Cllnlcal. Chstry. Measurement of sugar probes in serum: an alternative to urine measurement in intestinal permeability testing. SIMoN. C. FLEMING,l*. ANDREW.
Clinical
42:2
Chemistry
445-448
Cllnlcal
General Chstry
(1996)
Measurement of sugar probes in serum: an alternative to urine measurement in intestinal permeability testing C.
SIMoN
The
percentage
urine
after
dose
oral
assessing incomplete an
lactulose
and
sugars.
for
mannitol
We
the
test
jejunal
sensitive
enteropathy.
The
and
mannitol with
phy,
in
mined
in urine
(r
=
interchangeably. lose
collection It also
reduces
to 90
source
complete
and
samples
oral for
suspected
gluten-
at
90
miii
and
the
an
may
be particularly
the
time
spent
on the
mannitol
chemistry
#{149} chromatography,
Determination
lactulose
molecules
after
oral
disease
has been
bowel
mucosa
assess
respectively, ratios
of these
The
serum
and
and
passive
permeation, across
the
sugars
of intestinal
mannitol,
lactu-
supplied
by Sigma
urine
hydroxide
(500
JR
5 h
H
120
obtained
pediatric
#{149}
detection sugar
used
small
in a timed
urine
sample
glucose
active
mucosa.
has
form
in
are
transport,
Expressing provides
limited
quantifying carbohydrates the widespread use of
Department
of Clinical
2 Gastrointestinal Royal Received
Unit,
correspondence
Cornwall July
Hospital 10, 1995;
Biochemistry, Glasgow to this (Treliske), accepted
Royal
the
an index
in urine these tests.
University
of Newcastle
Infirmary,
Glasgow,
author, Truro December
at: Department TRI
compare
histology
lactulose
of test
its results
of jejunal
column,
Co.,
zinc
IRA
sugars.
with
biopsy
urine
specimens.
and
HPLC
Dionex
UK
(Camberley,
analysis
and
sample
sodium
Amberlite
5-sulfosalicylic
system PAl
a PAl
Surrey,
described
with (250
guard
resins acid
a pulsed 40
X
column UK).
preparation
as previously
Mannitol,
were
UK.
a Carbopac and
UK.
(anhydrous),
C1,
Poole,
and were
Poole,
acetate
400
BDH,
exchange
were
The
anion-
supplied
by
chromatographic
procedures
are
minor
modifications
with
electro-
mm)
essentially
the [4, 5].
and The
upon
Tyne.
deionized
with
helium
added
and
of Clinical
Chemis-
3LJ, UK. Fax 14-I 872-79224.
the
445
20
(968 mm,
solution
this
was
with
thorough
mixing
final
eluent
composition
zinc
added
at 34.5
NaOH
and
eluent
1 with
kPa.
0.5
sparging of
A final
2 in proportions system.
were
was
for
5
hydroxide
5 mm
of deionized eluents
was
sparged
to give
NaOH
concentration acetate
degassed
acetate
sodium
gIL
mmol/L eluent
eluent zinc
and
for a further
Both
was
I)
zinc
mixed
360
helium.
cm
mol/L
0.5
of 500
A second with
mmol/L
eluent
18 Mohm of
28 mL and
acetate.
for 20 mm
mL, 3 mL
was thoroughly
To
chromatographic
I, 1995.
water for
mm.
helium
UK.
PREPARATION
After
sparged
try,
the
and
Chemical
and
detector,
mmolIL
Address
method
g/L),
chemical
same
permeability. difficulty
in
intestinal
3-0-methyl
intestinal
to collect
particularly
to assess
in various and
ability
to measure
(6-0-ca-D-galatopyranosyl-D-glucopyranose)
ELUENT
Lactulose,
an
ingestion
melibiose
of nonmetabolized
administration
small
after
be used
from
#{149} electrochemical
excretion
of
Nevertheless,
Materials and Methods
children.
investigation
#{149}
liquid
of the
[1-3]. to
the and
a method
samples
(4-0-13-D-galactopyranosyl-D-fructofuranose)
to
in young
celiac
#{149}
of the urinary
permeability
used
samples,
A quaternarv-gradient TERMS:
diseases
is in the
urine
Lactulose
nun.
