Graft bile analysis for predicting post-transplant outcomes: A literature review and a protocol for a novel biomarker

Abstract

Liver transplantation (LT) remains the definitive treatment for end-stage liver disease, yet early detection of graft dysfunction and rejection is still challenging. Conventional blood-based markers provide systemic information but lack hepatic specificity. Bile, directly secreted by hepatocytes and cholangiocytes, represents an organ-specific biofluid with diagnostic potential for assessing graft viability and early complications. This minireview examined the biochemical, immunological, and molecular features of bile as biomarkers in LT, focusing on pH, bicarbonate, glucose, lactate, bile acids, and proteomic/lipidomic profiles in relation to ischemia-reperfusion injury, early allograft dysfunction, and ischemic-type biliary lesions. The integration of bile-based parameters into ex situ perfusion and post-transplant monitoring, supported by omics technologies and predictive modeling, was also discussed. Building on these insights, we designed a single-center prospective study (ClinicalTrials.gov: NCT03882164) evaluating biliary tacrolimus (TACbile) as a predictor of acute rejection after LT. Paired daily TACbile and plasma tacrolimus levels are measured in recipients with Kehr T-tubes to define a blood-bile ratio of tacrolimus. The primary endpoint was the predictive accuracy of blood-bile ratio of tacrolimus for biopsy-proven rejection; secondary outcomes include associations with early allograft dysfunction. Bile-based biomarkers, particularly TACbile, may revolutionize graft monitoring and personalize immunological surveillance after LT.

Key Words: Bile; Biomarkers; Early allograft dysfunction; Tacrolimus; Protocol

Core Tip: Early detection of graft dysfunction and rejection after liver transplantation remains challenging. This minireview highlighted bile as an organ-specific biofluid directly reflecting hepatocellular and cholangiocyte function. We summarized the biochemical, metabolic, and molecular components of bile as predictive markers of graft viability during machine perfusion and in the perioperative period. Building on these insights, we presented a prospective protocol assessing biliary tacrolimus concentration and introducing the blood-bile ratio of tacrolimus as a novel index for early, noninvasive detection of rejection. Integrating bile biomarkers into transplant workflows may enable real-time, graft-specific monitoring and improve post-transplant outcomes.

INTRODUCTION

Liver transplantation (LT) is a life-saving treatment for end-stage liver disease, yet ischemia-reperfusion injury (IRI), early allograft dysfunction (EAD), and biliary complications continue to impair graft outcomes despite major advances in surgical technique, perioperative care, and immunosuppression[1,2]. Current blood-based biomarkers capture systemic alterations but provide limited insight into the local hepatobiliary events that precede clinical deterioration[3]. As a result there is a growing interest in bile as an organ-specific biofluid capable of reflecting early functional and structural changes within the graft.

Bile is actively secreted and modified by hepatocytes and cholangiocytes, making it a direct readout of graft metabolism, transporter activity, and cholangiocyte integrity. In contrast to serum parameters, biliary constituents can signal localized injury, such as IRI-induced cholangiocyte dysfunction, impaired canalicular transport, or altered bile acid (BA) homeostasis, before systemic markers change. Similarly, perfusate biomarkers obtained during machine perfusion (MP) provide valuable ex situ metabolic information but do not fully capture the complexity of in vivo biliary physiology after implantation[4,5].

The advent of hypothermic oxygenated perfusion (HOPE), normothermic MP (NMP), and related technologies has expanded opportunities for real-time bile collection and analysis[6,7]. During MP parameters such as bile pH, bicarbonate concentration, glucose reabsorption, and BA composition correlate with graft viability and the risk of ischemic-type biliary lesions (ITBL)[8]. Parallel advances in metabolomics, proteomics, and lipidomics have enabled high-resolution characterization of bile, revealing molecular signatures linked with inflammation, epithelial injury, and metabolic dysfunction. Despite these advances clinical translation remains limited[9]. Studies differ substantially in sampling technique (e.g., Kehr T-tube vs MP aspirates), analytical platforms, and endpoint definitions, limiting the comparability and external validity of published findings. A more integrated framework is needed to synthesize biochemical, metabolic, and omics-based evidence and to clarify how bile biomarkers can guide perioperative decision-making.

