Multiple Sclerosis and Related Disorders (]]]]) ], ]]]–]]]
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/msard
Association of vitamin D deficiency with RNFL thickness in MS individuals without history of optic neuritis Cecilie Fjeldstada,n, Anette S. Fjeldstadb,1, Joseph P. Weirc,2, Gabriel Pardoa a
OMRF Multiple Sclerosis Center of Excellence, 820 NE 15th Street, Oklahoma City, OK 73104, USA Department of Neurology, University of Utah, Imaging & Neurosciences Center at Research Park, 729 Arapeen Drive Rm 1172, Salt Lake City, UT 84108, USA c Department of Health, Sport, and Exercise Sciences, 1301 Sunnyside Avenue, Robinson center 104D, University of Kansas, Lawrence, KS 66045, USA b
Received 30 August 2013; received in revised form 11 March 2014; accepted 17 March 2014
KEYWORDS
Abstract
Multiple sclerosis; Optic neuritis; Vitamin D; Retinal nerve fiber layer; Optical coherence tomography
Vitamin D deficiency has been associated with both increased risk and severity of Multiple Sclerosis (MS) as it has a modulating effect on the immune process that causes inflammation/demyelination and axonal damage. Optical Coherence Tomography (OCT) offers a quick, reliable and non-invasive way to assess the Retinal Nerve Fiber Layer (RNFL) and identifies axonal loss generated by either direct inflammation or from neurodegeneration. Objective: To determine the association of vitamin D and RNFL in MS patients without a history of Optic Neuritis (ON) by comparing RNFL thickness in patients that are vitamin D deficient with those having normal serum levels. Method: The cohort of 76 MS patients underwent OCT testing to assess the RNFL thickness and macular volume, and measurement of serum 25-OH Vitamin D level. Vitamin D deficiency was defined as o30 ng/ml and sufficiency as Z30 ng/ml. Results: A total of 131 eyes were divided in two groups: vitamin D deficient (n=86 eyes, mean=17.7 ng/ml) and vitamin D sufficient (n=45 eyes, mean=40.3 ng/ml). Twenty one eyes had previous ON and were excluded from this analysis. Vitamin D deficiency was identified in 66% of the participants. RNFL thickness was similar for the vitamin D deficient and sufficient groups (85.5 vs 86 μm respectively, p=0.89). Significant differences were present for age with the deficient group being younger (42 years vs 51 years, p=0.005) and having shorter disease duration (7.5 years vs 11.4 years, p=0.006).
n
Corresponding author. Tel.: +1 405 271 8001x58089; fax: +1 405 271 2319. E-mail addresses: [email protected] (C. Fjeldstad), [email protected] (A.S. Fjeldstad), [email protected] (J.P. Weir). URL: http://omrf.org/MSCenter (C. Fjeldstad). 1 Tel.: 801 585 8021. 2 Tel.: +1 758 864 0784. http://dx.doi.org/10.1016/j.msard.2014.03.001 2211-0348/& 2014 Published by Elsevier B.V.
Please cite this article as: Fjeldstad C, et al. Association of vitamin D deficiency with RNFL thickness in MS individuals without history of optic neuritis. Multiple Sclerosis and Related Disorders (2014), http://dx.doi.org/10.1016/j.msard.2014.03.001
2
C. Fjeldstad et al. Conclusion: Vitamin D deficiency is not associated with thinning of RNFL or macular volume in MS eyes unaffected by ON. This finding suggests the role of vitamin D in modulating the severity of MS is not exerted through an influence on neurodegeneration. & 2014 Published by Elsevier B.V.
1. 1.1.
Introduction Multiple Sclerosis (MS)
MS is one of the most common neurological diseases causing disability in young adults. The development of MS is believed to be multifactorial, induced by a combination of genetic predisposition and environmental factors resulting in an immune-mediated inflammation with secondary demyelination and axonal loss. In addition to acute clinical deficits seen as a direct consequence of inflammatory tissue injury, there is a component of progressive disability that is secondary to a neurodegenerative process.
1.2.
Vitamin D deficiency
Vitamin D deficiency has received attention as one of the potential environmental factors that increases the risk for developing MS (Kragt et al., 2009; Munger et al., 2006). The main sources of vitamin D are diet and exposure to ultraviolet B rays (vitamin D3) from the Sun (Ascherio et al., 2010). Vitamin D supply from food is however limited, and is mostly found in fatty fish, and fortified foods. The normal serum level range of vitamin D has been widely discussed in recent years. The Institute of Medicine considers o12 ng/ml of vitamin D as deficient; 12–20 ng/ml insufficient; Z20 ng/ml sufficient, with potential adverse effects at levels 450 ng/ml (Medicine, 2010). Others have reported levels lower than 30 ng/ml to be deficient (Bischoff-Ferrari, 2009), and normal levels to be Z 30 ng/ ml (Carlson and Rose, 2013). 1.2.1. The influence of vitamin D in MS The influence of vitamin D in MS is indirectly identified by the increased prevalence of the disease in regions of higher latitude where there is less amount of sunshine available for vitamin D production and synthesis in a given year due to seasonal effect (Orton et al., 2011). Clinical studies have shown an increased incidence of MS associated with inadequate levels of vitamin D (Kragt et al., 2009). Conversely, high circulating levels of 25(OH) vitamin D are associated with a lower risk of developing MS (Munger et al., 2006). Studies have consistently demonstrated a strong positive correlation between vitamin D deficiency and subsequent relapse and/or disability in individuals with MS (Ganesh et al., 2013). Low levels of vitamin D have been documented during acute exacerbations when compared to periods of clinical inactivity (Soilu-Hanninen et al., 2005). Low levels of vitamin D have also been linked with other conditions such as depression (Knippenberg et al., 2011), cognitive difficulties (Llewellyn et al., 2010), and diseases related to abnormal immune modulation such as lupus erythematosus,
rheumatoid arthritis, and type 1 diabetes mellitus (Haussler et al., 2013). Low levels of vitamin D not only favor the development of autoimmune diseases in genetically predisposed individuals, but can also increase the severity of the clinical manifestations, suggesting that vitamin D has an immunomodulatory effect on inflammation and an immunoregulatory effect on the autoimmune process (Antico et al., 2012).
