The finding of an early biomarker means that liver failure prediction can be made, and the biomarker can be used to show the effects of management. Although there have been reports of positive effects of portal inflow modulation such as the use of a somatostatin analogue or terlipressin and splenectomy after major liver resection, there is, to date, no definite treatment for liver failure besides liver transplantation3,4,5. The purpose of this study was to find liver failure biomarkers that can be identified earlier than the conventional liver biomarkers.
This study is the first to investigate metabolomic characterisation for mortality prediction following hepatectomy using a pig model. Untargeted metabolic profiling systematically characterised metabolomic differences in blood serum according to remnant liver volume after resection. Further, we constructed a biomarker model of five serum metabolites that discriminated mortality after hepatectomy with high sensitivity and specificity over all time points under investigation.
The mechanism underlying liver failure after liver resection and dysfunction of small-size grafts is related to shear stress and sinusoidal injury1. Although efforts have been made to lower portal pressure and shear stress1,6,7,8,9, reports on biomarkers related to liver failure and dysfunction are currently inadequate. In clinical settings, the conventional markers of liver function include serum bilirubin, PT, AST, and ALT. AST and ALT are indicators of past injury, not indicators of present function. Even the combination of PT and total bilirubin, which are known as the most reliable predictive conventional markers, only has sensitivities of 14% and 19% on postoperative days 1 and 3, respectively2. The sensitivity increases to 59% on postoperative day 5. Additionally, clinical manifestations, such as ascites and neurological status, vary in the early postoperative period. Postoperative delirium, anaesthesia, and analgesia can affect neurological status, and intraoperative lymph node dissection and systemic volume status can affect the amount of ascites. Therefore, new biomarkers are needed for early detection, and we focused on liver metabolomes in this study10. To determine early biomarkers of liver failure, we used 70% and 90% liver resection models. A 70% liver resection is within the limits of safe resection, while a 90% resection is associated with liver failure leading to death11,12. To determine the biomarkers with the capability to discriminate patients with failing livers and exclude those with liver regeneration and in recovery after resection, we compared the 70% resection model with the liver failure model. Further, we sought to determine biomarkers that do not discriminate between the sham operation and 70% resection models. PT and ALT, which are conventional markers, were discriminative after 14 postoperative hours, while total bilirubin and AST did not distinguish between the 70% and 90% resection groups at every time point. This pattern is consistent with that reported in another study, which showed that PT was the only discriminative parameter with a value of approximately 1.5 (INR) in the 90% liver resection model13. PT has also been used to predict liver failure in clinical settings. However, PT alone is not enough for liver failure prediction. Bilirubin level on postoperative day 5 can be used in conjunction with PT to better predict liver failure; however, this only increases the prediction sensitivity to 59%2.
Based on untargeted metabolic profiling using GC-TOF MS, we observed that the primary metabolic physiology was clearly distinguishable according to the different volumes of remnant liver after hepatectomy. The profiles of all three groups prior to open surgery were analogous to that of the sham operation group postoperatively. Additionally, the 90% hepatectomy group was clearly distinguished from the other groups, whereas the 70% hepatectomy and sham groups had similar profiles. This suggests that open surgery alone has minimal effect on the blood metabolic profiles, and primary metabolic physiology can clearly distinguish between the different remnant liver volumes after hepatectomy.
The metabolic physiology in the 90% hepatectomy group was best characterised by differential regulation in bioenergetics and amino acid metabolism that was systematically identified using an integrative multivariate statistical model. A previous study that used nuclear magnetic resonance spectroscopy reported that energy metabolism was significantly downregulated by partial hepatectomy14, and this is consistent with our findings. The levels of two glycolysis intermediates, glucose and fructose-6-phosphate were lower in the 90% hepatectomy group than in the survival groups. In contrast, lactic acid level was significantly higher in the 90% hepatectomy group than in the survival groups, and it has been proposed that this is associated with acute and chronic hepatic insufficiency in patients15. There was also significant alteration of the TCA cycle, and this resulted in aberrant levels of citric acid, fumaric acid, and malic acid. It was recently reported that a significant decline in metabolite levels was found in the first half of the TCA cycle16. The OPLS-DA model revealed that the metabolites contributed most to the metabolic features of the 90% hepatectomy group.
