Validation of a Liquid Chromatography-Mass Spectrometric Assay for Tacrolimus in Liver Biopsies After Hepatic Transplantation: Correlation With Histopathologic Staging of Rejection

Abstract

The aims of this work were both to validate a sensitive and specific method to quantify tacrolimus (TAC) in liver biopsies after hepatic transplantation and to evaluate the predictive value of either tissue or blood TAC concentrations for rejection in 146 adult patients under a TAC-based immunosuppression. Trough blood levels were monitored daily during the hospital stay by immunoassay. Liver biopsies were routinely performed at day 7 posttransplantation. The tissue assay was developed by liquid chromatography-mass spectrometry. The limit of quantification was 5 pg/mg, with intra- and interassay precision ranging from 3.9% to 14.3% and 4.7% to 15.9%, respectively. The extraction efficiency was approximately 80%. TAC found in liver biopsies ranged from less than 5 up to 387 pg/mg. Blood TAC levels ranged from 2.7 to 19.3 ng/mL. Tissue levels displayed excellent correlation with liver histopathologic BANFF rejection score, whereas blood levels did not. Clinically significant rejections (BANFF scores> or = 6) were characterized by mean TAC tissue and blood concentration of 13.1 pg/mg and 7.6 ng/mL, respectively, whereas these mean values became, respectively, 74.9 pg/mg (P < 0.05) and 7.1 ng/mL (not significant) for nonclinically significant rejection episodes (BANFF < 6). In this study, hepatic tissue TAC concentrations were distributed in a wider range and displayed a significantly better correlation with the severity of the organ rejection than predose blood levels. A tissue TAC concentration less than 30 pg/mg is 89% sensitive and 98% specific to discriminate clinically significant cellular rejection. Further studies are required to better understand the factors affecting TAC distribution within liver tissue (such as carrier proteins and cytochrome genetic polymorphism, liver function, age, hepatic blood flow, type of organ transplanted, time posttransplantation) and to define its value in the treatment of liver allograft rejection.

An UPLC–MS/MS method coupled with automated on-line SPE for quantification of tacrolimus in peripheral blood mononuclear cells

“…Measured amounts of TAC from each sample were standardized and converted in concentrations using their sample-specific MCV [28][29][30], obtained for each sample through the automated cell counter. This represents a strong novelty point respect to the previous methods [15][16][17][18]31], because the standardization of analytical results on the basis of the real cell volume in the single sample removes an important source of bias. Moreover, this allowed a correct comparison and correlation between intra-PBMC and whole-blood concentrations of TAC.…”

1199G>A and 2677G>T/A polymorphisms of ABCB1 independently affect tacrolimus concentration in hepatic tissue after liver transplantation

“…These studies, however, exclusively investigated the influence of genetic polymorphisms on Tac blood concentration and/or dose requirement. It has been recently reported that hepatic concentrations appear to be a better marker of histological rejection than trough blood concentrations [4]. In this study, Capron et al showed that Tac hepatic concentrations varied markedly among patients and were well correlated with the histological rejection score (Banff score), whereas no relation was found between the Banff score and trough blood concentrations.…”

“…The protocol of the present pharmacogenetic study was appended retrospectively to the previously described protocol of Capron et al [4]. Liver donors (n = 150) were all cadavers and all recipients had Tac-based immunosuppressive therapy.…”

“…It is, however, not always clear whether trough blood concentrations are related to the clinical outcome as some studies have reported conflicting results [25][26][27][28][29][30]. Furthermore, as mentioned above, it has been recently suggested, in liver transplant recipients, that Tac hepatic concentration constitutes a better marker of the outcome of the graft than trough blood concentration [4]. Therefore, the purpose of this study was to investigate the effect of the donor genotype for 13 different polymorphisms in biotransformation enzymes (CYP3A5 and CYP3A7), in their regulatory proteins (PXR) as well as in transporter proteins (P-gp and OATP-C) on Tac pharmacokinetics in hepatic transplantation and more specifically on Tac hepatic concentrations during the first week after surgery.…”

Adverse Effects of Drugs and Toxins on the Liver

“…Liver biopsy during drugand toxin-induced liver injury is of course not commonly used for this purpose. However, development of liquid chromatography-mass spectrometric (LC-MS/MS) assays for tissue specimens of tacrolimus from liver biopsy have been shown to be better correlated to histopathologic rejection scores than conventional immunoassays of ordinary blood concentrations (Capron et al, 2007). With further development of highly sensitive LC-MS/MS assays, specific determination of substances with metabolites can hopefully be useful in the context of drug-and toxin-induced liver injury.…”