1.3.
Individuals with MS
More than 50% of individuals with MS will experience Optic Neuritis (ON) during the course of their disease. ON often results in thinning of the Retinal Nerve Fiber Layer (RNFL) (Costello et al., 2008) as a consequence of direct inflammatory damage to the optic nerve. This retinal layer corresponds to the non-myelinated axons of the ganglion cells that ultimately group together to form the optic nerve. This loss of volume of the RNFL has also been documented in individuals with MS even in the absence of previous episodes of ON (Fjeldstad et al., 2011) suggesting a disease-related neurodegenerative process, that induces axonal loss independent from direct inflammatory injury. Optical Coherence Tomography (OCT) offers a quick, reliable, and non-invasive way to assess volume of the retinal layers.
1.4.
Objective of this study
The main objective of this study was to investigate the association of vitamin D levels with RNFL thickness in MS individuals without previous episodes of ON. Our hypothesis is that individuals with deficient vitamin D levels will show thinner RNFL compared to the group with normal serum level due to the decreased immune modulating effect of vitamin D favoring MS related neurodegeneration in optic nerves that have not been affected by inflammation.
2. 2.1.
Materials and methods Study design and patient population
This cross-sectional study included 76 individuals with MS as defined by the 2010 McDonald criteria (Polman et al., 2011). Females constituted 74% of the participants. Relapsing remitting MS was the clinical type in 90%, secondary progressive MS in 7%, and the rest had either primary progressive MS or clinically isolated syndrome. Because of the potentially confounding effects of ON on the analyses, we eliminated eyes with history of previous ON. For those individuals without ON, we calculated the average of the scores from both eyes. Twenty one subjects had previous ON
Please cite this article as: Fjeldstad C, et al. Association of vitamin D deficiency with RNFL thickness in MS individuals without history of optic neuritis. Multiple Sclerosis and Related Disorders (2014), http://dx.doi.org/10.1016/j.msard.2014.03.001
Vitamin D deficiency with RNFL thickness in MS
3
in one eye and in those cases we used the value from the unaffected eye for the analyses. All patients were recruited from the Oklahoma Medical Research Foundation (OMRF) Multiple Sclerosis Center of Excellence and all testing was part of routine clinical assessments. Previous occurrence of ON was determined by the treating physician based on historical data and clinical examination. Exclusion criteria included history of neuromyelitis optica, diabetes, glaucoma, retinal diseases, high refractive error, current vitamin D supplementation, and active MS relapse. The protocol was approved by the OMRF Institutional Review Board and all participants signed an informed consent prior to testing.
2.2.
Table 1
Deficient (n = 50) Age (years) MS duration (years) Vitamin D (ng/ mL) RNFL (mm) Superior (mm) Nasal (mm) Inferior (mm) Temporal (mm) Central (mm) Average (mm)
Vitamin D, 25(OH)D measurement
Serum 25(OH) vitamin D levels were measured in ng/ml. The deficiency level was considered as o30 ng/ml, and sufficient level as 30–100 ng/ml. All samples were collected into a Serum Separator Tube (SST) and spun at 1800 rpm for 15 min using a Damon/IEC Division centrifuge (model: IEC-HN-5) (Ramsey, Minnesota). The samples were refrigerated until testing with the ADVIA Centaur Vitamin D Total Assay (Siemens Healthcare Diagnostics, Tarrytown, NY, USA). All assay reagents were used and stored following manufacturer's instructions.
2.3.
2.4.
Blood draw month November– February March–June July–October
Statistical analyses
The results of all descriptive analyses are reported as mean7standard error for the two groups. For descriptive purposes, the subjects were dichotomized into two groups based on vitamin D deficiency criteria described above. The relationship between vitamin D levels and RNFL thickness was quantified using bivariate regression. Further, hierarchical multiple regression models were constructed with both age and duration of MS included as covariates. A simple between-subjects ANOVA was conducted to determine if seasonal variation had an effect on vitamin D levels. Age was also included as a potential covariate. Secondary analyses involved examining group differences (deficient vs. sufficient) for a variety of variables. Mean differences for secondary analyses were conducted using independent t tests. Statistical significance level was defined as a po0.05.
Sufficient (n = 26)
Pvalue
42.671.7 7.570.7
51.472.6 11.471.3
0.005 0.006
17.771.0
40.371.6
N/A
85.571.5 105.172.3 69.371.6 113.472.6 55.271.5 261.273.7 272.672.1
86.072.9 105.074.0 73.873.0 111.074.2 54.572.4 251.878.7 270.573.0
0.89 0.98 0.15 0.62 0.79 0.25 0.58
N
Vitamin D (ng/ mL) 31.772.4
26 32 18
20.072.2 26.272.9
n #
,
n
Mean7Standard Error (SE). N/A; Non-Applicable; RNFL; Retinal Nerve Fiber Layer, Superior; Superior Quadrant, Nasal; nasal Quadrant, Inferior; Inferior Quadrant, Temporal; Temporal Quadrant, Central; Central Macular Thickness, Average; Average Macular Thickness. n Indicates significantly greater than March–June (Tukey HSD). # Indicates significantly greater than July–October (Tukey HSD).
