Metal-induced oxidative stress level in patients with fibromyalgia syndrome and its contribution to the severity of the disease: A correlational study

Abstract

BACKGROUND:

Fibromyalgia syndrome (FMS) is an extra-articular rheumatological disease characterised by widespread chronic musculoskeletal pain. Metal-induced oxidative stress contributes to the severity of FMS.

AIMS:

First, this study evaluated the association between plasma levels of toxic heavy metals and essential metals with oxidative stress (OS) markers. Second, the OS markers and metal contents were correlated with the disease severity by assessing the Fibromyalgia Impact Questioner Revised (FIQR) and tender points (TP).

METHOD:

A total of 105 FMS patients and 105 healthy controls of similar age and sex were recruited. OS parameter such as lipid peroxidation (LPO), protein carbonyl group (PCG), nitric oxide (NO) and essential metals such as zinc (Zn), magnesium (Mg), manganese (Mn), copper (Cu) and toxic heavy metals such as aluminium (Al), arsenic (As), lead (Pb) were estimated.

RESULTS:

Levels of LPO, PCG, NO ( $p<$ 0.001) and Cu, Mn, and Al ( $p<$ 0.001), were significantly higher, and Mg ( $p<$ 0.001) and Zn ( $p<$ 0.001) were significantly lower in patients compared to controls. A positive association was observed between OS parameters, FIQR and TP with Cu, Al and Mn. A significant negative association was observed between Zn and Mg with FIQR, TP and OS parameters.

CONCLUSION:

Heavy metals such as Al induce OS parameters and decrease the levels of essential trace elements such as Mg and Zn, which may be responsible for the severity of FMS.

Keywords

Lipid peroxidation oxidants essential trace elements

1. Introduction

Fibromyalgia is an extra-articular rheumatological disease characterized by widespread pain, stiffness, and tenderness of the muscles, tendons and joints. The pathophysiology and etiology of fibromyalgia has not yet been clarified. It seems to be the outcome of complex interaction of multiple factors, such as, genetic, neuroendocrine, immune, psychosocial, environmental behavioural and even metal toxicity [1, 2]. Chronic exposure to metals occurs mainly through metal ion releases from environmental pollutants such as industrial waste and also from jewellery, vaccines, coated medical pills, food, coins and cigarette smoke which contain nickel, cadmium, titanium dioxide, iron oxide, manganese, mercury, lead and arsenic. Non-essential and toxic metals such as lead (Pb), arsenic (As), and aluminium (Al) cause metal poisoning when they enter the metabolic cycle whereas magnesium (Mg), zinc (Zn), manganese (Mn) and copper (Cu) are essential trace elements and non-enzymatic antioxidants and participates as cofactors in various metabolic and signalling pathways. Increased levels of toxic metals and imbalance of trace elements may be involved in the pathophysiology of FMS and may be responsible for its symptoms [3, 4]. Previous studies have shown that toxic metals trigger the production of free radicals, leading to oxidative stress and depletion of glutathione as well as influencing the metabolism of metallothioneins such as iron, copper, cadmium, chromium, mercury, nickel, vanadium are capable of producing reactive oxygen radicals, which leads to DNA damage, lipid peroxidation and protein carbonylation and other effects. Studies have shown evidence that toxic metals possesses oxidative nature and are also known to stimulate the production of inflammatory messengers known as cytokines in the immune system causing immense pain and oxidative stress which may have an important role in the pathophysiology of FMS [5, 40]. Oxidative stress is the outcome of imbalance between reactive oxygen species (ROS) and antioxidants in the cell. The oxidative effects of ROS are usually neutralized by catalase, superoxide dismutase, glutathione reductase, peroxidase and non-enzymatic antioxidants such as vitamin E, vitamin A, vitamin C and uric acid [6]. ROS accumulation leads to cellular damage due to lipid peroxidation (LPO) and is also responsible for the oxidation of protein (protein carbonylation) leading to the production of toxic aldehyde metabolites malondialdehyde (MDA) and 4-hydroxynoneal (HNE) that may cause oxidative tissue damage [7]. Metal toxicity also increases nitric oxide (NO), a highly diffusible and labile, gaseous messenger, involved in various biological functions such as vasodilatation, modulation of nociception, immune function and so on. It has been shown to modulate levels of ROS in a variety of cells.

