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Distribution of Ascorbate in the Anterior Bovine Eye

From The National Hospital Pharmacy, Oslo, Norway.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
PURPOSE. To analyze the ascorbate distribution in the anterior eye wall to better understand the functional significance of this compound in the eye.

METHODS. Ascorbic acid was determined by high-performance liquid chromatography using an LC-10 system (Shimadzu, Kyoto, Japan). Bovine eye samples were used.

RESULTS. The highest ascorbate concentration was observed in the corneal epithelium, with significantly higher values in the central (1.56 mg/g) than in the peripheral (1.39 mg/g) area. The ascorbate content was similar in the corneal stroma (0.22 mg/g), the Descemet’s membrane (DM)/endothelium (0.22 mg/g), and the aqueous humor (0.21 mg/ml). By comparison, the sclera (0.15 mg/g) and the conjunctiva (0.11 mg/g) showed lower values, as did the lacrimal gland (0.09 mg/g) and the serum (0.0008 mg/ml).

CONCLUSIONS. (1) Peak ascorbate concentration was observed in the central corneal epithelium covering the pupillary area. This is compatible with the idea that the ascorbate may act as an UV filter shielding internal eye structures from radiation damage. (2) The ascorbate concentration in the corneal stroma and DM/endothelium was as high as in the aqueous humor, and it is suggested that the aqueous humor plays a key role in the distribution of ascorbate to the anterior eye wall.

	Introduction

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Since the first report of significant amounts of ascorbate in the aqueous humor,¹ high concentrations have been observed in many parts of the eye,² with peak values in the corneal epithelium.^{3 4} It has turned out, however, that the ascorbate content is higher in diurnal than in nocturnal mammals, both in the aqueous humor^{5 6 7} and in the corneal epithelium.⁸ Indeed, the amount of ascorbate in different ocular compartments seems adjusted to the suggested ambient radiation dose at each particular level, and from these observations it has been deduced that the ascorbate acts as a UV filter protecting the eye from radiation damage.

During recent years, some experimental support has been presented for this hypothesis. Reddy et al.,⁹ who compared the effect of UV radiation on DNA strand breaks in the lens epithelium of rat and guinea pig, concluded that high levels of ascorbate in the aqueous humor of diurnal animals may protect the lens against UV radiation under physiological conditions. Furthermore, it has been shown that the most efficient radiation in cell killing in cultured rabbit lens epithelium is the 297-nm wavelength,¹⁰ i.e., just those rays most likely to be absorbed by the aqueous ascorbate in vivo.⁵ However, Williams and Delamere¹¹ have pointed out that the lack of antioxidant protection due to low ascorbate in the nocturnal aqueous might be compensated for by the high activity of a peroxidase enzyme.

Many questions remain unanswered concerning the significance of ascorbate for the cornea and its subjacent structures. Of particular interest is how it gets to the cornea. A better understanding of the ascorbate distribution in the cornea and its surrounding tissues may provide some clues as to its functional aspects, so we decided to analyze the ascorbate concentration in different parts of the anterior eye. The bovine eye was chosen as the model because of its size, availability, and significant ascorbate content.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fresh bovine eyes were collected at the abattoir. For practical reasons, the time span between killing and enucleation could not be reduced below 15 minutes. With subsequent time-consuming preparation procedures added, the total time from death to frozen specimen was up to 2.5 hours in the most detailed experiments. Two different pilot studies were therefore performed to test the stability of the cornea and its surrounding tissues/fluids during the preparation period. When not otherwise specified, paired sampling was performed from the single eye throughout. Eyes deliberately stored before preparation were kept in closed plastic bags at room temperature to reduce the loss of water and to prevent reduced ion pump activity in the anterior eye. The bovine cornea is oval; the longest and shortest diameters were 29.1 ± 0.1 and 22.7 ± 0.1 mm (10 eyes), respectively.

Pilot Studies
(a) The amount of postmortem ascorbate loss was estimated by comparing the ascorbate content in en bloc specimens of cornea and conjunctiva/sclera at 15 minutes and 2.5 hours after death, respectively. Such crude specimens were collected readily within a few minutes of the desired time by opening the cornea at the external limbal demarcation with a razor blade. The specimen was cut free with a pair of scissors, and a 5-mm broad rim of the adjacent conjunctival/scleral tissue was isolated. The specimens were then immediately incubated en bloc (see below), there being no further time-consuming procedures.

(b) The aqueous humor, tear fluid, and limbal blood vessels are putative sources of ascorbate to the cornea, and their ascorbate concentrations were analyzed accordingly. The aqueous humor was tested 15 minutes, 1, 2, and 3 hours after death. Blood was collected from living animals and lacrimal gland from 30 to 60 minutes postmortem.

