The Refractive Indexes Of Glucose Solutions Simulating The Aqueous And Vitreous Humor.

Abstract
A study was performed in early 2021 to quantify the correlation of in situ glucose concentrations in the aqueous and vitreous humor on their refractive indexes. Previous research has already shown that glucose concentrations have a noticeable effect on the solution’s refractive index but at a wider range of concentrations not found in the human eye. It measured the refractive indexes of nine glucose solutions at 35° C using an automatic refractometer. The results show a linear correlation between the refractive index and glucose concentrations near in situ environments revealing that fluctuations in glucose concentrations in the aqueous and vitreous humor can affect normal eyesight.
Introduction
Glucose levels are the cause behind many symptoms related to diabetes. One of these symptoms is the impairment of vision. Diabetes is an ongoing issue that affects the lives of nearly a tenth of the world’s population. To improve the lives of diabetics, many studies have sought alternatives to the invasive method of monitoring blood glucose levels: the finger blood test. One such alternative involved measuring the refractive index of the aqueous humor to determine the glucose concentration of the aqueous humor, which could be converted into the blood glucose concentration [1]. Although the effect of glucose concentrations on the refractive index of the aqueous humor has been studied, it has paid insufficient attention to the effect of glucose concentrations on the refractive capability of the aqueous and vitreous humor and eyesight. A study by Jan Kokavec et al. revealed that diabetic patients possessed a higher concentration in vitreous humor than non-diabetic patients [2]. Another study by WF Schrader et al. reveals a similar increase of glucose content of the aqueous humor in diabetic patients versus non-diabetic patients [3]. Using this information, the difference of refractive indexes of the humors between diabetics and non-diabetics can be found. This study set out to determine the correlation of glucose solutions and their refractive indexes to the concentrations found in the aqueous and vitreous humor.
Method and Materials
The chemical agents used for the solutions are reagent grade glucose and reagent grade distilled water. The glucose is weighed on cellulose weighing paper and a BSM-120.4 Analytical balance which is accurate to 0.0001 grams. The glucose solution was mixed using an SH-2 magnetic stirrer and glass beakers. A Reichert AR6 series automatic refractometer was used to examine the refractive index of the solution and a VWR model 1104 heated water circulator was used to control the temperature of the sample while the refractive index was measured on the refractometer. Refer to (Figure 1) for a labeled image of the laboratory setup.
The range of glucose concentrations to be tested was determined from the glucose concentrations of the aqueous and vitreous humor found from their studies by Kokavec and Schrader respectively (Table 1) [2,3]. Using stoichiometry, the mass of glucose needed could be calculated from the required concentration of glucose and the chosen volume of solution. The chosen volume of water to be used was 250 ml so that the required mass of glucose is large enough to be calculated on the analytical balance. Before each trial, the analytical balance was calibrated with a 100.0000-gram weight, and the refractometer was calibrated using distilled water. A study by Yuko Iguchi, et al. showed that the mid-vitreous temperature of a pre-surgery patient was 33.0° ± 1.3° Celsius and another study by Donald R. May, et al. measured the mean temperature of the anterior chamber in rabbits was 32.5° C [4,5]. The chosen temperature for each sample was 35° ± 0.5° C to simulate a similar environment and easily control the sample temperature during each trial.
During the preparation, each solution was stirred for ten minutes at maximum speed using the magnetic stirrer. Dry, plastic pipettes were used to transfer the solution to the well of the refractometer and the well temperature would equilibrate for five minutes before reading the refractive index.
Results
Eight solution samples were tested, each sample was tested with three replicates following the same testing procedure to get the mean value of the refractive index. Reagent grade distilled water was used as a control. All tests were performed at the in situ temperature of 35° ± 0.5° C. Test results were summarized in (Table 2).
To graph the data, the average refractive index was plotted as a function of the measured concentration. The “measured concentration” used for each data point was calculated from the mass of glucose measured by the analytical balance and the volume of water used to make the solution. The correlation between the glucose concentration and the refractive index was determined by using a linear regression line of the mean values (Figure 2).
Figures and Tables

Table 1. The table shows the concentrations of different solutes in the vitreous humor. Adapted from “Biochemical analysis of the living human vitreous,” by J. Kokavec, et al, 2016, Clinical & Experimental Ophthalmology, 44, p. 597-609. Copyright 1999-2021 by John Wiley & Sons, Inc.

