Comparison of two dosage regimens of the substrate for the [13C]methacetin breath test

The [¹³C]methacetin breath test ([¹³C]MBT) – a valuable non-invasive tool dedicated to the assessment of the liver metabolic capacity – still needs standardisation. The aim of this study was to check whether currently used dosage regimens of [¹³C]methacetin provide concordant [¹³C]MBT results in subjects with an atypical body constitution. Healthy volunteers: low body mass<55 kg (eight women), and high body mass>95 kg (eight large body frame men) were recruited. They underwent [¹³C]MBT on separate days, taking in random order [¹³C]methacetin: a fixed 75 mg dose (FX75), or a 1 mg kg^?1 body mass-adjusted dose (BMAD). Samples of expiratory air for ¹³CO₂ measurement were collected over 3 h. The maximum momentary ¹³C elimination in breath air occurred earlier and was higher following BMAD than with FX75 in the low body mass females (T _max 14.6±1.0 min vs. 22.1±2.4 min, p=0.019; D _max 41.9±2.9 % dose h^?1 vs. 36.6±3.6 % dose h^?1, p=0.071). In the high body mass men, T _max remained unchanged, whereas D _max was slightly higher with BMAD compared to FX75 (21.5±3.2 min vs. 23.0±3.0 min; 38.5±2.9 % dose h^?1 vs. 32.3±2.5 % dose h^?1). It is concluded that in subjects with a body constitution outside the general population average, the dosage of the substrate may affect some results of the [¹³C]MBT. The dosage-related differences appear, however, to be insignificant if the result of the [¹³C]MBT is reported as a cumulative ¹³C recovery in breath air.     

13C-Leucin-Atemtest zur Beurteilung der anabolen Verwertung von diätetischen Aminosäurengemischen bei Phenylketonurie

It is essential to establish whether and how environmental factors affect the reliability of [¹³C]methacetin breath test (¹³C-MBT). In 12 healthy volunteers (smokers), a standard ¹³C-MBT with 75 mg [¹³C]methacetin was performed twice in random order: on a control day without smoking and on another day with smoking two cigarettes antecedently. A considerable flattening of the curve of the momentary ¹³C recovery within the expiratory air was observed when the ¹³C-MBT was performed after smoking. The maximum of the momentary ¹³C recovery, D _max, decreased from 37.20±2.58 to 25.39±2.29% dose/h (p=0.00052). Moreover, the time to reach D _max was prolonged after cigarette smoking (26.5±3.1 vs. 16.5±1.9 min, p=0.0199). The curve of the cumulative ¹³C recovery on the cigarette smoking day appeared to be shifted downwards, and statistically significant differences relative to the control situation were found between the 24th and 75th minute following [¹³C]methacetin administration. Smoking cigarettes immediately prior to the ¹³C-MBT diminishes the ability of the liver to handle methacetin, and hence a possibility of such an interaction should be excluded in order to interpret the results of the test correctly.     

Exact profiles of 13CO2 recovery in breath air after per oral administration of [13C]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 [¹³C]methacetin breath test (¹³C-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 [¹³C]methacetin. Samples of expiratory air for ¹³CO₂ measurement were collected up to 48 h after intake of the substrate. A maximum momentary ¹³CO₂ 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 ¹³C 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 ¹³C 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 ¹³C-MBT results.     

Analysis of 13C-mixed Triacylglycerol in Stool by Bulk (Ea-IRMS) and Compound Specific (GC/MS) Methods

Abstract

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.     

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.     

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.     

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.     

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.     

Evaluation of Water Removal and Memory Effect in 13CO2 Breath Tests by Isotope Ratio Mass Spectrometry

The usefulness of different ways of water removal in off-line sample preparation of human breath samples for ¹³CO₂ breath tests was examined and compared. Cryogenic water trapping and water removal with common desiccants like silicagel blue, Mg(ClO₄)₂, and molecular sieves were checked for reliability and reproducibility. With silicagel blue and Mg(ClO₄)₂ memory effects for ¹³C content were observed. The use of molecular sieve 4 Å and 5 Å led to tremendous carbon isotope fractionation. Molecular sieve 3 Å was found to be an excellent alternative to the established use of Mg(ClO₄)₂ and of cryogenic water trapping.     