probe
tests
timed
deterand
alternative useful
[4].
in such
developed
in serum
The
can
of mannitol
acceptable
in urine
detection
determination
atro-
postdose.
methods
electrochemical
simultaneous
neonates.
therefore
measurements
pulsed and
LAKER1
between
villous ratios
two
detennination
with
of error accurately
we describe
F.
molecules
and
have
rapid
probe
mannitol
Here
of lactuwell
those
and
MICHAEL
with
the
children
We
to
of HPLC
has enabled
young
of test and
discriminated best
of
volunteers
of concentrations and
lead describe
ingestion
healthy
ratio
can We
determination
0.88),
INDEXING
the
inherent
collection
molecules.
lactulose:mannitol
provides
and
to
urine
with
The
in serum
sugar
biopsy
being well
multiple
after
biopsy
discrimination agree
of
to
serum
a normal
results
(PED)
in serum
applied
undergoing
subjects
introduction
of
simultaneous
subjects lose
in
method
The
and
RUSSELL,2
excreted
probe
the
I.
ROBIN
mannitol
timed
of sugar
DUNCAN,2
as a noninvasive
permeability.
or inaccurately method
and
is used
intestinal
in estimation HPLC
ANDREW
of lactulose
ingestion
small
errors
FLEMING,l*
and water stored
a 1.5
was under
of 120 mmol/L achieved
of 33.3:66.7
by (byvol)
mixing on the
446
SAMPLE
Fleming
et al.:
Sugar
volume,
the urine
probes
in serum
to test
Depending
diluted
between
of 1 mL. in deionized mixed
on collection 1:2 and
Then
1 mL water)
taking
with
resin to
and
deionized
and
mixture
tL
JR
of
of
the
250
120
H
and
volume.
supernate
IRA
400
was
injected
onto
the
volume
of serum
standard
(melibiose
250
ice-cold
5-sulfosalicylic
the
plasma
samples
were
were
removed. then
g/L)
injected
onto
200
added
20
and
.tL
of
L
of
mm,
the
CHROMATOGRAPHIC
expressed
a flow
were
rate
samples,
of
the
NaOH
1 mLlmin was
the
mixed
of this
super-
to maintain using
reference
the
were
detected
a gold
working
electrode.
(0-0.5
s), the
reduction
at
25 #{176}C. After a 5-mm
stability
of the
with
a pulsed
assay
potential
was
and
s), and
has
period
internal
was
0.05
to 0.5 s. Peak
to
125
recovery Overall
analyte
were
Ten
AND
-0.01
V s), the
the
testinal
34.2
disease,
were
28 Caucasians,
14.5
years),
acted
as control
were
and
the
tee of Glasgow
with
subjects
water
to drink.
On
a pretest
urine
solution,
which
dissolved
in 300
samples
were
mm
within
analysis.
Urine
aliquot analysis.
and
blood
0.5
mL of
this
sample.
contained mL
(osmolality
from Serum
30 mm,
was
removed,
was collected of thimerosal was
excretion
and
and
from
ratios
are shown
in Table
and
89%
between
1.8%
lactulose
[4, 5]. The 40
mg/L
and
and 105%
and
8.5%
for
serum. for
all
at various
was
11.8%
Mean
serum
90,
and
± 6.2%)
(SD
lactulose:mannitol
with
a mean
concentrations 120
mm
and
postingestion
1. SUBJECTS
underwent
the
procedure
failed
were
graded
as having
of celiac
no
total
with
an with
was
villous
One
increase
atrophy
(flat
subject
biopsy
biopsy), had
showed
normal
mild
with
an
a diag-
slight
villous The
shortening
lymphocytes.
biopsy
in 2
with
lymphocytes.
in interepithelial
all had
but
Six subjects
consistent
further
increase
18 subjects
procedure, obtained.
in interepithelial
an abnormal
a patchy
biopsy
biopsy
lymphocytes
disease.
with subject
villi
a jejunal
and
in interepithelial
Table and
allowed
only
provided the
test
1. Serum
and
calculated after
urine
results.
concentrations
Iactulose:mannltoi
ingestion
ratio
by control
of The
Therefore,
-20
into
Urine Serum
Mannitoib 11.8
times
(n
=
0.15
L:M ratio
(0.09)
(2.53)
10).
