Serum concentrations of the 20 standard amino acids were assessed in 200 ME/CFS patients included in clinical trials led by Haukeland University Hospit al and 102 healthy controls. The initial analyses showed a distinct pattern when comparing amino acids with different entry points for oxidation via the mitochondrial pyruvate-TCA cycle axis (Figure 1). Therefore, amino acids were assigned to one of three categories, in agreement with the established paradigm for the major routes of amino acid catabolism (26):

Figure 1 Hypothetic mechanism of ME/CFS linked to amino acid catabolism. According to this model, ME/CFS is caused by immune interference with an unidentified target, potentially a signaling factor, which ultimately causes metabolic dysfunction and induction of secondary rescue mechanisms. We hypothesize that aberrant PDK and SIRT4-mediated inhibition of PDH, and consequent obstruction(s) in central energy metabolism, occurs early during ME/CFS pathogenesis, followed by metabolic adaptations serving to maintain ATP production. The result will be increased consumption of amino acids not depending on PDH to fuel oxidative metabolism via the TCA cycle. Such a mechanism would expectedly change the serum amino acid profile in patients, depending on the different entry stages of the amino acids into the catabolic pathway. Accordingly, for the purpose of serum amino acid profiling, the 20 standard amino acids were assigned into three categories: category I amino acids that may convert to pyruvate (i.e., dependent on PDH; Gly, Ser, Thr, Cys, Ala); category II amino acids that may metabolize to acetyl-CoA and fuel the TCA cycle (i.e., independent of PDH; Lys, Leu, Ile, Phe, Tyr, Trp); and category III amino acids that are anaplerotic and serve to replenish TCA cycle intermediates (i.e., independent of PDH; His, Pro, Met, Val, Glu + Gln = Glx, Asp + Asn = Asx). The asterisk indicates amino acids that were significantly reduced in serum of ME/CFS patients compared with healthy controls in this study (see Table 1 and 2).

Category I amino acids are converted to pyruvate, and therefore depend on PDH to be further oxidized. These are alanine (Ala), cysteine (Cys), glycine (Gly), serine (Ser), and threonine (Thr).

Category II amino acids enter the oxidation pathway as acetyl-CoA, which directly and independently of PDH fuels the TCA cycle for degradation to CO2. These are isoleucine (Ile), leucine (Leu), lysine (Lys), Phe, tryptophan (Trp), and tyrosine (Tyr).

Category III consists of anaplerotic amino acids that are converted to TCA cycle intermediates, thereby replenishing and supporting the metabolic capacity of the cycle. In this category, we included only amino acids that do not simultaneously belong to categories I or II. Arginine (Arg) was excluded from these analyses for reasons described in Methods. This category consisted of methionine (Met) and valine (Val), which are converted to succinyl-CoA; histidine (His), Gln, Glu (Gln + Glu = Glx), and proline (Pro), which are converted to α-ketoglutarate; and asparagine (Asn) and aspartate (Asp), (Asn + Asp = Asx), which are converted to fumarate or oxaloacetate.

This classification represents a simplified model. Several amino acids may have alternative degradation pathways with varying importance depending on the physiological context. The amino acids in categories I and III are solely glucogenic, whereas the amino acids in category II are ketogenic or both ketogenic and glucogenic. The serum concentration of Ala is highly influenced by its role in the glucose-Ala cycle, which serves to traffic amino groups from peripheral tissues to the liver via blood and was therefore analyzed separately.

Amino acids providing pyruvate as substrate for PDH (category I). There were no significant differences in mean serum levels of category I amino acids (Cys, Gly, Ser, Thr) between nonfasting ME/CFS patients and nonfasting healthy controls, except for Ala, for which there was a small but statistically significant reduction in patients (P = 0.027, Cohen’s d = 0.28) (Table 1). In stratified analyses, there were no differences for Gly and Cys in female ME/CFS patients compared with female healthy controls, but there were slight reductions in the mean concentrations of Ser and Thr (P = 0.010, Cohen’s d = 0.39, and P = 0.010, Cohen’s d = 0.40, respectively) (Table 1). In men, there were no differences in these amino acids between ME/CFS patients and healthy controls (Table 1). The sum of serum concentrations of these 4 category I amino acids (Ala excluded) is shown, with no differences in mean serum levels between ME/CFS patients and healthy controls (Figure 2A), nor in stratified analyses by sex (Figure 2B).

