Acta Neurol Belg DOI 10.1007/s13760-015-0512-2
ORIGINAL ARTICLE
Low vitamin D levels in healthy controls and patients with autoimmune neuromuscular disorders in Greece Elisabeth Chroni1 • Nikolaos Dimisianos1 • Anna Rostedt Punga2
Received: 6 April 2015 / Accepted: 7 July 2015 Ó Belgian Neurological Society 2015
Abstract Normal autoimmune function is dependent on adequate levels of activated vitamin D, 25 hydroxy vitamin D [25(OH)D]. A recent study presented deficiency of 25(OH)D levels in Swedish MG patients. We aimed to study 25(OH)D levels in patients with MG and autoimmune polyneuropathies (PNP) at a southern latitude in Greece. Plasma levels of 25(OH)D were analyzed in Greek patients with MG (n = 19), immune-mediated PNP (N = 11) and in 30 Greek healthy age- and sex-matched controls. Ten MG patients received supplementation with vitamin D3. The MG Composite Score (MGC) and MG quality of life assessed disease severity in MG patients, whereas the INCAT Disability Scale assessed clinical features in the PNP patients. MG patients with and without vitamin D3 supplementation had higher 25(OH)D levels (mean 58.8 ± 16.3 and 62.0 ± 22.4 nmol/L, respectively) than PNP patients (mean 42.1 ± 11.5 nmol/L, p = 0.01) and healthy controls (mean 45.7 ± 13.8 nmol/L, p = 0.01). Plasma 25(OH)D levels was lower with age in all groups. There were no correlations between 25(OH)D and disease duration, MGC score, or INCAT score. Vitamin D deficiency was found in all Greek patient groups and healthy controls. Levels of 25(OH)D were higher in MG patients with as well as without vitamin D supplementation compared to healthy controls, whereas CIDP/GBS patients had levels similar to controls.
& Anna Rostedt Punga [email protected] 1
Department of Neurology, School of Medicine, University of Patras, Patras, Greece
2
Department of Clinical Neurophysiology, Institute of Neuroscience, Uppsala University Hospital, Uppsala University, 75185 Uppsala, Sweden
Keywords Myasthenia gravis Guillain–Barre´ syndrome Vitamin D Autoimmune polyneuropathy
Introduction Vitamin D has been attributed important functional roles in regulating the immune response [1], through action on the nuclear vitamin D receptor on lymphocytes, monocytes, dendritic cells, etc. [2]. The possible beneficial role of vitamin D on the prevention and/or treatment of autoimmune diseases is thought to be accomplished by restraining adaptive immune responses through inhibiting pro-inflammatory cytokine production and enhancement of regulatory T-cell function [1, 2]. Insufficient or deficient levels of 25 hydroxy vitamin D [25(OH)D] have been reported in a variety of autoimmune disorders such as systemic lupus erythematosus [3], multiple sclerosis [4], rheumatoid arthritis [5], type 1 diabetes mellitus [6], and narcolepsy [7]. Myasthenia gravis (MG), Guillain– Barre´ syndrome (GBS), and chronic inflammatory demyelinating polyneuropathy (CIDP) are all immunemediated diseases of the peripheral nervous system, in which autoreactive CD4? T-cells stimulate the production of autoantibodies. Further, there is a link between corticosteroid treatment, which is a common regimen for autoimmune neuromuscular diseases, and vitamin D deficiency [8]. Askmark et al. [9] recently identified vitamin D deficiency in Swedish MG patients as well as improvement of fatigue after vitamin D supplementation. Further, 25(OH)D levels were very low in Swedish patients with GBS and CIDP [10]. Nevertheless, vitamin D status may vary between countries, since vitamin D levels depend on dietary habits, sun exposure (latitude), skin type, and the use of sun-protecting agents [11].
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The aim of this study was to assess levels of 25(OH)D in Greek patients with autoimmune neuromuscular diseases, including MG and autoimmune PNP, and compare them with healthy individuals from the same area of South-western Greece, a country with high sun exposure.
Ethics
Materials and methods
Blood sampling and CLIA
Patients and controls
Blood from patients and controls was collected in EDTA tubes, and plasma was collected after centrifugation and stored in -80 °C freezer until the day of processing. Quantitative determination of total 25(OH)D in plasma was performed with a LIASONÒ analysis (DiaSorin Inc, Stillwater, MN, USA) through CLIA (chemiluminescence immunoanalysis), at Uppsala University Hospital. Severe 25(OH)D deficiency was defined as \25 nmol/L, mild deficiency as 25–74 nmol/L, and optimal level as 75–250 nmol/L. Samples from 10 subjects (5 patients and 5 controls) were re-tested in Patras, Greece, to assess interlaboratory agreement.
