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Plasma citrulline levels are increased in patients with multiple sclerosis
Journal of the Neurological Sciences 387 (2018) 174–178
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Plasma citrulline levels are increased in patients with multiple sclerosis a
b
a,c
Maxime Vande Vyver , Roel Beelen , Jacques De Keyser , Guy Nagels ⁎ Anne-Marie Van Binstf, Christiaan Verborghg, Miguel D'haeseleera,d,
a,d,e
T
,
a
Department of Neurology, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Center for Neurosciences, Brussels, Belgium Department of Cardiovascular and Thoracic Surgery, Onze-Lieve-Vrouw Ziekenhuis, Campus Aalst, Aalst, Belgium Department of Neurology, Universitair Medisch Centrum Groningen, Groningen, The Netherlands d Nationaal Multiple Sclerose Centrum, Melsbroek, Belgium e Faculté de Psychologie et des Sciences de l'Education, Mons, Belgium f Department of Radiology, Universitair Ziekenhuis Brussel, Brussels, Belgium g Department of Aenesthesiology, Universitair Ziekenhuis Brussel, Brussels, Belgium b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Multiple sclerosis Citrulline Myelin basic protein Nitric oxide Jugular vein
Background: Multiple sclerosis is an inflammatory demyelinating disorder of the central nervous system (CNS). Myelin basic protein (MBP), which is one of the main compounds of CNS myelin, appears to be hypercitrullinated in the brain of patients with MS. We hypothesized that MS is associated with an increased release of citrulline from the brain. Methods: Twenty-five patients with MS, 25 controls without neurological disease (CwND) and 25 subjects with non-MS cerebral white matter lesions were included in this study. Groups were matched for age and gender. Clinical MS disability measures were recorded by means of Expanded Disability Status Scale (EDSS) scores and Multiple Sclerosis Severity Scores (MSSS). Citrulline was assessed in plasma obtained from an antecubital peripheral vein (PV) in all participants. Additional internal jugular vein (IJV) samples were examined in 10 patients with MS and 10 CwND. Twelve patients with MS underwent brain magnetic resonance imaging to determine total brain and T2 fluid-attenuated inversion recovery lesion volume. Results: Median [IQR] PV citrulline levels were increased in patients with MS (50.47 [86.61] μM), as compared to CwND (33.58 [43.65] μM, P = 0.042) and subjects with non-MS cerebral white matter lesions (32.41 [28.86] μM, P = 0.006). Citrulline IJV levels and IJV/PV ratios were comparable between patients with MS and CwND. No significant correlations were found between PV citrulline levels and any of the clinical, nor radiological, disease measures. Conclusion: PV plasma levels of citrulline are elevated in patients with MS but this does not seem to result from an augmented release from the brain. Increased plasma citrulline may be a promising new biomarker in MS but the origin and significance need to be further elucidated.
1. Introduction
gene is located on chromosome 18 but alternative exon splicing leads to the expression of multiple isoforms, varying in length and molecular weight [4]. Subsequently, MBP can undergo several posttranslational modification steps, one of which is the conversion of arginine to citrulline residues by peptidylarginine deiminase (PAD) enzymes [5]. A 18.5 kD (170 amino acid residues) uncitrullinated protein, termed MBP-C1, is the dominant subtype in mature CNS myelin of healthy individuals. Myelin isolated from the brain of subjects with MS appears to contain a higher proportion of MBP-C8, a variant in which 6 out of 19 arginine residues are converted to citrulline, as compared to control samples [6,7], and one study using 1H-magnetic spectroscopy suggested that, as a consequence, total citrulline concentrations are elevated in
Multiple sclerosis (MS) is a chronic demyelinating and degenerative disease of the central nervous system (CNS) affecting over 2 million people worldwide [1]. Myelin covers most axons in the brain and spinal cord to form a protective lipid- and protein-rich sheath which, among other functions, facilitates nerve conduction and provides trophic support. Myelin basic protein (MBP) is one of the most abundant proteins in CNS myelin [2]. The exact underlying pathophysiology of MS remains obscure but demyelination involves T-cell mediated inflammatory responses directed against MBP [3]. The pool of MBP in the human brain is very diverse. The coding
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Corresponding author at: Department of Neurology, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, Brussels, Belgium. E-mail address: [email protected] (M. D'haeseleer).
https://doi.org/10.1016/j.jns.2018.02.025 Received 16 December 2017; Received in revised form 11 February 2018; Accepted 15 February 2018 Available online 16 February 2018 0022-510X/ © 2018 Published by Elsevier B.V.
Journal of the Neurological Sciences 387 (2018) 174–178
M.V. Vyver et al.
respective CwND (10/25) were individuals who underwent central venous catheter placement prior to scheduled cardiac surgery or pacemaker placement at the UZ Brussel. All endovascular procedures were performed by the same experienced cardiovascular surgeon in the patients with MS, or aenesthesiologist in the controls. Correct IJV localization was radiographically controlled in every case. Blood samples were centrifuged and plasma was stored at −80 °C as soon as possible. Plasma citrulline, arginine and glutamine levels were determined by high-performance liquid chromatography. NO is an unstable gas which is rapidly oxidized to its breakdown products (NOx) nitrite and nitrate [13], and can therefore only be indirectly assessed. NOx levels were measured colorimetrically after reduction of nitrate to nitrite by nitrate reductase with a commercially available kit (Enzo Life Sciences®; Zandhoven, Belgium). Plasma samples obtained at the OLVZ Aalst were transported to the laboratory of the UZ Brussel on dry ice by car and, as a result, rapid storage at −80 °C was not always feasible. Because NOx still have a relatively short half-life in plasma and stability of measurement has only been demonstrated for delayed processing up to 48 h [14,15], we excluded the OLVZ Aalst samples from the NOx analysis.
