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\begin{document}

\title{Circulating metabolites are associated with persistent elevations of ALT
in patients with chronic hepatitis B with complete viral suppression}



\author[1]{Jian Sun}%
\author[1]{Dekai Zheng}%
\author[1]{Changhao Cheng}%
\author[2]{Yanhua Tang}%
\author[1]{Zhixin Fang}%
\author[1]{Xuelian Gao}%
\author[1]{Yuchuan Chen}%
\author[1]{* Qiuhong}%
\author[1]{Kaifeng Wang}%
\author[1]{Heqi Zhou}%
\author[1]{Zhixian Lan}%
\affil[1]{Southern Medical University Nanfang Hospital Guangdong Provincial Key Laboratory of Viral Hepatitis Research}%
\affil[2]{First Affiliated Hospital of University of South China}%


\vspace{-1em}



  
  \date{June 11, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Background \& Aims}: Hepatitis B virus (HBV) can be completely
suppressed after antiviral treatment; however, some patients with
chronic hepatitis B (CHB) still exhibit elevated alanine
aminotransferase (ALT) levels and sustained disease progression. The aim
of this study was to provide novel insights into the mechanism and
potential predictive biomarkers of persistently elevated ALT (PeALT) in
patients with CHB after complete viral inhibition. \textbf{Methods}: CHB
Patients with undetectable HBV DNA at least 12 months after antiviral
treatment were enrolled from a prospective, observational cohort.
Correlations between plasma metabolites and the risk of elevated ALT
were examined using multivariate logistic regression. \textbf{Results}:
Of the 1238 patients with CHB who achieved complete viral suppression,
40 (3.23\%) had PeALT levels during follow-up (median follow-up: 2.42
years). Additionally, 40 patients with persistently normal ALT (PnALT)
levels were matched 1:1 as controls. Ser-Phe-Ala (variable importance in
projection {[}VIP{]} = 4.28), Lys-Ala-Leu-Glu (VIP = 4.49),
3-methylhippuric acid (VIP = 3.04), 3-methylxanthine (VIP = 2.62), and
7-methylxanthine (VIP = 3.35) were identified as critical differential
metabolites between the two groups and independently associated with
PeALT risk. Ser-Phe-Ala and Lys-Ala-Leu-Glu levels could be used to
discriminate patients with PeALT from those with PnALT. Furthermore,
\emph{N}-acetyl-l-methionine (NALM) demonstrated the strongest negative
correlation with ALT levels. NALM supplementation alleviated liver
injury and hepatic necrosis induced by carbon tetrachloride in mice.
\textbf{Conclusions}: Changes in circulating metabolites may contribute
to PeALT levels in patients with CHB who have achieved complete viral
suppression after antiviral treatment.%
\end{abstract}%



\sloppy


\textbf{Circulating metabolites are associated with persistent
elevations of ALT in patients with} \textbf{chronic hepatitis B with
complete viral suppression}

Dekai Zheng\textsuperscript{1, 2}, Changhao Cheng\textsuperscript{1, 2},
Yanhua Tang\textsuperscript{3}, Zhixin Fang\textsuperscript{1, 2},
Xuelian Gao\textsuperscript{1, 2}, Yuchuan Chen\textsuperscript{1, 2},
Qiuhong You\textsuperscript{1, 2}, Kaifeng Wang\textsuperscript{1, 2},
Heqi Zhou\textsuperscript{1, 2}, Zhixin Lan\textsuperscript{1, 2}, Jian
Sun\textsuperscript{1, 2}

\textsuperscript{1} State Key Laboratory of Organ Failure Research,
Guangdong Provincial Key Laboratory of Viral Hepatitis Research;
Guangdong Provincial Clinical Research Center for Viral Hepatitis;

\textsuperscript{2} Key Laboratory of Infectious Diseases Research in
South China, Ministry of Education; Department of Infectious Diseases,
Nanfang Hospital, Southern Medical University, Guangzhou, China

\textsuperscript{3} Pulmonary and Critical Care Medicine, The First
Affiliated Hospital, Hengyang Medical School, University of South China,
Hengyang, China.

\textbf{Correspondence details}

Jian Sun, Department of Infectious Diseases, Nanfang Hospital, Southern
Medical University, Guangzhou, China. Email: sunjian@smu.edu.cn

Dekai Zheng, Changhao Cheng and Yanhua Tang contributed equally to this
work.