Within this context the present minireview examined the biochemical, immunological, and molecular characteristics of bile and their association with graft viability during MP and the early post-LT period. Building on this evidence, we introduced a prospective protocol evaluating biliary tacrolimus (TACbile) and the blood-bile ratio of tacrolimus (BBRT), a mechanistically grounded biomarker incorporating hepatic metabolism (CYP3A4/5), canalicular transport [ATP-binding cassette sub-family B member 1 (ABCB1)/ATP binding cassette subfamily C member 2 (ABCC2)], and inflammation-mediated transporter modulation. Presenting the BBRT rationale early provides a unifying framework linking bile composition, transporter biology, and personalized immunosuppression strategies.

BILE PRODUCTION AND ITS LINK TO GRAFT VIABILITY

Bile production reflects the coordinated activity of hepatocytes, cholangiocytes, and the biliary transport machinery, making it a sensitive indicator of graft function after LT. Hepatocytes generate primary bile through active secretion of BAs, phospholipids, and bicarbonate into the canalicular lumen, a process dependent on ATP-consuming transporters such as ABCB11 (bile salt export pump), ABCB1, and ABCC2[10]. Cholangiocytes subsequently modify this primary bile through bicarbonate secretion, glucose reabsorption, and water/electrolyte transport, all of which are highly susceptible to IRI. During IRI mitochondrial dysfunction, oxidative stress, and sterile inflammation impair canalicular transport, reduce ATP availability, and disrupt cholangiocyte ion channels. These mechanisms contribute to early alterations in bile pH, bicarbonate concentration, glucose handling, and BA composition. Even subtle disruptions in transporter activity or epithelial integrity can therefore produce measurable changes in bile composition before systemic biomarkers or clinical parameters deteriorate[11].

MP platforms have further elucidated the association between bile quality and graft viability. In HOPE improvements in mitochondrial redox state correlate with the generation of more alkalinized, bicarbonate-rich bile, reflecting restored cholangiocyte function[12]. Conversely, during NMP low bile pH, impaired glucose reabsorption, and aberrant BA patterns are associated with post-transplant cholangiopathy, particularly in donation after circulatory death (DCD) grafts[13]. Although these relationships are increasingly recognized, clinical application remains variable due to heterogeneity in sampling techniques, analytical platforms, and the criteria used to define biliary complications[14]. A more unified framework describing how bile parameters reflect hepatobiliary injury may improve their integration into graft assessment during MP and the early post-LT period.

BIOCHEMICAL BILIARY BIOMARKERS

Biochemical constituents of bile, including pH, bicarbonate concentration, glucose reabsorption, lactate, and basic enzymatic markers, offer direct insight into the integrity and function of the biliary epithelium. These parameters reflect cholangiocyte viability, canalicular transporter activity, and early hepatobiliary injury, making them among the most accessible and widely studied biliary biomarkers in LT (Table 1).

Table 1 Summary of bile biomarkers and clinical significance.

Biomarker	Source	Normal range	Abnormal indication	Associated risk	Ref.
Bile pH	Cholangiocytes	> 7.5	A decrease in pH is associated with cholangiocyte injury	ITBL	Watson et al[7]
Bicarbonate	Cholangiocytes	> 18 mmol/L	A decrease in HCO3- is associated with reduced ductal secretion	ITBL	Sasaki et al[19]
Bile glucose	Reabsorbed by ducts	< 1 mmol/L	An increase in glucose is associated with reabsorption failure	Cholangiocyte injury	Watson and Pessin[29]
Lactate	Metabolic stress	< 5 mmol/L	An increase in lactate is associated with anaerobic metabolism	Graft dysfunction	Zhong et al[34]
Bile acids (CA, CDCA, etc)	Hepatocytes/metabolism	Varied	Hydrophobic shift is associated with cytotoxicity	Biliary epithelial apoptosis	Huang et al[10]
Proteins (e.g., IL-6, L-FABP)	Proteomic profiling		An increase in markers is associated with inflammation/apoptosis	Ductal necrosis, inflammation	Junior et al[30]
Lipids (e.g., PC, LPC)	Lipidomics		A decrease in PC and increase in LPC is associated with membrane damage	Biliary leak, ITBL	Zijlstra et al[41]

ITBL: Ischemic type biliary lesion; CA: Cholic acid; CDCA: Chenodeoxycholic acid; IL-6: Interleukine-6; L-FABP: Liver-type fatty acid-binding protein; PC: Phosphatidylcholine; LPC: Lysophosphatidylcholine.

