Clinical Chemistry 43: 518-522, 1997;
(Clinical Chemistry. 1997;43:518-522.)
© 1997 American Association for Clinical Chemistry, Inc.
Application of isotope-selective nondispersive infrared spectrometry (IRIS) for evaluation of [13C]octanoic acid gastric-emptying breath tests: comparison with isotope ratio–mass spectrometry (IRMS)
Peter Schadewaldt1,a,
Bernd Schommartz2,
Gregor Wienrich3,
Herbert Brösicke4,
Ralf Piolot1 and
Dan Ziegler1
1
Diabetes Forschungsinstitut,
2
Nuklearmedizinische Klinik, and
3
Kinderklinik, Heinrich-Heine-Universität Düsseldorf, D-40225 Düsseldorf, Germany.
4
Kinderklinik, Humbold-Universität zu Berlin,
D-13353 Berlin, Germany.
a Address correspondence to this author at: Diabetes Forschungsinstitut, Klinische Biochemie Auf'm Hennekamp 65, D-40225 Düsseldorf, Germany. Fax ++49-211-3382-603; e-mail schadewa{at}uni-duesseldorf.de
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Abstract
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Suitability of isotope-selective nondispersive infrared spectrometry
(IRIS) for evaluation of [13C]octanoic acid
gastric-emptying breath test was assessed and compared with standard
isotope ratio–mass spectrometry (IRMS). The estimated bias of IRMS and
IRIS measurements of baseline-corrected 13CO2exhalation amounted to ± 0.1 and ± 0.6 
values
(n = 360), respectively. In breath tests performed on 60 diabetic
patients, the gastric emptying parameters were calculated by nonlinear
regression analysis of the time course of 13CO2exhalation: half-emptying time
(t1/2,breath, 90 ± 39 min), lag
phase (tlag,breath, 34 ± 27 min), and
gastric emptying coefficient (GEC, 2.9 ± 0.5). A reasonable
linear correlation was found between the two methods
(y = IRIS, x = IRMS) with respect to
values (y = 0.35 + 0.92x,
r = 0.985, Sy|x
= ±0.6, n = 1116) and a rather good agreement of the computed
gastric emptying parameters was obtained
(t1/2,breath: y =
0.99x + 4.06, Sy|x =
±6.3; tlag,breath: y =
0.97x + 0.96, Sy|x =
±3.4; GEC: y = 0.97x - 0.01,
Sy|x = ±0.09).
Key Words: indexing terms: diabetes mellitus • exhalation rate • delayed gastric emptying
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Introduction
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Delayed gastric emptying is a relatively frequent complication in
a variety of diseases (see ref. 1 for review), e.g., in
functional dyspepsia (2)(3), AIDS
(4), and diabetes mellitus (5)(6)(7).
Successfull treatment of delayed gastric emptying by prokinetic drugs
has been described (2)(8)(9)(10)(11).
Diagnosis of impaired gastric emptying and control of therapy is
generally based on the assessment of gastric emptying of solids because
disturbances of solid emptying precede impairment of liquid emptying
(1). For the measurement of gastric emptying, various
methods have been described. 99mTc-colloid-based
scintigraphy is considered the reference method
(7)(12)(13)(14); other procedures are real-time
ultrasonography (9)(11), radiopaque method
(15), metal sphere method (10), and
sulfamethiazole capsule method (13).
Previously, a noninvasive [13C]octanoic acid
gastric- emptying breath test was developed by Ghoos et al., based on
the analysis of the time course of 13C-label enrichment in
13CO2 in the exhaled air (16). For
sensitive and accurate measurement of
13CO2/12CO2 ratio in
breath samples, isotope ratio–mass spectrometry (IRMS) is commonly
used as an established standard method
(17).1
Some studies indicate that infrared diode laser
spectroscopy (18), infrared heterodyne ratiometry
(19), and particularly infrared spectroscopy
(20)(21)(22)(23)(24) may prove to be alternative, far more simple, and
easy-to-operate optical methods for 13CO2
analysis in breath samples under routine laboratory conditions.
A newly designed (25) nondispersive infrared spectrometer
for 13CO2 measurements in breath samples (IRIS)
has now become commercially available. In the present study we examined
the applicability of the IRIS method as compared with standard IRMS for
evaluation of the [13C]octanoic acid breath test
parameters.
