Lipid profile analysis in spinal trauma patients shows severe distortion of AA/DHA after injury

Abstract

Event Abstract Back to Event Lipid profile analysis in spinal trauma patients shows severe distortion of AA/DHA after injury Danuta Radzioch1, 2*, Juan B. De Sanctis3, Gabriella Wojewodka1, 2, Mohan Radhakrishna1, 4, Ioli Makriyianni4, Stefan Parent5, 6, Jean Ouellet1, 7 and Samuel David1, 2 1 McGill University, Canada 2 Montreal General Hospital Research Institute, Canada 3 Central University of Venezuela, Venezuela 4 Montreal General Hospital, McGill University Hospital Center, Canada 5 Université de Montréal, Canada 6 Hôpital Sacré-Coeur de Montréal, Canada 7 Shriners Hospital Canada, Canada ABSTRACT Studies have shown fatty acid abnormalities in various neurological disorders with increased arachidonic acid (AA), an omega-6 pro-inflammatory fatty acid, and decreased levels of docosahexaenoic acid (DHA), an omega-3 fatty acid which reduces inflammation. Using a mouse model of spinal contusion injury, we previously showed rapid increases in AA/DHA ratio in the plasma and spinal cord of mice after spinal cord injuries. We found that treatment with the drug fenretinide corrected the fatty acid defects and improved recovery in mice. In this study, we hypothesized that AA and DHA may also be impaired in patients following trauma to the spine. Here we report preliminary results from our study including 23 spinal cord injury patients and 8 spinal fracture patients. Plasma samples were taken at various times post injury: 1, 2, 3, 8-16 and 25-49 days. We assessed the levels of AA, DHA and malondialdehyde (MDA), a marker of lipid peroxidation. The greatest changes in fatty acids occurred immediately after spinal cord trauma with increased AA and decreased DHA in both types of injuries. Thus the AA/DHA ratio was distorted towards a more pro-inflammatory status. Additionally, MDA increased after spinal cord injury which indicated higher levels of peroxidation. At later time points, the fatty acid levels returned to normal and peroxidation decreased as well. Our results provided confirmation for our hypothesis that AA/DHA ratio is distorted in the plasma immediately after spinal cord injuries and may be a factor in contributing to further neuronal damage. This is important as our data suggest that immediate application of fenretinide after a spinal injury in the rodent model might decrease secondary damage and improve neurological outcomes. INTRODUCTION Injury to the spinal cord leads to functional impairments due to loss of neurons, glia, myelin and the disruption of axonal pathways. This functional loss is permanent after spinal cord injury (SCI) because of the limited capacity of the CNS for regeneration and self-renewal (1). Several studies indicate that individuals with various neurological disorders have increased levels of arachidonic acid (AA), the omega-6 polyunsaturated fatty acid (PUFA) which causes inflammation, and decreased levels of docosahexaenoic acid (DHA), the omega-3 PUFA which reduces inflammation (2-4). These two fatty acids are also involved in many other biological functions in addition to inflammation. After SCI, PUFAs appear to be important regulators of the onset and resolution of the inflammatory response (5). AA is metabolized by cyclooxygenases and lypooxygenases giving rise to prostaglandins, thromboxanes and leukotrienes, which are potent pro-inflammatory mediators (6). Conversely, DHA, with its anti-inflammatory properties, plays a role in the resolution of inflammation by generating lipoxins, protectins and resolvins of the D series which actively terminate inflammation (7-9). In addition, DHA also has potent free radical scavenging and anti-oxidant properties (10). We hypothesized that the levels AA and DHA would be affected in patients with spinal traumas. METHODS Study Protocol. Patients with an isolated spinal cord injury (SCI) or isolated spinal fracture (SF) were recruited from the Montreal Sacré-Coeur Hospital (Montreal, Qc, Canada) and the Montreal General Hospital (Montreal, Qc, Canada) emergency departments. Here we report preliminary data on 23 SCI and 8 SF patients. The study was approved by the institutional review boards for both study sites. Patients and family members were asked to give consent to participate in the study. The inclusion criteria for isolated SCI included: up to 65 years of age, complete American Spinal Injury Association (ASIA) A grade of injury, incomplete (ASIA) B, C or D. The inclusion criteria for isolated SF included: up to 65 years of age, ASIA E (no spinal cord deficit), thoracic, cervical, and lumbar burst fractures with no spinal cord injury that have had conservative treatment or surgical intervention. Exclusion criteria included: older than 65 years of age, presence of tumors, other major trauma such as in long bones (femur, tibia, fibula, humerus, ulna, radius), traumatic