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C-methacetin breath test reproducibility study reveals persistent CYP1A2 stimulation on repeat examinations.

AIM
To find the most reproducible quantitative parameter of a standard (13)C-methacetin breath test ((13)C-MBT).


METHODS
Twenty healthy volunteers (10 female, 10 male) underwent the (13)C-MBT after intake of 75 mg (13)C-methacetin p.o. on three occasions. Short- and medium-term reproducibility was assessed with paired examinations taken at an interval of 2 and 18 d (medians), respectively.


RESULTS
The reproducibility of the 1-h cumulative (13)C recovery (AUC(0-60)), characterized by a coefficient of variation of 10%, appeared to be considerably better than the reproducibility of the maximum momentary (13)C recovery or the time of reaching it. Remarkably, as opposed to the short gap between consecutive examinations, the capacity of the liver to handle (13)C-methacetin increased slightly but statistically significantly when a repeat dose was administered after two to three weeks. Regarding the AUC(0-60), the magnitude of this fixed bias amounted to 7.5%. Neither the time gap between the repeat examinations nor the gender of the subjects affected the (13)C-MBT reproducibility.


CONCLUSION
(13)C-MBT is most reproducibly quantified by the cumulative (13)C recovery, but the exactitude thereof may be modestly affected by persistent stimulation of CYP1A2 on repeat examinations.


RESULTS:
The reproducibility of the 1-h cumulative 13 C recovery (AUC0-60), characterized by a coefficient of variation of 10%, appeared to be considerably better than the reproducibility of the maximum momentary 13 C recovery or the time of reaching it. Remarkably, as opposed to the short gap between consecutive examinations, the capacity of the liver to handle 13 C-methacetin increased slightly but statistically significantly when a repeat dose was administered after two to three weeks.
Regarding the AUC0-60, the magnitude of this fixed bias amounted to 7.5%. Neither the time gap between the repeat examinations nor the gender of the subjects affected the 13 C-MBT reproducibility.
CONCLUSION: 13 C-MBT is most reproducibly quantified by the cumulative 13 C recovery, but the exactitude thereof may be modestly affected by persistent stimulation of CYP1A2 on repeat examinations.

INTRODUCTION
There is no surprise that non-invasive diagnostic procedures in medicine attract much attention from either side participating in the search of any cause of malfunctioning of the organism-patients would definitely prefer methods sparing them the necessity of encroaching the body integrity, like for example a liver biopsy, whereas physicians too would prefer to avoid inflicting pain on their patients. A brilliant idea of per oral administration of 13 Cenriched substrates in order to get information on the metabolic efficiency or functional mass of the liver is a practical answer to the requirement outlined above. Continuous research work has brought about considerable progress and nowadays it is possible to assess by means of 13 C breath tests the microsomal, the cytosolic, and the mitochondrial function of the liver [1][2][3][4] . A look at pertinent literature shows that during the past decade, from among the compounds applied for microsomal 13 C breath tests, 13 C-methacetin has steadily been making its way to be recognized as the most frequently used substrate. A number of features support its usefulness as a functional liver probe: a fast metabolism to acetaminophen and 13 CO2 by cytochrome P450 1A2 (CYP 1A2), safety at low doses applied for a breath test, and a low cost [1][2][3][4] . Accordingly very promising results on its diagnostic usefulness were obtained in patients with chronic hepatitis C virus infection [5,6] , primary biliary cirrhosis [7,8] , non-alcoholic steatohepatitis [9] , and various stages of liver cirrhosis [10][11][12][13][14] , including those awaiting a liver transplantation [15] .
A vital asset of any measurement or diagnostic method used in medical practice is an ability to provide reproducible results. Quite surprisingly a search of data on the reproducibility of the 13 C-methacetin breath test ( 13 C-MBT) revealed this item as being almost a completely blank research area. We decided therefore to search in this prospective study for a quantitative parameter which would offer the best reproducibility for a standard 13 C-MBT.

Subjects
Twenty healthy non-obese subjects, 10 female and 10 male were invited to enter the study; their mean age was 25 years (range 21-31 years), and their average body mass index was 22.38 ± 0.64 kg/m 2 . During a screening interview the participants declared themselves as being in full health according to the World Health Organisation criteria [16] . Among the participants three (1 male and 2 female) were habitual cigarette smokers. All the subjects categorically denied a systematic use of significant amounts of alcoholic beverages. None of the volunteers did take any medication except for oral hormonal contraception which was used by 4 female. Exclusion criteria to attend the study comprised a history of surgery affecting the digestive tract anatomy, with the exception of appendectomy, current use of any drugs which might affect gastrointestinal motility, and pregnancy. In every subject the normal structure and size of the liver was confirmed by means of an ultrasonographic examination performed by a qualified investigator.
The study was conducted in accordance with the Helsinki Declaration, and every volunteer gave written consent to participate after getting information as to the aim, protocol and methodology of the project. During the introductory interview the subjects were instructed not to eat any food with a naturally increased 13 C content, such as products made of maize, cane sugar, pineapple, or kiwi fruit for 48 h preceding the examination [17][18][19] .

