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Patients were recruited in the Parkstad Clinic in Amsterdam, The Netherlands. They were diagnosed with CFS according to the CBO guideline (45). These are based on the Fukuda criteria (1), with the exclusion criteria of Reeves (3). In the Parkstad Clinic, 250 CFS patients are seen on a regular basis. From these, 150 were randomly selected to receive a letter requesting their voluntary participation. A total of 109 agreed to participate. Three of the participants were not patients of the Parkstad Clinic, making a total of 112 (see Figure 1 for flow scheme). The patients completed a questionnaire on their health, recent non-chronic medication use, smoking habits, supplement use, and pregnancy and lactation. Exclusion criteria were use of medication that may affect thyroid function (e.g., T4, antiarrhythmic drugs, such as amiodarone or corticosteroids), pregnancy, breastfeeding, and menstruation during urine collection. Other exclusion criteria were (biochemical) abnormalities that are excluded according to the CBO guideline and not demonstrated at the time of diagnosis, e.g., severe obesity [body mass index (BMI) > 35 kg/m²], infection [high-sensitive C-reactive protein (hsCRP) > 10 mg/L and white blood cells (WBC) > 10 × 10⁹/L], anemia [hemoglobin (Hb) < 7.0 mmol/L in women and < 8.0 mmol/L in men], hyperthyroidism [TSH below reference range with FT3 and/or free thyroxine (FT4) above reference range (46)], thyroid hormone resistance [elevated FT4 with non-suppressed TSH (47)], hypothyroidism (TSH above upper limit of reference range with FT4 below reference range), and subclinical hypothyroidism [TSH above reference range with normal FT4 (46)]. Weights and lengths were measured on the spot. Data on age were obtained from interviews in the Dutch language.

A total of 119 age- and sex-matched apparently healthy controls were recruited by advertisement in the city of Groningen, The Netherlands. Health was self-reported with the aid of a health checklist filled out before inclusion. Primary exclusion criteria were the use of any chronic medication, menstruation during urine collection, severe obesity (BMI > 35 kg/m²), and both pregnancy and breastfeeding. Incidental use of analgesics and short-term medication (e.g., antibiotics, more than 4 weeks ago) were allowed. Secondary exclusion criteria were infection (hsCRP > 10 mg/L and WBC > 10 × 10⁹/L), anemia (Hb < 7.0 mmol/L in women and < 8.0 mmol/L in men), hyperthyroidism [TSH below reference range with FT3 and/or FT4 above reference range (46)], thyroid hormone resistance [elevated FT4 with non-suppressed TSH (47)], hypothyroidism (TSH above upper limit of reference range with FT4 below reference range), and subclinical hypothyroidism [TSH above reference range with normal FT4 (46)]. Data on age were obtained from interviews in the Dutch language. Weight and height were self-reported.

All patients and controls received a verbal and written explanation of the objectives and procedures and all provided us with written informed consent. The study was in agreement with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008. The protocol was approved by the University Medical Center Groningen (UMCG) Medical Ethical Committee (NL44299.042.13, METc 2013/154, dated August 12, 2013).

The calculation of the sample size (i.e., 100 subjects per group) was based on the correlation coefficient of a comparable population using different steps [for more information, see Ref. (48)]. For this, we used the correlation coefficient found by Girvent et al. (49) for the association of both inflammatory markers CRP and IL-6 with rT3, choosing the highest (i.e., r = 0.75 for rT3 vs. CRP). In this study, subjects with NTIS were compared with patients without euthyroid sick syndrome, both undergoing surgery. Assuming a 95% confidence interval (CI) of (0.59, 0.79), we estimated the sample size using IBM SPSS Statistics (version 20), with the obtained formula, where the n (sample size) appeared inside the Euler number exponent (e). We anticipated 20% exclusion based on abnormal laboratory data, and therefore aimed at the initial inclusion of 120 patients and controls.

We gathered information about supplement intake (vitamin D and fish oil) from 71/98 CFS patients. Users were defined as supplementing themselves either with multivitamins and/or other supplements containing that specific nutrient.

Subsequently, we performed a sensitivity analysis applying stricter exclusion criteria for possible signs of (low-grade) inflammation (“selected groups 1 and 2,” see Results and Figure 1). In the first sensitivity analysis, both CFS patients and controls with BMI > 30 kg/m² and/or hsCRP > 5 mg/L were excluded. In the second one, we also excluded subjects with hsCRP > 3 mg/L and/or with WBC > 10 × 10⁹/L.

