Abstract

Objectives:

   Plasma bile acid (BA) has been widely studied as pathophysiological
   factors in chronic liver disease. But the changes of plasma BA level in
   lean metabolic dysfunction-associated fatty liver disease (MAFLD)
   remains unclear. Here, we clarified the BA metabolic characteristics of
   lean MAFLD and explored its significance and mechanism as a marker.

Methods:

   We employed ultra-performance liquid chromatography tandem mass
   spectrometry based on BA metabonomics to characterize circulating bile
   acid in lean MAFLD patients. Explore its significance as serum
   biomarkers by further cluster analysis, functional enrichment analysis,
   and serum concentration change analysis of differential BAs. Evaluation
   of diagnostic value of differential BAs by ROC analysis.

Results:

   A total of 65 BAs were detected and 17 BAs were identified which showed
   different expression in the lean-MAFLD group compared with the normal
   group. Functional annotation and enrichment analysis of KEGG and HMDB
   showed that differential BAs were mainly related to bile acid
   biosynthesis, bile secretion, cholesterol metabolism, and familial
   hypercholangitis, involving diseases including but not limited to
   cirrhosis, hepatocellular carcinoma, chronic active hepatitis,
   colorectal cancer, acute liver failure, and portal vein obstruction.
   ROC analysis displayed that the 6 BA metabolites (GCDCA-3S, GUDCA-3S,
   CDCA-3S, NCA, TCDCA, and HDCA) exhibited well differential diagnostic
   ability in discriminating between lean MAFLD patients and normal
   individuals with an area under the curve (AUC) ⩾0.85.

Conclusions:

   We delineated the characteristics of BA level in patients with lean
   MAFLD, and identified 6 potential plasma BA biomarkers of lean MAFLD.

   Keywords: Metabolic dysfunction-associated fatty liver disease, bile
   acid profile, LC-MS/MS, metabonomics, serum biomarkers

Plain Language Summary

   Analysis of serum bile acid profile characteristics and identification
   of new biomarkers in fatty liver disease accompanied by metabolic
   abnormalities in people with normal weight based on the technology of
   high-resolution mass spectrometry

   Objectives: The physique of lean MAFLD patient is normal or even
   leaner. They often does not pay enough attention to the onset of fatty
   liver disease. Plasma bile acids (BAs) have been extensively studied as
   pathophysiological actors in chronic liver disease. But the changes of
   plasma BA level in fatty liver disease accompanied by metabolic
   abnormalities in people with normal weight remains unclear. Here, we
   clarified the BA metabolic characteristics of lean MAFLD and explored
   its significance and mechanism as a marker. Methods: we employed an
   advanced mass spectrometry technology to characterize circulating bile
   acid in lean lean MAFLD patients. To explore its significance as a
   marker by bioinformatics methods, such as cluster analysis, functional
   enrichment analysis, and relative content change analysis of
   differential BAs. Evaluation diagnostic accuracy and determine
   threshold points of BAs by Receiver Operating Characteristic analysis.
   Results: A total of 65 BAs were detected and 17 BAs were identified
   which showed different expression in the lean MAFLD group compared with
   the normal group. Bioinformatics analysis showed that differential BAs
   were mainly related to bile acid biosynthesis, bile secretion,
   cholesterol metabolism, and familial hypercholangitis, involving
   diseases including but not limited to cirrhosis, hepatocellular
   carcinoma, chronic active hepatitis, colorectal cancer, acute liver
   failure, and portal vein obstruction. ROC analysis displayed that the
   six BA metabolites (GCDCA-3S, GUDCA-3S, CDCA-3S, NCA, TCDCA and HDCA)
   exhibited well differential diagnostic ability in discriminating
   between lean MAFLD patients and normal individuals with an area under
   the curve (AUC) ≥ 0.85. Conclusions: We delineated the characteristics
   of BA level in patients with lean MAFLD, and identified six potential
   plasma BA biomarkers of lean MAFLD. This strategy provided broad
   clinical application prospects for disease assessment.

Introduction

   Metabolic dysfunction-associated fatty liver disease (MAFLD) once
   called non-alcoholic fatty liver disease (NAFLD). In addition to fatty
   denaturation and lipid accumulation in hepatocytes, NAFLD is often
   accompanied by cardiovascular and metabolic diseases such as
   hypertension, atherosclerosis, obesity, and diabetes.

   Given the importance of metabolic abnormalities in this disease, NAFLD
   has now been renamed as MAFLD in order to reflect the disease
   characteristics and risks more appropriately.^ [35]1 With the
   prevalence of metabolic syndrome (insulin resistance, obesity,
   diabetes, etc.), MAFLD has become the main cause of chronic liver
   disease worldwide in the past decades. The progression of MAFLD can be
   described by the level of inflammatory activity and fibrosis stage,
   instead of simple fatty liver and steatohepatitis. Without therapeutic
   intervention, some MAFLD patients may subsequently progress to
   cirrhosis and eventually develop into hepatocellular carcinoma (HCC).
   The pathogenesis of MAFLD progression remains to be fully elucidated.
   MAFLD development depends on multiple hepatic insults via several
   different pathways.^ [36]2 Among them, intestine-liver axis interaction
   and abnormal BA metabolism cannot be ignored.

