Real-Time Assessment of Liver Function During Sub-Normothermic Machine Perfusion by a Non-Invasive 13C Based Test

doi:10.21203/rs.3.rs-1450501/v1

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Real-Time Assessment of Liver Function During Sub-Normothermic Machine Perfusion by a Non-Invasive ¹³C Based Test

https://doi.org/10.21203/rs.3.rs-1450501/v1

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Background

Assessment of liver quality during machine perfusion (MP) is under intensive investigation. Gold standard methods either are of limited validity or evaluation is time-consuming. Hence, herein we investigate a novel real-time method to estimate liver function during sub-normothermic MP.

Methods

Three healthy porcine livers and one non-allocable human liver were subjected to 24h of SNMP with either Custodiol® or Custodiol MP as perfusate. Additional to liver enzymes, Flavin mononucleotide (FMN) and High-mobility group box 1 (HMGB-1) as well-established markers of liver quality, a novel ¹³C-Methacetin based method was evaluated. Readouts of ϭ¹³CO₂ plots generated by the ¹³CORLab Prototype were calculated and compared to established parameters.

Results

The newly established method mirrored the pathological findings as well as the increasing changes in liver enzymes, HMGB-1 and FMN levels bearing the advantage of being a real-time, non-invasive test which is applicable on site.

Conclusion

This novel method is an easy to implement and reliable tool for quick on-site estimation of liver function during liver SNMP.

Methacetin

liver function

SNMP

Liver transplantation (LT), the best option to improve patient’s quality of life in a broad spectrum of end stage liver diseases, has evolved tremendously during the last decades. With the steadily increasing gap between demand and supply the use of extended criteria donor (ECD) organs and new preservation technologies is gaining importance [1]. Static cold storage (SCS) is, due to its cost effectiveness and simplicity, still the method of choice when it comes to organ preservation. In the last few years machine perfusion (MP) gradually gained importance [2, 3]. MP allows improved characterization of the ECD organs prior to implantation [4]. Although a variety of promising methods to estimate over-all liver function during MP [5] are available most of them are time-consuming and hardly applicable if it comes to real-time decision processes.

A ¹³C-Methacetin based test evaluates the P450-dependent metabolic activity of the liver [6, 7] by measuring the ¹³CO₂ exhaled after substrate (¹³C-Methacetin) application thereby granting an estimation of liver function [8, 9]. This method has been described in a wide range of liver diseases to be a potential non-invasive tool to estimate over-all liver function [10]. One study reported its successful use in diagnosing liver injury in terms of chemotherapy-associated side effects during the treatment of colorectal liver metastasis [11] and another one during LT [12]. It has been argued that one major limitation of the early breath tests using ¹³C-Methacetin is the varying blood supply of the liver [13] but in terms of MP of an isolated organ, this argument might lose its strength. Furthermore, a very recent proof of concept study reported the feasibility of ¹³CO₂ measurement in normothermic MP [14]. This study aimed to investigate for the first time the feasibility to use a ¹³C-Methacetin based test to assess liver function during sub-normothermic MP (SNMP) which has been reported to grant improved energy contents compared to normothermic MP in livers [15].

Characteristics of human donor liver

The liver used in this study originated from a 47 years old male, brain-dead donor with a history of smoking and excessive alcohol use (for liver parameters prior to organ retrieval see Table 1). Brain death was diagnosed after a prolonged period of cardiac arrest followed by reanimation with the cause of death not further specified. Cold ischemia time prior to start of SNMP was 2h26m.

Table 1

Liver parameters of liver donor prior to explantation.
	Time before organ retrieval		Normal range
	24h	15h	Normal range
ALT (U/l)	255	235	0–35
AST (U/l)	520	593	0–30
LDH (U/l)	1364	1549	50–300
GGT (U/l)	296	287	0–35
AP (U/l)	103	111	0-150
CRP (mg/dl)	30.19	316.8	0–1

Change of liver parameters during SNMP

Levels of liver enzymes increased during SNMP in all porcine organs reflecting a decrease in organ quality (Fig. 1). Although the enzyme levels decreased after perfusate renewal at 6h of perfusion, the levels reached or even exceeded pre-renewal values at end of perfusion.

A similar picture is shown when human liver parameters were investigated (Fig. 2).

