Vitamins C and E treatment combined with exercise modulates oxidative stress markers in blood of patients with fibromyalgia: A controlled clinical pilot study

Abstract

We aimed to investigate effects of vitamins C and E (VCE) supplementation with exercise (EX) on antioxidant vitamin and lipid peroxidation (LP) levels in blood of patients with fibromyalgia (FM). A controlled study was performed on blood samples from 32 female FM patients and 30 age-matched controls. The patients were divided into three groups namely EX (n = 10), VCE (n = 11), and EX plus VCE (n = 11) after taking basal blood samples. After 12 weeks of EX and VCE supplementation, blood samples were taken once more from the patients. LP levels in plasma and erythrocytes were higher in the patients at baseline than those in controls, whereas LP levels were lower in the VCE and EX groups at the end of 12 weeks than those at baseline. Plasma concentrations of vitamins A and E and reduced glutathione were lower in the patients than those in controls and their concentrations were increased by VCE and EX. Glutathione peroxidase activity in erythrocytes was increased by VCE supplementation, with or without EX. Concentrations of β-carotene in the groups did not change with treatment. Despite the measured effects on anti-oxidative mechanisms, FM symptoms were not improved by the treatments. In conclusion, VCE with EX may protect against FM-induced oxidative stress by up-regulation of an antioxidant redox system in the plasma and erythrocytes of patients with FM. Such protective effects of VCE in the patients seemed to be greater in combination with EX than EX alone.

Keywords::

• Antioxidant redox system
• exercise
• fibromyalgia
• local hypoxia
• oxidative stress
• vitamin E

Introduction

Erythrocytes and muscle may be vulnerable to oxidative stress induced by fibromyalgia (FM) and become exposed to reactive oxygen species (ROS) such as superoxide radical, hydrogen peroxide, and hydroxyl radical that are continuously generated via the auto-oxidation of hemoglobin (Hb) and polyunsaturated fatty acids (PUFAs) (Nazıroğlu et al. 2004; Çimen 2008). Lipid peroxidation (LP) causes injury to cell and intracellular membranes and may lead to cell damage and subsequently cell death (Akyol et al. 2004; Nazıroğlu 2007a; Kovacic and Somanathan 2008). There are various antioxidant mechanisms in the erythrocytes and muscle which neutralize the harmful effects of ROS; but with hypoxia, the loss of efficiency of the antioxidant mechanisms and alterations in the mitochondrial electron transport system result in increases in free radical formation (Altindag and Celik 2006; Özgöçmen et al. 2006a,b; Nazıroğlu 2007b). Glutathione peroxidase (GSH-Px) catalyzes the reduction of hydrogen peroxide to water. GSH-Px can also remove organic hydroperoxides (Kovacic and Somanathan 2008). GSH is a hydroxyl radical and singlet oxygen scavenger and participates in a wide range of cellular functions (Rayman 2000). Vitamin E (α-tocopherol) is the most important antioxidant in the lipid phase of cells. Vitamin E acts to protect cells against the effects of free radicals, which are potentially damaging byproducts of the body's metabolism (Nazıroğlu 2007a). Vitamin C (ascorbic acid), as well as being a free radical scavenger, also transforms vitamin E to its active form (Frei et al. 1989). Vitamin A, retinol, serves as a prohormone for retinoids and is involved with signal transduction at cytoplasmic and membrane sites (Finaud et al. 2006). Erythrocyte and muscle ascorbic acid and α-tocopherol concentrations are extremely low compared with body tissues such as liver and kidney (Frei et al. 1989; Çimen 2008).

FM is characterized by long-lasting, widespread pain and generalized tenderness, often accompanied by fatigue. The prevalence of FM ranges from 1 to 3% in the general population (Özgöçmen et al. 2006b), and the condition is more common among females than males. The etiology and pathogenesis of FM are not clearly understood and this makes the disease a frustrating condition for both patients and physicians (Mannerkorpi 2005). However, pathophysiology of FM may be related to local hypoxia due to disturbed microcirculation function. According to this theory, vasoconstriction occurs in the skin at tender points in patients with FM, supporting the hypothesis that FM is related to local hypoxia in the skin at upper tender points (Jeschonneck et al. 2000). It is well known that hypoxia may result in both ROS production and decreased antioxidant vitamin concentrations in different inflammatory diseases (Kökçam and Nazıroğlu 2002; Kamanli et al. 2004).

