143
Clinica Chimica Acta, @ Elsevier
Scientific
60 (1975) Publishing
143-145 Company,
Amsterdam
-
Printed
in The Netherlands
CCA 684 1
OLIGOSACCHARIDES STORAGE DISEASES
IN URINE OF PATIENTS
I. RAPID DETECTION
BY THIN-LAYER
WITH GLYCOPROTEIN
CHROMATOGRAPHY
R. HLJMBEL and M. COLLART
State Pediatric Clinic, Luxembourg (Received
September
(Luxembourg)
13, 1974)
Summary Thin-layer chromatography of urinary oligosaccharides was undertaken in patients affected with various glycoprotein storage diseases and showed the presence of compounds characteristic for each disease. Introduction A group of inherited metabolic storage diseases sharing characteristics with mucopolysaccharidosis and sphingolipidosis, has been studied in recent years. It includes mucolipidosis and glycoprotein storage diseases, namely mannosidosis [l], fucosidosis [ 21, and aspartylglucosaminuria [ 31. In these diseases an accumulation of oligosaccharides has been found in tissues [4-71 therefore an excretion of these compounds in the urine can be expected. Norden et al. [8] were able to demonstrate the excretion of a mannose-tetrasaccharide in patients affected with mannosidosis and Palo et al. [9] found glycoasparagine metabolites in addition to aspartylglucosamine in the urine of their patients; identical findings were made by Jenner and Pollit [lo]. We reported on the isolation and full characterization of keratan-sulfate in urine of patients affected with GMl-gangliosidosis Type I and with fucosidosis [ 111. In the present paper, we demonstrate the presence of typical oligosaccharides in the urine of these patients using a simple chromatographic technique. Materials and Method Urine specimens biochemical findings vided by Dr Durand Pollitt (Sheffield), Dr
from 10 patients diagnosed on clinical, pathological and as having a glycoprotein storage disease, were kindly pro(Genova), Dr Maroteaux (Paris), Dr Palo (Helsinky), Dr Toudic (Brest), and Dr van Hoof (Louvain).
144
Thin-layer chromatography was performed on 204 urine samples which are directly applied as a 1.5 cm line on a 20 cm X 20 cm silicagel plate (Merck 5715). 10 pg lactose are used as standard. After thoroughly drying the urine sample (this is an important step for good separation) chromatography is developed overnight in a freshly prepared mixture of n-butanollacetic acid/water (100 : 50 : 50 v/v) as shown by Palo et al. [12]. After drying, the plate is sprayed with a freshly prepared solution of orcinol (0.2 g/100 ml) in sulfuric acid (20 g/100 ml), and heated for 10 minutes at 100°C. The chromatograms are then examined by transparency. Results Control urine chromatograms show the presence of spots with an Rf close to that of lactose or higher (Fig. 1). The pathological specimens are characterized by the presence of unknown spots with a Rf lower than those taken in normal specimens. Fig. 2 shows our results. It appears that each disease is characterized by the presence of one or more additional compounds with a specific Rf. In mannosidosis there is no doubt as to the existence of a compound present in high concentrations which stains redbrown with orcinol, and with a lower Rf than lactose. In aspartylglucosaminuria, the abnormal band described by Palo et al. [12] and identified as glycoasparagines, was also observed in this study. In fucosidosis a brown-yellow band is clearly seen besides other compounds with a lower Rf. Keratan-sulfate remains at the origin. None of the above-mentioned compounds could be detected in urine specimens from patients with mucopolysaccharidosis of different types, mucolipidosis or sulfatidosis.
Fig. 1. Thin-layer chromatogram standard).
of urinary oligosaccharides normal urine specimens (at the right lactose
Fig.
2.
Type
Thin-layer
chromatograms
I; M, mannosidosis;
of
urinary
oligosaccharides
A, aspartylglucosaminurlia;
pathological
F. fucosidosis;
urines.
