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Systemic Alterations in Plasma Proteins from Women with Chronic Widespread Pain Compared to Healthy Controls: a Proteomic Study

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Systemic Alterations in Plasma Proteins from Women with Chronic Widespread Pain Compared to Healthy Controls: a Proteomic Study Journal name: Journal of Pain Research Article Designation: ORIGINAL RESEARCH Year: 2017 Volume: 10 Journal of Pain Research Dovepress Running head verso: Wåhlén et al Running head recto: Plasma proteomics in women with CWP open access to scientific and medical research DOI: http://dx.doi.org/10.2147/JPR.S128597 Open Access Full Text Article ORIGINAL RESEARCH Systemic alterations in plasma proteins from women with chronic widespread pain compared to healthy controls: a proteomic study Karin Wåhlén Abstract: Chronic widespread pain (CWP) is a complex pain condition that is difficult to treat. Patrik Olausson The prevalence of CWP approximates ~10% of the general population, with higher prevalence Anders Carlsson in women. Lack of understanding of molecular mechanisms has been a challenge for diagnosis Nazdar Ghafouri and treatment of chronic pain. The aim of this study was to explore the systemic protein changes Björn Gerdle in CWP compared to those in healthy controls (CON). By applying 2-dimensional gel electro- phoresis, we analyzed the protein pattern of plasma samples from women with CWP (n 16) Bijar Ghafouri = and healthy women (n=23). The proteomic data were analyzed using multivariate statistical Pain and Rehabilitation Centre, models, and altered proteins were identified using mass spectrometry. The proteome analysis Department of Medical and Health Sciences, Linköping University, was further validated by gel-free Western blot. Multivariate statistical data analysis of quantified For personal use only. Linköping, Sweden proteins revealed 22 altered proteins in women with CWP, compared to CON group. Many of the identified proteins are previously known to be involved in different parts of the comple- ment system and metabolic and inflammatory processes, e.g., complement factor B, vitamin D-binding protein, ceruloplasmin, transthyretin and alpha-2-HS-glycoprotein. These results indicate that important systemic protein differences exist between women with CWP and healthy women. Further, this study illustrates the potential use of proteomics to detect biomarkers that may provide new insights into the molecular mechanism(s) of chronic pain. However, further larger investigations are required in order to confirm these findings before it will be possible to identify proteins as potential pain biomarkers for clinical use. Keywords: inflammation, biomarker, painomics, complement system, GC protein Journal of Pain Research downloaded from https://www.dovepress.com/ by 85.228.200.17 on 22-May-2017 Introduction Chronic widespread pain (CWP) is a complex pain condition that is difficult to treat. The prevalence of CWP in the European population is ~10%,1–3 whereof 2–4% is further diagnosed with fibromyalgia syndrome (FMS).4–6 According to the American College of Rheumatology (ACR) criteria from 1990, CWP is defined as pain present in at least 3 of 4 body quadrants, including axial skeleton pain, and persists for at least 3 months.7 Individuals diagnosed with CWP/FMS often experience tiredness, sleeping disorders and other psychological and somatic disorders, e.g., irritable bowel syndrome and stiffness 1,8–11 Correspondence: Karin Wåhlén in muscle and joints. There is evidence that CWP/FMS is more prominent among Pain and Rehabilitation Centre, women with increased age, obesity, depression and low physical activity.4,12 Department of Medical and Health Central alterations in the pain matrix in the brain and central hyperexcitability are Sciences, PAINOMICS® Laboratory, Linköping University, SE-581 85 known to be part of the underlying mechanism in CWP/FMS.13–15 There are several Linköping, Sweden studies suggesting that peripheral factors and alterations could have a prominent role Tel +46 10 103 4155 Email [email protected] and therefore contribute to the maintenance of pain and increased pain sensitivity in submit your manuscript | www.dovepress.com Journal of Pain Research 2017:10 797–809 797 Dovepress © 2017 Wåhlén et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work http://dx.doi.org/10.2147/JPR.S128597 you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Powered by TCPDF (www.tcpdf.org) 1 / 1 Wåhlén et al Dovepress CWP/FMS, e.g., alterations in muscle16–19 and peripheral were used for classification of FMS.7 The recruitment process nociceptors.20–22 However, the molecular mechanisms behind resulted