Effect of UV-A and UV-B Irradiation on the Metabolic Profile of Aqueous Humor in Rabbits Analyzed by ¹H NMR Spectroscopy

Abstract

purpose. This study was conducted to investigate metabolic changes in aqueous humor from rabbit eyes exposed to either UV-A or -B radiation, by using ¹H nuclear magnetic resonance (NMR) spectroscopy and unsupervised pattern recognition methods.

methods. Both eyes of adult albino rabbits were irradiated with UV-A (366 nm, 0.589 J/cm²) or UV-B (312 nm, 1.667 J/cm²) radiation for 8 minutes, once a day for 5 days. Three days after the last irradiation, samples of aqueous humor were aspirated, and the metabolic profiles analyzed with ¹H NMR spectroscopy. The metabolic concentrations in the exposed and control materials were statistically analyzed and compared, with multivariate methods and one-way ANOVA.

results. UV-B radiation caused statistically significant alterations of betaine, glucose, ascorbate, valine, isoleucine, and formate in the rabbit aqueous humor. By using principal component analysis, the UV-B–irradiated samples were clearly separated from the UV-A–irradiated samples and the control group. No significant metabolic changes were detected in UV-A–irradiated samples.

conclusions. This study demonstrates the potential of using unsupervised pattern recognition methods to extract valuable metabolic information from complex ¹H NMR spectra. UV-B irradiation of rabbit eyes led to significant metabolic changes in the aqueous humor detected 3 days after the last exposure.

Ultraviolet radiation is an environmental factor known to increase the risk of developing an irreversible opacification of the lens (cataract). ¹ Recent depletion of stratospheric ozone causes increased irradiance of solar UV radiation of shorter wavelength, especially UV-B radiation (280–315 nm). Ocular exposure to UV-B rays is found to be the primary environmental risk of cortical and perhaps posterior subcapsular cataract. ^{2 3 4 5} These lens opacities are presumably a result of increased oxidative damage caused by UV-B irradiation. ^{2 6} Evidence for UV-B cataractogenesis on the basis of oxidative damage is found both in animal models and human studies. ^{7 8 9}  

Although several studies suggest a causal relationship between UV-B radiation and cataractogenesis, ⁸ the effects of UV-A radiation (315–400 nm) and the development of cataract is still highly debated. ¹⁰ Recently, it has been found that UV-A radiation causes inactivation of several protective and metabolic enzymes in human lens systems. ¹¹ Moreover, exposure to UV-A of two times the threshold value has confirmed that UV-A also is cataractogenic. ¹ The UV-A radiation seems to affect the lens in the same manner as UV-B radiation, but in higher doses and in stronger dependence on photosensitizers for damaging effects. ¹² In cornea, representing a major protective layer against UV radiation, significant differences have been found related to antioxidant enzyme systems between UV-A– and -B–exposed eyes. ¹³ However, the exact mechanism of either UV-A or -B radiation in the formation of cataract is not well understood. ¹⁴  

The composition of the aqueous humor is suggested to play a protective role in the pathogenesis of cataract, acting as a UV filter against both UV-A and -B radiation. ¹⁵ This filtering effect is mainly related to its high ascorbate concentration, operating as an antioxidant. ^{6 16 17 18} A previous study on the aqueous humor of cat, rabbit, monkey, and guinea pig found that the ascorbate level is mainly reduced in cataractous eyes. ¹⁶ In addition, some amino acids seem to contribute to the absorption of UV-rays in the aqueous humor of rabbits. ¹⁶ Among other metabolites, glucose level is not found to differ in human aqueous humor from aphakic and cataractous eyes. As glucose is used within the eye, lactate is produced as a result of glycolysis. However, no significant difference is found in lactate concentration when normal, cataractous, and aphakic aqueous humor are compared. ¹⁵  

The purpose of this study was to identify differences between the effects of UV-A and -B radiation on the metabolic profile of rabbit aqueous humor. The results might contribute to a better understanding of the mechanisms by which UV-A and/or -B radiation induces cataractogenesis. In particular, it seems important to elucidate the contribution of UV-A radiation to the development of UV cataract. 

