns are asymptomatic, although the
accumulation starts in the fetal period forming a pathological base
[4]. Development of clinical signs for MPS varies with some
patients showing severe disease in the first few years of life. Ther-
apeutic approaches toward MPS patients have offered various
treatment options represented by exogenously supplied enzymes
that are deficient (enzyme replacement therapy, ERT, approved
for MPS I, II, and VI, with clinical trials for other types of MPS in
progress) [5–8], by hematopoietic stem cell transplantation (HSCT)
for severe MPS forms, suggesting that enzymes from the donor
⇑
Corresponding author. Fax: +39 59 2055548.
E-mail address: volpi@unimo.it (N. Volpi).
Abbreviations used: AA, anthranilic acid or 2-aminobenzoic acid; AUC, area under
1
the curve; CETAB, cetyltrimethylammonium bromide; CE, capillary electrophoresis;
CR, creatinine; CS, chondroitin sulfate; DMB, 1,9-dimethylmethylene blue; DS,
dermatan sulfate; ESI, electrospray ionization; ERT, enzyme-replacement therapy;
Fd, fluorescence detection; GAG, glycosaminoglycan; Gal, D-galactosamine; GalNAc,
N-acetyl-D-galactosamine; GlcN, D-glucosamine; GlcNAc, N-acetyl-D-glucosamine; KS,
keratan sulfate; HA, hyaluronic acid or hyaluronan; HexNAc, N-acetylhexosamine;
HPCE, high-performance CE; HS, heparan sulfate; HSCT, hematopoietic stem cell
transplantation; MPS, mucopolysaccharidoses; MS, mass spectrometry.
0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2010.12.016

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Hexosamines in urine of MPS patients / G.V. Coppa et al. / Anal. Biochem. 411 (2011) 32–42
33
bone marrow can reduce GAG storage [9]. Finally, experimental
gene therapies have also been performed [10]. Generally, clinical
benefits of ERT for MPS include improved growth and quality of life
[8,9]. However, for treatment to be successful, patients need to be
treated earlier in the course of their disease and early identification
of the clinically asymptomatic subjects requires screening by
means of specific and sensitive tests.
tandem mass techniques have also been used to analyze GAG oli-
gosaccharides and mono- and disaccharides in biological samples
[2,20,27]. However, this approach generally requires time-consum-
ing and complex preparative and labeling measures before sample
analysis, in addition to very expensive equipment. An HPLC-ESI-
MS/MS method able to detect nanomolar amounts of HS-, DS-,
and KS-derived disaccharides in MPS patient serum and plasma
has been reported [26,28]. This approach requires the digestion
of sample GAGs to disaccharides with various lyases prior to anal-
ysis, which is potentially costly and time-consuming. Moreover, a
new reversed-phase HPLC-ESI-MS/MS assay for the determination
of the urinary intact HS- and DS-derived di- to pentasaccharides
has been recently described for MPS diagnosis [29]. A main draw-
back of this analysis is the incapacity to determine high-molecular-
mass urinary GAGs demonstrated to be present in high amounts in
MPS urines [18,19], as well as the time-consuming and complex
preparation of samples and derivatization with 1-phenyl-3-
methyl-5-pyrazolone. Finally, all these methods are not appropri-
ate for mass screening since they are excessively costly for sample
analysis due to the high cost of the equipment and they have never
been found able to correlate analytical data with the clinical diag-
nosis and severity of MPS signs and symptoms.
In our preliminary data, we present a high-throughput proce-
dure for the determination of urinary Hexs performed by means
of CE and HPLC for possible application in the early diagnosis of
MPS. Important correlations between analytical response and clin-
ical diagnosis and the severity of the disorders were also observed.
Finally, results from this study may be useful for newborn screen-
ing in preventive public health programs, as well as for a more
accurate follow-up of treatment and new possible therapeutic
interventions.
GAGs are structurally complex, sulfated, linear polymers [11]
constituted by repeating disaccharides with an N-acetylhexosamine
(HexNAc), N-acetyl-
D
-galactosamine (GalNAc), or N-acetyl-
D-gluco-
samine (GlcNAc) (or lower percentages of N-sulfo-
D
-glucosamine in
HS), as one of the sugars and hexuronic acid, glucuronic acid, or idu-
ronic acid, with the exception of KS which contains galactose
instead, as the alternating sugar. As a consequence, according to
the nature of the hexosamine residue, they are classified into two
groups: glucosaminoglycans with HA, KS, HS, and heparin, and
galactosaminoglycans composed of CS and DS. Hexs are common
monosaccharides forming more complex carbohydrates such as
oligo- and polysaccharides, and they are determined to obtain struc-
tural information and quantitative evaluations. Generally, for ana-
lytical purposes, GalN and GlcN are produced by controlled
chemical degradation of the (oligo)polysaccharides, labeled with a
suitable tag [12], and separated by means of HPLC in fluorescence
[13,14] or capillary electrophoresis (CE) with laser-induced fluores-
cence [15] or UV detector [16,17].
