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Urinary excretion of sialic acid-containing saccharides in systemic lupus erythematosus.

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1137
URINARY EXCRETION OF
SIALIC ACID-CONTAINING SACCHARIDES IN
SYSTEMIC LUPUS ERYTHEMATOSUS
C. PETER J. MAURY, CLARA SJOBLOM, and OTTO WEGELIUS
Urinary sialic acid-containing trisaccharides, total sialic acid, and serum sialic acid were studied in 17
patients with systemic lupus erythematosus (SLE) and
in 15 healthy controls. The urinary excretion of sialyllactose, measured by a gas chromatographic method,
was significantly greater in patients with SLE (37.4 f
21.4 mg/24 hours, SD) than in the control subjects (13.7
f 3.8 mg/24 hours, P < 0.001). The mean excretion of
sialyl-N-acetyllactosamine (16.6 & 8.5 mg/24 hours)
and total sialic acid (82.5 f 29.4 mg/24 hours) was also
greater in the SLE group than in the controls (8.7 f 2.8
and 58.0 f 16.0 mg/24 hours, respectively; P < 0.01).
Serum levels of sialic acid were correspondingly higher
in the SLE patients (84.4 f 20.4 mg/100 ml) than in the
controls (63.7 f 6.5 mg/100 ml, P c 0.001). Urinary excretion of sialyl-lactose correlated positively with clinical disease activity (Pc 0.001) and with anti-DNA antibody levels (P < 0.05). On the average, patients with
moderate or severe disease excreted three times more
sialyl-lactose than did those with mild or inactive disease. Our results suggest that the excretion of sialyloligosaccharides reflects disease activity in SLE.
Low molecular weight sialic acid-containing saccharides, structurally related to milk ohgosaccharides
and to tissue glycoproteins and glycolipids, are norexcreted in human urine (1-3)* It is generally
thought that these substances are metabolites Of tissue
-~
From the Fourth Department of Medicine and the Department of Medical Chemistry, University of Helsinki, Finland.
C. Peter J. Maury, MD: Assistant Professor of Medicine;
Clara Sjbblom, MB; Otto Wegelius, MD: Professor of Medicine.
Address reprint requests to Dr, p, Maury, Fourth Departmerit of Medicine, Unioninkatu 38, SF-00170 Helsinki 17, Finland.
Submitted for publication November 3, 1980; accepted in revised form February 4, 1981.
Arthritis and Rheumatism, Vol. 24, No. 9 (September 1981)
glycoconjugates, but their exact origin is unknown. Results of previous studies suggest that they have an endogenous origin (2,4) and that they are involved in the
general response of the organism to tissue injury and inflammation (3).
Recently, the urinary output of two sialyl-oligosaccharides was shown to be markedly increased in active rheumatoid arthritis (5,6). We report here the urinary excretion pattern of sialic acid-containing
trisaccharides, as well as the total serum and urinary
levels of sialic acid, in patients with systemic lupus erythematosus (SLE).
PATIENTS AND METHODS
We studied 17 women with SLE, all of whom fulfilled
the American Rheumatism Association criteria (7). Table 1
shows the clinical features of the patients. All had normal
serum creatinine levels. Patients 1-10 had mild disease, some
having only the serologic markers for SLE, while patients I116 had moderate and patient 17 severe disease. The clinical
grading of the disease activity was based on the seventy of
symptoms, organ manifestations, and the findings in routine
laboratory tests. The data were obtained from the case records. The patients were not selected because of particular organ system involvement.
At the time of serum and urine sampling, some patients were receiving drugs in addition to those shown in
Table 1. Patients 1 and 3 were receiving clonidine; patients 2,
3, 11, and 15 furosemide; patient 1 hydrochlorothiazide; patient 2 spironolactone, metoprolol, and pratsozine; patient 6
phenobarbital; and patient 11 thyroxine,
Fifteen healthy women served as controls.
