Tohoku
J. exp.
Med.,
1979,
127, 17-24
Glucose-Containing Human
Oligosaccharides
in Normal
Urine
MASAYUKI TOKUNAGA, MASAHIKO ENDO and ZENSAKU YOSIZAWA*
Department of Orthopedic Surgery and *Department of Biochemistry, Tohoku University School of Medicine, Sendai 980
TOKUNAGA,
„Ÿ
charides
in
Normal
human
M.,
urine
1 •~ 2 (Cl
form)
was
then
resulting
effluent
hexose
contents
excreted age
sex.
galactose intact
treated
with
The
into
and
in
chromatography its
reduction
Dowex
subfractions
(SFs. that
with
a
from
SF2
was
and
similar
(1),
2
(H+
and form)
containing
Sephadex 1-5).
neutral The
Comparison
individuals pattern
acid
the and
G-10.
of
regularly regardless
characterized after
17-24
(CPC),
50 •~
healthy
urine
before
of
Oligosac
127
effluent
column
indicated
isolated
1978, chloride
of
a
5
subfractions
Glucose-Containing Med.,
concentrated
through
oligosaccharides
paper
exp.
columns
succession.
A
Z.
J.
cetylpyridinium
through
divided
the
YOSIZAWA, Tohoku
gel-filtered
Component by
one
in
and
Urine.
passed
was of
neutral or
was
was
saccharides
M.
Hunan
CPC-supernatant Dowex
ENDO,
Normal
as
of
glucosyl
hydrolysis
of
product.„Ÿglucosylgalactose;
the
urinary
oligosaccharides
Since the presence of mucosubstances (glycosaminoglycans and glycoproteins) in normal human urine was first recognized by Morner (1895), numerous investiga tions have been carried out on these substances in urine under normal and pathological conditions, specifically, concerning with mucopolysaccharidosis and other connective tissue diseases (Bourrillon 1972; Dorfman and Matalon 1976; Lundblad 1977). These studies, however, dealt mostly with glycosaminoglycans and glycoproteins. Also, the presence of oligosaccharides in normal human urine has been reported (Lundblad and Berggard 1962; Miettinen 1963; Lundblad 1968; Lundblad et al. 1973; Lundblad and Svensson 1973; Norden et al. 1973; Kofoed et al. 1976; Lundblad 1977), but the results seemed not to be satisfactory. Since the meta bolism of the carbohydrate-containing substances in tissues may reflect not only on urinary glycosaminoglycans and glycoproteins, but also on oligosaccharides in urine, further investigation is necessary to elucidate the nature of the urinary oligosaccharides. This paper reports the results of the study on the urinary oligosaccharides of 20 healthy subjects. MATERIALS Materials. with
synthetic
Received
Urine penicillin
was
collected as
for publication,
a preservative
March
AND METHODS
separately
from during
8, 1978. 17
the
20
healthy
collection
Japanese for
24
and hr.
kept
Specific
at gravity
5•Ž
18 of
M. Tokunaga the
24-hr
When of
urine
the
distilled
6ml
10%
To
a
by
(1967),
of
column
onto
was
column
(1 •~
15
a
subjected
with
to
water
280nm
at of
a
of
of
Dowex
of
with
shown
a
Figs.
et
1 and
Cl
of
study.
of
of
1956).
The
form) applied
the
and the onto a
effluent
was
concentrate
Sephadex
G-10
collected
hexose
.
in
effluent
were
CPC-supernatant
and
were
was
, eluting
Absorbance
at
each
fraction
was
then
divided
into
2.
1. A representative profile of gel-filtration on Sephadex G-10 of neutral saccharides obtained from normal human urine. Vo , void volume; o-o, absorbance at 490 nm of the phenol-H,80, reaction for hexose; ?-? , absorbance at 280nm. Paper
chromatography.
analyzed
by
paper
The
subfractions
chromatography
obtained
before
and
after
by the
the
above
hydrolysis
gel
with
-filtration N HCl
1
were at
or
the
precipitates
materials
, H+ then
was
70cm)
2ml
added to
present
above
form),
content
al.
the
.
volume
were
saccharide-containing
(1.8 •~
The
(Dubois
in
column
one
The
mesh
effluent
neutral
Fractions
the
same
according
Other
(200-400
mesh,
The
for
Tokyo.
