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Journal of Clinical Microbiology, February 2001, p. 798-800, Vol. 39, No. 2
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.2.798-800.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Relationship between Susceptibility to Hemolytic-Uremic Syndrome
and Levels of Globotriaosylceramide in Human Sera
Shinobu
Watarai,1
Kenji
Yokota,2
Tana,1
Takumi
Kishimoto,3
Tomohosa
Kanadani,4
Kazuhisa
Taketa,5 and
Keiji
Oguma2,*
Laboratory of Veterinary Immunology,
Department of Veterinary Science, College of Agriculture, Osaka
Prefecture University, Sakai, Osaka,1
Department of Bacteriology, Okayama University Medical
School,2 Okayama Rosai
Hospital,3 and Okayama National
Hospital,4 Okayama, and Ibara City
Hospital, Ibara,5 Japan
Received 19 May 2000/Returned for modification 24 August
2000/Accepted 9 November 2000
 |
ABSTRACT |
The relationship between differential susceptibility to
hemolytic-uremic syndrome (HUS) and levels of globotriaosylceramide (Gb3) in serum was studied in patients infected with
verotoxin-producing Escherichia coli (VTEC). The serum Gb3
levels in patients with HUS were lower than these in diarrheal patients
without subsequent HUS or in patients without clinical symptoms,
indicating that individuals with a lower content of serum Gb3 show a
higher incidence of HUS following VTEC infection.
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TEXT |
Verotoxin (VT)-producing
Escherichia coli (VTEC) produces either VT1 or VT2 or both
(8). Epidemiological data show a strong correlation
between these VTs and the development of hemolytic-uremic syndrome
(HUS) (7, 14). However, it is well recognized that not all
patients who have VTEC-associated enterocolitis develop HUS
(2), and the nature of the underlying host susceptibility is not understood. It has been reported that a nonimmunoglobulin fraction of human serum (lipoproteins) shows VT-neutralizing activity (1), and in human serum, neutral glycosphingolipids
(GSLs), including globotriaosylceramide (Gb3), which is recognized as the functional receptor for VTs (3, 10-12), are closely
associated with serum lipoproteins (4). These findings
raise the possibility that the heterogeneity of Gb3 content in the
serum might be related to susceptibility to VT, leading to HUS.
Therefore, we compared levels of the neutral GSLs glucosylceramide
(GlcCer), lactosylceramide (LacCer), Gb3, and globotetraosylceramide
(Gb4) in sera of patients with HUS, with appropriate controls.
Serum samples.
Serum samples were obtained from Okayama
National Hospital (Okayama, Japan), Okayama Rosai Hospital (Okayama,
Japan), and Ibara City Hospital (Ibara, Japan). Blood was collected
from 12 HUS patients in the acute phase (group A), 11 patients who had VTEC-associated diarrhea without development of HUS (group B), and 12 VTEC-infected patients who had no obvious gastrointestinal symptoms
although they ate the same type of food as the other patients and
showed relatively high serum antibody titers against the organism
(group C). Blood samples were allowed to clot at 4°C, and following
centrifugation (1,600 × g, 5 min), the sera were
collected and stored at 
20°C. Serum samples obtained were used for
the extraction of neutral GSLs within 1 week. Six serum samples were
also obtained from HUS patients in group A at the convalescent phase
(group D) as described above. Clinical data for the patients are
summarized in Table 1.
Extraction, purification, and quantitative analysis of neutral GSLs
from serum samples.
Each serum sample (1.5 to 3.0 ml) was placed
in a flask, and ethanol was added to a concentration of 70%. The
mixture was stirred vigorously at room temperature for 1 h and
filtered through filter paper. The insoluble portion was reextracted
sequentially with 10 volumes each of 70% ethanol, chloroform-methanol
(2:1 [vol/vol], 1:1 [vol/vol], and 1:2 [vol/vol], and
chloroform-methanol-water (30:60:8 [vol/vol/vol]) at 50°C for
1 h and filtered. The filtrates (ethanol, chloroform-methanol, and
chloroform-methanol-water extracts) were combined and dried by rotary
evaporation. The dried lipid extracts were washed with acetone followed
by diethyl ether to remove neutral lipids and glycerophospholipids.
Crude sphingolipids, which were insoluble in acetone followed by
diethyl ether, were dissolved in a minimal amount of
chloroform-methanol (1:1, vol/vol) and then incubated at room
temperature for 3 h to cleave the ester-containing lipids after
adjusting the pH of the solution to 12 with 1 N sodium methylate. The
solution was neutralized with 1 N acetic acid in methanol and dialyzed
against distilled water. The dialysate was concentrated by evaporation
to dryness, and the residue was dissolved in a minimal amount of
chloroform-methanol-water (30:60:8, vol/vol/vol) and applied to a
DEAE-Sphadex A-25 column (acetate form) by the method of Ledeen et al.