Depending on MPS types and regardless of the severity of the
clinical findings, different high-molecular-mass GAGs [18,19] as
well as fragments [2,20] are accumulated in tissues and excreted
in biological fluids, in particular blood and urine. As a consequence,
a single analytical approach able to quantitatively measure total
abnormal amounts of GAGs in urine and qualitative differences be-
tween the various classes of these macromolecules as well as mod-
ifications related to the different types of MPS would be desirable.
Furthermore, a similar laboratory approach would be useful to per-
form newborn screening in preventive public health programs for
early detection of disorders, diagnosis, and treatment of these met-
abolic congenital disorders, thus leading to a significant reduction
in terms of death, disease, and associated disabilities. Finally, a
more accurate follow-up of treatments and new possible therapeu-
tic interventions may be monitored by this new analytical
application.
Materials and methods
Materials
D-(+)-GlcN hydrochloride, D-(+)-GalN hydrochloride, D-ribose,
anthranilic acid (2-aminobenzoic acid, AA), and sodium cyano-
borohydride were from Sigma-Aldrich. CS A from bovine trachea,
DS from porcine intestinal mucosa, and HS from beef spleen were
from Sigma-Aldrich. Standard HA/CS/DS unsaturated disaccharides
were purchased from Sigma-Aldrich.
Enzyme-activity assays based on cultured fibroblasts, leuko-
cytes, plasma, or serum are definitive for a specific MPS disorder
and are considered the gold standard for diagnosis. However, none
of the several different approaches developed to this aim, such as
the direct multiplex assay of lysosomal enzymes in dried blood
spots by tandem mass (MS/MS) spectrometry (MS) [21] or a mul-
tiplexed immune-quantification assay of lysosomal proteins from
dried blood spots on filter paper [22], are useful for a early diagno-
sis of MPS in newborn screening due to complex procedures and
laboratory equipment. Furthermore, to date, no genotype–pheno-
type correlations are evident due to the large number of mutations
and limited by the rarity of the disorders [23].
All the other reagents were analytical grade generally supplied
by Sigma-Aldrich.
Control subjects and MPS patients
Normal subjects and patients affected by MPS type I (Hurler,
Hurler-Scheie, Scheie), MPS II (severe and mild forms), MPS III,
MPS IV, and MPS VI (Table 1) were registered in the Pediatric Divi-
sion, Department of Clinical Sciences, Polytechnic University of
Marche, Presidio Salesi, Ancona, Italy.
The diagnosis of MPS was performed on the basis of the patho-
logical pattern of urinary GAGs and the enzymatic deficiency. The
different forms of the MPS I (Hurler, Hurler-Scheie, Scheie) and
MPS II (severe and mild forms) were established on the basis of
the peculiar clinical signs and of the presence or not of mental
retardation.
To date, there are several established procedures to diagnose
MPS by the evaluation of the accumulated GAGs, such as total uri-
nary GAGs measurement using dye-binding assay generally with
1,9-dimethylmethylene blue (DMB) [24,25] or electrophoresis on
cellulose acetate [8]. However, dye-binding assays are generally
used to obtain a quantitative evaluation but they are incapable of
identifying individual GAGs and electrophoresis is unable to eval-
uate related polysaccharide structures and characteristics useful
for the characterization of specific modifications. A sandwich ELISA
method has also been developed for HS and KS quantitative evalu-
ation on blood and urine [26]. However, DS measurement has not
been developed along with the incapacity to evaluate its (and HS
and KS) composition. Electrospray ionization (ESI) MS and the
In all subjects the parents gave informed consent for the collec-
tion of urinary samples.
Urine sample collection
Urine samples were collected from healthy volunteers and sub-
jects affected by various forms of MPS at the Department of Clinical

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34
Hexosamines in urine of MPS patients / G.V. Coppa et al. / Anal. Biochem. 411 (2011) 32–42
Table 1
were injected automatically, using the pressure injection mode, in
which the sample is pressurized for 5 s. Electrophoresis was per-
Characteristics of the healthy subjects and various MPS-affected patients.
formed at 15 kV (about 60 lA) using normal polarity. Peak areas
were recorded and calculated using the Beckman Gold V810 soft-
Subjects
(number)
Female/
male
Mean age (years)
ware system.
