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Application of isotope-selective nondispersive infrared spectrometry (IRIS) for evaluation of [[sup.13]C]octanoic acid gastric-emptying breath tests: comparison with isotope ratio-mass spectrometry (IRMS).

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 [5-7]. Successfull treatment of delayed gastric emptying by prokinetic drugs has been described [2, 8-11].

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. [sup.99m]Tc-colloid-based scintigraphy is considered the reference method [7,12-14]; other procedures are real-time ultrasonography [9,11], radiopaque method [15], metal sphere method [10], and sulfamethiazole capsule method [13].

Previously, a noninvasive [[sup.13]C] octanoic acid gastric-emptying breath test was developed by Ghoos et al., based on the analysis of the time course of [sup.13]C-label enrichment in [sup.13]C[O.sub.2] in the exhaled air [16]. For sensitive and accurate measurement of [sup.13]C[O.sub.2]/[sup.12]C[O.sub.2] ratio in breath samples, isotope ratio-mass spectrometry (IRMS) is commonly used as an established standard method [17]. (5)

Some studies indicate that infrared diode laser spectroscopy [18], infrared heterodyne ratiometry X19], and particularly infrared spectroscopy [20-24] may prove to be alternative, far more simple, and easy-to-operate optical methods for [sup.13]C[O.sub.2] analysis in breath samples under routine laboratory conditions.

A newly designed X25] nondispersive infrared spectrometer for [sup.13]C[O.sub.2] 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 [[sup.13]C] octanoic acid breath test parameters.

Subjects and Methods

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-Universitat Dusseldorf.

Twenty healthy volunteers (11 male, 9 female, mean age 35.4 [+ or -] 11.9 years, weight 73.4 [+ or -] 13.2 kg, height 173 [+ or -] 11 cm) and 62 diabetic patients (31 male, 31 female, mean age 37.0 [+ or -] 15.9 years, weight 70.3 [+ or -] 13.3 kg, height 173 [+ or -] 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].

[[sup.13]C]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 [[sup.13]C] octanoic acid (1-[sup.13]C, 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 [sup.13][O.sub.2] measurement were collected in 1.5-L breath bags (Tecobag; Tesseraux Container, Burstadt, Germany) every 10 min during the first hour and thereafter in 15-min intervals. Control (baseline) samples were collected before the test meal. For [sup.13]C[O.sub.2] 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 [sup.13]C[O.sub.2] IN BREATH

Measurement of [sup.13]C[O.sub.2] 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 [sup.13]C[O.sub.2] 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 [sup.13]C enrichment in breath C[O.sub.2] were read out as baseline-corrected [DELTA][delta] 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 [sup.13]C[O.sub.2] after ingestion of [[sup.13]C] octanoic acid (determined as 08 values) was used to calculate the extra [sup.13]C[O.sub.2] exhalation rate assuming a mean endogenous C[O.sub.2] production of 5 mmol x [min x [m.sup.2] [(body surface)].sup.-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 [sup.13]C[O.sub.2] 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 = [at.sup.b] x [e.sup.-ct] and y = m x [(1- [e.sup.-kt]).sup.[beta]], 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, [beta], and a, respectively: half emptying time [[t.sub.1/2,breath], = (1/k) x ln(1-[2.sup.-1/[beta]]) - 66], lag phase [[t.sub.lag,breath] = (1/k) x 1n([beta])-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.

[ILLUSTRATION OMITTED]

In general, results are presented as means [+ or -] 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.