brain injury, Type B or C pelvic fractures, Cauda Equina Syndrome, pregnancy, metabolic diseases and more than 72 hours post injury. Ten healthy volunteers were recruited and consented as controls for the fatty acid analysis. Sample collection and PUFA analysis. Blood samples were collected at 1, 2, 3, 8-16 and 25-49 days in EDTA coated tubes. Samples were spun at 3000 rpm at 4°C for 10 min for plasma isolation. For PUFA analysis, 100 µl of plasma was added to 900 µl of 1 mM butylated hydroxyanisole (BHA) in a 2:1 chloroform/methanol solution. Lipids were isolated according to the Folch method (11). AA and DHA were assessed by gas chromatography as previously described (12, 13). We assessed lipid peroxidation by indirectly measuring malondialdehyde (MDA) using the thiobarbituric acid reactive species (TBARS) assay (13). RESULTS AND DISCUSSION Using a mouse model of spinal contusions we previously demonstrated that spinal traumas have severe effects on PUFA levels. We found increases in AA and decreases in DHA localized to the spinal cord tissue which were also present systemically in blood plasma. When treated with the semi-synthetic retinoid fenretinide, the mice were protected from the changes in fatty acids and had better recovery of locomotor function (14). We found similar effects on PUFAs in our patient groups after spinal injury. In both SCI and SF patient groups, AA levels were higher than HC levels at day 1 (Figure 1A) and DHA levels were lower than normal values (Figure 1B). Overall, the AA/DHA ratios were also abnormally high. With time, the PUFA levels normalized at the 8 – 16 days post-injury time points. Lipid peroxidation was higher in both patient groups compared to HC values and normalized 1 to 2 weeks after injury, similar to PUFA kinetics. In general, patients were in a greater state of inflammation on day 1 with higher AA and MDA, and lower DHA values. Over time, these levels returned to normal due to the possible effect of current treatments on oxidative stress and improve the AA/DHA ratio to a more anti-inflammatory state. We did not observe any differences in fatty acids between SCI and SF patients thus far although MDA levels in SF patients tended to be higher on day 1 than in SCI patients. The pathway downstream of AA, namely the cyclooxygenase-2 enzyme that generates various eicosanoids, has been shown to contribute to the inflammation-induced secondary damage and functional loss after SCI. Inhibition of COX-2 results in improvement in functional recovery, reduced lesion size and an increase in viable tissue in mild forms of SCI (15, 16). Neuroinflammation was also shown to be modulated by various metabolites of phospholipase A2 (PLA2) affecting inflammation and demyelination. PLA2 enzymes catalyze the hydrolysis of fatty acids at the sn-2 position in phospholipids and thus give rise to the release of fatty acids, such as AA, and the production of lysophospholipids, such as lysophosphatidylcholine (LPC) and have been shown to play a role in SCI (17). PUFAs in general are susceptible to lipid peroxidation due to their high content of double bonds. DHA in particular, having more double bonds than AA, is affected by high states of oxidative stress (18). Reducing MDA, thus increases DHA levels, which was demonstrated by our results. As SCI and SF patients suffered from defects in PUFAs following spinal trauma, they may both benefit from therapies, such as fenretinide, to protect neurons from the consequences of initial PUFA imbalance which in case of SCI trauma frequently leads to irreversible damage of neurons and subsequently to paralysis. CONCLUSION Both SCI and SF patients suffer from defects in PUFAs and higher rates of lipid peroxidation following spinal trauma. The imbalances in the AA/DHA ratio return to normal after 1 to 2 weeks post-injury. Patients may benefit from anti-inflammatory and anti-oxidant therapies, such as fenretinide, to preserve PUFA levels and recover the balance between the levels of pro-inflammatory AA and the anti-inflammatory DHA. FIGURE LEGEND Figure 1. PUFA and lipid peroxidation levels in plasma following spinal trauma. PUFA and lipid peroxidation levels were measured in spinal cord injury patients (SCI, red) and spinal fracture (SF, blue). A) Arachidonic acid (AA) levels. Significant differences were observed in SCI patients between day 1 vs. 8 – 16 days, and vs. 25 – 49 days. For the SF group, day 1 values were significantly different from 8 – 16 days. SCI values at day 1 were significantly different from healthy controls (HC, grey shading) (ANOVA, p < 0.0001). B) Docosahexaenoic acid (DHA) levels. Significant differences were observed in SCI patients between day 1 vs. 8 – 16 days, and vs. 25 – 49 days (ANOVA, p < 0.0001). C) Arachidonic acid/docosahexaenoic acid ratios (AA/DHA). Significant differences (p < 0.05) were observed in the SCI group between day 1 vs. 8 – 16 days, and vs. 26 – 49 days. SF patients had significant differences between day 1 values vs. 8 – 16 days. At day 1, the AA/DHA ratio in the SCI group was significantly higher than HC (ANOVA, p < 0.0001). D) Malondialdehyde (MDA). Significant differences were found in SCI patients between day 1 vs. 8 – 16 days (ANOVA, p = 0.0065). Grey shading represents min and max ranges for HC values and the dotted horizontal line represents the mean of the HC group.