cerebral white matter [8]. MBP hypercitrullination in MS is most likely mediated by increased PAD2 and 4 enzymatic activity and decreases the net positive charge of the protein, resulting in an impaired capacity to interact with negatively charged lipid compounds. These processes potentially lead to a less compact structure of the myelin sheath and increased susceptibility to proteolytic degradation [5]. Reliable body fluid biomarkers for MS diagnosis and treatment response monitoring are currently still lacking. Plasma citrulline levels have never been investigated within this context. In this study, we hypothesized that demyelination in MS leads to an increased release of citrulline from the brain to the circulation. 2. Methods 2.1. Study design and main objectives We performed a prospective exploratory study at the Universitair Ziekenhuis (UZ) Brussel (Brussels, Belgium) and the Onze-Lieve Vrouw Ziekenhuis (OLVZ) Aalst (Aalst, Belgium) to investigate peripheral vein (PV) and internal jugular vein (IJV) plasma levels of citrulline in patients with MS and control subjects without neurologic disease (CwND). Plasma arginine, nitric oxide (NO) and glutamine levels were contemporaneously assessed since these agents are closely related to the metabolism of citrulline [9]. The difference in PV plasma citrulline levels between both groups was selected as the primary endpoint of this study. Significant alterations in patients with MS were verified against a second control group of individuals with non-MS cerebral white matter lesions to increase specificity. The study was approved by the ethics committees of both centers and conformed to the Declaration of Helskini principles. All participants were recruited from April 2011 to March 2014, and provided a written informed consent.
2.3. Magnetic resonance imaging MS patients recruited at the UZ Brussel (15/25) underwent additional brain MRI in supine position using a 3 T machine (Philips Achieva; Best, The Netherlands). The protocol contained, among others, two 3D sequences: a magnetization prepared 3D T1 TurboFLASH (TR 7.8 ms; TE 3.8 ms; TI, 970 ms; flip angle 8°, 240 FOV) and a fat-saturated 3D FLAIR (TR 8000 ms, TE 260 ms, 240 FOV). We used MSmetrix® software (Icometrix; Leuven, Belgium - coveted CE mark and FDA approved) to measure FLAIR laesion load, whole brain and grey matter volumes [16]. Volumetric analyses could not be performed in 3 patients due to technical issues.
2.2. Subjects and blood sampling Twenty-five patients with MS (relapsing-remitting: 12; secondary progressive: 10; primary progressive: 3), according to the 2010 revised McDonald criteria [10], 25 CwND and 25 subjects with non-MS cerebral white matter laesions (ischaemic leukoencephalopathy: 19; migraine: 2; antiphospholipid syndrome: 1; post-chemotherapy: 1; idiopathic: 1) were included in this study. All groups were a priori matched for age and gender. Individuals known with chronic kidney disease were excluded. Eleven MS patients received immunomodulatory drugs (interferon β-1a: 7; interferon β-1b: 2; glatiramere acetate: 1; natalizumab: 1). All MS patients were clinically stable without evidence of an exacerbation within 3 months prior to inclusion. The diagnosis of nonMS cerebral white matter laesions was based on a historic brain MRI, available in the medical records of these individuals, demonstrating at least 3 punctate or 2 confluent areas of T2-weighted hyperintensity in the cerebral periventricular and/or subcortical white matter. All respective brain scans were reviewed by the investigators and declared as non-suggestive of MS. Participants fasted for a minimum of 8 h before blood was drawn from an antecubital PV. All blood samples were collected between 8 and 10 am. Recruitment took place at the UZ Brussel for 15/25 patients with MS (neurology department), 25/25 CwND (healthy volunteers and aenesthesiology department, see below) and 25/25 subjects with nonMS cerebral white matter laesions (neurology department). In 10 patients with MS and 10 CwND an additional blood sample was collected from the right IJV, simultaneously with the PV blood draw. These patients with MS (10/25) were recruited at the Department of Cardiovascular and Thoracic Surgery of the OLVZ Aalst, where they were actively seeking treatment with dilatation angioplasty, for socalled chronic cerebrospinal venous insufficiency [11,12], on their own initiative and specific request. The investigators were never involved in this therapeutic decision process. MS diagnosis was confirmed based on clinical grounds and access to the patient's medical records. The
2.4. Disability and disease progression in MS Clinical disability was measured with the Expanded Disability Status Scale (EDSS) in all patients with MS [17]. Rate of progression of clinical disability was measured with the Multiple Sclerosis Severity Score (MSSS) [18]. This score is based on the combination of EDSS score and disease duration. Both EDSS scores and MSSS range from 0 to 10. Higher scores indicate, respectively, more disability and faster progression of disability. 2.5. Statistical analysis All statistical analyses were performed with SPSS® (Version 24.0, IBM; Amonk, NY USA). PV plasma citrulline, arginine and glutamine values from the total cohort of 75 subjects did not pass the KolmogorovSmirnov test for normal distribution and therefore we expressed them as median [IQR]. Amino acid levels obtained in IJV plasma were presented likewise for reasons of uniformity. All other ordinal and continuous data showed a normal distribution and were presented as mean ± SD. Group differences were assessed using Mann-Whitney U tests. Spearman's rank correlation was used for correlation analyses. All reported P values are two-tailed and declared statistically significant at the 0.05 level. 3. Results Demographics, clinical data and PV plasma results of all participants are presented in Table 1. Citrulline PV levels were significantly increased in patients with MS, as compared to CwND (P = 0.042) and individuals with non-MS white matter laesions (P = 0.006). Arginine PV levels were decreased in patients with MS, as compared to CwND, but this difference did not reach statistical significance (P = 0.128). In 175
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Table 1 Demographics, clinical data and peripheral venous plasma results of all participants.
Sex, male/female Mean (SD) age, years Mean (SD) EDSS score Mean (SD) disease duration, years Mean (SD) MSSS Disease course, RR/SP/PP Median (IQR) PV citrulline, μM Median (IQR) PV arginine, μM Median (IQR) PV glutamine, μM Mean (SD) NO, μM
MS
CwND
nMSWML
10/15 50 (9) 3.8 (2.6) 12 (8) 4.8 (2.9) 12/10/3 50.5 (86.5) 20.9 (21.5) 250.5 (401) 266.2 (68.4)a
11/14 51 (15)
10/15 54 (10)
33.6 (43.7) 24.9 (185.4) 160.4 (522.2) 227.8 (47.5)
32.4 (28.9) 7.1 (7.5) 147.7 (68.7)
Table 3 Characteristics of patients with multiple sclerosis who underwent brain magnetic resonance imaging.
