Serum prolidase enzyme activity and oxidative status in patients with fibromyalgia

Abstract

Objective

This study was performed to investigate serum prolidase enzyme activity and oxidative stress in patients diagnosed with fibromyalgia (FM).

Methods

The study population consisted of 40 patients with a previous diagnosis of FM and 30 healthy subjects. We measured serum prolidase enzyme activity, total antioxidant status (TAS), total oxidative status (TOS), oxidative stress index (OSI), and paraoxonase-1 (PON-1) levels.

Results

On average, FM patients were diagnosed within 3.2 years of symptom onset, and patients had a mean of 14 tender points. There were no significant differences between patients and controls in age, body mass index, serum TAS, or PON-1 levels. However, patients with FM demonstrated higher serum prolidase activity, TOS, and OSI than the control group. Serum prolidase activity was positively correlated with serum TOS, OSI, and visual analog scale pain and fatigue scores. No correlation was found between serum prolidase activity and FM duration or the average number of tender points.

Discussion

Our results demonstrate a previously unreported association between serum prolidase enzyme activity and FM. Increased prolidase activity may contribute to the pathogenesis of FM, and measuring serum prolidase enzyme activity may be a useful FM biomarker.

Keywords:

• Prolidase
• Oxidative status
• Fibromyalgia

Introduction

Fibromyalgia (FM) is a chronic musculoskeletal syndrome characterized by diffuse pain, fatigue, stiffness, and tenderness at specific anatomic sites termed ‘tender points’.^1,2 FM is more commonly diagnosed in women than in men, and its etiology remains unknown.^1,3 Alterations in collagen metabolism may contribute to FM pathogenesis. Nerve endings located in tender points have a distinctive histological appearance involving organized collagen matrix layers, which may indicate abnormal collagen metabolism at these sites. Specifically, trapezius biopsies from FM patients exhibit a characteristic collagen cuff that surrounds axons, which has been associated with myofibril disorganization contributing to post-exercise pain and stiffness.⁴ The authors of a previous study suggested that understanding collagen metabolism in FM may be central to understanding its etiology.⁵

Prolidase is an enzyme that participates in collagen metabolism and is expressed in both human and animal tissues. It participates in the final, rate-limiting step of collagen catabolism.⁶ Prolidase activity increases when extracellular matrix remodeling occurs, which necessitates a high rate of collagen turnover.⁷ In a culture system, increased prolidase activity was triggered by increased fibroblast density.⁸ Altered prolidase activity is also observed in the settings of oxidative stress and diseases characterized by fibrosis.⁹

Oxidation is a chemical reaction that transfers electrons or hydrogen atoms from a substance to an oxidizing agent, and this can cause cell damage or death. Antioxidants terminate this reaction by removing free radicals and inhibiting various oxidation reactions. When antioxidant levels are reduced, oxidative stress can result in cell structural damage and impaired cell function.¹⁰ Oxidative stress has been implicated in FM pathophysiology.^1,11,12 Plasma lipid peroxidation (LP) and protein carbonylation both generate reactive oxygen species (ROS) and are increased in patients with FM. Oxidative stress can cause endothelial dysfunction, fibroblast proliferation, and increased type I collagen synthesis.¹³ In addition, ROS can induce secretion of inflammatory and fibrogenic cytokines.¹⁴ There is evidence that FM patients have alterations in muscle metabolism and structure that are reflective of oxygenation abnormalities and/or heightened oxidative stress. Muscle and neural tissue are especially sensitive to free radical damage,¹⁵ and aberrant oxygenation has been reported in trigger point areas, which could induce dysregulated pain processing by the central nervous system.⁹ There are strong relationships among oxidative stress, major depression,¹⁶ pain,^1,11,17 and fatigue,¹⁶ which is commonly observed in FM patients.

Although the contribution of oxidative stress to FM pathophysiology has been assessed in several investigations, the role of serum prolidase in patients with FM has not been studied. The purpose of this study was to investigate serum prolidase enzyme activity and oxidative stress in patients with FM.

