Poster presentation and IBS Forum presentation given at Digestive Disease Week (DDW) in Orlando, May 2013. It details norms and the reason for using a paired 13C-glucose test to adjust for individual habitus and metabolic states. Results are expressed as a ratio, called the COEFFICIENT OF GLUCOSE OXIDATION for the (CGO) for the target substrate of interest, in this case, sucrose [ CGO-SUCROSE ]. Ideally, if sucrose digestion, at the luminal mucosal level is normal, the products of digestion, will be catabolized like the comparative glucose substrate. The ratio would be 1:1 and the CGO-Sucrose would approximate 1 (100% efficiency). An abnormal CGO is defined as 75% or less and could be attributed to sucrase deficiency, poor gastric emptying, fructose receptor deficiency or patient error (didn't drink all the substrate). The test takes 2.5 hour to complete and is processed using the identical technology as the H. pylori 13C-urea breath test. The accuracy of the biphasic 13C-sucrose/glucose breath test is still being evaluated, but it appears that it should be accurate in identification of patients whose biopsies show sucrase activity values less than 25 uM/min/gm protein.

1. Development of a New Bi-Phasic 13C-
Sucrose/Glucose Breath Test
for the Diagnosis of Sucrase-Isomaltase
Deficiency
Baylor College of Medicine1 – CNRC2 -Texas Children's Hospital Nutrition and GI Section3
Antone R. Opekun1, 3, Christina E. Lecea1,2, Bruno Chumpitazi1,1,3, Buford L. Nichols2,3, Mark A. Gilger1,3
Depts. of 1Medicine and 2Pediatrics, Gastroenterology Sections, at Baylor College of Medicine and Texas Children’s Hospital, Houston, Texas, U.S.A.
Abstract
Study Objectives
Congenital sucrase-isomaltase
deficiency (CSID) is a disorder
causing symptomatic maldigestion of
sucrose (SUC). It is autosomal-
recessively inherited. More than 43
variants have been identified with at
least 8 of the exonic amino acid
exchanges causing hypomorph or null
alleles. Practical screening tests do
not yet exist and the diagnosis
requires EGD with biopsy for sucrase
assay; therefore sucrase deficiency
is probably under diagnosed. Earlier
breath tests (BT) have been proposed,
but not perfected. Our goal was to
develop an easy and accurate 1-day
13C-stable isotope substrate-based
screening. BT. METHODS: Eleven
children (aged 11m-15y; 6m / 5f) with
proven CSID (sucrase <7 EU/g)
underwent 13C-sucrose BT (20 mg in
excipient) and repeat testing on day
2 with comparative 13C-glucose (GLU)
dosing to critically adjust for
habitus. Therefore, oxidation results
were expressed as a change in
isotopic enrichment ratio between the
reference GLU and the test SUC
substrates [Coefficient of Glucose
Oxidation (CGO)] and 13
children/control subjects (aged 8m-
13y, 4m / 9f) who underwent EGD with
biopsy for clinical reasons (BxC)
showed normal sucrase activity (>25
EU/g) and BT. It was postulated that
the 60’ or 75’ time-points might be
best. Thirty-four healthy,
asymptomatic and historically
sucrose-tolerant children (ASTC)
underwent 2-day/2-stage 13C-SUC/GLU BT
and results were compared with data
from the BxC subjects. A sub-set of
19 ASTC subjects repeated BT using a
modified 1day-biphasic 13C-SUC/GLU BT
in which the second, serial 13C-GLU
comparative substrate was super-dosed
(5X) to negate any residual SUC
effects. Delta enrichment measures
attributed to the GLU were adjusted
(X/5) before the sucrose CGO was
calculated. The results between the
2-day and 1-day test methods were
compared and the best diagnostic
time-point and cut-off were
determined based on 99% confidence
interval (C.I.). RESULTS: The CSID
patients had very low 2-day/2-stage
13C-sucrose CGO for both 60’ & 75’
time-points (0.25±0.19 vs. 0.36±0.29,
NSD) and CSID differed from BxC
subjects (60’:1.31±0.31; 75’:1.31±0.27;
p<0.01). 13C-SCGO enrichment values
for BxC subjects did not differ from
those obtained from ASTC subjects at
60’ (1.31±0.31 vs. 1.02±0.52, p=0.08),
but improved at the 75’ time point
(1.27±0.27 vs. 0.95±0.36, p<0.01).
