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Metabolic abnormalities reported in patients with multiple sclerosis include a decrease in cell membrane fatty acid C20:4n-6. The aim of the present study was to investigate whether this decrease was associated with abnormalities in the prostaglandin E2 pathway in patients with multiple sclerosis.
The study population included 31 patients with multiple sclerosis and 30 healthy controls. Peripheral blood mononuclear cell membrane fatty acids were measured by gas chromatography, secretory-phospholipase A2, and prostaglandin E2 with enzyme-linked immunosorbent assays and C-reactive protein with a Beckman auto-analyser.
Prostaglandin E2 was increased in patients (545.5 pg/mL; quartile range 585.1 pg/mL) and controls (248.2 pg/mL; quartile range 183.6 pg/mL; P = 0.0018). Phospholipase A2 was inversely associated with C20:4n-6 in patients and controls, respectively (P = 0.0398 and P = 0.0182). C-reactive protein showed a positive association with phospholipase A2 in patients (P = 0.0006), and an inverse association with prostaglandin E2 in controls (P = 0.0006).
The increase in prostaglandin E2 concentration in plasma from patients with multiple sclerosis was possibly enhanced by the positive association between the C-reactive protein and phospholipase A2 concentrations present in patients; that is, active stimulation of the prostaglandin E2 pathway, which can possibly explain decreases in membrane n-6 fatty acid C20:4n-6 reported in cell membranes from patients. It is not clear from the results of the present study whether this denotes chronic inflammation in patients, but could be expected to contribute to central nervous system damage reported in patients with multiple sclerosis.
Polyunsaturated fatty acid (PUFA) metabolic abnormalities have been well documented in peripheral blood cells from patients with multiple sclerosis (MS).[1-4] The disease presents with neuronal demyelination and plague formation, and abnormal immune cell activity has been reported in brain tissue from patients.[5-7] PUFA are precursors for the active metabolites eicosanoids, which mediate the inflammatory response, and include prostaglandins (PG), leukotrienes (LT), thromboxanes (TX) and platelet-activating factor (PAF).[8-10] The prostanoids are low in uninflamed tissue, but increase during acute inflammation, and are also found in chronic inflammatory lesions. Because of the relatively high amount of PUFA C20:4n-6 in immune cell membrane phospholipids, it is the major precursor fatty acid to be released by phospholipase A2 (PLA2) for the metabolism of eicosanoids including prostaglandin E2 (PGE2).[9, 12-14] PGE2 synthesis is controlled by the availability of non-esterified fatty acid C20:4n-6 and cyclooxygenase-2 (COX2).[9, 15] PGE2 is generally recognized as a mediator of active inflammation; but, depending on the different stages of the immune response, might suppress the production of pro-inflammatory mediators, and might also enhance its own production, resulting in its predominance at late/chronic stages of immunity.[11, 15] The ability of PGE2 to limit type 1 (cytotoxic) immunity is crucial for the host self-preservation. However, it is counterproductive during infections with intracellular pathogens, as this type of infection cannot be eliminated with enhanced PGE2 concentrations and the establishment of immunosuppression. Altered prostaglandin production, and an increase in PLA2 activity have been associated with acute and chronic inflammation and neurological disorders, such as MS.[9, 14, 16] However, there is a scarcity of literature on the concentrations of PLA2 and PGE2 in blood from patients with MS. Hofman et al. reported immune cells from MS brain lesions that stained positive for prostaglandin E (PGE), which was not found in normal brain tissue.
Inflammatory activation in the central nervous system has been well documented, and as measured by the C-reactive protein (CRP) concentration, has been reported to show a correlation with infectious episodes, clinical relapse and gadolinium-enhanced magnetic resonance imaging of the brain and spinal cord in patients with MS. This group has reported a positive correlation between the CRP concentration and the Expanded Disability Status Scale (EDSS) in patients with MS. The CRP is an inflammatory marker of which the stimulus might include infection, and in this regard proposed contributing factors to the disease etiology include autoimmunity and infectious agents.[20, 21] We have also shown an inverse correlation between the CRP concentration and C20:4n-6 in peripheral blood mononuclear cell (PBMC) membranes, and similarly with C20:4n-6 in red blood cell (RBC) membranes from patients with MS, suggesting a relationship between the CRP concentration and membrane PUFA in patients with MS.