The purpose of this case control study was to examine whether metals are correlated in inducing oxidative stress markers and the severity of FMS. The paper is organised as follows: first, we analysed the level of oxidative stress parameters such as LPO, protein carbonyl group and NO and metals such as As, Pb and Al as well as the role of Zn, Mg, Mn and Cu in patients and controls. Then we correlated metal contents and oxidative stress with FIQR total score and tender points. Further attention was paid to the correlation between metal content-induced oxidative stress parameters.

2. Material and methods

2.1 Ethics statement

The study protocol was approved by the Institutional Ethics Committee at King George’s Medical University (ethical approval ref. code: XL VI ECM/A-P9). Eligible patients signed an informed consent form prior to participating in the study.

2.2 Design of the study and sampling

This is a case control study. The selection process of this study was purposive sampling. We recruited 105 adults with fibromyalgia syndrome (FMS) who attended the outpatient clinic at the Department of Rheumatology, King George’s Medical University, Lucknow, India, as well as 105 healthy controls with no apparent diseases. The study was conducted between 2012 and 2015.

The sample size was calculated by determining the mean value of oxidative stress markers such as malondialdehyde level (MDA) indicator of lipid peroxidation, protein carbonyl groups (PCG) which reflects protein peroxidation, NO (nitric oxide) and erythrocyte glyceraldehydes phosphate dehydrogenase (GAPHD) based on original published studies on patients with FMS [30]. The sample size of the study was calculated by using the Snedecor and Cochran sample size formula [8].

2.3 Inclusion and exclusion criteria

Patients and control selection was based on the inclusion criteria. The study consisted of individuals of both genders aged between 18 to 60 years. We only included patients who met the American College of Rheumatology (ACR) 1990 criteria for FMS [9] and healthy individuals without FMS or any other disease. Individuals with inflammatory autoimmune diseases, diabetes mellitus, renal disease, known heart diseases, using any kind of antioxidant, known co-morbid conditions such as rheumatoid arthritis, SLE and other endocrine disorders, both in the control group and fibromyalgia group, were excluded. Smokers and those using any oral contraceptives as well as psychiatric patients and multiple myeloma patients were were also excluded, as these factors can influence the oxidative and antioxidative parameters.

In patients with fibromyalgia, depression was measured by the Center for Epidemiologic Studies Depression Scale (CES-D), the overall impact of fibromyalgia with the Fibromyalgia Impact Questionnaire Revised (FIQR), and the number of tender points at 18 standard sites on the body by applying a 4 kg pressure with the thumb on specific body points. Tender points were also scored as 0–4 points. (0-no pain, no reaction, 1-painful but no reaction, 2-painful but minimal reaction, 3-painful with irritation, 4-painful with excessive irritation) [9].

Laboratory parameters included estimation of NO by the Nitric Oxide Assay Kit, Protein carbonyl groups by Protein Carbonyl Assay Kit and malondialdehyde (MDA) as a marker for LPO was measured through the method described by Ohkawa et al. [10] in the plasma of FMS patients and an equal number of healthy controls.

2.4 Biochemical assay

2.4.1 Estimation of lipid peroxide levels

Blood plasma (0.2 ml) was mixed with 0.5 ml glacial acetic acid. Subsequently, 0.5 ml of 8% sodium dodecyl sulphate was added to the above reaction mixture. After mixing well, 1.5 ml of 0.8% tertiary butyl alcohol (TBA) solution was added. The reaction mixture was kept in a boiling water bath for 1 h. After cooling to room temperature, 3.0 mL of n-butanol was mixed, the reaction mixture was then centrifuged at 10000 $\times$ g for 15 min. The absorbance of clear butanol fraction obtained after centrifugation was read at 532 nm by UV-VIS double beam spectrophotometer (model no. 2203, serial no. 053, Systronics Instrumental Co., Hyderabad, India, year of purchase 2010). An appropriate standard made up of 2.5 nmol malondialdehyde was run simultaneously.

2.4.2 Protein carbonyl assay and nitric oxide assay

The estimation of protein carbonyl and nitric oxide was done by using the Abcam Protein Carbonyl Assay kKit and Abcam Nitric Oxide Assay kit.