Subsequently, the ascorbate distribution in the various parts of the anterior eye wall was analyzed in four different experiments, each of which was run twice. Separately isolated, fresh tissues were used for each run.

Experiment I
The ascorbate contents in the central versus the peripheral corneal epithelium were compared. The central area was defined by a corneal trephine 9 mm in diameter placed center to center with the pupil, the peripheral area outside reaching to the limbus. The respective specimens were then rubbed off with a diamond knife or a microscope slide.

Experiment II
The central epithelium was further analyzed separately. In this experiment, the central epithelium was defined as the circular area covering the total pupil. A plastic template imitating the pupil’s form, which is oval in the bovine eye, was created. This template, formed by two parallel sides 6 mm apart closed by two half-circles with the top points 12 mm apart, was placed on the epithelium to indicate the area of study. The template was subsequently circumscribed with a diamond knife, and the marked area was rubbed off. Because paired sampling was impossible, the number of eyes was doubled. Two separate series were run, one with the template corresponding to the pupil and one with the template rotated 90° to it. In the following, these specimens are referred to as "horizontal" and "vertical," respectively.

Experiment III
Anterior eye wall tissue was analyzed. The total epithelium was rubbed off, and a limited aqueous sample aspirated before the cornea was cut out along the external limbus with a pair of scissors. Finally, the corneal stroma and the Descemet’s membrane (DM)/endothelium were collected. Subsequently, the adjacent 5 mm broad rim of conjunctival and scleral tissues was isolated separately.

Experiment IV
To analyze the cornea in more detail, central and peripheral specimens were prepared. The epithelium was again subdivided into a central and a peripheral area using the 9 mm trephine and collected as described above, before the stroma was arbitrarily subdivided into two separate sheets with a spatula from a limbal incision. A limited sample of aqueous was then aspirated, the cornea cut out along the external limbus, and a central corneal tissue button (9 mm across) punched out from behind. Each of these corneal specimens was separated into anterior, posterior stroma, and DM/endothelium. These were the most time-consuming experiments.

Aliquots of aqueous humor were mixed 50/50 with precooled metaphosphoric acid (50 g/l deionized water) before storage. For serum and tissue specimens, different volumes (0.5–1.5 ml) of metaphosphoric acid of 100 g/l water were used, depending on the size of the specimens. The epithelial, conjunctival, DM/endothelial, and lacrimal gland specimens were homogenized as far as possible with a Teflon pestle in the original vials (10 ml disposable glass containers without any coating) for 1 minute, before being frozen for storage in the same containers. Because the corneal stroma and sclera were assumed to resist this treatment and because homogenization with a metal pestle would induce ascorbate oxidation, specimens containing these elements were frozen slowly (-5–10°C) and thawed three times in the refrigerator to create a spongious tissue accessible to the incubation solution. Mincing such specimens by cryosectioning also was tested in separate experiments. However, a ceramic knife was too blunt, and a metal knife reduced the amount of ascorbate by some 50% (not shown). Specimens were stored at -35°C and analyzed within 2 weeks.

The high-performance liquid chromagraphy (HPLC) system consisted of a Shimadzu LC-10 system (Kyoto, Japan) using a SPD-M10AVP detector, a SIL-10A autoinjector, an LC-10AS pump, degasser, and LC-10 software. The chromatographic conditions were adapted from Rodriguez et al.¹² The column used was a Supelcosil LC-18, 250 x 4.6 mm, 5-µm particle size (Supelco, Bellafonte, PA), with a small guard column containing the same material. The mobile phase consisted of HPLC-grade water acidified to pH 2.2 with sulfuric acid (isocratic method). The flow rate was 1.0 ml/min, with the detection performed at 243 nm. The column was washed regularly in grade water.

The specimens were thawed to room temperature and centrifuged at 12,000g for 10 minutes, and the supernatants were injected (20 µl) in triplicate into the HPLC apparatus from injector glass vials without further dilution. The analyst was not informed about the type and order of the specimens. Standard curves were obtained after triplicate injection of 0.01, 0.1, 0.25, and 0.5 mg/ml of analytical grade ascorbic acid dissolved in 10% metaphosphoric acid. The correlation coefficient of the standard curves was always greater than 0.999, and linearity was shown to be between 0.005 and 0.5 mg/ml. Minimum detection limit of the standard was found to be 10 ng/ml. The variation coefficient of the injection repeatability was less than 1%, while interday variation coefficient of identical bovine corneal samples was 13%.