Experimental protocol
Every subject underwent three examination sessions, held on separate days. In half of the volunteers the two first sessions were taken 2-4 d apart, and the third one was pursued 2-3 wk later. In the other half of the subjects the schedule was inversed, i.e. the first two examination measurements were taken 2-3 wk apart, followed by a third one 2-4 d after the second one. The assignment of the order of intervals separating the sessions (short-long or long-short) was randomized. Accordingly, the assessment of the short-term reproducibility involved 20 pairs of examinations separated by a median 2 d break (range 2-4 d), whereas the medium-term reproducibility was evaluated on 20 pairs of the most distant examinations, i.e. taken at a median interval of 18 d (range 17-23 d).
The volunteers came to the laboratory in the morning, after a 12-h overnight fast and abstaining from cigarette smoking (if applicable). In the female volunteers the examinations were taken always within the same phase of their menstrual cycle. After a 15-min rest in a sitting position, necessary for stabilization of the metabolism, a basal sample of the exhaled air was collected. At the time point designated "0" the subjects took 75 mg 13 C-methacetin (code INC590P, Euriso-Top SA, Saint-Aubin, France; according to the certificate of analysis, the manufacturer guarantees ≥ 99% isotopic 13 C atom enrichment determined by proton NMR) orally dissolved in 200 mL unsweetened black tea. Samples of expiratory air for 13 CO2 measurement were collected at 3, 6,9,12,15,18,21,24,27,30,40,50, 60, 75, 90, 105, 120, 150 and 180 min [20] . The procedure of collecting the breath air was standardized: the subjects took in breath and held it for 10 s, then steadily blew the air into a special aluminium covered plastic bag of 1.1 L capacity (Fischer Analysen Instrumente GmbH, Leipzig, Germany) equipped with a mouthpiece and an unidirectional valve [21,22] . The bag was closed with a plastic cork immediately at the end of the exhalation and stored for analysis. The subjects remained seated quietly and fasted throughout the whole period of the collection of the samples.

Measurement of 13 CO2 and derivation of the breath test parameters
Concentrations of 13 CO2 in the probes of the exhaled air were measured with the use of a non-dispersive isotopeselective infrared spectrometry apparatus (IRIS, manufactured by Wagner Analysen Technik Vertriebs GmbH, Hamburg, Germany; a model equipped with 16 ports for simultaneous mounting of bags with air samples was used). Following the procedures described previously [20,23,24] , curves of the momentary and cumulative recovery of 13 C in the exhaled air relative to the administered oral dose of 13 C-methacetin were constructed within the time domain of 0 to 180 min. Subsequently the following parameters were derived: Dmax -the maximum momentary 13 C recovery in expiratory air, Tmax -the time elapsing from the intake of the substrate until the occurrence of the Dmax, and AUCi -the cumulative 13 C recovery within Table 1 Reproducibility of the 13

C-methacetin breath test
a time span i, which was calculated by integration of the respective part of the momentary 13 C recovery curve.

Statistical analysis
The data were subjected to the Bland and Altman statistic for calculation of repeatability coefficients and check for a proportional or fixed bias [25,26] . In addition the reproducibility of breath test parameters was expressed in terms of the coefficient of variation for paired examinations, CVp [27,28] . Sets of absolute values of individual differences in paired measurements were compared with the use of the paired or unpaired t test (where appropriate) in order to check if factors such as the time gap between the examinations, or the gender of the subjects affected the reproducibility. Other statistical methods comprised the use of a repeated measures analysis of variance (R_ANOVA) followed by a post hoc check on the significance of differences between means, which was accomplished with the Tukey's honest significant difference test [29] . Statistical significance was set at the P < 0.05 level, two-tailed. The results are presented as mean ± SE. All statistical analyses were performed with the use of Statistical 6.0 software [30] .

RESULTS
Similar basal concentrations of 13 CO2 in the expiratory air were observed on the three study days, amounting to -25.79‰ ± 0.25‰, -25.44‰ ± 0.21‰ and -25.59‰ ± 0.22‰. After oral intake of the 13 C-methacetin solution an expected biphasic course of 13 CO2 content within the exhaled air was observed, characterized by a rapid rise followed by a less steep decline. R_ANOVA indicated that when referred to the basal situation, the rise in breath 13 CO2 was statistically significant between the 6th and the 150th minute ( Figure 1).

