Plasma lipidomics and fungal peptide-based community analysis identifies distinct signatures for early mortality in acute liver failure

Abstract

Background/aims: Acute liver failure (ALF) has high mortality predominantly due to compromised immune system and increased vulnerability to bacterial and fungal infections.

Methods: Plasma lipidome and fungal peptide-based community (mycobiome) analysis were performed in discovery cohort (ALF=40, healthy=5) and validated in a validation cohort of 230 patients with ALF using high-resolution-mass-spectrometry, artificial neural network (ANN) and machine learning (ML).

Results: Untargeted lipidomics identified 2,013 lipids across 8 lipid group. 5 lipid-species-phosphatidylcholine (PC)[15:0/17:0], PC[20:1/14:1], PC[26:4/10:0], PC[32:0] and TG[4:0/10:0/23:6]-significantly differentiated ALF-NS (FC>10, P<0.05, FDR<0.01). Mycobiome alpha/beta diversity was significantly higher and showed 4 phyla and >20 species significantly dysregulated in ALF-NS linked with lipid metabolism, fatty acid elongation in ER, and others (P<0.05). Lipid and mycobiome diversity values in ALF-NS were strongly correlated (r2>0.7, P<0.05). Multi-modular correlation network showed striking associations between lipid, fungal peptide modules, and clinical parameters specific to ALF-NS (P<0.05). Cryptococcus amylolentus CBS6039 and Penicillium oxalicum 1142 directly correlated with phosphatidylcholine, triglycerides, and severity in ALF-NS (r2>0.85, P<0.05). POD-fungus and POD-lipids showed direct association with infection, necrosis, and hepatic encephalopathy (Beta>1.2, P<0.05). POD-lipid (AUC=0.969 and HR=1.99 [1.02-2.04]) superseded POD-fungus and severity indices for early-mortality prediction. Finally, significant increase in PC (15:0/17:0) level showed highest normalized importance, and ANNs and ML predicted early mortality with >95% accuracy, sensitivity, and specificity. Interestingly, fungal surveillance protein Clec7a was significantly downregulated (>2-fold), leading to a notable increase in fungal infection-mediated choline/phosphatidylcholine and associated enzymes (FC>1.5; Kennedy cycle). This contributed to phosphatidic acid-mediated hyper-inflammation in ALF-NS.

Conclusion: In ALF, the plasma lipidome and mycobiome are dysregulated. Increased circulating phosphatidylcholine could stratify ALF predisposed to early mortality or require emergency liver transplantation.

Keywords: DILI; Fungal infections; Lipidome; Liver failure; Omics; Phosphatidylcholine.

Figure 1.

(A) Design of the study. (B) Relative lipidome abundance associated with 8 lipid groups in the study groups (ALF-S, ALF-NS, Healthy control) (P<0.05). (C) Volcano plot showing differentially expressed lipids in baseline plasma samples from ALF vs. Healthy and NS vs. S groups (FC>1.5, P<0.05, FDR<0.01). Upregulated expression is shown in red, and downregulated expression is shown in green (P<0.05). (D) PLS-DA and heatmap showing clear segregation of the Healthy (green), ALF survivor (red), and ALF non-survivor (dark blue) groups on the basis of lipid species signatures. (E) Debiased sparse partial correlation of lipid groups (red, positive correlation; blue, negative correlation; P<0.05). (F) AUC (1) value for predicting ALF-NS (POD>80%) based on the panel of the five best lipids. ALF, acute liver failure; PLS-DA, partial least squares discriminant analysis; NS, non-survivors; FC, fold change; FDR, false discovery rate; AUC, area under the curve; POD, probability of detection; S, survivors.

Figure 2.

(A) Relative fungal peptide–based taxonomic classification abundance in the study groups at the phylum and genus levels (P<0.05, FDR<0.01). (B) Alpha-diversity indices (Shannon/Simpson indices) and principal coordinate analysis (beta-diversity) in the fungal peptide–based community found in the study groups (P<0.05) at the feature level. (C) Correlations between clinical parameters and different fungal peptide phyla, along with their expression status (red bar=upregulated, blue=downregulated). (D) Cluster of Orthologous Groups function analysis of fungal peptides in the study groups. (E) LDA of fungal peptides in the study groups (P<0.05). FDR, false discovery rate; LDA, linear discriminant analysis.

