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.     

13C-aminopyrine demethylation is decreased in cirrhotic patients with normal biochemical markers

This study determined the rates of ¹³C-aminopyrine metabolism in patients with varying degrees of liver cirrhosis as defined by clinical scores. Twenty-five cirrhotic patients and 18 healthy subjects underwent a ¹³C-aminopyrine breath test. The cumulative per cent dose recovery (cPDR) of ¹³C on breath expressed as a percentage of the administered dose at 2 h was significantly lower in cirrhotic patients than in healthy subjects (median: 1.7% versus 9.0%; p<.0001). Significant inverse associations between cPDR at 2 h and the model for end-stage liver disease score, Child–Pugh score, international normalised ratio and bilirubin (all p<.05), but not alanine aminotransferase or alkaline phosphatase were observed in the cirrhotic patients. Taking each biochemical marker independently, cirrhotic patients with normal biochemistry had a significantly lower cPDR at 2 h than healthy subjects (all p<.05). Differences in ¹³C-aminopyrine metabolism were evident in cirrhotic patients with less severe disease and may mark hepatic dysfunction when conventional biochemical markers appear unchanged.     

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.     

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.     

[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.     

Preparation of Naturally 13C-Rich Free Fatty Acids (FFA) from Corn Oil and their Application in Metabolic Studies of Lactating Cows

Abstract

When the ¹³C/¹²C ratios in lactose and milk fat of normally fed healthy cows are compared to ¹³C/¹²C in feed a metabolic depletion of ¹³C in the milk constituents becomes obvious.

Due to a carbon isotope effect in the decarboxylation step of pyruvate to Acetyl-Co-A resulting in a ¹³C depleted acetate, depot fat and milk fat are especially reduced in ¹³C relative to ¹²C.

Under pathophysiological conditions after longer fasting periods or in ketosis, long chain fatty acids from depot fat are metabolized and their ¹³C depleted carbon atoms enter the citric acid cycle via Acetyl-Co-A. Then the intermediary products of this cycle become ¹³C depleted too and with a drain on the oxalacetate pool by gluconeogenesis ¹³C depleted carbon atoms are channeled into lactose.

This hypothesis is apt to explain a ¹³C/¹²C decrease in lactose of ketotic cows relative to healthy controls. It is supported by the observation that ¹³C-rich fatty acids from corn oil dosed orally to a ketotic cow increase the ¹³C/¹²C ratios in lactose. So a crossing-over of carbon atoms from long chain fatty acids into glucose and lactose seems highly probable.     

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.     

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.     

Scrutiny of 13C-phenylalanine breath test reproducibility

We evaluated the reproducibility of the ¹³C-phenylalanine breath test (¹³C-PheBT). On three separate days, 21 healthy volunteers (11 F and 10 M) underwent ¹³C-PheBT with 100 mg l-[1-¹³C]phenylalanine taken orally. Short-term reproducibility was evaluated with paired examinations taken 3 days apart; paired examinations separated by 23 days (median) served for the medium-term reproducibility assessment. Expiratory air was sampled at 19 points throughout 3?h. Determined limited reproducibility of the ¹³C-PheBT must be taken into consideration while interpreting the results of this diagnostic tool. The results of this study imply the following conclusions: (i) From among the three parameters examined, the cumulative ¹³C recovery area under the curve (AUC) offers much better reproducibility than the maximum momentary ¹³C recovery in the expiratory air (D_max) or the time to reach the maximum momentary ¹³C recovery (T_max) (ii) Collection of the breath air samples for 2?h results in a much better reproducibility of AUC, than for 1?h only; (iii) Reproducibility of ¹³C-PheBT is affected neither by the duration of the time gap between repeated tests nor by gender; (iv) Comparison with data obtained formerly reveals that reproducibility of the ¹³C-PheBT is worse than either that of of the ¹³C-methacetin (¹³C-MBT) or the ¹³C-alpha-ketoisocaproic acic (¹³C-KICA-BT) breath tests. This finding will have to be taken into consideration while interpreting the results of this diagnostic tool.     

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.     