Approximately 50 mL of blood were collected by venipuncture in the non-fasting state in three types of tubes (EDTA anticoagulated, lithium–heparin anticoagulated, and serum separator). Samples were processed within 2 h after collection. Twenty-four-hour urine samples were collected and their volumes measured. Samples were stored at −20°C and sent to the participating laboratories [UMCG, laboratory of Special Chemistry and Radiochemistry, Academic Medical Center in Amsterdam (AMC), Medical Laboratories, Reinier de Graaf Groep Diagnostisch Centrum, Delft, and European Laboratory of Nutrients (ELN), Bunnik].

EDTA-whole blood was used for the measurement of routine hematological parameters [Hb, hematocrit, WBC, red blood cells (RBC), and thrombocytes] with a Sysmex XN-9000 Hematology Analyzer (Sysmex Nederland BV, Etten Leur, The Netherlands). The remainder of the EDTA blood was separated into thrombocyte-rich plasma and an RBC pellet by centrifugation for 10 min at 1,800 g. RBC were washed three times with 0.9% NaCl and resuspended to an about 50% hematocrit. After washing, 200 µl of the RBC suspension was transferred to a teflon-sealable “Sovirel” tube containing 2 mL of methanol-6 mol/L HCl (5:1 v/v), 1 mg butylated hydroxytoluene (antioxidant), and 50 µg 17:0 (internal standard). In this ready-to-transmethylate mixture, FAs are stable at room temperature and in the dark for months (50). After centrifugation (10 min, 1,800 g) of the thrombocyte-rich EDTA-plasma, we aliquoted the isolated thrombocyte-poor EDTA plasma and stored it in 2 mL cryovials at −20°C. Lithium–heparin whole blood (1.5 mL) was aliquoted for measurement of elements. The remainders of the lithium–heparin anticoagulated blood and the coagulated blood sample were centrifuged for 10 min at 1,800 g. The resulting plasma and serum were isolated, transferred to 2 mL cryovials, and stored at −20°C until analysis.

Red blood cell–FA compositions were determined by capillary gas chromatography/flame ionization detection in the UMCG, using previously described procedures (50). RBC-FA contents were expressed in g/100 g FA (g%). Tryptophan and kynurenine were measured in EDTA-plasma by LC–electrospray ionization–MS/MS as previously described (51). Serum 25(OH)D2 and 25(OH)D3 [together referred to as 25(OH)D] were determined by isotope dilution-online solid-phase extraction liquid chromatography–tandem mass spectrometry (ID-XLC-MS/MS) in the UMCG (52). Plasma MMA was measured by LC-MS/MS according to Nelson et al. (53). Serum iron, ferritin, hsCRP, total cholesterol (TC), and LDL- and HDL-C were measured using a Roche Modular P module (Roche, Almere, The Netherlands). Vitamin B12, folate, TSH, FT4, and FT3 were assayed by electrochemiluminescent immunoassay on the Roche Modular E170 Analyzer. Serum TT4 and TT3 were measured using an Architect i2000SR (Abbott Diagnostics, Hoofddorp, The Netherlands). Serum antithyroglobulin antibodies and antithyroid peroxidase antibodies were measured with an Immulite 2000 (Siemens, The Netherlands). Plasma rT3 was measured by in-house RIA (54) at the AMC, The Netherlands. Plasma homocysteine was analyzed in the UMCG by competitive protein binding assays with the use of an immunochemistry analyzer (IMX; Abbott Diagnostics, Hoofddorp, The Netherlands).

Whole blood- and lithium–heparin plasma selenium, copper, magnesium and zinc and iodine in urine were measured using ICP-MS 7700x (Agilent, Amstelveen, The Netherlands) in the ELN. Selenium, copper, magnesium and zinc contents in RBC were calculated from their concentrations in plasma and whole blood, using hematocrit values for correction. Plasma zonulin (active form) concentrations were measured using the K5600 ELISA kit (Immundiagnostik AG, Bensheim, Germany). The quantification of 8-iso-prostaglandin F2-isoprostanes in urine was performed by GC-tandem-MS using a two-step derivatization and a selective solid-phase extraction protocol with HLB and Silica columns as described by Zhao et al. (55). The tryptophan/kynurenine ratio was calculated. This ratio may be decreased during inflammation (56, 57).