Cirrus optical coherence tomography
The subjects in this study had scans performed through undilated pupils using the Cirrus HD-OCT (Model 4000) (Carl Zeiss Meditec, Dublin, CA, USA) which acquires data at a rate of 27,000A-scans per second with resolution of 5 mm in order to identify the retinal layers and measure the RNFL and macular volumes (Zeiss, 2008) using the Optic Disc Cube 200 200 and Macular Cube 512 128 scan protocols respectively. Peripapillary RNFL thickness was measured in mm for each eye as a total average (3601) and by quadrants (superior, nasal, inferior and temporal). Average and central macular volumes, measured in mm3, were also analyzed and recorded. Only high quality scans reaching signal strength of Z6 (of maximum 10) were included in the analyses. All HD-OCT scans were performed by a single experienced technician.
Subject characteristics.
3. 3.1.
Results Analyses
A total of 76 subjects, of which 50 (66%) were vitamin D deficient (mean 17.7 ng/ml) and 26 vitamin D sufficient (mean 40.3 ng/ml) were included in the analyses (Table 1). Subjects with vitamin D deficiency were younger than the vitamin D insufficient group (Table 1). Mean MS disease duration at the time of blood draw was 7.5 years for the deficient group and 11.4 years for the sufficient group. The zero order relationship between vitamin D levels and average RNFL was not significant (r= 0.016, p= 0.89). The relationship is shown in Figure 1. To examine if the relationship between vitamin D and RNFL thickness was moderated by age and/or MS duration, we built a hierarchical multiple regression model with age and MS duration entered as covariates (in that order) prior to entering vitamin D concentration into the model. The correlation matrix for the variables in the model are shown in Table 2. Both age and MS duration were significantly correlated with RNFL thickness although the variance accounted for was modest (r2 =0.06 and r2 = 0.19 for age and MS duration respectively). At step 1 of the regression analysis, the resulting regression equation was RNFL ¼ 95:788–0:233 ðageÞ
Please cite this article as: Fjeldstad C, et al. Association of vitamin D deficiency with RNFL thickness in MS individuals without history of optic neuritis. Multiple Sclerosis and Related Disorders (2014), http://dx.doi.org/10.1016/j.msard.2014.03.001
4
C. Fjeldstad et al.
RNFL Thickness (μ μM)
150
100
50
0 0
20
40 60 Vitamin D (ng/mL)
80
Figure 1 Bivariate relationship between vitamin D concentration and RNFL thickness. The relationship was not significant (r = 0.016, p=0.89).
Group 1 had blood drawn done between November and February; Group 2; March–June and Group; July–October. A significant difference in levels of vitamin D and the time of year of blood draw was detected (p= 0.003). Subsequent pairwise comparisons were made using the Tukey post hoc test, and indicated that Group 1 had significantly higher vitamin D concentrations than both Groups 2 and 3. Group 3 was greater than Group 2. However, the omega squared from the ANOVA was ω2 = 0.12, indicating that while there were seasonal variations in vitamin D concentrations, the effects were modest. Including age as a covariate did not change the relationship of blood draw and vitamin D status.
4. 4.1.
Table 2 Correlation matrix for variables in the multiple regression model. Avg. RNFL Age Avg. RNFL 1.0 Age 0.238n MS duration 0.432n Vitamin D 0.016 n
MS duration Vitamin D
1.0 0.472n 1.0 0.247n 0.276n
1.0
Significant at po0.05.
which as indicated from the correlation analysis above, was statistically significant (p= 0.038). The inclusion of MS duration at step 2 resulted in a significant increment in explained variance (increment in R2 = 0.131, p= 0.001). The resulting regression equation was RNFL ¼ 95:009 0:041ðageÞ 0:851ðMS durationÞ: However, the inclusion of vitamin D into the model did not result in a significant increment in R2 (increment in R2 = 0.013, p= 0.28). These results indicate that vitamin D is not associated with RNFL thickness in individuals with multiple sclerosis. It should be noted that a priori we chose to force age into the model prior to MS duration. Forcing MS duration into the model prior to age removes the influence of age such that the increment in R2 associated with age is not significant. However, this has no influence on the effect of vitamin D in the model. We would have liked to have also examined race/ethnicity as covariates however the sample was primarily white, non-hispanic or latino (n= 71). The number of non-white subjects (n hispanic= 1; n black nonhispanic or latino = 3; n black with hispanic or latino = 1) was too small to allow an analysis. Finally, to examine if there was a relationship between vitamin D and the presence or absence of optic neuritis, a point-biserial correlation analysis was performed between these variables. The point biserial correlation was not significant (r= 0.014, p= 0.37).
3.2.
Seasonal variation, age and vitamin D levels
The subjects were divided into three separate groups based on when in the season blood draw for vitamin D was done.
Discussion Vitamin D serum levels
Vitamin D serum levels have been studied in relation to different autoimmune diseases to include MS, and insufficient levels have been associated with an increased risk of developing the disease and having a worse clinical severity. The direct effect of this deficiency on the two pathophysiologic processes responsible for MS manifestations, namely inflammation/demyelination and neurodegeneration, has not been well defined. Since the optic nerve can be affected by either one of these processes, and the event of inflammation (optic neuritis) can be easily identified historically and by clinical examination, we used OCT to measure the thickness of the RNFL in eyes not affected by ON. In the absence of direct demyelinating/inflammatory events of the optic nerve or other identifiable risk factors, increased thinning of the RNFL in MS individuals can be assumed to be induced by a neurodegenerative process. We have previously identified that eyes of individuals with MS and no history of ON had significant decreased overall RNFL thickness (89.1 mm) compared to healthy controls (98 mm) (po0.05) (Fjeldstad et al., 2011). The rate of RNFL volumetric loss in MS patients without a history of ON has been documented as being approximately 2 mm annually. This in contrast to the lower rate of 0.1 mm loss seen in healthy controls (Fjeldstad et al., 2012). It has been postulated that vitamin D has a neuroprotective effect that reduces the risk of developing MS and attenuates the degree of disease activity (Goldberg et al., 1986; Kragt et al., 2009; Munger et al., 2006).