Changes of liver tissue during SNMP

In the pig livers, no inflammation or impairment of liver lobe structure was detected immediately after organ retrieval. Only the liver of pig one remained without any sign of necrosis throughout the whole period of SNMP, whereas for pig 2 and 3 the first signs of cellular necrosis are reported after 6h of SNMP and remained evident in similar levels until the end of the 24h perfusion. Figure 3 depicts an overview of the liver tissues prior to SNMP start and after 24h of SNMP.

In the initially steatotic human liver no signs of inflammation or impairment of liver lobe structure was seen during the whole period of SNMP whereas cellular necrosis became evident as early as 1h after onset of SNMP but did not increase with continuation of perfusion (Fig. 4, Table S1).

Liver quality and cell damage

HMGB-1 levels are increasing in the human non-transplantable liver whereas seem to remain stable during perfusion of the healthy porcine livers (Fig. 5 upper panel). Increasing FMN levels in pig 1 and 3 (Fig. 5 lower panel) indicate a decrease in organ quality over time, which was not yet evident microscopically in that extent in the pathological evaluation of the liver tissue (especially for pig 1). Unfortunately, determination of FMN in Albumin containing Custodiol MP® is not possible due to high background fluorescence masking specific FMN signals.

ϭ¹³CO₂ curves as non-invasive, real-time measure of liver function during SNMP

The decrease in size of ¹³C-Methacetin response curves (Fig. 6) as a surrogate of impaired liver function mirrors the decrease in liver viability depicted by an increase of liver enzymes in Fig. 1 and Fig. 2 as well as the impairment in liver tissue quality found in the pathological evaluation of the tissue (Fig. 3 and Fig. 4). FMN and HMGB-1 levels reflect the histopathological findings as well (Fig. 5).

For calculation of curve characteristics (depicted in Fig. 7), outliers (Liver pig 1 M2 and M4: spikes above the maximum of the curve; Liver pig 2: M4 as a whole; Human liver: M2 and M3: spikes above maximum of the curve) were excluded to minimize non-liver related bias.

The decrease in absolute AUC, DOBmax and maximum ϭ¹³CO₂ complete the picture represented by liver enzymes, pathological evaluation and FMN values. Time to DOBmax increased in all pig organs but tended to decrease in the human non-transplantable liver which is in exceptionally bad condition and almost stopped responding to substrate during SNMP. As a shorter time to reach maxima mirrors more active cells, this parameter might be another valuable tool to estimate liver function in terms of organs of initially good quality.

As for SNMP, due to decreased metabolic activity, widely used parameters in normothermic MP might be of limited validity in context of liver function, implementation of the ¹³CORLab in combination with the novel ϭ¹³CO₂ ¹³C-Methacein readouts might pave the way to more efficient and faster assessment of liver function for this approach.

HMGB1 levels, as marker of hepatocyte death[16, 17], have been reported to increase upon hepatic IRI starting about 1h after reperfusion suggesting that it is an early marker of IRI[18]. HMGB1 is released by necrotic cells and its extracellular appearance in ischemic injury mediates liver injury[19]. The stable HMGB-1 levels in porcine organs might be explainable by generally good conditions of the organs at SNMP start whereas the obvious increase in the human non-allocable liver reflects its poor condition upon onset of SNMP decreasing with persistence of the perfusion. The mitochondrial injury marker FMN has already shown to be a surrogate for graft survival[20, 21]. Unfortunately, it was not possible to determine FMN levels in all four organs, as Albumin in Custodiol MP® produced too high background fluorescence for discriminatory FNM determination. For non-Albumin containing Custodiol®, FMN determination revealed increasing levels over time, suggesting ongoing mitochondrial damage during SNMP. This is in accordance with increasing liver enzyme levels and pathological evaluation reporting onset of liver damage of various extent throughout SNMP.

The read-outs calculated from the novel ϭ¹³CO₂ ¹³C-Methacein response curves mirror the findings described above irrespective of the perfusate. These findings are in accordance with results reported for the similar ¹³C-Methacetin based method in normothermic MP[14]. In combination with HMGB1 as well as FNM measurement, this ¹³C-Methacetin based method proves to be a fast and reliable method to estimate liver function during SNMP. Additional to these very promising proof-of principle results future in-depth studies, including more organs, are necessary to investigate in if the read-outs presented herein simply add to the pool of markers as quick pre-screening method or even, at least partly, replace non-sufficient gold standard surrogates.