Thirty patients with autoimmune neuromuscular disorders: MG (N = 19; 13 women; mean age 46.7 ± 20.0 years), Guillain–Barre´ Syndrome (GBS; N = 3; 1 woman; mean age 51.3 ± 25.2 years), and chronic inflammatory demyelinating polyneuropathy (CIDP; N = 8; 4 women; mean age 58.7 ± 13.4 years) were included in the study. Patients were examined either when they were hospitalized or came for follow-up visits in the outpatient clinic at the Neurology Department of Patras University Hospital (PUH) in South-western Greece. Patients were recruited in the study from May until December 2012. 19 patients had a diagnosis of MG, according to the diagnostic criteria of the Myasthenia Gravis Foundation of America (MGFA) [12], including objective muscle fatigue and neurophysiological evidence of disturbed neuromuscular transmission in combination with detection of antibodies against acetylcholine receptors (AChRs) or muscle-specific tyrosine kinase (MuSK). Muscle fatigue of MG patients was assessed according to the MG Composite Score (MGC) [13] and each patient was given a total score ranging from 0 (no myasthenic weakness) to maximum 50 points (worst possible myasthenic weakness in all examined muscles). Patients also filled out the MG questionnaire (MGQ), a tool for quality of life assessment [14]. Ten MG patients already received vitamin D3 supplementation at the time of blood collection (400–800 IU/day) since at least 6 months, while 9 were unsupplemented. The remaining 11 patients were diagnosed with GBS (n = 3) and CIDP (n = 8), according to the respective criteria [15, 16]. Disability in GBS/CIDP patients was assessed using the INCAT score [17]. None of the GBS/CIDP patients received vitamin D3 supplementation. Demographic and clinical characteristics of all patients are presented in Tables 1 and 2. Thirty age- and sex- matched healthy subjects, recruited by local information at the Patras University Hospital, served as controls. Blood collection of controls was performed during the same time period as for the patients. Healthy controls did not receive supplemental vitamin D3.
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All subjects signed written informed consent prior to inclusion into the study. The study protocol was approved by the Ethics Committee of PUH (no of approval: 37/25-12012).
Statistical analysis The non-parametric Mann–Whitney test was used for assessment of median values in continuous variables of independent samples. Spearman’s rank order correlations were applied to measure associations between two continuous variables. The hypothesis that 25(OH)D levels differ between the following groups: healthy controls, MG patients without D3 supplementation, MG patients with supplementation, and GBS/CIDP patients without supplementation was tested with one-way ANOVA. All analyses were performed using GraphPad Prism v.5.03 (GraphPad, San Diego, CA, USA) and the significance level was set at p \ 0.05.
Results Patient clinical characteristics and treatment status Of the 19 MG patients, 2 had purely ocular MG while the rest had generalized MG. Sixteen patients were AChR antibody seropositive (AChR?), whereas 3 patients who were seronegative for AChR antibodies were seropositive for MuSK antibodies (MuSK?). MG patients without vitamin D3 supplementation had MGC and MGQ scores similar to those with vitamin D3 supplementation (MGC mean score 12.1 vs. 10.1, n.s., MGQ: mean score 20.9 vs. 28.6, n.s.). The mean age (48.9 vs. 44.6 years, n.s.) and disease duration (5.4
Acta Neurol Belg Table 1 Demographic and disease characteristics of patients with myasthenia gravis (MG), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and Guillain–Barre´ syndrome (GBS) included in the study Patient ID
Sex (m/f)
Age (years)
Diagnosis
Duration (months)
Month of sample
PRD (mg/d)
IS/IMD
Vitamin D suppl
Vitamin D (nmol/L)
P01
F
34
MG-Musk
P02
M
51
CIDP
P04
F
50
MG
P06
F
46
CIDP
P07
F
16
MG
P09
F
30
MG
P10
F
55
MG
P12
F
80
GBS
0.5
P13
M
65
CIDP
P14 P15
M F
33 36
GBS MG-ocul
P16
F
32
MG
60
September
10
IVIG
400
70
P17
M
49
MG
96
October
25
AZA
No
27
P19
F
15
MG
24
October
20
No
400
69
P20
F
73
CIDP
72
November
10
CSP, IVIG
No
45
P21
M
77
CIDP
36
November
No
AZA, IVIG
No
42
P22
F
41
CIDP
108
November
No
IVIG
No
31
P23
M
52
MG-ocul
6
November
10
No
800
60
P25
F
59
MG
2
November
10
No
400
70
P26
M
50
CIDP
24
November
No
No
No
30
P27 P28
F M
82 41
MG GBS
72 0.5
December December
10 No
No IVIG
No No
75 46
P29
M
54
MG
36
December
20
No
No
57
P30
F
20
MG-Musk
8
December
20
AZA
400
65
P31
F
82
MG-Musk
18
December
40
IVIG
800
22
P32
F
61
MG
264
June
10
No
No
59
P33
F
37
MG
84
June
10
No
No
58
P34
M
67
CIDP
48
September
15
RTX, IVIG
No
52
P35
M
67
MG
12
September
No
No
No
31
P36
M
44
MG
12
October
No
No
No
72
24
June
15
IVIG
800
50
8
July
20
IVIG
No
28
84
August
5
AZA, IVIG
400
43
24
September
25
RTX, IVIG
No
26
0.5
September
15
No
No
90
4
September
10
IVIG
No
89
156
September
30
IVIG
400
70
September
No
IVIG
No
57
8
September
No
IVIG
No
50
1 24
September September
No 15
IVIG No
No 800
56 60
M male, F female, MG MG patients with AChR antibodies, MG-Musk MG with positive anti-Musk antibodies, MG-ocul ocular MG, AchEI achetylcholinesterase inhibitor, PRD prednisone, AZA azathioprine, CSP cyclosporine, RTX rituximab, IS immunosuppressants, IMD immunomodulatory treatment, IVIG intravenous immunoglobulin
vs. 3.5 years, n.s.) were also comparable between groups. Treatment with pyridostigmine (MestinonÒ, 180–240 mg/day), prednisone (PrezolonÒ, 5–40 mg/day), immunosuppressants, and IVIG are presented in Table 1. 25(OH)D levels in patients and controls Almost all patients and healthy controls (57 out of 60) had low 25(OH)D levels in the range of mild to severe
