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Urinary sialic acid levels in aspartylglycosaminuria
219
Clinica Chimica Acta, 109 (1981) 219-223 @ Elsevier/North-Holland Biomedical Press
CCA 1616
URINARY
C. PETER
SIALIC ACID LEVELS
J. MAURY
Department of Medical Chemistry Helsinki and Research Department, (Finland) (Received
IN ASPARTYLGLYCOSAMINURIA
May 28th,
and Fourth Rinnekoti
Department Institution
of Medicine, University of for Mentally Retarded, Espoo
1980)
Summary Urinary sialoglycoconjugates were studied in 22 patients with inherited deficiency of l-aspartamido-fl-N-acetylglucosamine amidohydrolase (aspartylglycosaminuria), in eight obligate heterozygotes, and in age- and sex-matched control subjects. Total sialic acid excretion was significantly higher in the patients (38.3 k 17.7 pmol/mmol creatinine, mean + S.D.) than in the matched controls (17.7 k 7.3 pmol/mmol creatinine, p < 0.001). The sialic acid output in the heterozygotes did not differ from that of the controls. Gel filtration studies revealed that the increase in urinary sialic acid in aspartylglycosaminuria is of bound type and confined to the low molecular mass region. A linear positive correlation was found between the output of sialic acid and glycoasparagine in the individual patients (r = 0.77, p < 0.001). The amount of sialylated metabolites excreted in urine did not correlate with the severity of clinical manifestations in aspartyl-glycosaminuria.
Introduction Reduced or absent activity of the glycoprotein degrading lysosomal amidase, l-aspartamido-fl-N-acetylglucosamine amidohydrolase (EC 3.5.1.26) is the basic enzymatic defect in aspartylglycosaminuria (AGU) [l-4]. The incidence of AGU in Finland is estimated to be of the order of 1 in 26 000 [5] and more than 130 cases have hitherto been diagnosed. In fact, AGU is one of the most common metabolic causes of infantile-onset mental retardation in Finland. It is inherited in an autosomal recessive mode [ 51. The disease is, on both morphological and biochemical criteria, a generalized storage disorder. Intracytoplasmic
Correspondence to Dr. P. Maury, Department of Medical Chemistry, University of Helsinki, Siltavuorenpenger 10 A. SF-00170 Helsinki 17. Finland.
220
vacuoles are seen in epithelial and mesenchymal cells [6]. Glycoasparagines accumulate in neural and visceral tissues [7-91 and are excreted in the urine [ 10-141. The main storage compound is N-acetylglucosaminyl-asparagine (2-acetamido-l-N-(4-L-aspartyl)-2-deoxy-~-D-glucopyranosyl~ine, GlcNAcAsn). In order to evaluate the extent of the excretion of sialylated metabolites in AGU, urinary sialic acid levels were measured in 22 patients with AGU and in 8 obligate heterozygotes, as well as in age- and sex-matched control subjects. The results have been correlated to urinary glycoasparagine levels and severity of clinical manifestations. Material and methods Urine samples Urine was collected from 22 patients with AGU and from 8 obligate heterozygotes (parents of AGU patients), as well as from age- and sex-matched control subjects. The samples were stored at -20°C until used. The cooperation of Dr. Aula in arranging AGU-urine samples is gratefully acknowledged. Analytical
methods Sialic acid was assayed essentially as described by Svennerholm [15]. After anion exchange chromatographic purification (Dowex-1, CH,COO-, Fluka AG), sialic acid was measured by the resorcinol method as modified by Miettinen and Takki-Luukkainen [16]. Sialic acid values are expressed as N-acetylneuraminic acid. Protein was measured by a modified Lowry method [17]. Sialyloligosaccharides [ 181 and N-acetylglucosaminyl-asparagine [ 121 were assayed by gas chromatography as described before. Gel filtration was performed on a Sephadex G-25 (Pharmacia) fine column, which was eluted by 10 mmol/l pyridyl acetate buffer. Gas chromatography was carried out on a Perkin-Elmer Model 900 instrument equipped with hydrogen flame ionization detectors. The columns were 2.2% SE-30 and 2.2% OV-101 on Gas Chrom Q (Applied Science Lab.). Nitrogen was used as carrier gas.
