Department of Anatomy and Cell Biology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA
Address correspondence and reprint requests to Dr Maria Irene Givogri, Department of Anatomy & Cell Biology, College of Medicine, University of Illinois at Chicago, 808 S. Wood St. M/C 512, Chicago, IL. USA. E-mail: firstname.lastname@example.org
Multiple sclerosis (MS) is considered an autoimmune demyelinating disease of the CNS and myelin-derived glycolipids are one of the targets of this autoimmune attack. In this study, we examined for the first time the plasma distribution of sulfatide isoforms. Sulfatides with long-chain (C24 : 0 or C24 : 1) and short-chain (C16 : 0 or C18 : 0) fatty acids were quantified in plasma of relapsing–remitting MS patients by ultra-high-performance liquid chromatography tandem mass spectrometry. We found that C18 : 0 and C24 : 1 sulfatide plasma levels positively correlated with the Expanded Disability Status Scale. C16/C18 : 0 and C16/C24 : 0 ratios also correlated with the age and the time since last relapse. Healthy women showed higher levels of C16 : 0 sulfatide than healthy men; however, this gender difference disappeared in MS patients. Our data underline the potential use of sulfatides as biomarkers in relapsing–remitting MS and points to a possible association with the higher susceptibility of women to develop MS.
Sulfatides are glycolipids highly enriched in myelin that have been associated with multiple sclerosis (MS). In this study, we have found a positive correlation between levels of specific sulfatides in plasma and increased disability in patients with relapsing-remitting MS. These findings underline the potential use of these molecules as biomarkers for MS.
ultra-high-performance liquid chromatography tandem mass spectrometry
Multiple sclerosis (MS) is an inflammatory disease characterized by central demyelination and axonal loss (Compston and Coles 2008). An autoimmune response against myelin glycolipids has been implicated with the course of MS, underlining the potential use of these molecules as biomarkers for the susceptibility, activity and progression of MS (Kanter et al. 2006; Quintana et al. 2012).
Sulfated galactosylceramides (sulfatides) are glycolipids highly enriched in myelin and also involved in other biological processes in non-neural tissues such as the immune system (Takahashi and Suzuki 2012). Sulfatides are heterogeneous sulfated-galactosylceramides with variable lengths of the fatty acid chain in the ceramide moiety (Fig. 1). Different isoforms exhibit a tissue-dependent distribution and myelin is highly enriched in sulfatides with long-chain fatty acids (Ishizuka 1997; Marbois et al. 2000; Isaac et al. 2006). Although the biological function of sulfatides species is not completely known, recent studies showed the importance of their fatty-acid composition in the control of autoimmune diseases (Jahng et al. 2004; Subramanian et al. 2012).
MS patients exhibit an enhanced antibody response against sulfatides, however, the role of these antibodies in MS has not been elucidated (Kanter et al. 2006; Quintana et al. 2008). Specificity of anti-sulfatide antibodies against different sulfatide isoforms is not known. However, it has been shown that sulfatide-reactive T cells exhibit isoform-dependent recognition against sulfatides (Blomqvist et al. 2009). Furthermore, total levels of sulfatides are increased in the CSF of MS patients, and strikingly, also in CSF of their healthy siblings (Haghighi et al. 2012, 2013). These findings led to postulate that a ‘Glycosphingolipid endophenotype’ may contribute to the identification of individuals at risk of developing MS. Despite these observations, and the importance of sulfatides as modulators of autoimmunity, the distribution and abundance of different sulfatides species in the plasma of MS patients has not been studied.
To address this, sulfatide isoforms with long-chain (C24 : 0 or C24 : 1) and short-chain (C16 : 0 or C18 : 0) fatty acids were analyzed in plasma samples from relapsing–remitting MS (RRMS) patients using ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS).
Materials and methods
Patient and control samples
Plasma samples were obtained from a total of 14 RRMS patients (seven women and seven men) and 14 healthy controls (seven women and seven men) after patient consent (Table 1). The study was approved by the Office for the Protection of Research Subjects at University of Illinois at Chicago and the Department of Defense Human Research Protection Office as research not involving human subjects in accordance with 32 CFR 291,102(f). MS patients were diagnosed with RRMS according to the revised McDonald criteria (Polman et al. 2011). RRMS is defined as patients undergoing attacks characterized by neurological dysfunction (relapse period), followed by periods of partial or full recovery (period of remission). Our study included samples that best matched age and gender between controls and RRMS patients. All patients were in remission at the time of blood collection, without any other non-MS medical condition and were not under any other medication except those for treating MS symptoms (Table 1). RRMS samples matched treatment and Expanded Disability Status Scale (EDSS, a method of quantifying disability in MS, with step from 0 to 10; step 1–4.5 as fully ambulatory and step 5–9.5 with impairment to ambulation) (Table 1). Approximately 10 mL of blood was collected in EDTA tubes (BD, Franklin Lakes, NJ, USA) to isolate Peripheral blood mononuclear cells using a Ficoll–Paque protocol. Briefly, blood was diluted 1 : 1 in sterile 2 mM EDTA phosphate-buffered saline to decrease viscosity, and then loaded onto a pre-made Ficoll-Paque gradient (GE Healthcare Biosciences, Pittsburgh, PA, USA). Supernatants, free of Peripheral blood mononuclear cells and erythrocytes, were collected and stored at −80°C until sulfatide were extracted.