Figure 3.

(A) Fungal alpha diversity (Shannon diversity index) correlates with the alpha diversity of circulating lipids (R²>0.8, P<0.05). (B) Linear regression of Shannon-Fungus and Shannon-Lipid with the clinical complications of ALF patients. (C) Weighted lipid correlation network analysis heatmap showing module–trait relationships, represented as mean values for each group. The color scale on the right indicates correlations from −1 (green) to 1 (red). (D) Weighted fungal peptide-based-taxonomic-classification correlation network analysis heatmap showing module–trait relationships represented as mean values for each group. The color scale on the right indicates correlations from −1 (green) to 1 (red) and the fungal LCA (F_turquoise and F_grey) correlation with the metabolic pathway (R²>0.5, P<0.050. (E) MMCC plot of lipid and fungal modules. The red rectangle shows a direct correlation between the lipid and fungal modules. (F) Correlation between the lipid-group module specific to ALF-NS and the metabolic pathway (R²>0.5, P<0.05). ALF, acute liver failure; LCA, lowest common ancestors; MMCC, multi-modular-correlation-cluster; NS, non-survivors.

Figure 4.

(A) Correlation between lipid species specific to ALF-NS and the metabolic pathway (R²>0.5, P<0.05). (B) Fungal peptides positively correlated with lipids (blue bar=positive correlation, R²>0.5, P<0.05). (C) Venn diagram shows lipid species and 4 fungal phyla common in ALF-NS. (D) Alluvial plot of fungal phyla with 4 common lipid species specific to ALF-NS. (E) Clinical correlations between fungal modules and lipid modules and the POD of the top fungal peptides and lipids (POD>80%, R²>0.5, P<0.05). (F) KEGG pathway analysis of upregulated lipid species specific to ALF-NS. ALF, acute liver failure; NS, non-survivors; POD, probability of detection; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 5.

(A) Panel of the top 5 indicators of lipid species and top 6 indicators of fungal peptides selected based on RF value, FC>1.5, and P-value<0.05 for calculating POD non-survivors. (B) Plot showing clinical correlations (liver functionality and lipid profile) with POD (r²>0.85, P<0.05). (C) Linear regression comparison of POD-lipid and POD-fungus against clinical complications in ALF patients. (D) Multivariate AUROC analysis against severity indices (MELD, KCH) and POD (lipid and fungus) (P<0.01). (E) Univariate and multivariate analyses of clinical parameters, top lipid species and fungal peptides, and POD (lipid and fungus) were conducted. POD-lipid had the best results, with a hazard ratio of 1.99. (F) Assessment of 30-day survival based on POD-lipid (25% cutoff) in ALF patients. RF, random forest; FC, fold change; POD, probability of detection; MELD, model for end-stage liver disease.

Figure 6.

(A) Schematic representation of the validation study conducted using two methods, HRMS-LC/MS and machine learning, on validation cohort 1 (plasma) and validation cohort 2 (disease control, SAH. (B) Quantitative assessment of the top 5 lipid species in validation cohort 1 indicates significant upregulation in ALF-NS (P<0.05). (C) Quantitative assessment of the top 5 lipid species in validationcohort 2, comparing the baseline plasma of SAH patients (n=200) with that of ALF patients (P<0.05). (D) Accuracy and kappa values of the different ML models tested for indicator identification in the study groups. (E) The accuracy, specificity, sensitivity, and p-values of five different lipid species, individually and together, in the validation cohort, along with a confusion matrix for the different ML models. (F) Normalized importance of the top 5 lipid species and clinical parameters according to an artificial neural network model. ALF, acute liver failure; ML, machine learning; NS, non-survivors; SAH, severe alcohol-associated hepatitis.

Figure 7.

(A) The CLR protein Clec7a is downregulated in the ALF-NS group compared with the ALF-S group (P<0.05). (B) Bar graphs show that the levels of phosphatidic acid and phosphatidylcholine are significantly increased in the ALF-NS group (P<0.05). (C) Graphical abstract shows that fungal infection contributes to hyperinflammation by increasing the level of plasma phosphatidylcholine. ALF, acute liver failure; NS, non-survivors; S, survivors.