Statistical calculations Linear regression analysis of the relationship between sialic acid and glycoasparagine levels was performed. Statistical significances were estimated by Student’s t test. Results
Urinary sialic acid levels The urinary excretion of total sialic acid in 22 AGU patients, 8 obligate heterozygotes and in age- and sex-matched control subjects is presented in Table I. The output of sialic acid was significantly higher in the AGU patients (38.3 + 17.7 pmol sialic acid/mm01 creatinine, mean ?S.D.) than in the controls (17.7 * 7.3 /_fmol/mmol creatinine, p < 0.01). The output was similar in females and males. The urinary sialic acid excretion in the obligate heterozygotes (13.4 f 3.6) did not significantly differ from that of matched controls (14.2 f 4.6). The output of sialic acid/creatinine was higher in the younger subjects (both patients and controls) than in the older ones.
221
TABLE
I
URINARY GOTES
EXCRETION AND
OF
IN MATCHED
GiWUP
SIALIC
ACID
CONTROL
SEX
IN
22
AGU
PATIENTS,
IN
8
OBLIGATE
HETEROZY-
SUBJECTS Urinary
Age
sialic
acid
(yrs) ~mol/mmol
creatinine
mg/24
h
AGLJpatients 1
M
3
55.2
37.5
2
M
6
37.5
39.2
3
M
8
44.9
26.8
4
F
10
79.0
95.9
5
F
12
31.7
64.3
6
.M
15
36.6
28.6
7
M
20
70.7
88.2
8
M
25
13.6
53.7
9
F
27
53.5
41.0
10
F
27
23.2
46.9
11
F
28
64.0
72.6
12
M
30
20.8
94.3
13
M
32
42.3
94.3
14
F
33
34.7
64.4
15
M
34
28.6
110.7
16
M
35
52.7
69.8
17
M
36
23.8
78.8
18
M
36
22.3
65.9
19
F
38
27.9
60.0
20
F
39
17.9
36.8
21
F
40
32.9
72.7
22
M
40
27.7
Mean
+ S.D.
Controls Mean
26.1
forAGUpatients
Mean
t 17.7
25.7
i 11.9
17.7
+
7.3
42.0
f
6.4
13.4
e
3.6
+
4.1
14.2
e
4.6
for heterozygotes
Mean
e S.D.
*p <
0.001
(n =
1.
*
Gel
filtration
acetate
buffer,
on
25
20
15
FRACTION Fig.
k 23.6
8)
37.5
1’0
pyridyl
63.5
heterozygotes(n = 8)
i S.D.
Controls
38.3
(n = 22)
+ S.D.
Obligate
55.5
c 12.0
NUMBER
a Sephadex
pH
5, and
G-25
fractions
column
(53
of 4 ml were
X 2 cm). coIIected.
The
column
Fractions
was were
eluted analyzed
with
10
mmol/l
for sialic
acid.
222
.
I
25
URINARY Fig.
2.
patients
Linear with
50
SIALIC
regression
15
100
ACID “W2dh analysis
of
the
relationship
between
urinary
sialic
acid
and
GlcNAc-Am
in 22
AGU.
Fractionation of urinary sialic acid AGU urine (Case 13, Table I) and the urine of a matched control were subjected to gel filtration on Sephadex G-25 (Fig. 1). The increase in sialic acid in AGU is clearly confined to fractions 15-18, which represent the low molecular mass glycopeptide, glycoasparagine and oligosaccharide region. Quantitative analysis of sialyl-lactose and sialyl-N-acetyllactosamine did not reveal differences between the patient and the control subject. Correlation between sialic acid and GlcNAc-Asn output A linear positive correlation (r = 0.77, p < 0.001) was found between the daily urinary excretion of total sialic acid and GlcNAc-Asn in AGU patients (Fig. 2). Discussion Several factors indicate that the increase in sialic acid in AGU is due to the excretion of sialylated glycoasparagines: (1) High sialic acid levels were accompanied by high GlcNAc-Asn levels; a linear positive correlation was noted between these two variables. (2) The distribution of sialic acid in gel chromatography indicated that the increase in sialic acid in AGU-urine is confined to the glycoasparagine region. (3) The excretion of sialyl-lactose and sialylN-acetyllactosamine was normal. (4) The presence of sialylglycoasparagines in AGU-urine has been described [ 11,191. Assuming that the increased sialic levels noted in this study would entirely be due to the excretion of one major sialylated glycoasparagine, N-acetylneuraminyl-N-acetyl lactosaminyl-asparagine [ 191, it can be calculated that the sialylated derivative could maximally represent lo-15% of the amount excreted as free GlcNAc-Asn. With respect to the catabolic pathways of glycoproteins the origin of the sialylated glycoasparagines is unclear. The possibility that the accumulating end-product GlcNAc-Asn could function as a saccharide acceptor should in this context be taken into account.