Statistics
Ascorbate concentrations are presented as means ± SD. Student’s t-test (two-sided) was used.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Pilot Studies
(a) The ascorbate contents of the en bloc cornea and conjunctiva/sclera specimens collected 15 minutes after death were 0.33 ± 0.03 and 0.107 ± 0.004 mg/g, respectively. The concentrations were reduced by 15.2% and 11.2%, respectively, in tissues analyzed 2.5 hours after death. Six eyes were used.

(b) An ascorbate content of roughly 0.2 mg/ml in the aqueous humor remained unchanged from 15 minutes to 1, 2, and 3 hours after death (6 eyes in each group). The lacrimal gland (30–60 minutes postmortem) and serum (from living animals) values were considerably lower [0.09 ± 0.03 mg/g (3 samples) and 0.0008 ± 0.001 mg/ml (3 samples), respectively].

Experiment I
The ascorbate concentration in the central (9 mm diameter trephine) versus peripheral epithelium was tested. Two separate runs, each with six paired specimens, were performed on different days and showed essentially the same results. The mean ascorbate concentrations were 1.58 ± 0.22 and 1.28 ± 0.09 mg/g in the central and peripheral corneal epithelium, respectively. This difference is statistically significant (P < 0.001). The central and peripheral epithelium taken together showed 1.31 ± 0.08 mg/g.

Experiment II
The ascorbate distribution in the central epithelial area was tested in more detail by comparing horizontal and vertical specimens. Two runs, each with 12 eyes, showed mean ascorbate concentrations of 1.22 ± 0.13 and 1.23 ± 0.17 mg/g, respectively, i.e., the values from the horizontal and vertical areas were not significantly different (P = 0.9).

Experiment III
The ascorbate distributions in the anterior eye segment were analyzed in two separate runs (6 + 6 eyes) showing essentially the same results: corneal epithelium 1.33 ± 0.15 mg/g, corneal stroma 0.19 ± 0.03 mg/g, DM/endothelium 0.22 ± 0.05 mg/g, aqueous humor 0.19 ± 0.04 mg/ml, conjunctiva 0.11 ± 0.02 mg/g, and sclera 0.15 ± 0.02 mg/g. As seen, the stroma, DM/endothelium, and aqueous humor were similar and significantly lower than the epithelium (P < 0.001). In addition, the scleral concentration was lower than the stromal, and the conjunctival lower than the scleral (P < 0.008). By weight, the epithelium, stroma, and DM/endothelium made up 14%, 75%, and 11% of the total cornea, respectively. For purposes of comparison with the pilot results, pooled values of the total cornea and conjunctiva/sclera were calculated at 0.34 ± 0.03 and 0.14 ± 0.02 mg/g, respectively.

Experiment IV
The cornea was separated into a central (9 mm diameter trephine) and a peripheral part and then analyzed with regard to ascorbate content in the epithelium, anterior stroma, posterior stroma, and DM/endothelium in each of these pieces. The aqueous humor also was tested. As in experiment II, the two separate runs (6 + 6 eyes) showed similar results, and taken together the following values were obtained: central cornea: epithelium (1.56 ± 0.18 mg/g), anterior (0.21 ± 0.04 mg/g) and posterior (0.18 ± 0.03 mg/g) stroma, and DM/endothelium (0.19 ± 0.04 mg/g); peripheral cornea: epithelium (1.39 ± 0.11 mg/g), anterior (0.24 ± 0.02 mg/g) and posterior (0.20 ± 0.02 mg/g) stroma, and DM/endothelium (0.23 ± 0.02 mg/g). The aqueous humor showed 0.21 ± 0.02 mg/ml. Again there was a significant difference in ascorbate concentration between the central and peripheral epithelium (P = 0.01), whereas central and peripheral concentrations were essentially the same in the stroma and in the DM/endothelium. The ascorbate contents of the epithelium, stroma, and DM/endothelium as separate layers were calculated at 1.41 ± 0.11, 0.22 ± 0.02, and 0.22 ± 0.02 mg/g, respectively, and taken together the pooled value of the total cornea was 0.36 ± 0.04 mg/g.

A short version of the ascorbate content in various anterior eye structures is highlighted in Figure 1 .

View larger version (14K): [in this window] [in a new window]   Figure 1. Ascorbate concentrations in various regions of the anterior bovine eye. The data are expressed as milligrams per gram tissue, except for the aqueous humor and serum, which are milligrams per milliliters.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The HPLC technique was performed according to well-established strategies¹² and has proved reliable in a previous work.⁸ In addition, all aqueous humor samples, i.e., specimens without extraction bias, came out with predictable results. Together, these factors confirm the reliability of the HPLC procedure.