The method for measuring urinary sialyl-lactose and
sialyl-N-acetyllactosamine has been described elsewhere (2,4).
Briefly, the urinary samples (usually 1-2 ml) were diluted and
passed through a Column system consisting of a Dowex-50
(H') mounted on a Dowex-1 (CH,COO-) column (0.9 X 3.0
MAURY ET AL
1138
Table 1. Clinical features of 17 women with SLE
-~
__
Duration
of disease
(years)
Patient
no. (age)
Mild disease
l(42)
2 (33)
3 (46)
4 (45)
5 (36)
7 (47)
8 (39)
9 (36)
10 (22)
Moderate disease
I 1 (42)
I2 (34)
13 (24)
14 (59)
15 (27)
16 (36)
Severe disease
17 (27)
~~
c3
C4
Anti-DNA
antibody
mg/litert
0.09
0.06
0.12
0.13
0.27
0.17
0.39
0.3 I
0.14
0.35
I1
2
3
ND
42
4
3
3
3
3
-~
~
Urinary
protein
gm/liter$
Drugs
34
55
40
20
70
40
22
30
206
213
29 I
2 15
240
319
279
234
350
78
0.46
0.78
0.77
0.74
0.60
I .08
0.88
I .07
0.63
0.84
2.7
2.5
3.4
5.9
8.9
4.2
I94
262
226
204
276
1,091
0.27
0.50
0.66
0.98
0.60
0.66
0.04
0.04
0.07
0.16
0.23
0.15
30
15
55
14
I2
I .7
0.5
2.9
P
P, A
-
15
64
31
34
45
38
82
9
-
P
P. c
p, D
3
95
3.3
555
0.32
0.06
I60
-
10
7
I
7
4
-~
-
3.8
6.0
4.7
5.7
4.3
4.6
11.4
4. I
9.0
2.5
I2
I
2
9
4
0.3
2
7
3
6
6(19)
Serum complement
gni/liter*
B-leukoB-plaieESR
cytes
lets xlO”/
mm/hour
X IOv/liter
liter
~-~
9
19
P
5.2
P, A, I
.
~
* Normal values: C3 0.55-1.20 gm/liter and C4 0.20-0.50 gm/liter.
t Normal values < 5 mg/liter. N D
= not determined.
+ Ail patients had normal serum creatinine levels. - = Albustix negative.
5 Antiinflammatory and analgesic drugs: P = prednisone: I
poxyphene.
=
indomethacin; A
cm). After being washed with water, the acidic substances
were eluted from the Dowex-1 column with 1M pyridyl acetate buffer. pH 4.4. The sialyl-linkages were then split by a
mild acid hydrolysis (0.025M H,SO,, 80°C I hour). The liberated neutral saccharides were purified by anion exchange
chromatography. trimethylsilylated, and then analyzed by
gas-liquid chromatography (Perkin-Elmer Model 900 gas
chromatograph equipped with hydrogen flame ionization detectors). Columns were 2.2% SE-30 on Gas Chrom Q (Applied
Science Laboratories). Melibiitol (prepared from melibiose by
borohydride reduction) was used as an internal standard. This
method permits calculation of the total amount of sialyl-lactose and sialyl-N-acetyllactosamine. Values are expressed as
monosialyl-lactose and monosialyl-N-acetyllactosamine.
Serum sialic acid was measured by the resorcinol
=
acetylsalicylic acid; C
=
cyclophosphamide; D
.
~
Urine
Number
-- .-
Control
SLE
* Mean f SD.
t P 0.01.
4 P 0.001.
*
*
15
17
. ..-
- .
Sialic
acid
58.0 f 16.0
82.5 f 29.41
dextrapro-
method (8) as modified by Miettinen and Takki-Luukkainen
(9). Urinary sialic acid was determined after ion-exchange
purification in the same way (10). Serum and 24-hour urine
samples were stored at -20°C until assayed.