   In HFC diet induced obese mice model, A muciniphila efficiently
   increased mitochondrial oxidation and BA metabolism in the
   intestine-liver axis by regulating the metabolism of L-aspartic acid,
   and improved steatosis and inflammation in MAFLD.^ [37]3 Vitamin C and
   vitamin D3 may be an effective method for treating MAFLD by regulating
   gut microbiota and BA metabolism via the intestine-liver axis, which
   may be a potential drug target for future MAFLD interventions.^ [38]4
   The latest research has found that 3-succinylated cholic acid derived
   from Bacteroides uniformis strains has been shown to be negatively
   correlated with liver injury in MAFLD patients.^ [39]5 BA are the main
   component of bile. It can not only promote the digestion and absorption
   of lipids, but also have important physiological signals and metabolic
   regulation effects. The quantitative detection of the BA profiles plays
   an important role in assessing liver disease.^[40]6,[41]7 There were
   significant differences in BA profiles among patients with chronic
   liver disease of different causes which suggested that BA profiles has
   clinical potential in distinguishing liver injury types.^ [42]8
   Compared with healthy individuals, there was no difference in total
   bile acid (TBA) levels among NAFLD patients, but BA composition changed
   significantly.^ [43]9 Animal experiments showed significant changes in
   BA levels in the enterohepatic circulation of non-alcoholic
   steatohepatitis, and the effects could be corrected by diet.^ [44]10 A
   certain study has shown that primary BA is associated with follow-up
   liver-related events among NAFLD patients, suggesting that BA
   metabolism may predict the prognosis of NAFLD.^ [45]11 Together,
   increasing evidence suggested a close relationship between BA plasma
   concentrations and MAFLD patients.

   It was noteworthy that “lean” or “non-obese” MAFLD has been extensively
   reported among in Asian populations in recent years and there were few
   studies on BA metabolism in lean MAFLD since its redefinition in 2020.
   MAFLD patients in the advanced stage were usually accompanied by
   lobular inflammation and liver fibrosis. Here, we assessed the changes
   in serum BA spectrum inMAFLD progression patients with normal BMI with
   the aim of explore appropriate serum BA markers to diagnose lean MAFLD
   by LC-MS/MS technology and metabonomics.

Materials and Methods

Sample collection

   This study was a retrospective study. From January to June 2023, serum
   samples of 15 diagnosed lean MAFLD patients (lean-MAFLD group) and 15
   healthy controls (Normal group) were collected from Nanjing Jinling
   Hospital. The diagnosis of MAFLD was made according to the diagnostic
   criterion proposed in 2020, emphasized the role of systemic metabolic
   disorders in triggering liver diseases.^ [46]1 In this study, enrolled
   MAFLD patients with normal BMI (18.5-23.9), serum TG ⩾ 1.70 mmol/L, and
   hepatic steatosis through abdominal ultrasound scan, were accompanied
   by diabetes or arterial hypertension for at least 5 years and failure
   of receive standardized medical treatment within the past 6 to
   12 months. The exclusion criteria were as follows: Other chronic liver
   disease (including, but not limited to viral hepatitis, autoimmune
   liver diseases and malignancy); medical history of drugs affecting
   liver function; hazardous alcohol intake. Patients were screened for
   the presence of lobular inflammation and fibrosis by liver biopsy upon
   additional informed consent. Histopathological diagnosis of all liver
   tissue samples was conducted by experienced pathologists. The Scheuer
   scoring system was adopted as the histological standard of Liver
   inflammation (G0~G4) and fibrosis (S0~S4). The grades of liver
   inflammation were classified into the following 5 stages: G0, no
   inflammation; G1, portal inflammation; G2, mild piecemeal necrosis or
   acidophil bodies; G3, moderate piecemeal necrosis and severe focal cell
   damage; and G4, widely bridging necrosis and lobular structural
   abnormalities. Liver fibrosis was scored as follows: S0, no fibrosis;
   S1, portal tracts expansion and portal fibrosis without septa; S2,
   normal lobular structural and portal fibrosis with rare septa; S3,
   disordered lobular structure and numerous septa without cirrhosis; and
   S4, early or confirmed cirrhosis. Hepatic steatosis was scored on a
   4-point scale (F0~F3). The percentage of fat in liver parenchyma: F0,
   <5%; F1, 5%~33%; F2, 34%~66%; F3, >66%. Fasting serum from 5 mL
   peripheral venous blood was collected from each participant and stored
   in a −80° C refrigerator.

   The research protocol was approved by the ethics committees of Nanjing
   University School of Medicine Affiliated Jinling Hospital. This study
   was conducted according to the ethical guidelines of the Declaration of
   Helsinki. Participants willingly agreed to participate in the study and
   written consents were taken.

BAs detection and quantification by LC-MS/MS

   Bile acids contents were detected by MetWare
   ([47]http://www.metware.cn/) based on the AB Sciex QTRAP 6500 LC-MS/MS
   platform.

   Sample preparation and extraction Samples (50 μL) were extracted with
   200 μL methanol/acetonitrile(v/v = 2:8). 10 μL 13 kinds internal
   standard mixed solution (1 μg/mL) was added into the extract as
   internal standards (IS) for the quantification. Put the samples at
   −20°C for 10 minutes to precipitated protein. Then centrifugation for
   10 minutes (12 000 r/min, and 4°C), the supernatant was transferred to
   clean plastic microtubes. The extracts were evaporated to dryness,
   reconstituted in 100 μL 50% methanol (V/V) for further LC-MS/MS
   analysis.

   The HPLC and ESI-MS/MS conditions based on references.^[48]12,[49]13