Standardizing the ¹³C-Methacetin Breath Test: A Call for Clinical Integration in Liver Function Testing

Abstract

Background/Objectives: The ¹³C-Methacetin Breath Test (MBT) is a non-invasive tool to evaluate hepatic microsomal function via exhaled ¹³CO₂, reflecting cytochrome P450 1A2 (CYP1A2)-mediated metabolism. Despite decades of evidence demonstrating its utility in diagnosing cirrhosis, stratifying liver disease severity, and predicting outcomes, MBT adoption remains limited due to methodological inconsistencies and variable diagnostic thresholds. This review aimed to summarize MBT data in adults and assess its diagnostic and prognostic performance. Methods: A literature review was conducted using PubMed, Web of Science, and Scopus. Eligible studies included those applying oral or intravenous methacetin with defined reference values or diagnostic cutoffs. Outcomes of interest were percent dose recovery (PDR), cumulative PDR (cPDR), and LiMAx^® values. Due to heterogeneity in protocols, units, and endpoints, results were synthesized narratively. Results: Healthy individuals typically demonstrated rapid metabolism (e.g., cPDR30 10–15%), whereas cirrhotic patients showed significantly reduced values (e.g., cPDR30 ≈ 1%). Diagnostic cutoffs varied widely (<0.35% to <8%), reflecting methodological and population differences. MBT reliably identified advanced liver disease but showed inconsistent sensitivity for early-stage fibrosis and metabolic dysfunction-associated steatotic liver disease. Variability in dosing, timing, measurement duration, and analytic technique limited cross-study comparability. Conclusions: MBT is a validated, dynamic marker of liver function with both diagnostic and prognostic relevance. However, inconsistent protocols and thresholds hinder its clinical implementation. Standardization of MBT procedures, reference ranges, and reporting metrics is essential. A harmonized protocol (“MBT-60”), supported by multicenter validation, demographic stratification, and direct comparison with structural and serologic liver tests, is recommended to facilitate MBT integration into routine hepatology practice.

1. Introduction

Accurate assessment of hepatic function is critical for the diagnosis, risk stratification, and management of liver disease. Conventional serum biomarkers, such as aminotransferases or bilirubin, primarily reflect hepatocellular injury rather than true metabolic capacity, while imaging-based methods like transient elastography evaluate structural stiffness rather than dynamic functional reserve. Consequently, there is growing recognition of the need for direct, quantitative tools that assess the liver’s metabolic performance in vivo.

The ¹³C-Methacetin Breath Test (MBT) is a non-invasive method for assessing hepatic microsomal function by measuring ¹³CO₂ exhalation after administering ¹³C-labeled methacetin (N-[4-methoxy-¹³C-phenyl]-acetamid) [1,2,3]. In the liver, methacetin is O-demethylated by cytochrome P450 1A2 (CYP1A2) to acetaminophen and ¹³CO₂, providing a quantitative marker of functional hepatocyte mass [1,4].

Due to its safety, low cost, and simplicity, MBT has been proposed as a clinical liver function test [3,5,6]. Over decades, studies have confirmed its ability to distinguish between healthy and impaired liver function in adults, particularly in conditions like cirrhosis, viral hepatitis, and metabolic dysfunction-associated steatotic liver disease (MASLD), as well as the stratification of disease severity. Patients with advanced liver disease consistently show reduced ¹³CO₂ exhalation compared to healthy controls [7,8].

Despite robust evidence, MBT has not been widely adopted in standard clinical hepatology, mainly due to inconsistent reference ranges and diagnostic thresholds across studies. This variability complicates the interpretation of results and the establishment of universally accepted “normal” values and diagnostic cutoffs. Reported cutoffs vary from <1% cumulative ¹³CO₂ recovery at early time points [9] to <20%/h peak recovery depending on protocol specifics and clinical endpoints [10]. This heterogeneity stems from methodological differences (e.g., oral vs. intravenous dosing, substrate amount, sampling intervals, test duration) and diverse patient populations.

