Free Radical Biology and Medicine; 29, Issue 12, 1252-59 Dec 15, 2000 Specific oxidative alterations in vastus lateralis muscle of patients with the diagnosis of chronic fatigue syndrome Stefania Fulle(a), Patrizia Mecocci(b), Giorgio Fano(c), Iacopo Vecchiet(d), Alba Vecchini(e), Delia Racciotti(d), Antonio Cherubini(b), Eligio Pizzigallo (d), Leonardo Vecchiet (c), Umberto Senin(b) and M. Flint Beal(f). (a) Lab. Interuniversitario di Miologia, Dip. Biologia Cellulare e Molecolare, Universita di Perugia, Perugia, Italy (b) Inst. Gerontologia e Geriatria, Universita di Perugia, Perugia, Italy (c) Lab. Interuniversitario di Miologia, Dip. Scienza del Farmaco, Universita "G.D'Annunzio," Chieti, Italy (d) Ist. Malattie Infettive, Universita "G.D'Annunzio," Chieti, Italy (e) Ist. Biochimica e Chimica Medica, Universita di Perugia, Perugia, Italy (f) Department of Neurology and Neuroscience, Weill Medical College of Cornell University and the New York Hospital-Cornell Medical Center, New York, NY, USA Address correspondence to: Dr. M. Flint Beal, Chairman, Neurology Department, New York Hospital-Cornell Medical Center, 525 East 68th Street, New York, NY 10021, USA; Tel: (212) 746-6575; Fax: (212) 746-8532; email:fbeal@mail.med.cornell.edu Received 13 April 2000; revised 21 July 2000; accepted 24 August 2000. Available online 13 December 2000. Abstract Chronic fatigue syndrome (CFS) is a poorly understood disease characterized by mental and physical fatigue, most often observed in young white females. Muscle pain at rest, exacerbated by exercise, is a common symptom. Although a specific defect in muscle metabolism has not been clearly defined, yet several studies report altered oxidative metabolism. In this study, we detected oxidative damage to DNA and lipids in muscle specimens of CFS patients as compared to age-matched controls, as well as increased activity of the antioxidant enzymes catalase, glutathione peroxidase, and transferase, and increases in total glutathione plasma levels. From these results we hypothesize that in CFS there is oxidative stress in muscle, which results in an increase in antioxidant defenses. Furthermore, in muscle membranes, fluidity and fatty acid composition are significantly different in specimens from CFS patients as compared to controls and to patients suffering from fibromyalgia. These data support an organic origin of CFS, in which muscle suffers oxidative damage. Author Keywords: Chronic fatigue syndrome; Oxidative stress; Muscle; Antioxidants; Free radicals Introduction Chronic fatigue syndrome (CFS) is a clinical condition characterized by long-lived, disabling fatigue associated with deficits in short-term memory, impairments in concentration, sleep disturbances, and skeletal muscle pain. The etiology of the disease is still unknown, but several studies suggest that an active viral infection and an immunological disorder may play fundamental roles. Onset of CFS after an acute or subacute viral infection, associated with mild fever, sore throat, muscular weakness, headache, migratory arthralgia, and other "flu-like" symptoms, is typical [1]. The prevalence of CFS ranges from seven to 267 cases per 10,000. The disease is more frequent in young adults and in white females [2]. Although some researchers consider CFS a psychological rather than a physical illness [3], several alterations support an organic origin of this disease. Many, although not all, CFS patients have atypical circulating lymphocytes and immune complexes [4], and increased concentrations of C-reactive protein and beta-2 microglobulin [5], which suggests a chronic low-level activation of the immune system. This might be the response to a subclinical viral infection, since antibodies to Coxsackie B virus and Epstein Barr virus are commonly observed in these patients [6 and 7]. Psychological factors have an important role in CSF: two-thirds of CFS patients suffer from a major depressive illness. These findings do not exclude the possibility of an organic origin