Chronic fatigue syndrome (CFS), also known as Myalgic Encephalomyelitis (ME), is a debilitating disease affecting 0.2–0.4% of the population in the UK ( NICE, 2007 ). CFS has a significant impact on the quality of life of patients with key symptoms including severe fatigue and post-exertional malaise ( Hvidberg et al., 2015 ; Winger et al., 2015 ). The mechanisms behind the aetiopathogenesis of CFS are yet to be elucidated. The lack of knowledge of the mechanisms behind the disease contribute to difficulty in obtaining a consensus on diagnostic criteria and the development of widely effective treatments.

The XF e 96 analyser (Agilent Technologies) was used to investigate the activity of individual mitochondrial respiratory chain complexes using specific substrates. The protocol used in this study is described by Salabei, Gibb & Hill (2014) and the mix, wait and measure times provided by Agilent Technologies (0.5 min/0.5 min/2 min) ( Agilent Technologies, 2016 ). Seeding densities for PBMCs and myotubes were used as described previously ( Tomas et al., 2017 ; Rutherford, 2016 ). Myotubes were seeded at a density of 3 × 10 3 cells per well while PBMCs were seeded at 5 × 10 5 per well. On the day of experiments, experimental medium was prepared by supplementing DMEM with 1mM pyruvate, 2 mM L-glutamine and 1 mM glucose. The pH of the media was adjusted to 7.4 with 0.1M NaOH and warmed to 37 °C. One hour before running the experiment, media was removed from each well of the XF e 96 and replaced with 180 µl of prepared medium and incubated for one hour at 37 °C with no CO 2 . Mannitol and Sucrose (MAS) buffer (70 mM sucrose, 220 mM mannitol, 10 mM potassium phosphate monobasic, 5 mM magnesium chloride, 2 mM HEPES, 1 mM EGTA) was prepared. A 4 mg/ml fatty acid free bovine serum albumin (BSA) solution was created by adding BSA to MAS to create MAS-BSA buffer. The medium on the plate was replaced with 180 µl of MAS-BSA 10 min prior to the plate being loaded into the machine. Oxygen consumption rate (OCR) of cells was measured at 12 points throughout the assay. Three basal readings were made before the first injection containing a mix of the substrate(s) of interest, ADP, FCCP and saponin. Three subsequent readings were made and then the second injection, containing oligomycin, was added to the cells. Another three readings of OCR were made and the final injection of either rotenone or potassium azide was added to the cells, and a final three OCR readings recorded. Saponin concentration was optimised independently for myotubes and PBMCs and the damage to mitochondria caused by saponin was also assessed using cytochrome C ( Data S1 ). The optimal concentration of saponin for myotubes was determined to be 25 µg/ml, while the optimum saponin concentration for PBMCs was 2.5 µg/ml. Data were normalised for protein concentration following a bicinchoninic acid (BCA) assay (Fisher Scientific, Loughborough, UK) conducted according to manufacturer’s instructions.

PBMCs were separated using Histopaque® as described by Tomas et al. (2017) . The PBMCs used in these experiments were frozen at −80 °C in freezing medium (50% RPMI-1640, 40% FBS, 10% DMSO) and revived and plated the day before experiments. Wells of a 96-well seahorse plate were coated with poly-D-lysine, to aid in the attachment of cells, and left to air-dry for 2 hours prior to the plating of cells. Following revival of cells, PBMCs were seeded at a density of 5 × 10 5 cells per well in quadruplicate in the poly-D-lysine coated 96-well seahorse plate and incubated overnight in RPMI-1640 (supplemented with 10% FBS and 1% penicillin-streptomycin) at 37 °C and 5% CO 2 .

Succinate was used to analyse complex II mediated respiration and (A) state 3 respiration, (B) state 4 respiration, and (C). RCR were measured. TMPD & ascorbate were added to investigate complex IV activity and (D) state 3 respiration, (E) state 4 respiration, and (F) RCR were measured. Glutamine & malate allowed respiratory activity supported by glutaminolysis to be measured and (G) state 3 respiration, (H) state 4 respiration, and (I) RCR were recorded. Pyruvate & malate were used to investigate complex I mediated respiration and (J) state 3 respiration, (K) state 4 respiration, and (L) RCR were measured. Palmitoyl-l-carnitine was added to assess respiratory activity supported by fatty acid oxidation and (M) state 3 respiration, (N) state 4 respiration, and (O) RCR were recorded. Groups were compared using student’s t -tests. Control n = 6; CFS n = 13.