* represents significant differences between the time point and day 1 within the patient group as per the ad hoc post-tests. † represents significant differences between the patient group at day 1 and HC as per the ad hoc post-tests. Statistical analysis was done using the Kruskal-Wallis non-parametric ANOVA. P values listed are for the overall ANOVA test. Figure 1 References 1 David S, Lacroix S Molecular approaches to spinal cord repair Annu Rev Neurosci 2003;26:411-40. 2 Richardson AJ Omega-3 fatty acids in ADHD and related neurodevelopmental disorders Int Rev Psychiatry 2006;18:155-72. 3 Orr SK, Bazinet RP The emerging role of docosahexaenoic acid in neuroinflammation Curr Opin Investig Drugs 2008;9:735-43. 4 Michael-Titus AT Omega-3 fatty acids and neurological injury Prostaglandins Leukot Essent Fatty Acids 2007;77:295-300. 5 Farooqui AA, Horrocks LA, Farooqui T Interactions between neural membrane glycerophospholipid and sphingolipid mediators: a recipe for neural cell survival or suicide J Neurosci Res 2007;85:1834-50. 6 David S, Greenhalgh AD, Lopez-Vales R Role of phospholipase A2s and lipid mediators in secondary damage after spinal cord injury Cell Tissue Res 2012;349:249-67. 7 Ariel A, Serhan CN Resolvins and protectins in the termination program of acute inflammation Trends Immunol 2007;28:176-83. 8 Serhan CN Novel chemical mediators in the resolution of inflammation: resolvins and protectins Anesthesiol Clin 2006;24:341-64. 9 Serhan CN, Yacoubian S, Yang R Anti-inflammatory and proresolving lipid mediators AnnuRevPathol 2008;3:279-312.:279-312. 10 Endres S, von Schacky C n-3 polyunsaturated fatty acids and human cytokine synthesis Curr Opin Lipidol 1996;7:48-52. 11 Folch J, Lees M, Sloane Stanely GH A simple method for the isolation and purification of total lipides from animal tissues J Biol Chem 1957;226:497-509. 12 Schlenk H, Gellerman J Esterification of Fatty Acids with Diazomethane on a Small Scale Anal Chem 1960;32:1412-14. 13 Oborna I, Wojewodka G, De Sanctis JB, et al. Increased lipid peroxidation and abnormal fatty acid profiles in seminal and blood plasma of normozoospermic males from infertile couples Hum Reprod 2010;25:308-16. 14 Lopez-Vales R, Redensek A, Skinner TA, et al. Fenretinide promotes functional recovery and tissue protection after spinal cord contusion injury in mice J Neurosci 2010;30:3220-6. 15 Lopez-Vales R, Garcia-Alias G, Guzman-Lenis MS, et al. Effects of COX-2 and iNOS inhibitors alone or in combination with olfactory ensheathing cell grafts after spinal cord injury Spine (Phila Pa 1976) 2006;31:1100-6. 16 O'Banion MK, Kyrkanides S, Olschowka JA Selective inhibition of cyclooxygenase-2 attenuates expression of inflammation-related genes in CNS injury Adv Exp Med Biol 2002;507:155-60. 17 Lopez-Vales R, Ghasemlou N, Redensek A, et al. Phospholipase A2 superfamily members play divergent roles after spinal cord injury FASEB J 2011;25:4240-52. 18 Halliwell B, Chirico S Lipid peroxidation: its mechanism, measurement, and significance Am J Clin Nutr 1993;57:715S-24S; discussion 24S-25S. Keywords: Polyunsaturated fatty acid (PUFA), Arachidonic Acid, docosahexaenoic acid, spinal cord injury (SCI), Spinal Fractures, Spinal Trauma, Malondialdehyde, Lipid Peroxidation, clinical study Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Radzioch D, De Sanctis JB, Wojewodka G, Radhakrishna M, Makriyianni I, Parent S, Ouellet J and David S (2013). Lipid profile analysis in spinal trauma patients shows severe distortion of AA/DHA after injury. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01183 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 31 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Danuta Radzioch, McGill University, Montreal, Canada, danuta.radzioch@mcgill.ca Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Danuta Radzioch Juan B De Sanctis Gabriella Wojewodka Mohan Radhakrishna Ioli Makriyianni Stefan Parent Jean Ouellet Samuel David Google Danuta Radzioch Juan B De Sanctis Gabriella Wojewodka Mohan Radhakrishna Ioli Makriyianni Stefan Parent Jean Ouellet Samuel David Google Scholar Danuta Radzioch Juan B De Sanctis Gabriella Wojewodka Mohan Radhakrishna Ioli Makriyianni Stefan Parent Jean Ouellet Samuel David PubMed Danuta Radzioch Juan B De Sanctis Gabriella Wojewodka Mohan Radhakrishna Ioli Makriyianni Stefan Parent Jean Ouellet Samuel David Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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