Potential Biomarkers of Fatigue Identified by Plasma Metabolome Analysis in Rats

<div><p>In the present study, prior to the establishment of a method for the clinical diagnosis of chronic fatigue in humans, we validated the utility of plasma metabolomic analysis in a rat model of fatigue using capillary electrophoresis-mass spectrometry (CE-MS). In order to obtain a fatigued animal group, rats were placed in a cage filled with water to a height of 2.2 cm for 5 days. A food-restricted group, in which rats were limited to 10 g/d of food (around 50% of the control group), was also assessed. The food-restricted group exhibited weight reduction similar to that of the fatigued group. CE-MS measurements were performed to evaluate the profile of food intake-dependent metabolic changes, as well as the profile in fatigue loading, resulting in the identification of 48 metabolites in plasma. Multivariate analyses using hierarchical clustering and principal component analysis revealed that the plasma metabolome in the fatigued group showed clear differences from those in the control and food-restricted groups. In the fatigued group, we found distinctive changes in metabolites related to branched-chain amino acid metabolism, urea cycle, and proline metabolism. Specifically, the fatigued group exhibited significant increases in valine, leucine, isoleucine, and 2-oxoisopentanoate, and significant decreases in citrulline and hydroxyproline compared with the control and food-restricted groups. Plasma levels of total nitric oxide were increased in the fatigued group, indicating systemic oxidative stress. Further, plasma metabolites involved in the citrate cycle, such as cis-aconitate and isocitrate, were reduced in the fatigued group. The levels of ATP were significantly decreased in the liver and skeletal muscle, indicative of a deterioration in energy metabolism in these organs. Thus, this comprehensive metabolic analysis furthered our understanding of the pathophysiology of fatigue, and identified potential diagnostic biomarkers based on fatigue pathophysiology.</p></div

Random forest analysis of the plasma metabolome.

<p>The mean decrease in accuracy from the random forest analysis was used to rank metabolites according to their prognostic importance for fatigue status. The 15 most important metabolites among three groups (control, food-restricted, and fatigued) or two groups (fatigued and a combined control and food-restricted group) are shown in A and B, respectively.</p

Relative concentrations of plasma metabolites related to the TCA cycle, urea cycle and proline metabolism, and BCAA metabolism.

<p>Relative amounts of each metabolite in the control (C, <i>n</i> = 5), food-restricted (R, <i>n</i> = 5), and fatigued (F, <i>n</i> = 6) groups are shown and expressed as a percent of the control group. Data are presented as mean ± S.D. ^†<i>P <</i> 0.05, significantly different than the control group. ^§<i>P <</i> 0.05, significantly different between the food-restricted and fatigued group.</p

Score and loading plots of principal component analysis of plasma metabolome CE-MS data.

<p>(A) PCA score plot of PC2 versus PC1. The control group (<i>n</i> = 5), food-restricted group (<i>n</i> = 5), and fatigued group (<i>n</i> = 5) are shown as black, green, and red circles, respectively. Black ellipses represent the 90% confidence intervals for each group. (B) PCA loading plot of PC2 versus PC1. The data were analyzed after being mean-centered and variance scaled.</p

Two-way hierarchical clustering heatmap of plasma metabolome data.

<p>Each column shows the metabolic pattern of individual animals in the control group (<i>n</i> = 5), food-restricted group (<i>n</i> = 5), and fatigued group (<i>n</i> = 6). The amount of each metabolite in individual samples is expressed as relative value obtained by the auto-scaling method and is represented by the color scheme in which red and blue indicate high and low concentrations of metabolites, respectively.</p

NOx content in the plasma.

<p>Plasma NOx content in the control (C, <i>n</i> = 5), food-restricted (R, <i>n</i> = 6), and fatigued (F, <i>n</i> = 6) groups are shown. Data are presented as mean ± S.D. ^†<i>P <</i> 0.05, significantly different from the control group. ^§<i>P <</i> 0.05, significantly different between the food-restricted and fatigued group.</p

Plasma metabolite levels in fatigued animals.

<p>Data are means ± SD (n = 4–6 per group). Rats were kept in normal cage (control), had food restriction (10 g/day, food-restriction) in normal cages, or kept in cages filled with water to the height of 2.2 cm (fatigue) for 5 days.</p><p>^†<i>P</i> < 0.05 and FDR < 0.1, significantly different from the control group (ANOVA followed by Tukey’s HSD post hoc test).</p><p>^§<i>P</i> < 0.05 and FDR < 0.1, significantly different between the food-restricted group and the fatigue group (ANOVA followed by Tukey’s HSD post hoc test).</p><p>Plasma metabolite levels in fatigued animals.</p