The
(200-400
vacuo.
2
diluted
overnight.
of
50 •~
centrifugation
the
stirring
used
(20ml)
Dowex
measured.
phenol-H2S04 as
was
by
with
or
5•Ž
Ltd.,
water.
25ml/hr.
was
under at
matter
diluted
urine
kept
portion
of
through
rate
was
(CPC)
Chemicals,
1 •~ 2
gel-filtration
insoluble
it
pooled
was
distilled
in
of
supernatant
A
volume
fraction
5 subfractions
the
15cm)
80ml
small
flow
each
determined
Fig.
with
the
mixture
Nakarai
(1 •~
1.020,
of
oligosaecharides.
cm)
to
the and
from
removal
chloride
and
column
washed
concentrated was
a
the
exceeded
(300ml)
centrifugation,
Fractionation applied
after urine
portion
CPC was purchased from commercial sources. was
the
cetylpyridinium
Ferrante
removed
measured of
aqueous
of Di
were
then
gravity
water.
of
method
was
specific
et al.
2
hr.
Paper
chromatography
was
carried
out
on
Toyo
filter
paper
No . 51
with
n-butanol-pyridine-water
(6:4:3 ng
paper
were
hydrogen
located phthalate
Reduction
of
by
staining
,
by
volume)
technique with
at
and
18•Ž
alkaline
n-butanol-acetic
for
silver
20-30
reagent
hr or
NaBH4
and
(14 •~
50cm)
acid-water .
The
(4:1:5
substances
ninhydrin
on
reagent
or
component the
A.
reaction
To
a
portion
mixture
(1ml)
was
kept
at
of
component
room
A
added
1ml
was
column
a
then
column with
(1 •~
destroyed
40ml
12cm)
with
of
of
distilled
Dowex
1ml
of
50
(200-400
30%
acetic
water
.
Borate
mesh
acid
in
the
, H+
and
effluent
form), was
of 0
o Excess
the solution
nto
aniline
wa
temperature
vernight. reductant
, by volume) by the descendi the
reagent. s
.5M
100•Ž f
followed removed
was
applied i
by
washing
by
the
the
repeat ed evaporation to dryness withmeth
anol volume
of
water.
in
vacuo.
The
residue
was
then
dissolved
in
a
small
Urinary
Fig.
2.
Representative
profiles
obtained
from
sorbances
at
case
20,
Glucose-Containing
3
normal
490
nm
of
Oligosaccharides
of
gel-filtration
on
human
urines.
Vo,
the
phenol-H2S04
Sephadex See
G-10 Fig.
reaction
19
of
neutral
saccharides
1; •¢-•¢, •ü-•ü, •œ-•œ,
for
hexose
of
ab
case
4,
case
13
and
respectively.
RESULTS
Fractionation of oligosaccharides Urine
from
in
Materials
of
Dowex
20
and 50 •~
2
Subsequently, by
gel-filtration
at
Since at
of void
No.
subfractions 43-46;
form)
and
in
the
tube
1
Nos.
Dowex
2.
two at
from
tube 47-50;
Nos.
tube
tube
be
or
peaks, No. 11
SF2, Nos.
seen
52.
form),
51 to
No.
tube 51-57,
52 57
Nos. as
in
these
succession. was
were and
the
figures,
often
the
was
observed. sucrose
divided SF3,
the
peak
authentic
were
in
of
small
39-42;
shown
a column
patterns in
peaks or
described
saccharides
Another
small
tube No.
31-38;
SF5,
(Cl neutral
can 51
as
through
Representative
As No.
CPC
passed
1 •~ 2
G-10.
tube
these
with
then
containing
and at
gel-filtrated
fractions SFl,
of
Sephadex
located
was
separately was
effluent
Between
glucose
treated
a column
Figs.
was
volume.