Bile pH

Bile alkalinization is primarily driven by cholangiocyte secretion of bicarbonate through cystic fibrosis transmembrane conductance regulator-dependent and anion exchanger 2-dependent mechanisms[15,16]. Reduced bile pH is one of the earliest indicators of cholangiocyte dysfunction during IRI. Studies in both hypothermic and NMP consistently show that bile pH < 7.4 is associated with impaired ductal secretory function, mitochondrial dysfunction, and increased risk of ITBL, particularly in DCD grafts[17,18].

Bicarbonate concentration

Bicarbonate secretion forms the basis of the bicarbonate umbrella, a key defense mechanism protecting cholangiocytes from hydrophobic BAs. Low biliary bicarbonate levels correlate with higher susceptibility to epithelial injury, reduced ductal alkalinization, and increased risk of postoperative ischemic cholangiopathy[19]. During HOPE, recovery of mitochondrial activity enhances bicarbonate-rich bile production, whereas insufficient bicarbonate secretion during NMP predicts subsequent biliary complications[20,21].

Glucose

Under physiological conditions cholangiocytes reabsorb glucose from bile via sodium-glucose cotransporters. Elevated biliary glucose, especially during NMP, is associated with impaired reabsorption due to cholangiocyte injury. While bile pH and bicarbonate are the primary viability metrics, glucose has emerged as a supplementary marker that reflects epithelial and transporter dysfunction[22].

Lactate and other metabolic indicators

Lactate accumulation in bile reflects metabolic stress and anaerobic glycolysis within biliary epithelial cells. Although less studied than pH or bicarbonate, elevated bile lactate levels during MP may signal inadequate mitochondrial recovery or ongoing epithelial injury. Additional biochemical parameters, such as lactate dehydrogenase or bilirubin concentration, have been investigated, but their clinical value is limited by nonspecificity and assay variability[23].

Clinical interpretation and limitations

Biochemical bile parameters are simple, accessible, and mechanistically grounded. However, their clinical integration is limited by heterogeneity in sampling techniques (MP aspirates vs T-tube), differences in perfusion protocols, and inconsistent reporting of thresholds. Furthermore, biochemical markers often reflect downstream injury and may require combination with metabolic or molecular readouts for improved discriminatory value[24].

METABOLIC BILIARY BIOMARKERS

Metabolic profiling of bile provides deeper insight into hepatobiliary function, epithelial integrity, and mitochondrial performance than simple biochemical measurements. Because bile formation depends on complex metabolic processes, including BA synthesis, glucose and amino acid (AA) transport, mitochondrial respiration, and oxidative balance, specific metabolic signatures can signal early graft dysfunction or cholangiocyte injury before clinical complications arise[25].

BAs

BAs are synthesized in hepatocytes and undergo extensive modification in the biliary tree. Their composition reflects both hepatocyte metabolic activity and cholangiocyte health. An increase in hydrophobic BAs or an altered hydrophilic/hydrophobic ratio has been associated with epithelial injury, impaired ATP-dependent transport, and increased risk of ITBL[26]. During NMP abnormal BA profiles correlate with impaired ductal viability while HOPE increases the proportion of hydrophilic BAs, reflecting improved mitochondrial recovery[27].

AA signatures

AA patterns in bile reflect metabolic stress, mitochondrial function, and local inflammation. Elevated aromatic AAs (e.g., phenylalanine, tyrosine) may indicate oxidative stress or impaired hepatocyte metabolism, whereas reduced levels of glutamine and serine can reflect mitochondrial dysfunction. Preliminary studies suggest that AA ratios may help discriminate between reversible IRI and evolving cholangiopathy[28].

Energy metabolism markers

Metabolites linked to energy homeostasis, such as ATP degradation products, acylcarnitines, and oxidative phosphorylation intermediates, provide valuable insight into mitochondrial function during MP[29]. High levels of acylcarnitines or purine metabolites suggest incomplete mitochondrial recovery and correlate with higher risk of biliary complications. These markers may complement bile pH and bicarbonate measurements, particularly in marginal or DCD grafts[30,31].

Oxidative stress metabolites

Reactive oxygen species-related metabolites, including oxidized glutathione, malondialdehyde, and lipid peroxidation products, can be detected in bile and reflect the severity of IRI[32]. Their quantification provides a molecular readout of oxidative injury that may precede structural ductal damage[33].

