Elevated blood lactate in resting conditions correlate with post-exertional malaise severity in patients with Myalgic encephalomyelitis/Chronic fatigue syndrome

Elevated blood lactate after moderate exercise was reported in some of patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We hypothesised that blood lactate could be also elevated in resting conditions. We aimed investigating the frequency of elevated lactate at rest in ME/CFS patients, and comparing characteristics of ME/CFS patients with and without elevated lactate. Patients fulfilling international consensus criteria for ME/CFS who attended the internal medicine department of University hospital Angers-France between October 2011 and December 2017 were included retrospectively. All patients were systematically hospitalised for an aetiological workup and overall assessment. We reviewed their medical records for data related to the assessment: clinical characteristics, comorbidities, fatigue features, post-exertional malaise (PEM) severity, and results of 8 lactate measurements at rest. Patients having ≥1 lactate measurement ≥2 mmol/L defined elevated lactate group. The study included 123 patients. Elevated (n = 55; 44.7%) and normal (n = 68; 55.3%) lactate groups were comparable except for PEM, which was more severe in the elevated lactate group after adjusting for age at disease onset, sex, and comorbidities (OR 2.47, 95% CI: 1.10–5.55). ME/CFS patients with elevated blood lactate at rest may be at higher risk for more severe PEM. This finding may be of interest in ME/CFS management.


Results
Among the 168 patients who were fulfilling inclusion criteria, we excluded 45 patients in whom 24 did not undergo lactate assessment, and 21 patients in whom results of lactate assay were incomplete, mainly due to blood sample haemolysis. Hence a total of 123 patients constituted the study population (Fig. 1). Table 1 presents the characteristics of the study population. The PEM assessment showed that the median PEM score on the CDC SI questionnaire was equal to 12 [7.5-12]. A more severe PEM (scores ≥ 12) were observed in 62 (50.4%) patients. Elevated blood lactate levels at rest ≥2 mmol/L on one or more measurements were observed in 55 (44.7%) patients, who constituted the elevated lactate group. Data concerning all lactate measurements can be found as Supplementary Table S1. Figure 2 shows that the overall lactate levels in the elevated lactate group were significantly higher (p < 0.0001) than those of the normal lactate group. Lactate values significantly differed between the 2 groups for each time point.
As summarised in Table 2, the comparison between the group of patients with elevated lactate levels and the group of those with normal lactate showed that the only significant difference between both groups was the