G,
GMl-gangliosidosis
C. controls.
Conclusion Thin-layer chromatography of urinary oligosaccharides demonstrates the presence of compounds characteristic of each disease and present in excess. They most likely originate from the inner core of the stored glycoproteins. Further analyses are necessary and particularly structural studies to identify them. The purpose of this communication is to indicate a simple and rapid method used for the detection of oligosaccharides in urine and serve as a screening procedure for glycoprotein storage diseases. References 1
B. Kjellman,
2
P. Durand.
I. Gamstorp, C. Borrone.
3
F.A.
Jenner
4
P.A.
Ockerman,
5
F. van
Hoof,
and
R.J.
A.
Brun.
P.A.
G. Della-Cella Pollitt.
J. Pediatr., in H.G.
Biochem. 75
Hers
Ockerman
and
and
B. Palmgren.
Phillippart,
J., 103
(1969)
and
M.
(1967)
Lancet,
J. Pediatr.,
i (1968)
75
(1969)
366
1198
48P
360
F. van
Hoof
(eds),
Lysosomes
and
Storage
Diseases,
Academic
Press,
1973 6
J. Palo
7
L.S.
8
N.E.
and
Wolfe,
H. Savolainen. R.G.
Norden,
Senior
A.
J. Chromatogr.. and
Lundblad,
N.M.K. S.
65
Ng Ying
Svensson,
(1972) Kin,
P.E.
447
J. Biol.
Ockerman
Chem.. and
249 S.
(1974)
Autio.
1828 J. Biol.
Chem.,
248
6210 9 10
J. Palo, R.J.
R.J.
Pollitt
11
R. Humbel,
12
J. Palo
and
Pollitt. and
M. Kathleen,
K.M.
Clin.
Pretty,
Chin
H. Savolainen,
M. Pretty
Biochem.
Acta. Clin.
52
(1974)
Chim.
and
J.. 141
H. Savolainen.
(1974)
141
173 Acta,
36
(1972)
431
Clin.
Chim.
Acta.
47
(1973)
69
(1973)
Clinica Chimica Acta, @ Elsevier
Scientific
60 (1975) Publishing
143-145 Company,
Amsterdam
-
Printed
in The Netherlands
CCA 684 1
OLIGOSACCHARIDES STORAGE DISEASES
IN URINE OF PATIENTS
I. RAPID DETECTION
BY THIN-LAYER
WITH GLYCOPROTEIN
CHROMATOGRAPHY
R. HLJMBEL and M. COLLART
State Pediatric Clinic, Luxembourg (Received
September
(Luxembourg)
13, 1974)
Summary Thin-layer chromatography of urinary oligosaccharides was undertaken in patients affected with various glycoprotein storage diseases and showed the presence of compounds characteristic for each disease. Introduction A group of inherited metabolic storage diseases sharing characteristics with mucopolysaccharidosis and sphingolipidosis, has been studied in recent years. It includes mucolipidosis and glycoprotein storage diseases, namely mannosidosis [l], fucosidosis [ 21, and aspartylglucosaminuria [ 31. In these diseases an accumulation of oligosaccharides has been found in tissues [4-71 therefore an excretion of these compounds in the urine can be expected. Norden et al. [8] were able to demonstrate the excretion of a mannose-tetrasaccharide in patients affected with mannosidosis and Palo et al. [9] found glycoasparagine metabolites in addition to aspartylglucosamine in the urine of their patients; identical findings were made by Jenner and Pollit [lo]. We reported on the isolation and full characterization of keratan-sulfate in urine of patients affected with GMl-gangliosidosis Type I and with fucosidosis [ 111. In the present paper, we demonstrate the presence of typical oligosaccharides in the urine of these patients using a simple chromatographic technique. Materials and Method Urine specimens biochemical findings vided by Dr Durand Pollitt (Sheffield), Dr
from 10 patients diagnosed on clinical, pathological and as having a glycoprotein storage disease, were kindly pro(Genova), Dr Maroteaux (Paris), Dr Palo (Helsinky), Dr Toudic (Brest), and Dr van Hoof (Louvain).