\textbf{Word count:} 3601

\textbf{Number of figures} (4) \textbf{and tables} (2)

\textbf{Abbreviations}

HBV, hepatitis B virus; ALT, alanine aminotransferase; CHB, chronic
hepatitis B; IL, interleukin; PeALT, persistently elevated ALT; CAP,
controlled attenuation parameter; LSM, liver stiffness measurement;
PnALT, persistently normal ALT; OPLS-DA, orthogonal projections to
latent structures discriminant analysis; VIP, variable importance in
projection; KEGG, Kyoto Encyclopedia of Genes and Genomes; PBS,
phosphate buffered saline; ROC, receiver operating characteristic; AUC,
area under the ROC curve; BMI, body mass index; TG, triglyceride;
NALM,\emph{N} -acetyl-l-methionine; 11-DHC, 11-dehydrocorticosterone;
3-Mx, 3-methylxanthine; 7-Mx, 7-methylxanthine; 3-MA, 3-methylhippuric
acid

\textbf{Ethics and Integrity Policies Statements}

\textbf{Conflict of interest disclosure}

All authors declare that they do not have conflict of interests relevant
to this manuscript.

\textbf{Funding information}

This study was supported by National Key Research and Development
Program of China (2022YFC2304800), National Natural Science Foundation
of China (U22A20274) and Guangdong Basic and Applied Basic Research
Foundation of Guangzhou Joint Fund (2022B1515120039).

\textbf{Acknowledgements}

The authors thank the nurses and patients for their cooperation.

\textbf{Data availability statement}

The data that support the findings of this study are available from the
authors, upon reasonable request.

\textbf{Ethics approval statement}

All of the survey protocols were approved by the Ethics Committee of
Nanfang Hospital (Guangzhou, China).

\textbf{Patient consent statement}

Written informed consent was obtained from all of the participants.

\textbf{Permission to reproduce material from other sources}

Not applicable

\textbf{Clinical trial registration}

Not applicable

\textbf{ORCID}

Dekai Zheng: 0000-0001-5783-3300

Jian Sun: 0000-0001-5320-227X

\textbf{Abstract}

\textbf{Background \& Aims} : Hepatitis B virus (HBV) can be completely
suppressed after antiviral treatment; however, some patients with
chronic hepatitis B (CHB) still exhibit elevated alanine
aminotransferase (ALT) levels and sustained disease progression. The aim
of this study was to provide novel insights into the mechanism and
potential predictive biomarkers of persistently elevated ALT (PeALT) in
patients with CHB after complete viral inhibition.

\textbf{Methods} : CHB Patients with undetectable HBV DNA at least 12
months after antiviral treatment were enrolled from a prospective,
observational cohort. Correlations between plasma metabolites and the
risk of elevated ALT were examined using multivariate logistic
regression.

\textbf{Results} : Of the 1238 patients with CHB who achieved complete
viral suppression, 40 (3.23\%) had PeALT levels during follow-up (median
follow-up: 2.42 years). Additionally, 40 patients with persistently
normal ALT (PnALT) levels were matched 1:1 as controls. Ser-Phe-Ala
(variable importance in projection {[}VIP{]} = 4.28), Lys-Ala-Leu-Glu
(VIP = 4.49), 3-methylhippuric acid (VIP = 3.04), 3-methylxanthine (VIP
= 2.62), and 7-methylxanthine (VIP = 3.35) were identified as critical
differential metabolites between the two groups and independently
associated with PeALT risk. Ser-Phe-Ala and Lys-Ala-Leu-Glu levels could
be used to discriminate patients with PeALT from those with PnALT.
Furthermore, \emph{N} -acetyl-l-methionine (NALM) demonstrated the
strongest negative correlation with ALT levels. NALM supplementation
alleviated liver injury and hepatic necrosis induced by carbon
tetrachloride in mice.

\textbf{Conclusions} : Changes in circulating metabolites may contribute
to PeALT levels in patients with CHB who have achieved complete viral
suppression after antiviral treatment.

\textbf{Abstract word count:} 244

\textbf{Keywords:} chronic hepatitis B; alanine aminotransferase;
metabolites; \emph{N} -acetyl-l-methionine

\textbf{Lay summary}

\begin{enumerate}
\tightlist
\item
  Alterations in the levels of circulating metabolites specific to
  patients with chronic hepatitis B (CHB) exhibiting persistently
  elevated alanine aminotransferase (PeALT) levels were identified.
\item
  ALT levels were most strongly negatively correlated with levels
  of\emph{N} -acetyl-l-methionine (NALM); Supplementation with NALM
  alleviated hepatocyte necrosis in a CCl\textsubscript{4}-induced mouse
  model of acute liver injury.
\item
  The identified key differential metabolites may serve as potential
  biomarkers and/or therapeutic targets for PeALT in patients with CHB.
\end{enumerate}

