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.