Figure 2 Serum amino acids in 153 nonfasting ME/CFS patients and 102 nonfasting healthy controls. (A) Amino acids converted to pyruvate (category I). (B) Amino acids converted to pyruvate (category I) by sex. (C) Amino acids converted to acetyl-CoA (category II). (D) Amino acids converted to acetyl-CoA (category II) by sex. (E) Anaplerotic amino acids that may replenish TCA intermediates (category III). (F) Anaplerotic amino acids (category III) by sex. (G) Serum levels of 1-MHis (marker of dietary protein intake). (H) Serum levels of the 1-MHis by sex. (I) Serum levels of 3-MHis (marker of endogenous protein catabolism). (J) Serum levels of 3-MHis by sex. P values from unpaired t tests (equal variances not assumed) or from ANOVA when comparing ME/CFS patients versus healthy controls by sex. For 1-MHis, Mann-Whitney test for independent samples or Kruskal-Wallis test was used. Effect sizes estimated from Cohen’s d tests. Error bars indicate mean and SD (median and interquartile range [IQR] for 1-MHis). ME, ME/CFS patients; HC, healthy controls.

Table 1 Serum levels of amino acids catabolized to pyruvate (category I), and to acetyl-CoA (category II), in nonfasting ME/CFS patients and healthy controls by sex

Amino acids fueling acetyl-CoA into TCA (category II). All the 6 amino acids in category II were significantly reduced in nonfasting ME/CFS patients compared with nonfasting healthy controls, with P values of less than 0.001 for Ile, Leu, Phe, and Tyr and P values of 0.001 and 0.009 for Lys and Trp, respectively (Table 1). The reductions in mean serum levels for all 6 amino acids were highly significant in women with ME/CFS compared with healthy women, with moderate effect sizes according to Cohen’s d, varying from 0.35 to 0.75. Comparing male ME/CFS patients to male healthy controls, there were no significant differences in category II amino acids, with a trend for reduction in mean serum level of Tyr in patients (P = 0.086, Cohen’s d 0.44) (Table 1). The sum of serum concentrations for the 6 category II amino acids was significantly lower in ME/CFS patients compared with healthy controls (Figure 2C) as well as in stratified analysis in women but not in men (Figure 2D). These effects were not caused by differences in statistical power due to higher number of women included compared with men (see Statistical analyses in the Methods).

Anaplerotic amino acids (category III). The mean serum levels of anaplerotic (category III) amino acids was significantly reduced in nonfasting ME/CFS patients relative to nonfasting healthy controls, with low to moderate effect sizes (Cohen’s d 0.28–0.60) (Table 2). For reasons explained in the Supplemental Methods (supplemental material available online with this article; doi:10.1172/jci.insight.89376DS1), the concentrations of Gln and Glu (Glx) and of Asp and Asn (Asx) were presented as the sum for the respective pairs. Reductions in anaplerotic amino acids were observed in female ME/CFS patients, but not in men, compared with controls (Table 2). The sum of serum concentrations for the anaplerotic amino acids was reduced for the entire cohort of ME/CFS patients compared with controls (Figure 2E). In separate analyses, there was a significant reduction in the sum of serum concentrations of anaplerotic amino acids in female ME/CFS patients, but not in male patients (Figure 2F).

Table 2 Serum levels of anaplerotic amino acids (category III), metabolites that may affect endothelial function, and markers of protein catabolism in nonfasting ME/CFS patients and healthy controls by sex

Comparison between nonfasting and fasting ME/CFS patients. The comparisons of serum concentrations of all amino acids, between the overnight fasting and nonfasting ME/CFS patients, by sex are shown in Supplemental Tables 1 and 2. Serum concentrations were lower in overnight fasting patients for category II amino acids Ile, Phe, Trp, and Tyr, in addition to Ala. There were trends for lower mean serum levels of Pro, Met, and Arg in serum of overnight fasting patients but no differences for the other amino acids or metabolites.