Based on previous experiments, ^{18 19 20 21 22 23} nuclear magnetic resonance (NMR) spectroscopy was chosen as a suitable method to study the chemical composition of rabbit aqueous humor. NMR spectroscopy is a fast and nondestructive technique, using only small sample volumes for the metabolic analyses. A large number of various metabolites can be detected and quantified in the same eye tissue or fluid as shown in previous studies. ^{18 21 22 23 24} Samples of aqueous humor can be analyzed directly, without any extraction methods, and the metabolites can be simultaneously detected and quantified in each sample. ^{18 19 20 25}  

A limiting factor in understanding the biochemical information from one- and two-dimensional ¹H-NMR spectra is the complexity of signals. This complexity and the presence of natural biological variation make it difficult to extract all the available information. Data reduction and pattern recognition techniques are therefore useful to access latent biochemical information present in the spectra. To our knowledge, the present study is the first to use NMR-based metabonomics on aqueous humor, interpreting changes induced by pathophysiological stimuli to the eye. NMR-based metabonomics is a modern method of measuring the “multiparametric metabolic response of living systems” and is well known from analysis of other biofluids. ^{26 27}  

Materials and Methods

Animal Experiments and Sample Preparation

The investigation was conducted according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Adult albino rabbits (2.5–3.0 kg) were used in the animal experiments, anesthetized intramuscularly with 2% xylazine hydrochloride (Rometar, 0.2 mL/kg; Spofa, Prague, Czech Republic) and 5% ketamine hydrochloride (Narkamon, 1.0 mL/kg; Spofa). The animals (n = 11) were divided into three experimental groups, one serving as an untreated control group (n = 3). Both eyes of four animals were exposed to UV-A radiation and both eyes of another four animals to UV-B radiation (UV-lamps; 366- and 312-nm wavelength, 6 W; Bioblock Scientific, Illkirch, France). The radiant energy was measured with a radiometer (VLX-3W; Cole-Parmer, Vernon Hills, IL) with a microprocessor equipped with two changeable sensors (UVA, UVB). The total dose per day of UV-A radiation was 0.589 J/cm² and of UV-B radiation was 1.667 J/cm². The distance between the source and the eye surface (0.03 m) and the exposure time (8 minutes, once daily in 5 days) was the same for both types of radiation. Only the corneal surface was irradiated, and the rest of the eye was protected. The animals were left untreated and killed intravenously with thiopental anesthesia (thiopental natricum; Spofa) on day 8. The eyes were enucleated, and the samples of aqueous humor were aspirated and frozen. Before the NMR analysis, the samples were lyophilized to reduce the water signal in the NMR analysis. The samples were further dissolved in a 500-μL solution of 0.25 mM sodium-3′-trimethylsilylpropionate-2,2,3,3-d₄ (TSP) in deuterium oxide (D₂O). 

NMR Spectroscopy

High-resolution NMR spectra were recorded on a NMR spectrometer (Avance DRX600, 14.1 T; Bruker Biospin GmbH, Rheinstetten, Germany), operating at 600.132 MHz for protons. ¹H spectra were recorded at 25°C. Water suppression was obtained with a 3.0-second presaturation pulse, followed by a 90° pulse angle for acquiring the one-dimensional proton spectra. Five hundred twelve transients recorded a spectral region of 6.6 kHz, and the free induction decays (FIDs) were collected with 32,000 points, giving an acquisition time of 3.5 seconds. The final repetition delay was 5.0 seconds. Chemical shift referencing in parts per million was performed relative to TSP at 0 ppm. Peak assignments were performed according to previous reports ^{18 26 28 29 30} and by spiking samples with authentic compounds. Homonuclear correlated spectra (COSY) and J-resolved spectra were also recorded to assign complicated coupling patterns. Metabolites were quantified by relating their peak area with the added standard TSP, and the peak integrals were calculated using special software (XWIN-NMR; Bruker BioSpin GmbH). The exact concentrations were found by referring the integrals to the weight of the original samples of aqueous humor. 

Multivariate Analysis

The NMR spectra were statistically analyzed by principal component analysis (PCA). The high-field area (0.5–4.7 ppm) of 22 ¹H NMR spectra from 11 rabbits was used as input in the PCA. This high-field area included most of the metabolites in a single spectrum of the rabbit aqueous humor. The dominating resonances of lactate (1.25–1.40 and 4.05–4.15 ppm) and water (4.7–5.0 ppm) were removed from the selected region. In this way, potential differences between the metabolites of lower intensities were easier to find. Variable reduction was achieved by averaging four times, and the results in this article are based on a matrix consisting of 22 samples × 11,746 variables. 

PCA was performed with full cross-validation, implying that the same samples are used both for calibration and validation. The PCA was also performed with mean centering, and the number of PCs to include in the cross-validation analysis was determined by the residual X-variance. The number of PCs that gave the minimal total X-variance was considered to be the required number of principal components. To find groupings and reveal the relationship between samples, the score plot from the various PCs was interpreted. Another important graphic representation, the loading profile, displayed the importance of each metabolite for the variation described by the PCs. 

