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.     

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.     

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.     

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.     

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.     

L-[1-13C]phenylalanine breath test in patients with chronic liver disease of different etiologies

The aim of this study was to compare the oxidation of l-[1-¹³C]phenylalanine (¹³C-PheOx) in patients with chronic liver failure due to different etiologies using l-[1-¹³C]phenylalanine breath test. Breath samples were collected before the administration of 100 mg l-[1-¹³C]phenylalanine, and every 10 min thereafter until completion of 1 h. Control subjects (n=9) presented a larger cumulative percentage of ¹³C dose recovery (CPDR) than patients (n=124) with chronic liver disease, regardless of the etiology (7.5±0.7 vs. 4.2±0.2, p=0.001). No differences in CPDR were found considering the Child-Pugh (CP) class or etiology: alcoholic (CP A=7.7±0.7, CP B=4.1±0.5, CP C=2.0±0.3), hepatitis C virus (CP A=5.4±0.5, CP B=4.0±0.2, CP C=2.2±0.3), hepatocellular carcinoma (CP A=5.5±1.6, CP B=3.6±1.8, CP C=2.2±1.0); or cryptogenic cirrhotic patients (CP A=7.4±1.5, CP B=4.4±0.4, CP C=2.1±0.7). Results confirm that ¹³C-PheOx decreases in patients with cirrhosis with respect to controls, notwithstanding the etiology.     

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.     

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.     

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.     

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.     

Non-invasive diagnosis of type 2 diabetes in Helicobacter pylori infected patients using isotope-specific infrared absorption measurements

Helicobacter pylori causes several gastrointestinal diseases and may also contribute to the development of type 2 diabetes (T2D). Several studies suggest that there might be a potential link between H. pylori infection and T2D, but it still remains the subject of debate. Here, we first report the cumulative effect of H. pylori infection and T2D by exploiting the excretion kinetics of ¹³C/¹²C and ¹⁸O/¹⁶O isotope ratios of exhaled breath CO₂ in response to an oral dose of ¹³C-enriched glucose in individuals with T2D and non-diabetic controls (NDC) harbouring the H. pylori infection. Using a high-resolution integrated cavity output spectroscopy (ICOS) technique in the infrared region, we observed that the isotopic fractionations of ¹³C and ¹⁸O in breath CO₂ are distinctly altered in H. pylori infected T2D patients as well as in H. pylori infected NDC. Several optimal diagnostic cut-off points of ¹³C and ¹⁸O isotopes of breath CO₂ were also determined which exhibited the diagnostic sensitivity and specificity of ～97?% and thus suggesting that breath ¹³C and ¹⁸O isotopes might be considered as potential biomarkers for the non-invasive assessment of the gastric pathogen prior to the onset of T2D. This may open a new diagnostic strategy for treating these common diseases in an alternative way.     

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.     

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.     

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.     

The effect of physical back-diffusion of 13CO2 tracer on the coupling between photosynthesis and soil CO2 efflux in grassland

Pulse labelling experiments provide a common tool to study short-term processes in the plant–soil system and investigate below-ground carbon allocation as well as the coupling of soil CO₂ efflux to photosynthesis. During the first hours after pulse labelling, the measured isotopic signal of soil CO₂ efflux is a combination of both physical tracer diffusion into and out of the soil as well as biological tracer release via root and microbial respiration. Neglecting physical back-diffusion can lead to misinterpretation regarding time lags between photosynthesis and soil CO₂ efflux in grassland or any ecosystem type where the above-ground plant parts cannot be labelled in gas-tight chambers separated from the soil. We studied the effects of physical ¹³CO₂ tracer back-diffusion in pulse labelling experiments in grassland, focusing on the isotopic signature of soil CO₂ efflux. Having accounted for back-diffusion, the estimated time lag for first tracer appearance in soil CO₂ efflux changed from 0 to 1.81±0.56 h (mean±SD) and the time lag for maximum tracer appearance from 2.67±0.39 to 9.63±3.32 h (mean±SD). Thus, time lags were considerably longer when physical tracer diffusion was considered. Using these time lags after accounting for physical back-diffusion, high nocturnal soil CO₂ efflux rates could be related to daytime rates of gross primary productivity (R²=0.84). Moreover, pronounced diurnal patterns in the δ¹³C of soil CO₂ efflux were found during the decline of the tracer over 3 weeks. Possible mechanisms include diurnal changes in the relative contributions of autotrophic and heterotrophic soil respiration as well as their respective δ¹³C values. Thus, after accounting for physical back-diffusion, we were able to quantify biological time lags in the coupling of photosynthesis and soil CO₂ efflux in grassland at the diurnal time scale.     