Effects of ergot alkaloids on liver function of piglets can be detected by the [13C]methacetin breath test irrespective of oral or intramuscular route of tracer administration

Ergot alkaloids (sum=total alkaloids, TA) originate from the phyto-pathogenic fungus Claviceps purpurea and might exert feed intake depressing and hepatotoxic effects on animals. The aim of the study was to evaluate TA effects on performance and liver function of piglets with the [¹³C]methacetin breath test and two routes of tracer administration (orally, p.o.; intramuscularly, i.m.). Two ergot batches were mixed into piglet diets resulting in 21 and 17 mg TA kg^?1 (Ergot-5 and -12, respectively) and compared with an ergot-free control diet. Feed intake was significantly depressed after feeding the ergot containing diets (p=<0.001). The time at maximum ¹³CO₂ exhalation (t _max) and the half-life (t _0.5) were not influenced by treatments and varied between 25 and 68 min after the p.o., and 28 and 62 min after the i.m. administration of [¹³C]methacetin, respectively. The cumulative ¹³C recovery (cPDR₃₀) was significantly lower due to feeding the diet Ergot-5 (6.6 %) compared with the Ergot-12 (8.8 %) and the control diet (9.7 %) irrespective of the route of tracer administration (p=0.044). As a discrimination of the diet effects through both tracer administration routes is possible, the i.m. application should be preferred in piglets as this causes less stress than the oral forced administration.     

The effect of resistant starches on fat oxidation in healthy adults as measured by a 13CO2-breath test

A resistant starch (RS) mixture (MIX) consisting of fibre of potatoes (FP) and wrinkled pea starch (WPS), and high amylose maize starch (HAMS) were supplemented in adults to evaluate their effects on fat oxidation by means of a ¹³CO₂-breath test. Sixteen subjects received a regular diet either without or with the supplementation of MIX and HAMS in randomised order. After administration of a [U-¹³C]algal lipid mixture, exhaled air was collected over 14?h in 0.5- and 1-h intervals. The ¹³C abundances were measured by nondispersive infrared spectroscopy. In comparison to the dry run (DR), supplementation with MIX and with HAMS increased the cumulative percentage dose recovery: (DR: 16.7?%, MIX: 16.9?%, HAMS: 18.0?%), but without statistical significance. The colonic degradation of MIX and HAMS to short-chain fatty acids tends to lower the formation of carbohydrate-derived acetyl-CoA and contributes to a postprandial lipid oxidation increase by using fat-derived acetyl-CoA as a compensatory fuel source.     

[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.     

The 13C bicarbonate method: an inverse end product method for measuring CO2 production and energy expenditure

We reconsider the principle of the ¹³C bicarbonate (NaH¹³CO₃) method (¹³C-BM) for the determination of the CO₂ production to obtain an estimate of energy expenditure (EE). Its mathematical concept based on a three-compartmental model is related to the [¹⁵N]glycine end product method. The CO₂ production calculated by the ¹³C-BM, R_aCO₂(¹³C) is compared to the result from the indirect calorimetry, RCO₂(IC). In an interspecies comparison (dog, goat, horse, cattle, children, adult human; body mass ranging from 15 to 350?kg, resting and fasting conditions) we found an excellent correlation between the results of ¹³C-BM and IC with RCO₂(IC)?=?0.703?×?R_aCO₂(¹³C), (R²?=?0.99). The slope of this correlation corresponds to the fractional ¹³C recovery (RF(¹³C)) of ¹³C in breath CO₂ after administration of NaH¹³CO₃. Significant increase in RF(¹³C) was found in physically active dogs (0.95?±?0.14; n?=?5) vs. resting dogs (0.71?±?0.10, n?=?17; p?=?.015). The ¹³C recovery in young bulls was greater in blood CO₂ (0.81?±?0.05) vs. breath CO₂ (0.73?±?0.05, n?=?12, p?<?.001) and in ponies with oral (0.76?±?0.03, n?=?8) vs. intravenous administration of NaH¹³CO₃ (0.69?±?0.07; n?=?8; p?=?.026). We suggest considering the ¹³C-BM as a ‘stand-alone’ method to provide information on the total CO₂ production as an index of EE.     