\textbf{Introduction}

Hepatitis B virus (HBV) infection is a global public health concern,
affecting approximately 300 million people and resulting in over 300,000
deaths every year due to cirrhosis, liver failure, or hepatocellular
carcinoma.\textsuperscript{1,2} Antiviral treatment with nucleoside
analogues can effectively inhibit HBV replication and delay disease
progression.\textsuperscript{3,4} Several pivotal registration studies
have shown that after five years of antiviral treatment with entecavir
or tenofovir, 94\%--99\% of patients achieved undetectable HBV DNA and
80\%--87\% of patients achieved normalization of alanine
aminotransferase (ALT),\textsuperscript{5-7} with approximately
13\%--20\% of patients still showing elevated ALT even after long-term
viral suppression.\textsuperscript{8} Our previous study also reported
that 34.8\% of patients with chronic hepatitis B (CHB) had complete
viral suppression and increased ALT during a follow-up of
18.36~months.\textsuperscript{9} Multiple studies have reported that
elevated ALT during antiviral therapy is associated with an increased
risk of liver events, cirrhosis, and hepatocellular cancer in patients
with CHB.\textsuperscript{10-13} Moreover, an increase in ALT inhibits
the accurate assessment of the disease stage and progression of
CHB.\textsuperscript{14} However, the reason and mechanism underlying
elevated ALT in patients with CHB after viral suppression have not been
fully explored.

Various studies have explored the reasons for elevated ALT during
antiviral therapy. An intermittent increase in ALT during antiviral
therapy is generally considered an immune response to HBV infection,
indicating changes in CHB disease
activity.\textsuperscript{15}Additionally, persistent ALT elevation is
typically associated with metabolic syndrome and liver
steatosis.\textsuperscript{16-18} However, the persistent elevation of
ALT in patients with CHB cannot be explained completely by these
factors. Numerous studies have recently found that metabolites play a
critical role in the development of liver
inflammation.\textsuperscript{19} For example,
3,4-dihydroxyphenylpropionic acid, a metabolite of gut microbiota, can
suppress macrophage activation and improve hepatic ischemia/reperfusion
injury via inhibiting histone deacetylase
activity.\textsuperscript{20}Additionally, the endogenous metabolite
phosphonic acid can alleviate acetaminophen-induced liver injury by
inducing the release of interleukin (IL-6) from adipose
tissue.\textsuperscript{21} A recent study showed that 2-oleoylglycerol
produced by triacylglycerol metabolism promotes western diet-induced
liver inflammation and fibrosis in mice through activating macrophages
and hepatic stellate cells.\textsuperscript{22} Overall, these findings
imply that metabolites possibly play a role in ALT elevation in patients
with CHB. Furthermore, alterations in circulating metabolites are
associated with disease activity and progression in patients with
CHB.\textsuperscript{23-25} A study with 10 years follow-up found that
dysregulation of methionine and branched-chain amino acid-related
metabolites were associated with progression of
CHB.\textsuperscript{23}In addition, lipid and bile acid-related
metabolites are associated with liver inflammation and fibrosis in
patients with CHB. However, the association between elevated ALT and
circulating metabolites in patients with CHB remains unclear.

In this study, we investigated the association between the levels of
circulating metabolites and the risk of persistently elevated ALT
(PeALT) in patients with CHB with complete viral suppression.
Additionally, the potential role of differential metabolites in liver
injury was primarily explored using animal models. Our aim was to
provide novel insights into the mechanism and potential predictive
biomarkers of PeALT in patients with CHB after complete HBV inhibition.

\textbf{Methods}

\textbf{Study participants}

This study was nested in a real-life prospective observational CHB
cohort (Search-B cohort: NCT02167503). The study design and inclusion
criteria for the Search-B cohort have been previously
described.\textsuperscript{26} Patients with CHB who achieved
undetectable HBV DNA levels for at least 12 months after antiviral
treatment at Nanfang Hospital between September 2015 and July 2017 were
enrolled in this study. Patients meeting any of the following exclusion
criteria were excluded: (1) coinfection with hepatitis C virus and
hepatitis D virus; (2) diagnosis of malignant tumor; (3) excessive
alcohol consumption (\textgreater{}21 units of alcohol per week for men
and \textgreater{}14 units of alcohol per week for women) in the
previous six months; (4) receiving medications with known effects on
ALT, such as Chinese traditional medicines, anti-tuberculosis drugs,
lipid-lowering drugs, nonsteroidal anti-inflammatory drugs, and
antibiotics; (5) diagnosis of systemic inflammatory diseases, diabetes,
and autoimmune liver disease; (6) HBV DNA [?] 20 IU/mL during follow-up;
and (6) loss of follow-up.

Written informed consent for clinical data and blood sample collection
was obtained from each patient during recruitment. The study protocol
was approved by the Ethics Committee of Nanfang Hospital (Guangzhou,
China).

