(Investigative Ophthalmology and Visual Science. 2000;41:2067-2069.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
UV Absorption by Uric Acid in Diurnal Bird Aqueous Humor
Amund Ringvold1,
Erlend Anderssen2 and
Inge Kjønniksen2
1 From the Eye Department and
2 The National Hospital Pharmacy, National Hospital, Oslo, Norway.
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Abstract
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PURPOSE. To analyze the components responsible for the UV absorbance in diurnal
bird aqueous humor.
METHODS. The absorbance studies were carried out using a Hitachi
spectrophotometer (U 2000). Uric acid was determined by
high-performance liquid chromatography (LC-10 system; Shimadzu, Kyoto,
Japan). Chicken and turkey eyes were examined.
RESULTS. The UV absorbance in chicken aqueous was largely accounted for by the
presence of protein, tryptophan, tyrosine, ascorbic acid, and uric
acid. Ascorbic acid was low (23 µmol/l). Uric acid was, on the other
hand, remarkably high (151 µmol/l) compared with that in mammals
(cattle, 16 µmol/l). Principally the same results were obtained in
chicken and turkey.
CONCLUSIONS. Uric acid is a significant UV-absorbing substance in the aqueous humor
of diurnal birds with its peak absorbance at 292 nm. The hypothesis
that the aqueous humor acts as a UV filter seems to be valid also for
the avian eye. However, in these eyes uric acid fulfills the role that
ascorbic acid does in mammals.
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Introduction
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The threshold energy for damage of the corneal epithelium by
UV-radiation is different in mammals and birds.1
2
Antarctic birds, penguins, and skua, which are normally exposed to high
UV-radiation for prolonged periods of time, show higher resistance to
the radiation than chickens and ducks. On the other hand, there is
apparently no systematic correlation between corneal transmission and
expected environmental exposure of the respective
species.2
Different from mammals, any details concerning
mechanisms minimizing UV damage in the avian eye have so far not been
presented. It is relevant for our purpose that in an early study on
reducing substances in various body fluids, the uric acid concentration
in the aqueous humor in fowl was reported to be well below that of the
serum (
157 µmol/l).3
However, this observation was
not ascribed any functional aspect.
In general, there are a number of compounds that absorb UV-radiation
and may induce photochemical damage to the tissue. The most common are
chromophores, nucleic acids, and proteins including various types of
enzymes. Damage is found at the molecular level and is reduced and
modified by the presence of quenchers. The eye is under permanent
photo-oxidative stress in diurnal species regardless of whether mammal
or bird. Because of the unique anatomic structure in this organ, its
UV-protective mechanisms may in part be different from those indicated
above. This field has been excellently reviewed by
Dillon.4
In mammals, there is a significantly higher concentration of ascorbate
in the aqueous humor of diurnal species than in that of nocturnal
species,5
6
7
and the differences are particularly
pronounced in the case of the corneal epithelium.8
Because
of the high molar absorptivity of ascorbate for UV-radiation, these
observations have been taken as an indication that this substance acts
as a UV filter for the eye. If this is correct, a similar mechanism may
be assumed in diurnal birds. It has been shown, however, that the
ascorbate content in the avian aqueous is low,9
which
implies that either the hypothesis presented for mammals is wrong or
that the material responsible for the aqueous UV absorption is
different between mammals and birds. To evaluate these questions we
decided to examine the aqueous humor from diurnal birds
spectrophotometrically and to analyze the components responsible for
the absorbance.
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Methods
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Chicken.
Chicken eyes were obtained at the local abattoir. The slaughter process
was by electric shock, debleeding, and decapitation before aspiration
of the aqueous humor from the anterior chamber with a 30-gauge cannula.
Four collections, each of roughly 5 ml volume, were transported on ice
to the laboratory and either analyzed immediately or stored at -35°C
for up to 2 weeks. In addition to spectrophotometry, the contents of
protein, tyrosine, tryptophan, and ascorbate were examined.
Another four specimens, each collected from 6 eyes, were analyzed fresh
for UV absorbance and uric acid content. To check for any possible
change in absorbance induced by the slaughter process at the abattoir,
these animals were killed with a moderate blow to the head before
decapitation. Spectrophotometry showed principally the same results in
the two groups.
Turkey.
Two different collections, each of approximately 5 ml volume, were
obtained at the abattoir and processed as above.
Cattle.
Individual aqueous samples were collected from bovine eyes for uric
acid determination. In addition, blood samples were collected from all
three species. All procedures conformed to the ARVO Statement for the
Use of Animals in Ophthalmic and Vision Research.
Total protein was determined using a micro protein assay (procedure No.
610; Sigma), and ascorbic and uric acids by the high-performance liquid
chromatography (HPLC) technique.8
The uric acid peak,
which was recorded at 299 nm, was identified by spiking, alternatively
by uricase-deletion, and spectral characteristics. Injection of
allantoin did not generate any peak at this wavelength. The amino acid
concentrations were obtained using an automatic amino acid analyzer
(Eppendorf/Biotronik LC 3000). In mock aqueous, total protein was added
as chicken albumin. In addition to ascorbic acid (Riedel-de
Haën AG), Sigma products were used. The absorbance studies were
carried out using a Hitachi spectrophotometer (U 2000). Quartz cuvettes
with 2 mm light pathway and phosphate buffer (0.1 M, pH 7.3) were used
throughout.