The level of ascorbate is remarkably stable in the aqueous humor from stored eyes. A previous report¹³ revealed that the ascorbate decline in bicarbonate–Ringer medium with EDTA is negligible after 90 minutes even when the solution is well aerated, and we found that the ascorbate level in aqueous stored intraocularly was stable for 3 hours. The ascorbate loss from the tissues during preparation was estimated at 15% for the most time-consuming experiments (experiment IV). This is a moderate reduction, and it is noteworthy that the results from experiment IV match the comparable figures obtained by shorter preparation (experiments I and III). We therefore conclude that the postmortem ascorbate loss during this study was a minor problem that does not discredit our results.

As to the tissue extraction procedure of ascorbate, different approaches were used. The epithelium, a purely cellular tissue, was easily homogenized, and so there is unlikely to be any extraction bias for these specimens. The corneal stroma and sclera, on the other hand, contain for the most part collagen, which made homogenization with nonmetallic equipment impossible. This is why the freeze–thaw procedure was chosen. The fact that similar concentrations were obtained from stromal tissue extracted in one piece (experiment III) compared to stromal tissue subdivided before extraction (experiment IV) indicates that the results are reliable. The figures are useful at least as minimum values.

The corneal epithelium contained significantly more ascorbate than the stroma in both experiment III and experiment IV, and so our study supports previous reports showing an ascorbate concentration mechanism in the corneal epithelium.^{3 4 8} However, the main observation in the present study is that the level of ascorbate in the central region of the epithelium is 12% to 23% higher than in the periphery (experiments I and IV). This is not entirely unexpected, since the corneal epithelial distribution of all small solutes concentrated from the aqueous would be higher centrally because of diffusion toward the limbus and loss to the blood. However, two aspects do not fit in with this view: (1) The bovine cornea is oval, and diffusion toward the limbus should tend to create an oval form of the central high-loaded field. According to experiment II, the field with the highest concentration is circular. (2) Diffusion toward the limbus also should be traceable within the stroma. Experiment IV did not indicate any difference in ascorbate concentration in central versus peripheral stroma. However, regardless of which mechanism is responsible, the fact remains that the highest epithelial ascorbate concentration covers the pupil. One cannot exclude that the UV-absorbing ability of ascorbate may help minimize radiation damage in subjacent eye structures.

How does the ascorbate get to the cornea? Principally, three sources are possible: the aqueous humor, the tears, and the conjunctival blood vessels.

The high ascorbate content in the aqueous humor, its proximity to the cornea, and the suggested ascorbate pump in the corneal endothelium^{14 15} make this fluid a likely source for the cornea. It has to be kept in mind that the DM/endothelium in the present study represents mixed specimens in which the endothelium made up only about 1/10 by volume (as judged from bovine corneal sections, not shown), and so the endothelial concentration could have been higher than that reflected in the numbers.

The ascorbate content in bovine tears also is unknown, but the lacrimal gland revealed much higher values than the serum (0.09 mg/g and 0.0008 mg/ml, respectively). By analogy, in humans, considerably higher ascorbate values have been found in tears than in plasma, in basal tears even within the range of aqueous humor values.¹⁶ The possibility that the corneal epithelium may receive some ascorbate from the tears therefore cannot be excluded. However, the main concentration mechanism in the epithelium is not confined to the superficial cells, which are preparing for desquamation. The basal layer is a more reasonable entrance to the epithelium, and these cells are reliant on supplies from the stroma and aqueous humor.

	Acknowledgements

 
The authors thank Diana de Besche, Eli Gulliksen, and Astrid Østerud for technical assistance.

	Footnotes

 
Submitted for publication April 8, 1999; revised August 16, 1999; accepted September 3, 1999.

Commercial relationships policy: N.

Corresponding author: Amund Ringvold, Eye Department, National Hospital, University of Oslo, Pilestr. 32, N-0027 Oslo, Norway. a.d.ringvold{at}klinmed.uio.no

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Harris, LJ (1933) Chemical test for vitamin C, and the reducing substances present in tumour and other tissues Nature 132,27-28
2. Heath, H. (1962) The distribution and possible functions of ascorbic acid in the eye Exp Eye Res 1,362-367
3. Pirie, A. (1946) Ascorbic acid content of cornea Biochem J 40,96-100
4. Reim, M, Seidl, M, Brucker, K. (1978) Accumulation of ascorbic acid in the corneal epithelium Ophthalmic Res 10,135-139
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6. Reiss, GR, Werness, PG, Zollman, PE, Brubaker, RF (1986) Ascorbic acid levels in the aqueous humor of nocturnal and diurnal mammals Arch Ophthalmol 104,753-755[Abstract]
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