To address this, consistent, evidence-based reference values are urgently needed. Some researchers advocate using the lower 95% confidence limit in healthy adults as a threshold; for instance, Lalazar et al. [10] reported a mean peak percent dose recovery (PDR) of 35% ± 9%/h (range 20–60%/h), suggesting ~20%/h as the minimum normal value. Others use time-specific cutoffs, such as <8% cumulative recovery at 30 min as reduced metabolic capacity [11,12,13] or ≤0.55% at 20 min in high-risk cirrhosis linked to poor prognosis [9,14]. Additionally, a more recent variant of the test, the LiMAx^® (Liver Maximum Capacity) test, utilizes intravenous methacetin and quantifies real-time ¹³CO₂ output [15]. The normal reference value (>315 µg/kg/h) was initially defined by the manufacturer (Humedics GmbH, Berlin, Germany) and has since been validated in multicenter cohorts involving both healthy controls and surgical patients [16,17]. However, the oral MBT remains more practical and accessible in non-surgical and outpatient settings, providing complementary information on intestinal absorption, first-pass metabolism, and systemic elimination.

These discrepancies highlight the need for harmonization of reference ranges. Standardizing MBT protocols and thresholds would clarify interpretation and facilitate broader clinical use. In contrast to previous reviews, significant advances have occurred in methodology and understanding of disease-specific metabolism, particularly in the context of MASLD, circadian variability, and age- and sex-specific reference ranges [18,19,20]. These advances now allow for a more integrated understanding of how physiological, demographic, and methodological factors influence MBT outcomes and justify a renewed call for standardization. The aim is therefore to consolidate recent findings, identify sources of heterogeneity, and propose a unified oral MBT protocol (“MBT-60”) as a step toward global harmonization of liver function testing in clinical practice.

2. Methods

This review represents an expert narrative synthesis of the published evidence on the MBT in adults. A structured literature search was conducted in PubMed, Web of Science, and Scopus to identify key studies from the early 2000s through April 2025 reporting MBT performance, reference ranges, diagnostic thresholds, and prognostic applications. Foundational studies from the 1990s were also screened for seminal reference ranges, though priority was given to the past 10–20 years of peer-reviewed hepatology/gastroenterology journals to avoid outdated methods. The search was designed to capture representative and methodologically relevant studies rather than exhaustively cataloging all publications. Search terms included combinations of “¹³C” or “¹³C” with “methacetin breath test”, “normal values”, “reference range”, “threshold”, “cut-off”, “liver function”, “cirrhosis”, “hepatitis”, “NAFLD (non-alcoholic fatty liver disease)”, “MASLD”, etc. Study inclusion was based on scientific relevance, methodological clarity, and the contribution to understanding MBT physiology and clinical utility.

As this review followed a narrative approach, no formal PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) framework [21], flow diagram, or risk-of-bias assessment was applied. The synthesis focused on identifying consistent methodological patterns, highlighting sources of heterogeneity, and summarizing key developments in test standardization and clinical interpretation. This approach allows a more nuanced discussion of the evidence base and facilitates expert comparison across diverse study designs.

The inclusion criteria for studies were: firstly, MBT studies in adults (≥18 years) using oral or intravenous methacetin; secondly, quantitative MBT outcomes (e.g., PDR, cumulative percent dose recovery (cPDR), delta-over-baseline (DOB) values, or related metrics like LiMAx^® in µg/kg/h; and thirdly, the reported reference values or diagnostic thresholds for healthy or diseased livers. Both observational and interventional studies were included, along with reviews or meta-analyses reporting baseline MBT values. Exclusion criteria included pediatric studies, animal research, or studies using other ¹³C substrates (e.g., ¹³C-methionine, ¹³C-aminopyrine).

From included studies, data were extracted on sample size, demographics, liver disease type/severity, MBT protocol (dose, route, measurement technique, sampling schedule), and key outcomes (normal ranges, mean values, cutoffs, sensitivity/specificity). Subgroup analyses explored heterogeneity. Although a few studies noted testing time, none systematically addressed circadian variation; thus, diurnal effects were inferred from external evidence (e.g., CYP1A2 activity).

3. Results

3.1. ¹³C-MBT Performance in Healthy Adults

Across studies, healthy adults consistently exhibit rapid hepatic methacetin metabolism with early and robust ¹³CO₂ exhalation, reflecting intact microsomal CYP1A2 activity. Median peak ¹³CO₂ elimination rates typically range between 30% and 40% of the administered dose per hour (PDRmax), and cumulative recovery at 30 min (cPDR30) lies between roughly 10% and 15% [7,11,13]. Despite variation in protocol duration and reporting format (PDR, DOB, or cPDR), the overall pattern is stable: healthy individuals show rapid early 13CO₂ peaks (~20–40 min) and approximately one-third of the administered ¹³C label is recovered within the first 1–2 h of testing.