Effect of alcohol consumption on whole-body protein turnover in healthy adults

The aim of the study was to investigate the whole-body protein turnover, either before or after continuous, moderate ethanol-induced oxidative stress by red wine consumption over a relatively short period in healthy volunteers. Ten healthy adults received an individual regular diet over 20 days. After 10 days, the subjects consumed 0.4 ml ethanol kg^?1 day^?1 as red wine together with dinner over a 10-day period. After 8 and 18 days, respectively, a ¹⁵N-labelled yeast protein was administered in a dosage of 4.2 mg kg^?1 body weight. Urine and faeces were collected over 48 h, respectively. The ¹⁵N-enrichment was measured by isotope ratio mass spectrometry, whereas the protein flux rates were calculated by a three-compartment model. The whole-body protein turnover without/with red wine consumption amounted to 3.73±0.6 and 3.49±0.6 g kg^?1 day^?1 (not significant), respectively. Moderate alcohol consumption does not induce significant short-term changes in the whole-body protein turnover of healthy adults.     

The metabolic effect of resistant starch and yoghurt on the renal and faecal nitrogen and ammonia excretion in humans as measured by lactose-[15N2]ureide

Resistant starch (RS) and Lactobacillus acidophilus yoghurt (LC1) were supplemented simultaneously in healthy adults to evaluate the effect on the urinary and faecal nitrogen and ammonia excretion by means of lactose-[¹⁵N₂]ureide (¹⁵N-LU) degradation. Nineteen subjects received a regular daily diet either without or with supplementation of an RS-LC1-mixture composed of fibre of potatoes (RS type 1), wrinkle pea starch (RS type 2), and LC1 over a 20-day period in randomised order. Thereafter, ¹⁵N-LU was administered together with breakfast. Urine and faeces were collected over a period of 48 and 72 h, respectively. The ¹⁵N abundances were measured by isotope ratio mass spectrometry. The intake of the pre- and probiotic mixture composed of RS of type 1, type 2 and of LC1 significantly lowered the colonic generation and the renal excretion of toxic ¹⁵NH₃ and functioned as an ammonia shift from urinary to faecal ¹⁵N excretion when using ¹⁵N-LU as a xenobiotic marker.     

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.     

Dietary fibre-rich resistant starches promote ammonia detoxification in the human colon as measured by lactose-[15N2]ureide

Three resistant starches (RSs), namely fibre of potatoes (FP), wrinkle pea starch (WPS), and high amylose maize starch (HAMS) with different dietary fibre contents, were supplemented in adults to evaluate their effects on urinary nitrogen and ammonia excretion as well as on faecal nitrogen excretion by means of lactose-[¹⁵N₂]ureide (¹⁵N-LU) degradation. Twenty subjects received a regular diet either without or with the supplementation of FP, WPS, and HAMS in a randomized order. After administration of ¹⁵N-LU, urine and faeces were collected over 48 and 72 h, respectively, whereas blood was collected after 6 h. The ¹⁵N-abundances were measured by isotope ratio mass spectrometry. In comparison to the dry run, supplementation with RS significantly lowered renal ¹⁵N-excretion (dry run: 43.2?%, FP: 34.6?%, WPS: 37.9?%, HAMS: 36.4?%) as well as the corresponding ¹⁵NH₃-excretion (dry run: 0.08?%, FP: 0.06?%, HAMS: 0.05?%), clearly indicating a reduced colonic nitrogen generation at high dietary fibre intake.