47,
was
CPC-supernatant
on
shown
(SF): SF4,
The
concentrated
hexose
authentic
tube
Japanese
gel-filtration
are peak
located
(H+
the
fractionated
largest
healthy
Methods.
into tube
5
Nos.
figures.
Comparison of hexose content of subfractions Ratios specimens are shown groups, regularly
of the
hexose
are shown in Fig.
3.
contents
in Table Although
of SF1,
1, and those some
difference
it was not significant. These excreted neutral oligosaccharides
of age or sex.
SF2
and
of children,
SF3
to
that
adult-male
was observed
of
SF5
of
20
and adult-female
between
these
three
data indicated that healthy individuals in urine with a similar pattern regardless
20
M. Tokunaga
et al.
TABLE1. Ratios of hexosecontents of SF1 (1), SF2 (2) and SF3 (3) to that of SF5 of individuals
Fi g. 3. Ratios oz hexose contents of SF1 (1), SF2 (2) and SF3 (3) to that of SF5 of children , adult -male and adult-female
Separation of glucose-containing oligosaceharide (component A) The and in
above
after Fig.
4
contain
(A,
B).
to
be
indicated
pecifically, e follows:
subfractions
hydrolysis As
were
with can
be
oligosaccharides.
seemed was
five
the
1 seen
the
SF1,
the
at
SF2
and of
SF3 SF2
components A,
a
disaccharide
by
100•Ž
figure
, major whereas SF1
hydrolysate
oligosaccharides, Component
in
examined
HCl
However
monosaccharides, that
N
, these
paper 2
hr
chromatography .
The
subfractions
components of SF3, hetero -oligosaccharides
contained contained A
for
, B and composed
glucose mainly C
-containing glucose
in Fig. 5 of glucos
results
are
were
indicated
SF4 .
shown to
and
SF5
Moreover,
it
oligosaccharides
and were e
before
and
galactose supposed galactose;
.S
(Fig to
. 5). th Of
be com
as
Urinary
Fig.
4.
Paper
and
after
carried
Glucose-Containing
chromatograms (B) out
as
acid described
The
substances
were
Gal,
galactose;
Ga1N,
G1cUA,
of
in
(1), (1N
the
located
glucuronic
text, by
SF2
(2),
HCl, using
(3), 2
SF4
hr).
(4)
and
Paper
with Foe,
alkaline
(5)
(6:4:3,
silver
L-fucose;
SF5
Glc,
before
chromatography
n-butanol-pyridine-water
staining
galactosamine;
SF3
100•Ž,
21
reagent.
glucose;
by Man,
GlcN,
(A) was
volume). mannose;
glucosamine;
acid.
ponent B, a trisaccharide a hetero-oligosaccharide Of the three
SF1
hydrolysis
Oligosaccharides
composed of fucose, glucose and galactose; component C, containing glucosamine, galactosamine, hexoses and fucose.
components,
graphy as described from SF3.
component
in Materials
A was isolated and
by preparative
Methods.
paper
This compound
chromato
was also isolated
Characterization of component A Component (Fig.
6).
Component the
reduction
A migrated
Acid A
hydrolysate was product
with of
reduced was
a similar
component
with hydrolyzed
pattern A
NaBH4
as with
to
lactose
contained described I N
HCl
on
paper
glucose in at
and
Materials 100•Ž
for
chromatogram galactose and
2 hr.
(Fig.
Methods, Paper
6). and chro
22
M. Tokunaga
Fig.
et al.
5. (2)
Paper and
100•Ž,
Fig.
(2H)
2 hr).
carried Fuc,
chromatograms
after
out GlcN, 4;
Malt,
of
acid
Paper as
before
(1 N
chromatography
described
GlcUA,
SF2
hydrolysis
Man,
in Gal,
Fig.
HCl, was
4.
