Excess alcohol intake in alcohol use disorder patients triggers liver diseases including steatosis, fibrosis, cirrhosis, and even cancer, which are termed as alcohol-associated liver disease (ALD) [1,2]. However, the susceptibility to ALD differs in individuals due to genetic variants and metabolic phenotypes [3,4]. The findings and clinical merits of ALD biomarkers have been recently reported [5,6], but the lipidomic characteristics of patients with alcoholic intemperance and the biomarkers for ALD susceptibility are still unclear. Herein, we presented two serum lipid biomarkers NAPhe 22:4;O and LPC 16:0. Patients with elevations of these two biomarkers resisted ALD after long-term excess drinking, in contrast, lower levels indicated more susceptibility to ALD.

We recruited 83 alcohol use disorder (AUD) participants and 138 Non-AUD controls in the Brain Disease Hospital affiliated to Zhengzhou University. Informed consent was obtained from all participants. The study was approved by the ethics committees of the hospital (approval ID: KS2024-001-01). AUD was tested by the Alcohol Use Disorders Identification Test (AUDIT score ≥20, Supplementary Material). The fatty liver degree was identified by B-mode ultrasound. The criteria were: None: normal echotexture of the liver; Mild: a slight and diffuse increase of liver echogenicity but normal diaphragm and portal vein wall; Moderate: a moderate increase of liver echogenicity with slightly impaired appearance of diaphragm and portal vein wall; Severe: a marked increase of liver echogenicity with poor or no visualization of diaphragm and portal vein wall. The clinical characteristics were summarized in Supplementary Table 1. The ALD was diagnosed by AUDIT ≥20, moderate/severe fatty liver, and increases in serum lipid indicators, alanine aminotransferase or aspartate aminotransferase. The lipidomic investigation was used to identify differential-ly lipid molecules between Non-AUD controls and AUD patients (Methods, Supplementary Material). The potential biomarkers were further verified between the none/mild fatty liver subgroup and the moderated/severe fatty liver subgroup in the AUD group (Fig. 1A).

Long-term excess alcohol intake distinctly altered the lipidomic profile (Fig. 1B). The permutation plot indicated the model was appropriate (Fig. 1C). Based on the criteria of variables important to projection value >1, P<0.05, 95 lipid molecules were identified (Fig. 1D, Supplementary Table 2). To investigate the correlation of these molecules with ALD in AUD patients, only 3 lipids, NAPhe 22:4;O, LPC 16:0, and Cer 21:2;O2/38:6, showed statistical significance between none/mild fatty liver subgroup and moderate/severe subgroup of AUD patients (Fig. 1E). Furthermore, NAPhe 22:4;O and LPC 16:0 showed statistical significance of AUC (Fig. 1F). Interestingly, the levels of NAPhe 22:4;O and LPC 16:0 were elevated by alcohol intake in none/mild ALD patients but not in moderated/severe ALD patients, meaning that patients with elevations of these two biomarkers were resistant to ALD, while no change indicated the susceptibility to ALD. However, NAPhe 22:4;O and LPC 16:0 were not altered in the subgroups of Non-AUD, which strengthened their specificities for AUD (Supplementary Fig. 1). We then analyzed the joint AUC of NAPhe 22:4;O and LPC 16:0 to enhance the predicting efficacy of ALD susceptibility (Fig. 1G, Combination AUC=0.7282, P<0.01), which has the potential as a diagnostic tool for ALD susceptibility in alcohol intemperance patients.

Alcohol metabolism primarily occurs in the liver through the action of oxidoreductases, transferases, and hydrolases. These metabolic pathways generate acetaldehyde and NADH in the liver, disturb fatty acid metabolism, and ultimately lead to the accumulation of fat in the liver and liver lesions. However, different AUD patients responded differently to the alcohol intemperance, as some having none/mild fatty liver while others having moderate/severe fatty liver. Our findings revealed that the elevation of NAPhe 22:4;O and LPC 16:0 in AUD patients reflected a compensatory metabolic adaptation to long-time, excess alcohol intake. Importantly, these biomarkers remained unchanged in moderate/severe fatty liver subgroups and non-AUD controls, indicating that their upregulation was specific to individuals with preserved metabolic resistance to ALD. NAPhe affects ligases and hydrolases, playing a role in regulating intracellular lipid metabolism and inflammatory responses in the liver [7]. LPC (lysophosphatidylcholine) affects hydrolases and isomerases. Higher level of LPC is associated with the synthesis and secretion of lipoproteins, preventing fat accumulation and alleviating the severity of fatty liver disease [8]. Hence, the increases in NAPhe 22:4;O and LPC 16:0 might be the compensatory responses, which present the enhanced metabolic ability for alcohol and the resistance to ALD in patients with alcoholic intemperance (Fig. 1H). Further recruitment of more AUD patients and the experimental explorations were needed for elucidating the molecular mechanism of ALD susceptibility in AUD patients.