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Subjects and Methods
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subjects
Informed consent was obtained from all subjects entering the
study. The present data were obtained in gastric-emptying tests
performed in clinical studies on diabetic gastroparesis. The clinical
outcome of the studies and the medical implications will be reported
elsewhere. The study protocol was in accordance with the Helsinki
Declaration as revised in 1983 and had been approved by the
Ethik-Kommission of the Heinrich-Heine-Universität
Düsseldorf.
Twenty healthy volunteers (11 male, 9 female, mean age 35.4 ±
11.9 years, weight 73.4 ± 13.2 kg, height 173 ± 11 cm) and
62 diabetic patients (31 male, 31 female, mean age 37.0 ± 15.9
years, weight 70.3 ± 13.3 kg, height 173 ± 9 cm) recruited
from the inpatient clinic of the Diabetes Forschungsinstitut were
investigated. Insulin-dependent patients received a subcutaneous
injection of the usual insulin 30 min before the test meal. The insulin
dose was adjusted according to the fasting blood glucose concentrations
and the meal modification (7).
[13c]octanoic acid breath test
Breath tests were performed as previously described by Ghoos et
al. (16). In short, overnight fasted healthy subjects and
diabetic patients ingested a test meal between 0800 and 0830 within 10
min. The meal comprised a scrambled egg with the yolk mixed with 0.1 mL
of [13C]octanoic acid (1-13C, 99%;
Promochem, Wesel, Germany) and yolk and egg white fried separately, two
slices of white bread, and 5 g of margarine (42% carbohydrate,
18% protein, 40% fat; ~250 cal), followed by 150 mL of mineral
water. During the following 4-h test period the subjects stayed in a
sitting position in a separate test room. Breath samples for
13CO2 measurement were collected in 1.5-L
breath bags (Tecobag; Tesseraux Container, Bürstadt, Germany)
every 10 min during the first hour and thereafter in 15-min intervals.
Control (baseline) samples were collected before the test meal. For
13CO2 analysis by IRMS, aliquots were withdrawn
from each bag and filled in duplicate into 10-mL evacuated tubes
(Vacutainer Tube; Becton Dickinson, Heidelberg, Germany) by means of a
50-mL syringe (Perfusor-Spritze, B.Braun, Melsungen, Germany) equipped
with a three-way stopcock.
analysis of 13co2 in breath
Measurement of 13CO2 in exhaled air
was carried out by standard mass spectrometric methods with a Finnigan
MAT (Bremen, Germany) Model 251 isotope ratio–mass spectrometer as
previously described (7).
IRIS was performed with the infrared spectrometer at our disposal (from
Wagner Analysentechnik, Worpswede, Germany). The principle of this
recently developed new device for 13CO2
measurement in gaseous samples has been described [23;
Scheme 1]. Breath samples were transferred directly from the breath
bags into the measuring cuvettes by means of a pump. About 300 mL of
gas are needed for thorough flushing of cuvettes and reliable
measurement. All data for 13C enrichment in breath
CO2 were read out as baseline-corrected values, as
these are used for the diagnostic evaluation of breath tests
(26).
calculations and statistics
Evaluation of the breath tests was performed as outlined by Ghoos
et al. (16). The increase in breath
13CO2 after ingestion of
[13C]octanoic acid (determined as values) was used
to calculate the extra 13CO2 exhalation rate
assuming a mean endogenous CO2 production of 5 mmol ·
[min · m2 (body surface)]-1
(27). Body surface was estimated by using the
weight–height formula given by Haycock et al. (28).
Results are expressed as the percentage of
13CO2 recovery per min and cumulatively over
the test period of 4 h.
As suggested by Ghoos et al. (16), the data were used for
mathematical curve fitting. Nonlinear regression analysis as provided
by the SOLVER procedure of the Excel 5.0 program was used, applying the
formulas y = atb ·
e-ct and y = m · (1-
e-kt)ß, respectively
((13); see (15) for
discussion). According to Ghoos et al. (16), the following
three main breath test parameters of gastric emptying were computed
from the coefficients k, ß, and a, respectively: half emptying time
[t1/2,breath = (1/k) ·
ln(1-2-1/ß) - 66], lag phase
[tlag,breath = (1/k) · ln(ß) - 66], and
gastric emptying coefficient [GEC = ln(a)].