Serum concentrations of C3 and C4 were measured by
nephelometry and the use of specific antisera (Behringwerke
AG). Anti-dsDNA antibody levels were determined by the
Farr technique. Levels of circulating DNA-immune complexes were measured by serum binding of radiolabeled
dsDNA after DNase digestion (12).
Standard deviation was used to express dispersion.
The significance of the differences between mean values was
tested by use of the Student’s t-test, and linear regression analysis by the least square method was used to study the relationship between two variables.
Table 2. Urinary excretion of total sialic acid, sialyl-lactose, and sialyl-N-acetyllactosarnine. and the
serum concentration of total sialic acid in patients with SLE and control subjects*
.
=
S i aI y I lactose
mg/24 hours
.~
13.7 f 3.8
37.4 f 21.4+
SialylN-acetyllactosamine
8.7
16.6
* 2.8
* 8.5t
-
Serum
sialic acid
mg/100 ml
63.7 f 6.5
84.4 r 20.4$
-
SIALIC ACID-CONTAINING SACCHARIDES IN SLE
1139
mine was also significantly higher in the patient group
(P < 0.01). Differences between the patient and control
groups in the mean serum and urinary content of total
sialic acid were also significant (Table 2). Excretion of
sialyl-lactose was greatest in patient 17 (Figure 1) who
had very active disease (Table 1) with hypocomplementemia, and raised levels of anti-DNA antibodies and circulating DNA-immune complexes.
Mean age and the mean duration of disease were
similar between patients with moderate or severe disease and those with mild or inactive disease (Table 3).
Urinary content of sialyl-lactose and sialyl-N-acetyllactosamine was significantly higher in patients with moderate or severe disease than in those with mild disease
(P < 0.001 and P < 0.05, respectively). Patients with
moderate or severe disease had lower complement and
leukocyte levels, higher concentrations of anti-DNA
antibody, and a more accelerated erythrocyte sedimentation rate (ESR) than those with mild disease, although
the differences were not statistically significant.
Linear regression analysis showed that the urinary excretion of sialyl-lactose correlated positively
with disease activity (P < 0.001) (Figure 2), as well as
with anti-DNA antibody levels (P < 0.05) (Figure 3).
Excretion of sialyl-N-acetyllactosamine and sialyl-lactose also correlated positively (Figure 4).
8C
7c
60
50
40
30
20
Con t ro Is
SLE
Figure 1. Urinary excretion of sialyl-lactose in 17 women with SLE
and in 15 female controls.
RESULTS
Mean urinary output of sialyl-lactose was significantly higher (P< 0.001) in the patients than in the controls (Table 2). Mean output of sialyl-N-acetyllactosa-
DISCUSSION
In SLE several organ systems are involved, clinical manifestations vary, and alternating relapses and remissions is the natural course. These characteristics
have prompted the search for laboratory parameters
that would serve as markers of the clinical activity of
Table 3. Urinary sialyl-lactose and sialyl-N-acetyllactosamine;serum levels of C3, C4, anti-DNA antibodies, and leukocytes; and the ESR in
patients with inactive/mild or moderate/severe SLE*
Urine
SLE group
(no.)
Moderatehevere
(7)
*0
Mean f SD.
t Not significant.
P < 0.001.
P < 0.05.
1No. = 9.
