Towards an Inhalative 13C Breath Test Method

Abstract

Customary ¹³CO₂ breath tests—and also ¹⁵N urine tests—always start with an oral administration of a test substrate. The test person swallows a stable isotope labelled diagnostic agent. This technique has been used to study several pathophysiological changes in gastrointestinal organs. However, to study pathophysiological changes of the bronchial and lung epithelium, the inhalative administration of a stable isotope labelled agent appeared more suitable to us. [1-¹³C]Hexadecanol and [1-¹³C]glucose were chosen. Inhaled [1-¹³C]hexadecanol did not yield ¹³CO₂ in the exhaled air, but [1-¹³C]glucose did. To study the practicability of the [1-¹³C]glucose method and the reproducibility of the results, 18 inhalation tests were performed with healthy subjects. In 6 self-tests, the optimum inhalative dose of [¹³C]glucose was determined to be 205 mg. Using the APS aerosol provocation system with the nebulizer ‘Medic Aid’ (Erich Jaeger Würzburg), a 25% aqueous solution was inhaled. Then, breath samples were collected at 15 min. intervals and analysed for ¹³CO₂. 75–120min after the end of inhalation a well-reproducible maximum δ¹³C value of 6‰ over baseline (DOB) was detected for 12 healthy probands.

Speculating that the pulmonary resorption of the [¹³C]glucose is the rate-limiting step of elimination, decompensations in the epithelium ought to be reflected in changed [1-¹³C]glucose resorption rates and changed ¹³CO₂ output.

Therefore, we speculate that the inhalation of suitable ¹³C-labelled substrates will pave the way for a new group of ¹³CO₂ breath tests aiding investigations of specific pathophysiological changes in the pulmonary tract, such as inflammations of certain sections and decompensations of cell functions.     

Comparison between the oral and intravenous L-[1-13C]phenylalanine breath test for the assessment of liver function

To simplify the L-[1-13C]phenylalanine breath test which is used to assess liver function the tracer is usually given orally, and CO2 production rate is estimated. In 12 healthy volunteers and 10 liver cirrhotics we compared the oral approach with i.v. tracer administration combined with measurement of individual CO2 production rate. The 13CO2/12CO2 enrichment was assessed by isotope-ratio mass spectrometry. After i.v. [1-13C]phenylalanine application exhaled 13C recovery per minute peaked within 10 minutes (controls: 0.17 +/- 0.06%; cirrhotics: 0.05 +/- 0.02%, p < 0.01). The oral approach yielded comparable separation between 30-60 minutes, with average peak values being 0.18 +/- 0.03% and 0.06 +/- 0.03% (p < 0.01), respectively. Variable gastrointestinal resorption kinetics after oral application probably causes this difference.     

Production of 13C labelled pea flour for use in human digestion and fermentation studies

Stable isotope breath tests offer a new approach to the study of digestion and fermentation of carbohydrates in man. In this study, 13C labelled peas were grown by pulsing 250 ml 13CO2 into a sealed growth chamber. A second pulse was added to a portion of the peas to increase the 13C enrichment. This generated pea flour with an enrichment of 2.36 at.% excess (range 2.09-2.71 n = 3) and 8.64 atom % excess (range 7.37-9.78 n = 3) respectively. This represented incorporation of an absolute yield of 3.8% of the 13CO2 into peas in the 'once-labelled' treatment and 7.5% in the 'twice-labelled' treatment. Ingestion of a mixture of the labelled pea flour (300 mg) by two volunteers generated measurable 13CO2 excretion for breath test analysis. The profile of breath 13CO2 enrichment increased to a maximum within three hours after consuming the pea flour followed by a decrease almost back to baseline by 13 hours. Breath 13CO2 appeared to rise again after this apparent nadir at 13 hours until the end of the sampling period. Mathematical analysis of the data suggested that two peaks best described the profile of breath 13CO2 up to 13 hours. A third peak was necessary to describe the late rise in breath 13CO2 enrichment. This use of 13C enriched pea flour may provide a useful non invasive method for measurement of digestion and fermentation in vivo.     

