Characterization of Salivary and Plasma Metabolites as Biomarkers for HCC: A Pilot Study

Abstract

(1) Background: The incidence of hepatocellular carcinoma (HCC) is rising, and current screening methods lack sensitivity. This study aimed to identify distinct and overlapping metabolites in saliva and plasma that are significantly associated with HCC. (2) Methods: Saliva samples were collected from 42 individuals (HCC = 16, cirrhosis = 12, healthy = 14), with plasma samples from 22 (HCC = 14, cirrhosis = 2, healthy = 6). We performed untargeted mass spectrometry on blood and plasma, tested metabolites for associations with HCC or cirrhosis using a logistic regression, and identified enriched pathways with Metaboanalyst. Pearson's correlation was employed to test for correlations between salivary and plasma metabolites. (3) Results: Six salivary metabolites (1-hexadecanol, isooctanol, malonic acid, N-acetyl-valine, octadecanol, and succinic acid) and ten plasma metabolites (glycine, 3-(4-hydroxyphenyl)propionic acid, aconitic acid, isocitric acid, tagatose, cellobiose, fucose, glyceric acid, isocitric acid, isothreonic acid, and phenylacetic acid) were associated with HCC. Malonic acid was correlated between the paired saliva and plasma samples. Pathway analysis highlighted deregulation of the 'The Citric Acid Cycle' in both biospecimens. (4) Conclusions: Our study suggests that salivary and plasma metabolites may serve as independent sources for HCC detection. Despite the lack of correlation between individual metabolites, they converge on 'The Citric Acid Cycle' pathway, implicated in HCC pathogenesis.

Keywords: biomarkers; cancer; hepatocellular carcinoma; liver disease; metabolomics.

Figure 1

Untargeted metabolomics of saliva samples from individuals with HCC, cirrhosis, and healthy livers. (A) Volcano plots depicting the fold change and false discovery rate for metabolites associated with HCC compared to cirrhosis, HCC compared to healthy livers, and HCC compared to non-HCC (cirrhosis and healthy livers). Metabolites significantly associated with each outcome are highlighted in red and FDR p of 0.1 is marked with a red dotted line. (B) Boxplots depicting the relative abundance of each metabolite significantly associated with HCC (FDR p < 0.1) for all pairwise comparisons.

Figure 2

Untargeted metabolomics of plasma samples from individuals with HCC, cirrhosis, and healthy livers. (A) Volcano plots depicting the fold change and false discovery rate for metabolites associated with HCC compared to cirrhosis, HCC compared to healthy livers, and HCC compared to non-HCC (cirrhosis and healthy livers). Ten metabolites with an FDR p < 0.1 (red dotted line) are highlighted. (B) Ten boxplots depicting the relative abundance of the metabolites that are significantly associated with HCC in at least one pairwise comparison.

Figure 3

Comparison of metabolites between saliva and plasma samples. (A) Scatterplot depicting the correlation between each metabolite in saliva and plasma samples. Malonic acid, the only metabolite with an FDR p < 0.1, is highlighted in red. (B) The relative abundance of malonic acid is significantly correlated between saliva and plasma samples. (C) Heatmap depicting FDR p values for pathway analysis performed on metabolites associated with HCC (p < 0.05) in saliva and plasma samples. FDR p values are displayed for significant pathways. (FDR p > 0.1).