This is the first time to monitor in real time liver function mandatory for the definition of a graft suitable for transplantation. Our study revealed for the first time that this ¹³C-Methacetin method is a very promising non-invasive real-time measure of liver quality changes during SNMP which is worth to be subjected to further investigation.

Organs included

Three livers of healthy pigs procured by standard explantation procedures, from animals used for another study (approved the Austrian federal ministry for education, science and research BMBWF-66.010/0112-V/3b/2019) by our group were used in accordance with 3Rs, the principles of laboratory animal care, and the Austrian national laws. The study was carried out in compliance with the ARRIVE guidelines.

Additionally, one human liver, rejected for transplantation due to biopsy proven steatohepatitis by all centers it was offered to, procured from a multi organ donor was used for the experiments according to the institutional ethics committee of the Medical University of Graz (30–493 ex17/18) approval.

Organ preparation and back table procedure

After procurement of the porcine organs the livers were used for MP after 3h of SCS (pig 1) or immediately after explantation (pig 2 and pig 3). For MP the A. hepatica (8F, Arterial cannula; Organ Assist, Xvivo, Groningen, Netherlands) and V. porta (25F, LiA portal cannula; Organ Assist, Xvivo, Groningen, Netherlands) are cannulated by fixation with a 2 − 0 Vicryl (Ethicon, Johnson & Johnson Medical N.V., Belgium) ligature. Furthermore, to collect the produced bile the common bile duct was cannulated with a polyurethane Nutrifit feeding tube (8F, 125cm length; Vygon, Paris, France) which was fixed by means of a 2 − 0 Vicryl (Ethicon, Johnson & Johnson Medical N.V., Belgium) ligature. Immediately prior to perfusion start, the organ was weighted and subsequently flushed with 2L of ice cold Custodiol® (Dr. Franz Köhler Chemie GmbH, Bensheim, Germany).

The human, non-allocable liver was used immediately after delivery to the study team. Cannulation and back-table procedure was done in the same way as described above.

Sub-normothermic machine perfusion and sampling

For liver perfusion the Liver Assist® (Organ Assist, Xvivo, Groningen, Netherlands) machine is used in combination with the LiA disposable set (Organ Assist, Xvivo, Groningen, Netherlands). The 24h perfusion protocol was adapted from [22]. The system was pre-filled with 4l of perfusion solution (Custodiol® or Custodiol MP®, Dr. Köhler Chemie, Bensheim, Germany) supplemented with Penicillin/Streptomycin (Sigma-Aldrich; Merck, Darmstadt, Germany) and Amphotericin B (Gibco; Thermo Fisher Scientific, Vienna, Austria) to a final concentration of 40,000 U/l, 0.04 mg/l and 1 mg/l respectively. Machine set-up was done according to the manufacturer’s protocol with the temperature set to 21°C and oxygenation with 100% O₂ at a constant flow rate of 1l/min. After 6h of perfusion, 2L of perfusate were replaced by 2L of fresh perfusate.

Sampling (perfusate and tissue) was performed immediately prior to application of ¹³C-Methacetin and 1h after substrate application. Perfusate samples were shock frozen in liquid nitrogen and stored at -80°C until further use. Tissue samples were fixed in 4% Formaldehyde solution for later histological evaluation.

Non-invasive assessment of liver function by the ¹³CORLab Prototype

The prototype ¹³CORLab (ArgosMed, Karlsruhe, Germany) works on basis of isotope-selective CO₂ determination enabling the detection of ¹²CO₂ and ¹³CO₂ separately. The machine automatically calculates ¹³CO₂ in ppm as well as ϭ¹³CO₂ in ‰ plotting it in real-time on the machine’s display. The latter is calculated as follows:

R depicts the ratio of concentration of ¹³CO₂ to ¹²CO₂ and R_PDB being the Pee Dee Belemnite (R_PDB = 0.0112372 [23])

The ¹³CORLab machine was connected to the air outlet of the LiverAssist® Perfusion machine (Fig. 8, yellow arrows) while all other potential air outlets were tightly closed and the oxygen supply was set to a flow rate of 1l/min. The prototype equilibrated for one hour to establish a stable baseline (BL) prior to addition of 4% ¹³C-Methacetin application (M1) in an organ weight-based manner. Addition of ¹³C Methacetin (8ml/kg of liver weight) was repeated at hours 6 (immediately after renewal of perfusate, M2), 12 (M3), and 18 (M4) immediately after routine sampling.