This consistency across independent cohorts supports the robustness of MBT kinetics in normal physiology. However, absolute reference intervals remain partly method-dependent. Differences in analytical technique and in sampling windows account for most of the observed numerical spread. Consequently, composite ranges (e.g., cPDR30 ≈ 10–15%, cPDR60 ≈ 20–30%, DOB15 ≈ 15–25‰) provide a more reliable benchmark than single cut-points. However, these values are dependent on the method and protocol used, and should be interpreted in context, as summarized in

Table 1. Overview of Representative Studies on the ¹³C-Methacetin Breath Test (MBT) for Liver Function Assessment.

Study	Population	Methodology	Test Protocol & Metrics	Notes	Outcome & Findings
Banasch et al., 2011 [22]	Metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction-associated steatohepatitis (MASH)	Nondispersive isotope-selective infrared spectrometry	Oral 2 mg/kg body weight 8-h fasting every 10 min for 1,5 h cPDR1.5 h	MASLD/MASH-specific cohort, variable fibrosis stages	Detects mitochondrial dysfunction in MASLD, predicts advanced stages and reduces biopsy necessity
Braden et al., 2005 [12]	Chronic hepatitis C virus (HCV)	Isotope ratio mass spectrometry	75 mg oral overnight fasting 5/10/15/20/25/30/40/50/60 min DOB15 min, cPDR30 min	HCV-specific cohort, variable fibrosis stages	Liver function declines with HCV severity; distinguishes early cirrhotic (Child A) from non-cirrhotic stages, but not early fibrosis
Ciccocioppo et al., 2003 [23]	Healthy elderly adults	Isotope ratio mass spectrometry	75 mg oral 12-h fasting every 15 min for 1 h cPDR15, 30 min	Demographic effects, limited sample sizes	Age-related changes; lower MBT capacity in the elderly, indicates impaired liver function with aging
Dinesen et al., 2008 [24]	Chronic HCV	Nondispersive isotope-selective infrared spectrometry	75 mg oral overnight fasting 0/15 min DOB15 min	Comparator tests varied, different fibrosis staging methods	Predicts fibrosis/cirrhosis more reliably than biochemical tests
Festi et al., 2005 [25]	Chronic liver disease	Isotope ratio mass spectrometry	75 mg oral 8-h fasting every 10 min for 2 h cPDR60 min	Dual-substrate protocol, variable etiology and stages of fibrosis/ cirrhosis	Assesses quantitative functional liver mass, distinguishes different chronic liver disease stages, and correlates these with liver function tests, serum bile acids, and Child–Pugh scores
Fierbinteanu-Braticevici et al., 2013 [26]	MASLD, MASH	Infrared isotope ratio spectrometer	75 mg oral overnight fasting 0 and every 10 min for 1 h cPDR60 min, PDR10 min	Stage-dependence, metabolic confounding	MBT values decrease in MASLD with steatosis and fibrosis correlating with histologic severity; follow-up tool
Fontana et al., 2021 [27]	Acute liver failure (ALF) and non-acetaminophen acute liver injury (ALI)	BreathID®	75 mg oral 6-h fasting 15/60/75 min cPDR20 min, PDRpeak	Acute setting, timing crucial	Helps risk-stratify critically ill patients with ALF and non-acetaminophen ALI, estimates hepatic recovery, and may reduce unnecessary transplants
Goetze et al., 2007 [9]	Chronic HCV	BreathID® Isotope ratio mass spectrometry (IRMS)	75 mg oral 8-h fasting 0/90 min BreathID® one sample/3 min IRMS one sample/10 min DOB, DOBmax	Device/platform effects, specific etiology	BreathID® and IRMS show good agreement in differentiating fibrosis grades in HCV
Goetze et al., 2020 [14]	Chronic HCV (7-year follow up)	BreathID®	75 mg oral 8-h fasting 0/60 min, every 5–10 min PDRpeak	Prospective, long-term, specific etiology	BreathID® is at least as effective as biopsy in predicting liver deterioration leading to transplantation or death in chronic HCV patients with moderate liver function