Other modified amino acids. Based on a study that showed endothelial dysfunction in ME/CFS patients (27), we measured serum concentrations of amino acids and derivatives known to affect endothelial function. Arg, asymmetric dimethylarginine (ADMA), and homoarginine (hArg) showed similar serum levels between ME/CFS patients and controls both in women and men (Table 2). For symmetric dimethylarginine (SDMA), there was a significantly reduced mean serum level in women with ME/CFS, but not in men, compared with healthy controls. 1-methylhistidine (1-MHis), a marker of dietary protein intake from mainly animal and meat sources (28), showed no differences between ME/CFS patients and controls, neither in women nor in men (Table 2 and Figure 2, G and H). Concentrations of 3-methylhistidine (3-MHis), a marker of endogenous protein catabolism (e.g., muscle atrophy) (28, 29), were significantly higher in men with ME/CFS compared with healthy men (P = 0.003 by t test, Cohen’s d = 0.80), while there was no corresponding difference in 3-MHis between female ME/CFS patients and healthy women (Table 2 and Figure 2, I and J).

Amino acids and their relationship to other ME/CFS patient characteristics. Among nonfasting women with ME/CFS, a correlation matrix demonstrated highly significant bivariate correlations for all combinations of the category II amino acids (Ile, Leu, Lys, Phe, Trp, Tyr) (P < 0.001, with Pearson’s r varying from 0.44 to 0.93 (Supplemental Table 3). Among the male ME/CFS patients, similar patterns of correlations were seen (Supplemental Table 3). In contrast, fewer and weaker correlations were found between category I and category II amino acids. However, for Thr there were several marked correlations to category II amino acids, possibly reflecting that Thr has alternative oxidation routes that are linked to the TCA cycle without being converted to pyruvate. Furthermore, there were highly significant correlations for all combinations of category II and category III amino acids but fewer significant correlations between category I and category III amino acids (Supplemental Table 4).

Sex appeared to be an important factor in interpretation of the results, with significant reductions of mean serum levels of category II and III amino acids evident in female ME/CFS patients. To evaluate the possibility that observed changes in amino acid patterns could be caused by other confounding factors, we performed correlation analyses and ANOVA to compare amino acid levels in serum and clinical variables (age, BMI, level of physical activity, ME/CFS severity, and ME/CFS disease duration). Increasing age correlated significantly with increasing levels of Cys, Gly, Ile, Leu, Lys, and Tyr among women with ME/CFS but only with Cys among men with ME/CFS (Supplemental Table 3). There were no significant correlations between serum levels of amino acids and physical activity, assessed as number of steps registered electronically per 24 hours in the ME/CFS group, except for a significant negative correlation with Cys in women (Supplemental Table 3). Among women with ME/CFS, there were significant positive correlations between BMI and Cys, Ile, Leu, Phe, and Tyr and significant negative correlations between BMI and Ser and Thr (Supplemental Table 3). To exclude the possibility that differences in BMI could account for the observed differences in serum amino acid levels in women with ME/CFS, the patients were assigned to four BMI groups, ranging from underweight to obese (Supplemental Tables 5 and 6). When comparing these groups, there were significant associations between higher BMI and higher serum levels of the category II amino acids Leu, Phe, Tyr, and Ile. However, among female ME/CFS patients, in all BMI groups including obese, the mean serum levels of these amino acids were lower than in healthy women (Supplemental Table 5). In men with ME/CFS, no significant associations between BMI groups and serum amino acids belonging to category I or II were seen, except for a significant positive association between Cys with BMI (Supplemental Table 5). The corresponding analyses comparing BMI groups and amino acids from category III and for modified amino acids are shown in Supplemental Table 6.

