Correlation between 25 (OH) D levels in aqueous humor and the patients with ARC

Abstract

Objective

Age-related cataract (ARC) is an eye disease characterized by lens opacity. This study analyzed the correlation between 25 hydroxyvitamin D [25 (OH) D] levels and Interleukin 6 (IL-6) levels in aqueous humor from the patients with ARC, as well as between 25 (OH) D levels and lens opacity, and explored the effect of 25 (OH) D on the pathogenesis of ARC.

Methods

A retrospective analysis was performed, which included the clinical data from 120 ARC patients and 40 healthy individuals. They were divided into three groups based on the objective scattering index (OSI): 40 healthy individuals (control group), 84 patients with the early cataract (EC group), and 36 patients with the mature cataract (MC group). An electrochemical luminescence method was used to detect the levels of 25 (OH) D and IL-6 in aqueous humor, the OSI of the eye was measured using a visual quality analysis system. The correlation between the changes in the above indicators and ARC was analyzed.

Results

The OSI and IL-6 levels in the control group, EC group, and MC group increased sequentially (all p < .001), while the levels of 25 (OH) D showed an opposite trend (all p < .001).

Conclusion

The decrease in 25 (OH) D levels is related to the occurrence and development of ARC, and the relationship between 25 (OH) D deficiency and the pathological mechanism of ARC needs further research.

Keywords

Introduction

At present, the aging of the population has led to the increasing incidence rate of cataract. Age related cataracts (ARC) can lead to blindness or visual impairment in adults over the age of 50.¹ The mechanism of ARC is currently unclear. Lens epithelial cells maintain the homeostasis of the lens, and oxidative stress-induced cell apoptosis is believed to be the cellular basis for cataract occurrence. The level of apoptosis of lens epithelial cells in ARC patients is significantly higher than that in healthy individuals, which can lead to lens opacity.² The visual quality analysis as a non-invasive operation, can distinguish the degree of lens opacity in ARC patients and detect changes in aquoeus in normal eyes.³ The latest research showed that vitamin D, as a steroid hormone, had beneficial effects on the musculoskeletal system and several other body systems. It is one of the key control factors for systemic inflammation, oxidative stress, and mitochondrial respiratory function.⁴

Lanosterol plays an important role in the synthesis of vitamin D. Vitamin D is synthesized from 7-dehydrocholesterol in terrestrial animals through lanosterol. 7-dehydrocholesterol, converted from cholesterol, is considered the only true precursor of vitamin D. These precursors are converted into corresponding D-group vitamins through ultraviolet radiation.⁵ Lanosterol exhibits a short-term and reliable reversal effect in early cortical cataracts, which can reduce the severity of cataracts,⁶ indicating that the changes in lanosterol may affect the synthesis of vitamin D, thereby affecting the pathological progression of cataracts.

25-hydroxyvitamin D [25 (OH) D] and its active hormone form, 1, 25-dihydroxyvitamin D [1, 25 (OH) D], had an inhibitory effect on inflammation and excessive oxidative stress. However, vitamin D deficiency can enhance oxidative stress and systemic inflammatory response, and damage mitochondrial function,⁴ which may lead to lens damage and the formation of cataracts.⁷ In this study, we explored the effect of 25 (OH) D on the pathogenesis of ARC by analyzing the levels of 25 (OH) D in 120 ARC patients.

Materials and methods

Research subjects

A retrospective analysis was conducted on the clinical data of 120 ARC patients and 40 healthy individuals who visited Affiliated Hospital of Shaanxi University of Traditional Chinese Medicine, First People's Hospital of Xianyang city and Affiliated Hospital of Xi'an medical advanced college in Shaanxi Province from September 2019 to September 2021. Among them, 50 were diagnosed with mid nucleus cataract, 23 with cortical cataract, and 47 with posterior subcapsular cataract. According to the Objective Scattering Index (OSI), the study subjects were divided into three groups: 40 control group, 84 early cataract patients (EC group), and 36 mature cataract patients (MC group). Inclusion criteria: The diagnosis and classification of ARC complied with the lens opacity classification systemⅢ (LOCSⅢ) grading.⁸ ARC patients were all monocular cataracts with no history of secondary cataract, congenital cataracts, or any other eye diseases, and all study subjects had no history of acute infectious diseases, kidney diseases, autoimmune diseases, or other systemic diseases.