The evaluation of the repeatability of the 13C-ketoisocaproate breath test for assessing hepatic mitochondrial function

The ¹³C-ketoisocaproate (¹³C-KICA) breath test (BT) has been recently proposed as a non-invasive test for assessing hepatic mitochondrial function. Results of the ¹³C-KICA BT can be expressed as different parameters. However, the best parameter for expressing the ¹³C-KICA BT result is uncertain which hinders use of the BT in routine clinical practice. We have investigated the repeatability of different parameters of ¹³C-KICA BT. Thirteen healthy adult subjects (5 men and 8 women) underwent a ¹³C-KICA BT on two occasions separated by a gap of approximately 30 days. There were no significant differences between the repeated measurements for all the test parameters over 30 days. Furthermore, the Bland Altman statistics showed no fixed or proportional bias for any of the test parameters. The cumulative ¹³C-dose enrichment over 60 min had the lowest within-subject variability of 12% compared to all other test parameters. The cumulative ¹³C-dose enrichment over 60 min could be a very useful parameter for the ¹³C-KICA BT to detect impaired hepatic mitochondrial function in patients with chronic liver diseases.     

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.     

13C/12C-Untersuchungen an Tumorzellen

Abstract

The metabolism of tumor-cells differs in many ways from normal (healthy) cells. One of the major differences is the high glycolytic activity in tumor-cells with the subsequent formation of lactate from glucose, even in the presence of oxygen. The question whether this high rate of glycolysis has any effect on the ¹³C/¹²C-relation of the cells is examined in experiments with a tumor-cell line (HT29) and in specimens of human breast cancer.

The HT29 cells show a clear decrease in ¹³C content compared to their culture medium (Δδ = 3.28‰).

Tissue from human breast cancer has more ¹³C than normal breast tissue taken from the same patient ((Δδ = 2.74‰). But the content of fat is much higher in the normal tissue and its δ-value is negatively correlated with its fat content. It is concluded that the difference between normal and tumor tissue is due to the heterogeneous composition of the normal tissue samples.     

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.     

Production of 13C Labelled Pea Flour for Use in Human Digestion and Fermentation Studies

Abstract

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 250ml 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.     

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.     

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.     

13C and 15N Abundances in the Sediment Core (VER 92/1-St-10-GC2) from Northern Lake Baikal

Abstract

Carbon and nitrogen stable isotope compositions of organic matter, TOC/TN ratio, and manganese concentration in a sediment core that was collected in northern part of Lake Baikal (VER92ST10-GC2, water depth at 922 m, about 3 m long) were investigated to elucidate the origin of the sedimentary organic matter and its associated environmental factors.

The sediment core was composed of mainly two parts: turbidite sections and other sections. Constant δ¹³C and δ¹⁵N values of the turbidite sections were observed (- 26.8 ±0.2 ‰ for δ¹³C and 3.2 ± 0.1 ‰ for δ¹⁵N) throughout the core. The higher δ¹³C in turbidite sections (about - 27 ‰) than that of the other sections (- 31 to - 29 ‰) was clearly observed, and δ¹⁵N was different between turbidite sections (about 3‰) and other sections (3 to 5 ‰). δ¹³C of other sections was close to that of pelagic phytoplankton, indicating that sediment other than turbidite sections is composed of autochthonous components. The variation of stable isotopes in other sections may be possibly caused by the changes in either phytoplankton growth rate or contribution ratios of terrestrial to aquatic plants for δ¹³C. Either denitrification or fluctuation of δ¹⁵N in pelagic phytoplankton can be the cause of variable δ¹⁵N in other sections.     

Assignment of the 1H and 13C Spectra of 5-Methylthio-4H-1-(p-R1-phenyl)-3a-(p-R2-phenyl)-3a,4-dihydro (1,2,4) oxadiazolo(4,5-a), 1,5-benzodiazepines using 2D NMR Spectroscopy.

The ¹H NMR spectra of the title benzodiazepines derivatives is highly congested because all the protons are in aromatic enviroment so many proton signals remain overlap even 300 MHz or higher fields. With this in mind, the assignment of the ¹H and ¹³C spectra of these compounds obtained using COSY, NOESY, HMQC and HMBC experiments is reported.