For the investigation of the pathogenesis of the “low-T3 syndrome,” we measured FT3/FT4, TT3/TT4 and rT3/TT3 ratios. For the investigation of the underlying etiology of the “low-T3 syndrome,” we calculated the following variables of thyroid metabolism: standard TSH index (sTSHi), in order to quantify the thyrotropic function of the pituitary (58); the sum activity of deiodinases [structure parameter inference approach (SPINA)-GD] as a variable for deiodination function (59); the secretory capacity of the thyroid gland (SPINA-GT), as an evaluation of thyroid secretory status (59); and the ratios of TT3/FT3 and TT4/FT4 as evaluations of protein binding of thyroid hormones. The sTSHi was calculated as TSHi = (TSH − 2.70)/0.676 (58). SPINA-GD and -GT were calculated as SPINA-GD = [β₃₁ × (K_M1 + FT4) × TT3]/(α₃₁ × FT4) and SPINA-GT = [β_T × (D_T + TSH) × TT4]/(α_T × TSH). Constants in the equations were as follows: β₃₁ = 8 × 10^–6/s, K_M1 = 5 × 10^–7 mol/L, α₃₁ = 0.026/L, β_T = 1.1 × 10⁻⁶/s, D_T = 2.75 mU/L, and α_T = 0.1/L (59, 60). The rT3/TT3 ratio was also calculated as a proxy for a metabolic shift. For the latter, we also calculated the %TT4, %TT3, and %rT3 by dividing their concentrations by the sum of TT4 + TT3 + rT3 and adjusting to 100%. Zinc/copper, TC/HDL-C and eicosapentaenoic acid (EPA)/arachidonic acid (AA) ratios were also calculated. A proxy for hepatic DNL (DNL liver) was calculated according to Kuipers et al. (61) (sum of RBC 16:0, 16:1ω7, 18:1ω7, 20:1ω7, 18:1ω9, 20:1ω9, and 22:1ω9). The omega-3 index, RBC-EPA + docosahexaenoic acid (DHA) (RBC-EPA + DHA) was calculated.

Statistical analyses were performed with IBM SPSS Statistics 23 SPSS Inc., Chicago, IL, USA. Mann–Whitney U-tests were used for the evaluation of between-group differences in the distribution. The Chi-square tests were used for the evaluation of between-group differences in nominal variables. Odds ratios were calculated to quantify the strength of the presence of low T3 in the different groups. Correlation analyses were performed using Spearman’s Rho for non-parametric variables.

Characteristics of the 98 CFS patients and the 99 controls are shown in Table 1. The CFS patients (21 males, 77 females) had a median age of 43 years (range 21–69), median height of 172 cm (149–198), median weight of 68 kg (48–118), and median BMI of 22 kg/m² (18–34). The age- and-sex-matched healthy controls (23 males, 76 females) had a median age of 39 years (19–65), median height of 173 cm (156–193), median weight of 70 kg (47–100), and a median BMI of 23 kg/m² (18–33). The above anthropometric characteristics exhibited no between-group differences.

The strength of the above findings for the whole group was tested with “sensitivity analyses.” For this goal, we created “selected subgroups 1 and 2” by exclusion of subjects with the most prominent signs of (metabolic) inflammation, defined as relatively high hsCRP and BMI.

The “low T3 syndrome” encountered in a subgroup of CFS patients bears clinical chemical similarity with NTIS features. Both syndromes are biochemically characterized by low TT3 and FT3 together with normal/high-normal FT4 and normal TSH, at least in the mild and moderate forms of NTIS (36). The clinical disparity relates to the underlying severity of the diseases that are usually linked to NTIS, as opposed to the chronicity and less life-threatening nature of CFS (66). NTIS is a typical feature of critically ill patients in intensive care units, although similar changes in the HPT-axis have been observed during calorie restriction and in patients with non-critical chronic diseases, such as heart failure, chronic obstructive pulmonary disease, and diabetes mellitus (67), also referred to as mild, or atypical forms of NTIS (36, 67). All of these conditions, especially calorie restriction, might find a common denominator in an adaptive response aiming at saving energy and body protein in order to outstay any potential acute stress stimulus (68–70). Through coordinated changes in thyroid hormone metabolism, transport and receptors, NTIS might mechanistically reflect a cytokine-orchestrated allostatic condition that is remote from the well-known homeostatic negative feedback regulation of the HPT axis (71).