Age,
Yr
Serum complement,
Duration
SialylSialyl-Nof disease,
lactose,
acetylyr
mg/24 hours lactosamine
* 4.9
gm/liter
C3
c4
0.79 f 0.20
0.20 f 0.12
Anti-DNA
antibodies,
mg/liter
ESR,
mm/hour
36.5 f 9.7
4.6 f 3.8
35.6 f 12.lt
6.7 f 4.7t 59.2 f 15.0$ 21.7 f 10.1s 0.57 f 0.24t 0.1 1 f 0.07t 42.1 f 54.4t 55.6
22.2 f 6.3
13.0
8.2 f 13.07 33.9 f 18.3
* 25.2t
Leukocyte
count,
x 10g/liter
5.6 f 2.7
4.4 f 2.3t
MAURY ET AL
1140
4
3
t
e
2
2
l-
0
a
W
m
a
w
2
'
a a/
1
/
D
,'
/
a-
a
/
/
a
a
1
20
40
60
80
10
URINARY SIALYL-LACTOSE mg124h
URINARY SIALYL-LACTOSE mg/24h
Figure 2. Linear regression analysis of the relationship between urinary sialyl-lactose and clinical disease activity (arbitrary units 0-4) in
17 patients with SLE. y = 0 . 0 3 ~+ 0.34; r = 0.83, P < 0.001.
Figure 4. Linear regression analysis of the relationship between urinary sialyl-lactose and sialyl-N-acetyllactosamine in 17 patients with
SLE. .v = 0 . 2 3 ~+ 7.85; r = 0.59, P < 0.05.
SLE. Measurements of circulating immune complexes
(12- 15), complement components (1 6- 18), and antiDNA antibodies (19-20) have been reported to be useful as such markers, but conflicting results have also
been published (2 1,22). Moreover, some common laboratory measures, such as the ESR (23) and C-reactive
protein (24), may correlate poorly with disease activity.
In the continued search for a reliable indicator of
disease activity in SLE, we studied another type of compound, urinary low molecular weight sialylated saccharides. The output of sialyl-lactose and sialyl-N-acetyllactosamine was significantly higher in patients with
SLE than in sex-matched controls. Linear regression
analysis revealed a close positive relationship between
the excretion of sialyl-lactose and clinical disease activity, as well as a weaker, but still significant, correlation
between sialyl-lactose excretion and anti-DNA antibody levels. Sialyl-lactose excretion did not correlate
significantly with serum levels of C3 and C4. Urinary
content of sialyl-lactose was significantly higher in the
patients with moderate or severe disease than in those
with mild or inactive disease. The other laboratory results followed the expected pattern: patients with moderate or severe disease had lower complement and leukocyte levels, higher anti-DNA levels, and a more
accelerated ESR than patients with mild or inactive disease, observations that corroborate the clinical grading.
However, between these two patient groups, only the
difference in sialyl-oligosaccharideexcretion was significant. This might be due to the small number of patients
studied or possibly to the fact that of the laboratory
variables tested, changes in sialyl-oligosaccharide levels
best reflected disease activity.
The increase in urinary output of sialyl-oligosaccharides was accompanied by increased excretion of
total sialic acid and by raised levels of serum sialic acid.
Changes in total sialic acid levels were significant, but
this is more difficult to interpret, since total sialic acid
200
-
'
F
9
150
t
0
0
m
Iz
a
a
100
z
?
I-
z
a
50
(
URINARY SIALYL-LACTOSE m g / 2 4 h
Figure 3. Linear regression analysis of relationship between sialyllactose and anti-DNA antibodies in 17 patients with SLE. y = 1 . 0 5 ~
17.3; r = 0.57, P < 0.05.
-
SIALIC ACID-CONTAINING SACCHARIDES IN SLE
represents the sum of a heterogenous group of sialylated
compounds.
Increased excretion of sialic acid-containing trisaccharides is not a phenomenon specific for SLE but
seems to be a general response of the organism to tissue
injury and inflammation (3,5,6,25).In fact, our results
are consistent with the view that sialyl-lactose and sialyl-N-acetyllactosamine might behave like acute phase
reactants. Changes in the urinary content of these trisaccharides are characteristically rapid; in acute myocardial infarction increases in urinary sialyl-lactose and
sialyl-N-lactosamine precede changes in serum glycoproteins and ESR (25).
ACKNOWLEDGMENT
The skillful technical assistance of Mrs. L. Kuivalainen is gratefully acknowledged.
REFERENCES
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1141
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