Normothermic Machine Perfusion of Explanted Human Metabolic Livers: A Proof of Concept for Studying Inborn Errors of Metabolism

Abstract

The human liver plays a central metabolic role; however, its physiology may become imbalanced in inborn errors of metabolism (IEM), a broad category of monogenic disorders. Liver transplantation has been increasingly used to improve patient metabolic control, especially in diseases related to amino acid metabolism, such as urea cycle disorders and organic acidurias, to provide enzyme replacement. Ex vivo liver normothermic machine perfusion (NMP) techniques have recently been developed to increase the number of transplantable grafts and improve transplantation outcomes. This study used seven NMP of explanted livers from patients with IEM undergoing transplantation as models to investigate disease-related liver metabolism and function. The perfused livers demonstrated positive viability indicators and disease-specific targeted metabolomics providing the proof-of-principle that our ex vivo model expresses the biochemical disease characteristics and responds to therapeutical intervention in a unique "physiological" milieu, offering an ideal tool to study novel treatments, in a setting closely mirroring human disease.

Keywords: machine perfusion; metabolic liver disease; pediatric liver transplantation.

FIGURE 1

Hemodynamic and vitality parameters in the perfusate during normothermic machine perfusion. (A) Percent of liver perfusion provided by portal vein (PV) and hepatic artery (HA); (B) Perfusate flows through PV and HA (mean ± standard deviation); (C) PV pressure (mean ± standard deviation); (D) HA pressure (mean ± standard deviation); (E) Lactate levels (mean ± standard deviation); (F) Glucose levels (mean ± standard deviation).

FIGURE 2

Functional and hematological parameters measured during normothermic machine perfusion. (A) Aspartate aminotransferase (AST); (B) Alanine aminotransferase (ALT); (C) Lactate dehydrogenase (LDH) in ASA livers (mean ± standard deviation) and in one PA liver; (D) Total bilirubin (mean ± standard deviation); (E) Factor V (mean ± standard deviation); (F) Hemoglobin (Hb) (mean ± standard deviation).

FIGURE 3

Livers histology before (T0) and after (T8) normothermic machine perfusion. (A) PA‐2: Preserved architecture with intact, viable hepatocytes; no apoptotic bodies (hematoxylin & eosin [H&E] staining; 10×); (B) CPS‐1: No pathological modifications at both T0 and T8 (H&E, 10×); (C) CPS‐1: No glycogen storage inside the hepatocytes (PAS, 10×).

FIGURE 4

Metabolic parameters measured in the perfusate during normothermic machine perfusion. Top figures display median and interquartile range values of all measurements during perfusion (*p < 0.05; **p < 0.01; ***p < 0.001); bottom figures show the measurements at the different time points (mean ± standard deviation). (A) Ammonia levels in propionic acidemia (PA) and in urea cycle defects (UCDs) liver perfusions (top), variations during perfusion of PA, argininosuccinic aciduria (ASA) and carbamoyl phosphate synthetase 1 (CPS1) livers (bottom); (B) Urea nitrogen (BUN) levels in PA and in UCDs liver perfusions (top), variations during perfusion (bottom); (C) Glutamine levels in PA and in UCDs liver perfusions (top), variations during perfusion (bottom); (D) Glutamate levels in PA and in UCDs liver perfusions (top), variations during perfusion (bottom); (E) Alanine levels in PA and in UCDs liver perfusions (top), variations during perfusion (bottom); (F) Arginine levels in PA, ASA and CPS liver perfusions (bottom), variations during perfusion (bottom).

FIGURE 5

Metabolic parameters measured in the perfusate during normothermic machine perfusion. In A, C and D top figures display median and interquartile range values of all measurements during perfusion (*p < 0.05; **p < 0.01; ***p < 0.001); bottom figures show the measurements at the different time points (mean ± standard deviation). (A) Citrulline levels in propionic acidemia (PA), argininosuccinic aciduria (ASA) and carbamoyl phosphate synthetase 1 (CPS1) liver perfusions (top), variations during perfusion (bottom); (B) Argininosuccinic acid levels during perfusion of ASA livers (mean ± standard deviation); (C) Methylcitrate (MCA), 3‐hydroxy‐propionate (3OH propionate), propionyl‐glycine (PG) and propionyl‐carnitine (C3) levels in PA liver perfusions (top), variations during perfusion (bottom); (D) Glycine levels in PA and UCDs livers perfusions (top), variations during perfusion (bottom); (E) Glycine and PG levels during perfusion of PA‐4 liver after pre‐treatment (T0) of the perfusate solution with supraphysiological concentration of glycine.

FIGURE 6

Schematic representation of ex vivo Normothermic Machine Perfusion of explanted metabolic livers. The flow chart illustrates sample collection of perfusate, bile fluid, hepatic and bile biopsies. NMP, normothermic machine perfusion; SCS, static cold storage.