2.4.3 Protein carbonyl assay

Sample preparation: Dissolve samples in dH2O and centrifuge to spin down any insolubles. Dilute samples with dH2O to approx. 10 mg/ml protein. If the protein is very diluted, it can be concentrated using a 10 kDa spin filter (ab93349). Use 100 $\mu$ l of sample containing approximately 0.5–2 mg protein per assay. Include a reagent background control by using 100 $\mu$ l of dH2O alone.

1.
Add 100 $\mu$ l DNPH to each sample, vortex and incubate for 10 min at room temperature.
2.
Add 30 $\mu$ l of TCA to each sample, vortex, place on ice for 5 min, spin at maximum speed for 2 min, remove and discard supernatant without disturbing pellet.
3.
Add 500 $\mu$ l of cold acetone to each tube and wash the pellet. 30 seconds in a sonicating bath is typically sufficient to effectively disperse the pellets. Place at $-$ 20 ${}^{\circ}$ C for 5 min then centrifuge for 2 min and carefully remove the acetone.
4.
Add 200 $\mu$ l of Guanidine solution and sonicate briefly. Most proteins will be resolubilized easily at this point. If your protein is resistant to resolubilization sonicate for a few seconds then let the solution sit at 60 ${}^{\circ}$ C for 15–30 min. Spin very briefly to pellet any unsolubilized material and transfer 100 $\mu$ l of each sample to the 96-well plate.
5.
Measure OD at $\sim$ 375 nm in a microplate reader.

Table 1
Levels of oxidative stress parameters and levels of toxic heavy metals and trace elements among FMS patients and healthy controls (Mann-Whitney U test)

Variables Median (Min.-Max.) $P$ value

Cases $N=$ 105 Controls $N=$ 105

Oxidative stress parameters

LPO ( $\mu$ mol/mg protein) 32.37 (6.83–45.01) 11.56 (8.00–27.54) $<$ 0.001*

NO ( $\mu$ mol/mg protein) 62.59 (0.75–458.65) 13.00 (8.00–20.00) $<$ 0.001*

Protein carbonyl ( $\mu$ mol/min/mg protein) 153.93 (43.69–714.31) 25.65 (20.43–115.84) $<$ 0.001*

Metals

Arsenic ( $\mu$ g/dl) 0.00 (0.00–50.00) 0.00 (0.00–0.00) 0.156

Copper ( $\mu$ g/dl) 106.00 (0.00–3348) 71.00 (9.00–136.00) $<$ 0.001*

Magnesium ( $\mu$ g/dl) 2184.0 (865.00–3931.00) 2699.0 (78.00–8921) $<$ 0.001*

Manganese ( $\mu$ g/dl) 1.00 (0.00–42.00) 0.00 (0.00–1.00) $<$ 0.001*

Lead ( $\mu$ g/dl) 0.00 (0.00–0.00) 0.0 (0.00–0.00) 1.000

Zinc ( $\mu$ g/dl) 51.00 (0.00–15.00) 192.00 (47.00–1620.00) $<$ 0.001*

Aluminium ( $\mu$ g/dl) 15.00 (0.00–159.00) 7.00 (0.00–159.00) $<$ 0.001*

LPO, Lipid peroxidation; NO, Nitric oxide; Toxic heavy metals: Arsenic, lead and aluminium; Trace elements: Magnesium, manganese, copper and zinc. $P$ value $p<$ 0.01 is considered significant; * Statistically significant by Mann-Whitney U test.

2.4.4 Nitric oxide assay

Variables	Median (Min.-Max.)	$P$ value
	Cases $N=$ 105	Controls $N=$ 105
Oxidative stress parameters
LPO ( $\mu$ mol/mg protein)	32.37	(6.83–45.01)	11.56	(8.00–27.54)	$<$ 0.001*
NO ( $\mu$ mol/mg protein)	62.59	(0.75–458.65)	13.00	(8.00–20.00)	$<$ 0.001*
Protein carbonyl ( $\mu$ mol/min/mg protein)	153.93	(43.69–714.31)	25.65	(20.43–115.84)	$<$ 0.001*
Metals
Arsenic ( $\mu$ g/dl)	0.00	(0.00–50.00)	0.00	(0.00–0.00)	0.156
Copper ( $\mu$ g/dl)	106.00	(0.00–3348)	71.00	(9.00–136.00)	$<$ 0.001*
Magnesium ( $\mu$ g/dl)	2184.0	(865.00–3931.00)	2699.0	(78.00–8921)	$<$ 0.001*
Manganese ( $\mu$ g/dl)	1.00	(0.00–42.00)	0.00	(0.00–1.00)	$<$ 0.001*
Lead ( $\mu$ g/dl)	0.00	(0.00–0.00)	0.0	(0.00–0.00)	1.000
Zinc ( $\mu$ g/dl)	51.00	(0.00–15.00)	192.00	(47.00–1620.00)	$<$ 0.001*
Aluminium ( $\mu$ g/dl)	15.00	(0.00–159.00)	7.00	(0.00–159.00)	$<$ 0.001*

