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Are higher blood mercury levels associated with dry eye symptoms in adult Koreans? A population-based cross-sectional study

Objectives The purpose of this study was to investigate whether blood mercury concentrations associated with the presence of dry eye symptoms in a nationally representative Korean population. Methods Population-based prospective cross-sectional study using the heavy metal data set of the 2010–2012 Korean National Health and Nutrition Examination Survey (KNHANES). A total of 4761 adult Koreans were the eligible population in this study. Of the 7162 survey participants, 2401 were excluded because they were <19 years of age, there were missing data in the heavy metal data set, or they had diabetes, rheumatoid arthritis, thyroid disease, asthma, depression and/or under-the-eye surgery. Blood mercury levels were measured on the day the participants completed a questionnaire regarding the presence of dry eye symptoms (persistent dryness or eye irritation). The population was divided into low and high groups by median level (4.26 and 2.89 µg/L for males and females, respectively). Results Self-reported dry eye symptoms were present in 13.0% of the cohort. Participants with dry eye symptoms were significantly more likely to have blood mercury levels exceeding the median than those without dry eye symptoms (45.7% vs 51.7%, p=0.021). Logistic regression analysis showed that, after adjusting for age, gender, education, total household income, smoking status, heavy alcohol use, sleep time, perceived stress status, total cholesterol levels and atopy history, dry eye symptoms were significantly associated with blood mercury levels that exceeded the median (reference: lower mercury group; OR, 1.324; 95% CI 1.059 to 1.655; p<0.05). Conclusions High blood mercury levels were associated with dry eye symptoms in a nationally representative Korean population.


INTRODUCTION
Dry eye syndrome is the most common disease for which patients visit ophthalmologists. Large epidemiological studies show that the prevalence of dry eye at various ages ranges from 5% 1 to >35%. 2 It is currently regarded as tear film instability and ocular surface inflammation with ocular surface damage. 1 Dry eye symptoms include dryness, sensation of foreign body or burning, photophobia, pain and visual symptoms. These symptoms affect daily living activities, including important tasks such as driving. 2 Dry eye syndrome is most commonly diagnosed using symptom questionnaires because they yield reliable diagnoses. 3 An international dry eye workshop reported that the mostly consistent risk factors of dry eye syndrome included older age, female gender, postmenopausal oestrogen therapy, ocular surface surgery history, antihistamine medications, connective tissue disease, radiation therapy, haematopoietic stem cell transplantation, vitamin A deficiency, hepatitis C infection and androgen deficiency. 4 Mercury is a ubiquitous metal in the environment that adversely affects health after absorption. Organic/inorganic/metal mercury can enter the body through various routes, including the gastrointestinal tract, skin and respiratory tract. After entry, it accumulates from the blood in the brain, kidney, liver, hair, eye and skin. 5 Ionic mercury in the blood is transported to the retina pigment epithelium, where it accumulates in Strengths and limitations of this study ▪ This study identified the blood mercury levels and dry eye symptoms of a nationally representative population in South Korea. ▪ The study population was large and the weights were considered. ▪ Potential confounders were excluded. ▪ The cross-sectional design of the study means that this association cannot be interpreted as a cause-effect relationship.
photoreceptors, the plexiform layer and ganglion cells, and is also found in the vitreous in animal studies. [6][7][8] In human studies, chronic exposure to methylmercury can affect eye movement. 9 With regard to the conjunctiva, one previous report demonstrated in rabbits that metallic mercury in the conjunctiva increased lymphocytes and macrophages and was also associated with increased amounts of altered mucus. 10 Since inflammation has a prominent role in the development and amplification of the signs and symptoms of dry eye syndrome, 11 we hypothesised that altered mucus and conjunctiva inflammation by exposure to mercury might affect dry eye syndrome. However, there are few studies that have investigated the association between dry eye and blood mercury level. This question was addressed by performing this prospective cross-sectional study of a general nationwide population in South Korea that is exposed to low levels of environmental mercury.