4.2. Relationship of vitamin D deficiency to total and sectorial RNFL We examined the relationship of vitamin D deficiency to total and sectorial RNFL thickness and average and central macular thickness attempting to identify a correlation between RNFL volume loss and vitamin D levels that would implicate vitamin D deficiency as a conditioning factor in MS neurodegeneration. Our results showed that there was no relationship between vitamin D levels and the extent of retinal axonal loss as quantified with RNFL using OCT. The inclusion of age and MS duration as covariates did not alter this effect.
Please cite this article as: Fjeldstad C, et al. Association of vitamin D deficiency with RNFL thickness in MS individuals without history of optic neuritis. Multiple Sclerosis and Related Disorders (2014), http://dx.doi.org/10.1016/j.msard.2014.03.001
Vitamin D deficiency with RNFL thickness in MS
4.3. Role of vitamin D with the increasing evidences With the increasing evidence from genetic, epidemiologic and clinical studies supporting the role of vitamin D as an immune modulator (Carlson and Rose, 2013), studies providing further insight into the role vitamin D supplementation in the development and severity course of MS are warranted. A potential way of measuring disease progression in MS is quantification of retinal axonal loss over time as measured by OCT. It is reliable, quick, inexpensive, and offers a quantitative measure of changes in neuronal loss and axonal damage over time.
4.4.
Limitations of this study
Limitations of this study include the cross sectional design which limits the causal inferences that can be made, to include potential previous vitamin D supplementation even though current intake was an exclusion criteria. The seasonal variation in vitamin D levels is a potential confounder, but the magnitude of the effect was modest. All patients came from the same center and all had a diagnosis of MS, but we did not control for all other autoimmune diseases and that may have biased the results.
4.5.
Hypothesis
Although our hypothesis that RNFL thickness was associated with vitamin D concentrations was not demonstrated, further attention is warranted to examine to which extent vitamin D has as an immune modulating effect in MS.
Conflict of interest Dr. C. Fjeldstad, Dr. A.S. Fjeldstad, Dr. Weir and Dr. Pardo have no conflicts of interest to report.
Acknowledgment The study was self-funded by the Oklahoma Medical Research Foundation, MS Center of Excellence in Oklahoma City, OK.
References Antico A, Tampoia M, Tozzoli R, Bizzaro N. Can supplementation with vitamin D reduce the risk or modify the course of autoimmune diseases? A systematic review of the literature Autoimmun Rev 2012;12(2):127–36. http://dx.doi.org/10.1016/ j.autrev.2012.07.007. Ascherio A, Munger KL, Simon KC. Vitamin D and multiple sclerosis. Lancet Neurol 2010;9(6):599–612. http://dx.doi.org/10.1016/ S1474-4422(10)70086-7. Bischoff-Ferrari H. Vitamin D: what is an adequate vitamin D level and how much supplementation is necessary? Best Pract Res: Clin Rheumatol 2009;23(6):789–95. http://dx.doi.org/10.1016/ j.berh.2009.09.005.
5 Carlson NG, Rose JW. Vitamin D as a clinical biomarker in multiple sclerosis. Expert Opin Med Diagn 2013;7(3):231–42. http://dx. doi.org/10.1517/17530059.2013.772978. Costello F, YI Pan WH, Eggenberger E, Coupland S, Kardon RH. Tracking retinal nerve fiber layer loss after optic neuritis: a prospective study using optical coherence tomography. Mult Scler 2008;14:893–905. Fjeldstad AS, Carlson NG, Rose JW. Optical coherence tomography as a biomarker in multiple sclerosis. Expert Opin Med Diagn 2012;6(6):593–604. http://dx.doi.org/10.1517/17530059.2012. 719496. Fjeldstad C, Bemben M, Pardo G. Reduced retinal nerve fiber layer and macular thickness in patients with multiple sclerosis with no history of optic neuritis identified by the use of spectral domain high-definition optical coherence tomography. J Clin Neurosci: Off J Neurosurg Soc Aust 2011;18(11):1469–72. http://dx.doi. org/10.1016/j.jocn.2011.04.008. Ganesh A, Apel S, Metz L, Patten S. The case of vitamin D supplementation in multiple sclerosis. Mult Scler Relat Disord 2013;2(4):281–306. Goldberg P, Fleming MC, Picard EH. Multiple sclerosis: decreased relapse rate through dietary supplementation with calcium, magnesium and vitamin D. [Clinical Trial Research Support, Non-U.S. Gov't]. Med Hypotheses 1986;21(2):193–200. Haussler MR, Whitfield GK, Kaneko I, Haussler CA, Hsieh D, Hsieh JC, et al. Molecular mechanisms of vitamin D action. Calcif Tissue Int 2013;92(2):77–98. http://dx.doi.org/10.1007/s00223-0129619-0. I. o. Medicine. In F. a. N. Board, editor. Dietary Reference Intakes for Calcium and Vitamin D. Washington, DC; 2010. Knippenberg S, Bol Y, Damoiseaux J, Hupperts R, Smolders J. Vitamin D status in patients with MS is negatively correlated with depression, but not with fatigue. [Comparative StudyResearch Support, Non-U.S. Gov't]. Acta Neurol Scand 2011;124(3):171–5. http://dx.doi.org/10.1111/j.1600-0404. 