The method for the indirect determination of NO involves the spectrophotometric measurement of its stable decomposition products NO-3 and NO-2. This method requires that NO-3 first be reduced to NO-2 and then NO-2 determined by the Griess reaction. Briefly, the Griess reaction is a two-step diazotization reaction in which the NO-derived nitrosating agent, dinitrogen trioxide (N ${}_{2}$ O ${}_{3}$ ) generated from the acid-catalyzed formation of nitrous acid from nitrite (or autoxidation of NO) reacts with sulfanilamide to produce a diazonium ion which is then coupled to $N$ -(1-napthyl)ethylenediamine to form a chromophoric azo product that absorbs strongly at 540 nm. For quantification of NO-3 and NO-2 in extracellular fluids, we found that enzymatic reduction of NO-3 to NO-2 using a commercially available preparation of nitrate reductase to be a satisfactory method. Following the incubation, any unreacted NADPH is oxidized by addition of lactate dehydrogenase and pyruvic acic because reduced pyridine nucleotides (NADPH, NADH) strongly inhibit the Griess reaction. A known volume of premixed Griess reagent is the added to each incubation mixture, incubated for 10 min and the absorbance of each sample determined at 543 nm.

2.5 Metal content assay

Metal content was analysed in the plasma sample of the patients and control groups as follows: Plasma was diluted 10 times with 1% HNO ${}_{3}$ prepared in milli Q water. This diluted sample was analyzed by Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES, Optima 8000, Perkin Elmer, USA). The standards of metal Al, Pb, As, Mg, Cu, Zn and Mn were prepared by diluting stock solutions of studied elements (1000 mg/L) in 0.2% (v/v) nitric acid. The dilutent was obtained from Perkin Elmer (USA). Blank sample was run simultaneously for each set of samples.

2.6 Statistical analysis

Statistical Package for Social Sciences (SPSS) software, version 20.0 was used for all statistical analyses. The sample size for the study was calculated by using the Snedecor and Cochran sample size calculating formula [8]. The normal distribution of the variables was assessed using the Shapiro Wilk test at 5% level of significance. The data was not normally distributed so non-parametric tests were used. To assess significant differences in median, the Mann-Whitney U test was used for statistical analysis between patient and control groups for oxidative stress parameters such as LPO, PC group, NO and metal ion contents. Karl Pearson’s correlation coefficient (r) was performed to find the relation of association between metal contents, FIQR total score and tender points and also for establishing the relationship between oxidative stress parameters and metal content. The results were considered significant at $P$ value less than 0.001.

3. Results

The mean age of the FMS patients was 38.8 $\pm$ 10.1 years (range: 18–60 years) and the mean age of the healthy controls was 36.9 $\pm$ 9.5 years (range: 18–60 years). As expected, females were more affected than men (80% female and 20% male).

The Mann-Whitney U test shows a statistically significant difference in oxidative stress markers such as LPO, PC groups and NO among FM patients and controls (Table 1). In contrast, a significant difference in the median levels of essential metals such as Zn, Mg and Mn, Cu and non-essential toxic metals such as Al was found in the patients with FMS and in controls, as shown in Table 1. Furthermore, no significant changes were found in blood plasma for Ar and Pb in both groups (Table 1).