Study design
The study design of the present study is a populationbased prospective cross-sectional study using the heavy metal data set of the 2010-2012 Korean National Health and Nutrition Examination Survey.

Study population
Since 1998, the Korea Center for Disease Control and Prevention (KCDC) has conducted the Korean National Health and Nutrition Examination Survey (KNHANES) to assess the health and nutritional status of South Koreans. The survey employs a rolling sampling design to select representative population samples of non-institutionalised civilians. The fifth survey (KNHANES V) was performed between 2010 and 2012. Details concerning the sampling frames of participants are described elsewhere. 12 13 The heavy metal data set in KNHANES V was the source of the raw data in this study. Participants were excluded if their age was <19 years, data in the heavy metal data set were missing, they had diagnosed diabetes, rheumatoid arthritis, asthma, depression, thyroid disease, or they had a history of eye surgery. Written informed consent was obtained from all KNHANES participants by KCDC. Moreover, KCDC has released the data, which are anonymised. All data are publicly available.

Measurement of blood mercury concentrations
Blood mercury analysis was performed by the Neodin Medical Institute in Seoul; this laboratory has been certified by KCDC and the Ministry of Labor for the measurement of heavy metals. As part of the health examinations of each KNHANES participant, an experienced medical technician drew 3 mL of whole blood into an EDTA tube for trace element measurement. The mercury levels in the whole blood were measured using gold amalgam sampling methods with a Mercury analyser (DMA-80; Milestone, Bergamo, Italy). For external quality assurance, we followed several protocols, namely, those of the German External Quality Assessment Scheme (G-EQUAS; Friedrick Alexander University), US Centers for Disease Control and Prevention, and Korean National Institute of Environmental Research. Monthly internal quality assurance and control programmes were performed with three different reference materials (Bio-rad, G-EQUAS 7B, and G-EQUAS 1A). The coefficients of variation were 0.78-3.22%, 1.54-6.08% and 0.84-3.52% for Bio-rad, G-EQUAS 7B and G-EQUAS 1A, respectively. The lower limit of detection for mercury in the blood was 0.05 µL.
Definition of dry eye symptoms Since July 2008, the South Korean Ophthalmological Society has participated in KNHANES by performing ophthalmological interviews and examinations of the study participants. Participants were deemed to have dry eye symptoms if they responded positively to the questionnaire item that asked if the person had dry eye symptoms, namely, persistent dryness or sense of eye irritation. If the participant said that they only had these symptoms 'sometimes' or 'occasionally', they were deemed not to have dry eye.

Other variables
Education status was divided into four groups: ≤6, 7-9, 10-12 and ≥13 years of schooling. Total household income was divided into quartiles (Q), where Q1 and Q4 reflect the lowest and highest incomes of the population, respectively. Smoking status was categorised as nonsmoking, ex-smoker or current smoker. Heavy alcohol use was defined as consuming seven or more drinks (men) or five or more (women) on a single occasion on five or more days in a month. Sleeping time was categorised as sleeping <6 h or ≥6 h. Perceived stress and those diagnosed with atopic disease were classified as 'yes/no'. Cholesterol level was determined during fasting and categorised as <240 and ≥240 mg/dL. Anaemia was defined as follows: for males with a haemoglobin level<13 g/dL, pregnant females with a haemoglobin level <11 g/dL, and non-pregnant females with a haemoglobin level <12 g/dL.