Is there an advantage in normalising the results of the Helicobacter Pylori [13C]Urea breath test for CO2 production rate in children?

The urea breath test (UBT) is a non-invasive diagnostic test to detect the presence of Helicobacter pylori in the stomach, and is the simplest way to confirm eradication after treatment. The test is based on the capacity of H. pylori to secrete the enzyme urease, which hydrolyses urea to ammonia and carbon dioxide. The aim of this study was to determine whether there is an advantage in expressing the results of UBTs in terms of urea hydrolysis rate (UHR), rather than breath ¹³C enrichment alone. Retrospective analysis of data collected between 1995 and 2002 from 260 patients undergoing UBTs was performed. The cut-offs for positive tests using breath 30-minute enrichment (E30), UHR calculated using VCO₂ estimated from height and weight (H/WT) and VCO₂ estimated from weight only were determined using two-graph receiver operator characteristic (TG-ROC) analysis. The cut-off points were 3.5‰ or 38.7?ppm ¹³C excess, 7.04?µmol/h and 7.08?µmol/h, respectively. There was no advantage in expressing the results as UHR (θ₀, Theta-zero, where sensitivity?=?specificity?=?0.97 (UHR H/WT), 0.98 (UHR WT) and 1.00 (E30)) rather than breath ¹³CO₂ enrichment alone. Differences in the extent of H. pylori colonisation and urease activity are more important than variation in VCO₂ in determining breath ¹³CO₂ enrichment in the UBT.     

A Sensitive Isotope Selective Nondispersive Infrared Spectrometer for 13CO2 and 12CO2 Concentration Measurements in Breath Samples

We present a nondispersive infrared spectrometer (NDIRS) for the measurement of the ¹³CO₂/¹²CO₂-ratio in breath samples. A commercial NDIR spectrometer for CO₂ concentration measurements in industrial process control was modified using two separate optical channels for the ¹³CO₂ and ¹²CO₂ detection. Cross interference due to overlapping absorption lines of both isotopic gases was successfully eliminated. The sensitivity of this device is ± 0.4‰ of the ¹³CO₂/¹²CO₂-ratio in a range of 2.5 to 5% of total CO₂. This is sufficient for biomedical applications. Our spectrometer is small in size, cheap and simple to operate and thus a true alternative to isotope ratio mass spectrometers (IRMS). Several biomedical applications with breath samples were demonstrated and were compared in very good agreement with IRMS.     

A comparison of the reproducibility of the parameters of the 13C-aminopyrine breath test for the assessment of hepatic function

This study determined the within-subject and between-subject variability of different ways of expressing the results of the ¹³C-aminopyrine breath test (¹³C-ABT) and the effect of shortening the test duration. The ¹³C-ABT was conducted on three separate occasions in 10 healthy volunteers and on a single occasion in 22 patients with established liver cirrhosis. The within-subject variability of cumulative percentage dose recovered (cPDR), using measured CO₂ production rate (VCO₂), in the reference group over three trials was 15% over 120 min. Higher within-subject variability in cPDR would have been evident if the test was terminated at either 30 or 60 min. Substitution of predicted VCO₂ to calculate cPDR yielded comparable values at all time points. Significant differences between cirrhotics and reference group were evident after just 10 min using PDR/h, cPDR or enrichment (all P<0.05). The ABT demonstrates clinically acceptable reproducibility. Shortening of the duration may make the test more acceptable clinically, but it is associated with increasing imprecision.     