Holtmeier et al., 2006 [28]	Primary biliary cholangitis (PBC) (early stages)	Isotope ratio mass spectrometry	2 mg/kg body-weight oral overnight fasting, 0/10/20/30/60/90/120/150/180 min cPDR30 min	Cholestatic disease-specific dynamics, long sampling windows	Detects restricted function in early PBC stages without cirrhosis
Jara et al., 2015 [16]	Elective extra-abdominal surgery	LiMAx®	2 mg/kg body weight intravenous 3-h fasting 60 min continuous LiMAx (µg/kg/h)	Different administration, units and dosing against oral conventional MBT	Reliably measures maximal liver function capacity in healthy subjects; unaffected by general anesthesia, supporting its perioperative use
Kochel-Jankowska et al., 2013 [29]	PBC (early and late stages)	Nondispersive isotope-selective infrared spectrometry	75 mg oral 12-h overnight fasting 3/6/9/12/15/18/21/24/27/30/40/ 50/60/75/90/105/120/150/180 min Tmax, Dmax, D%t	Correlation rather than diagnostic cutoffs	Valuable bedside tool for PBC assessment; correlates with Mayo scores, differentiates cirrhotic from non-cirrhotic and PBC stages
Lalazar et al., 2009 [10]	Acute liver disease	BreathID®	75 mg oral 8-h fasting 0/60 min, every 2–3 min cPDR20, 30, 60 min, PDRpeak	Acute-care setting, device-specific cutoffs	Improves bedside decision-making in acute severe liver disease
Lock et al., 2010 [30]	Deceased-donor liver transplantation	LiMAx®	2 mg/kg body weight intravenous 3-h fasting 60 min continuous LiMAx (µg/kg/h)	Transplant-specific thresholds, intravenous route	Evaluates early postoperative graft function, detects critical complications within 24 h, and predicts reoperation needs post-liver transplantation; low LiMAx® readouts indicate graft dysfunction
Molina-Molina et al., 2020 [11]	Metabolic disorders and/or liver steatosis	Nondispersive isotope-selective infrared spectrometry	75 mg oral 8-h fasting 15/30 min DOB15 min, cPDR30 min	Metabolic confounding, BMI stratification	Subclinical dysfunction in obesity; lower values in overweight/obese individuals despite no advanced disease
Pfaffenbach et al., 1998 [7]	Liver cirrhosis	Nondispersive isotope-selective infrared spectrometry	75 mg oral 12-h fasting 5/10/15/20/30/40/50/60/80/120/ 150/180 min cPDRmax, 30, 60, 120, 180 min	Older infrared methodology, long sampling windows	Distinguishes between healthy subjects and patients with liver cirrhosis, suitable for quantitative liver function analysis
Portincasa et al., 2006 [31]	MASLD, MASH	Isotope ratio mass spectrometry	Oral 100 mg, overnight fasting, every 15 min for 2 h cPDR 30, 60, 120 min	Mitochondrial and microsomal substrates combined, metabolic confounding	Mitochondrial dysfunction in MASH aids non-invasive characterization and staging; higher exhalation rates in stages 0–III reflect increased cytochrome P450 activity
Razlan et al., 2011 [1]	Chronic liver disease	Nondispersive isotope-selective infrared spectrometry	75 mg oral, 8-h fasting 10/20/30/40/50/60/80/100/120 min cPDR40, 120 min	Variable etiology and stages of fibrosis/ cirrhosis	Poor predictive value for liver fibrosis, but accurately identifies advanced cirrhosis
Schneider et al., 2007 [32]	Different liver disease (cirrhosis focus)	Nondispersive isotope-selective infrared spectrometry	75 mg oral overnight fasting 0/15-min DOB0, 15 min	Simplified protocol, variable etiology	Simplification of MBT indicates reduced liver function in cirrhosis and increases practicality and cost-effectiveness, promoting clinical acceptance
Stockmann et al., 2009 [33]	Hepatic tumors and indications for hepatectomy	LiMAx®	2 mg/kg body weight intravenous 3-h fasting 60 min continuous LiMAx (µg/kg/h)	Perioperative context, intravenous route	Predicts preoperatively residual liver function after hepatectomy and is the only predictor of liver failure and mortality on the first postoperative day

, which highlights key aspects.