Table 1. Demographic, clinical features and plasma levels of sulfatides in RRMS patients and healthy controls
Demographic and clinical features of RRMS patients and healthy controls included in the study. All data are expressed as median (range). EDSS, Expanded Disability Status Scale; NA, not applicable. Treatments: interferon-β (IFN), glatiramer acetate (GA) and natalizumab (Nat). Sulfatide species (C16 : 0, C18 : 0, C24 : 0 and C24 : 1) are expressed as median (range) in nmol/L.
C16 : 0 sulfatide levels showed a statistical significant increase in women with respect to men in healthy control groups, ap = 0.02. p < 0.05 was considered statistically significant.
Sulfatides were extracted from plasma (0.1 mL) as previously described (Folch et al. 1957) with minor modifications. C12 : 0 sulfatide (0.5 μM) was added as an internal standard. Samples were mixed with 10 vol. of chloroform: methanol (2 : 1 v/v) and incubated at 22°C with agitation for 2 h. After filtration, 0.2 vol. of 0.9% NaCl was added and the mixture was vortexed and centrifuged at 900 g for 5 min. Lower phases were dried under nitrogen at 45°C and stored at −20°C until use or reconstituted in 60 μL of 5 mM ammonium formate in methanol, and 2 μL was injected into the UHPLC column. Four sulfatide species (C16 : 0, C18 : 0, C24 : 0 and C24 : 1) were analyzed using UHPLC-MS/MS on a Shimadzu Nexera/LCMS-8040 triple quadruple mass spectrometer (Kyoto, Japan). Sulfatides species were separated on a Waters Acquity UPLC C18 column (2.1 × 50 mm, 1.7 μm) (Milford, MA, USA) using a solvent gradient: 0–3 min, from 65%–83% B, 3–3.5 min, from 83%–95% B, held at 95% B for 1.5 min (solvent A: 5 mM ammonium formate in water, pH 4.5; and solvent B: acetonitrile). The flow rate was 0.6 mL/min with the column oven temperature at 45°C, and the autosampler temperature at 20°C. Analytes and internal standard were measured using negative ion electrospray mass spectrometry (Shimadzu) with collision-induced dissociation and selected reaction monitoring. The following selected reaction monitoring transitions were monitored: C12 : 0: m/z 722 to 97; C16 : 0: m/z 778 to 97; C18 : 0: m/z 806 to 97; C24 : 1: m/z 888 to 97; C24 : 0: m/z 890 to 97.
Sulfatide levels were analyzed by Kruskal–Wallis test (non-parametric). The Spearman rank correlation test was used to determine associations between plasma sulfatide levels and different clinical parameters (age, age at onset, time since last relapse and EDSS). p < 0.05 was considered significant. All analyses were performed using GraphPad Prism version 5.0d for Mac OS X, GraphPad Software (La Jolla, CA, USA).
Sulfatides from plasma of 14 RRMS and 14 healthy controls (Table 1) were measured by UHPLC-MS/MS (Fig. 1). Although, RRMS patients showed a more variable range of sulfatide levels than healthy controls, the distribution of sulfatides with short (C16 : 0 and C18 : 0) and long fatty-acid chains (C24 : 0 and C24 : 1) was not significantly different among the groups (Table 1).
We next examined whether plasma levels of sulfatide species correlated or not with demographic and clinical features (disease duration, the time since last relapse and the Expanded Disability Status Scale or EDSS). A positive and significant correlation of plasma levels of C18 : 0 (rs = 0.751, p = 0.002) and C24 : 1 (rs = 0.630, p = 0.016) sulfatides with the EDSS was determined (Fig. 2a and b). C24 : 1 is enriched in myelin and C18 : 0 appears primarily produced by neurons and astrocytes (Ishizuka 1997; Isaac et al. 2006). Therefore, these correlations may indicate a mobilization of these sulfatides from the brain into the plasma as disease progresses.