4. Discussion We are the first group to report increased plasma citrulline levels in patients with MS, presenting a new and potentially promising Table 2 Characteristics and results of subjects who underwent internal jugular vein cannulation.
Sex, male/female Mean (SD) age, years Mean (SD) EDSS score Mean (SD) disease duration, years Mean (SD) MSSS Disease course, RR/SP/PP Median (IQR) PV citrulline, μM Median (IQR) IJV citrulline, μM Median (IQR) IJV/PV citrulline
P
5/5 56 (7) 4.8 (1.8) 14 (8) 5.9 (2.3) 2/5/3 47.7 (17.6) 40.4 (4.4) 0.9 (0.3)
5/5 60 (13)
0.165
30.5 (14.3) 37 (10.7) 1 (0.4)
0.035 0.190 0.165
Mean (SD) age, years Mean (SD) EDSS score Mean (SD) disease duration, years Mean (SD) MSSS Disease course: RR/SP/PP
46 (10) 3.1 (2.7) 11 (7) 3.9 (2.8) 8/4/0
biomarker. PV plasma levels in our MS cohort were significantly elevated as compared to both CwND and subjects with non-MS white matter laesions, hereby increasing the specificity of this finding for MS. Previous studies reported normal fasting plasma levels of citrulline ranging between 25 and 40 μM [19–21], which corresponds to the values that were found in our control groups. We did not observe an elevated IJV/PV gradient in patients with MS, compared to CwND, suggesting that the peripheral venous increase does not result from a release from the hypercitrullinated MBP fraction in the brain. Our a priori hypothesis for this statement thus could not be confirmed and the source of elevated plasma citrulline in MS remains open for debate. Citrulline is an amino acid mainly found in watermelons (Citrullus vulgaris) from which the substance was first isolated in 1930 and obtained its name. Humans do not have corresponding tRNA but can incorporate citrulline in proteins by posttranslational modification [9]. Most of the circulating citrulline is generated from transformation of dietary glutamine and arginine by intestinal epithelium enterocytes [22]. Changes in enterocyte mass and function are associated with decreased plasma citrulline levels in patients with inflammatory bowel disease, short bowel syndrome and cancer-therapy mediated intestinal mucositis; [22,23] but none of these conditions were present in our CwND subjects. In addition, two studies comparing small bowel biopsies between healthy volunteers and MS patients found no difference in aspect or surface size of jejunal enterocytes [24,25]. Nutritional citrulline is present only in a few proteins and intake from a normal diet is low. Dietary variation does not seem to be a likely explanation for the observed differences in plasma citrulline levels between our groups since all blood samples were obtained after overnight fasting. Collins and co-workers demonstrated that even extremely high daily dietary intake of citrulline (approximately 2 g per day during 3 weeks) via water melon juice did not lead to increased plasma citrulline levels after 12 h of fasting, as compared to a control diet group [21]. In a similar experiment, fasting plasma levels of citrulline remained stable and comparable to placebo during a 14-day regimen of arginine-enriched enteral nutrition in injured patients [26]. High glutamine intake, on the other hand, can provoke elevations of plasma citrulline levels, but thus far this has always been accompanied by a concomitant rise in plasma glutamine [27,28], which was not observed in our study. Gut microbiome research is currently of great interest in MS, but a thorough literature search did not reveal any evidence of hyperproduction of citrulline, nor its precursors glutamine or arginine, by commensal flora in patients with MS. Citrulline released by the intestines escapes rapid clearance by the liver but is then largely taken up by the kidneys, where nearly 100% is metabolized by argininosuccinate synthetase and lyase to produce arginine. This arginine/glutamine-citrulline-arginine cycle avoids excessive uptake of exogenous arginine and glutamine by the liver and plays a key role in maintaining correct nitrogen and amino acid homeostasis [9,20]. Renal insufficiency would be expected to increase citrulline and decrease arginine concentrations in blood and therefore, as mentioned in the materials and methods section, we excluded patients with known kidney disease. In general, previous studies in
contrast, arginine PV levels of MS patients were significantly elevated when compared to subjects with non-MS white matter lesions (P < 0.001). Glutamine PV levels did not significantly differ between patients with MS and CwND (P = 0.662) or subjects with non-MS white matter lesions (P = 0.24). We found increased PV NO levels in the MS group, as compared to CwND, with a trend towards statistical significance (P = 0.083). In patients with MS, no significant correlations were found between PV plasma citrulline levels and EDSS scores (r = 0.038, P = 0.857), disease duration (r = −0.066, P = 0.755) or MSSS (r = 0.063, P = 0.766). EDSS scores significantly correlated with disease duration (r = 0.548, P = 0.005) and MSSS (0.844, P < 0.001). Ten patients with MS and 10 CwND underwent IJV catheterization as described above. Characteristics and plasma results of both subgroups are shown in Table 2. An increased IJV/PV ratio for plasma citrulline was not observed in patients with MS. Demographic data of the patients with MS (N = 12) who underwent brain MRI are presented in Table 3. Total FLAIR laesion volume was 17.33 ± 12.00 mL. Whole brain and total grey matter volume was 1293.50 ± 57.43 mL and 794.83 ± 51.86 mL, respectively. There were no significant correlations between any of these radiological volumetric parameters and PV citrulline plasma levels (r = 0.214, P = 0.505; r = −0.266, P = 0.403; r = −0.262, P = 0.411; respectively).
CwND
5/10
EDSS = Expanded Disability Status Scale, MSSS = Multiple Sclerosis Severity Score, RR = relapsing-remitting, SP = secondary progressive, PP = primary progressive.
MS = multiple sclerosis, CwND = controls without neurological disease, nMSWML = non-MS white matter laesions, EDSS = Expanded Disability Status Scale, MSSS = Multiple Sclerosis Severity Score, RR = relapsing-remitting, SP = secondary progressive, PP = primary progressive, PV = peripheral venous, NO = nitric oxide. a Only the samples from the 15 MS patients recruited at the Universitair Ziekenhuis Brussel were used for this analysis.
MS
Sex: male/female
MS = multiple sclerosis, CwND = controls without neurological disease, EDSS = Expanded Disability Status Scale, MSSS = Multiple Sclerosis Severity Score, RR = relapsing-remitting, SP = secondary progressive, PP = primary progressive, IJV = internal jugular vein, PV = peripheral vein.