3.2. ¹³C-MBT Responses Across Liver Disease Severity and Etiology

3.2.1. Cirrhosis and Advanced Chronic Liver Disease

In established cirrhosis, particularly Child–Pugh B/C, MBT values decline sharply and uniformly across studies, confirming its ability to quantify residual metabolic capacity. A 40-min cumulative recovery threshold around 0.35% achieved area under the receiver operating characteristic curve (AUROC) values > 0.9 for cirrhosis detection with sensitivities and specificities above 80% [1,12,34,35,36]. Simplified two-point protocols (baseline and 15-min sample) maintain comparable diagnostic accuracy (92.6% sensitivity, 94.1% specificity) [32], indicating that even abbreviated formats can identify severe functional loss. These findings highlight that MBT integrates multiple determinants of hepatic reserve including hepatocellular mass, perfusion, and enzyme function yielding a dynamic signal that surpasses static biochemical indices.

Collectively, high-quality evidence demonstrates that MBT is a strong rule-in/rule-out test for advanced disease. However, MBT performance in detecting early-stage disease such as moderate fibrosis or steatosis is less consistent [6,24,37]. Yet the magnitude of reduction varies with etiology: viral and alcohol-related cirrhosis typically show steeper declines than cholestatic disease, where microsomal function is partly preserved until late stages [28,29]. This reinforces the need for disease-specific interpretation rather than universal thresholds.

3.2.2. Early and Intermediate Disease Stages

Sensitivity for early-stage fibrosis or steatosis remains inconsistent. Some hepatitis C cohorts reveal moderate correlations between MBT indices and histological fibrosis grades [24], while others, especially in hepatitis B-dominant populations, fail to differentiate mild fibrosis from normal liver [1,37]. These discrepancies appear linked more to methodological than biological variation: shorter protocols and unstandardized endpoints attenuate diagnostic contrast, whereas extended (≥60 min) or continuous analyses enhance discriminatory power. When harmonized protocols are used, MBT sensitivity for moderate fibrosis improves substantially, supporting its potential as a functional complement to structural tests such as elastography.

3.2.3. Biphasic Metabolic Response in MASLD

Across metabolic dysfunction-associated steatotic liver disease (MASLD) cohorts, a coherent biphasic metabolic pattern emerges. In early disease, low-grade inflammation and oxidative stress appear to transiently stimulate CYP1A2 activity, yielding higher ¹³C-methacetin clearance rates [31]. This adaptive hypermetabolism likely reflects cytokine-mediated enzyme induction. As fibrosis and steatohepatitis progress, however, hepatocellular loss and mitochondrial impairment dominate, producing the marked hypometabolism characteristic of advanced MASLD [22,26,38,39].

Apparent inconsistencies among studies are thus reconcilable as stage-dependent phenomena (compensatory-to-exhaustive transition) rather than contradictions. For instance, cohorts enriched with obese but non-fibrotic individuals report near-normal or slightly elevated MBT values [11], whereas histologically advanced MASLD cohorts uniformly show suppression. Molina-Molina et al. further demonstrated that obesity alone reduces DOB15 values (≈12.7‰ vs. 19‰ in normal-weight controls), indicating subclinical impairment of microsomal capacity [11,40]. These findings emphasize the necessity of stratifying MBT results by fibrosis stage and metabolic status instead of averaging across heterogeneous populations.

3.2.4. Prognostic Performance

Beyond diagnosis, low MBT or LiMAx^® values predict adverse outcomes. A LiMAx^® threshold <140 µg µg/kg/h accurately forecasted postoperative liver failure with high specificity [17,33], while values <7% PDRpeak typify end-stage cirrhosis and may guide transplantation decisions [25,41]. These extreme values are primarily relevant in intensive care settings, not in routine ambulatory diagnostics. Thus, MBT not only reflects current function but also prognosticates decompensation and surgical risk. This prognostic utility is strongest at the extremes of impairment, whereas intermediate ranges still require contextual interpretation alongside standard clinical scores.