(± SD)
Lactuloseb
(6.2)
5.3
probes
0.02
NDC
(0.014)
-
30
66.9
(20.2)
0.91
(0.55)
0.014
(0.005)
60
105.7
(33.8)
1.27
(0.72)
0.013
(0.005)
blood
90
116.1
(37.6)
1.40
(0.57)
0.013
(0.006)
#{176}C until
120
109.5
(36.2)
1.90
(0.66)
0.020
(0.008)
90,
the
and
a container A 20-mL
-20
postingestlon
of sugar at various
subjects Mean
0
Blood
60,
at
at
and
in urine
between
at 30, 60,
All 28 subjects
remaining
of the
as preservative. stored
by
study
5 g of mannitol
separated
a 5-h period
(1 g/L)
mannitol
of 0.4 mg/L
(±0.09%),
respective
informed
mmollkg). at 30,
stored
compared
previously
of mannitol
0.15%
their
Commit-
drank
vein
and
over taken
±
none
subjects
then 696
a forearm
for
Time
10 g of lactulose
of water
collected
urine
reasons,
They
are
on the same
Ethical
the
were
of mannitol
described
ranged
of 0.02(±0.014).
10th
suspected
gave
(10 h), being
of the test
analysis
ranged
ratio
biopsy.
overnight
morning
postingestion.
samples with
the
ethical
groups
so
Results
(ANOVA).
limit
(L:M)
blunting
41.6
of the
biopsy
of the
intestinal
fasted
for
distribution,
groups.
SUBJECTS
lactulose
nosis
subjects
age
All
All subjects
For
underwent
study
(mean
jejunal
approval
Infirmary.
were
The
disease).
women
of gastroin-
investigation
test.
the
three
history
12 men
capsule
had
Royal
All subjects
and
permeability study
and
no
subjects.
(celiac
a Crosby
men
with
undergoing
enteropathy
underwent
control
years]
16 women
day as the intestinal consent,
seven
(SD)
who
gluten-sensitive
subjects
subjects,
± 6.6
the
concentrations.
increase
Caucasian
age
test
integra-
measured
PROTOCOL
healthy
[mean
a normal
mg/L
CVs
urinary
and
standardization.
SUBJECTS
120
heights
followed
been
a detection
GASTROINTESTINAL
tion
a consultant
permeability
to compare
of the
urine
with
CONTROL
chloride
V (0.51-0.64
V (0.65-0.75
-0.75
validity
is linear
Mean
detec-
was
intestinal
serum 1 mollL
silver/silver
+0.75
at
times.
potential
was
five with
electrochemical and
detection
potential
eluent
every wash
retention
electrode
The
oxidation
acetate
given
(formaldehyde by
Results
lactulose,
NaOH:zinc
groups
of variance
analytical
in serum
column.
the
the
anti-
test.
saline
assessed
± SD, and subjects
analysis
analytes.
with
columns
Analytes tor
eluted
formal and
methods
as mean
one-way
The
ANALYSIS
samples
subject
parametric
Analytical
The
the
ANALYSIS
for the
the
supernates
with
25 L
in
of
to precipitate
for
desalted again;
the
this,
ice
5 mm
was
centrifuged
to 200
To
on for
supernate and
added
was
standing at 900g
Each resin
was
(35
After
centrifuged
ion-exchange nate
acid
proteins.
was mg/L).
fixed
mmol/L)
unaware
STATISTICAL
we used A 200-FL
150
or nonsteroidal
result.
Results
internal
were
NaC1
alcohol 24 h before
were
column. Serum.
ingesting
for at least
biopsies
g/L,
histopathologist
with
Samples
drugs
Jejunal 100
mg/L
was desalted
avoided
inflammatory
were
to a volume
(melibiose,
(Amberlite
one-third
25
the
samples
water
standard
was added
up
centrifuged,
1:20
of internal
ion-exchange
C1)
permeability
All subjects
PREPARATION
Urine.
intestinal
#{176}C until
a
After
minutes b
Units Not
ingestion
of 10 g of lactulose
and
5 g
of
mannitol;
for serum.
are % excretion detected.
for urine
and mg/L
for
serum.