223
GlcNAc-Asn is excreted in the urine in a constant fashion from an early stage to an advanced stage of AGU and no direct correlation between the severity of clinical symptoms and the amount of GlcNAc-Asn excreted can be found [20]. As sialic acid excretion linearly correlated to the amount of GlcNAc-Asn excreted in the urine in the individual patients, it follows that the output of sialylated metabolites does not correlate with the severity of the clinical manifestations of the disease either. Increased urinary excretion of sialic acid is also seen in other lysosomal storage diseases, e.g. sialidosis [ 211, GM,-gangliosidosis [ 221 and Salla disease [ 231, as well as in sialuria [24] and various inflammatory and neoplastic conditions [see ref. 251, which limits the diagnostic value of total sialic acid measurements. However, properly interpreted, urinary sialic acid assay has a place in the screening of disorders of glycoconjugate metabolism. Acknowledgements The skillful technical assistance of Mrs. Liisa Kuivalainen is gratefully nowledged. Financial support was provided by the Finska Lakaresallskapet The Rinnekoti Saatiij.
ackand
References 1
Pollitt,
2
Palo.
R.J., J.,
Jenner.
F.A.
and
P..
Arstila,
Riekkinen.
Merskey,
H. (1968)
A.U..
Autio,
Lancet
S. and
ii. 253-255
Kivimlki,
T.
(1972)
Acta
Neuropathol.
(Berlin)
20.217-224 3
Aula.
P., NlntB,
U.,
4
Aula,
P.. Raivio,
K.
5
Autio,
S. (1972)
J. Ment.
6
Haltia.
M.,
J. and
I
Mary,
P. (1979)
Palo,
8
Maury,
P. and
9
Maury,
P. (1980)
10
Jenner,
11
Pollitt,
R.J.
12
Maury,
P. (1979)
13
Lundblad,
F.A.
and
PoIIitt.
Res.
Chem.
10,
Series
Genet.
4, 297-300
625-629
I, l-93
Neuropathol.
(Berlin)
Chim.
31,
243-255
Acta
108,
293-299
in press
(1967)
Biochem.
J. 103,48P-49P
(1974)
Biochem.
J. 141,
Clin.
Med.
P.K..
Acta
Clin.
Res.
1513-1515
Med.,
R.J.
S. (1973)
Pediatr.
Monograph
254,
CIin.
Clin.
J. Lab.
Autio.
S. (1975)
K.M.
Masson.
67,
Defic.
J. (1980)
Pretty,
and
S. (1976)
Autio.
J. Lab.
and
M.-L.
Autio,
J. Biol.
Palo,
A.,
Biochem.
Laipio, and
93,
Norden,
141-146
718-723 N.E.,
Svensson,
S..
&kerman,
P.-A.
and
Palo,
J. (1976)
Eur.
J.
209-214
14
Akasaki,
15
Svennerholm,
16
Miettinen,
17
Hartree.
18
Huttunen,
J.K.
19
Sugahara.
K.,
M..
Sugahara,
K.,
L. (1958) T.A.
E.F.
and
Chem.
I.,
Anal.
48,
T.A.
S.,
P. and
12,
547-554
I.-T.
Biochem.
Miettinen,
Funakoshi,
Aula,
Stand.
Takki-Luukkainen,
(1972) and
Funakoshi,
Acta
(1959)
Yamashina,
Acta
I. (1976)
Chem.
Stand.
FEBS
13,
Lett.
69,
191-194
856-858
422427
(1969)
Anal.
Funakoshi,
I..
Biochem.
Aula,
P. and
29,
441458
Yamashina,
I. (1976)
J. Biochem.
80.
195-
201 20
Aula,
21
Okada,
S.,
(1978)
Clin.