Collectively, the evidence delineates a predictable continuum: from accelerated or normal MBT kinetics in early metabolic dysfunction to profound suppression in advanced fibrosis and cirrhosis. Variation across studies stems chiefly from protocol heterogeneity rather than fundamental disagreement about physiological trends.

3.3. Methodological and Physiological Determinants of Variability

Comparative analysis reveals that much of the apparent inconsistency in MBT performance originates from methodological diversity. Differences in substrate dosing after various fasting time (fixed 75 mg vs. weight-based, e.g., 1–2 mg/kg is common in LiMAx^®), route (oral vs. intravenous bypassing gut absorption and first-pass metabolism), sampling interval (0–180 min), and analytical platform produce non-trivial shifts in absolute readouts [42,43,44]. A lack of consensus on time points contributes to threshold variability, highlighting the need to report multiple time points or full curves for cross-study comparability. Nonetheless, relative trends remain stable when conditions are standardized.

LiMAx^® test, increasingly used perioperatively for liver function assessment [30,44], provides continuous ¹³CO₂ monitoring via facemask for 60 min and reports results as µg/kg/h. While yielding higher absolute values, relative thresholds remain comparable to oral MBT. Other systems, like BreathID^®, offer continuous ¹³CO₂ analysis using nasal cannulas, improving comfort and temporal resolution [9,42]. Shorter test durations (10–30 min) capture early kinetics, portal flow extraction, and immediate CYP capacity, often sufficient for cirrhosis detection [6,32], while longer durations assess full metabolism [7], making 30 to 60-min endpoints practical for diagnostic yield. In addition, modern cost-effective infrared spectrometry closely mirrors ¹³CO₂/¹²CO₂ isotope-ratio mass spectrometry (IRMS) [7,45,46], indicating that analytic technology per se exerts minimal clinical impact once calibration is maintained [46].

By contrast, uncontrolled variables such as fasting state, circadian rhythm, menstrual cycle phase, smoking, meals or physical exercise affect CYP1A2 and CO₂ kinetics [18,19,23,25,47,48,49,50,51,52,53,54]. Supplemental oxygen increases ¹³CO₂ exhalation independently of liver function [30,51], possibly due to altered CO₂ production or elimination, rather than true metabolic shifts [54]. Few studies have explicitly reported and controlled for these confounders, contributing to inter-trial dispersion of “normal” values. Normalization inconsistency, such as the varying definitions of “normal range”, further complicates the interpretation of results, particularly with varying output metrics like percent of dose per hour or time to peak (TTP) versus cumulative recovery [7,11,55]. Despite differing units (‰, %/h, %, µg/kg/h), all reflect CYP1A2 activity and are intercorrelated. Future adoption of unified testing conditions would likely narrow reference intervals and strengthen reproducibility.

Methodological heterogeneity currently limits meta-analytic synthesis but does not undermine the central physiological signal. When standardized, MBT yields reproducible, clinically interpretable data with diagnostic and prognostic validity across etiologies. Overall, the collective evidence supports MBT as both a diagnostic and prognostic marker of hepatic functional reserve, with its full clinical potential contingent on methodological standardization and disease-stage-specific interpretation.

4. Discussion

This review synthesizes existing data to validate the ¹³C-MBT as a sensitive marker of liver function in adults. Healthy individuals show rapid methacetin metabolism, while those with liver disease, especially cirrhosis or advanced fibrosis, exhale significantly less ¹³CO₂. However, physiological, demographic, environmental, and methodological factors influencing the MBT’s clinical integration, presented in Figure 1.

Although reference values for healthy adults are broadly similar across studies, diagnostic cut-offs vary due to differences in test protocols, endpoints, and study goals. Diagnostic thresholds (e.g., cPDR30 <8%, cPDR20 ≤0.55%) are primarily derived from cross-sectional studies aiming to identify fibrosis or cirrhosis, optimized for sensitivity and specificity [9]. Prognostic thresholds (e.g., LiMAx^® <140 µg/kg/h or PDRpeak <7%/h) are instead based on outcome studies predicting survival, postoperative liver failure, or decompensation. These categories serve distinct clinical purposes and should not be used interchangeably.

