Patients who visited the CFS/ME Clinic Amsterdam and healthy sedentary controls were invited for the study. All patients fulfilled the criteria of Fukuda et al [1
] for CFS/ME and reported the start of symptoms after an infectious disease. Exclusion criteria were according to Fukuda et al [1
]. Contra indications for the CPET were mainly cardiac diseases, hypertension, or the inability to perform the exercise as in arthrosis of the knee. Medication was discontinued 2 weeks before the first test. All subjects performed a CPET on a cycle ergometer (Excalibur, Lode, Groningen, The Netherlands) according to our protocol: 3 min without activity, 3 min of unloaded pedaling, followed by pedaling against increasing resistance until exhaustion (RAMP protocol) and ended by 3 min pedaling with low resistance. The rate of work rate increase was estimated from history, physical examination, gender, weight and height. The participants performed symptom limited exercise tests as described by Wassermann et al. [13
]. Verbal encouragement to perform maximally was used during the last phase of incremental exercise. Exhaustion of the leg muscles was the limiting symptom in all participants. The V'E, V'O2
, V'CO2 and oxygen saturation were continuously measured (Metasoft). The ECG was continuously recorded and blood pressure was measured every 2 min. The CPET was repeated after 24 h. The Respiratory Exchange Rate (RER) was used for validation of the repeated CPET.
The exercise ECG of the subjects was analyzed (by FCV). The anaerobic threshold was determined by the V-slope method.
The participants completed questionnaires among others (not shown) about additional symptoms of CFS/ME (Centers for Disease Control and Prevention Symptoms Inventory - Dutch Language Version (CDC Symptom Inventory-DLV)) [20
]. The criterion for fatigue was that at least 4 CFS/ME symptoms must be ≥ 7.5 [20
All subjects were seen and an ECG was approved by the internist (RMK).
The results of the tests were not available to the participants or the investigators until after the last test was performed by the participant.
Before entry into the study, the nature of the study was explained to the participants and written consent was obtained. The STEG independent ethics committee approved the study. The trial was conducted in accordance with the Declaration of Helsinki (1996 revision) and under the principals of good clinical practice, as laid out in the International Conference on Harmonization document Good Clinical Practice Consolidated Guideline.
ATP synthesis assay of PBMC
PBMC were isolated from 20 mL of blood obtained before each CEPT and anti-coagulated with 0.18% EDTA as described in detail elsewhere [21
]. For cryostorage in liquid nitrogen, PBMC were suspended at 1 × 107
cells/mL phosphate-buffered saline, pH 7.4, containing 2 mM EDTA, 10% newborn calf serum and 10% dimethyl sulfoxide. To study mitochondrial function, PBMC were thawed and ATP production via reduction of complex I or II was determined exactly as described [22
] except that the cell concentration was decreased to only 5 × 104
cells per mL incubation medium. A small sample was used to determine citrate synthase (CS) activity according to Srere [23
] and protein concentration by the Bio-Rad DC protein assay (Bio-Rad Laboratories) with bovine serum albumine as a standard. The ATP synthesis rate was expressed as nmol ATP synthesized per 30 min per U citrate synthase (CS) or per mg protein.
Plasma creatine kinase (CK) is usually considered a marker of non-specific muscle damage. In the plasma the activity of CK was measured, as a surrogate measure of a lowered oxidative phosphorylation in skeletal muscle. The rationale of this came from early work by Driessen-Kletter et al. [24
]. In a group of seven patients with chronic external ophthalmoplegia a high negative correlation of (R = -0.988; P
= 0.0002) was found between plasma CK 24 h after exercise, and the activity of oxidative phosphorylation via reduction of complex I. Plasma CK was tested by the Clinical Chemistry Laboratory (AKC) of Erasmus MC.
Statistical analyses were conducted using the Statistical Package for the Social Sciences (17.0 for Windows, Chicago, Ill, US). Kolmogorov-Smirnov tests for normality showed that the data were normally distributed. The results were expressed as the mean ± standard deviation (SD). Differences between groups were tested with multivariate or repeated measures Analysis of Variance (ANOVA) where appropriate; correlations were tested with Pearson's correlation test.