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.     

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 (13)C-aminopyrine breath test ((13)C-ABT) and the effect of shortening the test duration. The (13)C-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 CO(2) production rate (VCO(2)), 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 VCO(2) 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.     

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.     

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.     

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.     

Metabolic effects of HAY's diet

The aim of the study was, to evaluate the metabolic effect of HAY's diet on protein turnover, fat oxidation, respiratory quotient, body fat and weight loss. Twelve healthy adults received an individually regular diet and thereafter a corresponding isocaloric and isonitrogenous 10-day HAY-diet. Protein turnover and 13C-fat oxidation were investigated after administration of [15N]glycine and an [U-13C]algae lipid mixture. The 15N and 13C enrichment in urine and breath were measured by isotope ratio mass spectrometry. The respiratory quotient was measured by indirect calorimetry. Body fat, total body water and lean body mass were estimated by bio-electric impedance analysis. HAY's diet led to a significantly higher 13C-fat oxidation (15.4 vs. 22.0% P < 0.01), corresponding to a lower respiratory quotient (0.88 vs. 0.81; P < 0.01), whereas the protein turnover remained constant in both diets (3.06 vs. 3.05 g/kg/day). HAY's diet did not reduce total body water, lean body mass, body fat and body weight (72.2 vs. 71.4 kg).     

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.     

Use of dual isotope tracers in biomedical research

Biomedical stable isotope studies involve administration of tracer and measurement of isotope enrichment in blood, urine, feces or breath. The aim of the studies is to gather quantitative information about a specific metabolic function. However, the measured isotope enrichment may be affected by other metabolic events than only this function. In this case, a correction is necessary. The best approach is to add a second tracer simultaneously which is known to be metabolised by all interfering metabolic events but not by the function of interest. This dual isotope approach also enables simultaneous measurement of two interrelated functions. A summary of selected applications involving dual isotope tracer studies is presented. The applications deal with energy expenditure (doubly labelled water technique), cholesterol absorption, starch and lactose digestion, fat digestion, bile acid metabolism and the combination of stable and radioactive carbon isotopes in breath testing.     

Effects of ergot alkaloids on liver function of piglets can be detected by the [(13)C]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 [(13)C]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 (13)CO(2) 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 [(13)C]methacetin, respectively. The cumulative (13)C recovery (cPDR(30)) 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.     

Metabolic Effects of HAY's Diet

Abstract

The aim of the study was, to evaluate the metabolic effect of HAY's diet on protein turnover, fat oxidation, respiratory quotient, body fat and weight loss. Twelve healthy adults received an individually regular diet and thereafter a corresponding isocaloric and isonitrogenous 10-day HAY-diet. Protein turnover and ¹³C-fat oxidation were investigated after administration of [¹⁵N]glycine and an [U-¹³C]algae lipid mixture. The ¹⁵N and ¹³C enrichment in urine and breath were measured by isotope ratio mass spectrometry. The respiratory quotient was measured by indirect calorimetry. Body fat, total body water and lean body mass were estimated by bio-electric impedance analysis. HAY's diet led to a significantly higher ¹³C-fat oxidation (15.4 vs. 22.0% P < 0.01), corresponding to a lower respiratory quotient (0.88 vs. 0.81; P < 0.01), whereas the protein turnover remained constant in both diets (3.06 vs. 3.05 g/kg/day). HAY's diet did not reduce total body water, lean body mass, body fat and body weight (72.2 vs. 71.4 kg).     

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 3.5/1000 or 38.7 ppm 13C excess, 7.04 micromol/h and 7.08 micromol/h, respectively. There was no advantage in expressing the results as UHR (theta0, 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.     

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.     

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.     

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.     

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.     

In vivo research with stable isotopes in biochemistry, nutrition and clinical medicine: an overview

Tracers and kinetic modelling provide the opportunity to follow the movement and to quantify the metabolic fates of biological compounds in vivo. For studies in children and for repeated studies in adults, the use of stable isotopically labelled substrates are preferable and safe. Measurement of isotopic enrichment in biological molecules is highly specific and can be extremely precise. This allows the development of models of biological system dynamics in cells and organs that are otherwise inaccessible for sampling. Applications in biochemistry, nutrition and clinical medicine show the potential of stable isotopes in vivo. Methodology is of paramount importance and includes the choice of bolus studies, (primed) continuous infusions, use of multiple tracers and use of isotopomer information and intrinsic labels. There is no limit to the number and kind of compounds that can be traced. Topics include intermediate metabolism (lipids, proteins and carbohydrates) using hydrogen, carbon and nitrogen labels. In clinical medicine, 13C-breath tests are available for detection of Helicobacter pylori ([13C]urea breath test), and for assessment of a variety of gastro-intestinal and hepatic functions (e.g. [13C]octanoate and mixed [13C]triglyceride breath tests for studies of gastric emptying and fat absorption, respectively). Because theoretical, methodological, physiological and practical aspects are intertwined, in vivo research with stable isotopes demands close collaboration between the clinical researcher and the chemist responsible for the mass spectrometry.     

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.     

Determination of 13C Enrichment by Conventional GC-MS and GC-(MS)-C-IRMS

Abstract

Isotopic enrichment of branched-chain L-amino acids (BCAA) and branched-chain 2-oxo acids (BCOA) in standard preparations and in human plasma samples withdrawn after oral loads with 1-¹³C labelled BCAA were measured on a conventional GC-MS system and an on-line GC-C-IRMS employing O-TMS quinoxalinol derivatives. It was concluded that the recently introduced GC-C-IRMS, owing to its high sensitivity, is the adequate analytical tool when tracer doses of stable isotope labelled compounds of low enrichment are to be used in biomedical in vivo studies.     

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.     

NMR determination of photorespiration in intact leaves using in vivo 13CO2 labeling

Solid-state 13C NMR measurements of intact soybean leaves labeled by 13CO2 lead to the conclusion that photorespiration is 17% of photosynthesis for a well-watered and fertilized plant. This is the first direct assessment of the level of photorespiration in a functioning plant. A 13C{31P} rotational-echo double-resonance (REDOR) measurement tracked the incorporation of 13C label into intermediates in the Calvin cycle as a function of time. For labeling times of 5 min or less, the isotopic enrichment of the Calvin cycle depends on the flux of labeled carbon from 13CO2, relative to the flux of unlabeled carbon from glycerate returned from the photorespiratory cycle. Comparisons of these two rates for a fixed value of the 13CO2 concentration indicate that the ratio of the rate of photosynthesis to the rate of photorespiration of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in soybean leaves is 5.7. This translates into a photorespiratory CO2 loss that is 21% of net CO2 assimilation, about 80% of the value estimated from Rubisco kinetics parameters. The ratio of rates is reduced at low external CO2 concentrations, as measured by net carbon assimilation rates. The carbon assimilation was determined from 13C-label spin counts converted into total carbon by the REDOR-determined isotopic enrichments of the Calvin cycle. The net carbon assimilation rates indicate that the rate of decarboxylation of glycine is not directly proportional to the oxygenase activity of Rubisco as is commonly assumed.