Effect of alcohol consumption on the liver detoxication capacity as measured by [13C]methacetin- and [methyl-13C]methionine-breath tests

The aim of this study was to investigate the hepatic microsomal and mitochondrial functions by using the 13CO2-breath test in healthy subjects either before or after the consumption of red wine. Fourteen adults received [13C]methacetin and [methyl-13C]methionine together with a standardised dinner. Expired air samples were taken over 6 h. After a wash-out period, the subjects consumed 0.4 ml ethanol/kg/day together with dinner over a 10-day period. Thereafter, 13C-tracer administration was repeated under identical conditions. The 13CO2-enrichments were measured by isotope ratio mass spectrometry. The mean cumulative percentage 13C-dose recovery (CPDR) after administration of [13C]methacetin and [methyl-13C]methionine either without or with red wine consumption amounted to 38.2+/-6.3 vs. 36.3+/-6.7% (p=0.363) and 9.5+/-3.3 vs. 8.8+/-2.5% (p=0.47), respectively. Moderate alcohol consumption does not induce significant short-term changes of the microsomal and the mitochondrial functions of the human liver in healthy subjects.     

Biomedical Application of an Isotope Selective Nondispersive Infrared Spectrometer for 13CO2 and 12CO2 Concentration Measurements in Breath Samples

A newly developed isotope selective nondispersive infrared (NDIR) spectrometer for the measurement of ¹³CO₂ and ¹²CO₂ concentrations in breath samples was applied as a low cost and very simple to operate alternative to isotope ratio mass spectrometry (IRMS). We used this device for several biomedical applications ([¹³C]urea breath test, [¹³C]leucine metabolism, [¹³C]methacetin catabolism of rats) and found that the results agree very well with IRMS.     

Determination of (13)CO(2)/(12)CO(2) Ratio by IRMS and NDIRS

Abstract Breath tests using (13)C-labelled substrates require the measurement of (13)CO(2)/(12)CO(2) ratio in breath gas samples. Next to isotope ratio mass spectrometry (IRMS), which is very sensitive but also complex and expensive, alternatively isotope selective nondispersive infrared spectrometry (NDIRS) can be used to determine the (13)CO(2)/(12)CO(2) ratio in expired breath. In this study we compared NDIRS- with IRMS-results to investigate whether the less expensive and very simply to operate NDIRS works as reliable as IRMS. For this purpose we applicated 1-(13)C-Phenylalanine to patients with advanced liver cirrhosis and healthy volunteers and took duplicated breath samples for IRMS and NDIRS at selected time points. Our data show a good correlation between these two methods for a small number of samples as required for simple breath tests. Longer series, where repeated measurements are required on the NDIRS instrument lead to a decreasing correlation. This indicates the superiority of IRMS concerning (13)CO(2)-kinetics over longer time periods.     

Imaging of (13)C-labeled glucose and sorbitol in bovine lens by (1)H-detected (13)C nuclear magnetic resonance spectroscopy

Bovine lenses were incubated in a solution containing [1-(13)C]glucose (50 mM) for 1, 2 and 4 days. Spectroscopic images of [1-(13)C]glucose and [1-(13)C]sorbitol were constructed using (1)H-detected gradient-enhanced heteronuclear multiple-quantum coherence (GE-HMQC) in a 2.0-tesla magnetic field. Accumulations of [1-(13)C]glucose and [1-(13)C]sorbitol were mainly observed at the periphery of the lens. Their distributions corresponded to the cortex. (1)H-detected (13)C nuclear magnetic resonance (NMR) spectroscopic imaging by GE-HMQC successfully demonstrated the distribution of [1-(13)C]glucose and [1-(13)C]sorbitol at the periphery of bovine lenses.     

Analysis of 13C-mixed triacylglycerol in stool by bulk (EA-IRMS) and compound specific (GC/MS) methods

This paper was presented in poster form at the 17th International Congress of Nutrition, August 27-31, Vienna, Austria (Annals of Nutrition & Metabolism 2001; 45(Suppl.1):349). Some of the data were also presented in poster form at the British Society of Gastroenterology Meeting, March 18-21, Glasgow, UK (Gut 2001; 48(Suppl.1):A91). The 13C-mixed triacylglycerol (MTG) breath test is used to measure intraluminal fat digestion. In normal digestion, 20-40% of the ingested 13C label is recovered in breath CO2. We aimed to identify the proportions of ingested label excreted in stool, as well as breath following ingestion of 13C-MTG by children with impaired exocrine pancreatic function and healthy controls. 13C enrichment of breath samples was measured by continuous flow isotope ratio mass spectrometry (IRMS) and cumulative percent dose recovered (cPDR) in 10 h was calculated. Total 13C of a faecal fat extract from each stool was measured by elemental analyser-IRMS, and 13C enrichment and concentration of the TBDMS derivative of octanoic acid was measured by GC/MS after hydrolysis of the fat extract. Stool 5-day cPDR was calculated. Mean breath cPDR was 35%. Mean cPDR in stool by combustion-IRMS and GC/ MS, respectively, was 0.8% and 1.0%. Therefore, the remaining 64% of the 13C label must remain in the body and variability in breath cPDR is due to postabsorptive rather than predigestive factors.     