Clinical interpretation and limitations

Metabolic biomarkers offer mechanistic insight into bile formation, mitochondrial activity, and biliary epithelial stress[34]. However, their current use is limited to research settings due to analytical complexity and variability in measurement platform [liquid chromatography-tandem mass spectrometry (LC-MS/MS), nuclear magnetic resonance, targeted metabolomics][35]. Standardization of assays, sampling protocols, and reference ranges is essential before these markers can be applied in clinical decision-making[36,37].

OMICS-DERIVED BILIARY BIOMARKER

Omics technologies, including proteomics, lipidomics, metabolomics, and transcriptomics, have expanded the analytical depth of bile assessment, revealing molecular signatures linked to IRI, cholangiocyte dysfunction, inflammation, and early graft failure. Unlike traditional biochemical measures, omics-based biomarkers characterize global biological networks and provide mechanistic insight into the pathways driving early graft dysfunction.

Proteomics

Proteomic profiling of bile identifies differentially expressed proteins associated with epithelial integrity, immune activation, and extracellular matrix remodeling[38]. Increased concentrations of cytokeratins, heat shock proteins, and inflammatory mediators reflect cholangiocyte injury and local inflammation. Low levels of bicarbonate transport-related proteins may correlate with impaired ductal alkalinization[39]. Proteomic signatures obtained during MP have been associated with postoperative biliary complications, suggesting that protein-level alterations may serve as early viability markers[40].

Lipidomics

Lipidomics enables the characterization of phospholipids, sphingolipids, and BA derivatives, providing insight into membrane integrity and lipid metabolism. Reduced phosphatidylcholine levels, central to protecting cholangiocytes from BA toxicity, have been associated with epithelial vulnerability and increased risk of cholangiopathy. Alterations in sphingolipid metabolism correlate with mitochondrial stress and apoptosis[41]. Lipidomic profiles may help distinguish reversible IRI from evolving biliary injury.

Metabolomics

Metabolomic analyses reveal pathways linked to oxidative stress, energy metabolism, AA turnover, and BA biosynthesis. Elevated oxidative stress markers, perturbations in tricarboxylic acid cycle intermediates, and imbalances in AA metabolism signal early mitochondrial dysfunction[42]. These metabolic fingerprints can identify grafts at higher risk of dysfunction during MP or early after LT.

Transcriptomics and microRNA

Although data are limited, transcriptomic and microRNA analyses suggest that bile contains nucleic acids reflective of cholangiocyte stress, immune activation, and transporter regulation. Upregulation of microRNAs involved in epithelial injury (e.g., miR-30 family) or downregulation of those supporting transporter activity (e.g., miR-122) may provide early warning of ductal dysfunction[43]. Further research is required to validate bile-derived nucleic acids as clinical biomarkers.

Multi-omic integration

Combining proteomic, lipidomic, and metabolomic data may improve the predictive accuracy of biliary biomarkers[44]. Multi-omic integration has the potential to identify composite signatures associated with cholangiopathy, mitochondrial dysfunction, or impaired ductal regeneration[39]. These approaches may also support the development of predictive modeling tools that incorporate machine learning to enhance graft viability assessment.

Clinical interpretation and limitations

Omics-derived biomarkers offer mechanistic richness but remain limited by methodological heterogeneity, including differences in sampling, storage conditions, analytical platforms, and data processing pipelines[32]. Small sample sizes, lack of external validation, and variable endpoint definitions further limit clinical translation. Standardized protocols and multicenter studies are essential before omics biomarkers can be reliably incorporated into graft evaluation during MP or early post-transplant care[45].

PREDICTIVE MODELING AND INTEGRATIVE BIOMARKERS

Predictive models incorporating bile-derived biomarkers have the potential to improve graft assessment both during MP and in the early post-transplant period[46]. While traditional viability criteria prioritize bile flow, pH, and perfusion parameters, emerging evidence suggests that combining biochemical, metabolic, and omics-derived markers may offer superior discriminatory power for predicting graft dysfunction and biliary complications[40].

MP predictive frameworks

During NMP models integrating bile pH, bicarbonate concentration, glucose reabsorption, and BA composition outperform single-parameter assessments when predicting ITBL[26]. Multivariable logistic regression approaches and early machine learning tools have demonstrated improved accuracy although their applicability is limited by small sample sizes and heterogeneous perfusion protocols. The incorporation of metabolic intermediates or proteomic signatures into these models may enhance viability assessment, particularly in DCD grafts.