Materials and methods

This study was conducted between February and May 2012 by the Physical Therapy and Rehabilitation Department at Dicle University in Turkey. We enrolled a total of 40 patients diagnosed with primary FM, according to the American College of Rheumatology criteria, who were between 18 and 50 years of age, had signed the informed consent form, and did not satisfy any of the exclusion criteria.¹⁸ Healthy hospital staff members (n = 30) served as control subjects and were frequency-matched for age and body mass index (BMI). Healthy controls had no signs or symptoms of FM. The entire study population (n = 70) was female. This study was approved by the ethics committee (Dicle University).

Exclusion criteria

Patients were excluded for the following reasons: inflammatory rheumatic disease or infectious or endocrine-related arthropathy; pregnancy or lactation; chronic disorders including coronary artery disease, diabetes mellitus, hypertension, dyslipidemia, or renal disease; malignancy; systemic or local infection in the previous 4 weeks; past or present psychiatric, neurological, autoimmune, or allergy-related disease; or regular use of analgesics, glucocorticoids, immunosuppressive agents, anti-depressants, anti-epileptic drugs, or medication use within 4 weeks of the beginning of the study. None of the patients or control participants had taken drugs or vitamin/nutritional supplements in the 4-week period before blood sample collection. All patients and controls were non-smokers and reported that they consumed a standard diet.

Routine blood tests, erythrocyte sedimentation rate (ESR), total antioxidant status (TAS), total oxidative status (TOS), oxidative stress index (OSI), paraoxonase-1 (PON-1), and prolidase activity were evaluated for all subjects. Demographic and clinical data were obtained from subject interviews, chart reviews, physical examinations, and questionnaires.

FM patients completed the Fibromyalgia Impact Questionnaire (FIQ)¹⁹ and Modified Fatigue Impact Scale (M-FIS).²⁰ The clinical severity or the current health status of FM patients was evaluated using the FIQ, which is a self-reported instrument comprising 10 items (physical impairment, feel good, work missed, interference with job, pain, fatigue, morning tiredness, stiffness, anxiety, and depression). Each item was standardized on a scale ranging from 0 to 10. The impact of fatigue was assessed using the M-FIS, which consists of 21 items including 10 related to mental fatigue and 11 related to physical and social fatigue.

All study subjects completed two visual analog scales (VAS) for pain and fatigue; these were scored on a 10-point scale, where 0 represents symptom absence and 10 represents the maximum impact.²¹ About 4 kg of force was applied via palpation to assess tender points.

Biochemical analyses

After collection, fasting blood samples were immediately centrifuged at 4000 rpm for 10 minutes. Serum samples were then transferred to Eppendorf tubes for storage at −50°C before analysis was performed.

Measurement of the TAS

TAS of serum was determined using a novel automated measurement method, developed by Erel.²² This method produces hydroxyl radicals, which are the most potent biological radical. The sequentially produced radicals, such as brown-colored dianisidinyl radical cations produced by the hydroxyl radical, are also potent. This method measures the antioxidant effect of the sample against the potent free radical reactions, which is initiated by the produced hydroxyl radical. The assay has excellent precision values of lower than 3%. The results are expressed as μmol Trolox equivalent (equiv.)/l.

TOS measurement

Serum TOS was determined using a novel automated measurement method developed by Erel.²³ Oxidants present in the sample oxidize the ferrous ion-o-dianisidine complex to ferric ion. The oxidation reaction is enhanced by glycerol molecules, which are abundantly present in the reaction medium. The ferric ion forms a colored complex with xylenol orange in an acidic medium. The color intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules present in the sample. The assay is calibrated with hydrogen peroxide, and the results are expressed in μmol H₂O₂ equiv./l).

OSI determination

The percent ratio of TOS level to TAS level was considered as the OSI, which indicates the degree of oxidative stress.^24,25 OSI was calculated according to the following formula: OSI (arbitrary unit) = TOS (μmol H₂O₂ equiv./l)/TAS (μmol Trolox equiv./l.