Liver function assessment with three (13)C breath tests by two-point measurements

In this study, we performed three breath tests - l-[1-(13)C ]phenylalanine breath test (PBT), l-[1-(13)C ] methionine breath test, and [(13)C]methacetin breath test (MethaBT) - in patients with chronic liver disease to determine the optimal timing of expired air collection for diagnosing chronic liver disease and evaluating the grade of fibrosis. The subjects were 61 adults with normal livers, 98 chronic hepatitis patients, and 91 liver cirrhosis patients. We investigated the relationships of breath test results with routine biochemical tests and the Child-Pugh score, as well as the diagnostic capacities of the breath tests for liver dysfunction/cirrhosis and grade of liver fibrosis. For the diagnosis of liver cirrhosis and correlations with liver fibrosis, the accuracy of the PBT at 30 min (PBT30) was similar to that of the MethaBT at 15 min (Metha15). For liver function assessment by two-point measurement with (13)C breath tests, we recommend the PBT30 and the Metha15.     

Assessment of cerebrovascular reactivity with functional magnetic resonance imaging: comparison of CO(2) and breath holding

Cerebral blood flow (CBF) and oxygenation changes following both a simple breath holding test (BHT) and a CO(2) challenge can be detected with functional magnetic resonance imaging techniques. The BHT has the advantage of not requiring a source of CO(2) and acetazolamide and therefore it can easily be performed during a routine MR examination. In this study we compared global hemodynamic changes induced by breath holding and CO(2) inhalation with blood oxygenation level dependent (BOLD) and CBF sensitized fMRI techniques. During each vascular challenge BOLD and CBF signals were determined simultaneously with a combined BOLD and flow-sensitive alternating inversion recovery (FAIR) pulse sequence. There was a good correlation between the global BOLD signal intensity changes during breath holding and CO(2) inhalation supporting the notion that the BHT is equivalent to CO(2) inhalation in evaluating the hemodynamic reserve capacity with BOLD fMRI. In contrast, there was no correlation between relative CBF changes during both vascular challenges, which was probably due to the reduced temporal resolution of the combined BOLD and FAIR pulse sequence.     

Liver function assessment with three 13C breath tests by two-point measurements

In this study, we performed three breath tests – l-[1-¹³C ]phenylalanine breath test (PBT), l-[1-¹³C ] methionine breath test, and [¹³C]methacetin breath test (MethaBT) – in patients with chronic liver disease to determine the optimal timing of expired air collection for diagnosing chronic liver disease and evaluating the grade of fibrosis. The subjects were 61 adults with normal livers, 98 chronic hepatitis patients, and 91 liver cirrhosis patients. We investigated the relationships of breath test results with routine biochemical tests and the Child–Pugh score, as well as the diagnostic capacities of the breath tests for liver dysfunction/cirrhosis and grade of liver fibrosis. For the diagnosis of liver cirrhosis and correlations with liver fibrosis, the accuracy of the PBT at 30 min (PBT30) was similar to that of the MethaBT at 15 min (Metha15). For liver function assessment by two-point measurement with ¹³C breath tests, we recommend the PBT30 and the Metha15.     

Exact profiles of (13)CO(2) recovery in breath air after per oral administration of [(13)C]methacetin in two groups of different ages

The aim of this study is to determine if age is a factor influencing the results of a [(13)C]methacetin breath test ((13)C-MBT). Two groups of healthy volunteers, each comprising six men and six women, but differing in average age (Y=young, 25.1+/-0.6 years, MA=middle-aged;, 46.0+/-2.1 years) orally took 75 mg [(13)C]methacetin. Samples of expiratory air for (13)CO(2) measurement were collected up to 48 h after intake of the substrate. A maximum momentary (13)CO(2) breath exhalation of 37.0+/-2.6%dose/h was observed at 18 min (median, range: 9-30 min) in the young subjects and of 38.4+/-2.5%dose/h at 18 min (median, range: 12-30 min) in the middle-age volunteers. The cumulative (13)C elimination in expiratory air was statistically significantly higher in the MA compared with the Y group as from 75 min up to 180 min, indicating a greater microsomal metabolic efficiency of the liver in the middle-aged healthy subjects. Gender, use of hormonal contraception, cigarette smoking, or body mass index did not modify the age-related effect on the cumulative (13)C elimination in breath air. The study results imply a necessity of composing control groups well matched with regard to the age structure for a proper interpretation of clinical (13)C-MBT results.     