Table 1a
Levels of oxidative stress parameters and levels of toxic heavy metals and trace elements among FMS patients and healthy controls (independent sample $t$ -test)

Variables	Mean (S.D.)				$P$ value
	Cases		Controls
Oxidative stress parameters
LPO ( $\mu$ mol/mg protein)	30.15	(8.28)	12.13	(3.55)	$<$ 0.01
NO ( $\mu$ mol/mg protein)	71.40	(5739)	13.60	(3.04)	$<$ 0.01
Protein carbonyl ( $\mu$ mol/min/mg protein)	157.82	(86.45)	28.70	(13.42)	$<$ 0.01
Metals
Arsenic ( $\mu$ g/dl)	0.54	(4.92)	0.00	(0.00)	0.26
Copper ( $\mu$ g/dl)	152.82	(37891)	7036	(18.90)	0.03
Magnesium ( $\mu$ g/dl)	3223.46	(2134.04)	2067.77	(600.97)	$<$ 0.01
Manganese ( $\mu$ g/dl)	2.09	(4.39)	0.24	(0.42)	$<$ 0.01
Lead ( $\mu$ g/dl)	0.00	(0.00)	0.00	(0.00)	–
Zinc ( $\mu$ g/dl)	266.50	(224.44)	46.14	(32.90)	$<$ 0.01
Aluminium ( $\mu$ g/dl)	26.19	(30.89)	10.94	(17.20)	$<$ 0.01

LPO, Lipid peroxidation; NO, Nitric oxide; Toxic heavy metals: Arsenic, lead and aluminium; Trace elements: Magnesium, manganese, copper and zinc. $P$ value $p<$ 0.01 and $p<$ 0.05 are considered significant.

Table 2

Karl Pearson’s product moment correlation between oxidative stress parameters and metal content with FIQR total score and tender points in fibromyalgia

Variables	FIQR total score (r)		Tender points (r)
Oxidative stress parameters
LPO (mmol/mg protein)	0	.79	0	.75
Protein carbonyl ( $\mu$ mol/min/mg protein)	0	.71	0	.65
NO	0	.56	0	.55
Metal content
Cu ( $\mu$ g/dl)	0	.145	0	.146
Mg ( $\mu$ g/dl)	$-$ 0	.349*	$-$ 0	.313*
Mn ( $\mu$ g/dl)	0	.245	0	.244
Zn ( $\mu$ g/dl)	$-$ 0	.529*	$-$ 0	.512*
Al ( $\mu$ g/dl)	0	.254	0	.283
Pb ( $\mu$ g/dl)	0	.00	0	.00
As ( $\mu$ g/dl)	0	.00	0	.00

FIQR $=$ Fibromyalgia Impact Questionnaire Revised; (r) $=$ Karl Pearson’s correlation coefficient; LPO: Lipid peroxidation; NO: Nitric oxide; Cu: Copper; Mg: Magnesium; Mn: Manganese; Zn: Zinc; Al: Aluminium; (-ve) denotes that r correlation could range between $-$ 1 and $+$ 1 with FIQR and TP; * $=$ Statistically significant; Pb and As have no association with FIQR and tender points.

Karl Pearson’s correlation (r) showed a significant relationship between oxidative stress parameters with FIQR total score and tender points (Table 2) as well as a relationship between metal content with severity of FMS symptoms (FIQR total score and tender points) (Table 2). A positive correlation of oxidative stress parameters and non-essential metal concentrations such as Al was observed with FIQR total score and tender points. However, a strong negative linear relationship of essential metals such as zinc was observed with FIQR total score and tender points, and a moderate negative linear relationship of magnesium was observed with FIQR total score and tender points. Moreover, a positive relationship of metal concentration was observed with oxidative stress markers (Table 3). However, a negative linear relationship was observed for oxidants (protein carbonyl and LPO) with zinc and magnesium, and a weak negative linear relationship was observed between nitric oxide and metal such as zinc and aluminium (Table 3).

Table 3

Karl Pearson’s product moment correlation between metal content and oxidative stress parameters in fibromyalgia

Metal content	Oxidative stress parameters
	Nitric oxide	Protein carbonyl ( $\mu$ mol/min/mg protein)	LPO (mmol/mg protein)
Cu ( $\mu$ g/dl)	0.128	0.066	0.167
Mg ( $\mu$ g/dl)	0.254	$-$ 0.377	$-$ 0.431
Mn ( $\mu$ g/dl)	0.113	0.222	0.186
Zn ( $\mu$ g/dl)	$-$ 0.314	$-$ 0.468	$-$ 0.460
Al ( $\mu$ g/dl)	0.313	0.180	0.168
Pb ( $\mu$ g/dl)	0.00	0.00	0.00
As ( $\mu$ g/dl)	0.00	0.00	0.00

LPO: Lipid peroxidation; Pb and As denote no association among the variables; (-ve) denotes that r correlation could range between $-$ 1 and $+$ 1.

