In the present study, we performed metabolomic analysis of the plasma in order to obtain a comprehensive view of changes in several metabolic pathways in patients with NAFLD, in order to identify disease related patterns and to identify biochemical perturbations. The data revealed significant changes in certain key pathways, specifically bile acids, glutathione metabolism, lipid and amino acid metabolism. The changes in the plasma metabolome were more evident in subjects with NASH than in individuals with hepatic steatosis. It should be underscored that the plasma metabolome, during the steady state, represents the sum of changes in several tissues and organs and may not reflect any specific organ system. This is particularly true in the case of NAFLD where, as a result of insulin resistance and changes in hormones and cytokines, a number of organ systems, i.e. adipose tissue and skeletal muscle, in addition to the liver, may be affected (1
The subjects in the present study were carefully selected in order to minimize confounding variables, and the plasma samples were obtained at rest following an overnight fast. Only biopsy-proven subjects with steatosis and steatohepatitis were included. We excluded subjects with “indeterminate” diagnosis. All subjects had normal plasma levels of B12 and folate. Other causes of liver disease were excluded by appropriate laboratory investigations.
The concentrations of glycocholate, taurocholate and glycochenodeoxycholate were markedly higher in subjects with NASH. Taurocholate and glycochenodeoxycholate were also significantly higher in subjects with steatosis when compared with controls. Direct measurement of bile acids confirmed these findings (unpublished data). To our knowledge, the higher concentration of bile salts in NASH and steatosis subjects has not been reported previously, and the precise mechanism that is responsible for this increase in concentration remains speculative. It could be the consequence of either a higher bile acid pool due to a higher rate of bile acid synthesis, result from increased peroxisomal and microsomal metabolism, could be caused by hepatocellular injury, or possibly be an adaptive response to the accumulation of triglycerides in the liver. A higher concentration of bile acids has been previously reported in subjects with hyperlipidemia (12
). The healthy liver is very efficient at capturing and removing bile acids from hepatic-portal circulation. When liver function is compromised, more bile acids appear in the circulation because the liver is not adequately removing them. Thus, the concentration of bile acids in the serum has been suggested as an indicator of early liver dysfunction, and is considered more sensitive than most traditional assays (13
). It is plausible that the accumulation of triglycerides in the liver or increased fatty acid oxidation compromises liver function, resulting in its inefficiency in bile acid uptake from the circulation.
The higher plasma levels of bile acids could also be related to the higher insulin resistance in subjects with NAFLD. The interaction between insulin, hepatic insulin receptors and bile acids is complex (14
). Experimentally induced diabetes in animal models has been shown to result in higher plasma bile acids which decreased following insulin therapy (15
). Insulin is a suppressor of sterol 12α hydroxylase (CYP8B) in the liver, the key enzyme for the synthesis of cholic acid (17
). Liver specific disruption of insulin receptors, and therefore hepatic insulin resistance, in mice, however, resulted in lower synthesis of bile acid and decreased expression of the bile acid synthetic enzyme CYP7b1 (18
). The data in the transgenic mice cannot be easily reconciled with the other studies cited above (15
Irrespective of the mechanism of the increase, a higher concentration of bile acids in individuals with NAFLD could modulate lipid homeostasis through activation of the farnesoid X receptor (21
), resulting in changes in VLDL metabolism and hepatic beta oxidation. Only future studies will delineate the role of bile acids in the alteration in hepatic metabolism observed in subjects with NAFLD.