of this disease, since depression is also characterized by specific neurotransmitter alterations. The most striking symptoms of the syndrome are muscle weakness and pain. In CFS patients, a deficiency of serum acylcarnitine has been found [8], which could induce abnormalities in mitochondrial activity. Reduced oxidative metabolism [9 and 10] and higher plasma lactate concentration [11] have been reported. An impairment of mitochondrial oxidative phosphorylation in CFS is supported by the observation that in a group of patients suffering from this disease there is marked increase of intracellular pH after moderate exercise and a lower rate of ATP synthesis during recovery [12]. These findings may explain the muscle wasting due to an increased catabolic pathway observed in CFS. Recently, CFS has been included in the group "low cystine and glutamine syndromes," which are characterized by a combination of abnormally low plasma levels of these two aminoacids, as well as reduced natural killer cell activity, and increased rates of urea production [13]. Some studies indicated that there wasn't a consistent correlation between CFS symptoms and changes in muscle morphology (fiber-type prevalence, fiber size, degenerative features, or mitochondrial abnormalities) and physiological contractile properties [14]. Recent studies contradict this statement [12]. Oxidative metabolism related to mitochondrial activity can cause increased production of reactive oxygen species (ROS), leading to oxidative damage that can be correlated with an increase in muscle fatigability during normal aging [15]. Both these latter conditions are also seen in the muscle specimens derived from CFS patients [16]. We therefore examined whether (i) skeletal muscle is a site of damage, which induces reductions of functional activity; and whether (ii) muscle damage is a consequence of impairment of the oxidant/antioxidant system. Methods The study population consisted of six patients with CFS (four males and two females; mean age 35.7 p/m 1.6 years) and three patients (one male and two females; mean age 48.2 p/m 1.7 years) suffering from fibromyalgia (FS), used only as a positive control for lipid analysis. All patients were selected at the Center for the Study of CFS of the University of Chieti on the basis of the CDC criteria [17]. Six healthy subjects who underwent orthopedic surgery and who were sex- and age-matched were used as controls. Biopsy from vastus lateralis muscle was done after a thorough clinical evaluation, including detailed manual muscle testing. The risks associated with the muscle biopsy are usually minimal; however, it was explained to the patient the discomfort of the postoperative period, the possible complications of the surgical procedure, and what we hoped to learn from the biopsy using informed consent. Biopsy specimens (0.01-0.5 g) were obtained from the vastus lateralis on the basis of the principles indicated by A. G. Engel and C. Franzini-Armstrong [18] and the samples were immediately frozen in liquid nitrogen for the following determinations. OXIDATIVE DAMAGE Muscle samples were minced with a pestle in liquid nitrogen, resuspended in 1.5 ml of buffer (0.1 M NaCl, 0.03 M Tris, and 0.01 M EDTA) and 15 mu-l of 10% butylated hydroxytoluene (BHT) as antioxidant, and then centrifuged at 5,000 x g. DNA was extracted from the pellet using the DNAzol extractor (Gibco BRL; Gibco; Grand Island, NY, USA) and solubilized in 8 mM NaOH. The purity and the amount of DNA thus obtained were assessed spectrophotometrically at 260 and 280 nm. The supernatant was used for malondialdehyde (MDA) and protein carbonyl measurements. 