Absence of fumaric acid as a fecal biomarker for non-alcoholic fatty liver disease

Abstract

The global rise in the prevalence of non-alcoholic fatty liver disease (NAFLD) poses a huge health burden worldwide. Metabolomics-based studies are increasingly emerging as a new direction for understanding disease pathogenesis and in discovering non-invasive biomarkers with pharmacotherapeutic potential. Methods: Fecal microbiota transplantation (FMT) was performed individually in a total of 24 male C57BL/6J mice (n=8 each in 3 groups) using fecal slurry from same number of healthy human donors, and donors with lean NAFLD (BMI <25 kg/m2) and obese NAFLD (BMI ≥30 kg/m2), with liver steatosis quantified with controlled attenuation parameter measurements by vibration-controlled transient elastography. Targeted metabolites such as short-chain fatty acids (SCFAs), amino acids and tricarboxylic acid (TCA) cycle-related metabolites were measured in feces from post-FMT mice using gas chromatography 7890B and 7010 triple quadrupole mass spectrometer. Results: FMT-Healthy mice had a significantly lower intrahepatic triglyceride level than FMT-Obese mice (48.80±4.46 mg/g vs. 68.51±4.18 mg/g, p=0.0061). Principal coordinates analysis of identified fecal metabolites demonstrated that the metabolomic profile in FMT-Healthy mice differed significantly from that in FMT-Obese mice (p=0.003). In terms of amino acids, FMT-Healthy mice had significantly higher concentrations of fecal alanine, valine, isoleucine, threonine, methionine, phenylalanine, lysine, and tyrosine than FMT-Obese mice (all p<0.05). For TCA cycle-related metabolites, FMT-Healthy mice, when compared to FMT-Obese mice, had significantly higher concentrations of fecal pyruvic acid (122.49±16.76 nmol/g vs. 78.00±6.21 nmol/g, p=0.0207) and fumaric acid (5.88±2.95 nmol/g vs 0.00±0.00 nmol/g, p=0.0002), with fumaric acid levels being completely undetectable (instrument detection limit ≤0.5fg) in all FMT-Lean and FMT-Obese mice. No significant differences were noted between FMT-Lean and FMT-Obese mice. Certain fecal SCFAs such as formic acid, acetic acid, isobutyric acid, valeric acid, caproic acid and total SCFAs levels were significantly higher in FMT-Healthy mice when compared to FMT-Obese mice (all p<0.05). Conclusion: Unique metabolomic signatures were noted in mice colonized with microbiota from human NAFLD patients. With its complete undetectability in FMT-lean and FMT-obese mice, fecal fumaric acid is considered as a strong potential biomarker associated to both lean and obese NAFLD.