in 19 CWP patients. As it has been reported previ- CWP are not fully elucidated. ously30 based on the concentration of lactate using power and Applying proteomics as an approach to study new bio- sample size calculation (a=0.05, power=0.8), it was found markers in different body tissues, from individuals with that 17 subjects in each group were needed. different diseases, has been used in several studies. The To confirm the individual eligibility, all participants (CON proteome of serum from rheumatoid arthritis (RA) patients,23 and CWP) received a standardized clinical examination. cerebrospinal fluid (CSF) from patients with neuropathic The examination was followed by a health questionnaire pain24 and the interstitial fluid of the trapezius muscle from concerning chronic pain,30 which included questions about patients with CWP/FMS25 have been investigated. A prelimi- pain intensity in the neck–shoulder and low back regions nary proteomic study of human serum from FMS patients using numeric rating scale (NRS, 0=no pain and 10=worst found upregulated levels of 3 proteins, α1-antitrypsin, trans- possible pain), anxiety and depressive symptoms (Hospital thyretin and retinol-binding protein 4, compared to healthy Anxiety and Depression Scale [HADS; cutoff values for both controls (CON).26 Further, the metabolic profile in serum subscales >10]), catastrophizing (Pain Catastrophizing Scale from women with localized and widespread pain has recently [PCS]; cutoff >37) and quality of life (QoL). been investigated, suggesting systemic changes in processes All participants signed a written consent before the start related to lipid metabolism and energy consumption.27 of the study, after receiving verbal and written informa- We have previously performed proteomics on muscle tion about the objectives and procedures of the study. The biopsies from patients with CWP/FMS and found protein study was approved by the Regional Ethical Review Board alterations involved in metabolic pathways, inflammation in Linköping, Sweden (Dnr: M10–08, M233–09, Dnr: and muscle recovery.28 Further, the pain intensity and pres- 2010/164–32) and followed the guidelines according to the sure pain threshold correlated significantly with specific Declaration of Helsinki. All methods were carried out in proteomic patterns.29 In this study, from the same cohort, we accordance with the approved ethical application. For personal use only. aimed to analyze the protein pattern, and potential new bio- markers, in plasma sample from female CWP/FMS patients Sample collection compared to healthy CON. The participants were asked not to take nonsteroidal anti- inflammatory drugs for 7 days and/or paracetamol medication Materials and methods for 12 hours prior to the sampling. Venous blood samples Subjects were collected in EDTA vacutainer and centrifuged at 1000× The recruitment process, including the inclusion and exclu- g for 15 minutes, plasma was collected, aliquoted and stored sion criteria for the patients with CWP and healthy CON, at −70°C until use. All samples were blinded before analy- has been described in detail previously.30 Briefly, according sis. There were some difficulties with blood sampling for 2 to the exclusion criteria, none of the included participants subjects, and the sample from 1 subject was not sufficient for Journal of Pain Research downloaded from https://www.dovepress.com/ by 85.228.200.17 on 22-May-2017 in either group used any type of anticoagulatory, opioid or further proteomic analysis. Therefore, plasma samples from steroidal medication. Further, if a medical history record of 16 women with CWP were included in this study. bursitis, tendonitis, capsulitis, postoperative conditions in the neck/shoulder area, previous neck trauma, disorder of Proteomics the spine, neurological disease, RA or any other systemic The procedure for 2-dimensional gel electrophoresis (2-DE) diseases, metabolic disease, malignancy, severe psychiatric has been described in previous studies.24,28 Briefly, 40 µL illness or pregnancy or difficulties understanding the Swed- plasma sample from each subject was depleted of albumin ish languages were present, participants were excluded from and immunoglobulin G (IgG) using ProteoPrep (Sigma- the study. The healthy CON group was recruited through Aldrich Co, St Louis, MO, USA), followed by protein con- local newspaper advertisement and consisted of 23 women centration measurement using 2D-Quant Kit (GE Healthcare, aged between 20 and 65 years. Female patients with CWP Little Chalfont, UK). Samples were further desalted with were recruited to this project by either being among former PD-10 columns (GE Healthcare)
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