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cannot clearly explain this finding, and as the non-MS white matter laesions group is quite heterogeneous, it is complicated to draw conclusions or formulate hypotheses. In conclusion, this is the first study providing evidence of elevated plasma citrulline levels in patients with MS. We could not demonstrate an augmented release from the hypercitrullinated MBP fraction in brain as a causal source and hypothesize that the excess citrulline originates outside the CNS due to increased iNOS activity. A complementary analysis in which citrulline, arginine and NOS activity are measured inside leukocytes, would be interesting to put this theory to the test. The significance of elevated plasma citrulline in MS patients remains another intriguing question, since we could not find a direct correlation with standard clinical (EDSS and MSSS scores) nor radiological (total brain and FLAIR laesion volume) measures of disease severity. It would therefore be of interest to investigate whether plasma citrulline levels are elevated and correlate with disease measures in more active patients (e.g. during relapses) and whether these levels are predictive for future disability. Increased plasma citrulline may have promising biomarker potential in MS but its exact origin and significance still needs to be elucidated.
patients with chronic kidney dysfunction showed much higher citrulline levels than those found in our subjects [19,29,30]. Besides the gut-kidney axis and post-translational protein deimination, citrulline can be generated via two other processes in the human body. First, citrulline acts as an intermediate in the urea cycle, where it is synthesized from ornithine and carbamyol phosphate by ornithine carbamoyl transferase. This reaction takes place in the mitochondria of the liver and under physiological circumstances all the generated citrulline is in situ converted to arginine by the same enzymes as in the kidney [9,20]. Very high levels of citrulline can be found in patients with argininosuccinate synthetase or lyase deficiency, but these conditions lead to substantially higher citrulline levels than those found in our MS cohort and result in a severe clinical disease phenotype. In contrast, heterozygotes typically do not present with overt symptoms and may only have slightly increased citrulline and ammonia levels [20,31]. We did not measure ammonia levels in our study and therefore it is theoretically impossible to exclude these disorders as a cause for the encountered citrulline differences. However, there is currently no evidence suggesting a relationship between high ammonia levels or urea cycle deficiencies and MS. Second, citrulline is a by-product of NO generation by nitric oxide synthase (NOS) from arginine [9]. Elevated levels of inducable NOS (iNOS) mRNA were found in MS laesions [32], and several studies demonstrated raised NO levels in cerebrospinal fluid and plasma of patients with MS, as compared to controls [33–36]. In our study, PV plasma NO levels were higher in MS patients than in CwND, with a trend towards significance. There is no clear answer on whether the increased NO production in MS originates solely in the CNS or involves an additional peripheral fraction. The latter hypothesis may be supported by evidence of increased NO production in blood leukocytes outside the CNS of patients with MS [37–40]. Excess citrulline generated by iNOS in the periphery could explain the decreased PV arginine levels, although not significant, and absence of an IJV/PV citrulline gradient, as observed in our MS cohort. There are several limitations to our study. First, neither the citrulline, arginine or glutamine plasma values were normally distributed. We used non-parametric statistical tests for groupwise comparisons, which are considered as more stringent procedures. Second, plasma levels of ammonia are lacking and kidney function was not assessed. As explained above, the probability of urea cycle abnormalities to be present in our group of MS patients was considered low and known kidney dysfunction was an exclusion criterion for study entry. Third, CwND did not undergo brain MRI, nor had revision of eventual previous imaging for the presence of incidental cerebral white matter laesions. Thus, there might be some overlap between both control groups, but we assume that this does not affect the interpretation of our main findings. Fourth, subanalyses involving MRI, NO and IJV measurements were performed in smaller sample sizes with an increased risk for missing significance due to type II error. Moreover, a release of citrulline from the brain of MS patients might not be reflected by the IJV/PV ratio for other reasons. We only included MS patients free of exacerbations for at least three months, while a more prominent cerebral MBP degradation would be expected during relapses. MBP destroyed in the spinal cord does not drain into the jugular veins, which could attenuate or abolish the gradient. We know that certain MBP fragments, such as peptide 45–89, survive a long time in circulation [41,42], and it is not known how fast these fragments are degraded in vivo. Since high performance liquid chromatography only detects free amino acids, citrulline enclosed in these partially degraded protein fragments could remain unnoticed in the IJV. Fifth, in citrulline and arginine assessment we had, respectively, four and one immeasurable low value. For arginine, the subject belonged to the CwND group and for citrulline one was part of the MS and the other three were CwND. We decided to enter the lower detection limit (0.0066 μM for citrulline and 0.0032 μM for arginine) as value, rather than zero. Sixth, plasma arginine levels in subjects with non-MS white matter laesions were significantly decreased when compared with patients with MS. We
Acknowledgements We thank all MS patients and controls subjects for participating in this study, Mrs. Annick Van Merhaegen-Wieleman for supporting recruitment, Mrs. Anke De Smet for blood sample processing and Mr. Yves Fierens for imaging assistance. Contributors MD and JDK contributed to the concept and study design. All authors were involved in data acquisition and analysis. MV and MD wrote the first draft of the manuscript. All authors contributed to the critical reviewing and approved the final version. Competing interests GN is a shareholder of Icometrix. No further particularities to declare. References [1] A. Compston, A. Coles, Multiple sclerosis, Lancet 359 (2002) 1221–1231. [2] J.M. Boggs, Myelin basic protein: a multifunctional protein, Cell. Mol. Life Sci. 63 (2006) 1945–1961. [3] T. Korn, Pathophysiology of multiple sclerosis, J. Neurol. 255 (2008) 2–6. [4] G. Harauz, N. Ishiyama, C.M.D. Hill, et al., Myelin basic protein — diverse conformational states of an intrinsically unstructured protein and its roles in myelin assembly and multiple sclerosis, Micron 35 (2004) 503–542. [5] G. Harauz, A.A. Musse, A tale of two citrullines — structural and functional aspects of myelin basic protein deimination in health and disease, Neurochem. Res. 32 (2007) 137–158. [6] M.A. Moscarello, D.D. Wood, C. Ackerley, et al., Myelin in multiple sclerosis is developmentally immature, J. Clin. Invest. 94 (1994) 146–154. [7] D.D. Wood, J.M. Bilbao, P. O'Connors, et al., Acute multiple sclerosis (Marburg type) is associated with developmentally immature myelin basic protein, Ann. Neurol. 40 (1996) 18–24. [8] K. Oguz, A. Kurne, A. Aksu, et al., Assessment of citrullinated myelin by 1H-MR spectroscopy in early-onset multiple sclerosis, AJNR Am. J. Neuroradiol. 30 (2009) 716–721. [9] L. Breuillard, C. Cynober, C. Moinard, Citrulline and nitrogen homeostasis: an overview, Amino Acids 47 (2015) 685–691. [10] C.H. Polman, S.C. Reingold, B. Banwell, et al., Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria, Ann. Neurol. 69 (2011) 292–302. [11] P. Zamboni, R. Galeotti, The chronic cerebrospinal venous insufficiency syndrome, Phlebology 25 (2010) 269–279. [12] P. Zamboni, R. Galeotti, E. Menegatti, et al., Chronic cerebrospinal venous insufficiency in patients with multiple sclerosis, J. Neurol. Neurosurg. Psychiatry 80 (2009) 392–399. [13] M. Kelm, Nitric oxide metabolism and breakdown, Biochim. Biophys. Acta 1411 (1998) 273–289. [14] H. Moshage, B. Kok, J.R. Huizenga, et al., Nitrite and nitrate determinations in plasma: a critical evaluation, Clin. Chem. 41 (1995) 892–896.