Statistical analysis
Since KNHANES is a stratified multistage clustered probability design, the sampling weights, strata and clusters were included in all survey analyses. Mercury in the blood was expressed as geometric mean with geometric SE. Participants were deemed to have high blood mercury levels if their levels exceeded the median level of the cohort, which was 4.26 and 2.89 μg/L for men and women, respectively. To determine whether dry eye symptoms associated with the categorical variables described above, Rao-Scott χ 2 analysis was performed. To determine whether dry eye symptoms were associated with continuous variables, survey regression analysis was performed in accordance with KNHANES statistical guidelines. 13 To evaluate the association between dry eye symptoms and blood mercury level, survey multiple logistic regression analysis was applied after adjustment for age, gender, education, household income, smoking status, heavy alcohol drinking, sleep time, perceived stress status, blood cholesterol levels and atopy history (SAS Syntax: PROC SURVEYREG). To determine the potential bias, the sensitivity test was done with the optimal cut-off level is the median±10% (ie, between the 45th (3.99 and 2.72 μg/L for males and females, respectively) and 55th (4.26 and 2.89 μg/L for males and females, respectively) centiles) as the ORs and p values for the relationship between dry eye symptoms and these alternative cut-offs. All statistical analyses were performed using SAS V.9.4 (Statistical Analysis Software Institute, Cary, North Carolina, USA).

RESULTS
KNHANES V was performed in 2010-2012. In 2010, 2011 and 2012, 8958, 8518 and 8058 participants were surveyed, respectively. The KNHANES V heavy metal data set consisted of the 7162 participants whose blood concentrations of heavy metals were determined. Of these 7162 participants, 2355, 2395 and 2412 were surveyed in 2010, 2011 and 2012, respectively. A total of 2401 participants were excluded because their age was <19 years (n=1112), there were missing data in the heavy metal data set (n=18), they had been diagnosed with diabetes (n=134), rheumatic arthritis (n=90), asthma (n=110), depression (n=171), thyroid disease (n=152), or they had a history of eye surgery (n=614). The remaining 4761 participants were included in this study.
The general characteristics, socioeconomic status and blood mercury levels of the study participants are shown in table 1. The mean age of the study population was 39.6±0.2 years. Men were disproportionately represented (59.7%). More than 30% were current smokers and 21% were heavy alcohol users. Thirteen per cent of participants reported having persistent dry eye symptoms.
Men with and without dry eye had median blood mercury levels of 4.530 and 4.250 µg/L, respectively. In women, these values were 2.985 and 2.867 µg/L, respectively. Table 2 compares the participants with and without dry eye symptoms in terms of various demographic and clinical variables. Women, non-smokers, non-heavy alcohol drinkers, participants who perceived that they were stressed, and participants with higher levels of blood mercury (defined as≥ median levels of the study population) were more likely to have dry eye symptoms than the comparators (all p<0.05). Table 3 shows the ORs and 95% CIs for the association between the presence of dry eye symptoms and the demographic and clinical variables. Simple survey logistic regression analyses showed that the presence of dry eye symptoms was associated with an age of 70 years or older, female gender, less education, not smoking, not being a heavy alcohol drinker, perceiving oneself to be stressed, and higher levels (ie, ≥ median values) of blood mercury (all p<0.05). In multiple survey logistic regression analysis, after adjusting for age, gender, education, total household income, smoking status, heavy alcohol use, sleep time, perceived stress, total cholesterol levels and atopy history, higher blood mercury levels (≥median value) were significantly associated with the presence of dry eye symptoms (reference: <median value; OR, 1.324; 95% CI 1.059 to 1.655; p<0.05) (table 3).
Moreover, when the multivariate survey logistic regression analysis on the whole cohort was repeated using the median−10% range (3.99 and 2.72 μg/L for men and women, respectively) or median+10% range (4.61 and 3.10 μg/L for men and women, respectively) values as the cut-off to discriminate between lower and higher blood mercury levels, similar associations between the presence of dry eye symptoms and higher blood mercury levels were observed as when the median values were used (table 4).