time
is 5 h for
urine,
Clinical
we grouped serum
the
and
two
urine
subjects
biopsy
The
18
mannitol
on this
histological
but were
and
basis.
compared
Because
changes,
these
with
the subjects
grouped
42,
No.
not
0.5
of
(±0.13%),
normal
12.6%
and
control
.
having 0.2
with
excretion
-
only
were
447
2, 1996
L:M ratio
results.
subjects
of the
evidence
measurements
minor
separately
abnormal
0.27%
on histological
sugar
had
considered
that
subjects
Cheinirtiy
biopsy
results
(±4.6%),
an L:M
ratio
subjects.
a lactulose of 0.021
The
had
eight
a urine
excretion
(±0.013),
subjects
similar
with
0.1
of to
0 05
abnormal 0.02
biopsy
results
lactulose
had
urine
0.65%
L:M
ratio
and
those
was
Results Table
(±0.26%),
with
at 60, 90, and
result
compared (P
higher
in the
90
and
serum P
probes
mm
0.002 Fig.
and
biopsy
at 60 mm, the
at various
among
the
groups
0.001).
The
two
and
group
0,001
=
ratio
occurred
that
at 90 mm
subjects
biopsy
normal
with
serum
other and
the
Fig.
only
and
serum
the
best
minor
L:M
90’
ratio
biopsy
ratios
the mean ratio
(#{149}l, and normal
results
in urine
and
serum
(60
and
90
mm
in control subjects, subjects with normal intestinal and subjects with abnormal intestinal biopsy results.
bars represent
Horizontal
at
biopsy
each group. Controls (01,
in
results
abnormal
(#{149}).
resulting 120 mm:
on their biopsy specimens were within the reference
from
all the
discrimination
postsugar
L:M ratio
1. Lactulose:mannitol
postingestion) biopsy results,
were
groups
at 60, 90, and
in urine
60’
significantly
at 90 mm.
indicating
L:M ratio
5h urine
with both
-
in
biopsy
0.03),
=
higher
0.002
shown
an abnormal in
than
(P P
L:M times,
subjects
0.005
postingestion are
were
with
subjects 0.001).
=
120 mm
0.01
The
control (P
concentrations
was significantly
1 shows
subjects
controls
postingestion
ratio
the
in serum
(± 3.4%),
(±0.10).
biopsies
in subjects
Lactulose
abnormal
120 L:M
=
with
for results
abnormal
120 mm
0.01).
=
than
0.146
at 60, 90, and and
of 9.0%
ratio
biopsy
concentrations
lower results
normal
normal
2. Mannitol
L:M
higher
with
for sugar
of mannitol
and
significantly
subjects
in subjects
excretion
ingestion
(P
histological
The
correlation
(at 90 mm) =
changes
for
the
between
was
L:M
good
ratio
shows
that
(mean
± 2SD)
had L:M ratios at 60 and 90 mm range of the healthy subjects.
the
(r
the 0.88).
=
in serum
limits
L:M
ratios
Fig.
2, which
at 90-mm
and
of agreement
are acceptable;
in urine 5-h
between
i.e., the
two
and
depicts
that serum
a bias
plot
urine
samples,
two
methods
the methods
can
be used
interchangeably.
Table
2. Serum and
mannltoi
concentrations lactuiose
In 26 subjects
ingestion
and urinary
excretion
respective
ratios
and their being
gluten-sensitive
enteropathy.
Normal (n =
biopsy
Mannltoib
± SD
Lactuloseb
Discussion Measurement
for
investigated Mean
Time poetlngestlon
of after
L:M
ratio
0.27
(0.13)
0.021
absorption
pathways
in both
with
provides
a sensitive
PED
analysis
(0.013)
of sugars
the
0
6.6
60
60.7
90 120 Abnormal biopsy (n = 8) Urine
(1.56)
source [6].
adults
use
loading of small
children specific
[1-4]. method
is well intestinal HPLC for
the
serum. in this
an accurate The
oral
and and
and
of error
to provide
children
1.29
(0.68)
0.024
(0.012)
Difference
53.6 (22.5)
1.58
(0.88)
0.028
(0.016)
0.1
51.2
1.72
(1.06)
0.037
(0.021)
(21.7)
in urine
after
investigation
of investigation
type
urine
of HPLC
collection, with
lies in
especially
PED
in
to determine
ND
ND#{176}
(20.1)
major
ability
young
Serum
urine
noninvasive
One (4.6)
in
in the
results 12.6
sugars
established
18)
Urine
of
in L:M ratio (serum
-
urine)
.