deficiency. Nevertheless, the 25(OH)D levels in MG patients were significantly higher than controls as well as PNP patients. Among the MG patients, 25(OH)D levels of unsupplemented (mean ± SD: 62.0 ± 22.4 nmol/L) and vitamin D3 supplemented patients (58.8 ± 16.3 nmol/L; n.s.) did not differ. 25(OH)D levels in GBS/CIDP patients (42.1 ± 11.5 nmol/L) were comparable to those of healthy controls (45.7 ± 13.8 nmol/L; Fig. 1). Mean levels of 25(OH)D for every month in all subjects are presented in
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Acta Neurol Belg Table 2 Demographics and disease characteristics of patients and controls Group MG
N
m/f
Age (years)
Duration (years)
MGQ
MGC
INCAT
25(OH)D (nmol/L)
9
2/8
48.9 ± 20.0
5.4 ± 6.9
20.9 ± 14.3
12.1 ± 8.1
NA
62.0 ± 22.4
MG suppl
10
4/5
44.6 ± 20.9
3.5 ± 4.2
28.6 ± 12.8
10.1 ± 6.1
NA
58.7 ± 16.3
CIDP/GBS
11
6/5
56.7 ± 16.2
2.5 ± 2.8
NA
NA
5.5 ± 3.6
42.1 ± 11.5
Controls
30
12/18
49.6 ± 18.1
NA
NA
NA
NA
45.7 ± 13.7
Values are given as mean ± SD MGQ Myasthenia Gravis questionnaire, MGC Myasthenia Gravis Composite Score, INCAT polyneuropathies disability scale
Fig. 2 Seasonal variation of 25(OH)D levels in all study subjects (mean values/month) Fig. 1 Plasma levels of 25(OH)D (in nmol/L) in patients and healthy controls. *p = 0.01
Discussion Fig. 2. There was a trend towards lowest values in May and highest values in July–August. The 25(OH)D levels from the 10 samples (5 patients and 5 controls, blood drawn during April) analyzed in Patras were consistent with the results from the Uppsala laboratory, supporting inter-laboratory agreement. Parathormone (PTH) levels were similar between controls (3.8 ± 2.3) and all patient subgroups (3.9 ± 1.9). Correlation between 25(OH)D levels, disease status, and demographic characteristics There was no correlation between 25(OH)D levels and disease severity scores (MGC, MGQ, INCAT), disease duration, or prednisone dosage. However, there was an inverse correlation between 25(OH)D levels and age, indicating lower vitamin D levels with increasing age. This correlation was consistent for both controls and patients, supplemented as well as unsupplemented (Fig. 3).
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Vitamin D is considered an important regulator of the autoimmune response, and in addition, two previous studies have shown low levels of vitamin D in Swedish MG and GBS/CIDP patients [10, 18]. The current study was designed to examine 25(OH)D levels in patients with MG and immune-mediated PNP in Greece, where the mean number of sunny hours per year (N = 2700) is greatly increased compared to Scandinavia (N = 1700) (Data obtained from the National Meteorological Service). Contrary to what could be expected, we found reduced levels of 25(OH)D also in Greek controls. The normal values in this Greek study are indeed lower than those reported for healthy Swedish subjects. Mild vitamin D deficiency was strikingly common in the Greek healthy control population. To our knowledge, there is a lack of epidemiological data regarding reference values of 25(OH)D levels in a healthy Greek adult cohort. The only available epidemiological study of vitamin D levels in Greece was performed in 178
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Fig. 3 Correlation (Spearman r) of plasma 25(OH)D levels with age in all study subjects (patients and controls)
children, revealing that even during summer months the mean levels of 25(OH)D were lower than 50 nmol/L [19]. It is somewhat intriguing that the 25(OH)D levels are not higher with more southern latitude, considering that the sun exposure is an important factor to activate vitamin D into 25(OH)D. Vitamin D deficiency has been globally recognized [20, 21], affecting an estimated 1 billion people world-wide. Deficiency in vitamin D seems to be common not only in countries of higher latitude (North America, Scandinavia, Central Europe) [22], but also in more southern countries, like Turkey, Lebanon, India, Australia, Saudi Arabia [20, 23, 24]. Incidentally, a recent study from Turkey, regarding the vitamin D status of patients with rosacea, showed lower levels of 25(OH)D in controls (mean 42.7 nmol/L) compared to rosacea patients (53.5 nmol/L, p = 0.04) [25]. An obvious explanation would be that people in sunny countries avoid sun exposure due to fear of skin cancer or use sun-protecting agents and garments. On the other hand, dietary habits differ between countries, with Scandinavians, for example, consuming larger amounts of oily fish (e.g., tuna, salmon, mackerel), with a mean daily consumption of 30–35 g/day [26]. The negative correlation of 25(OH)D levels with age is a consistent finding in various populations [22, 27, 28]. Several
factors have been offered as an explanation, including less exposure to sun light and malabsorption of food nutrients in older as compared to younger individuals. One possible explanation to the absence of correlation between age and 25(OH)D values in the previous Swedish study could be that older people in Sweden eat considerably more oily fish than younger individuals [26]. Additionally, dairy products such as butter and milk are supplemented with vitamin D3 in Nordic countries. According to the globally accepted reference values for vitamin D deficiency (\75 nmol/L), the majority of subjects in our study, including the MG patients who had higher vitamin D levels than the other two groups, showed mild to severe deficiency. Thus, although our data confirm the vitamin D deficiency in MG patients, it appears also that the healthy population of southern