2010.01447.x. Kragt J, van Amerongen B, Killestein J, Dijkstra C, Uitdehaag B, Polman C, et al. Higher levels of 25-hydroxyvitamin D are associated with a lower incidence of multiple sclerosis only in women. [Research Support, Non-U.S. Gov't]. Mult Scler 2009;15 (1):9–15. http://dx.doi.org/10.1177/1352458508095920. Llewellyn DJ, Lang IA, Langa KM, Muniz-Terrera G, Phillips CL, Cherubini A, et al. Vitamin D and risk of cognitive decline in elderly persons. [Comparative StudyRandomized Controlled Trial Research Support, N.I.H., Extramural, Research Support, Non-U. S. Gov't]. Arch Intern Med 2010;170(13):1135–41. http://dx.doi. org/10.1001/archinternmed.2010.173. Munger KL, Levin LI, Hollis BW, Howard NS, Ascherio A. Serum 25hydroxyvitamin D levels and risk of multiple sclerosis. [Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't]. J Am Med Assoc, 296; 2832–8. http://dx.doi.org/10.1001/ jama.296.23.2832. Orton SM, Wald L, Confavreux C, Vukusic S, Krohn JP, Ramagopalan SV, et al. Association of UV radiation with multiple sclerosis prevalence and sex ratio in France. [Comparative Study Research Support, Non-U.S. Gov't]. Neurology 2011;76 (5):425–31. http://dx.doi.org/10.1212/WNL.0b013e31820a0a9f. Polman CH, Reingold SC, Banwell B, Clanet M, Cohen JA, Filippi M, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. [Research Support, Non-U.S. Gov't]. Ann Neurol 2011;69(2):292–302. http://dx.doi.org/10.1002/ana.22366. Soilu-Hanninen M, Airas L, Mononen I, Heikkila A, Viljanen M, Hanninen A. 25-Hydroxyvitamin D levels in serum at the onset of multiple sclerosis. [Research Support, Non-U.S. Gov't]. Mult Scler 2005;11(3):266–71. Zeiss C. Cirrus HD-OCT user manual. In: Meditec C.Z., editor; 2008.
Please cite this article as: Fjeldstad C, et al. Association of vitamin D deficiency with RNFL thickness in MS individuals without history of optic neuritis. Multiple Sclerosis and Related Disorders (2014), http://dx.doi.org/10.1016/j.msard.2014.03.001
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/msard
Association of vitamin D deficiency with RNFL thickness in MS individuals without history of optic neuritis Cecilie Fjeldstada,n, Anette S. Fjeldstadb,1, Joseph P. Weirc,2, Gabriel Pardoa a
OMRF Multiple Sclerosis Center of Excellence, 820 NE 15th Street, Oklahoma City, OK 73104, USA Department of Neurology, University of Utah, Imaging & Neurosciences Center at Research Park, 729 Arapeen Drive Rm 1172, Salt Lake City, UT 84108, USA c Department of Health, Sport, and Exercise Sciences, 1301 Sunnyside Avenue, Robinson center 104D, University of Kansas, Lawrence, KS 66045, USA b
Received 30 August 2013; received in revised form 11 March 2014; accepted 17 March 2014
KEYWORDS
Abstract
Multiple sclerosis; Optic neuritis; Vitamin D; Retinal nerve fiber layer; Optical coherence tomography
Vitamin D deficiency has been associated with both increased risk and severity of Multiple Sclerosis (MS) as it has a modulating effect on the immune process that causes inflammation/demyelination and axonal damage. Optical Coherence Tomography (OCT) offers a quick, reliable and non-invasive way to assess the Retinal Nerve Fiber Layer (RNFL) and identifies axonal loss generated by either direct inflammation or from neurodegeneration. Objective: To determine the association of vitamin D and RNFL in MS patients without a history of Optic Neuritis (ON) by comparing RNFL thickness in patients that are vitamin D deficient with those having normal serum levels. Method: The cohort of 76 MS patients underwent OCT testing to assess the RNFL thickness and macular volume, and measurement of serum 25-OH Vitamin D level. Vitamin D deficiency was defined as o30 ng/ml and sufficiency as Z30 ng/ml. Results: A total of 131 eyes were divided in two groups: vitamin D deficient (n=86 eyes, mean=17.7 ng/ml) and vitamin D sufficient (n=45 eyes, mean=40.3 ng/ml). Twenty one eyes had previous ON and were excluded from this analysis. Vitamin D deficiency was identified in 66% of the participants. RNFL thickness was similar for the vitamin D deficient and sufficient groups (85.5 vs 86 μm respectively, p=0.89). Significant differences were present for age with the deficient group being younger (42 years vs 51 years, p=0.005) and having shorter disease duration (7.5 years vs 11.4 years, p=0.006).
n
Corresponding author. Tel.: +1 405 271 8001x58089; fax: +1 405 271 2319. E-mail addresses: [email protected] (C. Fjeldstad), [email protected] (A.S. Fjeldstad), [email protected] (J.P. Weir). URL: http://omrf.org/MSCenter (C. Fjeldstad). 1 Tel.: 801 585 8021. 2 Tel.: +1 758 864 0784. http://dx.doi.org/10.1016/j.msard.2014.03.001 2211-0348/& 2014 Published by Elsevier B.V.