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Plasma citrulline levels are increased in patients with multiple sclerosis a
b
a,c
Maxime Vande Vyver , Roel Beelen , Jacques De Keyser , Guy Nagels ⁎ Anne-Marie Van Binstf, Christiaan Verborghg, Miguel D'haeseleera,d,
a,d,e
T
,
a
Department of Neurology, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Center for Neurosciences, Brussels, Belgium Department of Cardiovascular and Thoracic Surgery, Onze-Lieve-Vrouw Ziekenhuis, Campus Aalst, Aalst, Belgium Department of Neurology, Universitair Medisch Centrum Groningen, Groningen, The Netherlands d Nationaal Multiple Sclerose Centrum, Melsbroek, Belgium e Faculté de Psychologie et des Sciences de l'Education, Mons, Belgium f Department of Radiology, Universitair Ziekenhuis Brussel, Brussels, Belgium g Department of Aenesthesiology, Universitair Ziekenhuis Brussel, Brussels, Belgium b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Multiple sclerosis Citrulline Myelin basic protein Nitric oxide Jugular vein
Background: Multiple sclerosis is an inflammatory demyelinating disorder of the central nervous system (CNS). Myelin basic protein (MBP), which is one of the main compounds of CNS myelin, appears to be hypercitrullinated in the brain of patients with MS. We hypothesized that MS is associated with an increased release of citrulline from the brain. Methods: Twenty-five patients with MS, 25 controls without neurological disease (CwND) and 25 subjects with non-MS cerebral white matter lesions were included in this study. Groups were matched for age and gender. Clinical MS disability measures were recorded by means of Expanded Disability Status Scale (EDSS) scores and Multiple Sclerosis Severity Scores (MSSS). Citrulline was assessed in plasma obtained from an antecubital peripheral vein (PV) in all participants. Additional internal jugular vein (IJV) samples were examined in 10 patients with MS and 10 CwND. Twelve patients with MS underwent brain magnetic resonance imaging to determine total brain and T2 fluid-attenuated inversion recovery lesion volume. Results: Median [IQR] PV citrulline levels were increased in patients with MS (50.47 [86.61] μM), as compared to CwND (33.58 [43.65] μM, P = 0.042) and subjects with non-MS cerebral white matter lesions (32.41 [28.86] μM, P = 0.006). Citrulline IJV levels and IJV/PV ratios were comparable between patients with MS and CwND. No significant correlations were found between PV citrulline levels and any of the clinical, nor radiological, disease measures. Conclusion: PV plasma levels of citrulline are elevated in patients with MS but this does not seem to result from an augmented release from the brain. Increased plasma citrulline may be a promising new biomarker in MS but the origin and significance need to be further elucidated.
1. Introduction
gene is located on chromosome 18 but alternative exon splicing leads to the expression of multiple isoforms, varying in length and molecular weight [4]. Subsequently, MBP can undergo several posttranslational modification steps, one of which is the conversion of arginine to citrulline residues by peptidylarginine deiminase (PAD) enzymes [5]. A 18.5 kD (170 amino acid residues) uncitrullinated protein, termed MBP-C1, is the dominant subtype in mature CNS myelin of healthy individuals. Myelin isolated from the brain of subjects with MS appears to contain a higher proportion of MBP-C8, a variant in which 6 out of 19 arginine residues are converted to citrulline, as compared to control samples [6,7], and one study using 1H-magnetic spectroscopy suggested that, as a consequence, total citrulline concentrations are elevated in
Multiple sclerosis (MS) is a chronic demyelinating and degenerative disease of the central nervous system (CNS) affecting over 2 million people worldwide [1]. Myelin covers most axons in the brain and spinal cord to form a protective lipid- and protein-rich sheath which, among other functions, facilitates nerve conduction and provides trophic support. Myelin basic protein (MBP) is one of the most abundant proteins in CNS myelin [2]. The exact underlying pathophysiology of MS remains obscure but demyelination involves T-cell mediated inflammatory responses directed against MBP [3]. The pool of MBP in the human brain is very diverse. The coding
⁎
Corresponding author at: Department of Neurology, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, Brussels, Belgium. E-mail address: [email protected] (M. D'haeseleer).
https://doi.org/10.1016/j.jns.2018.02.025 Received 16 December 2017; Received in revised form 11 February 2018; Accepted 15 February 2018 Available online 16 February 2018 0022-510X/ © 2018 Published by Elsevier B.V.