countries has a higher prevalence of vitamin D deficiency. It has been suggested that vitamin D has dual effects on autoimmune neuromuscular disorders through regulation of the autoimmune response and by directly activating the vitamin D receptors located on muscle fibers [29]. The Swedish study showed that vitamin D3 supplementation improved MGC score in most patients, indicating a beneficial response to active vitamin D in myasthenic muscle fatigue. In the present study, as well as in the former study though, there was no clear correlation between disease severity and vitamin D levels. Given the great clinical heterogeneity of the neuromuscular diseases, this finding cannot exclude that any given individual will benefit from vitamin D supplementation. Another notable point was that the Greek MG patients with vitamin D3 supplementation did not achieve statistically higher 25(OH)D levels than the unsupplemented patients. It has been suggested that active vitamin D is needed to keep the autoimmune response in balance by increasing the Treg function in order to suppress the effector T-cells autoreactivity [1], which could be the case at least in MG patients. Thus, enhanced consumption of vitamin D by the autoimmune response could possibly explain the failure of exogenous administration of vitamin D to raise its blood levels. Another explanation could be that the doses of 400–800 IU/day used in MG patients were too low to achieve a substantial increase. Recommendations for vitamin D3 supplementation are based mainly on bone health as an outcome (i.e., prevention of rickets, osteomalacia, fractures, osteoporosis) [30]. However, other health outcomes, such as autoimmune diseases, cancer, cardiovascular disorders, have not been addressed, since randomized clinical trials are still missing. Several studies on the effect of supplementation on 25(OH)D levels have shown that much higher doses than those usually prescribed are required in order to attain sufficient levels [31–33]. These doses are as high as 50–100 lg/d
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(2.000–4.000 IU/d), thus much higher than the doses received by the MG patients in the current study. Prior to the interpretation of these findings, methodological issues or biases were considered. Technical fallacies due to transport of blood samples from Greece to Sweden had to be excluded. Indeed, inter-laboratory agreement was assured by re-testing samples from 10 subjects in a Greek lab. A relative limitation of this study is the small number of subjects in each group/subgroup, which did not allow commenting on specific disease types separately. Hence, we believe that the cohorts are too small to draw major conclusions regarding the higher levels of 25(OH)D in MG compared to the other groups. Despite this fact, it is apparent that vitamin D deficiency is relatively common in the majority of Greeks examined; a finding that deserves confirmation in a larger cohort of south European population. The awareness of higher risk of vitamin D deficiency in the south European populations will alert doctors to search for this possible deficiency in patients with autoimmune disease and provide supplementation when needed. In summary, this is the first report on vitamin D status in patients with autoimmune neuromuscular disorders in a southern European country (Greece). We found 25(OH)D deficiency both in the control population as well as in patients with autoimmune neuromuscular disorders. Since the supplemented MG patients did not display statistically higher 25(OH)D levels, higher doses of vitamin D3 may be required in order to achieve optimal vitamin D levels. Acknowledgments This work was supported by Lars Hiertas memorial foundation and Selanders foundation (ARP). Compliance with ethical standards Conflict of interest The authors declare that they have no conflicts of interest. Ethical and informed consent All subjects signed written informed consent prior to inclusion into the study. The study protocol was approved by the Ethics Committee of PUH (No of approval: 37 /25-12012).
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4. Lucas RM, Ponsonby AL, Dear K, Valery PC, Pender MP, Taylor BV et al (2011) Sun exposure and vitamin D are independent risk factors for CNS demyelination. Neurology 76(6):540–548 5. Cutolo M, Otsa K, Uprus M, Paolino S, Seriolo B (2007) Vitamin D in rheumatoid arthritis. Autoimmun Rev 7(1):59–64 6. Bener A, Alsaied A, Al-Ali M, Al-Kubaisi A, Basha B, Abraham A et al (2009) High prevalence of vitamin D deficiency in type 1 diabetes mellitus and healthy children. Acta Diabetol 46(3):183–189 7. Carlander B, Puech-Cathala AM, Jaussent I, Scholz S, Bayard S, Cochen V et al (2011) Low vitamin D in narcolepsy with cataplexy. PLoS One 6(5):e20433 8. Skversky AL, Kumar J, Abramowitz MK, Kaskel FJ, Melamed ML (2011) Association of glucocorticoid use and low 25-hydroxyvitamin D levels: results from the National Health and Nutrition Examination Survey (NHANES): 2001–2006. J Clin Endocrinol Metab 96(12):3838–3845 9. Askmark H, Haggard L, Nygren I, Punga AR (2012) Vitamin D deficiency in patients with myasthenia gravis and improvement of fatigue after supplementation of vitamin D3: a pilot study. Eur J Neurol 19(12):1554–1560 10. Elf K, Askmark H, Nygren I, Punga AR (2014) Vitamin D deficiency in patients with primary immune-mediated peripheral neuropathies. J Neurol Sci 345(1–2):184–188 11. Kulie T, Groff A, Redmer J, Hounshell J, Schrager S (2009) Vitamin D: an evidence-based review. J Am Board Fam Med 22(6):698–706 12. Jaretzki A 3rd, Barohn RJ, Ernstoff RM, Kaminski HJ, Keesey JC, Penn AS et al (2000) Myasthenia gravis: recommendations for clinical research standards. Task Force of the Medical Scientific Advisory Board of the Myasthenia Gravis Foundation of America. Neurology 55(1):16–23 13. Burns TM, Conaway M, Sanders DB (2010) The MG composite: a valid and reliable outcome measure for myasthenia gravis. Neurology 74(18):1434–1440 14. Padua L, Evoli A, Aprile I, Caliandro P, Batocchi AP, Punzi C et al (2002) Myasthenia gravis outcome measure: development and validation of a disease-specific self-administered questionnaire. Neurol Sci 23(2):59–68 15. Van der Meche FG, Van Doorn PA, Meulstee J, Jennekens FG (2001) Diagnostic and classification criteria for the Guillain– Barre syndrome. Eur Neurol 45(3):133–139 16. Koski CL, Baumgarten M, Magder LS, Barohn RJ, Goldstein J, Graves M et al (2009) Derivation and validation of diagnostic criteria for chronic inflammatory demyelinating polyneuropathy. J Neurol Sci 277(1–2):1–8 17. Merkies IS, Schmitz PI (2006) Getting closer to patients: the INCAT Overall Disability Sum Score relates better to patients’ own clinical judgement in immune-mediated polyneuropathies. J Neurol Neurosurg Psychiatry 77(8):970–972 18. Askmark H, Haggard L, Nygren I, Punga AR (2012) Vitamin D deficiency in patients with myasthenia gravis and improvement of fatigue after supplementation of vitamin D3: a pilot study. Eur J Neurol 19:1554–1560 19. Lapatsanis D, Moulas A, Cholevas V, Soukakos P, Papadopoulou ZL, Challa A (2005) Vitamin D: a necessity for children and adolescents in Greece. Calcif Tissue Int 77(6):348–355 20. Holick MF (2007) Vitamin D deficiency. N Engl J Med 357(3):266–281 21. Ferder M, Inserra F, Manucha W, Ferder L (2013) The world pandemic of vitamin D deficiency could possibly be explained by cellular inflammatory response activity induced by the renin-angiotensin system. Am J Physiol Cell Physiol 304(11):C1027– C1039 22. McKenna MJ (1992) Differences in vitamin D status between countries in young adults and the elderly. Am J Med 93(1):69–77
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ORIGINAL ARTICLE
Low vitamin D levels in healthy controls and patients with autoimmune neuromuscular disorders in Greece Elisabeth Chroni1 • Nikolaos Dimisianos1 • Anna Rostedt Punga2
Received: 6 April 2015 / Accepted: 7 July 2015 Ó Belgian Neurological Society 2015
Abstract Normal autoimmune function is dependent on adequate levels of activated vitamin D, 25 hydroxy vitamin D [25(OH)D]. A recent study presented deficiency of 25(OH)D levels in Swedish MG patients. We aimed to study 25(OH)D levels in patients with MG and autoimmune polyneuropathies (PNP) at a southern latitude in Greece. Plasma levels of 25(OH)D were analyzed in Greek patients with MG (n = 19), immune-mediated PNP (N = 11) and in 30 Greek healthy age- and sex-matched controls. Ten MG patients received supplementation with vitamin D3. The MG Composite Score (MGC) and MG quality of life assessed disease severity in MG patients, whereas the INCAT Disability Scale assessed clinical features in the PNP patients. MG patients with and without vitamin D3 supplementation had higher 25(OH)D levels (mean 58.8 ± 16.3 and 62.0 ± 22.4 nmol/L, respectively) than PNP patients (mean 42.1 ± 11.5 nmol/L, p = 0.01) and healthy controls (mean 45.7 ± 13.8 nmol/L, p = 0.01). Plasma 25(OH)D levels was lower with age in all groups. There were no correlations between 25(OH)D and disease duration, MGC score, or INCAT score. Vitamin D deficiency was found in all Greek patient groups and healthy controls. Levels of 25(OH)D were higher in MG patients with as well as without vitamin D supplementation compared to healthy controls, whereas CIDP/GBS patients had levels similar to controls.
& Anna Rostedt Punga [email protected] 1
Department of Neurology, School of Medicine, University of Patras, Patras, Greece
2
Department of Clinical Neurophysiology, Institute of Neuroscience, Uppsala University Hospital, Uppsala University, 75185 Uppsala, Sweden
Keywords Myasthenia gravis Guillain–Barre´ syndrome Vitamin D Autoimmune polyneuropathy
Introduction Vitamin D has been attributed important functional roles in regulating the immune response [1], through action on the nuclear vitamin D receptor on lymphocytes, monocytes, dendritic cells, etc. [2]. The possible beneficial role of vitamin D on the prevention and/or treatment of autoimmune diseases is thought to be accomplished by restraining adaptive immune responses through inhibiting pro-inflammatory cytokine production and enhancement of regulatory T-cell function [1, 2]. Insufficient or deficient levels of 25 hydroxy vitamin D [25(OH)D] have been reported in a variety of autoimmune disorders such as systemic lupus erythematosus [3], multiple sclerosis [4], rheumatoid arthritis [5], type 1 diabetes mellitus [6], and narcolepsy [7]. Myasthenia gravis (MG), Guillain– Barre´ syndrome (GBS), and chronic inflammatory demyelinating polyneuropathy (CIDP) are all immunemediated diseases of the peripheral nervous system, in which autoreactive CD4? T-cells stimulate the production of autoantibodies. Further, there is a link between corticosteroid treatment, which is a common regimen for autoimmune neuromuscular diseases, and vitamin D deficiency [8]. Askmark et al. [9] recently identified vitamin D deficiency in Swedish MG patients as well as improvement of fatigue after vitamin D supplementation. Further, 25(OH)D levels were very low in Swedish patients with GBS and CIDP [10]. Nevertheless, vitamin D status may vary between countries, since vitamin D levels depend on dietary habits, sun exposure (latitude), skin type, and the use of sun-protecting agents [11].