Please cite this article as: Fjeldstad C, et al. Association of vitamin D deficiency with RNFL thickness in MS individuals without history of optic neuritis. Multiple Sclerosis and Related Disorders (2014), http://dx.doi.org/10.1016/j.msard.2014.03.001
2
C. Fjeldstad et al. Conclusion: Vitamin D deficiency is not associated with thinning of RNFL or macular volume in MS eyes unaffected by ON. This finding suggests the role of vitamin D in modulating the severity of MS is not exerted through an influence on neurodegeneration. & 2014 Published by Elsevier B.V.
1. 1.1.
Introduction Multiple Sclerosis (MS)
MS is one of the most common neurological diseases causing disability in young adults. The development of MS is believed to be multifactorial, induced by a combination of genetic predisposition and environmental factors resulting in an immune-mediated inflammation with secondary demyelination and axonal loss. In addition to acute clinical deficits seen as a direct consequence of inflammatory tissue injury, there is a component of progressive disability that is secondary to a neurodegenerative process.
1.2.
Vitamin D deficiency
Vitamin D deficiency has received attention as one of the potential environmental factors that increases the risk for developing MS (Kragt et al., 2009; Munger et al., 2006). The main sources of vitamin D are diet and exposure to ultraviolet B rays (vitamin D3) from the Sun (Ascherio et al., 2010). Vitamin D supply from food is however limited, and is mostly found in fatty fish, and fortified foods. The normal serum level range of vitamin D has been widely discussed in recent years. The Institute of Medicine considers o12 ng/ml of vitamin D as deficient; 12–20 ng/ml insufficient; Z20 ng/ml sufficient, with potential adverse effects at levels 450 ng/ml (Medicine, 2010). Others have reported levels lower than 30 ng/ml to be deficient (Bischoff-Ferrari, 2009), and normal levels to be Z 30 ng/ ml (Carlson and Rose, 2013). 1.2.1. The influence of vitamin D in MS The influence of vitamin D in MS is indirectly identified by the increased prevalence of the disease in regions of higher latitude where there is less amount of sunshine available for vitamin D production and synthesis in a given year due to seasonal effect (Orton et al., 2011). Clinical studies have shown an increased incidence of MS associated with inadequate levels of vitamin D (Kragt et al., 2009). Conversely, high circulating levels of 25(OH) vitamin D are associated with a lower risk of developing MS (Munger et al., 2006). Studies have consistently demonstrated a strong positive correlation between vitamin D deficiency and subsequent relapse and/or disability in individuals with MS (Ganesh et al., 2013). Low levels of vitamin D have been documented during acute exacerbations when compared to periods of clinical inactivity (Soilu-Hanninen et al., 2005). Low levels of vitamin D have also been linked with other conditions such as depression (Knippenberg et al., 2011), cognitive difficulties (Llewellyn et al., 2010), and diseases related to abnormal immune modulation such as lupus erythematosus,
rheumatoid arthritis, and type 1 diabetes mellitus (Haussler et al., 2013). Low levels of vitamin D not only favor the development of autoimmune diseases in genetically predisposed individuals, but can also increase the severity of the clinical manifestations, suggesting that vitamin D has an immunomodulatory effect on inflammation and an immunoregulatory effect on the autoimmune process (Antico et al., 2012).
1.3.
Individuals with MS
More than 50% of individuals with MS will experience Optic Neuritis (ON) during the course of their disease. ON often results in thinning of the Retinal Nerve Fiber Layer (RNFL) (Costello et al., 2008) as a consequence of direct inflammatory damage to the optic nerve. This retinal layer corresponds to the non-myelinated axons of the ganglion cells that ultimately group together to form the optic nerve. This loss of volume of the RNFL has also been documented in individuals with MS even in the absence of previous episodes of ON (Fjeldstad et al., 2011) suggesting a disease-related neurodegenerative process, that induces axonal loss independent from direct inflammatory injury. Optical Coherence Tomography (OCT) offers a quick, reliable, and non-invasive way to assess volume of the retinal layers.
1.4.
Objective of this study
The main objective of this study was to investigate the association of vitamin D levels with RNFL thickness in MS individuals without previous episodes of ON. Our hypothesis is that individuals with deficient vitamin D levels will show thinner RNFL compared to the group with normal serum level due to the decreased immune modulating effect of vitamin D favoring MS related neurodegeneration in optic nerves that have not been affected by inflammation.
2. 2.1.
Materials and methods Study design and patient population
This cross-sectional study included 76 individuals with MS as defined by the 2010 McDonald criteria (Polman et al., 2011). Females constituted 74% of the participants. Relapsing remitting MS was the clinical type in 90%, secondary progressive MS in 7%, and the rest had either primary progressive MS or clinically isolated syndrome. Because of the potentially confounding effects of ON on the analyses, we eliminated eyes with history of previous ON. For those individuals without ON, we calculated the average of the scores from both eyes. Twenty one subjects had previous ON
Please cite this article as: Fjeldstad C, et al. Association of vitamin D deficiency with RNFL thickness in MS individuals without history of optic neuritis. Multiple Sclerosis and Related Disorders (2014), http://dx.doi.org/10.1016/j.msard.2014.03.001
Vitamin D deficiency with RNFL thickness in MS
3
in one eye and in those cases we used the value from the unaffected eye for the analyses. All patients were recruited from the Oklahoma Medical Research Foundation (OMRF) Multiple Sclerosis Center of Excellence and all testing was part of routine clinical assessments. Previous occurrence of ON was determined by the treating physician based on historical data and clinical examination. Exclusion criteria included history of neuromyelitis optica, diabetes, glaucoma, retinal diseases, high refractive error, current vitamin D supplementation, and active MS relapse. The protocol was approved by the OMRF Institutional Review Board and all participants signed an informed consent prior to testing.