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respective CwND (10/25) were individuals who underwent central venous catheter placement prior to scheduled cardiac surgery or pacemaker placement at the UZ Brussel. All endovascular procedures were performed by the same experienced cardiovascular surgeon in the patients with MS, or aenesthesiologist in the controls. Correct IJV localization was radiographically controlled in every case. Blood samples were centrifuged and plasma was stored at −80 °C as soon as possible. Plasma citrulline, arginine and glutamine levels were determined by high-performance liquid chromatography. NO is an unstable gas which is rapidly oxidized to its breakdown products (NOx) nitrite and nitrate [13], and can therefore only be indirectly assessed. NOx levels were measured colorimetrically after reduction of nitrate to nitrite by nitrate reductase with a commercially available kit (Enzo Life Sciences®; Zandhoven, Belgium). Plasma samples obtained at the OLVZ Aalst were transported to the laboratory of the UZ Brussel on dry ice by car and, as a result, rapid storage at −80 °C was not always feasible. Because NOx still have a relatively short half-life in plasma and stability of measurement has only been demonstrated for delayed processing up to 48 h [14,15], we excluded the OLVZ Aalst samples from the NOx analysis.
cerebral white matter [8]. MBP hypercitrullination in MS is most likely mediated by increased PAD2 and 4 enzymatic activity and decreases the net positive charge of the protein, resulting in an impaired capacity to interact with negatively charged lipid compounds. These processes potentially lead to a less compact structure of the myelin sheath and increased susceptibility to proteolytic degradation [5]. Reliable body fluid biomarkers for MS diagnosis and treatment response monitoring are currently still lacking. Plasma citrulline levels have never been investigated within this context. In this study, we hypothesized that demyelination in MS leads to an increased release of citrulline from the brain to the circulation. 2. Methods 2.1. Study design and main objectives We performed a prospective exploratory study at the Universitair Ziekenhuis (UZ) Brussel (Brussels, Belgium) and the Onze-Lieve Vrouw Ziekenhuis (OLVZ) Aalst (Aalst, Belgium) to investigate peripheral vein (PV) and internal jugular vein (IJV) plasma levels of citrulline in patients with MS and control subjects without neurologic disease (CwND). Plasma arginine, nitric oxide (NO) and glutamine levels were contemporaneously assessed since these agents are closely related to the metabolism of citrulline [9]. The difference in PV plasma citrulline levels between both groups was selected as the primary endpoint of this study. Significant alterations in patients with MS were verified against a second control group of individuals with non-MS cerebral white matter lesions to increase specificity. The study was approved by the ethics committees of both centers and conformed to the Declaration of Helskini principles. All participants were recruited from April 2011 to March 2014, and provided a written informed consent.
2.3. Magnetic resonance imaging MS patients recruited at the UZ Brussel (15/25) underwent additional brain MRI in supine position using a 3 T machine (Philips Achieva; Best, The Netherlands). The protocol contained, among others, two 3D sequences: a magnetization prepared 3D T1 TurboFLASH (TR 7.8 ms; TE 3.8 ms; TI, 970 ms; flip angle 8°, 240 FOV) and a fat-saturated 3D FLAIR (TR 8000 ms, TE 260 ms, 240 FOV). We used MSmetrix® software (Icometrix; Leuven, Belgium - coveted CE mark and FDA approved) to measure FLAIR laesion load, whole brain and grey matter volumes [16]. Volumetric analyses could not be performed in 3 patients due to technical issues.
2.2. Subjects and blood sampling Twenty-five patients with MS (relapsing-remitting: 12; secondary progressive: 10; primary progressive: 3), according to the 2010 revised McDonald criteria [10], 25 CwND and 25 subjects with non-MS cerebral white matter laesions (ischaemic leukoencephalopathy: 19; migraine: 2; antiphospholipid syndrome: 1; post-chemotherapy: 1; idiopathic: 1) were included in this study. All groups were a priori matched for age and gender. Individuals known with chronic kidney disease were excluded. Eleven MS patients received immunomodulatory drugs (interferon β-1a: 7; interferon β-1b: 2; glatiramere acetate: 1; natalizumab: 1). All MS patients were clinically stable without evidence of an exacerbation within 3 months prior to inclusion. The diagnosis of nonMS cerebral white matter laesions was based on a historic brain MRI, available in the medical records of these individuals, demonstrating at least 3 punctate or 2 confluent areas of T2-weighted hyperintensity in the cerebral periventricular and/or subcortical white matter. All respective brain scans were reviewed by the investigators and declared as non-suggestive of MS. Participants fasted for a minimum of 8 h before blood was drawn from an antecubital PV. All blood samples were collected between 8 and 10 am. Recruitment took place at the UZ Brussel for 15/25 patients with MS (neurology department), 25/25 CwND (healthy volunteers and aenesthesiology department, see below) and 25/25 subjects with nonMS cerebral white matter laesions (neurology department). In 10 patients with MS and 10 CwND an additional blood sample was collected from the right IJV, simultaneously with the PV blood draw. These patients with MS (10/25) were recruited at the Department of Cardiovascular and Thoracic Surgery of the OLVZ Aalst, where they were actively seeking treatment with dilatation angioplasty, for socalled chronic cerebrospinal venous insufficiency [11,12], on their own initiative and specific request. The investigators were never involved in this therapeutic decision process. MS diagnosis was confirmed based on clinical grounds and access to the patient's medical records. The
2.4. Disability and disease progression in MS Clinical disability was measured with the Expanded Disability Status Scale (EDSS) in all patients with MS [17]. Rate of progression of clinical disability was measured with the Multiple Sclerosis Severity Score (MSSS) [18]. This score is based on the combination of EDSS score and disease duration. Both EDSS scores and MSSS range from 0 to 10. Higher scores indicate, respectively, more disability and faster progression of disability. 2.5. Statistical analysis All statistical analyses were performed with SPSS® (Version 24.0, IBM; Amonk, NY USA). PV plasma citrulline, arginine and glutamine values from the total cohort of 75 subjects did not pass the KolmogorovSmirnov test for normal distribution and therefore we expressed them as median [IQR]. Amino acid levels obtained in IJV plasma were presented likewise for reasons of uniformity. All other ordinal and continuous data showed a normal distribution and were presented as mean ± SD. Group differences were assessed using Mann-Whitney U tests. Spearman's rank correlation was used for correlation analyses. All reported P values are two-tailed and declared statistically significant at the 0.05 level. 3. Results Demographics, clinical data and PV plasma results of all participants are presented in Table 1. Citrulline PV levels were significantly increased in patients with MS, as compared to CwND (P = 0.042) and individuals with non-MS white matter laesions (P = 0.006). Arginine PV levels were decreased in patients with MS, as compared to CwND, but this difference did not reach statistical significance (P = 0.128). In 175
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Table 1 Demographics, clinical data and peripheral venous plasma results of all participants.