Composite biomarkers for early post-LT monitoring

In the early postoperative period, combining bile biochemical markers (e.g., pH, bicarbonate) with metabolic or proteomic indicators may improve detection of early cholangiocyte injury, subclinical inflammation, or evolving graft dysfunction[18]. Composite indices reflecting cholangiocyte transport, mitochondrial function, and oxidative stress may outperform isolated variables[26]. However, the absence of standardized thresholds and limited prospective validation remain major barriers.

Integrating omics into predictive models

The complexity of omics-derived data lends itself to machine learning-based analytical frameworks. Multi-omic signatures have been associated with early markers of epithelial injury and mitochondrial dysfunction, supporting the development of predictive algorithms that could classify grafts into risk categories before clinical complications emerge. Early exploratory models show promise but require larger datasets, harmonized analytical pipelines, and multicenter validation[23].

Methodological challenges

Predictive modeling in bile biomarker research faces several limitations. First, studies differ in bile sampling techniques, perfusion protocols, and measurement platforms, complicating the aggregation of datasets across centers. Second, small sample sizes reduce generalizability and limit the development of high-resolution predictive tools. Third, endpoint definitions, particularly for ITBL and biliary complications, vary substantially between studies, hindering model performance and external validation.

Future directions

Future frameworks should combine multi-parameter bile analysis with clinical and perfusion metrics to generate robust, interpretable predictive tools. Standardized bile collection procedures, harmonized analytical workflows, and multicenter datasets will be essential to refining models for clinical integration. These approaches lay the groundwork for advanced biomarkers such as the BBRT, which integrates metabolic, transport, and pharmacologic dimensions into a single mechanistically grounded predictive signal.

INTEGRATION OF BILE BIOMARKERS INTO MP WORKFLOWS

MP technologies have enabled direct, structured assessment of bile quality during ex situ graft preservation. These platforms offer unique opportunities to characterize biliary physiology before transplantation and to identify grafts at risk of dysfunction. Because bile production depends on cholangiocyte viability, mitochondrial activity, and intact transporter function, bile biomarkers collected during MP provide mechanistically grounded indicators of graft health.

HOPE

HOPE supports mitochondrial recovery by reversing succinate accumulation and reducing oxidative stress. Improvements in mitochondrial redox status translate into enhanced cholangiocyte function, reflected by more alkaline and bicarbonate-rich bile during the early reperfusion phase[25]. Even modest bile alkalinization during HOPE may indicate restoration of epithelial integrity[32]. However, HOPE typically generates limited bile volumes, and sampling is often sparse, restricting the range of measurable parameters[27].

NMP

NMP provides a physiologic environment that enables continuous bile production and real-time assessment of multiple biomarkers (Table 2). Bile pH, bicarbonate concentration, glucose reabsorption, and BA patterns are the most informative parameters during NMP[31]. Bile pH < 7.4, impaired glucose reabsorption, and abnormal BA profiles have been consistently associated with ITBL, particularly in DCD grafts[32]. Nevertheless, variability in perfusion duration, bile sampling schedules, and perfusate composition complicates interpretation across centers.

Table 2 Clinical viability criteria using bile during normothermic machine perfusion.

Parameter	Threshold	Predictive value	Ref.
Bile production (NMP)	> 30 mL over perfusion	Functional biliary system	de Jong et al[26]
Bile pH	> 7.5	Low risk of cholangiopathy	Watson et al[13]
Bile/perfusate glucose ratio	< 0.67	Preserved glucose reabsorption	Zelger et al[20]
Bicarbonate concentration	> 18 mmol/L	Functional ductal secretion	Ceresa et al[46]
Bile acid hydrophobicity index	Low hydrophobicity	Low risk of epithelial damage	Huang et al[10]

NMP: Normothermic machine perfusion.

Limitations of current MP bile assessment

Despite promising results bile biomarkers during MP are influenced by multiple variables[46]: Perfusate composition; perfusion pressure; oxygenation strategy; donor type; and cannulation technique. Moreover, bile composition during MP may not perfectly mirror in vivo physiology due to altered hormonal stimuli and absence of enterohepatic cycling[47]. These factors highlight the need for standardized sampling and reporting protocols.

Future opportunities

Future work should integrate MP bile biomarkers with perfusate metabolic markers and early post-transplant bile analysis to create unified viability frameworks. Standardization of bile sampling intervals, analytical platforms, and threshold definitions will be critical for improving reproducibility. Multi-parameter predictive models and mechanistically grounded biomarkers, such as the BBRT, may further enhance risk stratification and support personalized decision-making during graft preservation.