(9) to separate neutral GSLs and gangliosides. The neutral
GSLs were eluted with chloroform-methanol-water (30:60:8, vol/vol/vol)
and evaporated to dryness in vacuo.
The amount of lipid-bound hexose in the neutral GSL fraction was
determined by the orcinol method (
6). A neutral GSL
solution
containing 50 µl of serum extract was applied on
high-performance
thin-layer, chromatography (HPTLC) plate and developed
with chloroform-methanol-water
(65:35:8, vol/vol/vol). Neutral GSLs
were visualized by spraying
the plate with 0.2% orcinol in 2 N
H
2SO
4, followed by heating
in an oven at
120°C for 5 to 10 min. The spots were scanned for
quantitation with a
dual-wavelength TLC densitometer (CS-900;
Shimadzu, Kyoto, Japan) at a
wavelength of 520 nm. The amounts
of individual neutral GSLs were
calculated from the amount of
lipid-bound hexose in the neutral GSL
fraction on the basis of
the peak area ratios obtained from
densitometric scanning of the
HPTLC
plate.
Student's
t test was performed for statistical evaluation.
Results are expressed as the arithmetic mean with the standard
error of
the
mean.
Figure
1 shows an HPTLC profile of the
neutral GSLs from sera of patients 1, 13, and 24 from groups A, B, and
C, respectively.
The neutral GSLs in the sera from patients 13 (group
B) and 24
(group C) were composed of GlcCer, LacCer, Gb3, and Gb4. In
patient
1 of group A, GlcCer, LacCer, and Gb3 were shown to be the
major
constituents of the neutral GSLs in the serum. Visually, however,
Gb3 of patient 1 was a minor component compared with that in patients
13 and 24.
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FIG. 1.
TLC of neutral GSLs in sera from patients infected with
E. coli 0-157:H7. Lane 1, standard neutral GSLs GlcCer,
LacCer, Gb3, and Gb4; lane 2, neutral GSLs from serum of patient 1 (group A); lane 3, neutral GSLs from serum of patient 13 (group B);
lane 4, neutral GSLs from serum of patient 24 (group C). The bands
marked with arrows were stained brown with orcinol spray.
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In order to clarify the relationship between susceptibility to HUS and
the Gb3 content in the serum, the neutral GSL components
in the sera
from the patients in groups A, B, and C were quantitatively
analyzed. The amounts of neutral GSL components in the sera from
each
group are shown in Table
2.
The concentrations of GlcCer, LacCer, Gb3, and Gb4 in group A were
distinct from those in the other groups. The content of
GlcCer in group
A was suggestively lower than that found in group
B (
P < 0.071) and significantly lower than that in group C (
P < 0.002). The LacCer content was lower in group A than in group
B
(
P < 0.0086) or group C (
P < 0.0003).
Similarly, the amount
of Gb3 in group A was also less than that in
group C (
P < 0.0003)
and suggestively low relative to
that in group B (
P < 0.068).
Gb3 is synthesized from
LacCer. Thus, the low level of Gb3 in
group A can reflect a lower
LacCer content. Moreover, the content
of Gb4 in group A was lower than
that found in group B (
P < 0.0076)
or group C
(
P < 0.0002). This is consistent with the decreased
level of Gb3, which is the precursor of Gb4. The total amount
of
lipid-bound hexose was significantly lower in group A than
in either
group B (
P < 0.0022) or group C (
P < 0.0001). Neutral
GSL components in the serum samples from group D
were also analyzed
and compared with those in group A. The amounts of
GlcCer and
LacCer in group D were similar to those in group A. The
level
of Gb3 in group D was slightly lower than that in group A,
whereas
the Gb4 content was higher in group D than in group A
(
P < 0.007).
Gb4 is synthesized from Gb3 by the enzyme
-
N-acetylgalactosaminyl
transferase (
13).
Thus, the differences in serum Gb4 levels
between group A and group D
might be explained by differences
in expression of this enzyme between
the HUS patients in the acute
phase and those in the convalescent
phase. Higher
N-acetylgalactosaminyl
transferase activities
in group D may relate to the increased
Gb4 content. The total amount of
lipid-bound hexose was slightly
higher in group D than in group
A.
