Changes of liver metabolites following hepatectomy with ischemia reperfusion towards liver regeneration

Abstract Background Metabolome analysis is one of the omics which investigates the final product of a central dogma. Changes of liver metabolites during liver regeneration following hepatectomy (Hx) continue to remain unclear. The aim of the present study was to investigate the changes of liver metabolites following Hx with ischemia reperfusion (I/R) towards liver regeneration. Methods Twenty‐three patients who underwent Hx were enrolled in this study. Non‐tumor tissues were sampled immediately before and after Hx and a comparison was made between the liver samples taken before and after Hx using capillary electrophoresis (CE)—time‐of‐flight mass spectrometry (TOFMS) as metabolome analysis. Results The metabolic pathway showed that there was a significant increase in “lactate” following Hx. There was a significant decrease in metabolites only in the first half of the tricarboxylic acid cycle (TCA) cycle, and adenosine triphosphate (ATP) by anaerobiotic glycolysis did not occur in time for energy consumption of the Hx. Principal component analysis revealed remarkably different component profiles between the samples taken before and after Hx. One hundred and three metabolites were selected as critical metabolites for separating components. Valine and tryptophan increased significantly after Hx and they were regulated by resected liver volume, ischemic time and liver function. Conclusion The liver metabolites changed remarkably between before and after Hx. Especially, liver valine and tryptophan were increased.

spectrometry (CE-MS), and each method is able to analyze a different class of metabolites. 3,4 It has been well established that the liver can regenerate after hepatectomy (Hx). 5 The authors have previously reported that liver regeneration is a fundamental mechanism by which the liver responds to various injuries such as ischemia reperfusion (I/R) injury. [6][7][8] Although the molecular and cellular mechanism of liver regeneration after Hx is well understood, postoperative liver failure after Hx is still one of the critical problems in clinical settings.
There have been no reports about liver metabolites during liver regeneration using metabolome analysis. It was hypothesized that some key metabolites immediately after Hx could regulate or stimulate future liver regeneration. The aim of the present study was to investigate changes of liver metabolites following Hx with I/R towards liver regeneration using human Hx samples.

| Patients
Twenty-three patients who underwent Hx between April 2014 and March 2015 were enrolled in this study. Inclusion criteria for this study were: (i) primary Hx; (ii) <15% of indocyanine green retention test (ICG R15); (iii) subsegmentectomy or more extended Hx; (iv) no biliary reconstruction and lymph node dissection; and (v) no preoperative chemotherapy. Backgrounds of the patients are shown in Table 1

| Sample collection
Non-tumor tissues were sampled immediately before and after Hx.
The collected samples were quickly frozen at À80°C until sample preparation was completed.

| Metabolome analysis
Frozen tissue (c.a. 40 mg) was added to methanol (500 lL) containing internal standards (20 lmol L À1 each of methionine sulfone and D-camphor-10-sulfonic acid) and homogenized using a beads beater (TOMY Micro Smash MS-100R; Tomy Digital Biology, Tokyo, Japan) at 3000 rpm for 60 seconds. Then, both chloroform (500 lL) and Milli-Q water (200 lL) were added to the homogenate. The solution was thoroughly mixed, then centrifuged at 4600 g for 15 minutes at 4°C, and the aqueous fraction was centrifugally filtered through a 5-kDa-cut-off ultra-centrifugal filter unit (Ultrafree-MC-PLHCC-HMT; Human Metabolome Technologies Inc., Tsuruoka, Japan) to remove proteins. The filtrate was dried using an evacuated centrifuge and dissolved in Milli-Q water (50 lL) containing 200 lmol L À1 reference compounds (3-aminopyrrolidine and trimesic acid) prior to CE-MS analysis. CE-MS-based metabolomic profiling and data analysis were carried out essentially as described. 9-13

| Definitions of clinical parameters
Preoperative, postoperative and changes between pre-and postoperative valine and tryptophan were compared in terms of the following clinical parameters (resected liver volume, regeneration rate, ischemic time, FIB-4 index, and sarcopenia).
1. Resected liver volume: subsegmentectomy was grouped for the minor Hx group, and segmentectomy and lobectomy were grouped for the major Hx group.
2. Regeneration rate: liver volumes before Hx and 1 week after Hx were measured using a 3D simulation imaging system. Regeneration rate was defined as the volume increase of the remnant liver as compared with the preoperative volume. Calculations

| Metabolic map
In the pentose phosphate pathway, there were no significant differences between before and after Hx ( Figure 1A). The metabolic pathway showed that there was a significant increase in "lactate" after Hx. There was only a significant decrease in metabolites in the first half of the TCA cycle, and adenosine triphosphate (ATP) by anaerobiotic glycolysis did not occur in time for energy consumption of the Hx ( Figure 1B). This suggested that lipid metabolism might be more dominant than glucose metabolism after Hx.