DISCUSSION
To the best of our knowledge, this study on a large population-based nationally representative cohort in South Korea is the first study to investigate the association between dry eye syndrome and blood mercury levels. Our results show an association between dry eye symptoms and blood mercury levels after adjusting for the relevant risk factors.
Mercury is a toxic metallic element that can have a significant impact on human health, even when the exposure is relatively small. 14 Mercury pollution led to public health disasters in Minamata, Japan and in Iraq. 5 15 16 Although high levels of mercury exposure in the world have decreased, environmental exposure to mercury continues through seafood consumption, waste incineration, energy production and use of dental amalgams. 17 18 In Korea, the main source of mercury is likely to be the frequent consumption of fish, including shellfish: the epidemiological studies of Kim and Lee showed that, in Korea, people with a higher intake of fish and shellfish also have higher blood mercury levels. 19 20 Notably, a recent study on the health effects of mercury in American adults showed that the participants had a median blood mercury level of about 1.0 µg/L, which is much lower than that in the Korean population. 21 Since Koreans differ from Americans in their dietary habits, this observation suggests that the high blood mercury levels of Koreans reflects their diet.
Several hypothetical mechanisms may explain the association we observed between high blood mercury levels and dry eye symptoms. The first is that blood mercury accumulates in the eye and may alter the conjunctival mucus and induce conjunctival inflammation. 5 The gel-like tear layer over the ocular surface is composed of soluble mucus (which is secreted by conjunctival goblet cells) mixed with fluid and proteins secreted by the lacrimal glands. 22 23 This hydrophilic gel moves over the membrane mucins (glycocalyx) on the superficial corneal and conjunctival epithelial cells and serves as a medium to refresh the tear components and clear  debris. In dry eye syndrome, the mucins are altered 24 and inflammatory/immune cells on the ocular surface ( particularly CD4+ and CD17+ T cells) are activated or recruited to the conjunctival epithelium. 25 26 The inflammation on ocular surface promotes the corneal epithelial disease in dry eye syndrome and evokes dry eye symptoms such as persistent dryness or sense of eye irritation. The possibility that mercury induces dry eye Anaemia was defined as follows; for males with a haemoglobin level <13 g/dL, pregnant females with a haemoglobin level <11 g/dL, and non-pregnant females with a haemoglobin level <12 g/dL. The median cut-off levels that discriminated between participants with 'low' and 'high' blood mercury levels were 4.26 and 2.89 µg/L for males and females, respectively.
syndrome by promoting ocular surface inflammation is supported by a study on 12 rabbits that showed that metallic mercury in the conjunctiva increases the numbers of conjunctival lymphocytes and macrophages; it also associates with increased amounts of altered mucus. 10 A previous report demonstrated that the subcutaneous injection of mercury can develop an autoimmune syndrome with infiltration of mononuclear cells into the oral mucosa, salivary and lacrimal glands. 27 We speculate that mercury induced autoimmunity in the lacrimal glands might reduce tear production and induce conjunctival inflammation, resulting in dry eye syndrome. The second possible mechanism that explains the relationship between mercury and dry eye syndrome is that mercury depletes the conjunctivae of antioxidant proteins such as metallothioneins. These cysteine-rich low-molecular-weight proteins help to prevent tissue inflammation. 28 They are thought to bind heavy metals such as mercury in the blood, thereby removing heavy metals that can induce oxidative stress. 5 28 29 We speculate that a persistent, albeit low level, exposure of the conjunctiva to mercury, such as in our Korean population, may reduce the availability of free metallothioneins in the lacrimal glands and conjunctiva. This, in turn, may increase the susceptibility of the ocular surface to inflammation and dry eye symptoms. Experimental studies that test these potential mechanisms by which mercury may promote dry eye syndrome are warranted.
There seems to be an association between smoking status and dry eye symptoms. 30 31 However, in this study, smoking status is not statistically significant. The gender difference in smoking status may be the possible reason why the discrepancy remains. In tables 2 and 3, females were likely to have a higher association with dry eye symptoms. According to previous research on smoking status by gender, 32 the smoking rate in Korean females was much lower than that in Korean males. We speculate that different smoking status by gender in this population might cause these results.
This study has a few limitations. First, its cross-sectional design means that the association observed in this study cannot be interpreted as a cause-and-effect relationship. A large-scale population-based longitudinal study that can assess causality is warranted. Second, we did not adjust for other factors that could confound the relationship between mercury and dry eye symptoms, including the use of contact lenses or systemic drugs (including traditional oriental medicines). Third, we did not examine the effect of occupational exposure because KNHANES does not ask about occupation. Fourth, we used the median blood mercury concentration of our population as the cut-off level to distinguish participants with 'low' and 'high' blood mercury levels. Whether this is the best approach or not may be debatable. However, the sensitivity tests shown in table 4 indicated that the optimal cut-off level is the median±10% (ie, between the 45th and 55th centiles) as the ORs and p values for the relationship between dry eye symptoms and these alternative cut-offs were similar to those when the 50th centile was employed. Fifth, we could not analyse the relationship between physician-diagnosed dry eye and blood mercury levels. However, physician-diagnosed dry eye is not a good index for health outcomes for several reasons. First, mercury has a relatively short half-life in the blood due to its rapid distribution to other tissues while its halflife in the body is about 2 months. 5 33 We hypothesised, therefore, that questionnaire-determined dry eye symptoms would mirror recent exposure to mercury better than physician-diagnosed dry eye syndrome, which could reflect earlier exposure to mercury that has long ago left the circulation a long time ago. Second, the KNHANES design did not include a direct physical examination to define dry eye, namely using a slit-lamp. This is because the survey was large and its main purpose was to evaluate the prevalence of and risk factors for dry eye. If dry eye syndrome would be diagnosed according to standard guidelines, the association between mercury and dry eye symptoms observed in this study might be altered. To reduce this information bias, further evaluation with an objective tool for diagnosing dry eye syndrome should be incorporated into a nationwide survey in Korea.
This study also had several strengths. First, it was a large-scale study that was based on a nationally representative Korean population. Second, potential confounders reported in the literature were excluded or the analyses were adjusted for them.