P.. Maury,
P. and
Kate, Chim.
22
Strecker,
G. and
23
Renlund,
M.,
Eur. 24
Acta 25
Maury,
21,
J.,
Raivio,
Miura, Acta
101, Biserte,
K.
S.,
86,
(1980)
J. Inherit.
Yabuuchi.
H.,
Metab.
Nishigaki.
M,,
I. (1971)
M.A.,
Clin.
Lundblad,
A..
Chim. Aula,
Acta
Sot.
in press
Kobata,
33,
A.,
Chiyo,
H. and
Furuyama,
J.-I.
395401
P., Raivio.
K.O.,
Fontaine.
G.
Autio.
S. and
Koskela.
S.-L.
(1979)
245-250 G.,
Strecker.
Sci.
Fenn.
G.,
Spik.
G..
6149 P. (1972)
Dis..
159-167
Montreuil,
Chester.
J. Biochem.
MontreuiI,
T.,
Comment.
Biol.
51.
130
and
Farriaux,
J.-P.
(1968)
Clin.
Chim.
Clinica Chimica Acta, 109 (1981) 219-223 @ Elsevier/North-Holland Biomedical Press
CCA 1616
URINARY
C. PETER
SIALIC ACID LEVELS
J. MAURY
Department of Medical Chemistry Helsinki and Research Department, (Finland) (Received
IN ASPARTYLGLYCOSAMINURIA
May 28th,
and Fourth Rinnekoti
Department Institution
of Medicine, University of for Mentally Retarded, Espoo
1980)
Summary Urinary sialoglycoconjugates were studied in 22 patients with inherited deficiency of l-aspartamido-fl-N-acetylglucosamine amidohydrolase (aspartylglycosaminuria), in eight obligate heterozygotes, and in age- and sex-matched control subjects. Total sialic acid excretion was significantly higher in the patients (38.3 k 17.7 pmol/mmol creatinine, mean + S.D.) than in the matched controls (17.7 k 7.3 pmol/mmol creatinine, p < 0.001). The sialic acid output in the heterozygotes did not differ from that of the controls. Gel filtration studies revealed that the increase in urinary sialic acid in aspartylglycosaminuria is of bound type and confined to the low molecular mass region. A linear positive correlation was found between the output of sialic acid and glycoasparagine in the individual patients (r = 0.77, p < 0.001). The amount of sialylated metabolites excreted in urine did not correlate with the severity of clinical manifestations in aspartyl-glycosaminuria.
Introduction Reduced or absent activity of the glycoprotein degrading lysosomal amidase, l-aspartamido-fl-N-acetylglucosamine amidohydrolase (EC 3.5.1.26) is the basic enzymatic defect in aspartylglycosaminuria (AGU) [l-4]. The incidence of AGU in Finland is estimated to be of the order of 1 in 26 000 [5] and more than 130 cases have hitherto been diagnosed. In fact, AGU is one of the most common metabolic causes of infantile-onset mental retardation in Finland. It is inherited in an autosomal recessive mode [ 51. The disease is, on both morphological and biochemical criteria, a generalized storage disorder. Intracytoplasmic
Correspondence to Dr. P. Maury, Department of Medical Chemistry, University of Helsinki, Siltavuorenpenger 10 A. SF-00170 Helsinki 17. Finland.
220
vacuoles are seen in epithelial and mesenchymal cells [6]. Glycoasparagines accumulate in neural and visceral tissues [7-91 and are excreted in the urine [ 10-141. The main storage compound is N-acetylglucosaminyl-asparagine (2-acetamido-l-N-(4-L-aspartyl)-2-deoxy-~-D-glucopyranosyl~ine, GlcNAcAsn). In order to evaluate the extent of the excretion of sialylated metabolites in AGU, urinary sialic acid levels were measured in 22 patients with AGU and in 8 obligate heterozygotes, as well as in age- and sex-matched control subjects. The results have been correlated to urinary glycoasparagine levels and severity of clinical manifestations. Material and methods Urine samples Urine was collected from 22 patients with AGU and from 8 obligate heterozygotes (parents of AGU patients), as well as from age- and sex-matched control subjects. The samples were stored at -20°C until used. The cooperation of Dr. Aula in arranging AGU-urine samples is gratefully acknowledged. Analytical
methods Sialic acid was assayed essentially as described by Svennerholm [15]. After anion exchange chromatographic purification (Dowex-1, CH,COO-, Fluka AG), sialic acid was measured by the resorcinol method as modified by Miettinen and Takki-Luukkainen [16]. Sialic acid values are expressed as N-acetylneuraminic acid. Protein was measured by a modified Lowry method [17]. Sialyloligosaccharides [ 181 and N-acetylglucosaminyl-asparagine [ 121 were assayed by gas chromatography as described before. Gel filtration was performed on a Sephadex G-25 (Pharmacia) fine column, which was eluted by 10 mmol/l pyridyl acetate buffer. Gas chromatography was carried out on a Perkin-Elmer Model 900 instrument equipped with hydrogen flame ionization detectors. The columns were 2.2% SE-30 and 2.2% OV-101 on Gas Chrom Q (Applied Science Lab.). Nitrogen was used as carrier gas.
