Discussion
In this study, we revealed for the first time an association between the levels of circulating metabolites and the risk of PeALT in patients with CHB who have achieved complete HBV suppression. Additionally, supplementation with NALM was observed to improve liver injury caused by CCl4.
ALT is predominantly found in the liver cytoplasm, and its main physiological function is to catalyze the transformation of glutamic acid to alanine through transamination. Generally, 1% of damaged hepatocytes can enhance circulating ALT activity by at least one-fold. As a result, ALT levels are considered one of the most sensitive indicators of liver injury.29 Our results indicate that 3.23% of patients with CHB who have achieved HBV suppression still experience PeALT after excluding factors such as excessive alcohol consumption, medication, and HBV activity. Previous studies reported that even mild elevation of ALT is associated with an increased risk of liver complications and more severe histological inflammation in patients with CHB.30,31 Moreover, PeALT levels during antiviral therapy are independently associated with an increased risk of hepatocellular carcinoma in patients with CHB.11Therefore, clarifying the causes of elevated ALT in CHB patients has clinical significance.
Given the hub role of metabolites in the progression of liver disease, we investigated the effect of circulating metabolites on PeALT levels in patients with CHB. Metabolomic analyses indicated 17 upregulated and 23 downregulated metabolites in patients with CHB with PeALT compared to those in the PnALT group. Furthermore, the correlation between the identified differential metabolites and the risk of elevated ALT was evaluated. We found that even after adjusting for confounding factors, the top 10 differential metabolites were still significantly associated with the risk of elevated ALT, further indicating the critical role of circulating metabolites in PeALT.
Next, we investigated how plasma metabolites may cause an increase in ALT. Notably, many amino acid metabolism-related pathways, such as amino acid synthesis, D-amino acid metabolism, and arginine metabolism, were enriched for the differential metabolites. Multiple studies have shown that changes in circulating amino acids are associated with the progression of chronic liver disease. For example, citrulline, a metabolite produced by arginine metabolism, has been shown to improve non-alcoholic fatty liver in mice by protecting intestinal barrier function and preventing bacterial translocation to the liver.32 By contrast, a decrease in glycine synthesis promoted western diet-induced liver injury and inflammation in mice by inhibiting glutathione production.33 As a result, we assumed that plasma amino acids may be involved in the pathogenesis of PeALT. Further analysis revealed that levels of NALM, an amino acid derivative, showed the strongest negative correlation with both ALT and AST levels among the top 10 metabolites. This suggests that NALM plays a key role in the continuous increase in ALT levels. NALM is produced by the acetylation of methionine with acetic acid34 and contributes to the clearance of reactive oxygen species and prevents oxidative stress in vitro and in vivo. 35,36 Our results indicate that NALM can significantly improve CCl4-induced liver injury. Moreover, NALM can reduce CCl4-induced hepatocyte necrosis, contributing to the repair of liver injury. These results imply that decreased NALM levels may affect the body’s ability to repair liver damage, leading to a sustained elevation of ALT levels after acute inflammation.
In addition, circulating levels of 3-Mx and 7-Mx, derivatives of caffeine, were significantly lower in the PeALT group than in the PnALT group. Caffeine enters the liver after absorption in the gastrointestinal tract and is metabolized by cytochrome P450, producing a series of methylxanthine derivatives, including 3-Mx and 7-Mx.37-39 These two metabolites directly participate in regulating the inflammatory response through adenosine receptors on the surface of immune cells.40,41 Our results also showed a significant negative correlation between 3-Mx levels and the numbers of peripheral white blood cells and neutrophils. This suggests that 3-Mx may be involved in the negative regulation of the immune system. Moreover, we found that in the acute phase of liver injury, 3-Mx reduced the elevation of ALT and AST caused by CCl4, indicating a potential protective effect of 3-Mx against liver injury. Another important differential metabolite was 3-MA, which is a metabolite of toluene and xylene; its levels are commonly used to monitor exposure to these environmental pollutants.42,43 As a result, higher circulating levels of 3-MA in patients with PeALT than in those with PnALT may indicate greater exposure to toluene and xylene.44Exposure to these chemicals can lead to hepatocellular oxidative stress and liver damage in mice, which is possibly mediated by the metabolites of toluene and xylene.45,46 Our results indicated that 3-MA can cause a delayed recovery in ALT levels in mice with acute liver injury, which may have led to a sustained elevation in ALT levels. Therefore, more clinical and basic research should be conducted to verify the correlation between PeALT and exposure to toluene, xylene, and their metabolites. Overall, these key differential metabolites, particularly NALM, may serve as potential therapeutic targets for PeALT in patients with CHB; however, further in vitro and in vivo experimental validation is necessary.
This study has several limitations. First, widely targeted metabolomics was used to analyze all plasma metabolites comprehensively and systematically. The relative concentrations of metabolites can be monitored; however, their absolute quantification remains a challenge. Hence, targeted metabolomics should be used to validate our results in the future. Moreover, owing to the short follow-up time and small sample size, we were unable to observe correlations between differential metabolites and disease progression in CHB patients with viral suppression. Another limitation of the present study is that we included CAP and LSM as regional substitutes for hepatic steatosis and liver fibrosis rather than liver biopsy, however, liver biopsy is not feasible in real-life large-sample prospective observational cohort studies.
To our knowledge, this is the first study to uncover the role of metabolites in liver inflammation in CHB patients with complete viral suppression, based on a long-term follow-up cohort and case-controlled studies. The present study revealed alterations in circulating metabolite levels in patients with CHB with PeALT. This may help to reveal the mechanism of ALT elevation in these patients and provide therapeutic targets.