8-HYDROXY-2-DEOXYGUANOSINE (OH8DG) MEASUREMENTS After adjustment of pH to 8.5 with Hepes 0.1 M, nuclear DNA was digested with Dnase 1 (200 U/mg DNA), spleen exonuclease (0.01 U/mg DNA), snake venom exonuclease (0.5 U/mg DNA), and alkaline phosphatase (10 U/mg DNA) in 40 mM Tris HCl and 10 mM MgCl2, pH 8.5. The sample was analyzed using high-performance liquid chromatography (HPLC). Deoxyguanosine (dG) and OH8dG were measured using ultraviolet (UV) and electrochemical detection systems linked in series. We used a Waters (Milford, MA, USA) model 486 UV detector at 260 nm to measure dG and a coulometric ESA electrochemical detector (model 5100) with electrodes adjusted to 0.05 and 0.30 V, respectively, to detect OH8dG [15]. The mobile phase consisted of 50 mM KH2PO4 pH 5.5, with 9% methanol, delivered at 1 ml/min. Analysis was performed using a 15 cm 5 mu-m C18 Nikko Bioscience column (Tokyo, Japan). Results are expressed as OH^8dG/dG ratio that is the molar ratio between OH8dG and dG multiplied x 105. MDA MDA levels were measured using HPLC with fluorometric detection [19]. Briefly 50 mu-l of sample was added to 350 mu-l of KH2PO4 1 mM pH 3.0 with 10 mu-l of 18 mM BHT. After 10 min incubation at room temperature, 100 of 0.6% w/v 2-thiobarbituric acid (TBA) in double-distilled water was added and samples heated at 93C for 45 min. The reaction was stopped by putting samples on the ice for 10 min. After centrifugation of 9,500 x g for 5 min, the supernatant was injected into a HPLC system with a fluorometric detector (Waters 470) (Ex 530 nm, Em 552 nm). Separation was performed using a C18 Rainin Microsorb column (Woburn, MA, USA) (25 cm 5 mu-m) with a mobile phase of 50 mM KH_2PO_4 pH 7.0, methanol 40%, delivered at 1.8 ml/min. Assays were performed in triplicate and results are expressed as nmol of MDA per mg of protein. PROTEIN CARBONYL MEASUREMENTS Protein carbonyl content was measured by forming labeled protein hydrazone derivatives, using 2,4-dinitro-phenylhydrazine (DNPH), and they were then quantified spectrophotometrically [20]. Briefly, after protein precipitation with an equal volume of 1% trichloroacetic acid (TCA), the pellet was resuspended in 1 ml of DNPH 10 mM in 2 N HCl. Separate blanks were prepared by adding 1 ml of 2 N HCl without DNPH. Samples were left at room temperature for 1 h in the dark and vortexed every 15 min. An equal volume of 20% TCA was added and after centrifugation at 12,000 x g for 15 min at 4C, the pellets were washed three times with 1 ml of ethanol-ethylacetate mixture (1:1) to remove the free DNPH and lipid contaminants. The final pellet was dissolved in 1 ml of 6 M guanidine and kept at 37C for 1 h in a water bath with mixer. The solution was centrifuged for 15 min at 12,000 x g. The carbonyl content was determined from the absorbance at 370 nm using a molar absorption coefficient of 22,000 mol/l-1 cm-1. Results are expressed as nmol DNPH per mg protein. ANTIOXIDANT ENZYMES Preparation for cytosol enzymes assay: each sample was homogenized in a glass dounce maintained in a cold bath. Homogenization was carried out in 20 mM Na-phosphate buffer pH 7.0 + 100 mu-M phenylmethylsulfonyl fluoride (PMSF) (rate w/w 1/10) and centrifuged at 100,000 x g for 1 h. Protein content was determined according to the method of Lowry et al. [21]. Total glutathione content was measured in the resulting supernatant by the enzymatic method of Akerboom and Sies [22]. The pellets were dissolved in 1 M NaOH and the total protein content determined by the Lowry method. Enzyme activities were measured spectrophotometrically by continuous registration at room temperature (20C). CATALASE ACTIVITY Catalase activity was determined, according to Greenwald [23] by the decrease in absorbance due to H_2O_2 consumption (epsilon=0.04 mM-1 cm-1) measured at 240 nm. One milliliter of the final reaction mixture contained 100 mM Na-phosphate buffer pH 7.0, 12 mM H_2O_2 and 30-80 mu-g sample. SUPEROXIDE DISMUTASE Superoxide dismutase activity was determined according to L'Abbe and Fisher [24]. The assay mixture contained a final volume of 1 ml and was 20 mM Na_2CO_3 buffer pH 10.0, 50 mu-M cytochrome c, 1 mM mixture xanthine, and 15 mU/ml xanthine oxidase. As the activity of xanthine oxidase varies, the amount used was the one that produces a rate of cytochrome c reduction resulting in a change in A415 of 0.025 per