Many patients with FM report limitations in daily activities such as carrying objects, walking, and working with their arms. Patients with FM demonstrate reduced physical performance capacity at levels similar to or below that of sedentary women (Mannerkorpi 2005). Recent reports from Ireland and the UK indicate that physical exercise (EX) is beneficial for patients with FM (Sim and Adams 2002; Gowans and deHueck 2004), although a certain physiological role of physical EX on the antioxidant redox system in patients with FM has not been clarified so far. Effects of vitamins C and E (VCE) supplementation with/without EX on antioxidant redox system have not been studied in patients with FM, although recent studies remarked on the role of the vitamins in FM (Altindag and Celik 2006; Akkuş et al. 2009; Ali et al. 2009).

In the current study, we combined moderate doses of VCE with EX and tested its possible beneficial effect on the antioxidant defense system in patients with FM by evaluating LP and scavenging enzyme activity.

Subjects and methods

Healthy untreated controls and patients with FM

The Ethics Committee of the Medical Faculty of Suleyman Demirel University (SDU) approved the study plan by protocol No 2007-13. All subjects volunteered for the trial and they gave written informed consent. Patients with FM were recruited from outpatient clinics within the Medical Center of SDU. The controlled study was performed on 32 female patients and 30 control subjects. Ages of patients and controls are shown in Table I. Nurses and workers in the Medical Center of SDU formed the healthy untreated control groups in the study. The diagnosis of FM was based on the 1990 criteria of the American College of Rheumatology (Wolfe et al. 1990), specifically widespread pain and the presence of tenderness in at least 11 or more of 18 specific tender point sites. Differential diagnosis was made to exclude any other disease causing widespread pain such as hypothyroidism or rheumatological disorders. Routine blood tests, erythrocyte sedimentation rate, liver and kidney function tests and enzymes, thyroid hormone concentrations, and sex hormone profiles of the patients and controls were evaluated. Patients taking anti-inflammatory and systemic drugs or antioxidant vitamin supplements within the previous 6 months were excluded, as were patients with liver or thyroid diseases. None consumed alcohol and they were nonsmokers.

Table I.  Demographic values of patients with FM, experimental groups, and controls.

	Control (n = 30)	FM (n = 31)	FM+EX (n = 11)	FM+VCE (n = 11)	FM+EX+VCE (n = 10)
Age (years)	39.6 ± 5.8 (26–42)	40.1 ± 5.2 (27–43)	37.8 ± 8.7 (25–43)	40.5 ± 4.9 (28–42)	37.4 ± 4.0 (25–39)
Height (cm)	161.9 ± 4.8 (153–166)	160.9 ± 5.3 (152–166)	159.9 ± 4.2 (151–163)	161.2 ± 4.3 (153–166)	161.2 ± 3.4 (154–162)
BMI (kg/m^2)	23.6 ± 1.8 (20–25)	25.0 ± 6.1 (20–29)	26.6 ± 3.5 (23–29)	27.0 ± 2.8 (24–29)	26.0 ± 3.5 (23–29)

Notes: BMI, body mass index; FM, all fibromyalgia patients at baseline; sub-groups: FM+EX, fibromyalgia+exercise group; FM+VCE, fibromyalgia+VCE supplemented group; FM+EX+VCE, fibromyalgia+VCE + exercise group; n, number of subjects. Values are mean ± SD and (min–max).

Study groups and EX

Compared with vitamins A and D, both acute and chronic studies with human and animals have shown that VCE are relatively nontoxic, but not entirely devoid of undesirable effects. Massive doses of vitamin E (5000 mg/kg of diet) cause a coagulation defect associated with a vitamin K deficiency. Safety and tolerance of ascorbic acid in humans at levels as high as 10 g/day have been demonstrated. Risk of vitamin C toxicity is minimal and unlikely in humans because of limited intestinal absorption capacity and efficient renal elimination (McDowell 1989; Saremi and Arora 2009). Daily protective doses of VCE in adult subjects are 500 and 150 mg for 12 weeks, respectively (Steinberg and Chait 1998; Kayan et al. 2009). Hence, we preferred treatment with moderate oral doses of VCE in the controlled clinical experiment. The patients with FM were orally supplemented with vitamin E (DL-α-tocopheryl acetate; 150 mg/day) and vitamin C (ascorbic acid; 500 mg/day) in combination for 12 weeks.