All study subjects underwent routine eye examinations such as intraocular pressure (Goldmann tonometer), visual acuity, and slit lamp microscopy (Suzhou Six Vision Technology Co., china). Additionally, a 50-MHz ultrasound biomicroscopy (UBM; MD-320; MEDA Co., Tianjin, China) inspection was performed. As shown in Figure 1. Classification of lens opacity was based on OSI. An electrochemical luminescence analyzer (Roche COBASE411, Mannheim, Germany) was used to detect the levels of 25 (OH) D and Interleukin 6 (IL-6).

Figure 1.

Lens opacity images obtained with 50-MHz UBM. (a) showed a 46-year-old normal eye with no echo in the lens at the red circle (OSI: 0.2); (b) showed a 62-year-old eye with the early cataract, with a small amount of echo in the lens at the red circle (OSI: 2.3); (c) showed a 73- year-old eye with the mature cataract, with the strong echoes in the lens at the red circle (OSI: 7.6).

Sample collection

All patients were collected aqueous humor. After successful anesthesia of the affected eye surface, the eyelids were opened under a disinfectant cloth. Then the anterior chamber was punctured with a 0.45-mm needle, and the eyeball was gently pressed to allow aqueous humor to pass through the needle and enter a disposable syringe, obtaining 0.15-0.2 mL of aqueous humor. The aqueous humor was stored in a refrigerator at −20°C for testing.

Analysis of visual quality

Visual quality analysis as a non-invasive operation, was performed before aqueous humor collection.⁹ Under the premise of refractive correction, the visual quality analysis system II (Visiometrics SL, Tarrasa, Spain) based on the double-pass technique was used to measure the patient's eye OSI. OSI value of healthy individuals is lower than 1.0 (although in some cases, the results of healthy subjects are slightly higher than 1.0), OSI value of early cataract eyes is between 1.0 and 2.9, and OSI value of mature cataract eyes is greater than 3.7.

Statistical analysis

The data were analyzed using SPSS 21.0 statistical software (IBM Corp., Chicago, IL), and the Pearson method was used for correlation analysis. The measurement data were represented by the mean ± standard deviation ( $\bar{x}$ ± s), the data between the two groups were compared using an independent samples t test, and one-way analysis of variance and q test were used to compare data among multiple groups. The counting data was expressed as a percentage (%), and a chi square test was used to compare data between two groups. The difference was considered statistically significant at p < .05.

Results

Influencing factors of ARC

As shown in Table 1. Age, history of hypertension, and history of diabetes were the relevant influencing factors for the occurrence of ARC (all p < .05). The levels of total cholesterol and triacylglycerol, and sex for the patients with ARC were not significantly different from the control group (all p > .05).

Table 1.

Analysis of factors influencing ARC.

Items	Control group (n = 40)	ARC group (n = 120)	χ²/t values	p values
Male (n, %)	21 (52.50)	66 (55.00)	0.076	0.783
Age ( $\bar{x}$ ± s, years)	57.25 ± 6.01	66.89 ± 6.08	9.641	< .001
TC ( $\bar{x}$ ± s, mmol/L)	4.62 ± 0.63	4.76 ± 0.71	1.244	0.216
TG ( $\bar{x}$ ± s, mmol/L)	1.59 ± 0.23	1.63 ± 0.21	1.418	0.159
Hypertension (n, %)	11 (27.50)	61 (50.83)	6.599	0.010
Diabetes (n, %)	7 (17.50)	46 (38.33)	5.878	0.015

Note: ARC: age related cataracts; SBP: systolic blood pressure; TC: total cholesterol; TG: triacylglycerol.