Healthy group
83
27/56
3.96 0.38 (0.01–
13)
MPS I Scheie
MPS I Hurler-
Scheie
MPS I Hurler
MPS II mild
MPS II severe
MPS III
6
1
3/3
0/1
9.17 2.41 (1–15)
8.33
RP-HPLC analysis
All separations were performed on a Jasco HPLC 1500 Series
equipped with an integrated degasser, a quaternary pump, Rheo-
dyne injector with a 20-lL loop, a fluorescence detector Model
FP-1520, and software Jasco-Borwin release 1.5. For fluorescence
detection the excitation wavelength was set at 360 nm and emis-
7
4
7
12
7
1
4/3
0/4
0/7
6/6
1/6
1/0
1.70 0.27 (1–3)
4.25 0.48 (3–5)
2.86 0.72 (0.3–6)
5.68 1.17 (3–15)
4.98 1.29 (1–12)
2.5
MPS IV
MPS VI
sion at 425 nm. HPLC column was a 3-lm Gemini C18 110 Å
Data are reported as mean standard deviation. Minimum and maximum values
are illustrated in parentheses.
(4.6 Â 150 mm) from Phenomenex (Torrance, CA, USA) equipped
with a precolumn. According to Racaityte et al. [14], eluent A con-
tained 50 mM sodium acetate buffer, pH 4.1, in water, and eluent B
contained 20% eluent A in methanol. The gradient program was 0–
10 min 3% B isocratic, 10–35 min linear gradient 3–10% B, 35–
45 min linear gradient 10–100% B, 35–50 min 100% B isocratic. In
order to ensure the reproducibility from run to run, the column
was reequilibrated with 3% B for 5 min. The flow rate was
1.0 mL/min.
Sciences, Polytechnic University of the Marche, Presidio Salesi,
Ancona, Italy, and frozen at À20 °C for analytical investigation.
According to Andrade et al. [30] the stability of GAGs allowed urine
samples to be sent to the Department of Biology, University of
Modena & Reggio Emilia, Modena, Italy, for analyses without the
need to freeze samples, as this would not affect results.
Validation of the analytical methods
Sample preparation
Quantitative CE/UV and RP-HPLC-fluorescence detection (Fd)
method validations were established according to the guideline
on the validation of bioanalytical methods by the European Medi-
cines Agency (EMA) published in 2009 (guideline on the validation
of bioanalytical methods, committee for medicinal products for hu-
man use. European medicines agency, London, 2009), including
specificity, linearity, detection (LOD) and quantification (LOQ) lim-
it, precision, accuracy, recovery, and robustness tests. The detec-
tion limits were estimated as the quantity of GlcN and GalN
Stock solutions of GlcN or GalN standard were prepared by dis-
solving an accurately weighed amount of 50 mg in 5 mL (10 mg/
mL) of doubly distilled water. A series of standard solutions were
obtained by dilution of the stock solution in a standard volume
of water (200
The amount of 500
10,000g for 10 min, and 1 mL ethanol was added to 200
supernatant. After 2 h at À20 °C, samples were centrifuged at
10,000g for 10 min and pellets were dissolved in 500 L of freshly
l
L) and lyophilized [17].
L of urine samples was centrifuged at
L of
l
l
l
accurately prepared 4 M HCl. After 120 min at 110 °C, the samples
were lyophilized.
Optimal hydrolysis conditions were obtained by performing
various time courses of chemical treatment under different condi-
tions of HCl molarity, temperature, and times.
Table 2
Effect of acid concentration, temperature, and incubation time on hydrolysis of
standard GAGs, HS, CS, and DS, to produce hexosamines.
CS
DS
HS
GalN%
GalN%
GlcN%
HCl 1 M/60 min/110 °C
HCl 1 M/90 min/110 °C
HCl 1 M/120 min/110 °C
HCl 1 M/150 min/110 °C
0
5
14
14
0
3
12
15
0
3
8
Derivatization of hexosamines with AA
Lyophilized GlcN or GalN standard solutions or treated samples,
in the presence of internal standard ribose, were dissolved in 50
13
lL
HCl 2 M/60 min/110 °C
HCl 2 M/90 min/110 °C
HCl 2 M/120 min/110 °C
HCl 2 M/150 min/110 °C
0
11
17
35
0
14
24
47
0
64
97
98
1% fresh sodium acetate and 50 L of AA (30 mg) and sodium cya-
l
noborohydride (20 mg) dissolved in 1 mL of methanol–acetate–bo-
rate solution (120 mg sodium acetate and 100 mg boric acid in
5 mL methanol) [17]. Tubes were heated at 80 °C for 60 min. After
cooling to room temperature, the samples were analyzed by CE and
HPLC.
