Subnormothermic Machine Perfusion of Steatotic Livers Results in Increased Energy Charge at the Cost of Anti-Oxidant Capacity Compared to Normothermic Perfusion

Negin Karimian^{1

2

3}, Siavash Raigani^{4

5

6}, Viola Huang^{7

8}, Sonal Nagpal^{9

10}, Ehab O A Hafiz¹¹, Irene Beijert¹², Paria Mahboub^{13

14}, Robert J Porte¹⁵, Korkut Uygun^{16

17

18}, Martin Yarmush^{19

20

21}, Heidi Yeh^{22

23

24}

Affiliations

¹ Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. n.karimian284@gmail.com.
² Division of Transplant Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. n.karimian284@gmail.com.
³ Shriners Hospital for Children, Boston, MA 02114, USA. n.karimian284@gmail.com.
⁴ Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. sraigani@partners.org.
⁵ Division of Transplant Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. sraigani@partners.org.
⁶ Shriners Hospital for Children, Boston, MA 02114, USA. sraigani@partners.org.
⁷ Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. vhuang450@gmail.com.
⁸ Shriners Hospital for Children, Boston, MA 02114, USA. vhuang450@gmail.com.
⁹ Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. nsonal400@gmail.com.
¹⁰ Shriners Hospital for Children, Boston, MA 02114, USA. nsonal400@gmail.com.
¹¹ Electron Microscopy Research Department, Theodor Bilharz Research Institute, 12411 Giza, Egypt. dr_ehabosama@yahoo.com.
¹² Division of Hepatobiliary Surgery and Liver Transplantation, University Medical Center Groningen, 9700 Groningen, The Netherlands. ibie65552@gmail.com.
¹³ Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. paria.mahboub@gmail.com.
¹⁴ Shriners Hospital for Children, Boston, MA 02114, USA. paria.mahboub@gmail.com.
¹⁵ Division of Hepatobiliary Surgery and Liver Transplantation, University Medical Center Groningen, 9700 Groningen, The Netherlands. r.j.porte@umcg.nl.
¹⁶ Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. KUYGUN@mgh.harvard.edu.
¹⁷ Division of Transplant Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. KUYGUN@mgh.harvard.edu.
¹⁸ Shriners Hospital for Children, Boston, MA 02114, USA. KUYGUN@mgh.harvard.edu.
¹⁹ Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. MYARMUSH@mgh.harvard.edu.
²⁰ Shriners Hospital for Children, Boston, MA 02114, USA. MYARMUSH@mgh.harvard.edu.
²¹ Department of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854, USA. MYARMUSH@mgh.harvard.edu.
²² Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. hyeh@partners.org.
²³ Division of Transplant Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. hyeh@partners.org.
²⁴ Shriners Hospital for Children, Boston, MA 02114, USA. hyeh@partners.org.

PMID: 31652927
PMCID: PMC6918199
DOI: 10.3390/metabo9110246

Subnormothermic Machine Perfusion of Steatotic Livers Results in Increased Energy Charge at the Cost of Anti-Oxidant Capacity Compared to Normothermic Perfusion

Negin Karimian et al. Metabolites. 2019.

. 2019 Oct 24;9(11):246.

doi: 10.3390/metabo9110246.

Authors

Affiliations

¹ Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. n.karimian284@gmail.com.
² Division of Transplant Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. n.karimian284@gmail.com.
³ Shriners Hospital for Children, Boston, MA 02114, USA. n.karimian284@gmail.com.
⁴ Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. sraigani@partners.org.
⁵ Division of Transplant Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. sraigani@partners.org.
⁶ Shriners Hospital for Children, Boston, MA 02114, USA. sraigani@partners.org.
⁷ Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. vhuang450@gmail.com.
⁸ Shriners Hospital for Children, Boston, MA 02114, USA. vhuang450@gmail.com.
⁹ Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. nsonal400@gmail.com.
¹⁰ Shriners Hospital for Children, Boston, MA 02114, USA. nsonal400@gmail.com.
¹¹ Electron Microscopy Research Department, Theodor Bilharz Research Institute, 12411 Giza, Egypt. dr_ehabosama@yahoo.com.
¹² Division of Hepatobiliary Surgery and Liver Transplantation, University Medical Center Groningen, 9700 Groningen, The Netherlands. ibie65552@gmail.com.
¹³ Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. paria.mahboub@gmail.com.
¹⁴ Shriners Hospital for Children, Boston, MA 02114, USA. paria.mahboub@gmail.com.
¹⁵ Division of Hepatobiliary Surgery and Liver Transplantation, University Medical Center Groningen, 9700 Groningen, The Netherlands. r.j.porte@umcg.nl.
¹⁶ Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. KUYGUN@mgh.harvard.edu.
¹⁷ Division of Transplant Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. KUYGUN@mgh.harvard.edu.
¹⁸ Shriners Hospital for Children, Boston, MA 02114, USA. KUYGUN@mgh.harvard.edu.
¹⁹ Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. MYARMUSH@mgh.harvard.edu.
²⁰ Shriners Hospital for Children, Boston, MA 02114, USA. MYARMUSH@mgh.harvard.edu.
²¹ Department of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854, USA. MYARMUSH@mgh.harvard.edu.
²² Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. hyeh@partners.org.
²³ Division of Transplant Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA. hyeh@partners.org.
²⁴ Shriners Hospital for Children, Boston, MA 02114, USA. hyeh@partners.org.