CLINICAL TRANSLATION AND POST-TRANSPLANT INTEGRATION OF BILE BIOMARKERS

Despite increasing interest the integration of bile biomarkers into routine post-transplant management remains limited. Their potential lies in providing organ-specific, early indicators of cholangiocyte injury, transporter dysfunction, or evolving graft dysfunction, signals that often precede changes in serum markers or imaging findings[17]. However, methodological heterogeneity and limited prospective validation have slowed widespread adoption.

Early detection of cholangiocyte injury

Post-transplant cholangiopathies such as ITBL and anastomotic strictures frequently develop before clinical symptoms emerge[17]. Bile parameters, including pH, bicarbonate concentration, and glucose reabsorption, can reveal impaired cholangiocyte function early in the postoperative course. Elevated bile lactate, abnormal BA profiles, and specific metabolic signatures may further indicate ongoing mitochondrial stress or epithelial injury[18]. These early alterations can support timely intervention before irreversible ductal damage occurs.

Monitoring of IRI and EAD

Bile biomarkers may help distinguish reversible IRI from evolving EAD[1]. Abnormalities in bile alkalinization, bicarbonate secretion, and metabolic signatures, particularly those reflecting oxidative stress, are often detectable before conventional markers such as aspartate aminotransferase, alanine aminotransferase, bilirubin, or international normalized ratio rise[5]. Their integration may improve early postoperative risk stratification and support personalized postoperative monitoring.

Integration into clinical algorithms

While isolated parameters have limited predictive value, combining multiple bile biomarkers may enhance clinical decision-making. Composite indices integrating biochemical, metabolic, or omics-derived markers could complement imaging and laboratory tests in detecting early biliary complications[5]. Potential applications include guiding the timing of endoscopic retrograde cholangiopancreatography (ERCP), informing immunosuppressive modulation, or prioritizing patients for enhanced monitoring.

Barriers to translation

The application of bile biomarkers in the postoperative setting faces several challenges. Techniques for bile collection vary widely (T-tube, biliary drain, ERCP sampling), and sampling frequency is inconsistent. Analytical platforms differ in sensitivity and reproducibility, limiting cross-study comparability. Small sample sizes and variable endpoint definitions, particularly for post-transplant cholangiopathy, remain major issues[17]. Standardized collection, analysis, and reporting frameworks are necessary for clinical implementation.

Future role in personalized immunosuppression: Linking to BBRT

Bile biomarkers may support individualized post-transplant care by providing direct insight into biliary transporter activity and epithelial function. This concept aligns with emerging pharmacokinetic-pharmacodynamic biomarkers such as the BBRT[48]. Because tacrolimus handling depends on CYP3A4/5 metabolism, transporter activity (ABCB1/ABCC2), and local inflammation, BBRT may detect early alterations in hepatic and cholangiocytic function associated with rejection or overexposure. Its integration into clinical workflows could complement bile biochemical and metabolic markers, offering a more comprehensive assessment of graft health.

PROSPECTIVE PROTOCOL: TACBILE AND THE BBRT

Rationale

Tacrolimus disposition depends on hepatic metabolism (CYP3A4/5), canalicular transport (ABCB1/ABCC2), and biliary excretion[49]. Inflammation, IRI, and early immune activation downregulate these pathways, resulting in altered intrahepatic handling of the drug. Measuring TACbile offers a unique opportunity to assess local hepatobiliary function. The BBRT, defined as the whole-blood tacrolimus trough concentration divided by TACbile, integrates systemic exposure with biliary elimination. A rising BBRT may reflect impaired canalicular secretion or transporter downregulation, conditions often associated with early graft dysfunction or rejection[50].

Study design

This was a single-center, prospective observational study enrolling adult recipients with a T-tube or external biliary drainage in place[51]. Bile and blood samples were collected daily during the first postoperative week with additional sampling as clinically indicated. The primary aim was to evaluate whether TACbile and BBRT correlated with early graft outcomes, including rejection, biliary complications, and biochemical markers of hepatocellular or cholangiocytic injury.