Further, the patients were also assigned to groups of disease severity, disease duration, physical activity level, and quality of life. Serum levels of category I or category II amino acids were not associated with ME/CFS disease severity (Supplemental Table 7). A significant positive association between ME/CFS disease duration and serum amino acid level was found only for Phe in female patients (Supplemental Table 8). There were no significant associations between mean serum amino acid levels (category I and II) and physical activity, except for a negative association of serum Cys in female ME/CFS patients (Supplemental Table 9). There were significant associations between higher serum ADMA levels and higher physical activity (P = 0.036) and between higher serum level of the marker of dietary protein intake 1-MHis and higher level of physical activity (P = 0.024) in female ME/CFS patients, with a trend in male patients (P = 0.086) (Supplemental Table 10). The associations between quality of life assessed by “SF36mean5” and amino acids (categories I and II) are shown in Supplemental Table 11, with a significant association between a higher “SF36mean5” score and a higher mean serum level of the category I amino acid Ser (P = 0.023) and with trends for Gly (P = 0.056) and Thr (P = 0.065), but only in female ME/CFS patients.

PDH-related gene expression (mRNA) in PBMCs. To investigate whether the observed effects on the serum amino acid profile in ME/CFS patients could be explained by changes in PDH function, we compared mRNA levels of PDH-related genes in PBMCs from nonfasting ME/CFS patients and nonfasting healthy controls (Figure 3). We found significantly increased mRNA expression in ME/CFS patients of the inhibitory kinases PDK1 (P = 0.002), PDK2 (P = 0.022), and PDK4 (P = 0.006), whereas PDK3 was unchanged (Figure 3, A–D). Also, the mitochondrial lipoamidase and PDH inhibitor SIRT4 was significantly upregulated in ME/CFS patients (P = 0.013) (Figure 3K). Among the PPAR transcription factors, PPARδ (PPARD) was upregulated in PBMCs of ME/CFS patients (P = 0.001) (Figure 3F). There were no differences for PPARα (PPARA) (Figure 3E), the PPAR transcriptional target and peroxisomal fatty acid β-oxidation enzyme, acyl-coenzyme A oxidase 1 (ACOX1), or the transcription factor HIF1α (HIF1A) (Figure 3, H and L). Pyruvate dehydrogenase E1α (PDHA) mRNA was slightly upregulated in ME/CFS patients (P = 0.037) (Figure 3G). PPARγ (PPARG) mRNA expression in PBMCs was below the detection limit of the analysis (data not shown). Analyses of the mitochondrial pyruvate carriers (MPCs) revealed a borderline significant upregulation of MPC1 in ME/CFS patients compared with healthy controls (P = 0.046) but no difference for MPC2 (Figure 3, I and J).

Figure 3 Quantitative RT-PCR for mRNA expression levels in peripheral blood mononuclear cells (PBMCs) of ME/CFS patients and healthy controls. mRNA expression levels in PBMCs from 75 nonfasting ME/CFS patients and 43 nonfasting healthy controls, normalized according to coamplified internal β-actin (ACTB) in duplex qRT-PCR and calculated relative to the mean of healthy controls. (A) Pyruvate dehydrogenase kinase 1 (PDK1) mRNA in PBMCs from ME/CFS patients and healthy controls. Similar analyses are shown for (B) PDK2, (C) PDK3, (D) PDK4, (E) PPARAα (PPARA), (F) PPARδ (PPARD), (G) pyruvate dehydrogenase E1, subunit α (PDHA), (H) acyl-coenzyme A oxidase 1 (ACOX1), (I) mitochondrial pyruvate carrier 1 (MPC1), (J) MPC2, (K) sirtuin 4 (SIRT4), (L) HIF-1α (HIF1A), (M) PDK1 mRNA in PBMCs of ME/CFS patients versus sex, (N) PDK1 mRNA in PBMCs versus ME/CFS severity, (O) PDK1 mRNA in PBMCs versus ME/CFS duration, and (P) PDK1 mRNA in PBMCs versus steps (mean) per 24 hours in ME/CFS patients. P values were from Mann-Whitney U test for independent samples (A–N and P) and from Kruskal-Wallis test (O). Error bars indicate median with 95% CI. All samples in a qRT-PCR assay were run in triplicate on the same plate. Of 75 samples from patients and 43 from healthy controls, two samples for PDK1 and four samples for PDK4 were excluded due to unsuccessful amplification. For SIRT4, due to a low expression level, 11 samples from ME/CFS patients and 5 samples from healthy controls were excluded due to high SD (≥30%) among triplicates (see the Methods). Sensewear bracelet data for physical activity for 7 consecutive days were available from 62 of the 75 patients.

