Determination of vitamin D in tears of healthy individuals by the electrochemiluminescence method

Background Vitamin D is a fat‐soluble steroid hormone which can be converted into various forms and is of extreme physiological importance to our body. However, its functions and local metabolic pathways in some organs, such as the eye, have not yet been well studied. We aimed to verify the correlation between vitamin D levels in blood and tear fluid and the possibility of using tear fluid as a biological material for monitoring eye disorders in the future. Methods The electrochemiluminescence method was used to examine blood and tear samples collected with Schirmer test strips from 21 individuals without ocular disease. Results At the 95% confidence interval, mean tear fluid vitamin D = 37.8 ± 3.6 ng/mL, which is higher than the serum level, with a mean of 30.3 ± 7.7 ng/mL; Lin's concordance correlation coefficient = −0.018 (−0.174; 0.139), Pearson's coefficient = −0.070, and the Bland‐Altman coefficient = −11.12 (−30.40; 8.16). Results were obtained using the program Stata version 11.0. Conclusion It is possible to determine vitamin D levels in tear fluid using the electrochemiluminescence method, and as the results do not correlate with blood, there is possibility of using tear fluid as a biological matrix for detection of vitamin D, which may increase the possibilities of new studies in eye disorders.

Background: Vitamin D is a fat-soluble steroid hormone which can be converted into various forms and is of extreme physiological importance to our body. However, its functions and local metabolic pathways in some organs, such as the eye, have not yet been well studied. We aimed to verify the correlation between vitamin D levels in blood and tear fluid and the possibility of using tear fluid as a biological material for monitoring eye disorders in the future.

Methods:
The electrochemiluminescence method was used to examine blood and tear samples collected with Schirmer test strips from 21 individuals without ocular disease.
Results: At the 95% confidence interval, mean tear fluid vitamin D = 37.8 ± 3.6 ng/ mL, which is higher than the serum level, with a mean of 30.3 ± 7.7 ng/mL; Lin's con-  When the body senses the need to act on blood and bone calcium levels, part of this 25-hydroxyvitamin D is transported to the kidneys, where it will undergo the last metabolization process, becoming calcitriol (1,25-hydroxyvitamin D), the active form of vitamin D.
The presence of vitamin D metabolites in tears has also been shown, which possibly serves for the maintenance of the ocular cornea; however, the origin of vitamin D in tear fluid has not yet been clarified. 4 There are still few studies that examine the presence of vitamin D synthesis and pathways in the eye, and whether this actually plays any role in autocrine vitamin D synthesis function by ocular cells. Certain studies show the expression and functionality of vitamin D3 in human ocular barriers, indicating that vitamin D3 may be important in ocular barrier function and the maintenance of immunological privilege. 3 Vitamin D deficiency is commonly described as when the serum 25-hydroxyvitamin D level is less than 20 ng/mL, 5 which can lead to several types of health problems such as cardiovascular problems, hypertension, and ophthalmic disorders (eg, dry eye syndrome, a common tear film disease and ocular surface that causes discomfort, visual disturbance, tear film instability and potential damage to the ocular surface). 6,7 There is evidence of the difference between vitamin D levels in blood and tear fluid in the same organism, and this difference may be related to the principle and sensitivity of the methods used to measure 25-hydroxyvitamin D. 8,9 It has been found that the amount of this vitamin is higher in tear fluid than in blood in patients with eye diseases. In addition, some studies show that there are contradictions between the serum value of 25-hydroxyvitamin D with dry eye disorder 10,11 ; therefore the measurement of 25-hydroxyvitamin D in tears may be more relevant for ocular diseases, especially for ocular surface diseases. 12 Thus, this study aimed to verify the correlation between levels of vitamin D in blood and tear fluid and the possibility of using tear fluid as a biological material for monitoring eye disorders in the future.

| Samples and collection procedures
Blood and tear samples were collected from people without ocular disease. Tear samples were collected using OPHTHALMOS test strips used in the Schirmer tear test (STT), a method that evaluates whether the eye produces enough tear fluid to remain lubricated. The strips were placed in the lower eyelid pouches and removed after 5 minutes; afterward, they were placed in 0.5 mL plastic tubes containing 250 μL of saline solution. The tubes were stored at temperatures between 2°C and 8°C for a period of 24 hours (overnight) and analyzed later.
Blood samples were collected with a separating gel tube (yellow tube).
This study was performed in accordance with the ethical standards established by the Declaration of Helsinki and has been approved by Faculdade de Medicina do ABC Ethical Committee; informed consent was obtained from all individual participants included in the study.

| Dosage of vitamin D in tear fluid and blood
Quantitative determination of vitamin D from tear fluid and blood samples of the patients in this study was performed by the electrochemiluminescence technique using the Cobas ® e411 device, and its calibration curve was corrected by two calibrators with a low value of 1.32 ng/mL and a high value of 45.50 ng/mL.

| RE SULTS
In this study, 21 people were recruited; 21 blood samples and 29 tear samples were collected of which 16 samples were from the right eye and 13 from the left eye. Tear fluid was collected from both eyes of 8 people, while samples from the others were collected from only one eye.
It was observed that the mean of vitamin D is higher in tear fluid compared to blood. This fact was also observed when comparing vitamin D value between blood and tears from the left and right eye (Table 1).
When we evaluated the coefficient values of Lin, Pearson, and Bland-Altman (Tables 2 and 3

| D ISCUSS I ON
Currently, the role of vitamin D in the eye is still not very clear. Some studies examine the physiological functions of vitamin D in a single organ exposed directly to solar rays without being the skin, the eye. 13 Research on vitamin D testing can be performed by various types of laboratory methods, such as chemiluminescent microparticle immunoassay (CMIA) and ELISA. 12  Ocular barrier epithelial cells can convert vitamin D3 to its active form, and this synthesis has a significant value. The rate of this conversion is comparable to that of primary respiratory epithelial cells, bladder, and mammary epithelial cell lines. 3,16,17 This conversion rate is much higher in tear fluid than in the collecting duct cells of the human kidney. 19 The eye is an organ that is directly exposed to potentially harmful radiations such as ultraviolet radiation and blue light, thus the necessity of protective molecules, lutein, vitamin D, etc, to prevent or reduce possible damage to ocular tissues and cells. Since the habit of modern F I G U R E 3 Lin's correlation coefficient (A) and Bland-Altman (B) for vitamin D in serum and tear fluid of the left eye society of using electronic products such as smartphones, laptops, and television sets for long periods of time may increase the exposure to the radiation previously mentioned, we can therefore suppose that these acts can increase the synthesis of these protective molecules in the ocular region, leading to higher concentrations in tear fluid.
Vitamin D in the blood acts in various functions of different organs, converting into different forms and acting synergistically with other enzymes in their physiological actions. Therefore, the level this vitamin in the blood is more variable and may be lower in certain cases when compared to tear fluid, as the results of this work show.
In addition, studies on chronic renal diseases have shown the reduction of megalin expression due to reduction of 25(OH)D3 level and deficient autocrine VDR activation, 20