Correlation between tear fluid and serum vitamin D levels
© The Author(s). 2016
Received: 10 May 2016
Accepted: 19 August 2016
Published: 2 September 2016
Vitamin D deficiency is associated with a range of systemic diseases including ocular disorders. The objective of this study is to measure tear vitamin D levels and investigate the correlation between serum and tear vitamin D levels.
A total of 48 healthy volunteers without any systemic and ocular disease were recruited for this observational cohort study. Serum was collected using clot activator coated Vacutainer® Plus tubes. Tear fluid was collected using Schirmer’s strips. Serum and tear total 25-hydroxyvitamin D levels were measured by competitive chemiluminescent ELISA and the correlation between the levels were studied.
The measured serum 25-hydroxyvitamin D level ranged between 3.3 and 27.5 ng/ml (Mean ± SEM, 9.4 ± 0.7 ng/ml; Median 8.4 ng/ml). Significantly (p < 0.0001) higher level of 25-hydroxyvitamin D was detected in the tears (Mean ± SEM, 17.0 ± 1.6 ng/ml; Range 3.2–45.8 ng/ml; Median, 16.3 ng/ml) compared to serum. An average of ~2 fold (Mean ± SEM, 1.9 ± 0.2; Range 0.4–5.8; Median, 1.7) higher 25-hydroxyvitamin D was observed in tears compared to serum in the subjects. In addition, a positive correlation was observed between serum and tear 25-hydroxyvitamin D levels (r = 0.5595; p < 0.0001).
A higher level of 25-hydroxyvitamin D was observed in the tear fluid compared to that of the serum. It would be beneficial to consider tear vitamin D levels to study its role with reference to ocular surface diseases.
25-hydroxyvitamin D measured in the tear fluid was found to be higher than in the corresponding serum sample.
Vitamin D is well known for regulating calcium homeostasis, immune response, cellular proliferation and differentiation, angiogenesis, apoptosis and nociception [1, 2]. A high prevalence of vitamin D deficiency has been documented worldwide . The optimal vitamin D (total 25-hydroxyvitamin D) level ranges between 30 and 80 ng/ml in the serum, and deficiency/insufficiency of vitamin D is considered when total 25-hydroxyvitamin D is <20 ng/ml [3, 4]. Owing to its pleiotropic nature and ubiquitous expression of vitamin D receptor (VDR) in almost all cells and tissues , deficiency of vitamin D has been associated with a range of diseases, and supplementation of vitamin D has substantially improved the prognosis of these diseases . Vitamin D status in the serum is suitable for studying its association with systemic conditions. However, it would be beneficial to obtain tissue specific vitamin D status for localized conditions such as ocular surface disease. There is increasing evidence regarding extra-renal synthesis and tissue-specific effects of vitamin D3 in other tissues , including, the eye [8–11]. The cells and tissues in the eye are responsive to 1,25-dihydroxyvitamin D since VDR is present in the epithelium of the cornea, lens and ciliary body, corneal endothelium, retinal pigment epithelium, ganglion cell layer and photoreceptors of the human eye [8, 12]. Hence, vitamin D status is being associated with the incidence and severity of various ocular conditions, including myopia, age-related macular degeneration, diabetic retinopathy and uveitis . Serum 25-hydroxyvitamin D levels were also reported to be associated with some ocular surface conditions such as dry eye and allergic conjunctivitis [13–15]. In vivo reports provide evidence concerning the anti-inflammatory and immunomodulatory roles of vitamin D in the corneal region [16, 17]. The role of vitamin D in ocular surface conditions, mechanistic insights into the aetiopathology of vitamin D deficiency and beneficial effects of vitamin D supplementation in the management of ocular surface conditions are yet to be studied. Determining local vitamin D levels in the eye is essential to meet this knowledge gap. Therefore, the current study aims to quantify 25-hydroxyvitamin D in tear fluid and correlate its level with that in the serum.
Study design & clinical examination
The observational cohort study approved by the Narayana Nethralaya Institutional Review Board (Ref. No.: C/2015/05/05) was conducted in adherence to Indian Council for Medical Research (ICMR) guidelines and tenets of the Declaration of Helsinki. A total of 48 healthy volunteers were selected (after obtaining informed written consent) for the study after thorough clinical investigation at Narayana Nethralaya Eye Hospital, Bangalore, India to rule out any ongoing or recent ocular and/or systemic co-morbidity. In addition, contact lens wearers were not included in the study.
Serum and tear sample collection
Serum was isolated by centrifuging peripheral venous blood collected in BD Vacutainer® Plus Plastic Serum Tubes (BD, New Jersey, USA) with spray-coated silica as a clot activator and stored in −80 °C until further use. Tear fluid from the subjects were collected using sterile Schirmer’s strip (5 × 35-mm2; Tear Strips, ContacareOpthalmics and Diagnostics, India) by following Schirmer’s Test I procedure and stored at -80 °C in a sterile microcentrifuge tube until further use. Tear fluid was extracted from Schirmer’s strips by agitating small cut pieces of these strips in phosphate buffered saline (PBS) solution in a sterile microcentrifuge tube at +4 °C for 1.5 h. Tear fluid was then eluted by centrifugation and stored at −80 °C until further use. Schirmer’s strip based tear fluid collection was followed as it was reported to be suitable and comparable with capillary tube based tear fluid collection for downstream analysis .