Basic reproducibility assessment
As shown in Table 1, the Tmax exhibited rather an unsatisfactory reproducibility, whereas the Dmax displayed a fair reproducibility, supposedly sufficient from the point of view of diagnostic applications of the breath test. The reproducibility of the cumulative 13 C recovery relative to the administered substrate dose was strongly dependent on the time span included for the calculation of this parameter, and improved rapidly with inclusion of the subsequent measurement points (Figure 2). The AUC0-60 attained a CVp of 10%-identical in both the case of the    tion vs 21.03% ± 1.07% dose on the second administration, NS]. A graphical representation of the phenomenon observed is provided in Figure 4.

DISCUSSION
Without any doubt provision of reproducible measurement results is a feature of paramount importance, expected to be assured by methods designed for research and/or clinical applications in medicine [28,[31][32][33] . Breath tests performed with the use of stable isotopes, such as 13 C, cannot be considered an exception in this respect, especially because during the past decade their clinical use has been constantly growing [24,34,35] . An increase in the number of relevant scientific papers indicates that, from among the tests dedicated to assess the activity of cytochrome P450, known as the microsomal breath tests, the 13 C-MBT has lately taken the lead. Accordingly, Yaron Ilan in his very recent review [36] provides evidence-based arguments that the 13 C-MBT is a powerful tool to aid hepatologists in bedside decision making. While searching the literature we came across only one study which was aimed at the evaluation of the reproducibility of the 13 C-MBT. Petrolati et al [15] performed repeat measurements separated by an interval of 12 wk in 10 healthy volunteers. This scarcity of data encouraged us to undertake a systematic, prospective study on the reproducibility of the quantitative measures of a standard 13 C-MBT. Much effort was given in order to assure comparable conditions while performing the 13 C-MBT. Accordingly, all the examinations were started at the same time in the morning, a 15-min rest always preceded the collection of the basal samples of expiratory air, the procedure of taking the breath samples was standardized, and comfortable surroundings were provided to the volunteers so that they could stay relaxed while maintaining a sitting position throughout the examination. Moreover, the female participants were always examined in the same phase of the menstrual cycle. While designing the study protocol we decided to adopt the currently accepted mode of administration of 13 C-methacetin, namely a fixed oral dose of 75 mg [5,6,8,13,15,20,37,38] . It should be noted, however, that formerly other body mass adjusted dosage regimens were applied, encompassing 5 mg/kg in the pioneer work by Krumbiegel et al [39] , then 2 mg/kg [7,11,12,40] , as well as 1 mg/ kg [10,41] , and finally Iikura et al [42] applied in infants 0.5 mg/kg 13

C-methacetin.
Taking into account the investigative nature of our research, we generously took as many as 19 samples of breath air throughout 3 h. This approach appears to be precedential in nature, because for routine clinical use just a few measurement points are usually considered [15,42] . Nine samples were collected in the study by Zipprich et al [13] (at 10, 20, 30, 40, 50, 60, 80, 100 and 120 min after application of the substrate), and twelve samples of the expiratory air-every 15 min for 3 h of observation -were taken by Ciccocioppo et al [41] . A team of German researchers from Bochum collected thirteen samples-at 5, 10, 15, 20, short-or the medium-term reproducibility. The inclusion of data from beyond the 60 min did not bring about much refinement of the reproducibility of the AUC.
No statistically significant difference between the shortand medium-term reproducibility of the breath test parameters was found. Moreover, none of the parameters considered had a different short-or medium-term reproducibility in men and women.