minute without any added SOD. One unit of SOD was defined as the activity that inhibits the rate of cytochrome c reduction by 50%. GLUTATHIONE S-TRANSFERASE (GST) This enzyme is present in human skeletal muscle overall as mu+pi isoforms [25]. The activity was determined by using 1-cloro-2-4-dinitrobenzene (CDNB) as substrate according to Habig and Jakoby [26]. The assay was performed at 340 nm (epsilon=9.6 mM-1 cm-1) and a final volume of 1 ml contained 100 mM Na- phosphate buffer pH 6.5, 1 mM CDNB, 1 mM reduced glutathione (GSH), and a 30-80 mu-g sample. GLUTATHIONE REDUCTASE Glutathione reductase activity was determined by the rate of decrease in absorbance, induced by oxidation of NADPH, at 340 nm (epsilon=-6.22 mM-1 cm-1) [27]. The assay mixture contained in a final volume of 1 ml: 100 mM Na-phosphate buffer pH 7.0, 1 mM glutathione disulphide (GSSG), 60 muM NADPH, and 50-150 mu-g sample. GLUTATHIONE PEROXIDASE (GPX) Glutathione peroxidase activity was measured, according to Lawrence and Burk [28], by following the formation of GSSG using a coupled enzyme system with glutathione reductase. Oxidation of NADPH was recorded at 340 nm (epsilon=-6.22 mM-1 cm-1). The Se-dependent as well as the sum of Se-dependent and Se-independent activity (also having Gst activity) were determined by using respectively H_2O_2 and cumene hydroperoxide as substrates. A final volume of 1 ml contained: 100 mM Na-phosphate buffer pH 7.5, 1 mM EDTA, (1 mM NaN3 only for H_2O_2 assay), 2 mM GSH, 1 unit of glutathione reductase, 0.24 mM NADPH, 30-80 mu-g sample, and 0.6 mM H2O2 or 0.8 mM cumene hydroperoxide. GLUTATHIONE The sum of the reduced disulfide forms of glutathione were determined using a kinetic assay in which catalytic amounts of GSH or GSSG result in the continuous reduction of 5'5'-dithiobis (2-nitrobenzoic acid) (DTNB) by NADPH. The reaction rate is directly related to the concentration of glutathione at values up to about 2 mu-M. The formation of 5-thio-2-nitrobenzoate (TNB) was measured spectrophotometrically at 421 nm. LIPID ANALYSIS For total lipid extraction, 30 mg of human vastus lateralis muscle was cut up in small pieces. Then 5 ml of chloroform/methanol (C/M 2:1) supplemented with 0.01% butylated hydroxytoluene (BHT) as an antioxidant were added. The samples were homogenized with an Ultra-turrax homogenizer standing in ice. The samples were left at room temperature for 30 min, then passed through a cotton-wool filter and washed twice with 2 ml of C/M 2:1. Then 2.5 ml H2O plus 2.5 ml chloroform were added to the samples and the mixtures were centrifuged to separate them into two phases. The lower phase was washed three times with 3 ml of a solution constituted by chloroform/methanol/0.9% NaCl (30:470:480) according to Folch et al. [29]. The final extract was evaporated with a nitrogen flow at 30C. To separate the fatty acids from total lipids, the dry extract was passed through a transmethylation process. Six milliliters of 3% methanolic H2SO4 (w/v) were added to the dry residues and the samples were left at 70C for 2 h, then extracted three times with n-hexane. The composition of most abundant (higher than 2%) fatty acids were analyzed using gas chromatography with a spiral column (0.5 mm x 50 m), with H, air, and He as gas. The results are expressed as the molar percentage in the mixture according to established procedures [30]. MEMBRANE FLUIDITY The muscles were triturated in liquid N2 and homogenized in dounce with 0.25 M sucrose. The samples were centrifuged at 1,000 x g for 10 min; the supernatants were centrifuged at 12,000 x g for 10 min. The new supernatants were centrifuged at 100,000 x g for 90 min. The pellets were resuspended in 0.25 M sucrose at a concentration of 1 mg/ml. The protein concentration was determined according to Lowry et al. method [21]. We used 200 mu-g of protein for determination of fluorescence anisotrophy. Two microliters of 2 mM diphenylhexatriene (DHP), dissolved in tetrahydrofuran, was added to the sample. The suspension was