Subjects in the study were divided into two groups comprising healthy untreated controls (n = 30) and patients (n = 32). The patients were randomly divided into three subgroups as the EX group (FM+EX; n = 10), VCE supplemented group (FM+VCE; n = 11), and the VCE plus EX group (FM+EX+VCE; n = 11). Fasting blood samples at 08.00 h were taken from healthy untreated controls and patients with FM at baseline, before treatment was started. Fasting blood samples were again taken at 08.00 h from the patients with FM after 12 weeks of VCE therapy. The last dose of VCE and the last EX bout were at 12 h before blood collection.

Patients in the EX and VCE plus EX groups underwent an aerobic program on a treadmill. Each EX session lasted 30 min, including warm-up and cool-down periods. EX intensity was adjusted to heart rates equivalent to 70–85% of age-adjusted maximum heart rates (220 b.p.m. minus age in years). Heart rate and rhythm were monitored by Cardiovit AT-60 (Schiller, Baar, Switzerland). Aerobic EX was performed three times per week for 12 weeks.

Pain intensity

The patients' pain intensity was recorded on a visual analog scale (VAS), 100 mm in length. For pain intensity, the scale ends were assigned 0 = no pain and 100 = severe pain.

Blood collection and preparation of blood samples

After an overnight fast and with all morning medication omitted, venous blood (5 ml) was taken from an antecubital vein, using a monovette system of blood collection, into anticoagulation tubes containing sodium EDTA and protected against light. One milliliter of anticoagulated blood was used for hematological and sedimentation rate analysis. The remaining anticoagulated blood was separated into plasma and erythrocytes by centrifugation at 1500g for 10 min at +4°C. The erythrocyte samples were washed three times in cold isotonic saline (0.9%, w/v), then hemolyzed with a ninefold volume of phosphate buffer (50 mM, pH 7.4). After the addition of butylhydroxytoluol (4 μl/ml), hemolyzed RBC samples were stored at − 30°C for < 3 months pending measurement of LP, GSH, and GSH-Px values. One milliliter of plasma was used for the detection of thyroid hormones, kidney (urea, creatinine, and uric acid), and liver (total bilirubin, direct bilirubin, lactate dehydrogenase, gamma-glutamyl transpeptidase, and alkaline phosphatase) biochemical values (data not shown). The remaining plasma was used for immediate LP and antioxidant vitamin assay.

LP level determinations

LP levels in the plasma and erythrocytes samples were measured with the thiobarbituric acid reaction by the method of Placer et al. (1966). The quantification of thiobarbituric acid reactive substances was determined by comparing the absorption to the standard curve for malondialdehyde (MDA) equivalents generated by acid-catalyzed hydrolysis of 1,1,3,3 tetramethoxypropane. The values of LP in the plasma and erythrocytes were expressed as nmol/ml and μmol/Hb, respectively. Although the method is not specific for LP, measurement of the thiobarbituric acid reaction is an easy and reliable method, which is used as an indicator of LP and ROS activity in biological samples.

Reduced GSH, GSH-Px, and protein assay

The GSH content of the erythrocytes was measured at 412 nm using the method of Sedlak and Lindsay (1968). GSH-Px activities of erythrocytes were measured spectrophotometrically at 37°C and 412 nm according to the method of Lawrence and Burk (1976). Hb values of erythrocytes were determined according to the cyanmethemoglobin method of Cannan (1958).

Plasma vitamin A, vitamin E, and β-carotene analyses

Vitamins A (retinol) and E (α-tocopherol) were determined in the plasma samples by a modification of the method described by Desai (1984) and Suzuki and Katoh (1990). Plasma samples of about 250 μl were saponified by the addition of 0.3 ml of 60 % (w/v in water) KOH and 2 ml of 1% (w/v in ethanol) ascorbic acid, followed by heating at 70°C for 30 min. After cooling the samples on ice, 2 ml of water and 1 ml of n-hexane were added and mixed with the samples and then rested for 10 min to allow phase separation. An aliquot of 0.5 ml of n-hexane extract was taken and vitamin A concentrations were measured at 325 nm. Then reactants were added and the absorbance value of hexane was measured in a spectrophotometer at 535 nm. Calibration was performed using standard solutions of all-trans retinol and α-tocopherol in hexane.