\textbf{Data collection}

All subjects were followed up every six months. Baseline clinical and
laboratory data were obtained through physical examination,
questionnaire, routine blood test, FibroScan (controlled attenuation
parameter {[}CAP{]} and liver stiffness measurement {[}LSM{]}), and
virological examination. The participants were required to fast for at
least 10 h before blood collection. Plasma and serum were collected and
stored within 2 hours at -80 \selectlanguage{ngerman}°C. Serum ALT levels were measured using an
automatic biochemical analyzer (Olympus, Tokyo, Japan) according to the
standard protocol. HBV DNA levels and serological markers were measured
using the Roche COBAS TaqMan platform (lower limit of detection for HBV
DNA: 20 IU/mL) and the Elecsys immunoassay (Roche, Basel, Switzerland)
at the Hepatology Unit of Nanfang Hospital.

\textbf{Definitions}

Complete viral suppression was defined as HBV DNA levels below the
detection limit or undetectable for more than 12 months. ALT levels
exceeding 35 U/mL in males and 25 U/mL in females were defined as ALT
elevation.\textsuperscript{14} In this study, the serum ALT elevation at
all visits was defined as PeALT. If the ALT level was normal at all
visits, it was defined as persistently normal ALT (PnALT).

\textbf{Widely targeted metabolomics analysis}

Widely targeted metabolomics was performed as previously
described.\textsuperscript{27,28} Briefly, 50 \selectlanguage{greek}µ\selectlanguage{english}L of plasma was
transferred to an extraction solution (acetonitrile:methanol = 1:4, v/v)
containing internal standards and then centrifuged at 12,000 rpm for 10
min (4 \selectlanguage{ngerman}°C). The supernatant (180 \selectlanguage{greek}µ\selectlanguage{english}L) was collected for further analysis.
Metabolites were separated and identified using ultra performance liquid
chromatography (ExionLC AD, https://sciex.com.cn/), quadrupole-time of
flight (TripleTOF 6600, AB SCIEX), and triple quadrupole-linear ion trap
mass spectrometry (QTRAP\selectlanguage{ngerman}®, \emph{https://sciex.com/} ). The orthogonal
projections to latent structures discriminant analysis (OPLS-DA) model
and variable importance in projection (VIP) values were generated using
the R package MetaboAnalystR. Metabolites with p \textless{} 0.05, VIP \selectlanguage{english}[?]
1, and fold change \selectlanguage{english}[?] 1.2 or \selectlanguage{english}[?] 0.83 were considered significantly
differential metabolites. Pathway enrichment analyses for these
metabolites were performed using the Kyoto Encyclopedia of Genes and
Genomes (KEGG) pathway database (\emph{http://www.genome.jp/kegg} ).

\textbf{Animals and carbon tetrachloride (}
CCl\textsubscript{4})\textbf{-induced liver injury}

Eight-week-old C57BL/6 mice were purchased from SPF Biotechnology Co.,
Ltd. (Beijing, China) and bred in a specific pathogen-free facility at
the Experimental Animal Center of Nanfang Hospital. Food and water were
provided ad libitum under a 12/12 h light/dark cycle.

To induce acute liver injury, mice were administered one intraperitoneal
injection of 10 mL/kg of 5\% CCl\textsubscript{4} (Rhawn Co., Ltd.,
Shanghai, China) dissolved in corn oil, and the control group~was
administered one intraperitoneal injection of just corn oil (Macklin,
Shanghai, China). Mice were treated with metabolites or phosphate
buffered saline (PBS) at 2 h after injection of CCl\textsubscript{4}.
Several available differential metabolites were divided into
water-soluble and non-water-soluble metabolites. For the
non-water-soluble metabolites, mice with liver injury were administered
100 mg/kg of the corresponding metabolites by oral gavage. For
water-soluble metabolites, mice with liver injury were intraperitoneally
injected with 100 mg/kg of the corresponding metabolites. Blood was
collected 24 and 48 h after CCl\textsubscript{4} injection. All mice
were sacrificed 72 h after injection, and blood and liver samples were
collected. The protocols for animal experiments were approved by the
Institutional Animal Ethics Committee of the Experimental Animal Center
of Nanfang Hospital.

\textbf{Histological analysis}

The liver tissue of mice was fixed in 4\% paraformaldehyde, embedded in
paraffin, and sectioned into 4 \selectlanguage{greek}µ\selectlanguage{english}m-thick slices. For histological
analysis, the sections were stained with hematoxylin and eosin.