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The aim of this study was to assess levels of 25(OH)D in Greek patients with autoimmune neuromuscular diseases, including MG and autoimmune PNP, and compare them with healthy individuals from the same area of South-western Greece, a country with high sun exposure.
Ethics
Materials and methods
Blood sampling and CLIA
Patients and controls
Blood from patients and controls was collected in EDTA tubes, and plasma was collected after centrifugation and stored in -80 °C freezer until the day of processing. Quantitative determination of total 25(OH)D in plasma was performed with a LIASONÒ analysis (DiaSorin Inc, Stillwater, MN, USA) through CLIA (chemiluminescence immunoanalysis), at Uppsala University Hospital. Severe 25(OH)D deficiency was defined as \25 nmol/L, mild deficiency as 25–74 nmol/L, and optimal level as 75–250 nmol/L. Samples from 10 subjects (5 patients and 5 controls) were re-tested in Patras, Greece, to assess interlaboratory agreement.
Thirty patients with autoimmune neuromuscular disorders: MG (N = 19; 13 women; mean age 46.7 ± 20.0 years), Guillain–Barre´ Syndrome (GBS; N = 3; 1 woman; mean age 51.3 ± 25.2 years), and chronic inflammatory demyelinating polyneuropathy (CIDP; N = 8; 4 women; mean age 58.7 ± 13.4 years) were included in the study. Patients were examined either when they were hospitalized or came for follow-up visits in the outpatient clinic at the Neurology Department of Patras University Hospital (PUH) in South-western Greece. Patients were recruited in the study from May until December 2012. 19 patients had a diagnosis of MG, according to the diagnostic criteria of the Myasthenia Gravis Foundation of America (MGFA) [12], including objective muscle fatigue and neurophysiological evidence of disturbed neuromuscular transmission in combination with detection of antibodies against acetylcholine receptors (AChRs) or muscle-specific tyrosine kinase (MuSK). Muscle fatigue of MG patients was assessed according to the MG Composite Score (MGC) [13] and each patient was given a total score ranging from 0 (no myasthenic weakness) to maximum 50 points (worst possible myasthenic weakness in all examined muscles). Patients also filled out the MG questionnaire (MGQ), a tool for quality of life assessment [14]. Ten MG patients already received vitamin D3 supplementation at the time of blood collection (400–800 IU/day) since at least 6 months, while 9 were unsupplemented. The remaining 11 patients were diagnosed with GBS (n = 3) and CIDP (n = 8), according to the respective criteria [15, 16]. Disability in GBS/CIDP patients was assessed using the INCAT score [17]. None of the GBS/CIDP patients received vitamin D3 supplementation. Demographic and clinical characteristics of all patients are presented in Tables 1 and 2. Thirty age- and sex- matched healthy subjects, recruited by local information at the Patras University Hospital, served as controls. Blood collection of controls was performed during the same time period as for the patients. Healthy controls did not receive supplemental vitamin D3.
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All subjects signed written informed consent prior to inclusion into the study. The study protocol was approved by the Ethics Committee of PUH (no of approval: 37/25-12012).
Statistical analysis The non-parametric Mann–Whitney test was used for assessment of median values in continuous variables of independent samples. Spearman’s rank order correlations were applied to measure associations between two continuous variables. The hypothesis that 25(OH)D levels differ between the following groups: healthy controls, MG patients without D3 supplementation, MG patients with supplementation, and GBS/CIDP patients without supplementation was tested with one-way ANOVA. All analyses were performed using GraphPad Prism v.5.03 (GraphPad, San Diego, CA, USA) and the significance level was set at p \ 0.05.