13C]aminopyrine and [13C]caffeine breath test: influence of gender,cigarette smoking and oral contraceptives intake

The [13C]aminopyrine breath test ([13C]ABT) measures the global activity of cytochrome P450 in vivo and is a sensitive indicator of liver metabolic dysfunction. The present study aims to determine whether gender and cigarette smoking influence the results of [13C]ABT as well as to confirm the effect of oral contraceptive steroids (OCS) intake on this metabolic test. Hundred and ten healthy subjects, including men and women, smoker and non-smoker, women taking OCS or not, were phenotyped for CYP1A2 using the [13C]caffeine breath test and underwent a [13C]ABT. Both tests showed large inter-individual variations in accordance with that of CYP450 liver content. [13C]ABT was sensitive enough to point out a significant induction or inhibition related to cigarette smoking habits or OCS. The combined effect of smoking and OCS resulted in an overall unchanged metabolic activity. Consequently, the impact of the studied conditions on the [13C]ABT parameters must be considered by clinicians or clinical investigators.     

Determination of cholesterol absorption in humans: from radiolabel to stable isotope studies

Hypercholesterolemia is a major health risk. Dietary cholesterol absorption is one important factor affecting levels of plasma and tissue cholesterol. Considerable effort has thus been devoted to develop reliable in vivo clinical methodologies to determine dietary cholesterol absorption in humans. The present paper summarises radiolabelled experiments and major advances in stable isotope technologies to determine cholesterol absorption. Initially, direct methods employing gastro-intestinal intubation were developed. Later, indirect methods using oral-faecal cholesterol balance permitted calculation of cholesterol mass absorption. Once the use of radiolabelled [3H, 14C]cholesterol balance was developed in healthy humans, it was finally possible to distinguish exogenous and endogenous cholesterol. Non-invasive and safer stable isotope (2H, 13C, 18O) labelled cholesterol tracers then replaced radioisotopes for use in infants and adults. Stable isotopes and radioisotopes showed identical cholesterol kinetics. The most promising contemporary stable isotope assessment of cholesterol absorption is a dual stable isotope dual tracer approach based on simultaneous administration of oral and intravenous differentially labelled cholesterol tracers, followed by plasma sampling for 3-4 d. Online GC/Combustion/IRMS and GC/Pyrolysis/IRMS allow minimal amounts of dual stable isotope cholesterol tracers to be detected. Using the dual stable isotope dual tracer approach, the percent cholesterol absorption in adult volunteers has been determined to be 50-70%.     

Automated data processing of [1H-decoupled] 13C MR spectra acquired from human brain in vivo

In clinical 13C infusion studies, broadband excitation of 200 ppm of the human brain yields 13C MR spectra with a time resolution of 2-5 min and generates up to 2000 metabolite peaks over 2h. We describe a fast, automated, observer-independent technique for processing [1H-decoupled] 13C spectra. Quantified 13C spectroscopic signals, before and after the administration of [1-13C]glucose and/or [1-13C]acetate in human subjects are determined. Stepwise improvements of data processing are illustrated by examples of normal and pathological results. Variation in analysis of individual 13C resonances ranged between 2 and 14%. Using this method it is possible to reliably identify subtle metabolic effects of brain disease including Alzheimer's disease and epilepsy.     

Use of the optogalvanic effect (OGE) for isotope ratio spectrometry of 13CO2 and 14CO2

The use of isotopic carbon dioxide lasers for determination of carbon (and oxygen) isotope ratios was first demonstrated in 1994. Since then a commercial device called LARA, has been manufactured and used for Helicobacter pylori breath tests using (13)C-labelled urea. The major advantages of the optogalvanic effect compared with other infrared absorption isotope ratio measurement techniques are its lack of optical background and its high sensitivity resulting from a signal gain proportional to laser power. Continuous normalisation using two cells, a standard and sample, lead to high accuracy as well as precision. Recent advances in continuous flow measurement of (13)C/(12)C ratios of CO(2) in air and extensions of the technique to (14)C, which can be analysed as a stable isotope, are described.     

The first in vivo observation of (13)C-(15)N coupling in mammalian brain.