2.2.
Table 1
Deficient (n = 50) Age (years) MS duration (years) Vitamin D (ng/ mL) RNFL (mm) Superior (mm) Nasal (mm) Inferior (mm) Temporal (mm) Central (mm) Average (mm)
Vitamin D, 25(OH)D measurement
Serum 25(OH) vitamin D levels were measured in ng/ml. The deficiency level was considered as o30 ng/ml, and sufficient level as 30–100 ng/ml. All samples were collected into a Serum Separator Tube (SST) and spun at 1800 rpm for 15 min using a Damon/IEC Division centrifuge (model: IEC-HN-5) (Ramsey, Minnesota). The samples were refrigerated until testing with the ADVIA Centaur Vitamin D Total Assay (Siemens Healthcare Diagnostics, Tarrytown, NY, USA). All assay reagents were used and stored following manufacturer's instructions.
2.3.
2.4.
Blood draw month November– February March–June July–October
Statistical analyses
The results of all descriptive analyses are reported as mean7standard error for the two groups. For descriptive purposes, the subjects were dichotomized into two groups based on vitamin D deficiency criteria described above. The relationship between vitamin D levels and RNFL thickness was quantified using bivariate regression. Further, hierarchical multiple regression models were constructed with both age and duration of MS included as covariates. A simple between-subjects ANOVA was conducted to determine if seasonal variation had an effect on vitamin D levels. Age was also included as a potential covariate. Secondary analyses involved examining group differences (deficient vs. sufficient) for a variety of variables. Mean differences for secondary analyses were conducted using independent t tests. Statistical significance level was defined as a po0.05.
Sufficient (n = 26)
Pvalue
42.671.7 7.570.7
51.472.6 11.471.3
0.005 0.006
17.771.0
40.371.6
N/A
85.571.5 105.172.3 69.371.6 113.472.6 55.271.5 261.273.7 272.672.1
86.072.9 105.074.0 73.873.0 111.074.2 54.572.4 251.878.7 270.573.0
0.89 0.98 0.15 0.62 0.79 0.25 0.58
N
Vitamin D (ng/ mL) 31.772.4
26 32 18
20.072.2 26.272.9
n #
,
n
Mean7Standard Error (SE). N/A; Non-Applicable; RNFL; Retinal Nerve Fiber Layer, Superior; Superior Quadrant, Nasal; nasal Quadrant, Inferior; Inferior Quadrant, Temporal; Temporal Quadrant, Central; Central Macular Thickness, Average; Average Macular Thickness. n Indicates significantly greater than March–June (Tukey HSD). # Indicates significantly greater than July–October (Tukey HSD).
Cirrus optical coherence tomography
The subjects in this study had scans performed through undilated pupils using the Cirrus HD-OCT (Model 4000) (Carl Zeiss Meditec, Dublin, CA, USA) which acquires data at a rate of 27,000A-scans per second with resolution of 5 mm in order to identify the retinal layers and measure the RNFL and macular volumes (Zeiss, 2008) using the Optic Disc Cube 200 200 and Macular Cube 512 128 scan protocols respectively. Peripapillary RNFL thickness was measured in mm for each eye as a total average (3601) and by quadrants (superior, nasal, inferior and temporal). Average and central macular volumes, measured in mm3, were also analyzed and recorded. Only high quality scans reaching signal strength of Z6 (of maximum 10) were included in the analyses. All HD-OCT scans were performed by a single experienced technician.
Subject characteristics.
3. 3.1.
Results Analyses
A total of 76 subjects, of which 50 (66%) were vitamin D deficient (mean 17.7 ng/ml) and 26 vitamin D sufficient (mean 40.3 ng/ml) were included in the analyses (Table 1). Subjects with vitamin D deficiency were younger than the vitamin D insufficient group (Table 1). Mean MS disease duration at the time of blood draw was 7.5 years for the deficient group and 11.4 years for the sufficient group. The zero order relationship between vitamin D levels and average RNFL was not significant (r= 0.016, p= 0.89). The relationship is shown in Figure 1. To examine if the relationship between vitamin D and RNFL thickness was moderated by age and/or MS duration, we built a hierarchical multiple regression model with age and MS duration entered as covariates (in that order) prior to entering vitamin D concentration into the model. The correlation matrix for the variables in the model are shown in Table 2. Both age and MS duration were significantly correlated with RNFL thickness although the variance accounted for was modest (r2 =0.06 and r2 = 0.19 for age and MS duration respectively). At step 1 of the regression analysis, the resulting regression equation was RNFL ¼ 95:788–0:233 ðageÞ
Please cite this article as: Fjeldstad C, et al. Association of vitamin D deficiency with RNFL thickness in MS individuals without history of optic neuritis. Multiple Sclerosis and Related Disorders (2014), http://dx.doi.org/10.1016/j.msard.2014.03.001
4
C. Fjeldstad et al.
RNFL Thickness (μ μM)
150
100
50
0 0
20
40 60 Vitamin D (ng/mL)
80
Figure 1 Bivariate relationship between vitamin D concentration and RNFL thickness. The relationship was not significant (r = 0.016, p=0.89).
Group 1 had blood drawn done between November and February; Group 2; March–June and Group; July–October. A significant difference in levels of vitamin D and the time of year of blood draw was detected (p= 0.003). Subsequent pairwise comparisons were made using the Tukey post hoc test, and indicated that Group 1 had significantly higher vitamin D concentrations than both Groups 2 and 3. Group 3 was greater than Group 2. However, the omega squared from the ANOVA was ω2 = 0.12, indicating that while there were seasonal variations in vitamin D concentrations, the effects were modest. Including age as a covariate did not change the relationship of blood draw and vitamin D status.