Positive and significant correlations were also determined between the C16 : 0/C24 : 0 ratio and the time since last relapse (rs = 0.704, p = 0.005) and between the C16 : 0/C18 : 0 ratio and the age (rs = 0.815, p < 0.001) of RRMS patients (Fig. 2c and d). C16 : 0/C18 : 0 did not correlate with the age of healthy controls (rs = 0.278, p = 0.358) (Fig. 2d, white circles). When control and RRMS groups were analyzed by gender, plasma levels of C16 : 0 sulfatide was significantly different between women and men in healthy controls (Table 1, p = 0.02). This gender-related difference was absent in RRMS patients, maybe as a consequence of a more variable range of sulfatide levels in RRMS patients (Table 1, p = 0.14), which could underlie the alterations of sulfatides mobilization during the course of the disease.
This study presents for the first time the quantitative determination of four major sulfatide species (C16 : 0, C18 : 0, C24 : 0, C24 : 1) in the plasma of RRMS patients.
Our analysis showed that C18 : 0 and C24 : 1 sulfatides correlated with severity scores (EDSS) in RRMS patients. In addition, C16 : 0/C24 : 0 and C16 : 0/C18 : 0 ratios correlated with the time since last relapse and the age in RRMS patients. Previous studies have suggested that demyelination could induce an outflow of myelin-damage metabolites in plasma and urine (Shore et al. 1987; Nicholas and Taylor 1994). Our observations of higher plasma sulfatides may reflect CNS damage during MS and reveal the potential use of sulfatides as biomarkers for MS.
A previous study showed decreases of ~ 60% and ~ 25% in total sulfatides in plaques and normal adjacent white matter of MS patients, respectively (Marbois et al. 2000). More recent findings showed higher levels of sulfatides in CSF of MS patients, although analyses of individual sulfatides species were not reported (Haghighi et al. 2012). C18 : 0 and C24 : 1 sulfatides are species more abundant in neurons/astrocytes and myelin, respectively (Isaac et al. 2006). In all, these reports and the results presented in our study provide support to the hypothesis of an outflow of sulfatides as a bystander effect of demyelination/neurodegeneration.
Recent studies have shown the relevance of the fatty-acid moiety of sulfatides during the activation of type II natural killer T (NKT) cells (Blomqvist et al. 2009). NKT cells play an important role in autoimmune diseases and sulfatide-reactive NKT cells increase in peripheral blood of MS patients (Shamshiev et al. 1999). Our observations of higher levels of C18 : 0 or C24 : 1 in plasma in RRMS patients may contribute to the activation of NKT cells during relapses. Further experiments are needed to confirm this potential function.
Women are more susceptible to develop MS than men (from 2 : 1 to 3 : 1) (Voskuhl and Gold 2012). Recently, an increase in CSF levels of sulfatides was suggested as a risk factor for MS in healthy siblings of MS patients (Haghighi et al. 2013). Interestingly, C16 : 0 sulfatide appears to induce a faster and preferential secretion of pro-inflammatory cytokines in CD4+ T cells (Subramanian et al. 2012). As our study shows that healthy women have higher levels of C16 : 0 sulfatide in plasma than men, these findings may indicate an association with a higher susceptibility of women to develop autoimmune diseases such as MS. This gender-related difference was absent in RRMS patients. Moreover, C16 : 0/C24 : 0 and C16 : 0/C18 : 0 ratios correlated with the time since last relapse and the age in RRMS patients. Therefore, these differences may indicate that C16 : 0 sulfatide play a biological role in the pathogenesis of the disease.
Our observations indicate that plasma sulfatides levels change during RRMS, revealing their potential clinical relevance in this disease. Further investigations with larger cohorts of samples will address specific questions about the use of sulfatides species as biomarkers for susceptibility, course and progression of RRMS.
MS is a complex and multifactorial disease where several pathophysiological mechanisms are involved. The many attempts for the identification of biomarkers for MS and the relatively small number of them underlines the difficulty to formulate new biomarkers (Bielekova and Martin 2004; Katsavos and Anagnostouli 2013). We propose that sulfatides, in addition with other biomarkers such as neurofilaments and/or myelin basic protein, can improve the diagnostic and/or prognosis of RRMS.
In summary, our study reports for the first time the abundance of four sulfatide isoforms in RRMS plasma. Because of their relevance in autoimmune response and their positive correlation with EDSS, sulfatide isoforms C18 : 0 and C24 : 1 are potential candidates as biomarkers in MS.
Supported by DOD Grant contract number (W81XWH-11-1-0198) and in part by NMSS Grant (RG 4439-A-2) to M.I.G. We are grateful to Dr. D. L. Feinstein for generous feedback and for providing human plasma samples.
Conflict of interest
The authors declare no competing financial interests.