\textbf{Statistical analysis}

Data analysis was performed using SPSS (version 20.0; IBM Corp., Armonk,
NY, USA). The distribution of data was checked using the Shapiro--Wilk
test. Continuous data with a normal distribution were expressed as means
\selectlanguage{ngerman}± standard deviation; otherwise, data were expressed as medians with
interquartile ranges. Categorical data are presented as counts (\%).
Differences between groups were examined using the Student's t-test,
Mann--Whitney U test, and chi-square test. Correlations between the
differential metabolites and ALT levels were analyzed using Spearman's
correlation coefficient. Multivariate logistic regression was used to
assess the association between metabolites and the risk of PeALT. The
ability to distinguish between patients with PeALT and PnALT using
different metabolites was evaluated using the area under the receiver
operating characteristic (ROC) curve (AUC). The level of statistical
significance was set at \emph{p} \textless{} 0.05 (two-sided).

\textbf{Results}

\textbf{Demographic and clinical information of participants}

As shown in Figure 1A, 1709 patients with CHB who had undetectable serum
HBV DNA levels for at least 12 months after antiviral treatment were
screened. In total, 208 participants were excluded at baseline, and 263
participants were excluded during follow-up (median follow-up: 2.42
years). Finally, 1238 patients with CHB were included in the analysis.
Among these patients, 510 (41.19\%) experienced elevated ALT during
follow-up, 40 (3.23\%) showed PeALT at all visits, and only 55.57\%
maintained PnALT.

To further investigate the potential reason for elevated ALT, we matched
40 patients with PeALT and 40 with PnALT using propensity score
matching. Confounding factors, including sex, age, body mass index
(BMI), follow-up duration, serum triglyceride (TG) and cholesterol
levels, and CAP, were well-matched between these two groups. Baseline
demographics and clinical information of the patients are summarized in
Table 1. The serum ALT (49.5 {[}42, 64.5{]} vs. 21 {[}14, 25.75){]}) and
AST (31 {[}25.25, 38{]} vs. 20 {[}19, 25{]} levels were significantly
higher in the PeALT group than in the PnALT group. The dynamic changes
in ALT and AST levels are shown in Figure 1B and 1C. In addition to ALT
levels, the serum AST level in patients with PeALT was consistently and
significantly higher than that in patients with PnALT, indicating
sustained liver injury.

\textbf{Significant differences in circulating metabolites between the
PeALT and PnALT groups}

To investigate the role of circulating metabolites in PeALT, plasma
metabolomics was conducted, and 1051 metabolites were detected. Greater
than 60\% of the metabolites identified in the plasma belonged to the
following six categories: amino acids and their metabolites (20.85\%),
benzene and its substituted derivatives (12.61\%), organic acids and
their derivatives (11.75\%), heterocyclic compounds (10.62\%), fatty
acids (7.49\%) and alcohol and amines (5.69\%) (Figure 2A). OPLS-DA
showed a clear separation in the metabolite profiles of the PeALT and
PnALT groups (R2Y = 0.966, Q2 = 0.509, Figure 2B). The expression of 17
metabolites was significantly upregulated, whereas the expression of 23
metabolites was significantly downregulated in the PeALT group compared
to that in the PnALT group (Figure 2C). KEGG pathway analysis revealed
that the mTOR signaling pathway, drug metabolism-cytochrome
P\textsubscript{450}, amino acid biosynthesis, D-amino acid metabolism,
and central carbon metabolism in cancer were primarily enriched for the
differential metabolites (Figure 2D). For further analysis, we focused
on the top ten differential metabolites ranked by VIP, which reflected
the level of influence of the metabolites. Levels of Lys-Ala-Leu-Glu
(VIP = 4.49), 11-dehydrocorticosterone (VIP = 4.17),
L-beta-phenylalanine (VIP = 3.65), \emph{N} -acetyl-l-methionine (NALM)
(VIP = 3.28), 3-methylxanthine (3-Mx) (VIP = 2.62), 7-methylxanthine
(7-Mx) (VIP = 2.35), and Pro-His (VIP = 2.32) were significantly lower,
while levels of Ser-Phe-Ala (VIP = 4.28), 3-methylhippuric acid (3-MA)
(VIP = 3.04), and \selectlanguage{greek}α-\selectlanguage{english}ketoglutaric acid (VIP = 2.82) were significantly
higher in patients with PeALT than in patients with PnALT (Figure 2E and
2F).