Serum prolidase levels (U/l) were determined using a spectrophotometric method that measures the levels of proline produced by prolidase. The supernatant was diluted two-fold with physiologic saline (0.9% NaCl). Next, 25 µl of the mixture was preincubated with 75 µl preincubation solution (50 mmol/l Tris–HCl buffer (pH 7.0) containing 1 mmol/l glutathione and 50 mmol/l MnCl₂) at 37°C for 30 minutes. A total of 100 µl reaction mixture, which was composed of 144 mmol/l gly–pro (pH 7.8), was incubated with 100 µl preincubated sample at 37°C for 5 minutes. To stop the incubation reaction, 1 ml glacial acetic acid was added. After adding 300 µl Tris–HCl buffer (pH 7.8) and 1 ml ninhydrin solution (3 g/dl ninhydrin was dissolved in 0.5 mol/l orthophosphoric acid), the mixture was incubated at 90°C for 20 minutes and then cooled with ice. Absorbance was then measured at a 515-nm wavelength to determine proline content according to the method proposed by Myara et al.,²⁶ which is a modification of Chinard's method.²⁷ The intra- and interassay coefficients of variation were both lower than 7%.

Serum PON-1 levels (U/l) were measured with an Anthos Zenyth 200rt spectrophotometer (Biochrom Ltd., Cambridge, UK) via the modified Eckerson method. Initial paraoxon (diethyl 4-nitrophenyl phosphate) phosphate hydrolysis rates were determined by measuring liberated p-nitrophenol at 405 nm at 37°C (Sigma, St. Louis, MO, USA).²⁸

Statistical analyses

All data were expressed as the mean ± one standard deviation. Kolmogorov–Smirnov tests were performed to determine whether the data were normally distributed. Intergroup comparisons were performed using Mann–Whitney U tests and Student's t-tests. Relationships between variables were analyzed by either Pearson or Spearman correlation analyses depending on how the variables were distributed. Statistical significance was achieved if the P value was less than 0.05. All data were analyzed with SPSS statistical software package 18.0 for Windows (IBM Corp., Armonk, NY, USA).

Results

Demographic and clinical data, as well as blood laboratory results, are displayed in Table 1. The mean delay from symptom onset to FM diagnosis was 3.2 years, and the average number of tender points in FM patients was 14. The mean ages were 36.20 ± 7.97 years and 34.30 ± 6.19 years for FM patients and controls, respectively. No statistically significant difference was demonstrated between groups in terms of age (P = 0.266), BMI (P = 0.062), mean hematocrit level (P = 0.253), mean C-reactive protein (CRP) level (P = 0.486), mean ESR (P = 0.848), and mean white blood cell counts (WBC) (P = 0.191). However, the VAS pain and fatigue scores were significantly higher in patients with FM compared with the control group (P = 0.000).

Table 1. Demographics and clinical and laboratory results for FM patients and controls

	FM (n = 40)	Control (n = 30)	P value
FM duration (years)	3.21 ± 3.52	—
Tender points	13.7 ± 1.85	—
Age (years)	36.20 ± 7.97	34.30 ± 6.19	0.266
VAS (pain)	6.32 ± 1.62	0.93 ± 1.65	0.000
VAS (fatigue)	6.28 ± 1.65	1.80 ± 1.68	0.000
BMI (kg/m^2)	24.84 ± 2.95	23.80 ± 4.05	0.062
Mean hematocrit (%)	37.25 ± 4.30	36.25 ± 2.64	0.253
Mean CRP (mg/dl)	0.84 ± 0.38	0.94 ± 0.41	0.486
Mean ESR (mm/h)	8.91 ± 4.59	9.13 ± 4.65	0.848
Mean WBC (cells/μl)	7.083 ± 1.50	6.55 ± 1.68	0.191

FM, fibromyalgia; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; WBC, white blood cell count; VAS, visual analog scale; BMI, body mass index.

Serum prolidase activity (P = 0.004), TOS (P = 0.006), and OSI (P = 0.001) were all significantly increased in the FM group. Serum TAS levels were lower in FM patients, but this difference was not statistically significant compared with the control group (P = 0.069). Serum PON-1 levels were higher in the FM group, but this was not statistically significant (P = 0.451). Serum prolidase activities are shown in Fig. 1. TAS, TOS, OSI, and PON-1 levels are shown in Table 2.