4. 4.1.
Table 2 Correlation matrix for variables in the multiple regression model. Avg. RNFL Age Avg. RNFL 1.0 Age 0.238n MS duration 0.432n Vitamin D 0.016 n
MS duration Vitamin D
1.0 0.472n 1.0 0.247n 0.276n
1.0
Significant at po0.05.
which as indicated from the correlation analysis above, was statistically significant (p= 0.038). The inclusion of MS duration at step 2 resulted in a significant increment in explained variance (increment in R2 = 0.131, p= 0.001). The resulting regression equation was RNFL ¼ 95:009 0:041ðageÞ 0:851ðMS durationÞ: However, the inclusion of vitamin D into the model did not result in a significant increment in R2 (increment in R2 = 0.013, p= 0.28). These results indicate that vitamin D is not associated with RNFL thickness in individuals with multiple sclerosis. It should be noted that a priori we chose to force age into the model prior to MS duration. Forcing MS duration into the model prior to age removes the influence of age such that the increment in R2 associated with age is not significant. However, this has no influence on the effect of vitamin D in the model. We would have liked to have also examined race/ethnicity as covariates however the sample was primarily white, non-hispanic or latino (n= 71). The number of non-white subjects (n hispanic= 1; n black nonhispanic or latino = 3; n black with hispanic or latino = 1) was too small to allow an analysis. Finally, to examine if there was a relationship between vitamin D and the presence or absence of optic neuritis, a point-biserial correlation analysis was performed between these variables. The point biserial correlation was not significant (r= 0.014, p= 0.37).
3.2.
Seasonal variation, age and vitamin D levels
The subjects were divided into three separate groups based on when in the season blood draw for vitamin D was done.
Discussion Vitamin D serum levels
Vitamin D serum levels have been studied in relation to different autoimmune diseases to include MS, and insufficient levels have been associated with an increased risk of developing the disease and having a worse clinical severity. The direct effect of this deficiency on the two pathophysiologic processes responsible for MS manifestations, namely inflammation/demyelination and neurodegeneration, has not been well defined. Since the optic nerve can be affected by either one of these processes, and the event of inflammation (optic neuritis) can be easily identified historically and by clinical examination, we used OCT to measure the thickness of the RNFL in eyes not affected by ON. In the absence of direct demyelinating/inflammatory events of the optic nerve or other identifiable risk factors, increased thinning of the RNFL in MS individuals can be assumed to be induced by a neurodegenerative process. We have previously identified that eyes of individuals with MS and no history of ON had significant decreased overall RNFL thickness (89.1 mm) compared to healthy controls (98 mm) (po0.05) (Fjeldstad et al., 2011). The rate of RNFL volumetric loss in MS patients without a history of ON has been documented as being approximately 2 mm annually. This in contrast to the lower rate of 0.1 mm loss seen in healthy controls (Fjeldstad et al., 2012). It has been postulated that vitamin D has a neuroprotective effect that reduces the risk of developing MS and attenuates the degree of disease activity (Goldberg et al., 1986; Kragt et al., 2009; Munger et al., 2006).
4.2. Relationship of vitamin D deficiency to total and sectorial RNFL We examined the relationship of vitamin D deficiency to total and sectorial RNFL thickness and average and central macular thickness attempting to identify a correlation between RNFL volume loss and vitamin D levels that would implicate vitamin D deficiency as a conditioning factor in MS neurodegeneration. Our results showed that there was no relationship between vitamin D levels and the extent of retinal axonal loss as quantified with RNFL using OCT. The inclusion of age and MS duration as covariates did not alter this effect.
Please cite this article as: Fjeldstad C, et al. Association of vitamin D deficiency with RNFL thickness in MS individuals without history of optic neuritis. Multiple Sclerosis and Related Disorders (2014), http://dx.doi.org/10.1016/j.msard.2014.03.001
Vitamin D deficiency with RNFL thickness in MS
4.3. Role of vitamin D with the increasing evidences With the increasing evidence from genetic, epidemiologic and clinical studies supporting the role of vitamin D as an immune modulator (Carlson and Rose, 2013), studies providing further insight into the role vitamin D supplementation in the development and severity course of MS are warranted. A potential way of measuring disease progression in MS is quantification of retinal axonal loss over time as measured by OCT. It is reliable, quick, inexpensive, and offers a quantitative measure of changes in neuronal loss and axonal damage over time.
4.4.
Limitations of this study
Limitations of this study include the cross sectional design which limits the causal inferences that can be made, to include potential previous vitamin D supplementation even though current intake was an exclusion criteria. The seasonal variation in vitamin D levels is a potential confounder, but the magnitude of the effect was modest. All patients came from the same center and all had a diagnosis of MS, but we did not control for all other autoimmune diseases and that may have biased the results.
4.5.
Hypothesis
Although our hypothesis that RNFL thickness was associated with vitamin D concentrations was not demonstrated, further attention is warranted to examine to which extent vitamin D has as an immune modulating effect in MS.
Conflict of interest Dr. C. Fjeldstad, Dr. A.S. Fjeldstad, Dr. Weir and Dr. Pardo have no conflicts of interest to report.
Acknowledgment The study was self-funded by the Oklahoma Medical Research Foundation, MS Center of Excellence in Oklahoma City, OK.
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Please cite this article as: Fjeldstad C, et al. Association of vitamin D deficiency with RNFL thickness in MS individuals without history of optic neuritis. Multiple Sclerosis and Related Disorders (2014), http://dx.doi.org/10.1016/j.msard.2014.03.001