Sex, male/female Mean (SD) age, years Mean (SD) EDSS score Mean (SD) disease duration, years Mean (SD) MSSS Disease course, RR/SP/PP Median (IQR) PV citrulline, μM Median (IQR) PV arginine, μM Median (IQR) PV glutamine, μM Mean (SD) NO, μM
MS
CwND
nMSWML
10/15 50 (9) 3.8 (2.6) 12 (8) 4.8 (2.9) 12/10/3 50.5 (86.5) 20.9 (21.5) 250.5 (401) 266.2 (68.4)a
11/14 51 (15)
10/15 54 (10)
33.6 (43.7) 24.9 (185.4) 160.4 (522.2) 227.8 (47.5)
32.4 (28.9) 7.1 (7.5) 147.7 (68.7)
Table 3 Characteristics of patients with multiple sclerosis who underwent brain magnetic resonance imaging.
4. Discussion We are the first group to report increased plasma citrulline levels in patients with MS, presenting a new and potentially promising Table 2 Characteristics and results of subjects who underwent internal jugular vein cannulation.
Sex, male/female Mean (SD) age, years Mean (SD) EDSS score Mean (SD) disease duration, years Mean (SD) MSSS Disease course, RR/SP/PP Median (IQR) PV citrulline, μM Median (IQR) IJV citrulline, μM Median (IQR) IJV/PV citrulline
P
5/5 56 (7) 4.8 (1.8) 14 (8) 5.9 (2.3) 2/5/3 47.7 (17.6) 40.4 (4.4) 0.9 (0.3)
5/5 60 (13)
0.165
30.5 (14.3) 37 (10.7) 1 (0.4)
0.035 0.190 0.165
Mean (SD) age, years Mean (SD) EDSS score Mean (SD) disease duration, years Mean (SD) MSSS Disease course: RR/SP/PP
46 (10) 3.1 (2.7) 11 (7) 3.9 (2.8) 8/4/0
biomarker. PV plasma levels in our MS cohort were significantly elevated as compared to both CwND and subjects with non-MS white matter laesions, hereby increasing the specificity of this finding for MS. Previous studies reported normal fasting plasma levels of citrulline ranging between 25 and 40 μM [19–21], which corresponds to the values that were found in our control groups. We did not observe an elevated IJV/PV gradient in patients with MS, compared to CwND, suggesting that the peripheral venous increase does not result from a release from the hypercitrullinated MBP fraction in the brain. Our a priori hypothesis for this statement thus could not be confirmed and the source of elevated plasma citrulline in MS remains open for debate. Citrulline is an amino acid mainly found in watermelons (Citrullus vulgaris) from which the substance was first isolated in 1930 and obtained its name. Humans do not have corresponding tRNA but can incorporate citrulline in proteins by posttranslational modification [9]. Most of the circulating citrulline is generated from transformation of dietary glutamine and arginine by intestinal epithelium enterocytes [22]. Changes in enterocyte mass and function are associated with decreased plasma citrulline levels in patients with inflammatory bowel disease, short bowel syndrome and cancer-therapy mediated intestinal mucositis; [22,23] but none of these conditions were present in our CwND subjects. In addition, two studies comparing small bowel biopsies between healthy volunteers and MS patients found no difference in aspect or surface size of jejunal enterocytes [24,25]. Nutritional citrulline is present only in a few proteins and intake from a normal diet is low. Dietary variation does not seem to be a likely explanation for the observed differences in plasma citrulline levels between our groups since all blood samples were obtained after overnight fasting. Collins and co-workers demonstrated that even extremely high daily dietary intake of citrulline (approximately 2 g per day during 3 weeks) via water melon juice did not lead to increased plasma citrulline levels after 12 h of fasting, as compared to a control diet group [21]. In a similar experiment, fasting plasma levels of citrulline remained stable and comparable to placebo during a 14-day regimen of arginine-enriched enteral nutrition in injured patients [26]. High glutamine intake, on the other hand, can provoke elevations of plasma citrulline levels, but thus far this has always been accompanied by a concomitant rise in plasma glutamine [27,28], which was not observed in our study. Gut microbiome research is currently of great interest in MS, but a thorough literature search did not reveal any evidence of hyperproduction of citrulline, nor its precursors glutamine or arginine, by commensal flora in patients with MS. Citrulline released by the intestines escapes rapid clearance by the liver but is then largely taken up by the kidneys, where nearly 100% is metabolized by argininosuccinate synthetase and lyase to produce arginine. This arginine/glutamine-citrulline-arginine cycle avoids excessive uptake of exogenous arginine and glutamine by the liver and plays a key role in maintaining correct nitrogen and amino acid homeostasis [9,20]. Renal insufficiency would be expected to increase citrulline and decrease arginine concentrations in blood and therefore, as mentioned in the materials and methods section, we excluded patients with known kidney disease. In general, previous studies in
contrast, arginine PV levels of MS patients were significantly elevated when compared to subjects with non-MS white matter lesions (P < 0.001). Glutamine PV levels did not significantly differ between patients with MS and CwND (P = 0.662) or subjects with non-MS white matter lesions (P = 0.24). We found increased PV NO levels in the MS group, as compared to CwND, with a trend towards statistical significance (P = 0.083). In patients with MS, no significant correlations were found between PV plasma citrulline levels and EDSS scores (r = 0.038, P = 0.857), disease duration (r = −0.066, P = 0.755) or MSSS (r = 0.063, P = 0.766). EDSS scores significantly correlated with disease duration (r = 0.548, P = 0.005) and MSSS (0.844, P < 0.001). Ten patients with MS and 10 CwND underwent IJV catheterization as described above. Characteristics and plasma results of both subgroups are shown in Table 2. An increased IJV/PV ratio for plasma citrulline was not observed in patients with MS. Demographic data of the patients with MS (N = 12) who underwent brain MRI are presented in Table 3. Total FLAIR laesion volume was 17.33 ± 12.00 mL. Whole brain and total grey matter volume was 1293.50 ± 57.43 mL and 794.83 ± 51.86 mL, respectively. There were no significant correlations between any of these radiological volumetric parameters and PV citrulline plasma levels (r = 0.214, P = 0.505; r = −0.266, P = 0.403; r = −0.262, P = 0.411; respectively).