4. Discussion

There are several studies providing evidence of toxic interaction of metals in humans. In the current study, we discussed the association of metal content with oxidative stress in individuals with FMS and the severity of its symptoms. Leads (Pb), arsenic (As), aluminium (Al) are non-essential and toxic metals whereas magnesium (Mg) and zinc (Zn), copper (Cu) and manganese (Mn) are essential trace elements.

4.1 Magnesium, Zinc and oxidative stress

The levels of Cu, Mn and Al were significantly higher in our study, whereas the levels of Mg and Zn were significantly lower in the patient group compared to the control group. No significant difference in the level of Pb and As was found in the studied groups. The patients in our study were found free from the presence of Pb and As. In the studies of Eisinger et al. [11, 12] and Al-Khalifa et al. [13], lower levels of Mg and Zn were found in the FMS patients, compared to the healthy controls, which is similar to our study. Another study by Abraham and Flechas suggested that the mechanism of pain and fatigue in FMS patients was possibly due to Mg deficiency [14]. Several expressions of FMS such as fatigue, muscle weakness, paresthesias are similar to the symptoms of Mg deficiency [15]. In the current study we observed that the level of Mg was lower and the higher levels of NO in the patient group and normal levels of Mg and lower levels of NO in control groups. Low Mg level elevates the production of NO, which suppresses processes such as vasodilation and mitochondrial dysfunction [15, 16]. Sendur et al. [17] reported a significant negative association of Mg and Zn with FIQR and TP similar to that of current study, whereas Russell et al. showed a highly significant association between Zn levels and tender points [18]. Zinc deficiency has been related to tissue oxidative damage by increasing the level of lipid, proteins and DNA oxidation. Long term deficiency of Zn makes an organism more prone to oxidative stress induced injury such as formation of ROS, associated with lung damage and malondialdehyde formation in the liver [19] similar to our study, the levels of Zn were lower in the patients and levels of oxidative stress markers were higher in the FMS patients compared to the healthy control group. Contrary to our study, Prescott et al. reported no change in Mg level in patients with FMS [20].

4.2 Copper and oxidative stress

In the study by Al-Gebori et al. [21] and Eck et al. [22], a higher level of Cu was found in the patients with FMS compared to the healthy control group, which is similar to our study. Cu is required for the Ca absorption in bones to built and repair connective tissue. Excessive Cu accumulation creates problem in soft tissue of the body [23]. The copper induced toxicity generates from the formation of cupric and cuprous ion. Cuprous ion generates hydroxyl radicals which results in lipid peroxidation via Fenton reaction [24]. However, the deficiency of Zn is responsible for elevation in the level of Cu in plasma. This Zn/Cu imbalance is associated with any previous episode of viral infection or predisposition towards viral susceptibility in patients with chronic fatigue syndrome (CFS), a disorder closely linked to FMS [38]. These factors together influence the symptoms and severity of FMS such as pain and fatigue.

4.3 Aluminium and oxidative stress

In the FMS patients Al was found to be toxic as the level of Al was significantly higher in the present study in comparison to the healthy controls. Also, a significant association of Al was found with the symptom severity of the syndrome i.e. FIQR total score and TP. Fatima et al. [25, 39] did not find any significant association of Pb and Al with FIQR and TP in the FMS patients and controls. The result of their study was in contrast to the current study. The reduced levels of Mg and Zn and elevated levels of Al, Cu and Mn in patients with FMS make us to think that Mg deficiency may lead to Al toxicity. Mg suppresses the toxic effects of Al in the body, it is an important cofactor in the metabolic energy production pathway. Additionally, the use of Al utensils for cooking, storing and drinking purposes appears to be the cause of Al toxicity in the patients with FMS. Also, the majority of the population in our study belongs to poor or middle class families whose socioeconomic status is low and use Al utensils. Although metals possess rich coordination chemistry and redox properties, they have the ability to produce reactive radicals, which leads to DNA damage, lipid peroxidation, protein carbonylation and other effects [26, 27, 28].