Analysis of visual quality and levels of 25 (OH) D and IL-6

As shown in Table 2. The OSI and IL-6 levels in the control group, EC group, and MC group increased sequentially (all p < .001), while the levels of 25 (OH) D showed an opposite trend (all p < .001).

Table 2.

Analysis of levels of 25- (OH) D and IL-6, as well as OSI ( $\bar{x}$ ± s).

Items	Control group (n = 40)	EC group (n = 33)	MC group (n = 36)	F Values	p values
OSI	0.75 ± 0.32	2.44 ± 1.20^a	4.56 ± 1.36^a,b	111.750	< .001
25 (OH) D (ng/mL)	42.51 ± 22.74	25.26 ± 18.46^a	16.86 ± 12.97^a,b	19.485	< .001
IL-6 (pg/mL)	7.48 ± 2.18	15.31 ± 7.84^a	24.79 ± 9.79^a,b	31.648	< .001

Note: Compared with the control group.

^ap < .01; Compared with the control group.

^bp < .01; Compared with the EC group. (EC, early cataract; MC, mature cataract; OSI, Objective scattering index of the eye; 25 (OH) D, 25- hydroxyvitamin D; IL-6, interleukin-6).

Correlation analysis

As shown in Figure 2. The levels of 25 (OH) D in aqueous humor from ARC patients were negatively correlated with OSI and IL-6, respectively (all p < .05).

Figure 2.

Correlation Scatter Plot of 25 (OH) D (OSI, objective scattering index of the eye; 25 (OH) D, 25-dihydroxyvitamin D; IL-6, interleukin-6).

Multivariate logistic regression analysis

As shown in Table 3. Using 25 (OH) D levels, OSI, IL-6 levels, age, history of hypertension, and history of diabetes as variables (0 = control, 1 = ARC), the results of logistic regression analysis showed that a decrease in 25 (OH) D levels, an increase in OSI, an increase in IL-6 levels, and an increase in age were independent risk factors for the occurrence of ARC (all p < .05).

Table 3.

Multivariate logistic regression analysis on the occurrence of ARC.

Variables	B	S.E.	Walds	p vales	Exp (B)	95% C.I. for exp(B)
Il-6	0.143	0.055	6.666	0.010	1.154	1.035	1.285
25 (OH) D	−0.048	0.017	8.168	0.004	0.953	0.922	0.985
OSI	1.541	0.409	14.196	0.000	4.670	2.095	1 0.410
Age	0.139	0.052	7.003	0.008	1.149	1.037	1.273
Hypertension	0.371	0.672	0.305	0.581	1.450	0.388	5.410
Diabetes	0.224	0.758	0.087	0.768	1.251	0.283	5.527
Constant	−9.609	3.278	8.592	0.003	0.001

Note: OSI, objective scattering index of the eye; 25 (OH) D, 25-dihydroxyvitamin D; IL-6, interleukin-6; B, correlation coefficient; S.E., standard Error; C.I., confidence Interval.

ROC curve analysis

As shown in Figure 3. The area under the curve (AUC) for diagnosing ARC in 25 (OH) D levels in aqueous humor was 0.764 (95% CI: 0.682 - 0.846, p < .001). The sensitivity and specificity of 25 (OH) D in diagnosing ARC were 72.50% and 70.80%, respectively, and the cutoff was 27.01 pg/mL.

Figure 3.

ROC curve of 25 (OH) D levels in aqueous humor for diagnosing ARC (AUC, area under the curve).

Discussion

Oxidative stress causes the body to produce excessive peroxides, and the presence of a large amount of peroxides in the anterior chamber can promote apoptosis of lens epithelial cells, leading to lens opacity. Oxidative stress is considered the main pathogenesis of ARC.¹⁰ Lens opacity is a characteristic associated with ARC.¹¹ OSI has been proven to be closely related to the presence of cataracts and can directly and objectively quantify the changes in the lens of cataract patients.¹² OSI value of early cataract eyes is between 1.0 and 2.9, and OSI value of mature cataract eyes is greater than 3. But Other report suggested that the average OSI value for early cataracts was 3.7.^3,9 In this study, the OSI in the control group, early and mature cataract patients increased sequentially, indicating that as the condition progressed, the lens turbidity of patients continue to increase.