CONCLUSIONS
In conclusion, recent environmental exposure to mercury was associated with the presence of dry eye symptoms. To elucidate a causal relationship between mercury exposure and dry eye syndrome, further longitudinal and experimental studies are needed. Our observations also suggest that to reduce the prevalence of dry eye syndrome, public health measures that control the dietary and polluted air sources of mercury are needed. Table 4 Multiple survey logistic regression analysis of the association between dry eye symptom prevalence and blood mercury levels using median±10% range (45th, 50th and 55th percentile) mercury values as the cut-off The median-10% range cut-off levels that discriminated between participants with 'low' and 'high' blood mercury levels were 3.99 and 2.72 µg/L for males and females, respectively. The median cut-off levels that discriminated between participants with 'low' and 'high' blood mercury levels were 4.26 and 2.89 µg/L for males and females, respectively. The median+10% range cut-off levels that discriminated between participants with 'low' and 'high' blood mercury levels were 4.61 and 3.10 µg/L for males and females, respectively.
Acknowledgements The authors thank the Epidemiological Survey Committee of the Korean Ophthalmologic Society for conducting the KNHANES examinations and supplying the data for this study. The quality of language in this manuscript was improved with the assistance of the professional editing agency Bioedit.
Contributors S-HC drafted the manuscript and contributed to the data interpretation and statistical analyses. J-PM contributed to the study design, statistical analyses and interpretation of data.
Funding This work was supported by a grant from the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI14C1607).
Competing interests None declared.

Patient consent Obtained.
Ethics approval The use of publicly released data and the study protocol were approved by the Institutional Review Board of Seoul St. Mary's Hospital, Catholic University of Korea (approval ID: KC14EISI0877).
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement No additional data are available.
Open Access This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work noncommercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http:// creativecommons.org/licenses/by-nc/4.0/