The concentrations of β-carotene in plasma samples were determined according to the method of Suzuki and Katoh (1990). Two milliliter of hexane were mixed with 250 μl plasma. The concentration of β-carotene in hexane was measured at 453 nm in a spectrophotometer.

Quantification of ascorbic acid in the plasma samples was performed according to the method of Jagota and Dani (1982). The absorbance of the samples was measured spectrophotometrically at 760 nm.

Chemicals

All chemicals (cumene hydroperoxide, KOH, NaOH, thiobarbituric acid, 1,1,3,3 tetraethoxy propane, tris-hydroxymethyl-aminomethane, 5,5-dithiobis-2 nitrobenzoic acid, glutathione, and butylhydroxytoluol) were obtained from Sigma–Aldrich Chemical, Inc. (St Louis, MO, USA) and all organic solvents (n-hexane, ethyl alcohol) from (Merck Chemical, Inc., Darmstadt, Germany). All reagents were of analytical grade. All reagents except the phosphate buffers were prepared daily and stored at +4°C. The reagents were equilibrated at room temperature for half an hour before an analysis was initiated or reagent containers were refilled. Phosphate buffers were stable at +4°C for 1 month.

Statistical analyses

All results are expressed as means ± SD. Significance between values for control subjects and those for patients was assessed with unpaired Student's t-tests. Before and after treatment, values within groups were compared with paired Wilcoxon tests. Data were analyzed using the SPSS statistical program (version 9.05 software, SPSS, Inc., Chicago, IL, USA). p-values of less than 0.05 were regarded as significant.

Results

Demographic values for patients with FM and healthy untreated controls are shown in Table I. There were no significant differences in the values between controls and patients. Tender point number and pain intensity in patients with FM and in controls are shown in Table II and the values were significantly (Student's t-test; p < 0.001) higher in patients with FM than in controls. There were no statistically significant differences among the FM+EX, FM+VCE, and FM+EX+VCE groups (Table II).

Table II.  Tender point number and pain intensity in patients with FM and controls.

			FM+EX		FM+VCE		FM+EX+VCE
	Control (n = 30)	Total FM (n = 31)	Before (n = 11)	After (n = 11)	Before (n = 11)	After (n = 11)	before (n = 10)	After (n = 10)
Tender point number	2.3 ± 2.2	14.7 ± 1.9*	13.2 ± 1.7	15.4 ± 1.8	15.7 ± 1.5	15.0 ± 1.6	15.8 ± 8.7	14.3 ± 1.6
Pain intensity (VAS, cm)	10.0 ± 5.0	77.3 ± 9.4*	77.0 ± 3.8	78.0 ± 7.8	74.0 ± 3.5	75.0 ± 11.7	76.6 ± 5.5	79.0 ± 8.7

Notes: FM, all fibromyalgia patients at baseline; sub-groups: FM+EX, fibromyalgia+exercise group; FM+VCE, fibromyalgia+VCE supplemented group; FM+EX+VCE, fibromyalgia+VCE + exercise group. Values are mean ± SD and (min–max). n, number of subjects; Before, before treatment; After, after treatment. Values are mean ± SD. *p < 0.001 versus control, Student's t-test.

The mean LP values in plasma and erythrocytes of the groups are shown in Tables III and IV, respectively. The results showed that the LP levels in plasma (Student's t-test; p < 0.001) and erythrocytes (Student's t-test; p < 0.05) of patients with FM were significantly higher than those in the control group. Plasma LP levels in all three treatment groups were significantly decreased, compared with before treatment values, by 12 weeks of EX or VCE treatment, alone or combined (Wilcoxon test; p < 0.01; Table III). Erythrocyte LP levels in all three treatment groups also were significantly decreased by treatment (Wilcoxon test; p < 0.05, p < 0.01; Table IV).

Table III.  The effects of VCE and EX on plasma LP levels and antioxidant vitamin concentrations in patients with FM.