Spatial and seasonal variations in the stable C isotope composition of dissolved inorganic carbon and in physico-chemical water parameters in the Plitvice Lakes system

Plitvice Lakes waters were collected at 14 sampling points, including springs, tributaries and lakes, for the period 2002–2007. The results of the physical and chemical conditions of calcite precipitation as well as the δ¹³C values of dissolved inorganic carbon (DIC) were used to study the processes influencing calcite precipitation. Significant differences between spring, lake and stream waters as well as changes in the downstream direction were observed. The correlation between δ¹³C_DIC values and physico-chemical conditions for calcite precipitation showed that calcite precipitates in lake waters which are oversaturated with respect to CaCO₃ (I _sat values 4–10) and with δ¹³C_DIC values between?11.5 and?8.5 ‰. In spring waters, the δ¹³C_DIC values were more negative, from?14 to?12 ‰, and I _sat values of 1–2 indicated that equilibrium conditions for calcite precipitation were not attained. The downstream increase in δ¹³C_DIC correlated with the increase in the δ¹³C values of calcite in the lake sediments, suggesting that the freshwater calcite was mainly of autochthonous origin and precipitated within the water column in isotopic equilibrium with DIC.     

Combustibles,fuels and their combustion products: A view through carbon isotopes

Stable (i.e. non-radioactive) carbon-isotope composition (δ¹³C) in fuels has been extensively used as an indicator of the processes leading to the generation of their parent crude-oil. With the example of those used in Paris (France), this preliminary study isotopically characterizes fuels and combustibles, as well as the isotopic relations existing with their combustion by-products, i.e. gases (CO₂) and particles (bulk carbon). Results show that δ¹³C in fuels is clearly related to their physical state, with natural gas being strongly depleted in ¹³C while coal yields the highest δ¹³C, and liquid fuels display intermediate values. This relation is also valid for combustion gases, although δ¹³C values of combustion particles form a homogeneous range within which no clear distinction is observed. Combustion processes are accompanied by carbon-isotope fractionation (noted Δ¹³C) resulting from the combustion being incomplete. Carbon-isotope fractionation is strictly negative (Δ¹³C = ?1.3‰) during the formation of combustion gases, but generally positive in particle formation even if values close to zero are observed. Using simple mixing equations for describing the closed system formed by fuel, CO₂ and carbonaceous particles, we discuss the carbon budget for spark-ignition (unleaded gasoline) and diesel engines. Stable carbon isotopes corroborate the already-proved superior efficiency of diesel combustion mode compared with spark ignition, as carbon is preferentially transformed into CO₂.     

13C NMR Chemical Shifts of Heterocycles: 3∗. Empirical Substituent Effects in 2-Halomethyl-2-hydroxy-tetrahydrofurans and -5,6-tetrahydro-4H-pyrans

Abstract

Evaluation by empirically derived equations for the Substituent effect (α, β, γ, δ) on the ¹³C NMR chemical shifts for C-2, C-3, C-4, C-5, C-6, the halomethyl-substituted carbon (C-7) and the cyano or oxymic carbon (C-8) in 2-halomethyl-2-hydroxy-tetrahydrofurans 1a-c, 2, 3a, b, 4a and -5,6-tetrahydro-4H-pyrans 5a-c, 6a [with C-2-substituents (R²): CF₃, CCl₃ or CHCl₂, C-3-substituents (R³): CN, C(Me)=NOH, CH=NOMe, C(Me)=NOMe or CH=NOH], taking as reference the 2-trifluoromethyl-2-hydroxy-tetrahydrofuran (la), is reported. From the additivity properties of the α-, β-, γ-, δ-and ?-effects for each Substituent it is possible to predict the chemical shift of each carbon of the compounds 1–6.

     

Fast 13C-NMR Spectral Editing for Determining CHn Multiplicities

With an improved version of the ¹³C DEPTQ NMR experiment all carbon multiplicities (C_q, CH, CH₂ und CH₃) can be identified unequivocally and most conveniently in two experiments and at best with only one scan each. This simplifies the analysis of ¹³C-NMR data on a routine level in general. With higher sample amounts and/or exploiting the high sensitivity of cryogenically cooled ¹³C probeheads the efficiency of such investigations may be improved. This makes this method attractive for fast ¹³C analysis of small-to-medium sized molecules in high-throughput laboratories.