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.     

Towards an inhalative 13C breath test method

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. 75-120 min after the end of inhalation a well-reproducible maximum delta13C value of 6%o 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.     

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

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

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

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

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

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.     

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.     

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.     

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.     

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.     

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.     

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.     

A simple non-invasive method to detect and monitor hypercapnia: the sodium [13C]bicarbonate breath test

Arterial partial pressure of carbon dioxide (paCO(2)) is commonly evaluated by an invasive test, the arterial blood gas analysis (ABG). The sodium [(13)C]bicarbonate breath test (SBT) can potentially estimate arterial paCO(2). We studied 55 subjects with respiratory disorders and performed the ABG and the SBT to determine if the SBT can predict hypercapnia. The percentage of (13)CO(2) recovered in exhaled breath at 30 minutes (PDR(30)) alone was able to discriminate clinically significant hypercapnia (>53 mmHg) with a sensitivity of 82 % and specificity of 93 % (p<0.001). To evaluate the clinical utility of the SBT as a non-invasive substitute to repeated ABG, we monitored the progress of seven chronic obstructive pulmonary disease (COPD) patients on therapy with both the ABG and the SBT. The PDR(30) values from the SBT were able to correctly predict improvement or worsening of paCO(2) with 100 % accuracy. In conclusion, the SBT is a simple test that can be used in clinical practice to detect clinically significant hypercapnia and monitor COPD patients before and after therapy.     

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.     

Significance of diagnostic parameters in [(13)c]octanoic Acid gastric emptying breath tests*

Abstract Two novel characteristic parameters, the latency time (t (lat)) and the ascension time (t (asc)), are proposed for evaluation of non-invasive [(13)C]octanoic acid breath tests for assessment of the gastric emptying of solids. In breath tests performed in control subjects (n = 30) and diabetic patients (n = 100), the usefulness of these parameters was compared to conventional parameters, i.e., gastric half emptying-time t (1/2,b )) and lag phase (t (lag,b )). The proposed parameters were only loosely correlated (controls, r = 0.199; diabetics, 0.616). A strong correlation was found between the conventional parameters (controls, r = 0.891; diabetics, r = 0.962). Based on the conventional method, 36 patients were suspicious of delayed gastric emptying including 24 patients which exhibited a simultaneous delay in both parameters. Using the new parameters, a total of 46 patients were suspicious of delayed gastric emptying with 15 and 20 having isolated delay in t (lat) and t (asc), respectively. We conclude that the novel parameters may be more appropriate for examination of the different phases of gastric emptying and for evaluation of gastric emptying disturbances in diabetic patients than the parameters conventionally used for this purpose.     

Hepatic parenchymal enhancement at Gd-EOB-DTPA-enhanced MR imaging: correlation with morphological grading of severity in cirrhosis and chronic hepatitis

The aim was to clarify whether enhancement effects of the liver parenchyma in the hepatobiliary phase (HP) of gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced MR imaging were correlated with the morphological grading of the severity in cirrhosis. A total of 62 patients with chronic hepatitis or cirrhosis underwent Gd-EOB-DTPA-enhanced MR imaging. Relative enhancement (RE) of liver parenchyma was calculated from signal intensity (SI) measurements obtained at precontrast images (SIpre) and 20-min postcontrast HP images (SIpost) as: (SIpost-SIpre)/SIpre. Morphological MR grades of severity in cirrhosis were divided into four groups. Then, RE of liver parenchyma and morphologic MR grading were correlated. Regarding the morphologic severity of cirrhosis, the numbers of patients with MR grade 1, 2, 3 and 4 were 14 (23%), 7 (11%), 28 (45%) and 13 (21%), respectively. The mean REs of liver parenchyma in each group of MR morphologic grade 1, 2, 3 and 4 were 0.71±0.21, 0.62±0.16, 0.70±0.22 and 0.77±0.18, respectively. There was no significant correlation between the MR grading of morphologic severity and the RE of liver parenchyma at 20-min HP. Hepatic parenchymal enhancement in the HP of Gd-EOB-DTPA-enhanced MR imaging did not necessarily decrease according to the severity of morphologic changes in cirrhosis. This fact may suggest that the hepatic uptake of Gd-EOB-DTPA depends on the preserved hepatocytes function rather than the severity of morphologic changes in cirrhosis.     

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.     

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.     

Evidence for Increased Microsomal Hepatic Function in Thalassaemic Children as Detected by [13C]Aminopyrine Breath Test

Abstract

[¹³C] aminopyrine breath tests (AP-BT) were performed in 19 children suffering from homozygous β-thalassaemia major. In contrast to expectations an increased ¹³C elimination representing an increased AP demethylation was found in initial breath tests. Repeated AP-BT over several years indicated a significant decrease by time. Hypothyreoidism was proved to delay AP demethylation. Though some of the patients show slightly disturbed coagulation and most of them elevated transaminases and definite hepatic iron storage, no correlation of these laboratory parameters to ¹³C elimination in ¹³C AP-BT was found.     

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.     

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.