			FM+EX		FM+VCE		FM+EX+ VCE
	Control (n = 30)	Total FM (n = 31)	Before (n = 11)	After (n = 11)	Before (n = 11)	After (n = 11)	Before (n = 10)	After (n = 10)
LP (nmol/ml)	1.3 ± 0.2	3.4 ± 0.5**	3.6 ± 0.6	2.2 ± 0.3^‡	3.1 ± 0.5	2.1 ± 0.2^‡	3.6 ± 0.4	2.1 ± 0.1^‡
Vitamin A (μmol/l)	2.4 ± 0.2	1.5 ± 0.5*	1.6 ± 0.3	2.2 ± 0.3^†	1.8 ± 0.2	2.3 ± 0.2^†	1.4 ± 0.1	2.2 ± 0.2^†
Vitamin C (μmol/l)	0.9 ± 0.2	0.9 ± 0.1	0.9 ± 0.1	0.7 ± 0.2^†	0.9 ± 0.1	1.4 ± 0.2^†	0.9 ± 0.2	1.2 ± 0.3^†
Vitamin E (μmol/l)	7.3 ± 1.8	3.9 ± 05**	4.2 ± 0.6	5.8 ± 1.1^†	3.9 ± 0.4	6.0 ± 0.6^‡	3.5 ± 0.2	6.7 ± 0.9^¶
β-Carotene (μmol/l)	1.3 ± 0.3	1.1 ± 0.2	1.0 ± 0.2	1.0 ± 0.1	1.0 ± 0.2	1.0 ± 0.1	1.1 ± 0.1	1.0 ± 0.2

Note: FM, all fibromyalgia patients at baseline; sub-groups: FM+EX, fibromyalgia+exercise group; FM+VCE, fibromyalgia+VCE supplemented group; FM+EX+VCE, fibromyalgia+VCE+exercise group. Values are mean ± SD. n, number of subjects; Before, before treatment; After, after treatment. Significant differences between values at 12 weeks and baseline are indicated for the FM+EX, FM+VCE, and FM+EX+VCE groups. *p < 0.05 and **p < 0.001 vs. control, Student's t-test

^† p < 0.05

^‡ p < 0.01

^¶ p < 0.001 vs. before treatment, Wilcoxon test.

Table IV.  The effects of VCE and EX on erythrocyte LP levels, reduced GSH, and GSH-Px values in patients with FM.

			FM+EX		FM+VCE		FM+EX+VCE
	Control (n = 30)	Total FM (n = 31)	Before (n = 11)	After (n = 11)	Before (n = 11)	After (n = 11)	Before (n = 10)	After (n = 10)
LP (μmol/g Hb)	12.7 ± 2.9	14.8 ± 2.7*	14.9 ± 4.1	10.2 ± 2.1^†	14.1 ± 1.3	8.5 ± 2.2^‡	15.1 ± 2.0	7.3 ± 1.6^‡
GSH (μmol/g Hb)	12.4 ± 3.9	7.0 ± 1.5**	6.4 ± 1.4	8.1 ± 1.9^†	6.7 ± 1.4	11.2 ± 1.1^‡	7.9 ± 1.5	12.0 ± 1.3^‡
GSH-Px (IU/g Hb)	15.8 ± 3.4	15.6 ± 3.4	15.0 ± 2.8	15.6 ± 2.6	16.0 ± 3.4	18.2 ± 2.5^†	15.9 ± 4.1	18.7 ± 2.8^†

Notes: FM, all fibromyalgia patients at baseline; sub-groups: FM+EX, fibromyalgia+exercise group; FM+VCE, fibromyalgia+VCE supplemented group; FM+EX+VCE, fibromyalgia+VCE+exercise group. Values are mean ± SD. n, number of subjects; Hb: Before, before treatment; After, after treatment. Significant differences between values at 12 weeks and baseline are indicated for the FM+EX, FM+VCE, and FM+EX+VCE groups. *p < 0.05 and **p < 0.001 vs. control, Student's t-test

^† p < 0.05

^‡ p < 0.01 vs before treatment, Wilcoxon test.

GSH levels in the erythrocytes in the FM group were significantly (Student's t-test; p < 0.001) lower than those in the control group; GSH-Px activities were not significantly different between the control and FM groups (Table IV). Treatment for 12 weeks with EX alone (FM+EX) increased erythrocyte GSH levels (Wilcoxon test; p < 0.05), but not GSH-Px activity, compared to those before treatment (Table IV). Treatment for 12 weeks with VCE and VCE plus EX (FM+VCE and FM+EX+VCE groups) increased erythrocyte GSH levels and GSH-Px activities compared to those before treatment (Wilcoxon tests; p < 0.01 and p < 0.05).

