Interpretation of non-invasive breath tests using 13C-labeled substrates - a preliminary report with 13C-methacetin
© I. Holzapfel Publishers 2009
Received: 7 September 2009
Accepted: 1 October 2009
Published: 14 December 2009
Non-invasive breath tests can serve as valuable diagnostic tools in medicine as they can determine particular enzymatic and metabolic functions in vivo. However, methodological pitfalls have limited the actual clinical application of those tests till today. A major challenge of non-invasive breath tests has remained the provision of individually reliable test results. To overcome these limitations, a better understanding of breath kinetics during non-invasive breaths tests is essential. This analysis compares the breath recovery of a 13C-methacetin breath test with the actual serum kinetics of the substrate. It is shown, that breath and serum kinetics of the same test are significantly different over a period of 60 minutes. The recovery of the tracer 13CO2 in breath seems to be significantly delayed due to intermediate storage in the bicarbonate pool. This has to be taken into account for the application of non-invasive breath test protocols. Otherwise, breath tests might display bicarbonate kinetics despite the metabolic capacity of the particular target enzyme.
Keywordsliver function liver function test 13C-breath test methacetin cytochrome P450 1A2 LiMAx test
However, it remains somehow undefined which way actually provides the most valid and reliable test readout. The aim of this analysis was to explore the correlation between substrate and 13CO2 kinetics during the intravenous 13C-methacetin breath test to improve the analytic algorithms.
The experimental study was performed in healthy volunteers after approval by the faculties ethics review board. The persons were assessed by a specific breath test using 13C-methacetin as substrate for the hepatic cytochrome P450 1A2 system and thereby blood samples were drawn to determine the substrate kinetics. The methodology was based on the previously reported LiMAx test of Stockmann et al. . The substrate was administered into a peripheral vein as a bolus in a dose of 2 and 4 mg/kg body weight.
Breath sampling and analysis
An online protocol of breath analysis was applied, to enable a high sampling rate to enable kinetic analysis of breath recovery. Breath samples were automatically drawn and analyzed with a frequency of as approximately 1/min by a modified nondispersive isotope-selective infrared spectroscopy based device (FANci2db16, Fischer Analyseninstrumente, Leipzig, Germany). Exhaled breath was collected by a special two-way face mask. Mean baseline 13CO2/12CO2 ratio was recorded ten minutes before injection for the calculation of delta-over-baseline (DOB) 13CO2/12CO2 ratio values. The presented 13CO2/12CO2 ratio is standardized by the Pee Dee Belemnite standard . For each test, a total of 46 breath samples were automatically analyzed.
Blood sampling and analysis
Bloods samples were drawn from a peripheral vein before injection of the substrate, and after 30 seconds, 1, 2, 5, 10, 20, 30 and 60 minutes. Samples were taken in a standardized way. Firstly, 5 mL of blood were sampled and discarded. Secondly, a sample of 5 mL was taken in a serum tube for analysis. Finally, the catheter was flushed by 10 mL of 0.9% sodium chloride solution. Serum probes were centrifuged with 3,000 rpm for 4 minutes and the serum aliquot was taken into a separate tube. Probes were analyzed for the concentration of methacetin by high performance liquid chromatography (HPLC). The analysis was performed by a specialized pharmacologist, who was blinded from the breath test results. For sample preparation 50 μL serum were mixed with 100 μL of a acetonitrile methanol solution (1 : 1) and centrifuged 14,000 rpm for 8 minutes. Finally, 10 μL of each sample was applied to the analyzer. A commercial HPLC-Test-Kit for measurement of levetiracetam in serum (Chromsystems GmbH, Munich, Germany) was used for analysis. The Kit-conditions were modified for estimation of methacetin. Chromatography was performed with a LC-6B system (Shimadzu, Duisburg, Germany) at a flow rate of 1.5 mL/min, with UV-detection at 260 nm. The sensitivity was 0.5 μg/mL with proven test linearity up to a concentration of 100 μg/mL. The mean inter-assay variability for methacetin was 6.8%.
The pilot experiment was performed in a 34-year old male healthy volunteer without any history of hepatic or extra-hepatic disease. His healthy condition was confirmed by routine clinical biochemistry including a standard pattern of parameters (Aspartat-aminotransferase, alanine-aminotransferase, bilirubin, albumine, creatinine, urea, blood count, prothrombin time) and a standard history taking and clinical examination. The tests were performed in a resting position on two consecutive days.
Any protocol of breath analysis for dynamic breath test should aim to display the actual metabolism at its best. The literature has reported the successful differentiation between diseased and non-diseased groups by NBTs using 13C-labeled substrates [1, 2]. However, this is only a pre-condition for the successful implementation into clinical diagnostics. Individually reliable test results that prove superior prognostic power in comparison to preexisting diagnostic tests are required  and the different algorithms require further standardization for clinical application. If 13CO2 is not expired directly but retained inside the body during the active metabolism, this has to be taken into account for the methodology of breath sampling and the correct interpretation of test results. These preliminary results confirm the significant difference between serum kinetics of methacetin and the kinetics of 13CO2 in expired breath. Intravenous injection of 13Cmethacetin leads to a very early maximum of DOB values within less than 10 minutes, while the substrate levels have already decreased significantly from its maxima directly after injection. This could be interpreted that the physiological metabolism of 13C-labeled methacetin is extremely fast at the administered dosages. Moreover the 13CO2 excretion and thus breath recovery appears to be significantly delayed in comparison to the continuously rapid decrease of the substrate serum levels. The prolonged pulmonary excretion of 13CO2 over one hour strongly confirms that the quickly produced 13CO2 is not completely expired, but a certain magnitude is stored as bicarbonate inside different body compartments. As the 13C-methacetin breath test was meant to analyze cytochrome capacity and not individual bicarbonate kinetics, this phenomenon needs to be considered more thoroughly. As a consequence, protocols that determine test readouts from single time point breath samples could be significantly influenced by individual bicarbonate kinetics. In contrast, the online assessment analyzes a large number of breath samples - without any sampling bags or tubes - and thus could also determine the individual bicarbonate kinetics. As a result, the maximum of 13CO2 excretion can be accurately determined at an early point after injection and might be more closely connected to the fast in vivo methacetin metabolism (Figure 1). Nevertheless, these effects need to be further investigated and confirmed in larger numbers of healthy volunteers and liver diseased patients. In conclusion, accurate test results from NBTs could only be obtained, when other influencing factors such as the physiological serum kinetics of the substrate and the bicarbonate kinetics are taken into account in the development of suitable test protocols.
non-invasive breath tests
delta over baseline
high performance liquid chromatography.
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