Among the 19 ASTC subjects who also
completed the 1d-biphasic 13C-SUC/GLU
• Simple and complex
carbohydrates provide more
than
60% of the usual human
dietary energy needs.
• Disaccharides and
Oligosaccharides
(sucrose, lactose ,
maltose, maltotriose)
• Alpha-Limit Dextrins
(minimal 1-6 branches)
• Amylose (mostly straight
chains glucose (G) units)
•Amylopectin ( 1-4 & 1-6
branches after ~20 G )
• Glycogen (branches after
~8-10 G units)
• Oligosaccharides and
complex carbohydrates must be
completely hydrolyzed
to permit absorption via
luminal
enterocyte apical
transporters:
• SGLT1 absorbs glucose &
galactose
• GLUT5 absorbs fructose
• Dietary Fiber (is not
proximally digested ).
• Sucrase is the only brush
border disaccharidase that
digests sucrose, but it
also hydrolyzes starch 1-4
bonds.
Treem WR. J Pediatr. 1996;128(6):727-9.
Sucrose
• Sucrose is a non-reducing
disaccharide that requires
complete hydrolysis for
most absorption to occur.
FW: C12H22O11 MW: 342
Da
• Proportionally more C, by
atomic weight ((18Da) , than
2X glucose due to
glycoside bond (water loss,
2% FW).
• Due to the fructose
component, sucrose substrate
is
oxidized to CO2
approximately 1.16X faster
than
glucose (cellular
metabolism of fructose is
relatively
unregulated compared to
glucose).
• Minute quantities directly
diffuse into the body down
the
concentration gradient
to appear in the urine if not
first hydrolzyed by gut
sucrase (no renal sucrase).
• Malabsorption , due to
maldigestion, is associated
with
abdominal pain,
Materials:
• UL-13C-substrates (Sigma-Aldrich
Isotec Div., Miamisberg, OH).
Due to 13C -FW and hydroscopic
differences, 13C-glucose and
13C-sucrose doses were cross-
calibrated for 13C-content using an
in-vitro yeast fermentation
assay with measure of post-
reaction
13CO2 enrichments, then,
validated in multiple known normal
human controls to provide
basis for data corrections.
Atomic Enrichment: 13C-glucose
10.00 mg/dL demonstrates
an identical 13CO2 BT enrichment
signal as 13C-sucrose
9.50 mg/dL; a ≅ 5% difference of
DOB (‰) signal (cf=1.055).
2-day Breath Test
• 13C-sucrose 20 mg. in 20%
excipient (20 gm. / dL)
Methods:
•34 asymptomatic, historically
healthy preschool and
adolescent sucrose-tolerant
children were screened with an
8-hr timed urine collection
after consuming a sucrose load
and surveyed for the typical
reactogens associated with
sucrose maldigestion .
Subjects who demonstrated the
appearance of sucrose in the
urine by chromotography and/or
experienced abdominal
discomfort or excessive
flatulence or positive breath
hydrogen test were excluded
from the study ( n=1 ).
•All qualified subjects (n=33)
had 2-day serial 13C-sucrose
(SUC) and 13C-glucose (GLU)
breath testing and oxidation
results were compared. After
obtaining a 1.5L baseline
breath sample prior to
substrate ingestion, serial
breath collections were
obtained every 15 mins though
135 mins to observe for peak
13CO2-enrichment and onset of
isotopic depletion in breath.
BT enrichments were assayed
using a 13CO2 infrared
spectrophotometer (POCone®,
Otsuka Electronics, Japan).
DOB ‰ values were obtained for
each time-point.
•Oxidation results were
measured as a change in
isotopic enrichment ratio
between the reference GLU and
the test SUC substrates and
expressed as [sucrose
Coefficient of Glucose
Oxidation (CGO)]. This
adjusted for (blanked-out)
inter-subject differences in
habitus and variable effects
of total CO2-rate (metabolic
rate); therefore represented
isolated SUC mucosal digestive
capacity. A ratio was
calculated for each paired
outcome, sample means and
standards deviations were
Results
Conclusions
Introduction Sucrase-Isomaltase
Study Goal
• To perfect the 13C-sucrose
breath test to facilitate its
routine use in the
clinical and clinical research
setting
to assess mucosal
sucrase-isomaltase activity.
.
• To confirm the ideal dose
of 13C-sucrose (~98% APE).
• To identify the minimal
number samples of necessary to
facilitate an accurate
point-of-care test result.
• To identify the minimal
time point necessary to
estimate the peak rate
of sucrose digestion/oxidation
measured oxidation.