The mean concentrations of β-carotene, vitamin A, and VCE in plasma of patients with FM are shown in Table III. Vitamin A (Student's t-test; p < 0.05) and vitamin E (Student's t-test; p < 0.001) concentrations at baseline level were significantly lower in patients with FM than those in controls. In the three treatment groups, vitamin A concentrations were significantly higher after treatment than before treatment (Wilcoxon tests; vitamin A: p < 0.05; vitamin E: p < 0.05, p < 0.01, p < 0.001; Table III). The vitamin C concentrations in patients with FM before treatment were not significantly different from those in the control group. Plasma vitamin C concentration in the FM+EX group after EX for 12 weeks was significantly lower (Wilcoxon test; p < 0.05) than before EX (Table III). Vitamin C concentrations were higher after treatment in the FM+VCE (Wilcoxon test; p < 0.01) and FM+EX+VCE groups (Wilcoxon test; p < 0.05) than before treatment (Table III). Plasma concentrations of β-carotene were not significantly different among the groups.

Discussion

We found that LP levels in erythrocytes of patients with FM were increased compared those in with controls, and erythrocyte GSH values and plasma vitamins A and E concentrations were decreased. However, VCE supplementation with EX for 12 weeks decreased erythrocyte LP levels, whereas GSH-Px activities, GSH levels, and plasma vitamins A, C, and E concentrations were increased by the supplementations. FM is characterized by decreased erythrocyte GSH, and plasma vitamins A and E concentrations and increased plasma LP levels. A limited number of in vivo or in vitro studies of blood from patients with FM regarding the effects of antioxidant redox systems and LP levels in the pathogenesis of FM have been reported (Eisinger et al. 1994; Bagis et al. 2005; Altindag and Celik 2006; Özgöçmen et al. 2006a; Akkuş et al. 2009). To the best of our knowledge, the current study is the first to compare treatment with antioxidant vitamins with particular reference to oxidative stress and the antioxidant redox systems in plasma of patients with FM. Despite the improvements in antioxidant status, FM symptoms were not improved by the treatments.

The etiology and pathogenesis of FM are not clearly understood although it is characterized by the activation of local ischemic injury (Bagis et al. 2005; Altindag and Celik 2006; Özgöçmen et al. 2006a,b). LP levels, reflecting oxidative degradation products of membrane PUFAs, are known to be related to ROS actions (Nazıroğlu 2007a). In the present study, LP levels in plasma and erythrocytes in patients with FM were increased. Local ischemia leads to overproduction of ROS and interferes with the structure and ratio of PUFA (Yilmaz et al. 1997; Nazıroğlu et al. 2004; Finaud et al. 2006), leading to the loss of biological membrane fluidity.

Reports on the role of ROS-mediated oxidative damage in the etiology and pathogenesis of FM are scarce and controversial. Fassbender and Wegner (1973) reported that muscle tender points in FM result from local hypoxia although Lund et al. (1986) indicated abnormal oxygen pressure at the muscle surface above trigger points. Bengtsson et al. (1986) investigated oxidative metabolism and they found that adenosine diphosphatase and creatinine phosphate levels were decreased and adenosine monophosphatase and creatinine levels were increased in patients with FM. Similar to the LP results of our current study, Bagis et al. (2005), Altindag and Celik (2006), Özgöçmen et al. (2006b) and Akkuş et al. (2009) reported that LP levels in plasma of patients with active FM were higher than those in controls. In contrast, however, Eisinger et al. (1994) measured LP levels, protein carbonyls, and antioxidant in female patients with FM and found no difference in LP levels, as MDA, between controls and patients although they were able to show protein peroxidation in the patients.

Inherent to its function, skeletal muscle is continuously exposed to fluctuations in its redox environment as, during EX, ROS production by the mitochondrial respiratory chain increases. Therefore, the capability of skeletal muscle to respond to oxidative stress may not be surprising. In vitro experiments have demonstrated that skeletal myocytes adapt to oxidative stress by upregulation of antioxidant enzymes such as Mn- and Cu/Zn-superoxide dismutase, catalase, and GSH-Px (Altindag and Celik 2006; Özgöçmen et al. 2006a). This suggests that under some conditions (such as aging, poor sleep, or microtrauma) antioxidant capacity may be impaired, as it may result in more oxidative stress in skeletal muscle (Schachter et al. 2003). Potential triggers of oxidative stress in the muscle compartment include hypoxia and local sources of reactive oxygen and nitrogen species; skeletal muscle trophic state, contractility, and fatigability may be affected by oxidative stress, resulting in skeletal muscle dysfunction (Altindag and Celik 2006; Özgöçmen et al. 2006a).