Glc,
Ga1N,
See
maltose.
matogram of the acid hydrolysate of the reduced component A gave glucose as the reducing sugar (Fig. 7). Component A migrated faster than hydroxylysine and was not apparently stainable with ninhydrin, indicating absence of hydroxylysine and other amino acids. The above data indicated that component A was glucosyl galactose. DISCUSSION
Although the presence of oligosaccharides in normal human urine has been reported (Lundblad and Berggard 1962; Miettinen 1963; Lundblad 1968; Lundblad et al. 1973; Lundblad and Svensson 1973; Norden et al. 1973; Kofoed et al. 1976; Lundblad 1977), these substances were mostly hetero-oligosaccharides containing hexosamine or sialic acid. Therefore, little is known about the neutral disaccharides or trisaccharides without amino sugars in urine. In the present study, we found a neutral disaccharide (component A) and probably a trisaccharide (component B) and a hetero-oligosaccharide (component C) in normal human urine. Moreover, the data showed that healthy individuals regularly excreted the neutral oligosac
Urinary
Fig.
6.
Paper
(1N
HCl,
Gal,
Glc,
chromatograms 100•Ž, See
2 Fig.
Glucose-Containing
of
hr). 4;
Paper Lact,
component
A
Oligosaccharides
before
chromatography
(A) was
and
carried
23
after out
(AH) as
acid
hydrolysis
described
in
Fig.
4.
lactose.
Fig. 7. A paper chromatogram of the acid hydrolysate of the reduction product of com ponent A(ARH). Paper chromatography was carried out as described in Fig. 4. The substances were located with aniline hydrogen phthalate reagent.
charides in urine with a similar pattern regardless of age or sex. Component A isolated from SF2 was characterized as glucosylgalactose. This disaccharide is known to attach to hydroxylysine in collagen and basement membranes (Spiro 1967, 1969; Sato et al. 1975). Therefore, it might be a metabolite of these glycoproteins. Although the presence of glucosylgalactosylhydroxylysine in urine has been reported (Segrest and Cunningham 1970; Sato et al. 1976), no report was found about the excretion of glucosylgalactose in urine. Since the increased excretion of this disaccharide in patients with various connective tissue diseases was observed (Tokunaga et al., unpublished data), it might give a clue for the metabolism of collagen or related substances in the connective tissues. Acknowledgments We thank Professor interest throughout this
Eikichi work.
Wakamatsu,
Department
of Orthopedic
Surgery,
for his
24
M. Tokunaga
et al.
References
1)
2) 3) 4)
5) 6) 7)
8) 9) 10)
11) 12) 13) 14)
15)
16)
17) 18)
Bourrillon, R. (1972) Urinary glycoproteins, glycopeptides and related heterosac charides. In: Glycoproteins, edited by A. Gottschalk, BBA Library, Vol. 5, Elsevier, Amsterdam, pp. 909-925. Di Ferrante, N.M. (1967) The measurement of urinary mucopolysaccharides. Analyt. Biochem., 21, 98-106. Dorfman, A. & Matalon, R. (1976) The mucopolysaccharidosis. Proc. nat. Acad. Sci. USA, 73, 630-637. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. & Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Analyt. Chem., 28, 350 - 356. Kofoed, J.A., Barcelo, A.C. & Tocci, A.A. (1976) Further studies on gel filtration of urinary substances. Experientia, 32, 851-852. Lundblad, A. (1968) Isolation and characterization of a urinary oligosaccharide characteristic of blood group O(H)-secretors. Biochim. biophys. Acta, 165, 202-207. Lundblad, A. (1977) Urinary glycoproteins, glycopeptides, and oligosaccharides. In: The Glycoconjugates, edited by M.I. Horowitz & W. Pigman, Vol. 1, Academic Press, New York-London, pp. 441-458. Lundblad, A. & Berggard, I. (1962) Gel filtration of the low molecular weight carbohydrate components of normal urine. Biochim. biophys. Acta, 57, 129-134. Lundblad, A. & Svensson, S. (1973) The structure of urinary difucosylpentasaccharides, characteristic of secretors with blood-group A gene. Carbohyd. Res., 30, 187-189. Lundblad, A., Hallgren, P., Rudmark, A. & Svensson, S . (1973) Structures