CwND
5/10
EDSS = Expanded Disability Status Scale, MSSS = Multiple Sclerosis Severity Score, RR = relapsing-remitting, SP = secondary progressive, PP = primary progressive.
MS = multiple sclerosis, CwND = controls without neurological disease, nMSWML = non-MS white matter laesions, EDSS = Expanded Disability Status Scale, MSSS = Multiple Sclerosis Severity Score, RR = relapsing-remitting, SP = secondary progressive, PP = primary progressive, PV = peripheral venous, NO = nitric oxide. a Only the samples from the 15 MS patients recruited at the Universitair Ziekenhuis Brussel were used for this analysis.
MS
Sex: male/female
MS = multiple sclerosis, CwND = controls without neurological disease, EDSS = Expanded Disability Status Scale, MSSS = Multiple Sclerosis Severity Score, RR = relapsing-remitting, SP = secondary progressive, PP = primary progressive, IJV = internal jugular vein, PV = peripheral vein.
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cannot clearly explain this finding, and as the non-MS white matter laesions group is quite heterogeneous, it is complicated to draw conclusions or formulate hypotheses. In conclusion, this is the first study providing evidence of elevated plasma citrulline levels in patients with MS. We could not demonstrate an augmented release from the hypercitrullinated MBP fraction in brain as a causal source and hypothesize that the excess citrulline originates outside the CNS due to increased iNOS activity. A complementary analysis in which citrulline, arginine and NOS activity are measured inside leukocytes, would be interesting to put this theory to the test. The significance of elevated plasma citrulline in MS patients remains another intriguing question, since we could not find a direct correlation with standard clinical (EDSS and MSSS scores) nor radiological (total brain and FLAIR laesion volume) measures of disease severity. It would therefore be of interest to investigate whether plasma citrulline levels are elevated and correlate with disease measures in more active patients (e.g. during relapses) and whether these levels are predictive for future disability. Increased plasma citrulline may have promising biomarker potential in MS but its exact origin and significance still needs to be elucidated.
patients with chronic kidney dysfunction showed much higher citrulline levels than those found in our subjects [19,29,30]. Besides the gut-kidney axis and post-translational protein deimination, citrulline can be generated via two other processes in the human body. First, citrulline acts as an intermediate in the urea cycle, where it is synthesized from ornithine and carbamyol phosphate by ornithine carbamoyl transferase. This reaction takes place in the mitochondria of the liver and under physiological circumstances all the generated citrulline is in situ converted to arginine by the same enzymes as in the kidney [9,20]. Very high levels of citrulline can be found in patients with argininosuccinate synthetase or lyase deficiency, but these conditions lead to substantially higher citrulline levels than those found in our MS cohort and result in a severe clinical disease phenotype. In contrast, heterozygotes typically do not present with overt symptoms and may only have slightly increased citrulline and ammonia levels [20,31]. We did not measure ammonia levels in our study and therefore it is theoretically impossible to exclude these disorders as a cause for the encountered citrulline differences. However, there is currently no evidence suggesting a relationship between high ammonia levels or urea cycle deficiencies and MS. Second, citrulline is a by-product of NO generation by nitric oxide synthase (NOS) from arginine [9]. Elevated levels of inducable NOS (iNOS) mRNA were found in MS laesions [32], and several studies demonstrated raised NO levels in cerebrospinal fluid and plasma of patients with MS, as compared to controls [33–36]. In our study, PV plasma NO levels were higher in MS patients than in CwND, with a trend towards significance. There is no clear answer on whether the increased NO production in MS originates solely in the CNS or involves an additional peripheral fraction. The latter hypothesis may be supported by evidence of increased NO production in blood leukocytes outside the CNS of patients with MS [37–40]. Excess citrulline generated by iNOS in the periphery could explain the decreased PV arginine levels, although not significant, and absence of an IJV/PV citrulline gradient, as observed in our MS cohort. There are several limitations to our study. First, neither the citrulline, arginine or glutamine plasma values were normally distributed. We used non-parametric statistical tests for groupwise comparisons, which are considered as more stringent procedures. Second, plasma levels of ammonia are lacking and kidney function was not assessed. As explained above, the probability of urea cycle abnormalities to be present in our group of MS patients was considered low and known kidney dysfunction was an exclusion criterion for study entry. Third, CwND did not undergo brain MRI, nor had revision of eventual previous imaging for the presence of incidental cerebral white matter laesions. Thus, there might be some overlap between both control groups, but we assume that this does not affect the interpretation of our main findings. Fourth, subanalyses involving MRI, NO and IJV measurements were performed in smaller sample sizes with an increased risk for missing significance due to type II error. Moreover, a release of citrulline from the brain of MS patients might not be reflected by the IJV/PV ratio for other reasons. We only included MS patients free of exacerbations for at least three months, while a more prominent cerebral MBP degradation would be expected during relapses. MBP destroyed in the spinal cord does not drain into the jugular veins, which could attenuate or abolish the gradient. We know that certain MBP fragments, such as peptide 45–89, survive a long time in circulation [41,42], and it is not known how fast these fragments are degraded in vivo. Since high performance liquid chromatography only detects free amino acids, citrulline enclosed in these partially degraded protein fragments could remain unnoticed in the IJV. Fifth, in citrulline and arginine assessment we had, respectively, four and one immeasurable low value. For arginine, the subject belonged to the CwND group and for citrulline one was part of the MS and the other three were CwND. We decided to enter the lower detection limit (0.0066 μM for citrulline and 0.0032 μM for arginine) as value, rather than zero. Sixth, plasma arginine levels in subjects with non-MS white matter laesions were significantly decreased when compared with patients with MS. We
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