One-Way ANOVA and Multiple Comparisons

One-way ANOVA was performed on the concentrations of each metabolite to determine whether the irradiated samples (UV-A and -B) and the control group differed significantly. Significant results were further tested by a multiple-comparison method (Bonferroni) to identify the differing group or groups. The level of significance was set at P < 0.05. 

Results

Using both one- and two-dimensional ¹H NMR techniques, 21 different metabolites were assigned within the spectral region of 0 to 9.0 ppm. These metabolites are leucine, isoleucine, valine, lactate, alanine, lysine, acetate, acetone, glutamate, pyruvate, succinate, citrate, taurine, betaine, myo-inositol, glucose, ascorbate, tyrosine, histidine, phenylalanine, and formate. Figures 1A 1B and 1Cshow a representative ¹H NMR spectrum with the assigned metabolites from the aqueous humor of a UV-B–irradiated rabbit eye. 

The principal component analysis, including all 22 samples, demonstrated a clear pattern among the selected chemical shift regions (0–4.7 ppm). The score plot of the first principal component (PC1) and the second principal component (PC2), explaining 49% of the total variation in the NMR spectra, shows that UV-B–irradiated samples have a higher score for PC1 than the UV-A–irradiated samples and the control samples (Fig. 2) . This is illustrated by the diagonal dashed line inserted in the score plot, separating all but one of the UV-B–irradiated samples from the other experimental groups. The score plot shows no distinct grouping between the samples of the UV-A–irradiated eyes and the control eyes. Considering that UV-B–irradiated samples had a higher score for PC1, the loading profile of PC1 shows the importance of each metabolite for the variation described in the score plot. The loading profile of the first principal component, explaining 29% of the total variation in the NMR spectra, shows that the high scores for UV-B–irradiated eyes were a result of altered concentration of several metabolites (Fig. 3) . The UV-B–irradiated samples had relatively higher concentrations of betaine, acetate, and valine and relatively lower concentrations of ascorbate, citrate, pyruvate, and β-glucose. 

The ¹H NMR spectra of aqueous humor include areas where signals are complex and metabolites overlap each other, and these peaks are therefore not possible to quantify by straightforward integration. However, in this study, a selected group of 14 different metabolites was considered quantifiable, and the concentration levels of these metabolites, before and after exposure (UV-A and -B irradiation), are presented in Table 1 . The one-way ANOVA followed by the Bonferroni test showed that the concentration level of six different metabolites in the aqueous humor was significantly changed (P < 0.05) after exposure to UV-B radiation: glucose, ascorbate, betaine, valine, isoleucine, and formate. All metabolites, except ascorbate, increased in concentration after exposure to UV-B radiation, whereas the concentration level of ascorbate was considerably reduced after UV-B exposure. The concentrations of the remaining substances (i.e., alanine, lactate, tyrosine, histidine, acetone, acetate, pyruvate, and citrate) were not significantly changed compared with unexposed and UV-A–irradiated eyes. Analyzing the samples from the UV-A–exposed eyes, no significant difference in the metabolic profile of the aqueous humor was detected compared with the control animals (Table 1) . However, UV-A exposure initiated metabolic changes similar to those detected after UV-B radiation (Table 1) . 

The results obtained from the multivariate analysis and the quantifications by integration were close to identical for metabolites examined with both methods. However, there was a conflicting result from glucose. The loading spectra of PC1 showed a decrease in β-glucose due to UV-B irradiation (Fig. 3) , but the statistical analysis based on peak integrals showed a significant increase in the total amount of glucose (Table 1) . When we performed a new PCA from a smaller area (3.0–5.5ppm) and removed the dominating resonance of betaine, the results showed agreement with the results of the peak integrals. In this case, all resonances from glucose in the loading profile, both from α- and β-glucose, increased in the aqueous humor of UV-B–exposed eyes. In addition, the metabolic change of pyruvate and citrate did not reach significant level in the ANOVA. 

Discussion

The use of ¹H NMR spectroscopy revealed the metabolic profiles of aqueous humor from rabbit eyes exposed to UV-A or -B radiation. Detection of >20 different metabolites in each sample provided a large amount of metabolic information, and multivariate analysis was found useful in extracting this information from the ¹H NMR spectra. Our results showed that UV-B irradiation of the anterior segment of the rabbit eye had a significant effect on several metabolites in the aqueous humor, whereas exposure to UV-A radiation did not significantly change the metabolic profile compared with unexposed eyes. 