and serological activities of three oligosaccharides isolated from urines of nonstarved secretors and from secretors on lactose diet. Biochemistry, 12, 3341-3345 . Miettinen, T.A. (1963) Further studies on gel filtration of urinary mucosaccharides . Clin. chim. Acta, 8, 693-700. Morner, K.A.H. (1895) Untersuchung fiber die Proteinstoffe and die eiweissfallenden Substanzen des normalen Menschenharns. Scand. Arch . Physiol., 6, 332-437. Norden, N.E., Eriksson, 0., Hultberg, B. & Ockerman, P.A. (1973) Gel chromato graphic distribution of urinary carbohydrate compounds. Clin. chim. Acta, 44, 95-100. Sato, T., Munakata, H., Yoshinaga, K. & Yosizawa, Z. (1975) Chemical compositions of glomerular and tubular basement membranes of human kidney . Tohoku J. exp. Med., 115, 299-306. Sato, T., Saito, T., Yasuda, K., Kokubun, M., Yoshinaga, K . & Yosizawa, Z. (1976) Determination of 0-hydroxylysyl-glycosides (OHLG) in urine . Tohoku J. exp. Med., 118, 81-87. Segrest, J.P. & Cunningham, L.W. (1970) Variations in human urinary 0-hydroxylysyl glycoside levels and their relationship to collagen metabolism. J. din. Invest ., 49, 1497-1509. Spiro, R.G. (1937) The structure of the disaccharide unit of the renal glomerular basement membrane. J. biol. Chem ., 242, 4813-4823. Spiro, R.G. (1969) Characterization and quantitative determination of the hydroxy lysine-linked carbohydrate units of several collagens . J. biol. Chem., 244, 602-612.
J. exp.
Med.,
1979,
127, 17-24
Glucose-Containing Human
Oligosaccharides
in Normal
Urine
MASAYUKI TOKUNAGA, MASAHIKO ENDO and ZENSAKU YOSIZAWA*
Department of Orthopedic Surgery and *Department of Biochemistry, Tohoku University School of Medicine, Sendai 980
TOKUNAGA,
„Ÿ
charides
in
Normal
human
M.,
urine
1 •~ 2 (Cl
form)
was
then
resulting
effluent
hexose
contents
excreted age
sex.
galactose intact
treated
with
The
into
and
in
chromatography its
reduction
Dowex
subfractions
(SFs. that
with
a
from
SF2
was
and
similar
(1),
2
(H+
and form)
containing
Sephadex 1-5).
neutral The
Comparison
individuals pattern
acid
the and
G-10.
of
regularly regardless
characterized after
17-24
(CPC),
50 •~
healthy
urine
before
of
Oligosac
127
effluent
column
indicated
isolated
1978, chloride
of
a
5
subfractions
Glucose-Containing Med.,
concentrated
through
oligosaccharides
paper
exp.
columns
succession.
A
Z.
J.
cetylpyridinium
through
divided
the
YOSIZAWA, Tohoku
gel-filtered
Component by
one
in
and
Urine.
passed
was of
neutral or
was
was
saccharides
M.
Hunan
CPC-supernatant Dowex
ENDO,
Normal
as
of
glucosyl
hydrolysis
of
product.„Ÿglucosylgalactose;
the
urinary
oligosaccharides
Since the presence of mucosubstances (glycosaminoglycans and glycoproteins) in normal human urine was first recognized by Morner (1895), numerous investiga tions have been carried out on these substances in urine under normal and pathological conditions, specifically, concerning with mucopolysaccharidosis and other connective tissue diseases (Bourrillon 1972; Dorfman and Matalon 1976; Lundblad 1977). These studies, however, dealt mostly with glycosaminoglycans and glycoproteins. Also, the presence of oligosaccharides in normal human urine has been reported (Lundblad and Berggard 1962; Miettinen 1963; Lundblad 1968; Lundblad et al. 1973; Lundblad and Svensson 1973; Norden et al. 1973; Kofoed et al. 1976; Lundblad 1977), but the results seemed not to be satisfactory. Since the meta bolism of the carbohydrate-containing substances in tissues may reflect not only on urinary glycosaminoglycans and glycoproteins, but also on oligosaccharides in urine, further investigation is necessary to elucidate the nature of the urinary oligosaccharides. This paper reports the results of the study on the urinary oligosaccharides of 20 healthy subjects. MATERIALS Materials. with
synthetic
Received
Urine penicillin
was
collected as
for publication,
a preservative
March
AND METHODS
separately
from during
8, 1978. 17
the
20
healthy
collection
Japanese for
24
and hr.