• To estimate the normal
parameters (diagnostic cut-
off;
Delta-Over–Baseline,
DOB) for sucrose digestion and
13C-oxidation from sucrose as
a percentage of the reference
13C-oxidation from glucose,
defined as the sucrose
Coefficient of Glucose
Oxidation (Sucrose-CGO ).
Human IRB Approval: H10239
Written informed consent
was obtained for all
participants.Support
This study was supported in
part by an investigator-
initiated grant from QOL
Medical, LLC (Vero Beach,
Florida, USA)
Figure 1. Composite 13C-sucrose oxidation
data (‰) after substrate ingestion. There
were statistical differences between the
means at each time-point between 45 and 75
mins (ANOVA) and NSD between 75’ & 90”.
Subjectively, the primary hydrolysis rate
approximates a plateau enrichment after 60
to 75 mins when mean oxidation approximates
~13.3 Δ‰ (SD ±6.5; 95% CI = 11.1-15.5) and
depletion begins by 105 minutes. The 75 min
time-point was identified as the earliest
critical decision point.
Figure 2. Composite Coefficient of Glucose
Oxidation (CGO) data for 13C-sucrose oxidation
following substrate ingestions Primary hydrolysis
approximates plateau appeared after 60 or 75 mins
(mean CGO 60’ = 0.96; CGO 75’ = 0.91). ANOVA
revealed that the data obtained at the 45-min
time-point did not statistically differ from the
60-min time point, nor did the data obtained at 75
mins significantly differ from that obtained at 60
mins, though the SE was minimal at 75 mins. Using
ANOVA, data obtained at the 30-min time-points did
statistically differ from the 45- and 60 min time-
points (Tukey HSD Test p<0.05), suggesting that
the optimal point of maximum first-pass oxidation
had been clearly identified ( 75 mins).
Figure 3. The graphs demonstrate the similarities
of outcomes between the 2-day 13C-sucrose breath
test panel (CGO at the 75’ time-point) and the 1-
day biphasic 13C-sucrose CGO at the 75’ time-point.
The data were not statistically different (T-Test:
p= 0.95). The mean CGO for the two-day test was
0.96 (95% CI ±.18) vs. 0.98 (95% CI ±.11) for the
13C-sucrose biphasic breath test. The diagnostic
threshold (LL) was estimated to be 75%. The
outliers shown on the 2-day for the 13C-sucrose CGO
(2-day test) may be explained by slightly
different gastric-emptying rates for the
substrates on different days. The assumption was
that the rate of gastric emptying during the
single-day, biphasic breath test was relatively
uniform and may have contributed to the improved
(decreased) data spread).
• CSID-patients had very low
2-day 13C-Suc CGO for both
60’ & 75’ time-points
(0.25±0.19 vs. 0.36±0.29, NSD)
&
CSID significantly differed
from BxC subjects at
(60’:1.31±0.3 &
75’:1.31±0.3; p <0.01).
• 13C-SCGO enrichment values
for BxC subjects did not differ
from those obtained from
ASTC subjects at 60’ (1.31±0.31
vs. 1.02±0.52, p=0.08), but
improved at the 75’ time-point
(1.27±0.3 vs. 0.95±0.3, p
<0.01).
• A decision CGO threshold of
<75% for abnormal sucrose
digestion was identified
at the 75 min time-point (T-
optimal).
• The 1-day-biphasic SUC/GLU
breath test is feasible for
non-invasive mucosal
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
0 15 30 45 60 75 90 105 120 135 150
Enrichment(13CO2DOB)
Time (Minutes)
Mean 13C-Sucrose Oxidation for Preschoolers and Adolescents
(unadjusted, n = 33)
Upper Limit Mean
+ SEM
MEAN
Lower Limit Mean
- SEM
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
0 15 30 45 60 75 90 105 120 135 150
13CO2Enrichment(DOB)
Time (Minutes)
Mean Coefficient of Glucose Oxidation for
13C-Sucrose, 20 mg. for Preschoolers and Adolescents (n=33)
Standard Error
(UL)
MEAN, All; N=33
Standard Error
(LL)
0
0.5
1
1.5
2
2.5
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E
n
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h
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C
G
O
%
)
2-Day Test
13C-Sucrose CGO @ 75 mins
0
0.5
1
1.5
2
2.5
0 25 50 75 100
E
n
r
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h
m
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1-Day BiPhasic Test
13C-Sucrose CGO @ 75 mins
75%
Baylor College of Medicine