Results

For evaluation of repeatability, samples from several [[sup.13]C] octanoic acid gastric-emptying breath tests were subjected to repeated analysis of [sup.13]C[O.sub.2] by IRMS (n = 20) or IRIS (n = 20). [sup.13]C enrichment in the samples was evenly distributed and ranged from 0 to ~20 [DELTA][delta] 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 ([S.sub.y|x]) of [+ or -] 0.1 [DELTA][delta] values and [+ or -] 0.6 [DELTA][delta] 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 [+ or -] 2.1 min (0.6 [+ or -] 0.6 min), 2.6 [+ or -] 3.0 min (0.2 [+ or -] 0.2 min), and 0.03 [+ or -] 0.04 (0.01 [+ or -] 0.01), respectively. These values were equivalent to a mean relative deviaton of 2.7 [+ or -] 2.3% (0.8 [+ or -] 0.7%), 6.1 [+ or -] 5.8% (1.1 [+ or -] 1.9%), and 1.0 [+ or -] 0.9% (0.3 [+ or -] 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 [+ or -] 4.1 min, 2.6 [+ or -] 2.4 min, and 0.11 [+ or -] 0.07 (n = 60), respectively. The variation (-SD) of the IRIS results relative to the IRMS results (=100%) were [+ or -] 7.2%, [+ or -] 9.9%, and [+ or -] 3.2%, respectively. The overall means (and standard deviations) of the gastric emptying parameters of the investigated diabetic patients were practically identical (Table 2).

Discussion

The [[sup.13]C]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 [71 by using standard scintigraphic methods. For [sup.13]C[O.sub.2] 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 [[sup.13]C]urea breath tests [22-24].

In the present study, we evaluated the routine clinical use of a commercially available infrared spectrometer (IRIS) for the [[sup.13]C] octanoic acid gastric-emptying breath test procedure.

[FIGURE 1 OMITTED]

As is evident from the data presented, repeatability of IRIS [sup.13]C[O.sub.2] 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 [+ or -] 1.2 [DELTA][delta] values (i.e., [+ or -] 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 [sup.13]C[O.sub.2] content in expired air of overnight fasted subjects (SD about [+ or -] 0.7 [delta] value [261).

The statistical variability of the IRIS data was extenuated in the evaluation of [[sup.13]C] octanoic acid breath tests, where calculation of the gastric emptying parameters by nonlinear regression analysis was based on [sup.13]C[O.sub.2] analyses in multiple breath samples with a wide range of [sup.13]C-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 [+ or -] 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 [+ or -] 3 min (Table 1) and [+ or -] 6 min (Table 2), respectively.

[FIGURE 2 OMITTED]

Although the accuracy of IRIS determinations of [sup.13]C enrichment in breath C[O.sub.2] samples is notably lower than that of standard IRMS, the present results suggest that the IRIS method is suitable for the evaluation of the [[sup.13]C] 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.

We are indebted to M. Karallus, A. Pour Mirza, and to B. Teuber for assistance in performing the [[sup.13]C]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.

References

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PETER SCHADEWALDT, (1) * BERND SCHOMMARTZ, (2) GREGOR WIENRICH, (3) HERBERT BROSICKE; RALF PIOLOT, (1) and DAN ZIEGLER (1)

(1) Diabetes Forschungsinstitut, (2) Nuklearmedizinische Klinik, and (3) Kinderklinik, Heinrich-Heine-Universitit Dusseldorf, D-40225 Dusseldorf, Germany.

(4) Kinderklinik, Humbold-Universitat zu Berlin, D-13353 Berlin, Germany.

(5) Nonstandard abbreviations: IRMS, isotope ratio mass spectrometry; IRIS, isotope-selective nondispersive infrared spectrometry; and GEC, gastric emptying coefficient.

* Address correspondence to this author at: Diabetes Forschungsinstitut, Klinische Biochemie Auf'm Hennekamp 65, D-40225 Dusseldorf, Germany. Fax ++49-211-3382-603; e-mail schadewa@uni-duesseldorf.de.

This work contains part of the Thesis of B.S.

Received July 1, 1996; revised September 23, 1996; accepted October 4, 1996.
Table 1. Reproducibility of [[sup.13]C]octanoic acid gastric-emptying
breath test parameters.