Although results vary, the n-6 PUFA, including C20:4n-6 and the precursor fatty acid C18:2n-6, have been reported to be decreased in plasma and blood cells from patients with MS,[1, 3, 23] suggesting a possible role for these fatty acids in the inflammatory and/or infectious aspect of the disease. However, the association between fatty acid metabolic abnormalities and the disease etiology has not been fully elucidated. Therefore, the aim of the present study was to compare PLA2 and PGE2 concentrations in plasma from patients with MS and a healthy age- and sex-matched control group, and to investigate a possible association between these parameters and reported metabolic abnormalities in the n-6 PUFA in PBMC membranes from patients with MS. Because the CRP concentration has been reported to show associations with membrane PUFA,[4, 22] as well as with the disease status in patients with MS,[4, 18] results for the association studies were given for single variable analysis, as well as for multivariate analysis, adjusted for the CRP concentration and age.
Ethics approval for the present study was obtained from the Health and Applied Sciences Research Ethics Committee (HASREC) of the Cape Peninsula University of Technology (CPUT), Cape Town, South Africa. The study was carried out in accordance with the Declaration of Helsinki (1964; revised in Edinburgh 2000). Patients with MS were contacted and recruited through the MS Society, Western Cape Branch, South Africa. Written informed consent was obtained from all participants. Patient anonymity has been preserved.
The study population consisted of 31 Caucasian female patients with MS, 28 presented with relapsing remitting MS, one with primary progressive MS and two with secondary progressive MS, and 30 age-, sex- and race-matched control participants. The number of male patients (two) who responded to recruitment and who met the required criteria was insufficient for statistical analysis. The patients recruited were diagnosed by a neurologist based on the McDonald Diagnostic Criteria (revision in 2005), which specify that at least two different events (lesion formation in the central nervous system) must occur, separated in time as well as space, before diagnosis can be confirmed. Additional tests include cerebrospinal fluid (CSF) analysis and visual evoked potential (VEP) recordings, which are further used to confirm diagnosis.[24-27] The diagnosis of all patients recruited for the present study was confirmed by a neurologist based on clinical, laboratory and, specifically, magentic resonance imaging confirmation included in the diagnosis of MS. The median age of the patients was 51 years (interquartile range 23 years) and that of the control participants was 50 years (interquartile range 23 years), the median age at onset (diagnosis) of disease in patients was 37 years (interquartile range 20 years) and the median years since diagnoses was 7 years (interquartile range 11 years). The EDSS of the MS patients was 5.5 (interquartile range 3.5). Six of the patients were active disease cases, 11 had a relapse 5–12 months before the study and 14 had not relapsed for more than a year. Patients on any fatty acid supplementation, interferon or cortisone treatment were excluded from the study. A total of 10 patients were using non-steroidal anti-inflammatory drugs (NSAIDs), five patients were using immunosuppressive drugs and 16 patients were not using either.
Peripheral blood mononuclear cell (PBMC) membrane non-esterified (NEFA), and phosphatidylcholine (PC) and sphingomyelin (SM) esterified PUFA were measured by gas chromatography as previously described.[4, 29-31] Briefly, thin layer chromatography was used in the extraction and separation of the fatty acids, which were further converted to methyl esters and quantified by gas chromatography. PBMC membrane fatty acids were quantified in μg/mg protein.
Enzyme-linked immunosorbent assays
Enzyme-linked immunosorbent assays (ELISA) were used to determine the concentrations of PLA2 and PGE2 in serum fromparticipants. Secretory PLA2 (sPLA2) was investigated in the present study because of its relative abundance.[32, 33] Reagents for the determination of sPLA2 concentrations were ordered from Cayman Chemical Company (item no. 585000) and for PGE2 from Whitehead Scientific (Cape Town, South Africa, cat no: KGE004B). The ELISA assays were carried out and final concentrations calculated according to the respective manufacturer's instructions.
C-reactive protein (CRP) concentrations were determined in a routine chemical pathology laboratory, Tygerberg Hospital, on a Beckman nephelometer auto-analyser using reagents from Beckman, Cape Town, South Africa. The chemical pathology laboratory at Tygerberg Hospital defines the normal range for CRP in healthy subjects to be <10 μg/mL.