\textbf{Differential plasma metabolites are associated with PeALT risk}

We evaluated the correlations between the levels of differential
metabolites and clinical parameters. As shown in Figure 3A and 3B,
Ser-Phe-Ala and NALM had the strongest negative and positive correlation
with ALT and AST levels, respectively, implying that these two
metabolites may be associated with liver injury in patients with CHB.
Moreover, 3-Mx was negatively associated with white blood cells and
neutrophils. Multivariate logistic regression analysis further confirmed
that these metabolites were associated with PeALT risk, even after
adjusting for age, sex, BMI, low density lipoprotein, platelet, total
cholesterol, and TG (Table 2). These findings suggest that the
correlation between serum metabolites and the risk of elevated ALT
levels may be independent of BMI and metabolic syndrome. The ROC curve
revealed that Ser-Phe-Ala (AUC = 0.843, 95\% CI 0.75--0.93) and
Lys-Ala-Leu-Glu (AUC = 0.839, 95\% CI 0.744--0.934) can be used to
distinguish patients with PeALT from those with PnALT (Figure 3C). The
findings indicate that plasma metabolites are associated with PeALT risk
and may be potential biomarkers for diagnosing PeALT.

\textbf{NALM improves CCl\textsubscript{4}-induced liver injury and may
be a potential treatment option for PeALT in patients with CHB}

To explore the potential role of differential metabolites in ALT
elevation, we established a mouse model of liver injury by inducing
liver damage using CCl\textsubscript{4} to simulate the ALT elevation
observed in patients with CHB (Figure 4A). After 24 h of
CCl\textsubscript{4} stimulation, the serum ALT levels of mice
significantly increased, peaking at 48 h, and then decreasing
significantly at 72 h, indicating regression in liver injury. Notably,
we found that at 48 and 72 h after CCl\textsubscript{4} stimulation, ALT
levels in the CCl\textsubscript{4}+NALM (i.p.) group were significantly
lower than those in the CCl\textsubscript{4}+PBS (i.p.) group,
indicating that NALM supplementation alleviated the liver injury induced
by CCl\textsubscript{4} (Figure 4B). In addition, ALT levels in the
CCl\textsubscript{4}+3-Mx (i.g.) group were significantly reduced
compared to those in the CCl\textsubscript{4}+PBS (i.g.) group at 48 h.
At 72 h, the ALT levels in the CCL\textsubscript{4}+3-MA (i.g.) group
were significantly higher than those in the CCl\textsubscript{4}+PBS
(i.g.) group, indicating that 3-MA may hinder the regression of liver
injury (Figure 4C). Subsequently, we evaluated the roles of these
metabolites on AST levels. NALM, 7-Mx and 3-Mx significantly inhibited
the increase in AST caused by CCL\textsubscript{4} (Figure 4D and 4E).
Further pathological analysis suggested that NALM supplementation
significantly reduced CCl\textsubscript{4}-induced necrosis compared to
that in the CCl\textsubscript{4}+PBS (i.p.) group (Figure 4F and 4G).
Overall, NALM may improve liver injury, promote the recovery of liver
function, and be a potential treatment for PeALT in patients with CHB.

\textbf{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
CCl\textsubscript{4}.

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.\textsuperscript{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.\textsuperscript{30,31} Moreover, PeALT levels during
antiviral therapy are independently associated with an increased risk of
hepatocellular carcinoma in patients with
CHB.\textsuperscript{11}Therefore, 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.\textsuperscript{32} By contrast, a decrease in glycine synthesis
promoted western diet-induced liver injury and inflammation in mice by
inhibiting glutathione production.\textsuperscript{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 acid\textsuperscript{34} and
contributes to the clearance of reactive oxygen species and prevents
oxidative stress \emph{in vitro} and \emph{in vivo.}
\textsuperscript{35,36} Our results indicate that NALM can significantly
improve CCl\textsubscript{4}-induced liver injury. Moreover, NALM can
reduce CCl\textsubscript{4}-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.\textsuperscript{37-39} These two metabolites directly participate
in regulating the inflammatory response through adenosine receptors on
the surface of immune cells.\textsuperscript{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 CCl\textsubscript{4},
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.\textsuperscript{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.\textsuperscript{44}Exposure 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.\textsuperscript{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 \emph{in vitro} and \emph{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.

\textbf{Author contributions}

DK Zheng, CH Cheng and YH Tang contributed to statistical analyses and
manuscript writing. ZX Fang and YH Tang contributed to interpretation of
the data. YC Chen, QH You, and XL Gao helped with collecting tissues
from the mice. KF Wang, HQ Zhou, and ZX Lan provided advice and revised
the manuscript. J Sun contributed to the study concept and design,
critical revision of the manuscript, and administrative support. All of
the authors critically reviewed and approved the final version of the
manuscript.