The amyloidosis process caused by the increase of reactive oxygen species and excessive activation of inflammatory reactions exists in different regions of the anterior part of the eye, and is related to the pathological progression of eye diseases such as glaucoma and cataract.¹³ Research has found that an increase in age is associated with an increase in the levels of cytokines in the eyes of patients, such as IL-6, IL-8, and TNF-α.¹⁴ Elevated IL-6 levels were found in the aqueous humor of ARC patients.¹⁵ Our research also confirmed the views of the aforementioned literature on age and IL-6 analysis.

25 (OH) D is a neuroendocrine immune regulatory hormone that can regulate the balance of calcium and phosphorus, act on neutrophils, macrophages, and lymphocytes, and stimulate the expression of antimicrobial peptides, thereby affecting the body's inflammatory response and antimicrobial activity.¹⁶

Research suggested that long-term 25 (OH) D deficiency could promote the occurrence of oxidative stress, leading to the occurrence of chronic inflammation. Adequate levels of 25 (OH) D inhibited the release of inflammatory cytokines such as interleukin-6 and tumor necrosis factor- α.^17,18 A study on Behcet's disease reported an inverse relationship between 25 (OH) D and IL-6 ¹⁹, which was also confirmed in this study on ARC. A literature suggested that 25 (OH) D counteracted the formation and development of cataract through anti-inflammation and inhibition of oxidative stress.²⁰ Park et al.'s study for 16,086 cataract patients aged 40 or above showed a negative correlation between serum 25 (OH) D levels and the risk of nuclear cataract.²¹ However, Bao et al.'s study showed that serum 25 (OH) D levels were not associated with nuclear turbidity. But their exploratory analysis showed a negative correlation between serum 25 (OH) D levels and nuclear cataracts in women under 70 years old.²² Cho et al. believed that the high levels of 25 (OH) D in aqueous humor was related to diabetes cataract. They believed that studying patients with eye diseases should include measuring vitamin D levels in aqueous humor.²³ Among the 40 samples of cataract patients, the average concentration of 25 (OH) D was significantly lower than that of 72 patients with retinal diseases. Intraocular 25 (OH) D levels were not associated with serum 25 (OH) D levels.²⁴ In this study, our results showed that 25 (OH) D levels in aqueous humor continuously decreased with the progression of ARC. Forethermore, we also found a negative correlation between 25 (OH) D levels and OSI, suggesting that 25 (OH) D may be involved in the occurrence and development of ARC.

Conclusion

In this study, the analysis of ROC curve suggested that 25 (OH) D had good sensitivity and specificity in diagnosing ARC. In addition, a decrease in 25 (OH) D levels, an increase in OSI, an increase in IL-6 levels, and an increase in age were independent risk factors for the occurrence of ARC, the history of hypertension and diabetes were also relevant influencing factors of ARC. Therefore, the regulation of 25 (OH) D levels and the control of related influencing factors can help prevent and improve the occurrence and progression of ARC.

Limitations

The following deficiencies exist in this study. First, in this study, the selected sample size was not calculated. Second, a comparative analysis on 25 (OH) D levels in aqueous humor and serum of ARC patients was not conducted. Third, the information of age and gender for early and mature ARC patients was not provided.

Author contribution

XZ, GH, WQS and JYD conceived the study, participated in the design and coordination, and XZ wrote the manuscript. WQS, GH and JYD analyzed the data. All authors have read and approved the manuscript.

Footnotes

Acknowledgments

This study was approved by the medical ethics committee of Affiliated Hospital of Shaanxi University of Traditional Chinese Medicine, First People's Hospital of Xianyang city and Affiliated Hospital of Xi'an medical advanced college in Shaanxi Province. We thank Dr Sheng Wang from the Ophthalmology Center of First People's Hospital of Xianyang city for his suggestions and opinions on this manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethical statement

ORCID iDs

Gang Huang

Xiong Zhang

Appendix

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