Figure 1. Serum prolidase enzyme activity in patients with FM and control subjects.

Table 2. Serum TAS, TOS, OSI, and PON-1 levels in control and FM groups

	FM (n = 40)	Control (n = 30)	P value
TAS (μmol Trolox equiv./l)	1.45 ± 0.13	1.50 ± 0.12	0.069
TOS (μmol H2O2 equiv./l)	28.06 ± 14.85	20.69 ± 4.56	0.006
OSI (arbitrary unit)	19.50 ± 10.47	13.72 ± 2.78	0.001
PON-1 (U/l)	59.27 ± 21.39	55.30 ± 19.17	0.451

TAS, total antioxidant status; TOS, total oxidant status, OSI: oxidative stress index; PON-1, paraoxonase-1; FM, fibromyalgia.

Serum prolidase activity was positively correlated with TOS (P = 0.010, r = 0.305), OSI (P = 0.002, r = 0.410), VAS pain scores (P = 0.010, r = 0.300), and VAS fatigue scores (P = 0.020, r = 0.320). However, serum prolidase activity was negatively correlated with TAS (P = 0.003, r = 0.348). There was no correlation between serum prolidase activity and the average number of tender points (P = 0.618, r = 0.084) or between FM duration and prolidase activity (P = 0.086, r = 0.384). Notably, prolidase and all oxidative stress markers (TAS, TOS, OSI) were correlated with VAS fatigue score. These correlations are summarized in Table 3.

Table 3. Correlations between prolidase, TAS, TOS, OSI, PON-1, and clinical variables in control and FM groups

	Prolidase (U/l)	TAS (μmol Trolox equiv./l)	TOS (μmol H2O2 equiv./l)	OSI (arbitrary unit)	PON-1 (U/l)
Prolidase, n = 70		r = −0.348	r = 0.305	r = 0.410	r = −0.113
		P = 0.003	P = 0.010	P = 0.000	P = 0.350
Disease duration (year), n = 40	r = 0.384	r = −0.384	r = 0.180	r = 0.232	r = −0.351
	P = 0.086	P = 0.086	P = 0.435	P = 0312	P = 0.118
Tender points, n = 40	r = 0.084	r = − 0.018	r = −0.148	r = 0.063	r = 0.150
	P = 0.618	P = 0.913	P = 0.375	P = 0.709	P = 0.368
VAS (pain), n = 70	r = 0.300	r = −0.212	r = 0.235	r = 0.313	r = 0.058
	P = 0.010	P = 0.078	P = 0.050	P = 0.008	P = 0.632
VAS (fatigue), n = 70	r = 0.320	r = −0.248	r = 0.239	r = 0.364	r = 0.084
	P = 0.020	P = 0.041	P = 0.050	P = 0.002	P = 0.496
M-FIS, n = 40	r = 0.195	r = −0.212	r = 0.146	r = 0.317	r = 0.099
	P = 0.114	P = 0.085	P = 0.238	P = 0.009	P = 0.427
FIQ, n = 40	r = 0.208	r = −0.226	r = 0.262	r = 0.419	r = 0.140
	P = 0.091	P = 0.066	P = 0.032	P = 0.000	P = 0.258

FM, fibromyalgia; VAS, visual analog scale; FIQ, fibromyalgia impact questionnaire; M-FIS, modified fatigue impact scale; TAS, total antioxidant status; TOS, total oxidant status; OSI, oxidative stress index; PON-1, paraoxonase-1.

r = Pearson's correlation coefficient.