Clinicians must interpret MBT results contextually, distinguishing diagnostic from prognostic thresholds, with intermediate gradations possible. A two-threshold model, akin to liver enzymes with „normal” and „danger zone” levels, could enhance clinical utility. Importantly, MBT should not be used in isolation for high-stakes decisions like transplant listing; it must be integrated with other criteria, as opposed to relying exclusively on an absolute cut-off.

A major source of variability is lack of test protocols standardization, as previously mentioned (e.g., fixed vs. weight-based dosing, breath bags vs. continuous analyzers). Although commercial systems like BreathID^® and LiMAx^® have improved within-platform consistency, cross-platform discrepancies persist. The LiMAx^® system, approved in some countries [56], offers standardized cut-offs (315 and 140 µg/kg/h), supporting future harmonization. Circadian factors may contribute slightly to variability. While most studies occurred in the morning due to fasting protocols, drug metabolism is known to fluctuate diurnally [18]. If MBT were performed at night, healthy subjects might show lower values attributable to the circadian low phase of metabolism, potentially mimicking dysfunction.

A key finding is that MBT reliably distinguishes cirrhosis from normal liver function, often outperforming standard blood tests [1,41]. It offers a dynamic, noninvasive measure of functional reserve, sensitive to changes post-treatment or surgery [1,57]. However, MBT may be normal in diseases like cholestasis where metabolic capacity is preserved. Thus, it should be viewed as a targeted tool for assessing hepatocellular function—not a comprehensive liver test.

4.1. Limitations of Current Evidence

The current evidence base for the MBT is limited by several methodological inconsistencies and study-level constraints. First, comparing MBT results from different devices (e.g., 30-min infrared spectroscopy vs. 120-min mass spectrometry) creates incompatible data, hindering direct comparison and meta-analytic synthesis. Secondly, most studies also are small (n < 100), single-center, and observational with limited diversity and narrow inclusion criteria. Many rely primarily on young, healthy volunteers, overlooking age-related effects. Though some studies suggest older adults may have reduced MBT capacity [23], robust normative aging data are lacking. Third, the definition of “non-cirrhotic” controls is often inconsistent, with some cohorts including patients with early fibrosis, potentially skewing reference ranges downward. Few studies employ blinded or randomized designs, and only a minority provide detailed control for confounders such as body mass index (BMI), diabetes, or alcohol use. Geographic concentration also limits external validity, most early MBT data originate from European cohorts (notably Germany and Italy), where breath-testing technology was more accessible and sometimes manufacturer-supported. Underrepresentation of other regions may bias “normative” values, so global thresholds should be applied with caution. Fourth, potential circadian and environmental influences, though acknowledged, remain underexplored. Preliminary data suggest MBT shows acceptable reproducibility under controlled conditions (with ~10% variation) [53], but variability in real-world settings is not well studied. Fifth, publication bias must be considered: studies with positive or clinically useful MBT results are more likely to be published, while neutral or negative findings remain unreported. This asymmetry may have contributed to the slow clinical adoption of MBT, despite decades of research [47,58].

Despite these limitations, consistent trends emerge: MBT parameters reliably discriminate advanced fibrosis and cirrhosis across etiologies, with high reproducibility in standardized test settings. However, the variability in test execution and population characteristics underscores the need for harmonized study protocols and multicenter validation. The present synthesis therefore emphasizes comparative interpretation and pattern recognition over statistical pooling, in keeping with the narrative review framework.

4.2. Implications for Practice and Research

To facilitate the clinical utility of the MBT, future efforts must address not only methodological standardization but also economic and practical implementation barriers. We propose a stepwise implementation framework to harmonize protocols and reference values, as outlined in Figure 2. The process commences with the establishment of a consensus protocol (e.g., “MBT-60”) that defines key parameters: fasting status, a fixed 75 mg oral dose, breath sampling every 10 min over 60 min, infrared spectrometry analysis, and standardized reporting metrics such as cPDR30 and cPDR60. The proposed “MBT-60” protocol was selected to balance diagnostic efficiency and methodological consistency. Although several studies have shown that 30-min measurements (e.g., cPDR30) are highly discriminative for cirrhosis, extending sampling to 60 min captures both early and late phases of methacetin metabolism, providing a more complete assessment of total hepatic capacity. Moreover, aligning the duration with established intravenous systems such as LiMAx^® facilitates cross-study comparability. Shorter protocols remain valuable for rapid screening, whereas “MBT-60” ensures broader standardization across patient subgroups and testing platforms. Standardized protocols would enable data pooling and the creation of shared reference ranges. Similar harmonization is needed for continuous monitoring systems (e.g., BreathID^®) and intravenous methods (e.g., LiMAx^®). Multicenter trials using unified methodologies could then generate robust reference intervals.