left 1 h in the dark and then used as described in Shinitzky and Barenholz [31]. The fluorescence polarization (P) is inversely correlated with membrane fluidity and indicates the membrane rigidity: high values of P represent low membrane fluidity and vice versa. The statistical differences were tested by unpaired t test for each point and were automatically derived by the Prism-2 computer program (GraphPad Software, Inc.; San Diego, CA, USA). Results Figure 1 shows the levels of oxidative-damage markers as measured in skeletal muscle biopsies from CFS and healthy-matched controls. Results, expressed as mean p/m SD, showed that CFS patients had significant increases of OH^8dG (10.37 p/m 2.44 vs. 6.72 p/m 1.73, p<.05) and MDA (7.60 p/m 0.80 vs. 5.40 p/m 1.03, p<.01). No significant differences were found in protein carbonyl content (2.37 p/m 1.05 vs. 2.03 p/m 1.20). In Table 1 the activity of skeletal muscle antioxidative enzymes is reported. Se-dependent peroxidase was significantly increased from 21.88 p/m 13.31 to 61.18 p/m 22.99 nmol/min/mg (p<.01) in the muscle samples derived from CFS patients. The activity of soluble catalase, the glutathione-independent H2O2 scavenging enzyme, was significantly increased from 51.98 p/m 4.32 to 127.05 p/m 52.01 mu-mol/min/mg, p<.01. Gutathione mu and pi transferase, which utilizes the hydroperoxide lipids as substrate, was significantly increased from 145.2 p/m 13.10 to 238.00 p/m 50.73 nmol/min/mg, p<.05 in the CFS samples. Figure captions Fig. 1. The figure reports the oxidative damage in human skeletal muscle (vastus lateralis) expressed as relative values of OH^8dG (OH^8dG/10^5dG ratio), MDA (nmol/mg protein), and protein carbonyl (nmol DNPH/mg protein) in CFS patients and healthy age-matched controls. The values are the means p/m SD (n=6). *p <.05, **p <.01. Fig. 2. Analysis of fatty acids composition of total lipids in human skeletal muscle (vastus lateralis). (A) The percentage of variation of the most abundant fatty acids present in the mixture of total lipids of samples from patients with CFS or FS with respect to controls Base-lines (percent in the sample) 16:0 22.49 p/m 1.59; 16:1 5.94 p/m 1.63; 18:0 5.18 p/m 2.35; 18:1 50.13 p/m 4.63; 18:2 11.84 p/m 1.59; 20:4 2.09 p/m 0.71. (B) The specific activity (expressed as arbitrary units) of three enzymes involved in fatty acid elongation (elongase) or in desaturation (D-5 desaturase and D-9 desaturase), measured in CFS or FS patients and healthy- matched controls. The values are the means p/m SD (n=5). *p<.05, ** p<.01. Fig. 3. Membrane fluidity in human skeletal muscle (vastus lateralis). The fluores- cence polarization (P) of membrane, at different temperatures (15-40C), in CFS, FS, and healthy-matched controls. Also, the statistical analysis of the slope and the intercepts are reported. The values are the means p/m SD (n=5). Table Table 1. Activity of Human Skeletal Muscle Antioxidative Enzymes (See Methods and Details) -------------------------------------------------------------------------- Controls CFS p -------------------------------------------------------------------------- Catalase 51.98 p/m 4.32 127.05 p/m 52.01 <.01 (mu-mol/min/mgprot) (n=6) (n=6) Transferase (mu+pi) 145.25 p/m 13.1 238.00 p/m 50.73 <.05 (nmol/min/mg prot) (n=4) (n=4) Reductase 9.01 p/m 13.1 13.05 p/m 3.43 ns (nmol/min/ mg prot) (n=5) (n=5) Peroxidase (Se dependent) 21.88 p/m 13.31 61.18 p/m 22.99 <.01 (nmol/min/ mg prot) (n=6) (n=6) Glutathione (total) 3.40 p/m 2.49 7.53 p/m 1.79 <.05 (mu-mol/min/g tissue) (n=6) (n=6) Superoxide dismutase 13.88 p/m 4.40 15.11 p/m 3.49 ns (U/g prot) (n=6) (n=6) -------------------------------------------------------------------------- In this table it is reported the activity of the antioxidative enzymes catalase, peroxidase (Se dependent or independent), transferase, reductase, superoxide dismutase, and the amount of total glutathione in samples from CFS patients and healthy-matched controls. 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