Discussion
ME/CFS is characterised by persistent or recurrent fatigue and the occurrence of PEM, which is a key feature of this debilitating illness. Due to the heterogeneous character of ME/CFS and the absence of biological marker, many studies tried to subtype ME/CFS patients on the basis of clinical or biological variables. Mitochondrial dysfunction has been shown in some of ME/CFS patients [14][15][16][17][18][19][20][21][22] . In the current study we identified a group of ME/ CFS patients with elevated blood lactate in resting conditions and displaying more severe PEM. The demographic characteristics, clinical manifestations, and comorbid conditions of our population were comparable to that reported in prior studies. For instance, the age peak in the age range 30-39 (34% of patients) and the median age of disease onset of 34 [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41] years 23 , the higher prevalence of women and the median duration of illness 1 , and the family history of chronic fatigue syndrome (CFS) in one or more first-degree relatives (11% of patients) 24 were in accord with that previously reported. Nevertheless, the percentage of patients who identified at least one ME/CFS precipitating factor (70.7%) and that of those who reported an infectious trigger (47.2%) was somewhat lower than that of prior studies 24,25 .
One or more comorbidities were found in 76 (61.8%) patients, which corresponds to previous reports that found comorbidities prevalence between 50 and 65% 26,27 but higher prevalence was reported in one study 28 .
Blood lactate assay results showed elevated lactate levels in resting conditions in 55 (44.7%) patients. Lane et al. found elevated lactate concentrations in a proportion of CFS patients (31/96; 32%) after short periods of exercise below the predicted anaerobic threshold 19 . They also reported that patients displaying abnormal lactate responses to exercise had relative deficiency of mitochondria type I fibres on muscle biopsies, which may explain elevated lactate responses to exercise 32 , and low intracellular pH during recovery phase when examined by magnetic resonance spectroscopy of muscle 33 . Other teams showed elevated ventricular lactates in CFS patients pointing to metabolic dysfunction in these patients 16,17 . In the current study, almost half of patients had elevated blood lactate concentrations in resting conditions. To the best of our knowledge, our study is the first to show these results. Lactate values were significantly different between the 2 groups and this difference was observed for each time point. Nevertheless, this difference appeared to be more prominent at T2. A hypothesis that could explain, at least in part, the predominant elevation of lactate levels at T2 is that eating after 16-hour fasting would abolish the beneficial effect of fasting on mitochondrial functioning and redox signalling 34 , leading to a sort of rebound effect responsible for mitochondrial dysfunction with increased blood lactate concentration after breakfast. The positive effect of fasting was reported in FM patients 35 , which could be interesting to evaluate its impact in ME/ CFS patients.
When we classified our study population into two groups with or without elevated lactate to compare their phenotypic characteristics, we found that they only differ in the number of patients experiencing more severe PEM, which was significantly higher in patients with elevated lactate. This association persisted after adjusting for age at disease onset, sex, and comorbidities (OR 2.47, 95% CI: 1.10-5.55; p = 0.03). PEM severity was assessed in all patients by the standardised CDC SI self-reported questionnaire 36 , which includes an item designed to measure the presence, frequency, and intensity of PEM. The CDC SI is one of two tools proposed by the IOM 2 for PEM assessment. We used the median PEM score on the CDC SI questionnaire to set the cut-off score ≥12 defining more severe PEM in our study. www.nature.com/scientificreports www.nature.com/scientificreports/ Elevated blood lactate in resting conditions in ME/CFS patients was not reported before. In our study, patients did not display a state of extreme fatigue at the time of lactate assay, and strict resting conditions were followed carefully in all patients all over the period of different samples' collection during hospitalisation. As well, all patients received standardised meals. Thus, the rise of lactate levels in a proportion of patients could not be related to exertion or due to variation in caloric intake.
It is interesting to note that although the whole study population was suffering from high levels of fatigue and fatigue-related impairment, results of fatigue scales showed that fatigue severity was comparable between both groups; FS median score (elevated lactate group 24 [21][22][23][24][25][26][27][28] vs. normal lactate group 23 [17][18][19][20][21][22][23][24][25][26][27]; p = 0.14) and FSS median score = (elevated lactate group 5.6 [4.8-6.2] vs. normal lactate group 5.4 [5.1-6]; p = 0.90). In this way, elevated lactate levels were correlated with PEM severity, but not with fatigue severity. One can expect that patients who display severe PEM are suffering from high level of general fatigue, however some authors report www.nature.com/scientificreports www.nature.com/scientificreports/ that some of patients who experienced severe PEM did not report high fatigue levels because they were reducing their activities to stay within their energy envelope and so their fatigue levels may be low but they can experience exhaustion if they exceed their functional capacities or exposed to a PEM trigger 37,38 . Another possible explanation is that there is currently no gold standard measurement of fatigue, and a ceiling effect for FSS and FS was reported 39 so these scales would not accurately reflect fatigue severity.

Study's limitations.
Although data of our study were collected retrospectively, all patients were examined and diagnosed by the same physician, and all patients underwent a same standardised procedure in terms of clinical assessment including PEM, and laboratory investigations, especially lactate assay.
One source of weakness in this study was the lack of data concerning fatigue assessment as well its impact on function in some of our patients. However, these data were not statistically different in the 2 groups. As well, data concerning mode of onset of the disease were unavailable for a great number of patients and thus unexploitable. Finally, the MFIS scale that we used to measure the impact of the fatigue on function has been mainly validated in multiple sclerosis patients, but not in those with ME/CFS. Nevertheless, we have used this scale alongside two other scales, FS and FSS, which explore fatigue severity as recommended by the CDC-NINDS project 40 .
At the best of our knowledge, this is the first study that reports elevated blood lactate in resting conditions in a significant proportion of patients with ME/CFS. Patients who showed abnormal elevation of blood lactate at rest displayed more frequent severe PEM than those with normal lactate concentrations. This finding brings supplementary evidence for mitochondrial dysfunction in ME/CFS patients, and may contribute to a better understanding the illness. Subtyping ME/CFS patients adds to the growing body of evidence that ME/CFS is heterogeneous, and allows identifying patients with more risk for severe PEM who must adhere more closely to pacing strategies in order to avoid PEM occurrence and prevent disease exacerbation. Furthermore, our study allowed describing clinical and biological characteristics of a French population with MPE/CFS. Shedding light on these characteristics may improve knowledge and raise awareness of this public health issue.