On basis of the ϭ¹³CO₂ plots maximum value of ϭ¹³CO₂, maximum delta over baseline (DOBmax), time to DOBmax as well as the area under the curve (AUC) were calculated for each ¹³C-Methacetin response curve (M1-M4) separately in R (version 4.1.1) in combination with R Studio (Version 1.4.1717). The area under the curves (AUC) was calculated by using the trapezoid method with the DescTools (Andri Signorell et mult. al. (2021). DescTools: Tools for descriptive statistics. R package version 0.99.43) package.

Liver specific parameters

Aspartate aminotransferase (AST), Alanine Aminotransferase (ALT), γ-glutamyltransferase (GGT), alanine phosphatase (AP) and lactate dehydrogenase (LDH) were determined from frozen perfusate samples taken immediately prior to and 1h after each ¹³C-Methacetin application. While the porcine parameters were determined by means of the dry-chemistry analyzer Fuji Dri-Chem NX500V (Fujifilm Europe, Düsseldorf, Germany) human parameters were analyzed on a Cobas® 8000 analyzer (Roche Diagnostics GmbH, Mannheim, Germany) with reagents from the same manufacturer. Since the respective methods were not validate for this extraordinary sample matrix, we evaluated the analytical performance using a standard addition protocol (for details see supplemental methods).

Flavin mononucleotide (FMN) as marker for organ quality was determined based on a previously described method [24]. Briefly, a 7-point Standard curve ranging from 10-640ng/ml was prepared by dissolving Firboflavin-5’monophospate (Sigma Aldrich, Vienna, Austria) in the desired blank perfusate. For determination of FMN levels in black 96-well plates Fluorescence was recorded at 485/520nm (Ex/Em) in duplicate by means of a Fluostar Omega (BMG Labtech, Ortenberg, Germany) plate reader. High-mobility group box 1 (HMGB-1) as marker of liver cell damage was determined by a commercially available ELISA (human: Biomatik, Delaware, USA; porcine: ByBioSource, SanDiego CA, USA) according to manufacturer’s instructions by means of a Spectrostar Omega (BMG Labtech, Ortenberg, Germany) plate reader.

Tissue preparation and evaluation

Formalin fixed paraffin embedded liver samples were prepared in a standard manner. Liver sections (1.5µm thick) were de-paraffinized and stained with hematoxylin and eosin (H&E) for evaluation by an experienced pathologist with focus on changes in inflammation, cellular necrosis and structure of liver lobes over the period of SNMP (for details refer to supplementary material).

Graphic representation of data

Graphs were drawn by means of GraphPad Prism 9 for Windows (Version 9.1.2) or R (version 4.1.1) in combination with R Studio (Version 1.4.1717) with packages ggplot2 (H. Wickham. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York, 2016.), dplyr (Hadley Wickham, Romain François, Lionel Henry and Kirill Müller (2021). dplyr: A Grammar of Data Manipulation. R package version 1.0.7), ggpubr (Alboukadel Kassambara (2020). ggpubr: 'ggplot2' Based Publication Ready Plots. R package version 0.4.0), DescTools (Andri Signorell et mult. al. (2021). DescTools: Tools for descriptive statistics. R package version 0.99.43), tidyverse (Wickham et al., (2019). Welcome to the tidyverse. Journal of Open Source Software, 4(43), 1686, https://doi.org/10.21105/joss.01686)

Authors contributions:

Study design: B.L., P.S., P.S.; Data acquisition and analysis: B.L., J.W., L.R., T.N., A.A.; Study performance: B.L., J.W., L.R.; Manuscript preparation and editing: B.L., J.W., L.R., T.N., A.A., P.S, P.S.

Data availability: Raw Data is available at request from the corresponding author.

Additional Information and Competing interest:

The authors declare no competing interests.

Supplementary information is available online.

The study was carried out in compliance with the ARRIVE guidelines.

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No competing interests reported.

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Real-Time Assessment of Liver Function During Sub-Normothermic Machine Perfusion by a Non-Invasive ¹³C Based Test

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Abstract

Background

Methods

Results

Conclusion

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1. Introduction

2. Results

3. Discussion

4. Methods

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References

Additional Declarations

Supplementary Files

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