Sample collection and handling

Daily bile samples were collected immediately before the morning tacrolimus dose (C0 trough) to ensure pharmacokinetic consistency. To minimize contamination from bile stasis, the first few milliliters of drained bile were discarded, and the drainage system was flushed when appropriate. Bile samples were inspected for color, viscosity, and volume, and collection was standardized with early postoperative fasting/feeding protocols to limit dilution variability. Blood samples for tacrolimus trough concentrations were collected concurrently with bile sampling. Both samples were processed immediately and stored at -80 °C until analysis.

Analytical methods

Tacrolimus quantification in bile and whole blood was performed using validated LC-MS/MS methods. For bile tacrolimus concentrations were normalized to total protein content, chosen over bilirubin-based or volume-based correction because protein concentration is a more stable surrogate for bile compositional load, particularly in the early postoperative period when bile volume and bilirubin content fluctuate significantly. Additional biochemical and metabolic marker quantification were performed based on sample availability.

Endpoints

Primary endpoints included: (1) Correlation between TACbile, BBRT, and biopsy-proven acute rejection; (2) Prediction of biliary complications ITBL and strictures; and (3) Association with biochemical markers of graft dysfunction (aspartate aminotransferase, alanine aminotransferase, bilirubin, international normalized ratio). Secondary endpoints included trends over time (days 1-7) and comparisons between donor types (donation after brain death vs DCD) and graft preservation strategies (static cold storage vs HOPE).

Statistical analysis

Continuous variables were analyzed using mixed-effects models to account for repeated measurements. Missing daily bile values were handled using restricted maximum likelihood estimation with sensitivity analyses employing multiple imputation for non-random missingness. Logistic regression and time-to-event models explored associations between TACbile/BBRT and clinical outcomes. Predictive performance was assessed using receiver operating characteristic curves and cross-validation where applicable.

Expected impact

This protocol aimed to determine whether TACbile and BBRT can serve as early mechanistic biomarkers of graft dysfunction and immune activation. By integrating transporter biology, metabolic function, and pharmacokinetics[52-54], the BBRT may complement traditional serum biomarkers and enhance the personalization of early immunosuppression[55,56].

LIMITATIONS

The interpretation and clinical translation of bile-derived biomarkers are constrained by several methodological and conceptual limitations. First, studies differ substantially in sampling techniques, including whether bile is obtained from a T-tube, external drain, ERCP aspirate, or during MP (HOPE or NMP). These sampling approaches generate bile with different compositions, dilutions, and kinetics, limiting comparability across studies. Second, most available studies rely on small, single-center cohorts that reduce statistical power and restrict the development of robust multivariable or machine learning models[57]. Third, analytical platforms differ widely, ranging from biochemical assays to LC-MS/MS, nuclear magnetic resonance spectroscopy, and targeted or untargeted omics workflows, each with unique sensitivity, specificity, and preanalytical requirements. Differences in storage, processing, and normalization strategies further complicate dataset integration. Fourth, clinical endpoints are inconsistently defined, particularly for ITBL, EAD, and cholangiopathy. Variability in diagnostic criteria makes it difficult to validate biomarkers across centers and undermines the development of standardized thresholds. Finally, few biomarkers have undergone external or prospective validation, and multicenter studies remain scarce. The absence of harmonized sampling protocols, analytical pipelines, and endpoint definitions remains a major barrier to the clinical adoption of bile biomarkers. Addressing these limitations will require coordinated multicenter efforts, standardized methodologies, and integration of biochemical, metabolic, and omics-derived datasets into unified predictive frameworks.

CONCLUSION

Bile-derived biomarkers offer a unique and organ-specific window into hepatobiliary function, providing mechanistic insights that complement conventional serum tests and imaging[58]. Advances in MP, omics technologies, and targeted metabolic analysis have expanded the range of measurable biliary parameters and strengthened their potential role in graft assessment[59]. However, heterogeneity in sampling techniques, analytical platforms, and endpoint definitions continues to limit clinical translation[60].

The integration of biochemical, metabolic, and omics-derived data may enable more accurate prediction of biliary complications, EAD, and rejection. Within this evolving landscape the BBRT represents a promising mechanistically grounded biomarker that links transporter biology, epithelial function, and systemic immunosuppression. Its evaluation in a prospective clinical protocol may help clarify its role in personalized postoperative management. Future progress will depend on harmonized sampling protocols, multicenter validation, and the development of integrated predictive frameworks combining bile biomarkers with perfusion data and clinical metrics[61]. Such efforts may ultimately support more individualized immunosuppression strategies and more precise early graft monitoring, contributing to improved outcomes after LT[62].