[5-(13)C,(15)N]Glutamine, with (1)J((13)C-(15)N) of 16 Hz, was observed in vivo in the brain of spontaneously breathing rats by (13)C MRS at 4.7 T. The brain [5-(13)C]glutamine peak consisted of the doublet from [5-(13)C,(15)N]glutamine and the center [5-(13)C,(14)N]glutamine peak, resulting in an apparent triplet with a separation of 8 Hz. The time course of formation of brain [5-(13)C,(15)N]glutamine was monitored in vivo with a time resolution of 20-35 min. This [5-(13)C,(15)N]glutamine was formed by glial uptake of released neurotransmitter [5-(13)C]glutamate and its reaction with (15)NH(3) catalyzed by the glia-specific glutamine synthetase. The neurotransmitter glutamate C5 was selectively (13)C-enriched by intravenous [2,5-(13)C]glucose infusion to (13)C-label whole-brain glutamate C5, followed by [(12)C]glucose infusion to chase (13)C from the small and rapidly turning-over glial glutamate pool, leaving (13)C mainly in the neurotransmitter [5-(13)C]glutamate pool, which is sequestered in vesicles until release. Hence, the observed [5-(13)C,(15)N]glutamine arises from a coupling between (13)C of neuronal origin and (15)N of glial origin. Measurement of the rate of brain [5-(13)C,(15)N]glutamine formation provides a novel noninvasive method of studying the kinetics of neurotransmitter uptake into glia in vivo, a process that is crucial for protecting the brain from glutamate excitotoxicity.     

Compartmental approach for evaluation of plasma kinetics and (13)co(2)-exhalation after oral loading with L-[1-(13)c]leucine

Abstract A seven compartment model was applied for evaluation of oral L-[1-(13)C]leucine loading tests (38 μmol/kg body wt.) in healthy volunteers. The model comprises transport and absorption in stomach and gut into a central L-leucine-compartment which is connected to a protein compartment and to the compartment of the corresponding 2-oxo acid. CO(2) release from the latter occurs in a fast and a slow compartment into the central CO(2) compartment for exhalation. Using the fmins routine of MATLAB for parameter estimation, a good agreement was obtained between calculated and actually measured kinetics of (13)C-labelled metabolites and a mean in vivo L-leucine oxidation of 0.365 ± 0.071 μmol/kg per min (n = 5) was computed. Plausibility of the model was checked by predicting in vivo leucine oxidation rates from primed continuous infusion tests (priming: L-[1-(13)C]leucine, 5 μmol/kg; NaH(13)CO(2), 1.2 μmol/kg; infusion: L-[1-(13)C]leucine, 5 μmol/kg per h). In 5 tested volunteers, the experimental L-leucine oxidation rate amounted to 0.358 ± 0.105 μmol/kg per min versus predicted 0.324±0.099 μmol/kg per min. Possible causes for some observed intraindividual variations are discussed.     

13C basal abundance of expired CO2 -definition of pre-requisites for kinetic breath tests

A sufficiently stable rate of 13CO2 exhalation is necessary when the diagnostic 13CO2 breath tests are performed in healthy subjects and patients. The aim of the research was to define prerequisite conditions for kinetic breath tests in order to ensure a stable 13CO2 background. A 3-part protocol was developed. Part I: a study of the one-day variation of 13CO2 abundance in expired CO2 confirmed that shifts of the basal 13C abundance in breath are inherent in nature. Part II: a study of the variations of 13C enrichment after the ingestion of different meals and beverages showed that ingestion of food items containing C4 plant sugars, such as maize, induces a significant increase in isotopic abundance. Part III: a new test breakfast containing rice grain cereal, milk and orange juice was tested. This test meal induces no significant change on the basal 13CO2 abundance in healthy subjects. This new finding allows to avoid the fasting period normally required prior to a breath test which is sometimes difficult for children and pregnant women.     

[13C]Aminopyrine and [13C]Caffeine Breath Test: Influence of Gender,Cigarette Smoking and Oral Contraceptives Intake

Abstract

The [¹³C]aminopyrine breath test ([¹³C]ABT) measures the global activity of cytochrome P450 in vivo and is a sensitive indicator of liver metabolic dysfunction. The present study aims to determine whether gender and cigarette smoking influence the results of [¹³C]ABT as well as to confirm the effect of oral contraceptive steroids (OCS) intake on this metabolic test. Hundred and ten healthy subjects, including men and women, smoker and non-smoker, women taking OCS or not, were phenotyped for CYP1A2 using the [¹³C]caffeine breath test and underwent a [¹³C]ABT. Both tests showed large inter-individual variations in accordance with that of CYP450 liver content. [¹³C]ABT was sensitive enough to point out a significant induction or inhibition related to cigarette smoking habits or OCS. The combined effect of smoking and OCS resulted in an overall unchanged metabolic activity. Consequently, the impact of the studied conditions on the [¹³C]ABT parameters must be considered by clinicians or clinical investigators.