Results Patient clinical characteristics and treatment status Of the 19 MG patients, 2 had purely ocular MG while the rest had generalized MG. Sixteen patients were AChR antibody seropositive (AChR?), whereas 3 patients who were seronegative for AChR antibodies were seropositive for MuSK antibodies (MuSK?). MG patients without vitamin D3 supplementation had MGC and MGQ scores similar to those with vitamin D3 supplementation (MGC mean score 12.1 vs. 10.1, n.s., MGQ: mean score 20.9 vs. 28.6, n.s.). The mean age (48.9 vs. 44.6 years, n.s.) and disease duration (5.4
Acta Neurol Belg Table 1 Demographic and disease characteristics of patients with myasthenia gravis (MG), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and Guillain–Barre´ syndrome (GBS) included in the study Patient ID
Sex (m/f)
Age (years)
Diagnosis
Duration (months)
Month of sample
PRD (mg/d)
IS/IMD
Vitamin D suppl
Vitamin D (nmol/L)
P01
F
34
MG-Musk
P02
M
51
CIDP
P04
F
50
MG
P06
F
46
CIDP
P07
F
16
MG
P09
F
30
MG
P10
F
55
MG
P12
F
80
GBS
0.5
P13
M
65
CIDP
P14 P15
M F
33 36
GBS MG-ocul
P16
F
32
MG
60
September
10
IVIG
400
70
P17
M
49
MG
96
October
25
AZA
No
27
P19
F
15
MG
24
October
20
No
400
69
P20
F
73
CIDP
72
November
10
CSP, IVIG
No
45
P21
M
77
CIDP
36
November
No
AZA, IVIG
No
42
P22
F
41
CIDP
108
November
No
IVIG
No
31
P23
M
52
MG-ocul
6
November
10
No
800
60
P25
F
59
MG
2
November
10
No
400
70
P26
M
50
CIDP
24
November
No
No
No
30
P27 P28
F M
82 41
MG GBS
72 0.5
December December
10 No
No IVIG
No No
75 46
P29
M
54
MG
36
December
20
No
No
57
P30
F
20
MG-Musk
8
December
20
AZA
400
65
P31
F
82
MG-Musk
18
December
40
IVIG
800
22
P32
F
61
MG
264
June
10
No
No
59
P33
F
37
MG
84
June
10
No
No
58
P34
M
67
CIDP
48
September
15
RTX, IVIG
No
52
P35
M
67
MG
12
September
No
No
No
31
P36
M
44
MG
12
October
No
No
No
72
24
June
15
IVIG
800
50
8
July
20
IVIG
No
28
84
August
5
AZA, IVIG
400
43
24
September
25
RTX, IVIG
No
26
0.5
September
15
No
No
90
4
September
10
IVIG
No
89
156
September
30
IVIG
400
70
September
No
IVIG
No
57
8
September
No
IVIG
No
50
1 24
September September
No 15
IVIG No
No 800
56 60
M male, F female, MG MG patients with AChR antibodies, MG-Musk MG with positive anti-Musk antibodies, MG-ocul ocular MG, AchEI achetylcholinesterase inhibitor, PRD prednisone, AZA azathioprine, CSP cyclosporine, RTX rituximab, IS immunosuppressants, IMD immunomodulatory treatment, IVIG intravenous immunoglobulin
vs. 3.5 years, n.s.) were also comparable between groups. Treatment with pyridostigmine (MestinonÒ, 180–240 mg/day), prednisone (PrezolonÒ, 5–40 mg/day), immunosuppressants, and IVIG are presented in Table 1. 25(OH)D levels in patients and controls Almost all patients and healthy controls (57 out of 60) had low 25(OH)D levels in the range of mild to severe
deficiency. Nevertheless, the 25(OH)D levels in MG patients were significantly higher than controls as well as PNP patients. Among the MG patients, 25(OH)D levels of unsupplemented (mean ± SD: 62.0 ± 22.4 nmol/L) and vitamin D3 supplemented patients (58.8 ± 16.3 nmol/L; n.s.) did not differ. 25(OH)D levels in GBS/CIDP patients (42.1 ± 11.5 nmol/L) were comparable to those of healthy controls (45.7 ± 13.8 nmol/L; Fig. 1). Mean levels of 25(OH)D for every month in all subjects are presented in
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Acta Neurol Belg Table 2 Demographics and disease characteristics of patients and controls Group MG
N
m/f
Age (years)
Duration (years)
MGQ
MGC
INCAT
25(OH)D (nmol/L)
9
2/8
48.9 ± 20.0
5.4 ± 6.9
20.9 ± 14.3
12.1 ± 8.1
NA
62.0 ± 22.4
MG suppl
10
4/5
44.6 ± 20.9
3.5 ± 4.2
28.6 ± 12.8
10.1 ± 6.1
NA
58.7 ± 16.3
CIDP/GBS
11
6/5
56.7 ± 16.2
2.5 ± 2.8
NA
NA
5.5 ± 3.6
42.1 ± 11.5
Controls
30
12/18
49.6 ± 18.1
NA
NA
NA
NA
45.7 ± 13.7
Values are given as mean ± SD MGQ Myasthenia Gravis questionnaire, MGC Myasthenia Gravis Composite Score, INCAT polyneuropathies disability scale
Fig. 2 Seasonal variation of 25(OH)D levels in all study subjects (mean values/month) Fig. 1 Plasma levels of 25(OH)D (in nmol/L) in patients and healthy controls. *p = 0.01
Discussion Fig. 2. There was a trend towards lowest values in May and highest values in July–August. The 25(OH)D levels from the 10 samples (5 patients and 5 controls, blood drawn during April) analyzed in Patras were consistent with the results from the Uppsala laboratory, supporting inter-laboratory agreement. Parathormone (PTH) levels were similar between controls (3.8 ± 2.3) and all patient subgroups (3.9 ± 1.9). Correlation between 25(OH)D levels, disease status, and demographic characteristics There was no correlation between 25(OH)D levels and disease severity scores (MGC, MGQ, INCAT), disease duration, or prednisone dosage. However, there was an inverse correlation between 25(OH)D levels and age, indicating lower vitamin D levels with increasing age. This correlation was consistent for both controls and patients, supplemented as well as unsupplemented (Fig. 3).