144
Thin-layer chromatography was performed on 204 urine samples which are directly applied as a 1.5 cm line on a 20 cm X 20 cm silicagel plate (Merck 5715). 10 pg lactose are used as standard. After thoroughly drying the urine sample (this is an important step for good separation) chromatography is developed overnight in a freshly prepared mixture of n-butanollacetic acid/water (100 : 50 : 50 v/v) as shown by Palo et al. [12]. After drying, the plate is sprayed with a freshly prepared solution of orcinol (0.2 g/100 ml) in sulfuric acid (20 g/100 ml), and heated for 10 minutes at 100°C. The chromatograms are then examined by transparency. Results Control urine chromatograms show the presence of spots with an Rf close to that of lactose or higher (Fig. 1). The pathological specimens are characterized by the presence of unknown spots with a Rf lower than those taken in normal specimens. Fig. 2 shows our results. It appears that each disease is characterized by the presence of one or more additional compounds with a specific Rf. In mannosidosis there is no doubt as to the existence of a compound present in high concentrations which stains redbrown with orcinol, and with a lower Rf than lactose. In aspartylglucosaminuria, the abnormal band described by Palo et al. [12] and identified as glycoasparagines, was also observed in this study. In fucosidosis a brown-yellow band is clearly seen besides other compounds with a lower Rf. Keratan-sulfate remains at the origin. None of the above-mentioned compounds could be detected in urine specimens from patients with mucopolysaccharidosis of different types, mucolipidosis or sulfatidosis.
Fig. 1. Thin-layer chromatogram standard).
of urinary oligosaccharides normal urine specimens (at the right lactose
Fig.
2.
Type
Thin-layer
chromatograms
I; M, mannosidosis;
of
urinary
oligosaccharides
A, aspartylglucosaminurlia;
pathological
F. fucosidosis;
urines.
G,
GMl-gangliosidosis
C. controls.
Conclusion Thin-layer chromatography of urinary oligosaccharides demonstrates the presence of compounds characteristic of each disease and present in excess. They most likely originate from the inner core of the stored glycoproteins. Further analyses are necessary and particularly structural studies to identify them. The purpose of this communication is to indicate a simple and rapid method used for the detection of oligosaccharides in urine and serve as a screening procedure for glycoprotein storage diseases. References 1
B. Kjellman,
2
P. Durand.
I. Gamstorp, C. Borrone.
3
F.A.
Jenner
4
P.A.
Ockerman,
5
F. van
Hoof,
and
R.J.
A.
Brun.
P.A.
G. Della-Cella Pollitt.
J. Pediatr., in H.G.
Biochem. 75
Hers
Ockerman
and
and
B. Palmgren.
Phillippart,
J., 103
(1969)
and
M.
(1967)
Lancet,
J. Pediatr.,
i (1968)
75
(1969)
366
1198
48P
360
F. van
Hoof
(eds),
Lysosomes
and
Storage
Diseases,
Academic
Press,
1973 6
J. Palo
7
L.S.
8
N.E.
and
Wolfe,
H. Savolainen. R.G.
Norden,
Senior
A.
J. Chromatogr.. and
Lundblad,
N.M.K. S.
65
Ng Ying
Svensson,
(1972) Kin,
P.E.
447
J. Biol.
Ockerman
Chem.. and
249 S.
(1974)
Autio.
1828 J. Biol.
Chem.,
248
6210 9 10
J. Palo, R.J.
R.J.
Pollitt
11
R. Humbel,
12
J. Palo
and
Pollitt. and
M. Kathleen,
K.M.
Clin.
Pretty,
Chin
H. Savolainen,
M. Pretty
Biochem.
Acta. Clin.
52
(1974)
Chim.
and
J.. 141
H. Savolainen.
(1974)
141
173 Acta,
36
(1972)
431
Clin.
Chim.
Acta.
47
(1973)
69
(1973)