Measurement of serum and tear vitamin D
Total 25-hydroxyvitamin D (25-hydroxyvitamin D3 + 25-hydroxyvitamin D2) levels in the serum and tear fluid were measured by direct competitive chemiluminescent enzyme linked immunoassay – 25-hydroxyvitamin D ELISA Kit (Enzo Life Sciences, Switzerland). The kit was optimized and validated to detect serum vitamin D. As assays to detect tear 25-hydroxyvitamin D are unavailable, we adapted the above mentioned kit to quantify tear 25-hydroxyvitamin D. Since the tear fluid was eluted in PBS, we included additional PBS based 25-hydroxyvitamin D3 (Cayman Chemical, Ann Arbor, MI, USA) standards for the assays. 25-hydroxyvitamin D3 is the most predominant and endogenous form of vitamin D which is the major contributor towards measured total vitamin D - 25-hydroxyvitamin D levels. The accuracy of this modified assay results was evaluated by comparing results from an automated direct competitive chemiluminescent enzyme linked immunoassay (ADVIA Centaur® Vitamin D Total, Siemens) that detects both 25-hydroxyvitamin vitamins D2 and D3. The latter was chosen to validate our method because it was reported to have acceptable accuracy compared with LC-MS/MS . Furthermore, the levels of tear 25-hydroxyvitamin D were normalized to the amount of tear fluid collected for the measurements.
All statistical analyses were performed with GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA) and Stata 12.1 (StataCorp, Texas USA). Shapiro-Wilk normality test was used to check the distribution of the data set. Spearman correlations analysis, Wilcoxon matched-pairs signed rank test and Mann-Whitney test were used to analyse data sets that were not normally distributed. The agreement between the data sets was analysed using the Bland-Altman plot. The mean or median value of the individual groups was reported as Mean ± SEM or median (along with the range). Two-tailed p < 0.05 was considered to be statistically significant.
Serum vitamin D deficiency is associated with various systemic conditions and recently its relevance in ocular health and disease is being reported . There is growing evidence regarding 25-hydroxyvitamin D status in ocular surface diseases such as allergic conjunctivitis and dry eye [13, 14, 20]. However, there has been some rather contradicting observations with regards to 25-hydroxyvitamin D and dry eye [15, 21]. Nevertheless, all these reports have been based on serum 25-hydroxyvitamin D. We suggest that assessing 25-hydroxyvitamin D status in tears would hold more relevance in eye disease, especially in ocular surface conditions. Vitamin D (both 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3) was shown to influence corneal epithelial barrier function by regulating expression of occludin , and the ability of corneal epithelial cells to synthesize and metabolize vitamin D has also been documented [8, 9]. An in vivo study in rabbits showed that 25-hydroxyvitamin D can be measured in tears and its level increases in the tears following oral supplementation of vitamin D . In the current study, we have shown that 25-hydroxyvitamin D can be measured in human tears by competitive chemiluminescent immunoassay and that the 25-hydroxyvitamin D levels were significantly higher in the tears than in the serum. Similarly, another study reported significantly higher 25-hydroxyvitamin D levels in tears (71.8 ± 6.2 ng/ml) compared to serum (21.8 ± 11.3 ng/ml) in children (12.5 ± 2.5 years) using electro chemiluminescent immunoassay . The difference in the 25-hydroxyvitamin D levels between tear fluid and serum seem to vary between the current study and that reported by Goksugur SB et al. The primary reason underlying this difference could be related to the principle and sensitivity of the assay used to measure 25-hydroxyvitamin D [23, 24]. Moreover, we have modified the method to render more accurate measurement of 25-hydroxyvitamin D in tear fluid. The age difference between these study cohorts could also be considered as a possible factor contributing to the variation. It should also be noted that contrary to our observation and that of Goksugur SB et al., 25-hydroxyvitamin D levels in tears was lower than plasma in rabbits , this could once again be attributed to the technique adopted for quantifying 25-hydroxyvitamin D and possible inter-species differences. Since the human corneal epithelium is capable of synthesizing vitamin D , we speculate that the increased level observed in the tears could be due to vitamin D produced by the corneal epithelium following exposure of the eye to UVB rays present in sunlight. Unlike tear fluid, 25-hydroxyvitamin D in the saliva (another non-invasive source for measuring vitamin D) was reported to be manifold lower than that of the serum [25, 26]. Hence, more detailed studies are required to confirm increased 25-hydroxyvitamin D in tear fluid and understand the source that contributes to it.
The observations from the current study suggest that 25-hydroxyvitamin D is present in tear fluid and its level was found to be higher than in the corresponding serum in humans. It would be beneficial if studies investigating the association between tear vitamin D and ocular surface disease can determine its relevance with reference to disease severity and pathogenesis so as to inform the need for topical supplementation of vitamin D to ameliorate ocular surface diseases.
The study was supported by the Narayana Nethralaya Foundation, Bangalore, India. The authors would like to thank Dr. Harsha Laxmana Rao (Narayana Nethralaya Eye Hospital, Bangalore) and Dr. Abhijit Sinha-Roy (Narayana Nethralaya Foundation, Bangalore) for assistance with statistical analysis.
The study was supported by the Narayana Nethralaya Foundation, Bangalore, India.
SS, RS, AG – conception and design; analysis and interpretation; writing of the article; critical revision of the article; final approval of the article; statistical expertise; obtaining funding; literature search. KD, NP, NC, AA, AS – data generation and sample collection; literature search. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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