 Parameters of
 reproducibility
 (linear regression
 analysis)
 Gastric emptying
 parameters Mean

IRIS [t.sub.1/2, breath], min 100.7 [+ or -] 53.7
 [t.sub.lag, breath], min 42.8 [+ or -] 32.6
 GEC 2.90 [+ or -] 0.55

IRMS [t.sub.1/2, breath], min 79.0 [+ or -] 24.4
 [t.sub.lag, breath], min 24.8 [+ or -] 16.1
 GEC 3.33 [+ or -] 0.35

 Parameters of reproducibility
 (linear regression analysis)

 Slope Intercept

IRIS 0.997 [+ or -] 0.014 1.076 [+ or -] 1.572
 1.045 [+ or -] 0.025 -0.798 [+ or -] 1.366
 0.980 [+ or -] 0.021 0.049 [+ or -] 0.061

IRMS 1.006 [+ or -] 0.008 -0.065 [+ or -] 0.640
 1.004 [+ or -] 0.004 -0.014 [+ or -] 0.116
 0.998 [+ or -] 0.008 0.004 [+ or -] 0.026

 Parameters of reproducibility
 (linear regression analysis)

 r [SD.sub.y-values]

IRIS 0.9983 [+ or -] 3.240
 0.9946 [+ or -] 3.640
 0.9959 [+ or -] 0.050

IRMS 0.9995 [+ or -] 0.825
 0.9999 [+ or -] 0.277
 0.9995 [+ or -] 0.012

Data for gastric [t.sub.1-2, breath], [t.sub.lag, breath], and GEC
were obtained by curve fitting by using nonlinear regression analysis
as detailed in Subjects and Methods. Two analysis runs were performed
on the samples from independent breath tests with 20 diabetic patients
(10 male, 10 female; mean age 41.3 [+ or -] 16.6 years, weight 67.3
[+ or -] 10.8 kg, height 172 [+ or -] 10 cm) and 20 healthy volunteers
(see methods section), respectively. Results from the first and second
analysis were plotted on the x-and y-axes, respectively. Linear
regression analysis (least-square method) was used for statistical
evaluation. Data are presented as means [+ or -] SD (based on data
from the first analysis run), when applicable.

Table 2. Correlation of [[sup.13]C]octanoic acid gastric-emptying
breath test parameters as computed on the basis of IRMS and IRIS
[sup.13]C[O.sub.2] measurement.

 Mean

Gastric emptying
 parameters IRMS IRIS

[t.sub.1/2,
 breath], min 87.0 [+ or -] 38.7 90.4 [+ or -] 38.9
[t.sub.lag,
 breath], min 34.5 [+ or -] 27.3 34.4 [+ or -] 26.7
GEC 2.95 [+ or -] 0.50 2.85 [+ or -] 0.49

 Correlation parameters
 (linear regression analysis)

Gastric emptying
 parameters Slope Intercept

[t.sub.1/2,
 breath], min 0.994 [+ or -] 0.021 4.061 [+ or -] 2.006
[t.sub.lag,
 breath], min 0.973 [+ or -] 0.016 0.960 [+ or -] 0.717
GEC 0.971 [+ or -] 0.025 -0.006 [+ or -] 0.073

 Correlation parameters
 (linear regression analysis)

Gastric emptying
 parameters r [s.sub.y|x]

[t.sub.1/2,
 breath], min 0.987 66.271
[t.sub.lag,
 breath], min 0.992 63.432
GEC 0.982 60.093

Data for gastric [t.sub.1-2, breath], [t.sub.lag], breath, and GEC
were obtained by curve fitting by using nonlinear regression analysis
as detailed in Subjects and Methods. IRMS and IRIS measurements were
performed on samples from 60 independent breath tests with diabetic
patients and were plotted on the x-and y-axes, respectively (Fig. 2).
Linear regression analysis (least-square method) was used for
statistical evaluation. Data are presented as means [+ or -] SD, when
applicable.
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Title Annotation:Automation and Analytical Techniques
Author:Schadewaldt, Peter; Schommartz, Bernd; Wienrich, Gregor; Brosicke, Herbert; Piolot, Ralf; Ziegler, D
Publication:Clinical Chemistry
Date:Mar 1, 1997
Words:3813
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