Regarding the UV radiation doses used in the present study, the daily dose of UV-B (1.667 J/cm²) is reported to be close to threshold for developing permanent cataract after a single application on pigmented rabbit eyes. ³¹ The exposure time and the experimental conditions were the same for both UV-A and -B irradiation. With the chosen set-up, the daily dose of UV-A (0.589 J/cm²) was lower than the UV-B dose. This is due to the difference in energy between the UV-A and -B wavelengths. Compared with the threshold values reported for rabbit cornea, ³¹ the applied UV-A dose was below this level. However, because of a difference in the different study designs, the values reported in other studies are difficult to compare directly. In our study, the cumulative effect of repeated exposure for 5 days must be taken into account by the evaluation of the UV irradiation’s effect on the metabolic profile of aqueous humor. Compared with the doses calculated for solar UV-A and -B radiation reaching the human cornea, Zigman ³² reported average values of 3.4 J/cm² of UV-A and 105 mJ/cm² of UV-B during a 1-hour exposure. The daily UV-B dose applied in the present study is thus roughly equivalent to exposing the human cornea to approximately 16 hours of sunlight, whereas the daily UV-A dose is equivalent to approximately 10 minutes of sunlight. The most adverse UV radiation health effects were previously related to UV-B wavelengths below 315 nm. However, during the past few decades, the longer wavelength (UV-A) at a higher dose has been accepted as a risk factor. In the present study, UV-A radiation caused small metabolic alterations in the same directions as those detected after exposure to the UV-B wavelength (Table 1) . These findings may indicate that UV-A radiation has the same effects as UV-B radiation, but requires a higher energy level to induce significant results. 

A significant decrease in ascorbate concentration was observed in the aqueous humor after UV-B exposure (Table 1) . This finding is in agreement with the results of previous reports. ^{16 33} Ascorbate is known to scavenge free radicals in the aqueous humor, ³⁴ protect against UV-induced DNA damage to the lens, ³⁵ and minimize UV radiation by absorption and by suppressing fluorescence of radiation. ^{16 17 36} However, a disruption of the blood–aqueous barrier induced by UV-B radiation and resulting in a decrease of ascorbate level in the aqueous humor cannot be excluded. 

The total amount of glucose in the rabbit aqueous humor was found to increase significantly due to UV-B radiation. Previous experiments performed on cultured rabbit lenses showed that the initial effect from UV-B radiation was impaired permeability and transport problems in the lens membrane, affecting also glucose transport. ³⁷ In addition, an impairment of the blood–aqueous barrier due to an inflammatory response initiated by UV radiation could not be excluded. Because plasma contains a higher concentration of glucose than the aqueous humor, ¹⁵ a leakage of glucose from the plasma through a defective barrier would increase the level of glucose in aqueous humor. Generally, glucose from the aqueous humor is the main source of energy in the metabolism of the cornea and the lens. Inactivation of glycolytic enzymes after UV-B irradiation might impair glycolysis ^{12 37 38} and thereby contribute to an increase of glucose level in aqueous humor. UV exposure is previously found to deactivate both hexokinase in the lens ³⁹ and glucose-6-phosphate dehydrogenase (G6PDH) in porcine corneas. ⁴⁰  

An increase in concentration of betaine was observed after UV-B exposure. Betaine has been found to be a major metabolite in the liver, ^{41 42 43} in the mammalian placenta, in the renal medulla ⁴⁴ and is further known to stabilize macromolecules against physiological disturbance. ⁴⁵ Assignment of betaine in the NMR spectra of aqueous humor is a novel finding, corresponding to the unknown peak in a previous study. ¹⁸ A recent study reported a decrease in betaine concentration after UV-B exposure in rat lenses. ²³ The increase of betaine concentration in aqueous humor revealed in the present study may be a result of cell membrane disruption due to UV-B exposure ⁴⁶ causing betaine leakage from the lens to the aqueous humor. So far, the role of betaine in the eye and in relation to cataractogenesis is not known. 

The concentrations of the amino acids valine and isoleucine in the aqueous humor were found to increase after UV-B exposure, indicating impairment of protein synthesis and degradation. Furthermore, amino acids may function as both osmolytes ⁴⁷ and as antioxidants in the lens. ⁴⁸ Another metabolite with rather unknown importance is formate. UV-B exposure of the rabbit eye caused a significant increase in formate concentration in the aqueous humor. Formate has been detected in cell extracts of lymphocytes ⁴⁹ and in the eye in extracts of cornea and lens ²² and in intact samples of aqueous humor ¹⁸ and lens. ²³ A possible source of formate in mammalian systems may be formaldehyde, which rapidly oxidizes to formate. ⁵⁰ Formate is incorporated into synthesis of folic acid where the enzyme cobalamin (vitamin B₁₂) is included. Cobalamin inactivation in rats has been found to cause a significant accumulation of endogenous formate. ⁵¹ Recently, the use of cobalamin alone or in combination with other vitamins (B₃ and B₉) was found to protect cells against the damaging effects of UV light. ⁵² Therefore, the increase of formate concentration in the present study may have a relation to the damaging effects of UV light on these vitamins. Formate is further known as a toxic metabolite, ⁵³ inhibiting cytochrome oxidase activity, a component of the electron transport chain involved in ATP synthesis. ⁵⁴  