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Vitamin D is considered an important regulator of the autoimmune response, and in addition, two previous studies have shown low levels of vitamin D in Swedish MG and GBS/CIDP patients [10, 18]. The current study was designed to examine 25(OH)D levels in patients with MG and immune-mediated PNP in Greece, where the mean number of sunny hours per year (N = 2700) is greatly increased compared to Scandinavia (N = 1700) (Data obtained from the National Meteorological Service). Contrary to what could be expected, we found reduced levels of 25(OH)D also in Greek controls. The normal values in this Greek study are indeed lower than those reported for healthy Swedish subjects. Mild vitamin D deficiency was strikingly common in the Greek healthy control population. To our knowledge, there is a lack of epidemiological data regarding reference values of 25(OH)D levels in a healthy Greek adult cohort. The only available epidemiological study of vitamin D levels in Greece was performed in 178
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Fig. 3 Correlation (Spearman r) of plasma 25(OH)D levels with age in all study subjects (patients and controls)
children, revealing that even during summer months the mean levels of 25(OH)D were lower than 50 nmol/L [19]. It is somewhat intriguing that the 25(OH)D levels are not higher with more southern latitude, considering that the sun exposure is an important factor to activate vitamin D into 25(OH)D. Vitamin D deficiency has been globally recognized [20, 21], affecting an estimated 1 billion people world-wide. Deficiency in vitamin D seems to be common not only in countries of higher latitude (North America, Scandinavia, Central Europe) [22], but also in more southern countries, like Turkey, Lebanon, India, Australia, Saudi Arabia [20, 23, 24]. Incidentally, a recent study from Turkey, regarding the vitamin D status of patients with rosacea, showed lower levels of 25(OH)D in controls (mean 42.7 nmol/L) compared to rosacea patients (53.5 nmol/L, p = 0.04) [25]. An obvious explanation would be that people in sunny countries avoid sun exposure due to fear of skin cancer or use sun-protecting agents and garments. On the other hand, dietary habits differ between countries, with Scandinavians, for example, consuming larger amounts of oily fish (e.g., tuna, salmon, mackerel), with a mean daily consumption of 30–35 g/day [26]. The negative correlation of 25(OH)D levels with age is a consistent finding in various populations [22, 27, 28]. Several
factors have been offered as an explanation, including less exposure to sun light and malabsorption of food nutrients in older as compared to younger individuals. One possible explanation to the absence of correlation between age and 25(OH)D values in the previous Swedish study could be that older people in Sweden eat considerably more oily fish than younger individuals [26]. Additionally, dairy products such as butter and milk are supplemented with vitamin D3 in Nordic countries. According to the globally accepted reference values for vitamin D deficiency (\75 nmol/L), the majority of subjects in our study, including the MG patients who had higher vitamin D levels than the other two groups, showed mild to severe deficiency. Thus, although our data confirm the vitamin D deficiency in MG patients, it appears also that the healthy population of southern countries has a higher prevalence of vitamin D deficiency. It has been suggested that vitamin D has dual effects on autoimmune neuromuscular disorders through regulation of the autoimmune response and by directly activating the vitamin D receptors located on muscle fibers [29]. The Swedish study showed that vitamin D3 supplementation improved MGC score in most patients, indicating a beneficial response to active vitamin D in myasthenic muscle fatigue. In the present study, as well as in the former study though, there was no clear correlation between disease severity and vitamin D levels. Given the great clinical heterogeneity of the neuromuscular diseases, this finding cannot exclude that any given individual will benefit from vitamin D supplementation. Another notable point was that the Greek MG patients with vitamin D3 supplementation did not achieve statistically higher 25(OH)D levels than the unsupplemented patients. It has been suggested that active vitamin D is needed to keep the autoimmune response in balance by increasing the Treg function in order to suppress the effector T-cells autoreactivity [1], which could be the case at least in MG patients. Thus, enhanced consumption of vitamin D by the autoimmune response could possibly explain the failure of exogenous administration of vitamin D to raise its blood levels. Another explanation could be that the doses of 400–800 IU/day used in MG patients were too low to achieve a substantial increase. Recommendations for vitamin D3 supplementation are based mainly on bone health as an outcome (i.e., prevention of rickets, osteomalacia, fractures, osteoporosis) [30]. However, other health outcomes, such as autoimmune diseases, cancer, cardiovascular disorders, have not been addressed, since randomized clinical trials are still missing. Several studies on the effect of supplementation on 25(OH)D levels have shown that much higher doses than those usually prescribed are required in order to attain sufficient levels [31–33]. These doses are as high as 50–100 lg/d
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(2.000–4.000 IU/d), thus much higher than the doses received by the MG patients in the current study. Prior to the interpretation of these findings, methodological issues or biases were considered. Technical fallacies due to transport of blood samples from Greece to Sweden had to be excluded. Indeed, inter-laboratory agreement was assured by re-testing samples from 10 subjects in a Greek lab. A relative limitation of this study is the small number of subjects in each group/subgroup, which did not allow commenting on specific disease types separately. Hence, we believe that the cohorts are too small to draw major conclusions regarding the higher levels of 25(OH)D in MG compared to the other groups. Despite this fact, it is apparent that vitamin D deficiency is relatively common in the majority of Greeks examined; a finding that deserves confirmation in a larger cohort of south European population. The awareness of higher risk of vitamin D deficiency in the south European populations will alert doctors to search for this possible deficiency in patients with autoimmune disease and provide supplementation when needed. In summary, this is the first report on vitamin D status in patients with autoimmune neuromuscular disorders in a southern European country (Greece). We found 25(OH)D deficiency both in the control population as well as in patients with autoimmune neuromuscular disorders. Since the supplemented MG patients did not display statistically higher 25(OH)D levels, higher doses of vitamin D3 may be required in order to achieve optimal vitamin D levels. Acknowledgments This work was supported by Lars Hiertas memorial foundation and Selanders foundation (ARP). Compliance with ethical standards Conflict of interest The authors declare that they have no conflicts of interest. Ethical and informed consent All subjects signed written informed consent prior to inclusion into the study. The study protocol was approved by the Ethics Committee of PUH (No of approval: 37 /25-12012).
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