ethics.
The study was approved by ethics committee of the University hospital of Angers (2018/44) and was conducted in compliance with the Helsinki agreement. Data collection was approved by the French institutional authority for data protection (CNIL).

Study population and data collection.
We reviewed all medical records of patients attending the outpatient clinic of the internal medicine department of Angers University Hospital and diagnosed as having ME/ CFS between October 1, 2011 and December 31, 2017. The diagnosis of ME/CFS was established by the same physician after a systematic 3-day hospitalisation during which an aetiological workup and an overall assessment of fatigue features and PEM severity were realised for each patient. We enrolled retrospectively all adult patients aged ≥18 years who met the International Consensus Criteria (ICC) 2011 4 . In accordance with these criteria, patients with an identifiable medical condition that could account for chronic fatigue, and those with primary psychiatric disorders or substance dependence were excluded. Patients with diseases or drugs susceptible to be associated with elevated lactate levels were also excluded. According to ICC 2011, comorbidities such as FM, irritable bowel syndrome, Hashimoto's thyroiditis, and reactive depression did not constitute an exclusionary condition.
Epidemiological characteristics, fatigue features, and comorbidities were extracted from patients' medical records as well as reported patients' symptoms including the frequency and intensity of PEM.
The following biological data were also gathered for each patient: lactate assessment, immunological assay, infectious disease screen for HIV, hepatitis B and C, Lyme disease, enterovirus, Mycoplasma pneumoniae, Chlamydia pneumoniae, serum zinc, 25-hydroxyvitamin D, morning (8 am) and evening (8 pm) plasma cortisol, testosterone, iron studies, plasma vitamin B12 and serum folate, full blood count and differential, erythrocyte   The intensity score was converted into equidistant score (0 = symptom not reported, 1 = mild, 2.5 = moderate, 4 = severe). The frequency and intensity scores were then multiplied to create the PEM severity score ranging from 0-16. In the absence of validated threshold that defines PEM severity, we used the median PEM score on the CDC SI questionnaire to set the cut-off score ≥12 defining more severe PEM.
Lactate assay. The following procedure was applied for all patients included in the study: each patient was admitted at 3 pm (day 1). A peripheral venous catheter was inserted as soon possible after admission and patient was asked to follow resting and fasting conditions (drinking water was allowed till midnight) until next day (day 2). At day 2, eight blood samples were taken from each patient without tourniquet in resting conditions for at least 30 minutes and without preceding exercise 44   patients' grouping. According to blood lactate concentrations, we stratified patients into two groups; the first one included patients in whom ≥1 lactate measurements were ≥2 mmol/L, and the other one comported patients with normal lactate concentrations.
Statistical analysis. Categorical data were expressed as absolute number and percentage. Continuous data were expressed as median and interquartile (IQ). Data were compared using the Student t test for continuous variables and the χ² test or Fisher's test for categorical data. Lactate analyses were compared using 2 way-ANOVA and lactate values were presented as means with 95% confidence interval in Fig. 2. Post-hoc analyses for each lactate time point were realised after 2-way ANOVA with Bonferroni corrected t-tests. The multivariate analysis for testing the association of comorbidities and the group of elevated lactates were carried out using binomial logistic regression. The continuous variable Age was transformed into a categorical variable with a cut-off equal to the median of the whole population (< or ≥34 years). Odds-ratio (OR) were calculated with their 95% confidence interval (CI). A p value of < 0.05 was considered significant. The analyses were performed using the Graphpad Prism v6.01 software (GraphPad Software, Inc., La Jolla, CA 92037 USA).

Data availability
Data that support the findings of this study are available from the corresponding author upon reasonable request.