In conclusion, ¹H NMR spectroscopy is shown to be an efficient tool for investigation of the metabolic changes in the aqueous humor. The unsupervised pattern-recognition method, PCA, visualizes the patterns of metabolic differences between the normal and the UV-B–exposed aqueous humor. As shown in this study, ultraviolet rays with shorter wavelengths (UV-B) have larger influence than rays with longer wavelength (UV-A) on the metabolic profile of rabbit aqueous humor. The metabolic alterations suggest several effects from UV-B radiation: permeability and osmoregulatory problems (betaine, glucose, valine and isoleucine), oxidation (ascorbate, formate), and deactivation of glycolytic enzymes (glucose). The focus on the metabolic profile of aqueous humor seems to be an important approach in understanding the complete biochemical processes in the development of UV cataract. 

 Supported by Grant 148600/320 from The Norwegian Research Council.

 Submitted for publication July 6, 2004; revised November 8, 2004; accepted November 24, 2004.

 Disclosure: M.-B. Tessem, None; T.F. Bathen, None; J. Čejková, None; A. Midelfart, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

A representative 600-MHz ¹H NMR spectrum of aqueous humor from a UV-B–irradiated rabbit eye, divided into three spectral regions: (A) 0.70 to 3.1, (B) 3.1 to 4.7, and (C) 4.7 to 8.6 ppm.

Figure 2.

 

Plot of the scoring of the first versus the second principal component from the principal component analysis of the rabbit aqueous humor ¹H NMR spectra (49% of total variation). UV-B–irradiated samples are separated from the UV-A–irradiated samples and the control group (visualized with a diagonal dashed line).

Figure 3.

 

Loading profile of the first principal component from the principal component analysis of the rabbit aqueous humor ¹H NMR spectra (29% of total variation). The high score of UV-B–irradiated samples are a result of metabolic alteration in several metabolites.

Table 1.

 

Comparison of Metabolic Composition of Aqueous Humor from Control Animals and from UV-A– and UV-B–Exposed Rabbit Eyes

Metabolites	UV-A Irridated (n = 4) (μmol/g)	UV-B Irridated (n = 4) (μmol/g)	Control (n = 3) (μmol/g)	ANOVA P
Glucose	3.879 (1.493)	5.147 (1.051)	2.972 (0.780)	0.009*
Alanine	0.987 (0.246)	0.932 (0.248)	0.823 (0.265)	0.385
Ascorbate	0.848 (0.356)	0.392 (0.071)	1.073 (0.278)	0.000*
Lactate	14.223 (1.998)	16.911 (3.367)	14.002 (1.838)	0.052
Betaine	0.145 (0.052)	0.436 (0.384)	0.063 (0.021)	0.006*
Valine	0.184 (0.044)	0.323 (0.084)	0.177 (0.051)	0.000*
Isoleucine	0.072 (0.021)	0.142 (0.051)	0.053 (0.014)	0.001*
Formate	0.030 (0.008)	0.060 (0.013)	0.028 (0.010)	0.000*
Tyrosine	0.177 (0.045)	0.172 (0.037)	0.172 (0.045)	0.764
Histidine	0.104 (0.028)	0.136 (0.028)	0.106 (0.033)	0.106
Acetone	0.046 (0.008)	0.082 (0.030)	0.045 (0.010)	0.061
Acetate	0.173 (0.085)	0.300 (0.164)	0.157 (0.055)	0.325
Pyruvate	0.174 (0.124)	0.097 (0.083)	0.078 (0.057)	0.301
Citrate	0.395 (0.089)	0.317 (0.178)	0.372 (0.092)	0.418

 Data are expressed as mean with standard deviation in parenthesis.

*  Significant (P < 0.05) only for the UV-B group compared to control group. No significant results were detected for UV-B group versus UV-A group.

The authors thank Oddbjørn Sæther and Øystein Risa for advice and assistance with the NMR-analysis, and Čestmir Čejka for radiometric measurements.