In two diabetic patients, markedly delayed gastric emptying resulted in
a poor curve fitting and insignificant values for the gastric emptying
parameters. Therefore, the parameters of these patients were excluded
from the parameter evaluation.
In general, results are presented as means ± SD with the number
of determinations in parentheses. Linear regression analysis
(least-square method) was used for statistical comparison of
parameters.
Because of the limited volume of an individual breath, standard
procedures for evaluation of between- and within-run reproducibility
were not applicable with authentic breath samples. Therefore, two
consecutive series of measurements were performed on samples from a
representative number of breath tests. Statistical evaluation of
repeatability was carried out by using linear regression analysis.
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Results
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For evaluation of repeatability, samples from several
[13C]octanoic acid gastric-emptying breath tests
were subjected to repeated analysis of 13CO2 by
IRMS (n = 20) or IRIS (n = 20). 13C enrichment in
the samples was evenly distributed and ranged from 0 to ~20
values (data not shown). When the results from the first and second
analysis run were plotted on the x- and y-axes,
respectively, linear regression analysis of the data yielded standard
deviations of repeated IRMS [y = 0.034 (SD 0.013) +
0.999 (SD 0.001)x, r = 0.9997, n =
360] and IRIS [y = 0.076 (SD 0.074) + 0.984 (SD
0.009)x, r = 0.9857, n = 360]
measurements (Sy|x) of ± 0.1
values and ± 0.6 values, respectively.
In Table 1
, the gastric emptying parameters are listed as computed from
the IRIS and IRMS measurements, together with an analysis of parameter
reproducibility. The calculated bias of the duplicate determinations
with IRIS (IRMS results given in parentheses) of gastric half-emptying
time, lag phase, and GEC in the 20 breath tests amounted to 2.4 ±
2.1 min (0.6 ± 0.6 min), 2.6 ± 3.0 min (0.2 ± 0.2
min), and 0.03 ± 0.04 (0.01 ± 0.01), respectively. These
values were equivalent to a mean relative deviaton of 2.7 ± 2.3%
(0.8 ± 0.7%), 6.1 ± 5.8% (1.1 ± 1.9%), and
1.0 ± 0.9% (0.3 ± 0.3%), respectively, when related to
the individual gastric emptying parameters of the investigated
subjects.
For further evaluation of the IRIS method, IRIS and IRMS measurements
were performed in parallel with breath samples from a representative
number of [13C]octanoic acid gastric emptying breath
tests performed in diabetic patients (n = 60). When plotted, some
scatter of the individual data points around the regression line was
observed (Fig. 1
). However, when the data were used to compute the
gastric emptying parameters of the patients, a good agreement was
obtained between the results of both methods (Table 2
, Fig. 2
).When related to the gastric emptying parameters as estimated by
nonlinear regression analysis on the basis of IRMS measurements, the
bias of IRIS-based determinations of gastric half-emptying time (range
24–252 min), lag phase (range -23–140 min), and GEC (range 1.7–3.9)
in these breath tests amounted to 5.8 ± 4.1 min, 2.6 ± 2.4
min, and 0.11 ± 0.07 (n = 60), respectively. The variation
(±SD) of the IRIS results relative to the IRMS results (
100%)
were ± 7.2%, ± 9.9%, and ± 3.2%, respectively. The
overall means (and standard deviations) of the gastric emptying
parameters of the investigated diabetic patients were practically
identical (Table 2
).
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Figure 1. Comparison of IRMS and IRIS measurements of
13C-enrichment in CO2 in samples from
[1-13C]octanoic acid gastric-emptying breath tests.
Dashed line: regression line (least-square method;
y = 0.346 (SD 0.036) + 0.917 (SD 0.005)x,
r = 0.985, Sy|x =
0.597, n = 1116 from 60 breath tests).
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Table 2. Correlation of [13C]octanoic acid
gastric-emptying breath test parameters as computed on the basis of
IRMS and IRIS 13CO2
measurement.