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\textbf{Table 1.} Baseline characteristics of
participants\selectlanguage{english}\textsuperscript{+}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& \textbf{PnALT\textsuperscript{++}} & \textbf{PeALT} &
\emph{p}\selectlanguage{ngerman}\textbf{-value\textsuperscript{§}}\tabularnewline
\textbf{Male,} n {[}\%{]} & 32 {[}80{]} & 33 {[}82.5{]} &
0.775\tabularnewline
\textbf{Age, years} & 40.95 \selectlanguage{ngerman}± 10.78 & 40.82 \selectlanguage{ngerman}± 8.90 &
0.955\tabularnewline
\textbf{BMI, kg/m\textsuperscript{2}} & 24.44 \selectlanguage{ngerman}± 3.55 & 24.49 \selectlanguage{ngerman}± 3.18 &
0.949\tabularnewline
\textbf{CAP,} \textbf{dB/m} & 221.42 \selectlanguage{ngerman}± 41.86 & 220.6 \selectlanguage{ngerman}± 38.80 &
0.927\tabularnewline
\textbf{TG, mmol/L} & 0.93 {[}0.65--1.41{]} & 1.15 {[}0.7--1.61{]} &
0.394\tabularnewline
\textbf{TC, mmol/L} & 4.26 {[}3.54--4.85{]} & 4.64 {[}3.99--5.71{]} &
0.092\tabularnewline
\textbf{Follow-up time, years} & 2.26 {[}1.97--3.44{]} & 2.42
{[}1.67--3.48{]} & 0.634\tabularnewline
\textbf{Liver cirrhosis, n (\%)} & 17 {[}42.5{]} & 14 {[}35{]} &
0.491\tabularnewline
\textbf{HBeAg positive,} n {[}\%{]} & 5 {[}12.5{]} & 4 {[}10{]} &
0.723\tabularnewline
\textbf{ALT, U/L} & 21 {[}14--25.75{]} & 49.5 {[}42--64.5{]} &
\textbf{0.0001}\tabularnewline
\textbf{AST, U/L} & 20 {[}19--25{]} & 31 (25. 25--38) &
\textbf{0.0001}\tabularnewline
\textbf{LSM,} \textbf{Kpa} & 8.15 {[}6.125--14.07{]} & 7 {[}5.6--9.22{]}
& 0.172\tabularnewline
\textbf{ALB, g/L} & 44.6 {[}42.07--47.2{]} & 45.6 {[}44--47.1{]} &
0.368\tabularnewline
\textbf{AFP, ng/mL} & 3.29 \selectlanguage{ngerman}± 2.2 & 2.84 \selectlanguage{ngerman}± 1.53 & 0.288\tabularnewline
\textbf{GLU, mmol/L} & 5.2 {[}4.88--5.53{]} & 5.07 {[}4.89--5.36{]} &
0.600\tabularnewline
\textbf{HDL, mmol/L} & 1.04 {[}0.92--1.28{]} & 1.08 {[}0.94--1.29{]} &
0.507\tabularnewline
\textbf{LDL, mmol/L} & 2.69 \selectlanguage{ngerman}± 0.78 & 3.09 \selectlanguage{ngerman}± 0.73 &
\textbf{0.021}\tabularnewline
\textbf{WBC, 10\textsuperscript{9}/L} & 5.69 {[}4.99--6.68{]} & 5.9
{[}5.19--5.9{]} & 0.433\tabularnewline
\textbf{NEU, 10\textsuperscript{9}/L} & 3.29 {[}2.6--3.81{]} & 3.32
{[}2.85--4.4{]} & 0.433\tabularnewline
\textbf{PLT, 10\textsuperscript{9}/L} & 167.5 {[}105.2--210.2{]} & 212
{[}134--257.2{]} & \textbf{0.011}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{+}Data are depicted as means \selectlanguage{ngerman}± standard deviation,
median {[}interquartile range{]}, or counts.

\selectlanguage{english}\textsuperscript{++}Abbreviations: PnALT, persistently normal ALT; PeALT,
persistently elevated ALT; BMI, body mass index; CAP, controlled
attenuation parameter; TG, triglyceride; TC, total cholesterol; HBeAg,
hepatitis B e antigen; ALT, alanine aminotransferase; AST, aspartate
transaminase; LSM, liver stiffness measurement; ALB, AFP,
Alpha-fetoprotein; albumin; GLU, glucose; HDL, high density lipoprotein;
LDL, low density lipoprotein; WBC, white blood cell; NEU, neutrophil;
PLT, platelet.

\textsuperscript{SS}\emph{p} \emph{-} values \textless{} 0.05 are bolded.