kept
Specific
at gravity
5•Ž
18 of
M. Tokunaga the
24-hr
When of
urine
the
distilled
6ml
10%
To
a
by
(1967),
of
column
onto
was
column
(1 •~
15
a
subjected
with
to
water
280nm
at of
a
of
of
Dowex
of
with
shown
a
Figs.
et
1 and
Cl
of
study.
of
of
1956).
The
form) applied
the
and the onto a
effluent
was
concentrate
Sephadex
G-10
collected
hexose
.
in
effluent
were
CPC-supernatant
and
were
was
, eluting
Absorbance
at
each
fraction
was
then
divided
into
2.
1. A representative profile of gel-filtration on Sephadex G-10 of neutral saccharides obtained from normal human urine. Vo , void volume; o-o, absorbance at 490 nm of the phenol-H,80, reaction for hexose; ?-? , absorbance at 280nm. Paper
chromatography.
analyzed
by
paper
The
subfractions
chromatography
obtained
before
and
after
by the
the
above
hydrolysis
gel
with
-filtration N HCl
1
were at
or
the
precipitates
materials
, H+ then
was
70cm)
2ml
added to
present
above
form),
content
al.
the
.
volume
were
saccharide-containing
(1.8 •~
The
(Dubois
in
column
one
The
mesh
effluent
neutral
Fractions
the
same
according
Other
(200-400
mesh,
The
for
Tokyo.
The
(200-400
vacuo.
2
diluted
overnight.
of
50 •~
centrifugation
the
stirring
used
(20ml)
Dowex
measured.
phenol-H2S04 as
was
by
with
or
5•Ž
Ltd.,
water.
25ml/hr.
was
under at
matter
diluted
urine
kept
portion
of
through
rate
was
(CPC)
Chemicals,
1 •~ 2
gel-filtration
insoluble
it
pooled
was
distilled
in
of
supernatant
A
volume
fraction
5 subfractions
the
15cm)
80ml
small
flow
each
determined
Fig.
with
the
mixture
Nakarai
(1 •~
1.020,
of
oligosaecharides.
cm)
to
the and
from
removal
chloride
and
column
washed
concentrated was
a
the
exceeded
(300ml)
centrifugation,
Fractionation applied
after urine
portion
CPC was purchased from commercial sources. was
the
cetylpyridinium
Ferrante
removed
measured of
aqueous
of Di
were
then
gravity
water.
of
method
was
specific
et al.
2
hr.
Paper
chromatography
was
carried
out
on
Toyo
filter
paper
No . 51
with
n-butanol-pyridine-water
(6:4:3 ng
paper
were
hydrogen
located phthalate
Reduction
of
by
staining
,
by
volume)
technique with
at
and
18•Ž
alkaline
n-butanol-acetic
for
silver
20-30
reagent
hr or
NaBH4
and
(14 •~
50cm)
acid-water .
The
(4:1:5
substances
ninhydrin
on
reagent
or
component the
A.
reaction
To
a
portion
mixture
(1ml)
was
kept
at
of
component
room
A
added
1ml
was
column
a
then
column with
(1 •~
destroyed
40ml
12cm)
with
of
of
distilled
Dowex
1ml
of
50
(200-400
30%
acetic
water
.
Borate
mesh
acid
in
the
, H+
and
effluent
form), was
of 0
o Excess
the solution
nto
aniline
wa
temperature
vernight. reductant
, by volume) by the descendi the
reagent. s
.5M
100•Ž f
followed removed
was
applied i
by
washing
by
the
the
repeat ed evaporation to dryness withmeth
anol volume
of
water.
in
vacuo.