While there were sex-specific differences in mean serum levels of amino acids in ME/CFS patients, the increased mRNA levels in PBMCs from ME/CFS patients versus healthy controls were similar in men and women for PDK1 (Figure 3M), PDK4, PPARA, PPARD, and SIRT4 (Supplemental Figure 1, A, E, I, and M).

For PDK1 gene expression, there were significant associations with ME/CFS disease severity (higher PDK1 mRNA level in moderate/severe versus mild/mild-moderate groups), with ME/CFS disease duration (higher PDK1 mRNA level with increasing duration), and with physical activity level assessed as the mean steps per 24 hours (higher PDK1 mRNA level with lower activity) (Figure 3, N–P). The corresponding associations were not found for PDK4 mRNA (Supplemental Figure 1, B–D), for PPARA (Supplemental Figure 1, F–H), for PPARD (Supplemental Figure 1, J–L), or for SIRT4 (Supplemental Figure 1, N–P).

The mRNA expression level of PDK1 correlated highly with PPARD (r = 0.51, P < 0.0001) (Figure 4A), with SIRT4 (r = 0.38, P = 0.003) (Figure 4D), and weakly, but significantly, with PPARA (r = 0.26, P = 0.028) (Figure 4B). There was no significant correlation between PDK1 and PDK4 (r = 0.19, P = 0.12) (Figure 4C). Further, there were highly significant correlations between SIRT4 and PPARD (r = 0.60, P < 0.0001) and SIRT4 and PPARA (r = 0.52, P < 0.0001) but no significant correlation between SIRT4 and PDK4 (r = 0.20, P = 0.15) (Figure 4, E–G). These data were substantiated by a highly significant correlation between PPARA and PPARD mRNA levels (r = 0.75, P < 0.0001) (Figure 4H). For PDK1, PDK4, PPARA, or PPARD mRNA expressions, there were no significant correlations to age or to BMI (Supplemental Figure 1, Q–X). Among the 75 nonfasting ME/CFS patients analyzed for mRNA expression in PBMCs, there were no significant correlations between serum levels of category I or II amino acids and mRNA expression levels in PBMCs for PDKs, SIRT4, PPARA, or PPARD (data not shown).

Figure 4 Correlation analyses between mRNA expression levels in peripheral blood mononuclear cells (PBMCs) of ME/CFS patients. mRNA expression levels in PBMCs from nonfasting ME/CFS patients, normalized according to coamplified internal β-actin (ACTB) in duplex qRT-PCR and calculated relative to the mean of healthy controls. (A) Correlation of pyruvate dehydrogenase kinase 1 (PDK1) and PPARδ (PPARD) mRNA levels in PBMCs of ME/CFS patients. (B) Correlation of PDK1 and PPARα (PPARA). (C) Correlation of PDK1 and PDK4. (D) Correlation of PDK1 and sirtuin 4 (SIRT4). (E) Correlation of PDK4 and SIRT4. (F) Correlation of PPARD and SIRT4. (G) Correlation of PPARA and SIRT4. (H) Correlation of PPARD and PPARA. P values from Spearman correlation analyses. Of 75 samples from patients, two samples for PDK1 and four samples for PDK4 were excluded due to unsuccessful amplification. For SIRT4, due to a low expression level 11 samples from ME/CFS patients were excluded, due to high SD (≥30%) among triplicates (see the Methods). In three samples with SIRT4 mRNA data, PDK and PPAR mRNAs were not analyzed, leaving 61 samples for SIRT4 correlation analyses.