Discussion

The activity of different aspects of mitochondrial respiratory chain function were investigated by adding various combination of substrates and inhibitors. The effect of the different substrates on state 3 respiration, state 4 respiration and respiratory control ratio (RCR) was measured.

Five different combinations of substrates were investigated for their effects on myotubes and PBMCs—glutamine and malate; palmitoyl-l-carnitine; pyruvate and malate; succinate; TMPD and ascorbate. These were added to investigate respiration supported by glutaminolysis; respiration supported by fatty acid oxidation; complex I activity; complex II activity; and complex IV activity, respectively. Despite there being a lack of difference in OXPHOS between control and CFS cohorts shown in whole myotubes (G Rutherford, pers. obs., 2016) (Rutherford, 2016), these experiments aimed to investigate if more subtle and specific differences occurred in individual complexes. PBMCs were used to see if the specific location of abnormalities identified in whole cells reported previously could be pinpointed to specific complexes or pathways (Tomas et al., 2017).

In myotubes only two of the substrate combinations produced viable results. The addition of pyruvate and malate, palmitoyl-l-carnitine, and glutamine and malate produced negative values for OCR in both CFS (n = 6) and control myotubes (n = 6) (Fig. 3). The addition of the injected compounds did appear to have an effect on the OCR, but not the anticipated effect, and only achieved the result of producing more negative OCR readings with the addition of each injection and not the expected increase after the first injection. These experiments were repeated a number of times with similar traces produced each time. This suggests that this technique for measuring mitochondrial complex activity in permeabilised cells may not be appropriate for use in myotubes. Successful recordings of the effect of succinate, and TMPD and ascorbate were made in control and CFS myotubes. No difference in state 3 respiration, state 4 respiration, or RCR were seen between the control and CFS cohorts. This indicates that there are no abnormalities in respiratory activity linked to glutaminolysis, or complex IV activity in CFS myotubes. This is in agreement with previous research which, using 3 carboxyl-14C–labelled substrates, found there to be no difference between CFS and control skeletal muscle cell complex I, complex II + III, complex III, or complex IV activity (Smits et al., 2011). However, given the inconsistency of between substrate readings in this study, with some of the substrates giving successful readings and some not, and the large error bars shown on the traces for each of the substrates (including the substrates for which we could successfully derive state 3 & 4 respiration), the use of this technique to accurately record the activity of specific aspects of mitochondrial respiration in permeabilised myotubes should be questioned. Other techniques such as phosphorescence oxygen sensitive probes and spectrophotometric enzyme assays should be used to analyse the same samples in future experiments in order to determine if these techniques can provide more accurate results than those achieved here with the XFe96 and to see the consistency between techniques. Very few studies have previously been published using extracellular flux analysis to detect mitochondrial activity in permeabilised myotubes. One study used extracellular flux analysis and high resolution respirometry to determine the differences between the techniques (Boyle et al., 2011). The study showed traces from the Seahorse XFe24 to have higher levels of variability for each data point which supports the data reported here showing that extracellular flux analysis may not be the most suitable technique for detecting changes in mitochondrial complex activity in permeabilised myotubes.

In PBMCs, successful traces were recorded for all five combinations of substrates. State 3 respiration, state 4 respiration, and RCR of control (n = 6, succinate controls n = 4) and CFS (n = 13) PBMCs were compared. No difference was seen between the two cohorts in any of the substrates investigated. This suggests that the activity of complexes I, II and IV, and respiration supported by fatty acid oxidation or glutaminolysis do not differ in CFS PBMCs compared with healthy controls. These results are in agreement with a study by Lawson et al. (2016) who used spectrophotometric techniques to show that permeabilised CFS PBMCs do not differ from healthy controls in terms of complex I, complex II–III, or complex IV activity. The consistency of results between the different research groups, using different techniques, strengthens the evidence suggesting that there are no abnormalities in individual mitochondrial complex activity in CFS PBMCs. The lack of differences in PBMCs may suggest that the abnormalities found in whole cells shown previously is not due to abnormalities in the mitochondrial respiratory chain complexes but rather at different points of the respiration pathway such as movement of glucose into cells, AMPK abnormalities, or altered functioning of other mitochondrial enzymes (Tomas et al., 2017). However, caution must be used in interpreting these results on their own given the differences in OXPHOS observed in whole PBMCs (Tomas et al., 2017), as the results from whole cells reflect a more natural and physiologically relevant environment for the mitochondria. While relatively small sample sizes were used in this study, the consistency of the findings of this study with previous studies using different techniques to measure mitochondrial complex activity in myotubes and PBMCs in CFS patients adds validity to the results (Lawson et al., 2016; Smits et al., 2011).