STATISTICA 12 (StatSoft Southern Africa, Sandton, South Africa) was used to carry out all statistical analyses. Breakdown and one-way anova was used to analyze designs with a single categorical independent variable (median and interquartile range). The non-parametric Mann–Whitney U-test was used to determine statistical significance between independent variables. Linear regression analysis using log scores was used to determine the association between PLA2, PGE2 and fatty acids. Results are given for single variable analysis, as well as being adjusted for age and inflammation and/or infection as measured by the CRP.
Differences in the PLA2 and PGE2 concentrations between patients with MS and controls
The PGE2 concentration was significantly higher in patients with MS than in controls: patients: median 545.5 pg/mL (quartile range 585.1 pg/mL); controls: median 248.2 pg/mL (quartile range 183.6 pg/mL), P = 0.0018 (Fig. 1). There was no significant difference in the PLA2 concentration between patients and controls: patients: median 2810.0 pg/mL (quartile range 4780.0 pg/mL); controls: median 3440.0 pg/mL (quartile range 6270.0 pg/mL), P = 0.4345. The CRP levels in both controls and patients were within the normal range; that is, <10 μg/mL, and no significant differences were observed in the two groups, patients: median 3.80 μg/mL (quartile range 4.30 μg/mL); controls: median 3.40 μg/mL (quartile range 3.80 μg/mL), P = 0.2800.
Association between the PLA2 and PGE2 concentrations, and the CRP concentration, adjusted for age
As shown in Table 1, the CRP concentration, when adjusted for age, showed a positive association with the PLA2 concentration in patients with MS, but an inverse association with the PGE2 concentration in controls (respectively, b* = 0.6077; P = 0.0005, and b* = −0.6143; P = 0.0006 [PLA2: Fig. 2a,b and PGE2: Fig. 3a,b]). Results for single variable analysis are included in Table 1.
Table 1. Association between the phospholipase A2 and prostaglandin E2 concentrations, and C-reactive protein
Adjusted for age
Not adjusted for age
Controls n = 30
Patients with MS n = 31
Controls n = 30
Patients with MS n = 31
Phospholipase A2 (PLA2) and prostaglandin E2 (PGE2) were quantified in pg/mL plasma.The C-reactive protein (CRP) was quantified in μg/mL plasma.
MS, multiple sclerosis.
Associations between PBMC membrane n-6 PUFA and the PLA2 concentration
As shown in Table 2, in controls, when adjusted for age and the CRP concentration, the PLA2 concentration showed inverse associations with PBMC n-6 PUFA PC C18:2n-6, PC C20:3n-6, PC C20:4n-6 (Fig. 4a), PC C22:4n-6 and PC C22:5n-6 (respectively, b* = −0.4401; P = 0.0345, b* = −0.3971; P = 0.0338, b* = −0.4675; P = 0.0182, b* = −0.4663; P = 0.0069 and b* = −0.3714; P = 0.0320). In patients with MS, the PLA2 concentration was inversely associated with PBMC PC C20:4n-6 (Fig. 4b) only (b* = −0.5330; P = 0.0398). Results for single variable analysis are included in Table 2.
Table 2. Association between peripheral blood mononuclear cell membrane n-6 polyunsaturated fatty acids and the phospholipase A2 concentration
Adjusted for age and the CRP
Not adjusted for age and the CRP
Controls n = 25
MS n = 26
Controls n = 25
MS n = 26
Peripheral blood mononuclear cell membrane n-6 polyunsaturated fatty acids were quantified in μg/mg protein.
Association between PBMC membrane n-6 PUFA and the PGE2 concentration
As shown in Table 3, in controls, when adjusted for age and the CRP concentration, the PGE2 concentration showed a significant inverse association with PC C18:2n-6 in PBMC membranes (b* = −0.7295; P = 0.0041); whereas in patients with MS, no significant association was found. Results for single variable analysis are included in Table 3.
Table 3. Association between peripheral blood mononuclear cell membrane n-6 polyunsaturated fatty acids and the prostaglandin E2 concentration
Adjusted for age and the CRP
Not adjusted for age and the CRP
Controls n = 25
Patients with MS n = 26
Controls n = 25
Patients with MS n = 26
Peripheral blood mononuclear cell n-6 polyunsaturated fatty acids were quantified in μg/mg protein.