The
residue
was
then
dissolved
in
a
small
Urinary
Fig.
2.
Representative
profiles
obtained
from
sorbances
at
case
20,
Glucose-Containing
3
normal
490
nm
of
Oligosaccharides
of
gel-filtration
on
human
urines.
Vo,
the
phenol-H2S04
Sephadex See
G-10 Fig.
reaction
19
of
neutral
saccharides
1; •¢-•¢, •ü-•ü, •œ-•œ,
for
hexose
of
ab
case
4,
case
13
and
respectively.
RESULTS
Fractionation of oligosaccharides Urine
from
in
Materials
of
Dowex
20
and 50 •~
2
Subsequently, by
gel-filtration
at
Since at
of void
No.
subfractions 43-46;
form)
and
in
the
tube
1
Nos.
Dowex
2.
two at
from
tube 47-50;
Nos.
tube
tube
be
or
peaks, No. 11
SF2, Nos.
seen
52.
form),
51 to
No.
tube 51-57,
52 57
Nos. as
in
these
succession. was
were and
the
figures,
often
the
was
observed. sucrose
divided SF3,
the
peak
authentic
were
in
of
small
39-42;
shown
a column
patterns in
peaks or
described
saccharides
Another
small
tube No.
31-38;
SF5,
(Cl neutral
can 51
as
through
Representative
As No.
CPC
passed
1 •~ 2
G-10.
tube
these
with
then
containing
and at
gel-filtrated
fractions SFl,
of
Sephadex
located
was
separately was
effluent
Between
glucose
treated
a column
Figs.
was
volume.
47,
was
CPC-supernatant
on
shown
(SF): SF4,
The
concentrated
hexose
authentic
tube
Japanese
gel-filtration
are peak
located
(H+
the
fractionated
largest
healthy
Methods.
into tube
5
Nos.
figures.
Comparison of hexose content of subfractions Ratios specimens are shown groups, regularly
of the
hexose
are shown in Fig.
3.
contents
in Table Although
of SF1,
1, and those some
difference
it was not significant. These excreted neutral oligosaccharides
of age or sex.
SF2
and
of children,
SF3
to
that
adult-male
was observed
of
SF5
of
20
and adult-female
between
these
three
data indicated that healthy individuals in urine with a similar pattern regardless
20
M. Tokunaga
et al.
TABLE1. Ratios of hexosecontents of SF1 (1), SF2 (2) and SF3 (3) to that of SF5 of individuals
Fi g. 3. Ratios oz hexose contents of SF1 (1), SF2 (2) and SF3 (3) to that of SF5 of children , adult -male and adult-female
Separation of glucose-containing oligosaceharide (component A) The and in
above
after Fig.
4
contain
(A,
B).
to
be
indicated
pecifically, e follows:
subfractions
hydrolysis As
were
with can
be
oligosaccharides.
seemed was
five
the
1 seen
the
SF1,
the
at
SF2
and of
SF3 SF2
components A,
a
disaccharide
by
100•Ž
figure
, major whereas SF1
hydrolysate
oligosaccharides, Component
in
examined
HCl
However
monosaccharides, that
N
, these
paper 2
hr
chromatography .
The
subfractions
components of SF3, hetero -oligosaccharides
contained contained A
for
, B and composed
glucose mainly C
-containing glucose
in Fig. 5 of glucos
results
are
were
indicated
SF4 .
shown to
and
SF5
Moreover,
it
oligosaccharides
and were e
before
and
galactose supposed galactose;
.S
(Fig to
. 5). th Of
be com
as
Urinary
Fig.
4.
Paper
and
after
carried
Glucose-Containing
chromatograms (B) out
as
acid described
The
substances
were
Gal,
galactose;
Ga1N,
G1cUA,
of
in
(1), (1N
the
located
glucuronic
text, by
SF2
(2),
HCl, using
(3), 2
SF4
hr).