In this study, the compositions of neutral GSLs in groups A, B, and C
were quantitatively different. The most important point
is that the
level of Gb3 in the sera was significantly lower in
group A than in
group C (
P < 0.0003) and was suggestively low
relative
to that in group B (
P < 0.068). This suggests that
there
may be an association between the heterogeneity of Gb3 contents
in the sera and outcome of VT-associated HUS. During VTEC infection,
Gb3 in the serum should bind to circulating VTs and may reduce
the
amount of VTs binding to the target cells. Therefore, patients
with
lower serum Gb3 levels would show a higher incidence of HUS
following
VTEC infection. However, it is also possible that these
differences in
neutral GSLs in serum could reflect the change
of neutral GSL
composition in the serum associated with HUS. If
this was so, the
decreased neutral GSLs in group A would be expected
to increase on
recovery from the illness. The experimental results
indicated that
although the Gb4 level was higher in group D than
in group A, the
contents of GlcCer, LacCer, and Gb3 did not vary
significantly between
group A and group D, suggesting that the
lower neutral GSL contents are
not a consequence of VTEC infection.
Thus, quantitative differences in
the neutral GSLs of groups A,
B, and C may be due to innate differences
in the metabolism of
GSLs (
4,
5).
It is suggested that individuals with a lower content of serum Gb3 may
have a higher incidence of HUS following VTEC
infection.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Bacteriology, Okayama University Medical School, Shikata-cho, Okayama 700-8558, Japan. Phone: 81-86-235-7157. Fax: 81-86-235-7162. E-mail: kuma{at}med.okayama-u.ac.jp.
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REFERENCES |
| 1.
|
Bitzan, M.,
M. Klemt,
R. Steffens, and D. L. Müller-Wiefel.
1993.
Differences in verotoxin immunoglobulins and sera from healthy controls.
Infection
21:140-145[CrossRef][Medline].
|
| 2.
|
Cleary, T. G.
1988.
Cytotoxin-producing Escherichia coli and the hemolytic uremic syndrome.
Pediatr. Clin. N. Am.
35:485-501[Medline].
|
| 3.
|
Cohen, A.,
G. E. Hannigan,
B. R. G. Williams, and C. A. Linfwood.
1987.
Roles of globotriosyl- and galabiosylceramide in verotoxin binding and high affinity interferon receptor.
J. Biol. Chem.
262:17088-17091[Abstract/Free Full Text].
|
| 4.
|
Dawson, G.,
A. W. Kruski, and A. M. Scanu.
1976.
Distribution of glycosphingolipids in the serum lipoproteins of normal human subjects and patients with hypo- and hyperlipidemias.
J. Lipid Res.
17:125-131[Abstract].
|
| 5.
|
Dumontet, C.,
A. Rebbaa, and J. Portoukalian.
1992.
Kinetics and organ distribution of [14C]-sialic acid-GM3 and [3H]-sphingosine-GM1 after intravenous injection in rats.
Biochem. Biophys. Res. Commun.
189:1410-1416[CrossRef][Medline].
|
| 6.
|
Francois, C.,
R. D. Marshall, and A. Neuberger.
1962.
Carbohydrates in protein. 4. The determination of mannose in hen's-egg albumin by radioisotope dilution.
Biochem. J.
83:335-341.
|
| 7.
|
Karmali, M. A.,
M. Petric,
C. Lim,
P. C. Fleming,
G. S. Arbus, and H. Lior.
1985.
The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli.
J. Infect. Dis.
151:775-782[Medline].
|
| 8.
|
Karmali, M. A.
1989.
Infection by verocytotoxin-producing Escherichia coli.
Clin. Microbiol. Rev.
2:15-38[Abstract/Free Full Text].
|
| 9.
|
Ledeen, R. W.,
R. K. Yu, and L. F. Eng.
1973.
Gangliosides of human myelin: sialosylgalactosylceramide (G7) as a major component.
J. Neurochem.
21:829-839[CrossRef][Medline].
|
| 10.
|
Lingwood, C. A.,
H. Law,
S. Richardson,
M. Petri,
J. L. Brunton,
S. DeGrandis, and M. Karmali.
1987.
Glycolipiod binding of purified and recombinant Escherichia coli produced verotoxin in vitro.
J. Biol. Chem.
262:8834-8839[Abstract/Free Full Text].
|
| 11.
|
Lingwood, C. A.
1994.
Verotoxin-binding in human renal sections.
Nephron
66:21-28[Medline].
|
| 12.
|
Lingwood, C. A.
1996.
Role of verotoxin receptors in pathogenesis.
Trends Microbiol.
4:147-153[CrossRef][Medline].
|
| 13.
|
Makita, A., and N. Taniguchi.
1985.
Glycosphingolipids, p. 1-99.
In
H. Wiegant (ed.), New comprehensive biochemistry, vol. 10. Elsevier, Amsterdam, The Netherlands.
|
| 14.
|
Rowe, P.,
E. Orrbine,
G. Wells, and P. McLaine.
1991.
Epidemiology of the hemolytic-uremic syndrome in Canadian children from 1986-1988.
J. Pediatr.
119:218-224[CrossRef][Medline].
|
Journal of Clinical Microbiology, February 2001, p. 798-800, Vol. 39, No. 2
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.2.798-800.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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