A recent study identified 12,608 differentially methylated sites in peripheral blood mononuclear cells of 49 female CFS patients vs. 25 healthy female controls. These sites were predominantly involved in metabolism and to a lesser extent in neuronal cell development. Among these sites, 1,600 were related to a score of self-reported quality of life, while 13 sites were associated with glucocorticoid sensitivity in a subgroup of CFS patients with glucocorticoid hypersensitivity (72). In line with downregulated energy expenditure, recent CFS case–control studies of the metabolome revealed abnormalities in several metabolic pathways, including those reflecting mitochondrial metabolism, consistent with a hypometabolic state (73, 74). The lower proxy for DNL encountered in the currently studied CFS patients might fit into this picture, since hypothyroidism in mice has been shown to downregulate the rate limiting enzymes involved in DNL (75). In addition, induced hypothyroidism in humans for two weeks causes profound changes in FA metabolism (76). Another recent case–control study using metabolic profiling showed an altered serum amino acid profile in CFS patients, suggesting impaired mitochondrial pyruvate oxidation (74), a condition likely to reflect energy deficiency and excessive lactate production, with utilization of amino acids from endogenous protein as alternative TCA cycle substrate. The “low T3 syndrome” in a subgroup of CFS patients found in this study might be cause and consequence of the above noted epigenetic changes (72) and a driving force of the metabolic differences noted by others (73, 74) and by us. Through both genomic and non-genomic actions, T3 has profound impacts on mitochondria and metabolism (77), including several pathways regulating the expression of target genes contributing to mitochondrial biogenesis (78).

The association between low T3 and low hsCRP suggests that both CFS patients and controls with low FT3 are less responsive to inflammatory stimuli, which is in line with observations by others. In apparently healthy individuals, Hodkinson et al. (79) found, amongst others, that TT3 concentrations are positively related to monocyte phagocytic activity and expression of -6 (IL-6) by activated monocytes, while TT4 is positively related to CRP. Their data suggest that higher thyroid hormone concentrations within the normal range enhance innate and adaptive immunity by greater responsiveness to immune stimuli. Accordingly, Rozing et al. (80) showed that, although higher circulating levels of inflammatory markers were associated with lower levels of free serum FT3; higher serum FT3, but not higher TSH and FT4, are related to a higher production capacity of proinflammatory cytokines (IL-1β, IL-6, TNF-α) in whole blood of 85-year-old women and men, following ex vivo stimulation with LPS. They suggest a mutual association between T3 and proinflammatory cytokines, whereas T3 stimulates production of proinflammatory cytokines that subsequently diminish the conversion of T4 to T3. Finally, evidence of a diminished specific immune response has been found in patients with CFS. Investigating pokeweed mitogen-stimulated isolated peripheral blood mononuclear cells, Loebel et al. (81) found a deficient EBV-specific immune response in patients with CFS, possibly causing impaired EBV control. Taken together, it is possible that a subgroup of CFS patients with low FT3, but also controls with low T3, present a diminished responsiveness to immunologic stimuli.

Hypothyroidism is, among others, associated with a decrease in both metabolic and heart rates, oxygen consumption, body temperature and oxidation of glucose, FA, and amino acids. It has been estimated that 4–8% of genes are regulated by T3 in human skeletal muscle, rodent liver and a pituitary cell line (78). The encountered “low T3 syndrome” in our study resembles the thyroid hormone profile of a subgroup of hypothyroid patients receiving T4 monotherapy. Substitution with T4 is the currently recommended treatment of hypothyroid patients, like those with Hashimoto thyroiditis. Nevertheless, it is becoming increasingly clear that a subgroup of these patients experiences residual hypothyroid symptoms, including psychological and metabolic traces. These symptoms occur despite reaching a chemical euthyroid state, i.e., normal TSH (82, 83). In thyroidectomized rats, no single dose of T4 was able to simultaneously restore TSH, T4, and T3 in plasma and organs to normal levels (84). In so-called “euthyroid, yet symptomatic” patients, the basal metabolic rate and serum cholesterol, among others, are not fully restored and they are also likely to have both low TT3 and FT3. These findings of low T3 may be explained by a disrupted TSH-T3 shunt (41). The question whether they would benefit more from a T4 and T3 combination therapy or sustained-release T3 (85) is debated and subject of further research (82, 83). Hormone replacement therapy, notably T3, has also been suggested for severe NTIS (71, 86, 87).

In the NHANES cohort, 469 out of 9,981 participants with normal TSH were T4-treated. This subgroup of T4-treated subjects exhibited 10–15% higher TT4 and FT4, but 5–10% lower TT3 and FT3 and a 15–20% lower T3/T4 ratio, as compared to 469 carefully matched healthy controls (88). These apparently moderate differences suggest that the extra-thyroidal conversion of T4 to T3 during T4 monotherapy might be insufficient in some patients to restore serum T3 levels up to those normally maintained by an intact thyroid secreting 80% T4 and 20% T3 (82, 83, 88). A similar shift in the thyroid hormone profile was observed in the present study. However, the encountered deviations from thyroid hormone reference ranges and from controls are modest (Figure 2). It should in this context be noted that many biological effects of T3 depend on its cellular concentrations, which exhibit a complex relationship with the serum T3 concentration (89). A recent study with chemically induced hypothyroidism in rats showed a more severely reduced tissue T3 than serum FT3, averaging 1–6% of the baseline versus 30%, respectively. In addition, the extent of tissue T3 reduction, expressed as percentage of the baseline, was not homogeneous, with more serious reductions occurring in the order: liver = kidney > brain > heart > adipose tissue (90). In other words, the finding of slightly decreased circulating FT3 and perhaps also FT3 levels in the lower reference range may reflect the tip of the iceberg of the genuine T3 deficits in target tissues.