In this study we found a negative linear relationship of Zn and Mg for protein carbonyl and LPO. A strong negative correlation between Zn and NO and week positive relationship of NO with Al was found.

In the present study we also analysed the association of oxidative stress markers with FIQR and TP. The results indicated that patients in our study were under oxidative stress. We had found a positive correlation of oxidative stress markers such as LPO, protein carbonyl with FIQR and TP score. Eisinger et al. [29] and Bagis et al. [30] reported significant higher level of malondialdehyde (marker for LPO) and protein carbonyl in patients with FMS than in healthy controls. Hein and Franke [31] showed higher levels of pentosidine marker of oxidative stress in FMS. Cordero et al. [32] suggested the role of oxidative stress in headache symptoms associated with FMS. In another study by Fatima et al. [40], a significant association between body mass index (BMI) and FIQR was reported. The findings of these studies support the results of our study. Additionally, in the present study, we also showed a significantly positive association of NO with the severity of FMS (FIQR and TP). There are studies in support which showed increased levels of NO in patients with FMS [33, 34]. On the other hand there are other studies contradictory to our study, which represented lower levels of NO in FMS patients [35, 36]. NO is an important neurotransmitter which is involved in the spinal pain pathway [37]. The findings related to the role of trace elements, heavy metal and oxidative stress parameters in the pathophysiology of FMS are still conflicting. However, despite strong evidence in support of involvement of metal-induced oxidative stress in FMS, it is still difficult to determine whether oxidative stress is a cause or effect of primary fibromyalgia.

5. Conclusion

The involvement of metal toxicity and trace elements deficiency in FMS patients alters the oxidative stress parameters and effectively plays an important role in the etiopathogenesis and severity of FMS symptoms. An increase level of Al in FMS patients highlights the frequent environmental exposure to aluminium through drinking water, vegetables, air and also packaging and handling food in aluminium utencils. Al is considered one of the factors contributing to oxidative stress and may suppress non-enzymatic antioxidant Mg levels and Zn levels in FMS patients. The results provide evidence that toxic metals are capable of interacting with nuclear proteins and cellular lipids causing specific site damage such as lipid peroxidation, protein carbonyl group formation and also metal-driven formation of reactive nitrogen species such as nitric oxide.

However, complete knowledge of the biochemical and genetic events occurring at the cellular level that influence oxidative damage is required to guide future research. Supplementation of Mg and Zn according to the recommended dietary allowance and minimizing Al exposure and intake of antioxidants may benefit the treatment of FMS individuals.

5.1 Limitation

The limitation of the study is that food supplements and regular physical activities that influence oxidative stress could not considered. It would be interesting to study the effects of food supplements and regular physical activities in further research.

Footnotes

Acknowledgments

Funding to support this work was provided by the Indian Council of Medical Research, New Delhi, India (grant no. 5/4-ortho/2010-NCD-1).

Conflict of interest

There are no conflicts of interest regarding the publication of this article.

Ethical approval

Institutional Ethical Committee “King George’s Medical University”, ref. code: XL VI ECM/A-P9.

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Metal-induced oxidative stress level in patients with fibromyalgia syndrome and its contribution to the severity of the disease: A correlational study

Abstract

BACKGROUND:

AIMS:

METHOD:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Material and methods

2.1 Ethics statement

2.2 Design of the study and sampling

2.3 Inclusion and exclusion criteria

2.4 Biochemical assay

2.4.1 Estimation of lipid peroxide levels

2.4.2 Protein carbonyl assay and nitric oxide assay

2.4.3 Protein carbonyl assay

2.5 Metal content assay

2.6 Statistical analysis

3. Results

Table 1a Levels of oxidative stress parameters and levels of toxic heavy metals and trace elements among FMS patients and healthy controls (independent sample t -test)

4.1 Magnesium, Zinc and oxidative stress

4.2 Copper and oxidative stress

4.3 Aluminium and oxidative stress

5. Conclusion

5.1 Limitation

Footnotes

Acknowledgments

Conflict of interest

Ethical approval

References

Table 1a
Levels of oxidative stress parameters and levels of toxic heavy metals and trace elements among FMS patients and healthy controls (independent sample $t$ -test)