Results from the present study showed a highly significant increase in PGE2 concentrations in plasma from patients with MS. Although there is a scarcity of literature on PLA2 and PGE2 concentrations in plasma from these patients, Hofman et al. showed PGE present in brain tissue from patients with MS, which was not found in that of healthy controls. The presence of high concentrations of PGE2 in plasma can be expected to affect neuronal tissue as well, and in this regard, neuronal demyelination and plague formation have been well documented in these patients.[5-7]
High concentrations of PGE2 in plasma could possibly be a response to pathogens that the immune system cannot successfully eliminate, including exogenous herpesviruses, such as Epstein–Barr virus, which can establish latent infections in the central nervous system[34, 35]; and endogenous retroviruses, which have been reported to be actively expressed in brain tissue from patients with MS, and also in healthy controls.[36, 37] If infections cannot be cleared, it is possible that increased PGE2 concentrations in plasma from patients could suggest a chronic immune response, and in this regard, PGE2 can also mediate chronic inflammation. However, PGE2 can also limit type 1, which is cytotoxic immunity that is required during infections with intracellular pathogens, as this type of infection cannot be eliminated with enhanced PGE2 concentrations. It is possible, therefore, that an inappropriate immune response might be present in patients with MS.
Furthermore, results from the present study show that in healthy controls and patients, an inverse association existed between PLA2 and C20:4n-6 concentrations, suggesting increased release of the fatty acid for PGE2 production with increased concentrations of PLA2. However, in patients, there was also a positive association between the CRP and PLA2 concentrations, suggesting increased activation of the PGE2 pathway during inflammatory activation in patients with MS. This association was not present in healthy controls, but an inverse association between the CRP and PGE2 concentrations in controls suggested that during inflammatory activation in controls the PGE2 pathway might have been suppressed to allow for a different type of response from the immune system. In this regard, PGE2 can suppress the production of pro-inflammatory mediators.[11, 15] It is possible, therefore, that the inflammatory condition experienced by patients could be the result of a metabolic abnormality in this pathway, the cause is unknown as yet. In this regard, altered PG production and an increase in PLA2 activity have been associated with acute and chronic inflammation and neurological disorders, such as MS.[9, 14, 16, 38]
The results from the present study showed a changed profile for the associations of PLA2 and PGE2 with PBMC PUFA, suggesting that in patients with MS, membrane fluidity could have been affected as well. In controls, the PLA2 concentration was inversely associated with all the PC PUFA in PBMC membranes, whereas the PGE2 concentration was inversely associated with essential fatty acid C18:2n-6, suggesting that in control subjects, all PBMC membrane PUFA were sourced to adjust for PLA2 action, and that PGE2 synthesis eventually showed primary sourcing from essential fatty acid PC C18:2n-6. In contrast, in patients with MS, the PLA2 concentration was inversely associated with PBMC PC C20:4n-6 only, which could possibly explain why this PUFA has been reported decreased in PBMC membranes from patients with MS.[3, 23] The results from the present study suggest that C20:4n-6 was excessively sourced for PGE2 production in patients with MS. The rate of PGE2 synthesis is normally controlled by COX2 expression, but the availability of non-esterified fatty acid C20:4n-6 is also rate-limiting in the synthesis of PGE2.[9, 15] The essential fatty acid of the n-6 PUFA series, C18:2n-6, cannot be synthesized in the body, and must be sourced from plasma (dietary source) and also by PBMC membranes.[10, 39] These findings suggest a healthy redistribution and/or uptake of PUFA from plasma by PBMC membranes during PLA2 activation and/or action in controls, but in patients the release of C20:4n-6 might have been excessive and/or not sufficiently replaced. In this regard, decreases in the n-6 PUFAs in plasma, PBMC and RBC membranes from patients with MS have been reported repeatedly,[1, 3, 4, 22, 23] suggesting insufficient plasma sources and/or possibly excessive use elsewhere.
In conclusion therefore, we have shown a significant increase in PGE2 concentration in plasma from patients with MS, and that this increase was associated with the C20:4n-6 metabolic abnormalities previously reported in patients with MS. We have further shown that the increase in PGE2 concentrations could have been from an altered response to the inflammatory status in patients with MS as compared with that of healthy controls, measured against the associations between the CRP, and both PLA2 and PGE2 concentrations. It is therefore possible that the inflammatory condition experienced by patients might be the result of a metabolic abnormality in this pathway, cause/s unknown as yet.
We extend our sincere gratitude to the MS Society, Western Cape Branch, South Africa and Sister Treska Botha for the recruitment of patients. This study was funded by a grant from the University Research Fund of the Cape Peninsula University of Technology, South Africa. No conflict of interest declared.