HCl 4 M/60 min/110 °C
HCl 4 M/90 min/110 °C
HCl 4 M/120 min/110 °C
HCl 4 M/150 min/110 °C
79
89
103
97
85
93
98
95
101
102
73
101
HCl 6 M/60 min/110 °C
HCl 6 M/90 min/110 °C
HCl 6 M/120 min/110 °C
HCl 6 M/150 min/110 °C
86
96
84
53
72
103
95
93
75
42
12
Capillary electrophoresis
CE was performed on a Beckman HPCE instrument (P/ACE Sys-
tem 5000) equipped with a UV detector set at 214 nm. Separation
and analysis were performed on an uncoated fused-silica capillary
46
HCl 4 M/120 min/110 °C
HCl 4 M/120 min/100 °C
HCl 4 M/120 min/90 °C
HCl 4 M/120 min/80 °C
103
98
63
98
93
57
33
102
96
88
tube (50 lm i.d., 85 cm total length, and 65 cm from the injection
42
48
point to the detector) at 25 °C. The operating buffer was composed
of 150 mM boric acid and 50 mM NaH2PO4 buffered at pH 7.0 with
NaOH solution. Before each run, the capillary tube was washed
with 0.1 M NaOH for 1 min and doubly distilled water for 2 min,
and then conditioned with the operating buffer for 2 min. After
each run, the capillary was rinsed with 0.1 M NaOH for 1 min
and then conditioned with the operating buffer for 2 min. Samples
The absolute amount of both produced hexosamines from standard GAGs was
determined by performing hydrolysis and derivatization procedures and calculating
the response on hexosamine calibration curves. Values are reported as % hexos-
amines generated in comparison with theoretical expected values. Values are mean
of six independent analyses performed both by HPLC and by HPCE having an overall
coefficient of variation generally lower than 15%. In bold are underlined the opti-
mum obtained results.

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Hexosamines in urine of MPS patients / G.V. Coppa et al. / Anal. Biochem. 411 (2011) 32–42
35
producing a signal-to-noise ratio of 3:1 for LOD and 10:1 for LOQ.
The specificity of the two analytical techniques was determined
with migration time (MT) and peak area (PA) of the two Hex peaks
through the precision analysis assay. The calibration curves were
constructed from PA versus concentrations of GlcN/GalN standard.
Linear regression analysis was used to calculate the slope, intercept,
and correlation coefficient (r2) of the calibration curve (from 1 to
Statistics
Micrograms of Hexs were calculated by means of specific
calibration curves (not shown) and reported as microgram per
milligram creatinine (CR) determined according to Coppa et al.
[24] and de Lima et al. [25]. Data are expressed as means SD or
SE. Statistical analysis was performed by analysis of variance (AN-
OVA), Student–Newman–Keuls test, and Mann–Whitney U test as
appropriate by means of SPSS Statistics software Version 17.0 for
Windows. The statistical significance of differences was set at
P < 0.05.
20 lg of Hexs for CE and from 5 to 100 lg for RP-HPLC-Fd). The pre-
cision of the method was assessed by determination of Hexs with
five replicates (n = 5) of five different concentrations of standard
solutions. Intra- and inter-day precision and accuracy of the meth-
ods were estimated by relative standard deviation percentage (CV%)
from the analysis of freshly prepared solutions on 3 separate days.
For recovery of Hexs from urine, GlcN and GalN were added to
samples and the solutions were spiked with standards at three
Results
Validation of the analytical procedures applied to urine samples
concentration levels (5, 10, and 20
lg for CE and 10, 50, and
100 g for RP-HPLC-Fd) and then analyzed in comparison with
l
After a rapid (2-h) urine pretreatment procedure consisting in
precipitating complex heteropolysaccharides along with related
oligomers and fragments [11,17], free Hexs are generated by acidic
hydrolysis. The hydrolysis process involves two acid-catalyzed
steps, the hydrolysis of glycosidic linkage and the N-acetyl (and
the N-sulfo in minor percentages for HS) group (resulting in de-
N-acetylation or de-N-sulfonation), with the formation of the Hexs.
The standard GAGs, HS, CS, and DS, hydrolysis procedure was per-
formed using HCl after evaluating both the effect of the acid
samples with no external standards. The solutions were replicated
three times each, and the Hexs amounts determined were com-
pared to the theoretical amounts. The recovery ratio percentage
(REC%) and their CV% were calculated.
Robustness of analytical procedures was assessed by analysis of
the standards under different analytical conditions, in particular
temperatures, voltage, flow rate, and buffers composition no more
and no less than 10% of the adopted values.
Fig.1. (A) Examples of HPCE/UV electrophoregram and RP-HPLC-Fd chromatogram of GlcN and GalN derivatized with AA and obtained after the hydrolysis procedure of
urinary GAGs from (A) controls, (B) MPS III subjects, and (C) MPS VI patients. Ribose used as internal standard is out of the two panels having greater migration times (see also
Refs. [14–17]).