PMID: 31652927
PMCID: PMC6918199
DOI: 10.3390/metabo9110246

Abstract

There continues to be significant debate regarding the most effective mode of ex situ machine perfusion of livers for transplantation. Subnormothermic (SNMP) and normothermic machine perfusion (NMP) are two methods with different benefits. We examined the metabolomic profiles of discarded steatotic human livers during three hours of subnormothermic or normothermic machine perfusion. Steatotic livers regenerate higher stores of ATP during SNMP than NMP. However, there is a significant depletion of available glutathione during SNMP, likely due to an inability to overcome the high energy threshold needed to synthesize glutathione. This highlights the increased oxidative stress apparent in steatotic livers. Rescue of discarded steatotic livers with machine perfusion may require the optimization of redox status through repletion or supplementation of reducing agents.

Keywords: anti-oxidant; liver transplantation; machine perfusion; metabolomics; normothermic; steatosis; subnormothermic.

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Conflict of interest statement

Uygun is an inventor on pending patents relevant to this study and has a provisional patent application relevant to this study. Uygun has a financial interest in Organ Solutions, a company focused on developing organ preservation technology. Uygun’s interests are managed by the Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies.

Figures

**Figure 1**
Perfusion parameters during machine perfusion of steatotic livers. Steatotic livers had higher qualitative hepatic artery (a) and portal vein (b) flows during NMP, though this was not statistically significant. Arterial resistance (c) was significantly higher throughout the majority of three hours perfusion during NMP compared to SNMP. Portal vein resistance (d) was also higher during NMP but not statistically significant due to large variance. Random intercept mixed model analysis for longitudinal data was used. Data shown as mean ± SEM, * indicates p < 0.05. NMP, normothermic machine perfusion; SNMP, subnormothermic machine perfusion.

**Figure 2**
Functional outcome measures during machine perfusion of steatotic livers. NMP of steatotic livers resulted in evidence of higher metabolic activity as expected compared to SNMP. Venous lactate content (a) decreased during three hours of perfusion in both groups but at a fast rate during NMP. ALT (b) is qualitatively higher in steatotic livers during NMP compared to SNMP but did not reach significance. Glucose content in the perfusate (c) was significantly higher during NMP. Steatotic livers produced comparable volumes of bile during the first 2 h of perfusion, but production was significantly higher by the third hour compared to SNMP (d) Data shown as mean ± SEM, * indicates p < 0.05. NMP, normothermic machine perfusion; SNMP, subnormothermic machine perfusion; ALT, alanine aminotransferase.

**Figure 3**
Histologic differences of steatotic livers during NMP and SNMP. Representative H&E-stained liver sections from steatotic livers undergoing NMP and SNMP shown pre-perfusion and post-perfusion. No significant change in macrosteatosis was observed with either perfusion modality. (a) pre-perfusion biopsy of liver in SNMP group, (b) pre-perfusion biopsy of liver in NMP group, (c) biopsy after 3 hours of SNMP, (d) biopsy after 3 hours of NMP.

**Figure 4**
Energy cofactor changes during NMP and SNMP of steatotic livers. (a) ATP:ADP, (b) ATP:AMP, and (c) energy charge ratios at each hour of perfusion. Given the lower metabolic activity at subnormothermic temperatures, steatotic livers are able to regenerate ATP and energy charge to higher levels with oxygenated perfusion compared to NMP. Energy charge was calculated as (ATP + ADP*0.5)/(ATP + ADP + AMP). Within group comparisons at 60, 120, and 180 min are made to a pre-perfusion measurement (time = 0 min). * indicates p < 0.05 for random intercept mixed model comparison of longitudinal data to pre-perfusion levels. Data shown are mean ± SEM. NMP, normothermic machine perfusion; SNMP, subnormothermic machine perfusion.

**Figure 5**
Pathway enrichment analysis for metabolomic profiles of steatotic livers during NMP and SNMP. Pathway enrichment scores of metabolite subgroups at 180 min of perfusion compared to pre-perfusion concentrations between SNMP and NMP groups. NMP, normothermic machine perfusion; SNMP, subnormothermic machine perfusion.

**Figure 6**
Redox status during NMP and SNMP of steatotic Livers. (a) N-acetylcysteine, (b) reduced glutathione, and (c) oxidized glutathione ratios demonstrate depleted redox capacity within hepatocytes during SNMP compared to NMP. ** indicated p < 0.05. NMP, normothermic machine perfusion; SNMP, subnormothermic machine perfusion; x-axis represents fold change at 60, 120, and 180 min compared to pre-perfusion concentrations.

**Figure 7**
Glutathione degradation varies in steatotic livers during SNMP compared to NMP. (a) The glutathione cycle involves the degradation of glutathione into its base amino acids. Synthesis of glutathione involves three molecules of ATP. (b) Heatmap of the metabolites in the glutathione cycle compared between SNMP and NMP of steatotic livers. There is a significant depletion of the precursor metabolites in the synthesis arm of the cycle during SNMP compared to NMP, as well as a larger accumulation of 5-oxoproline. Reduced glutathione content in hepatocytes is decreased during NMP but depletion is near-total during SNMP. GSH, reduced glutathione; GSSH, oxidized glutathione; Cys-Gly, cysteinylglycine; γ-Glu-Cys, γ-glutamylcysteine; NMP, normothermic machine perfusion; SNMP, subnormothermic machine perfusion; x-axis represents fold change at 60, 120, and 180 min compared to pre-perfusion concentrations.

See this image and copyright information in PMC

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Subnormothermic Machine Perfusion of Steatotic Livers Results in Increased Energy Charge at the Cost of Anti-Oxidant Capacity Compared to Normothermic Perfusion

Affiliations

Subnormothermic Machine Perfusion of Steatotic Livers Results in Increased Energy Charge at the Cost of Anti-Oxidant Capacity Compared to Normothermic Perfusion

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

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