Beyond technical standardization, real-world integration requires addressing health-economic feasibility and system-level logistics. A major barrier, as noted by Afolabi et al., is the absence of cost-effectiveness data [58]. Future research should therefore include formal health-economic evaluations (e.g., cost-utility and budget-impact analyses) comparing MBT with existing diagnostic pathways such as FibroScan, serologic fibrosis panels, and liver biopsy. Demonstrating cost neutrality or superiority, particularly in reducing unnecessary biopsies or hospitalizations will be essential to support reimbursement and adoption by healthcare systems.

In parallel, implementation science approaches should be applied to identify barriers to adoption at the institutional and clinician levels, including staff training, workflow integration, device accessibility, and patient acceptance. Pilot implementation studies could test streamlined MBT workflows in outpatient hepatology settings, guided by frameworks such as RE-AIM (Reach, Effectiveness, Adoption, Implementation, Maintenance) [59].

Large-scale studies in healthy adults stratified by age, sex, and ethnicity are also needed to establish normative MBT values (e.g., 95% intervals for cPDR30/60), clarify demographic influences such as age-related metabolic decline [23], and define reliable diagnostic cutoffs. MBT should be directly compared to existing liver function tests and fibrosis markers within the same cohorts to assess its relative sensitivity and clinical value. Early evidence suggests MBT outperforms APRI (AST to Platelet Ratio Index) in detecting advanced fibrosis in hepatitis C [32]. Comparing MBT to tools like FibroScan could reveal complementary roles: MBT measures function, FibroScan measures stiffness. For instance, a normal MBT in a patient with borderline elastography (e.g., in MASLD) could support non-invasive monitoring, potentially avoiding liver biopsy. Conversely, a low MBT result warrants further investigation, given its strong predictive value for moderate to advanced fibrosis [60]. MBT may also help stratify risk in cirrhosis or heart failure patients and guide transplant prioritization or intensive management. In perioperative settings, LiMAx^® testing has shown promise in predicting post-hepatectomy liver failure more accurately than MELD (Model for End-Stage Liver Disease) or Child–Pugh scores [40]. MBT has also been shown to track treatment response for example, improving with weight loss in MASLD alongside histological improvement [61].

Further research should evaluate MBT performance at different times of day to determine the impact of circadian rhythms. If significant, this may necessitate time-specific testing windows or reference ranges. Investigating chronopharmacologic influences, such as timed CYP1A2 modulation, could refine testing protocols further. Although this framework focuses on adults, pediatric reference ranges are lacking and should be established to ensure consistency across age groups [30]. MBT may also hold value in acute liver failure and drug-induced liver injury [27]; acute-phase reference data would support its emergency use.

Finally, data sharing and collaborative infrastructure will accelerate progress. An international MBT consortium could pool raw test data for individual-patient meta-analyses, enable health-economic modeling, and harmonize disease-specific thresholds. By integrating economic evaluation, implementation science, and technical standardization, future research can transform MBT from a specialized diagnostic tool into a scalable, cost-effective component of routine hepatology practice.

5. Conclusions

The ¹³C-MBT is a sensitive, non-invasive tool for quantifying hepatic microsomal function, with strong diagnostic and prognostic utility, particularly in reliably distinguishing healthy from diseased states and advanced liver disease. Consistent patterns emerge across studies: healthy adults show high MBT values, while advanced liver disease results in markedly reduced readings. Despite consistent evidence, inconsistent protocols and variable thresholds have hindered widespread adoption. To realize the full clinical potential of MBT, coordinated efforts are required to unify test procedures, establish population-specific reference ranges, and integrate MBT alongside routine structural and serological liver assessments. A standardized framework supported by multicenter validation will facilitate its routine use in hepatology, enhancing diagnostic precision, risk stratification, and treatment monitoring in both chronic and acute liver diseases. The case for its clinical reintegration is compelling and increasingly actionable.