Discussion

Prolidase is a ubiquitous enzyme that catalyzes the rate-limiting step of collagen catabolism and functions in dietary protein degradation.⁴ Prolidase promotes collagen metabolism because it recycles proline for use in collagen biosynthesis, and this enzyme is dysfunctional in certain pathological conditions.^29–32 For instance, free radicals promote oxidative stress and alter prolidase activity, which prevents collagen fibril formation.³³ Duong et al.³⁴ observed a statistically significant difference in prolidase activity in keloid tissue compared with normal skin. Therefore, prolidase might be a useful marker in measuring severity of conditions characterized by fibrosis. Moreover, the extent of prolidase activity might have a role in FM pathogenesis; tissue biopsies of tender points exhibit collagen cuffs. Urinary hydroxyproline concentrations are significantly lower in FM patients compared with age- and sex-matched controls, indicating a systemic alteration in collagen metabolism.⁵ However, there is no literature describing serum prolidase activity in patients with FM. In this study, we observed significantly higher serum prolidase activity in FM patients compared to healthy controls. Additionally, serum prolidase activity was positively correlated with VAS pain and fatigue scores. However, there was no correlation between serum prolidase activity and the average number of tender points or FM duration.

Prolidase is a matrix metalloproteinase, and its activity is affected by oxidative stress.³⁵ Plasma prolidase activities are elevated in chronic tissue inflammation and positively correlate with TAS.³⁶ In this study, the serum prolidase activity was positively correlated with TOS and negatively correlated with TAS. However, we did not observe a relationship between serum prolidase activity and acute phase reactants.

Aberrant pain-regulating mechanisms at the central and peripheral nervous system levels contribute to FM pathophysiology.³⁷ As high oxidant levels were observed in low antioxidant settings in FM patients in this study, it is possible that oxidative stress might contribute to neuropathic pain in FM. OSI is the ratio of TOS to TAS and has been used as a novel indicator of oxidative stress extent.³⁸ A recently published study reported that plasma TAS in FM patients was significantly lower in healthy control subjects. Similarly, another group found that OSI values for FM patients were significantly higher than those for controls.⁹ Neyal et al.¹⁸ also described increased TOS and OSI in patients with FM. We found that in general, serum TAS was lower in FM patients, but this finding was not statistically significant. However, we observed significantly higher serum TOS and OSI levels in FM patients.

Located on high-density lipoprotein (HDL), the PON-1 enzyme hydrolyzes oxidized low-density lipoprotein (LDL). PON-1 also protects HDL from oxidation and is therefore protective against oxidative stress.³⁹ The antioxidant properties of PON-1 then facilitate HDL-mediated protection against atherosclerosis.^40,41 Altındag et al.⁴² reported that patients with FM had significantly lower PON-1 and arylesterase activities than healthy controls. We found that serum PON-1 levels were higher in the FM group compared with the control group; however, this difference was not statistically significant.

Acute phase reactants, such as CRP, ESR, and WBC, are important markers that may aid in making diagnoses and determining medical treatment efficacy for patients with inflammatory diseases. Cordero et al.⁴³ reported that inflammation plays a role in FM pathophysiology in some patients and concluded that oxidative stress and mitochondria are contributing factors for the development of inflammation. Another study found that pro-inflammatory cytokine levels were elevated in serum and biopsies from FM patients.⁴⁴ Another group described a positive correlation between plasma LP and clinical symptoms for VAS for total FIQ score in FM patients. In this study, we found that levels of acute phase reactants were not significantly different between controls and FM patients. Cordero et al.⁴⁵ reported a significant correlation between LP in blood mononuclear cells and clinical parameters in FM patients. They also described a positive correlation between plasma LP and clinical symptoms of FM. Here, we found that all oxidative stress markers assessed in plasma (TAS, TOS, and OSI) were correlated with VAS fatigue scores in FM patients.

Our study is a preliminary investigation designed to provide information about serum prolidase activity in patients with FM, and it has several limitations. We assessed a limited sample, and the study was cross-sectional in design. Also, it was not randomized. Assessing prolidase activity in terms of clinical findings would be more informative; however, a subgroup classification could not be done in these small study groups. Further prospective and randomized studies with larger samples are needed to clarify this issue.

Conclusion

Serum prolidase enzyme activity and oxidative stress were both significantly higher in patients with FM compared with healthy controls. These data demonstrate a previously unreported association between serum prolidase enzyme activity and FM. Increased prolidase activity may contribute to FM pathogenesis; however, prospective, randomized control trials are needed to elucidate this possible association and identify prolidase activity levels for diagnosing and treating patients with FM.