(4)
and
Paper
with Foe,
alkaline
(5)
(6:4:3,
silver
L-fucose;
SF5
Glc,
before
chromatography
n-butanol-pyridine-water
staining
galactosamine;
SF3
100•Ž,
21
reagent.
glucose;
by Man,
GlcN,
(A) was
volume). mannose;
glucosamine;
acid.
ponent B, a trisaccharide a hetero-oligosaccharide Of the three
SF1
hydrolysis
Oligosaccharides
composed of fucose, glucose and galactose; component C, containing glucosamine, galactosamine, hexoses and fucose.
components,
graphy as described from SF3.
component
in Materials
A was isolated and
by preparative
Methods.
paper
This compound
chromato
was also isolated
Characterization of component A Component (Fig.
6).
Component the
reduction
A migrated
Acid A
hydrolysate was product
with of
reduced was
a similar
component
with hydrolyzed
pattern A
NaBH4
as with
to
lactose
contained described I N
HCl
on
paper
glucose in at
and
Materials 100•Ž
for
chromatogram galactose and
2 hr.
(Fig.
Methods, Paper
6). and chro
22
M. Tokunaga
Fig.
et al.
5. (2)
Paper and
100•Ž,
Fig.
(2H)
2 hr).
carried Fuc,
chromatograms
after
out GlcN, 4;
Malt,
of
acid
Paper as
before
(1 N
chromatography
described
GlcUA,
SF2
hydrolysis
Man,
in Gal,
Fig.
HCl, was
4.
Glc,
Ga1N,
See
maltose.
matogram of the acid hydrolysate of the reduced component A gave glucose as the reducing sugar (Fig. 7). Component A migrated faster than hydroxylysine and was not apparently stainable with ninhydrin, indicating absence of hydroxylysine and other amino acids. The above data indicated that component A was glucosyl galactose. DISCUSSION
Although the presence of oligosaccharides in normal human urine has been reported (Lundblad and Berggard 1962; Miettinen 1963; Lundblad 1968; Lundblad et al. 1973; Lundblad and Svensson 1973; Norden et al. 1973; Kofoed et al. 1976; Lundblad 1977), these substances were mostly hetero-oligosaccharides containing hexosamine or sialic acid. Therefore, little is known about the neutral disaccharides or trisaccharides without amino sugars in urine. In the present study, we found a neutral disaccharide (component A) and probably a trisaccharide (component B) and a hetero-oligosaccharide (component C) in normal human urine. Moreover, the data showed that healthy individuals regularly excreted the neutral oligosac
Urinary
Fig.
6.
Paper
(1N
HCl,
Gal,
Glc,
chromatograms 100•Ž, See
2 Fig.
Glucose-Containing
of
hr). 4;
Paper Lact,
component
A
Oligosaccharides
before
chromatography
(A) was
and
carried
23
after out
(AH) as
acid
hydrolysis
described
in
Fig.
4.
lactose.
Fig. 7. A paper chromatogram of the acid hydrolysate of the reduction product of com ponent A(ARH). Paper chromatography was carried out as described in Fig. 4. The substances were located with aniline hydrogen phthalate reagent.
charides in urine with a similar pattern regardless of age or sex. Component A isolated from SF2 was characterized as glucosylgalactose. This disaccharide is known to attach to hydroxylysine in collagen and basement membranes (Spiro 1967, 1969; Sato et al. 1975). Therefore, it might be a metabolite of these glycoproteins. Although the presence of glucosylgalactosylhydroxylysine in urine has been reported (Segrest and Cunningham 1970; Sato et al. 1976), no report was found about the excretion of glucosylgalactose in urine. Since the increased excretion of this disaccharide in patients with various connective tissue diseases was observed (Tokunaga et al., unpublished data), it might give a clue for the metabolism of collagen or related substances in the connective tissues. Acknowledgments We thank Professor interest throughout this
Eikichi work.
Wakamatsu,
Department
of Orthopedic
Surgery,
for his
24
M. Tokunaga
et al.
References
1)
2) 3) 4)
5) 6) 7)
8) 9) 10)
11) 12) 13) 14)
15)
16)
17) 18)
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