Signs of oxidative stress in FM include high levels of oxidative damage to DNA, as seen in biopsy samples of patients with FM. Reduced oxidative metabolism and mitochondrial abnormalities in FM also support a mitochondrial defect as a contributor (Sprott et al. 2004). Moreover, because mitochondria supply energy to the cell through oxidative phosphorylation, the lower level of Adenosine Triphosphate that results from low mitochondrial activity may explain the low EX capacity and fatigue reported in patients with FM (Sprott et al. 2004).

In the current study, plasma vitamins A and E concentrations were higher in the FM plus EX group than those in the pre-treatment FM group, and erythrocyte LP levels were lower in the FM plus EX group than those in the FM group. During EX, free radicals may be produced in excess of the body's natural defenses (Nazıroğlu et al. 2004; Finaud et al. 2006). There are a few studies, in patients, demonstrating an increase in free nitrogen radicals due to exercising patients to exhaustion (Sim and Adams 2002; Gowans and deHueck 2004; Özgöçmen et al. 2006a). The biochemical mechanisms by which regular EX has beneficial effects are not well understood. Reports of increased production of nitrogen radicals during EX are also conflicting (Mannerkorpi 2005; McIver et al. 2006; Özgöçmen et al. 2006a). Although EX may cause LP production in cells, moderate EX has been suggested to be beneficial in the management of patients with FM (Sim and Adams 2002; Gowans and deHueck 2004; Özgöçmen et al. 2006a), perhaps by improving long-term metabolic control of the oxidative stress products.

Vitamin C has been shown to be an important antioxidant, to regenerate vitamin E through redox cycling, and to raise intracellular GSH levels (Nazıroğlu 2007a). Thus, vitamin C plays an important role in protein thiol group protection against oxidation. The GSH levels and GSH-Px activities in erythrocytes were significantly higher in the VCE and VCE plus EX supplemented groups than in the pre-treatment FM group. Taking into consideration the data given here, the observed increased levels of GSH and GSH-Px in the erythrocytes of the VCE and VCE plus EX groups indicate a role for vitamin C supplementation in normalizing GSH levels in FM. By contrast, EX alone did not increase GSH-Px level in erythrocytes of FM patients, although it increased GSH level, while decreasing plasma vitamin C level.

In the current study, vitamins A and E concentrations in the plasma of patients with FM were lower than those in controls, and LP levels in plasma were higher in the patients than in controls. Thus, plasma vitamins A and E concentrations in the FM patients may be decreased as a result of their action in inhibiting free radicals. Previously, Eisinger et al. (1994) measured plasma vitamins A and E concentrations in 28 patients with FM and 20 age-matched controls and they found that there were no significant differences in the vitamin concentrations in the patients. However, in the recent study of Akkuş et al. (2009), vitamins A and E concentrations in the plasma of patients with FM were lower than those in controls, and LP levels in plasma were higher in the patients than those in controls. Moreover, the vitamins A and E findings are supported by studies on inflammatory diseases such as Behcet's disease (Kökçam and Nazıroğlu 2002) and rheumatoid arthritis (Kamanli et al. 2004).

In conclusion, we have shown that increased LP is evident in patients with FM. These results are consistent with the underlying hypothesis that there is an imbalance between ROS production and the antioxidant defense system in the local ischemia of patients with FM. The co-administration of VCE with EX may act against FM by counteracting inhibition of the enzymatic and nonenzymatic antioxidant systems by FM. Moderate EX training and VCE supplementation may play a role in modulating FM-induced oxidative stress formation in blood of patients with FM. The results may aid treatment of FM, with VCE supplementation and/or EX, as well as providing leads to clarifying the etiology of FM.

Acknowledgements

We thank Associate Professor Dr Mustafa Öztürk (Department of Public Health, Medical Faculty, Suleyman Demirel University) for his help in statistical analysis of data in the manuscript. We also thank Dr John Russell (Editor in Chief, Stress) for improving the use of English in the manuscript.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.