Some features of CFS resemble those of a persistent response to environmental stress known as dauer (hypometabolic state). The cell danger response (CDR) is an evolutionarily conserved metabolic response, activated when a cell encounters a chemical, physical, or microbial threat that could injure or kill the cell (91). When the CDR is abnormally maintained, whole body metabolism and the gut microbiome become disturbed, the functionality of systems and organs becomes impaired and behavior is changed, resulting in chronic disease (91). Accordingly, the intestinal microbiota and virome have recently been implicated in CFS (92), while gene expression data show prominent roles for genes involved in immunity and defense (93). Psychological trauma, particularly during childhood, can also activate the CDR and produce chronic inflammation (91, 94). It has recently been shown that CFS patients are endowed with different psychological vulnerability factors, notably perfectionism and high moral standards (95). These may render them more susceptible to the psychological stress of current society, with possible effects on the immune system and thyroid axis (56, 62, 79, 80). Finally, the aforementioned case–control study by Naviaux et al. (73) showed that CFS patients present cellular metabolic responses similar to the evolutionarily conserved persistent response to environmental stress. Thus, the features of hypometabolism that characterize CFS may be a consequence of a persisting CDR, either or not inflammatory driven.

The current opinion on the causes of CFS may fit mechanistically into the presently encountered “low T3 syndrome.” We observed a shift from T3 toward rT3 in the investigated CFS patients, who exhibited lower T3/T4 ratios and higher rT3/TT3 ratios (Table 1) compared to controls. This shift toward rT3 in CFS patients was also apparent from their higher %rT3 and lower %TT3, when the sum of rT3, TT3 plus TT4 was adjusted to 100% (Table 1). These findings, as well as lower urinary iodine in CFS, may be in line with higher D3 activity. Low T3 levels in human organs have also been found in NTIS (87), but they are more likely to derive from deviant pathways of intracellular deiodination than from a seriously impaired entry of T3 into cells (87). Induction of D3 in muscle may occur in chronic inflammation (34), but D3 may also become induced by other factors, such as estradiol, progesterone, and growth hormone (96). Such mechanisms may be at the basis of CFS symptoms and may explain the lower urinary iodine excretion in CFS patients as compared with controls, although the latter also exhibited a relatively high prevalence of low iodine excretion (Table 1). Intracellular D3-catalyzed liberation of iodide from T4 and T3 may serve various antioxidant and defense functions that may potentially contribute to high intracellular “thyroid hormone consumption,” manifesting as the “low T3 syndrome” with negative iodine balance in the long term (67, 83, 97).

The lower SPINA-GD (step-up deiodinase activity) and SPINA-GT (thyroid secretory capacity) are likely to reflect thyroid allostasis responses, and the lower protein binding of thyroid hormones, as shown by the lower TT3/FT3 and TT4/FT4 ratios, may potentially result in higher metabolism/degradation of thyroid hormones. Thyroid allostasis-altered responses have been found in NTIS associated with cardiac disease (37), radiation enteritis (60) and enterocutaneous fistulas (98). The acute adaptation of thyroid hormone metabolism to critical illness may prove beneficial to the organism, whereas the more complex alterations associated with chronic illness frequently lead to type 1 thyroid allostasis (where energy demands exceed the sum of energy intake and energy mobilized from stores) (41).

Strength of the present case–control study is the performing of two sensitivity analyses to assess the robustness of the association of CFS with thyroid parameters and chronic (low-grade) metabolic inflammation. These analyses resulted in some differences, but the findings in thyroid parameters remained unchanged. We also calculated the %rT3, which may be a more sensitive marker for subtle metabolic shifts than concentrations and ratios per se.

Our study also has limitations. There was a lack of information on the duration of illness and patient characteristics at diagnosis. For instance, dependent on illness duration, different cytokine profiles in CFS patients have been reported (99). CFS is likely a heterogeneous disease with a common final pathophysiological pathway. The present findings are possibly in line with a common final pathway, but do not get us closer to the cause(s).