We performed a binary logistic regression, which showed that the linear combination of five metabolites (malic acid, methionine, tryptophan, glucose, and GABA) sensitively predicted mortality following hepatectomy. The model discriminated the 90% hepatectomy group from the survival groups at all time points under investigation. GABA level gradually decreased and was lowest at the last time point in the 90% hepatectomy group (p > 0.08). In our study, we found that GABA level had a pattern that is opposite to that in previous studies that reported an inhibitory effect on hepatic regeneration following partial hepatectomy in rat models17,18. However, it has also been reported that GABA plays a protective role in acute liver injury19 and hepatotoxicity20. The metabolites need to be considered to play pivotal roles determining the liver regenerative capacity following partial hepatectomy in future investigations. In contrast, the difference in methionine levels gradually increased with time in the 90% hepatectomy group. A previous study reported that methionine-enriched diets instigated liver injury in cystathione-β-synthase-deficient mice as the consequent accumulation of homocysteine impaired liver regeneration after partial hepatectomy in a rat model21. Increase in methionine levels was noted in cases of severe liver failure in clinical settings, but not in cases of chronic active hepatitis22,23. Serum methionine concentrations greater than 200 μmol/L were found only in patients with severe liver failure, but not in patients with compensated viral hepatitis23. Additionally, significantly higher methionine levels were observed concomitantly with signs of decompensation such as ascites, jaundice, and hepatic encephalopathy22. There are two possible mechanisms underlying high levels of serum methionine in a failing liver. One is the disruption of the methionine cycle and the other is compensatory reactions to protect from further liver damage. The methionine cycle is one of the vulnerable cycles in liver disease, and methionine level varies depending on liver function and degree of compensation. S-adenosylmethionine synthetase (SAMe) level is higher in the early stages of liver disease and leads to low methionine levels. However, SAMe level eventually decreases during the compensatory periods, and this in turn causes an increase in methionine levels during the liver failing process24. With regard to compensatory reactions after massive surgery, the level of 5-methylthioadenosine (MTA), which is a downstream derivative of SAMe, increases via increase in the SAMe level. MTA has the capacity to protect the liver by increasing glutathione level, improving membrane stability, and decreasing the activity of TNFα25,26. Consequently, higher MTA levels result in higher methionine levels. In a previous study, this process was found to occur more actively in the acutely failing liver, especially in the group that received a somatostatin analogue with protective effect on the remnant liver after liver resection27. Both mechanisms may occur concomitantly 48 hours after massive surgery. The exact cut-off level is yet to be determined, and further serial examinations will help in the prediction of prognosis and liver failure in such patients.
The kynurenine pathway for tryptophan catabolism mainly occurs in the liver; however, tryptophan level varies depending on liver function and availability of the rate-limiting enzyme (indoleamine 2,3-dioxygenase) induced by inflammatory response and kidney function28. A recent prospective study revealed that, in the 70% resection model with compensated liver function, serum tryptophan level increased with normal or mildly elevated indoleamine 2,3-dioxygenase level29. Conversely, in the 90% resection model with decompensated liver function, tryptophan level decreased with increase in indoleamine 2,3-dioxygenase level induced by other organs such as the kidney29. Given the similar inflammatory states in the 70% and 90% resection models, it is unclear if compensation determines the tryptophan level26,27,29.
As described, high malic acid level is associated with TCA cycle disorders, and elevated levels of fumaric acid may lead to increased malic acid production. Urea cycle dysfunction is also associated with malic acid accumulation as aspartic acid consumption is reduced30. The level of malic acid, unlike that of other TCA metabolites, is affected by the urea cycle. The compensatory action of high malic acid level may be due to fumaric acid, which generates oxaloacetate and aspartic acid to promote the transaminase process. However, in patients with urea cycle disorders, this may lead to malic acid accumulation.
This study is meaningful as liver biomarkers in patients with acutely failing livers differ compared to other metabolites which are determined using untargeted metabolic profiling. In the clinical setting, it should be considered that these metabolic markers (malic acid and methionine) may vary during the failing process because of compensatory reactions. However, these markers can be monitored in patients with acute liver failure after hepatectomy at 48 postoperative hours. It should also be kept in mind that underlying diseases in a patient may affect the methionine cycle. In patients with hepatocellular carcinoma, the levels of glutamine, malic acid, and methionine may be elevated due to urea cycle disorders30. Furthermore, human hepatitis B-related liver cirrhosis leads to increase in urine and serum methionine levels31. We used a 90% hepatectomy model that leads to definite liver failure to find the biomarkers. However, there are limitations regarding determination of the exact cut-off level of the biomarkers in patients with marginal liver function in the clinical setting. Further study is needed to determine and validate cut-off levels of metabolomics biomarkers.
In summary, time-dependent profiling of serum metabolome characterized the metabolic features specific to mortality following hepatectomy. Primary dysregulation was identified in central carbon-nitrogen metabolism including glycolysis, TCA cycle, and amino acid metabolism. Systematic prioritization based on OPLS-DA and binary logistic regression analysis proposed robust biomarker panels that can accurately predict the risk of mortality associated with hepatectomy.
Nevertheless, our data has some limitations including sample size. Biomarker study based on high-throughput molecular profiling (e.g., metabolomics) is conducted in an untargeted and hypothesis-free way32. This often limits sample size determination for appropriate statistical power. According to a univariate statistic (e.g., analysis of variance [ANOVA]), to meet a false discovery rate (FDR) of 0.2, the sample size of our study should be 80 per group.