+2SD
results 0.05-
9.02
(3.4)
0.65
(0.26)
0.146
U
(O.iO)z
Serum 6.0
(3.4)
0
ND
ND
27.8
(17.9)”
2.0
(±1.3)
0.109
(0.08y
90
27.6
(14.7)”
4.9
(±2.7)e
0.229
(0186)g
120
18.3
(16.6)”
3.0
(1.8)e
0.201
(0,162)f
a
After ingestion
of 10 g of lactulose
and 5 g of mannitol;
time
is
5
Units are % excretion Not detected.
for urine and mg/L
-2SD
-0.05
h for urine, -0.1
for serum.
0
0.02
#{176}
d-g
Significantly
results: d P
=
different
0.01,
P
1. #{149}
U
minutes for serum. #{176}
mean
U
#{149}
60
0.04
0.06
0.08
Mean L:M from 0.03,
control
‘P
=
group 0.002,
and subjects and
g
P
=
with
0.001.
normal
0.1
0.12
ratio
biopsy
Fig. 2. Bias plot between 90
mm
postingestion.
urine and serum
lactulose:mannitol
ratios
at
448
Fleming
probe
markers
ingestion to
resort
sugars
to in
normal
urine
the
at
various The
discriminates ratio
and
of sugars
and
ratio
of the
villous
correlates
of with atrophy.
well
with
can
be
methods
to test
Serum
the used
1. Travis S, Menzies and significance.
L:M
ratios
those
postingestion,
normal
can
results
and
mannitol
acceptable larly
with
in serum alternative in
young
of reducing
5 h to 90 mm.
with
only
ratio
and
urine.
here
show
children the
time
giving
minor
within
functional
assessment
2. Pearson ADJ, Eastham EJ, Laker MF, Craft AW, Nelson R. Intestinal permeability in children with Crohn’s disease and coeliac disease. Br Med J 1982;286:20-1.
3. Cobden
I, Dickinson Ri, Rothwell J, Axon ATR. Intestinal permeabil by excretion ratios of two molecules: results in coeliac Br Med J 1978;ii:1060.
with and
120
the
best
discrim-
histological the
villous
90,
disease.
mm
range
Kynaston
JA, Fleming
SC, Laker
quantification of mannitol, urine by HPLC with pulsed
changes
reference
4.
of
studies
of intestinal
MF, Pearson
ADJ. Simultaneous
3-0-methyl glucose, and electrochemical detection,
permeability.
Clin Cttem
lactulose in for use in
1993;39:453-6.
5. Fleming that
at 90 mm to urine
subjects at 60,
at 90 mm
an L:M
serum
between biopsies
ratio
subjects
presented
useful
advantage
normal
the
have
in both
The
distinguished with
However,
on biopsy
I. Intestinal permeability: Clin Scm 1992;82:471-88.
ity assessed
and
ination.
permeability
References
interchangeably. atrophy
intestinal
oral
the need
subjects
with
two
in serum
concentrations
between
in serum
results
after
without
of
those
probes
points
ratio
clearly
architecture
ratio,
time
of an L:M
collection.
serum
urine
serum
the calculation
mucosal
Moreover, 5-h
in
allows
et al.: Sugar
measurement
postoral
collection, or taken
and
neonates. for
of lactulose
loading
the
may It
provides
an
be particualso
investigation
has
the from
SC, Kynaston JA, Laker MF, Pearson ADJ, Kapembwa MS, Griffin GE. Analysis of multiple sugar probes in urine and plasma b high performance anion-exchange chromatography with pulsed electrochemical detection. Application in the assessment of intes tinal permeability in human immunodeficiency virus infection. Chromatogr 1993;640:293-7.
6. Weaver newbom.
LT, Arch
Laker
MF,
Dis Child
Nelson
R.
1984;59:236-41.
Intestinal
permeability
in
the