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Figure 2. Correlation of gastric half-emptying time
(t1/2,breath) in [1-13C]octanoic
acid gastric-emptying breath tests as computed on the basis of IRMS and
IRIS 13CO2 measurements in breath samples from
60 diabetic patients.
Nonlinear regression analysis was used for mathematical curve fitting
and parameter evaluation as detailed in Subjects and
Methods. Dashed line: regression line (see Table 2
for
data).
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Discussion
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The [13C]octanoic acid gastric-emptying breath
test is a noninvasive procedure for the assessment of gastric emptying
rate of solids and has been validated in healthy subjects
(16)(29) and diabetic patients
(7) by using standard scintigraphic methods. For
13CO2 analysis in diagnostic breath tests, IRMS
is generally applied as a well-established reference method. So far,
the principle of infrared spectrometry has been only used in a few
limited studies on the detection of Helicobacter pylori
infections by [13C]urea breath tests
(22)(23)(24).
In the present study, we evaluated the routine clinical use of a
commercially available infrared spectrometer (IRIS) for the
[13C]octanoic acid gastric-emptying breath test
procedure.
As is evident from the data presented, repeatability of IRIS
13CO2 measurements in breath test samples
was somewhat less superior than the expected excellent repeatability of
IRMS analyses. According to these data, IRIS data may be expected to
deviate up to about ± 1.2 values (i.e., ± 2SD, 95%
confidence level) from the IRMS results. This degree of deviation was
verified experimentally in the present study (Fig. 1
). It is
noteworthy, however, that neither the variability of repeated IRIS
measurements nor the deviations from the IRMS data exceeded the natural
fluctuations in the 13CO2 content in expired
air of overnight fasted subjects (SD about ± 0.7 value
(26)).
The statistical variability of the IRIS data was extenuated in the
evaluation of [13C]octanoic acid breath tests, where
calculation of the gastric emptying parameters by nonlinear regression
analysis was based on 13CO2 analyses in
multiple breath samples with a wide range of
13C-enrichment. Thus, the variability of gastric emptying
parameters as obtained in repeated IRIS determinations (Table 1
) and in
the comparative IRIS vs IRMS measurements (Table 2
) was far lower than
the inter- ((7)(16), Table 1
) and
intraindividual variability (7)(16) that had
been found in healthy subjects and patients; e.g., the reported gastric
half-emptying time in overnight fasted healthy subjects is ~80 min
and the intra- and interindividual standard deviation about ± 30
min ((7)(16), Table 1
), whereas the standard
errors of estimate of the half-emptying time in the repeated IRIS
analyses and in the comparison of the IRIS with the IRMS determinations
was ± 3 min (Table 1
) and ± 6 min (Table 2
), respectively.
Although the accuracy of IRIS determinations of 13C
enrichment in breath CO2 samples is notably lower than that
of standard IRMS, the present results suggest that the IRIS method is
suitable for the evaluation of the [13C] octanoic acid
gastric-emptying breath test procedure under routine clinical
conditions. In some cases of doubt, however, in which the gastric
emptying parameters are just on the border between normal and
pathological values, it may be advisable to reexamine the outcome by
repetition of the breath test.
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Scheme 1. Principle of setup of IRIS for analysis of
13CO2 in gaseous samples.
Cuvettes are filled by means of a pump. 13CO2
(upper channel) and 12CO2 (lower channel)
content-dependent light absorption are monitored separately in special
measuring/reference cuvette systems by appropriate microphone detectors
of the Lehrer–Luft type.
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Acknowledgments
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We are indebted to M. Karallus, A. Pour Mirza, and to B. Teuber for
assistance in performing the [13C]octanoic acid
breath tests, to A. Pfundstein for technical assistance in IRMS
measurements, to L. Bohne for some IRIS measurements, to K. Dannehl for
statistical advice, and to M. Haisch for advice with Scheme 1. The
support and valuable discussions of F.A. Gries and U. Wendel are
gratefully acknowledged.
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Footnotes
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This work contains part of the Thesis of B.S.
1 Nonstandard abbreviations: IRMS, isotope ratio–mass spectrometry; IRIS, isotope-selective nondispersive infrared spectrometry; and GEC, gastric emptying coefficient. 
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Abstract
Introduction