Bland and Altman statistics of reproducibility
Bland and Altman plots pertaining to representative parameters of the 13 C-MBT are provided in Figure 3. Bland and Altman statistics revealed no proportional bias in either Dmax or AUC0-60 ( Table 1). The same applied to the short-term reproducibility of Tmax, whereas analysis of its medium-term reproducibility showed that the slope of the linear regression of the between-day differences on the corresponding means of the paired measurements was statistically significantly different from zero ( Table 1).
The Bland and Altman statistic disclosed a fixed bias in the case of the medium-term reproducibility of either the Dmax or the AUC0-60. It means that the mean differences between the paired measurements taken a median of 18 d apart differed statistically significantly from zero. A closer look at the pertinent differences indicated that the ability of the liver to handle 13 C-methacetin increased when a repeat dose was administered after two to three weeks (Dmax: 34.60% ± 2.75% dose/h on the first administration vs 39.08% ± 2.77% dose/h on the second administration, P = 0.015; AUC0-60: 20.80% ± 0.99% dose on the first administration vs 22.27% ± 0.98% dose on the second administration, P = 0.017). No such effect was observed if two doses of 13   30, 40, 50, 60, 80, 100, 120, 150 and 180 min after administration of 13 C-methacetin [12] . One should mention that our frequent sampling of breath air (an aliquot was taken every 3 min during the first half hour) is similar to the new approach recently proposed by Lalazar et al [6,38] who introduced a "continuous" 13 C-MBT wherein samples of breath air are taken every 2-3 min and analysed with a laser-based device.
Our intention was to determine the length of the Tmax with the greatest exactitude possible. Nevertheless, with the CVp between 30.6% and 32.5%, the reproducibility of this parameter appeared to be a bit disappointing. Apparently the Tmax may therefore not be the most useful parameter for the purposes of clinical decision making.
On the other hand, we established on the basis of the within-subject study protocol that the repeatability of the Tmax was sufficient to discern a difference in its length as small as 4 min (P = 0.05 level, two-tailed, n = 20).   Dmax (CVp of 16%) showed better reproducibility, whereas the most reliable with regard to its repeatability appeared to be cumulative 13 C recovery in expiratory air. At this point it is important to emphasize that the reproducibility of the cumulative 13 C recovery was dependent on the time span considered for its calculation. Hence the CVp improved rapidly with inclusion of the subsequent measurement points, achieving a reasonable value of 10% for the period comprising the first 60 min following the administration of the substrate. Consideration of later measurement points offered only a small further improvement in the reproducibility of the AUC. Our finding provides an argument in support of limiting the time for sample collection to 1 h when performing the 13 C-MBT-which is a standpoint coherent with the procedure proposed lately by the research group headed by Yaron Ilan from Israel [6,38] .
Of great importance is the finding that in the case of each of the considered parameters, characterizing quantitatively the result of the 13 C-MBT, the reproducibility depended neither on the time gap between the repeat examinations nor on the gender of the subjects. Thus a convincing proof of reliability of this test has been obtained. Quite unexpectedly a thorough analysis of reproducibility, involving the application of the Bland and Altman statistic, disclosed the existence of a fixed bias in the case of the medium-term reproducibility of the Dmax and the AUC60. It was found that those two parameters increased statistically significantly if the repeat examinations were separated by a two-to three-week gap, which was not the case when the results of examinations taken at a close sequence were compared. A similar trend can, however, be discerned when looking at the results obtained by Petrolati et al [15] in their attempt to evaluate the reproducibility of the 13 C-MBT. They found that the 45-min cumulative 13 C recovery (AUC0-45) in breath air after peroral intake of 75 mg 13 C-methacetin amounted to 17.5% ± 2.8% dose and to 18.8% ± 4.3% dose on the first and the second occasion, respectively. Although the two means did not differ statistically significantly (P = 0.30), it is evident that the AUC0-45 determined on the second occasion increased by 7.4%. Quite a similar magnitude of increment was determined in our study-the AUC0-60 rose by 7.5% on the repeat measurement taken after two to three weeks (P = 0.017), whereas it remained unchanged with an average difference of -0.6% (NS) between measurements performed a median of 2 d apart. A delayed and persisting stimulation of a minor, but statistically significant, magnitude of the CYP1A2 capacity to metabolize methacetin would therefore be inferred. It remains unclear if the phenomenon disclosed would have a clinically relevant impact. It is rather obvious that not all patients with liver disease require performance of a series of 13 C-MBTs, which most likely may pertain to those awaiting liver transplantations [15] . At present one cannot predict whether a stimulation of CYP1A2 observed on repeated 13 C-MBT in healthy volunteers will occur also in patients with major impairment of the liver functional reserve. Nevertheless the error in estimating the cumulative 13 C recovery in breath air caused by a stimulation of CYP1A2 by 13 C-methacetin seems to be quite small, not exceeding 8% according to our results, as well as those obtained by Petrolati et al [15] .
Summing up, the study renders evidence that the 13 C-MBT provides satisfactorily reproducible results. Remarkably, the cumulative 13 C recovery appear to be the most reproducible quantitative index of the test, the computation of which over the first hour following administration of the substrate offers a reasonable 10% coefficient of variation, regardless of the time span separating the repeat measurements. One should be aware that the exactitude of this parameter may be modestly affected by a persistent stimulation of CYP1A2 on repeat examinations.

Background
Great progress has been made during the past two decades with respect to the diagnostic use of stable isotopes in hepatology -it is now possible to assess by means of 13 C breath tests the microsomal, the cytosolic, or the mitochondrial function of the liver. From the point of view of a patient those tests are particularly attractive as in many instances they may result in avoidance of the inconvenience of a repeat liver biopsy.