Statistical calculations Linear regression analysis of the relationship between sialic acid and glycoasparagine levels was performed. Statistical significances were estimated by Student’s t test. Results
Urinary sialic acid levels The urinary excretion of total sialic acid in 22 AGU patients, 8 obligate heterozygotes and in age- and sex-matched control subjects is presented in Table I. The output of sialic acid was significantly higher in the AGU patients (38.3 + 17.7 pmol sialic acid/mm01 creatinine, mean ?S.D.) than in the controls (17.7 * 7.3 /_fmol/mmol creatinine, p < 0.01). The output was similar in females and males. The urinary sialic acid excretion in the obligate heterozygotes (13.4 f 3.6) did not significantly differ from that of matched controls (14.2 f 4.6). The output of sialic acid/creatinine was higher in the younger subjects (both patients and controls) than in the older ones.
221
TABLE
I
URINARY GOTES
EXCRETION AND
OF
IN MATCHED
GiWUP
SIALIC
ACID
CONTROL
SEX
IN
22
AGU
PATIENTS,
IN
8
OBLIGATE
HETEROZY-
SUBJECTS Urinary
Age
sialic
acid
(yrs) ~mol/mmol
creatinine
mg/24
h
AGLJpatients 1
M
3
55.2
37.5
2
M
6
37.5
39.2
3
M
8
44.9
26.8
4
F
10
79.0
95.9
5
F
12
31.7
64.3
6
.M
15
36.6
28.6
7
M
20
70.7
88.2
8
M
25
13.6
53.7
9
F
27
53.5
41.0
10
F
27
23.2
46.9
11
F
28
64.0
72.6
12
M
30
20.8
94.3
13
M
32
42.3
94.3
14
F
33
34.7
64.4
15
M
34
28.6
110.7
16
M
35
52.7
69.8
17
M
36
23.8
78.8
18
M
36
22.3
65.9
19
F
38
27.9
60.0
20
F
39
17.9
36.8
21
F
40
32.9
72.7
22
M
40
27.7
Mean
+ S.D.
Controls Mean
26.1
forAGUpatients
Mean
t 17.7
25.7
i 11.9
17.7
+
7.3
42.0
f
6.4
13.4
e
3.6
+
4.1
14.2
e
4.6
for heterozygotes
Mean
e S.D.
*p <
0.001
(n =
1.
*
Gel
filtration
acetate
buffer,
on
25
20
15
FRACTION Fig.
k 23.6
8)
37.5
1’0
pyridyl
63.5
heterozygotes(n = 8)
i S.D.
Controls
38.3
(n = 22)
+ S.D.
Obligate
55.5
c 12.0
NUMBER
a Sephadex
pH
5, and
G-25
fractions
column
(53
of 4 ml were
X 2 cm). coIIected.
The
column
Fractions
was were
eluted analyzed
with
10
mmol/l
for sialic
acid.
222
.
I
25
URINARY Fig.
2.
patients
Linear with
50
SIALIC
regression
15
100
ACID “W2dh analysis
of
the
relationship
between
urinary
sialic
acid
and
GlcNAc-Am
in 22
AGU.