\textbf{Table 2.} Association between the levels of differential
metabolites and the risk of persistently elevated alanine
aminotransferase\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\textbf{Metabolites} & \textbf{Unadjusted} & \textbf{Unadjusted} &
\textbf{Unadjusted} & & \textbf{Adjusted\textsuperscript{+}}
&\tabularnewline
& \textbf{OR\textsuperscript{++}} & \textbf{95\% CI (OR)} &
\emph{p}\textbf{-value} & \textbf{OR} & \textbf{95\% CI (OR)} & \emph{p}
\selectlanguage{ngerman}\textbf{-value\textsuperscript{§}}\tabularnewline
Lys-Ala-Leu-Glu & 0.014 & 0.002--0.101 & \textbf{0.001} & 0.001 &
0.001--0.027 & \textbf{0.001}\tabularnewline
Ser-Phe-Ala & 5.423 & 2.505--11.743 & \textbf{0.001} & 5.53 &
2.425--12.626 & \textbf{0.001}\tabularnewline
11-Dehydrocorticosterone & 0.127 & 0.048--0.337 & \textbf{0.001} & 0.076
& 0.021--0.268 & \textbf{0.001}\tabularnewline
l-beta-Phenylalanine & 0.488 & 0.260--0.914 & \textbf{0.025} & 0.437 &
0.209--0.915 & \textbf{0.028}\tabularnewline
\emph{N}-acetyl-l-methionine & 0.018 & 0.002--0.161 & \textbf{0.001} &
0.015 & 0.001--0.169-- & \textbf{0.001}\tabularnewline
3-Methylhippuric acid & 1.647 & 1.164--2.332 & \textbf{0.001} & 1.778 &
1.172--2.698 & \textbf{0.007}\tabularnewline
\selectlanguage{greek}α-\selectlanguage{english}Ketoglutaric Acid & 11.819 & 2.770--50.424 & \textbf{0.001} & 24.23 &
3.732--157.343 & \textbf{0.001}\tabularnewline
3-Methylxanthine & 0.162 & 0.051--0.513 & \textbf{0.002} & 0.205 &
0.059--0.717 & \textbf{0.013}\tabularnewline
7-Methylxanthine & 0.223 & 0.082--0.609 & \textbf{0.003} & 0.268 &
0.091--0.792 & \textbf{0.017}\tabularnewline
Pro-His & 0.09 & 0.019--0.440 & \textbf{0.003} & 0.053 & 0.008--0.364 &
\textbf{0.003}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{+}Adjusted for age, gender, BMI, TC, TG, LDL, and PLT.

\textsuperscript{++}Abbreviations: OR, odds ratio; CI, Confidence
Interval.

\selectlanguage{ngerman}\textsuperscript{§}\emph{p} -values \textless{} 0.05 are bolded.

\selectlanguage{english}\textsuperscript{++}Abbreviations:

\textbf{Figure legends}

Figure 1. (A) Study~flowchart. Dynamic changes in alanine
aminotransferase (ALT) (B) and aspartate transaminase (AST) (C) levels
during follow-up. * \emph{p} \textless{} 0.05; ** \emph{p} \textless{}
0.01; ***\emph{p} \textless{} 0.001. PeALT, persistently elevated ALT;
PnALT, persistently nomal ALT.

Figure 2. Significant differences in circulating metabolites between the
PeALT and PnALT groups. (A) Total number and main categories of
identified metabolites. (B) OPLS-DA model and (C) Volcano plots of the
PnALT vs. PeALT groups. (D) KEGG analysis results of enriched
signaling~pathways. (E) The top 10 differential metabolites were ranked
by VIP values. (F) Relative intensity of the top 10 differential
metabolites between the PeALT and PnALT groups. **\emph{p} \textless{}
0.01; ***\emph{p} \textless{} 0.001. OPLS-DA, orthogonal projections to
latent structures discriminant analysis; KEGG, Kyoto Encyclopedia of
Genes and Genomes; VIP, variable importance in projection.

Figure 3. Differential plasma metabolites associated with PeALT risk.
(A) Correlations of the levels of differential metabolites with ALT
levels, and (B) clinical parameters. (C) ROC curve for differential
metabolites. *\emph{p} \textless{} 0.05; **\emph{p} \textless{} 0.01.
ROC, receiver operating characteristic.

Figure 4. NALM improves carbon tetrachloride-induced liver injury. (A)
Schematic diagram of the mouse experiment. (B, C) Comparison of serum
ALT levels in each group at 24, 48, and 72 h. (D, E) Comparison of serum
AST levels in each group at 72 h. (F, G) Representative images of
hematoxylin and eosin staining and quantification of necrotic areas in
the liver. Scale bar = 100 \selectlanguage{greek}µ\selectlanguage{english}m. *\emph{p} \textless{} 0.05;
**\emph{p}\textless{} 0.01; ***\emph{p} \textless{} 0.001. ''ns''
indicates not significant. NALM, \emph{N} -acetyl-l-methionine; 11-DHC,
11-dehydrocorticosterone; 3-Mx, 3-methylxanthine; 7-Mx,
7-methylxanthine; 3-MA, 3-methylhippuric acid.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3-(1)/Figure-3-(1)}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-4/Figure-4}

\end{center}
\end{figure}

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