| Principal components analysis
Principal components analysis showed remarkably different component profiles between before and after Hx ( Figure 2A). Out of a total of 267 metabolites, 103 metabolites that had a variable importance for projection (VIP) score of more than 1.0 were selected as critical metabolites for separating components ( Figure 2B). Table 2 shows all metabolites (VIP score >1.0) and Figure 2C shows hierarchical clustering of 103 metabolites. Valine (VIP score: 1.78957) and tryptophan (VIP score: 1.74943) were significantly up-regulated after Hx.

| Valine/tryptophan and clinical parameters
In the case of valine, much more resected volume and longer ischemic time affected its up-regulation. Furthermore, preoperative valine significantly increased in the high FIB-4 group whereas sarcopenia did not affect valine expression. Tryptophan was found to have similar tendencies to valine (Table 3).

| DISCUSSION
In the present metabolomics study: (i) the metabolic pathway showed that lipid metabolism might be more dominant than glucose metabolism after Hx; (ii) liver metabolites changed remarkably between before and after Hx; and (iii) liver valine and tryptophan were remarkably increased after Hx and they were regulated by resected liver volume, ischemic time and liver function.
It has already been reported that remnant liver metabolism after Hx switches to a predominant utilization of fatty acid as an energy source from glucose. 16,17 Within 24 hours after Hx, the uptake of free fatty acids in the residual hepatocytes was increased in order to acquire energy, and hepatocytes accumulated triglyceride (TG). [18][19][20] In the present study, glucose metabolism did not occur in time for energy consumption after Hx, and lipid metabolism might be dominant for producing ATP. These results were reasonable for previous reports.
Regarding liver metabolism during liver regeneration, there was a report on amino acid and glucose metabolism in the rat fulminant hepatic failure (FHF) model. 21 In that model, a new isolated perfused liver system in combination with a mass-balance model to generate a metabolic map was introduced. However, the number of metabolites was limited, and it was not a comprehensive analysis. Conversely, metabolome analysis is concerned with the comprehensive analysis of endogenous low-molecular-weight compounds in biological samples.
Valine, which is one of the branched-chain amino acids (BCAA), has been reported to stimulate the proliferation of hepatocytes by a dose dependent method in vitro. 22 Valine was also found to be most effective in vivo among three BCAA in the Hx model. Furthermore, valine was reported to increase serum free fatty acid, or liver triglyceride, and up-regulated liver fatty acid became a source of ATP production. 22 Valine, was also reported to have an antioxidative effect by down-regulating tumor necrosis factor (TNF) and up-regulating superoxide dismutase 2 (SOD2) towards human umbilical vein endothelial cells (HUVEC). 23 In contrast, tryptophan was the major source of serotonin production, and platelets were major carriers of serotonin in the blood.
In thrombocytopenic mice, a serotonin agonist reconstituted liver proliferation after Hx. 24 Tryptophan might stimulate liver regeneration after Hx by serotonin. Furthermore, tryptophan regulated reactive oxygen species (ROS) by inducing nuclear factor (erythroidderived 2)-like 2 (NF-E2-related factor 2 or Nrf2) in primary hepatocyte culture. 25 In our hierarchical clustering, valine and tryptophan significantly increased after Hx, and they were regulated by resected liver volume, ischemic time and liver function. Although further investigations are necessary, these phenomena might reflect the remnant liver's protective response for I/R injury.
It was already reported that the period immediately after Hx (0-6 hours after Hx) was critical for future liver regeneration. 5,6 In the present study, liver tissues were sampled immediately after Hx, and we hypothesized that metabolites immediately after Hx would affect future liver regeneration. However, there was a limitation of this study regarding the time-point after Hx, and several time-points after Hx might be necessary.
In conclusion, the present study identified the changes of liver metabolites in Hx with I/R towards liver regeneration. Liver