Table 2. This table shows the target and measured values of each controlled value and the refractive indexes and average refractive index of each trial.

Figure 1. Laboratory setup with all four instruments labeled.

Figure 2. The regression line of the average refractive index versus the measured concentration in mmol/L.
Discussion and Conclusion
The test results showed a positive linear association of the Average Refractive Index as a function of the Measured Concentration (Figure 2). An increased aqueous humor concentration would cause a smaller difference of index of refraction between the aqueous humor and the lens. This would cause light to bend at a lower angle as it goes through the lens. As the light leaves the lens into the vitreous humor, it will again bend at a smaller angle. A smaller angle of refraction would cause light rays to converge at a more posterior location. This shows that an increased glucose concentration in the aqueous humor and vitreous humor would cause hyperopia. Calculating the exact angular difference for the aqueous would require the exact refractive index of the cornea and the angle of incidence of the light ray. Calculating the difference for the vitreous humor would require the angle of incidence and the refractive indexes of the lens. Using data from Kokavec’s study and the formula of the regression line, it was determined that the mean difference of the refractive index of the vitreous humor of diabetics and non-diabetics was 4.6E-5 [2]. For the aqueous humor, using data from Schrader’s study, the calculated mean refractive index of the aqueous humor of diabetics and nondiabetics differed by 2.3E-6 [3]. A similar study focused on methods to optically determine glucose concentrations in the aqueous humor discovered “A 5mg/dl change in the glucose concentration of the aqueous humor changes na by approximately 10-5,” a slope very similar to the slope of the regression line when converted to mg/dl from mmol/l [1]. The anomalous result at 2 mmol/L was most likely caused by the improper measurement of water volume because of the limited accuracy provided by beakers and pipettes. Another source of error could be temperature fluctuations of each sample, because the allowed sample temperatures were 35° ± 0.5°, there was room for small amounts of refractive index fluctuations because of temperature.
Further research on the refractive properties of solutions made from different solutes would contribute to this study. As shown in Kokavec’s study, glucose was not the only solute whose concentration was affected when comparing the vitreous humor of diabetic and non-diabetic patients [2]. This study was limited to the testing of glucose because of the lack of availability of other chemicals and the complications which would be caused by attempting to perform refractive index research on ionic compounds. A thorough study on all components of the humors in the eyes of diabetics and non-diabetics could help professionals in the ophthalmic field to make life easier for diabetic patients.
Acknowledgements
Special thanks to Dr. Peter Li, Ms. Tracy Dawson, Mr. Darin Hallstrom, and my parents for their contributions that made this research project possible.
References

  1. Daly, D, G Clark, and K Kaouri. “Optical Measurement of Glucose Content of the Aqueous Humor.” Online Research @ Cardiff, May 18, 2018. http://orca.cf.ac.uk/111558/.
  2. Kokavec, Jan, San H Min, Mei H Tan, Jagjit S Gilhotra, Henry S Newland, Shane R Durkin, John Grigg, and Robert J Casson. “Biochemical Analysis of the Living Human Vitreous.” Clinical & Experimental Ophthalmology 44, no. 7 (2016): 597–609. https://doi.org/10.1111/ceo.12732.
  3. Schrader, WF, B Stehberger, and P Meuer. “The Concentration and Proportions of Lactate and Glucose Levels in Human Aqueous Humor and Blood.” Investigative Ophthalmology & Visual Science. The Association for Research in Vision and Ophthalmology, December 1, 2002. https://iovs.arvojournals.org/article.aspx?articleid=2420506.
  4. Iguchi, Yuko, Tetsu Asami, Shinji Ueno, Hiroaki Ushida, Ruka Maruko, Kazuhiro Oiwa, and Hiroko Terasaki. “Changes in Vitreous Temperature During Intravitreal Surgery.” Investigative Opthalmology & Visual Science 55, no. 4 (2014): 2344. https://doi.org/10.1167/iovs.13-13065.
  5. May, D. R., R. J. Freedland, S. Charles, C. Wang, and J. Bakos. “Ocular Hypothermia: Anterior Chamber Perfusion.” British Journal of Ophthalmology 67, no. 12 (1983): 808–13. https://doi.org/10.1136/bjo.67.12.808.

Availability of Data
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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