Fractionation of urinary sialic acid AGU urine (Case 13, Table I) and the urine of a matched control were subjected to gel filtration on Sephadex G-25 (Fig. 1). The increase in sialic acid in AGU is clearly confined to fractions 15-18, which represent the low molecular mass glycopeptide, glycoasparagine and oligosaccharide region. Quantitative analysis of sialyl-lactose and sialyl-N-acetyllactosamine did not reveal differences between the patient and the control subject. Correlation between sialic acid and GlcNAc-Asn output A linear positive correlation (r = 0.77, p < 0.001) was found between the daily urinary excretion of total sialic acid and GlcNAc-Asn in AGU patients (Fig. 2). Discussion Several factors indicate that the increase in sialic acid in AGU is due to the excretion of sialylated glycoasparagines: (1) High sialic acid levels were accompanied by high GlcNAc-Asn levels; a linear positive correlation was noted between these two variables. (2) The distribution of sialic acid in gel chromatography indicated that the increase in sialic acid in AGU-urine is confined to the glycoasparagine region. (3) The excretion of sialyl-lactose and sialylN-acetyllactosamine was normal. (4) The presence of sialylglycoasparagines in AGU-urine has been described [ 11,191. Assuming that the increased sialic levels noted in this study would entirely be due to the excretion of one major sialylated glycoasparagine, N-acetylneuraminyl-N-acetyl lactosaminyl-asparagine [ 191, it can be calculated that the sialylated derivative could maximally represent lo-15% of the amount excreted as free GlcNAc-Asn. With respect to the catabolic pathways of glycoproteins the origin of the sialylated glycoasparagines is unclear. The possibility that the accumulating end-product GlcNAc-Asn could function as a saccharide acceptor should in this context be taken into account.
223
GlcNAc-Asn is excreted in the urine in a constant fashion from an early stage to an advanced stage of AGU and no direct correlation between the severity of clinical symptoms and the amount of GlcNAc-Asn excreted can be found [20]. As sialic acid excretion linearly correlated to the amount of GlcNAc-Asn excreted in the urine in the individual patients, it follows that the output of sialylated metabolites does not correlate with the severity of the clinical manifestations of the disease either. Increased urinary excretion of sialic acid is also seen in other lysosomal storage diseases, e.g. sialidosis [ 211, GM,-gangliosidosis [ 221 and Salla disease [ 231, as well as in sialuria [24] and various inflammatory and neoplastic conditions [see ref. 251, which limits the diagnostic value of total sialic acid measurements. However, properly interpreted, urinary sialic acid assay has a place in the screening of disorders of glycoconjugate metabolism. Acknowledgements The skillful technical assistance of Mrs. Liisa Kuivalainen is gratefully nowledged. Financial support was provided by the Finska Lakaresallskapet The Rinnekoti Saatiij.
ackand
References 1
Pollitt,
2
Palo.
R.J., J.,
Jenner.
F.A.
and
P..
Arstila,
Riekkinen.
Merskey,
H. (1968)
A.U..
Autio,
Lancet
S. and
ii. 253-255
Kivimlki,
T.
(1972)
Acta
Neuropathol.
(Berlin)
20.217-224 3
Aula.
P., NlntB,
U.,
4
Aula,
P.. Raivio,
K.
5
Autio,
S. (1972)
J. Ment.
6
Haltia.
M.,
J. and
I
Mary,
P. (1979)
Palo,
8
Maury,
P. and
9
Maury,
P. (1980)
10
Jenner,
11
Pollitt,
R.J.
12
Maury,
P. (1979)
13
Lundblad,
F.A.
and
PoIIitt.
Res.
Chem.
10,
Series
Genet.
4, 297-300
625-629
I, l-93
Neuropathol.
(Berlin)
Chim.
31,
243-255
Acta
108,
293-299
in press
(1967)
Biochem.
J. 103,48P-49P
(1974)
Biochem.
J. 141,
Clin.
Med.
P.K..
Acta
Clin.
Res.
1513-1515
Med.,
R.J.
S. (1973)
Pediatr.
Monograph
254,
CIin.
Clin.
J. Lab.
Autio.
S. (1975)
K.M.
Masson.
67,
Defic.
J. (1980)
Pretty,
and
S. (1976)
Autio.
J. Lab.
and
M.-L.
Autio,
J. Biol.
Palo,
A.,
Biochem.
Laipio, and
93,
Norden,
141-146
718-723 N.E.,
Svensson,
S..
&kerman,
P.-A.
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