Effects of ME/CFS patient serum on mitochondrial respiration and lactate production in cultured skeletal muscle cells. In order to explain the hallmarks of ME/CFS symptoms, i.e., postexertional malaise and poor recovery, one would expect relevant metabolic defects to be operative in skeletal muscle cells. We were not able to measure energy metabolism directly in muscle tissue of our patients. However, to study the influence of possible blood-borne substances in ME/CFS pathophysiology, we investigated energy metabolism in cultured human skeletal muscle cells (HSMM) exposed to serum from 12 ME/CFS patients (including 3 patients with very severe disease and 6 patients with severe disease) and 12 healthy controls. The study was designed to assess mitochondrial respiration and lactate production by measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively, in presence of amino acids and glucose as substrates. For didactic purposes, the conditions mirroring different energetic states of the cells were defined as conditions I–IV, as explained in Methods.

Basal (resting) amino acid–driven mitochondrial respiration (condition I) was moderately increased in muscle cells exposed to ME/CFS serum for 6 days (Figure 5, A and B), and this effect was also present when glucose was added (condition II). Subsequent addition of the ATP synthase inhibitor oligomycin (condition III) demonstrated that nearly all respiratory activity was linked to ATP production, confirming that the integrity of the oxidative phosphorylation system was intact in cells cultured in the presence of ME/CFS serum. There was, however, a minor increase in the remaining OCR (i.e., leak activity). Next, administration of the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) revealed a significantly increased respiratory capacity in cells exposed to ME/CFS serum (condition IV). The data also indicated that ATP-linked respiration (difference condition II–III) and spare respiratory capacity (difference condition IV–II) were increased after exposure to ME/CFS serum (Figure 5E).

Figure 5 Effects of ME/CFS patient and healthy control serum on muscle cell metabolism. Rates of oxygen consumption and lactate production were measured simultaneously in cultures of human muscle cells (HSMM) after exposure (6 days) to serum from healthy individuals (n = 12) or ME/CFS patients (n = 12). Glucose (GLC), oligomycin (OLIGO), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and rotenone/antimycin A (ROT/AMA) were administered sequentially during the analysis to assess specific properties of cellular energy metabolism. The consequent energetic states of the cells were classified as follows: I Resting (AAs), II Resting (AAs+GLC), III Anaerobic strain, and IV Aerobic strain, as described in the Methods. (A) Recordings of oxygen consumption rate are shown for muscle cells preexposed to healthy control (black) and ME/CFS patient (red) serum. The substance additions and the resultant energetic conditions of the cells are indicated (conditions I–IV). (B) Statistical analysis of the data in A. (C) Recordings of the lactate production rate from the same experiment as A and B. (D) Statistical analysis of the data in C. (E) Specific descriptors of mitochondrial respiration were calculated as indicated, based on the data in A. (F) Specific descriptors of inducible lactate production were calculated as indicated, based on the data in C. The analysis was performed with 5 replicate wells for each serum sample and is representative of 3 separate experiments. Statistical comparisons between healthy controls and ME/CFS samples were performed by Mann-Whitney U test for independent samples.

The basal glycolytic rate (condition II) was similar in cells exposed to ME/CFS and control serum (Figure 5, C and D). However, there was a trend toward reduced glucose-induced rate in the cells cultured with ME/CFS serum (difference condition II–I) (Figure 5F). In contrast, the maximum glycolytic rate in presence of oligomycin tended to be slightly increased in cells exposed to ME/CFS serum (condition III), and this trend was also present after injection of CCCP (Figure 5, C and D). Further analysis of these data revealed that the lactate production caused by oligomycin (difference condition III–II), and by CCCP (difference condition IV–II), were significantly increased in cells exposed to ME/CFS compared with control serum (Figure 5F). Therefore, the cells exposed to ME/CFS serum displayed a metabolic change involving amplified lactate production under conditions of energetic strain.

In summary, serum from ME/CFS patients with severe disease was found to increase rates of mitochondrial oxidative metabolism and respiration in muscle cells, particularly under conditions of energetic strain. Additional experiments with shorter exposure showed that the effect of ME/CFS serum on mitochondrial respiration gradually increased depending on exposure time (data not shown).