Metabolomic analysis revealed significantly higher concentrations of γ-glutamyl peptides in subjects with both steatosis and steatohepatitis (), and lower concentrations of cysteine-glutathione disulfide. Targeted analysis showed that the plasma concentration of total glutathione was significantly lower in individuals with NAFLD (). Glutathione is the major antioxidant in the liver, which is also the primary source of plasma glutathione. A linear correlation between plasma and hepatic glutathione concentration has been reported in liver disease (24
), and a lower hepatic glutathione concentration has been reported in non-alcoholic steatosis (25
). Following its extrusion from hepatocytes, glutathione is degraded by glutamyl transpeptidase to form cystenyl glycine and glutamyl-amino acid (26
). The lower plasma concentration of glutathione and a higher concentration of glutamyl peptides suggest a high rate of hepatic glutathione turnover in subjects with NAFLD as a result of oxidant stress. The later may be the consequence of increased influx of fatty acid and higher rate of beta oxidation in the livers of individuals with NAFLD (27
). The higher plasma glutamate and cysteine concentration in subjects with NAFLD as compared with controls may also be the consequence of higher rate of turnover of glutathione, similar to that seen in insulin deficient state (30
). Studies using tracer isotopic measurements of glutathione kinetics will be required to confirm these observations.
The plasma concentrations of total carnitines were significantly higher in subjects with both steatosis and NASH. Amongst the 14 species of carnitines measured, short chain acylcarnitines (C3, C4 and C5) were significantly higher in individuals with NASH. Only butyrylcarnitine (C5) was significantly elevated in steatosis. Interestingly, we did not find any significant change in most of the fatty acids measured except eicosapentanoate (EPA; 20:5n3), docosahexanoate (DHA; 22:6n3), and 10-undecenoate (11:1n1), which were lower in subjects with NASH. These changes in plasma long chain fatty acids are similar to those described by targeted lipidomic analysis of hepatic tissue in subjects with NAFLD (31
). These observed changes may be caused by altered metabolism in the liver, the skeletal muscle and the contribution of gut microbes. In particular, changes in branched chain amino acid metabolism in the skeletal muscle may have resulted in higher levels of short chain acylcarnitines (32
). Further studies will be required to determine if increase dietary intake of these specific poly-unsaturated fatty acids can improve the outcome for individuals with NASH.
Several essential amino acids, leucine, isoleucine, valine, and phenylalanine, were elevated in subjects with NASH and not steatosis. The increase in essential amino acids suggests a higher rate of whole body protein turnover. In addition, the lack of increase in essential amino acids in individuals with steatosis suggests that changes in protein turnover may be a late event in the progression of steatosis to NASH, and may be modulated by other factors such as cytokines and inflammation, in addition to insulin resistance (33
Changes in non-essential amino acids, aspartate and glutamate, may be due to increased anaplerosis of amino acids into the TCA cycle, resulting in an increased cataplerosis to insure the required removal of the resulting carbon skeletons of these amino acids from the cycle.. The higher levels of glutamate in the plasma of subjects with NASH, in addition to higher glutathione turnover, could also be due to increased transamination of amino acids being degraded in the liver and skeletal muscle.
Steatosis vs. steatohepatitis
Plasma concentration of only a few biochemicals were significantly different between subjects with steatosis and steatohepatitis. The physiological significance of these changes is uncertain. If one considers steatosis and steatohepatitis as part of a continuum, the lack of any significant difference between the two groups in the metabolomic profile is not surprising.
Random Forest and Principal Component analysis
To assess the ability to classify subjects as healthy, with steatosis or with NASH, random forest analysis was performed using the entirety of the metabolomic data. An excellent separation of the healthy subjects and NAFLD subjects was achieved. However, the steatosis and NASH subjects were not readily distinguishable (). This is consistent with the result from Welch’s t test. Many metabolites were deemed to be statistical significance when either the steatosis group or the NASH group was compared to the healthy control group. Only a few metabolites were significantly different between the steatosis and the NASH groups. It is worth noting that the number of subjects in the steatosis group was rather limited (n = 11), and the statistical significance is impacted by the group size. In future studies, we are planning to increase the groups size to assess if significant differences in plasma metabolic profiles between steatosis and NASH can be detected. As shown in , a panel of markers which provided the most contribution to the separation of the healthy group and NASH group was discovered. Not surprisingly, these markers matched with the metabolites identified by the Welch’s t tests and described in this manuscript: glutathione metabolites and bile acids, amino acids, etc. These markers can be potentially used as diagnostic markers for NAFLD and for the assessment of therapeutic intervention in patients with NASH.