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Results
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The absorption of UV-radiation in the aqueous humor of diurnal
mammals is due to its content of protein, tyrosine, tryptophan, and
ascorbate. To find out whether this was also the case in birds, four
aqueous collections from chicken were analyzed with respect to these
components, and the mean values obtained (Table 1)
were used to mix a mock aqueous humor on a phosphate buffer basis. The
results can be seen in Figure 1A
, which shows the absorbance of one native chicken sample compared with
its matching mock aqueous sample. The marked difference between the two
curves (Fig. 1B)
indicates that in addition to protein, amino acids,
and ascorbate, there is some other material contributing significantly
to the total absorbance in chicken aqueous. Based on the tentative
conclusion that the difference might be accounted for by one single
component, various options were evaluated using a spectrum
collection.11
The best fit for the UV-B range (320–290
nm) turned out to be uric acid (as illustrated in Fig. 1B
), and this
finding singled out uric acid as a potential UV-absorbing substance in
the chicken eye.
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Figure 1. (A) Absorbance of native (upper curve:
 max, 282 nm) and mock chicken aqueous humor
(max, 269 nm; i.e., phosphate buffer
containing protein, tyrosine, tryptophan, and ascorbic acid according
to Table 1
). Note the marked difference in UV absorbance between the
curves. (B) The upper curve (max,
288 nm) i.e., the mock aqueous absorbance subtracted from the native
one in Figure 1
, imitates the absorbance of uric acid shown in
the lower curve (max, 290 nm; pH 7.3) at an
arbitrarily chosen concentration of 100 µmol/l. (C) UV
absorbance of one native chicken aqueous collection (upper
curve). Mock aqueous containing protein, tyrosine, tryptophan, and
ascorbate according to mean chicken values in Table 1
(lower
curve) was supplemented with 195 µmol/l uric acid (middle
curve), which was the content of this particular native sample.
The curves showed max at 288, 269, and 288 nm,
respectively. (D) UV absorbance of one native turkey aqueous
collection (upper curve). Mock aqueous containing protein,
tyrosine, tryptophan, and ascorbate according to mean turkey values in
Table 1
(lower curve) was supplemented with 167 µmol/l
uric acid (middle curve), which was the content of this
particular native sample. The curves showed
max at 281, 271, and 287 nm, respectively.
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To test this hypothesis, another four aqueous sample collections (each
pooled from 6 eyes) were analyzed spectrophotometrically and with
respect to their uric acid contents by HPLC (Table 1) . The
chromatograms revealed a marked peak with spectrum and retention time
in accordance with the uric acid standard. Subsequently, four identical
mock aqueous samples were created (see Table 1
), and each of them was
supplemented with one of the four observed uric acid concentrations.
The absorbance of these upgraded mock samples was then compared with
the equivalent native ones. All four setups showed principally the same
results (exemplified in Fig. 1C
). As can be seen in Figures 1A
1B
1C
, uric acid is to a large extent responsible for the photometric peak
in the UV-B range of native chicken aqueous.
To test whether this conclusion also applies in another species, a
similar setup was run for turkey. The results, exemplified in Figure 1D
, confirm the previous observation.
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Discussion
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The capacity for synthesis of ascorbic acid is apparently lost in
many birds,12
(i.e., any function in birds based on this
substance would be dependent on external supplies). Uric acid, on the
other hand, is the major end product of nitrogen metabolism in these
animals. It is present in ample amounts and readily available from
blood. In addition, uric acid shows the higher molar absorptivity of
the two with max at a longer wavelength (uric
acid in 0.067 M glycine buffer, pH 9.4; 12,500 at 292 nm versus
ascorbic acid in water or neutral solutions; 7,000 at 265
nm),13
14
indicating that it is a powerful UV-B absorber.
This fact should be considered when comparing uric and ascorbic acid
concentrations in the aqueous humor of birds and cattle (Table 1)
.
The observation of high ascorbic and low uric acid concentrations in
the aqueous of mammals, and vice versa in diurnal birds, brings us to
the conclusion that the aqueous humor acts as a UV-B filter in diurnal
birds just as it does in diurnal mammals but that the basis for the
absorption is different: In the avian eye, uric acid fulfills the role
that ascorbic acid does in mammals. Any of these antioxidants is likely
to deteriorate at physiologically relevant spectral irradiation of the
aqueous.15
It is noteworthy that evolution has taken
different tracks in the environmental adaptation of birds and mammals
in meeting their common need for a stable radiation barrier in the
anterior eye. In many diurnal birds the spectral range of vision is
extended into the UV by specific retinal receptors.16
However, with a spectral sensitivity maximum of 360 to 370 nm, this
vision is uninfluenced by the present observations.
It has previously been stated that in humans uric acid is merely a
metabolic waste elaborated only to be eliminated, and quite devoid of
any perceptible physiological effects.17
However, today we
know that the substance has antioxidant properties18
and
is being ascribed a number of important functions, including modulating
redox reactions and oxidative events in the blood, arterial wall, and
lung.19
20
Uric acid also accounts for the high reducing
agent on the extracellular surface of the corneal epithelium in
rabbits.21
It is, therefore, not surprising that it may
also have an important function in the avian eye.
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Acknowledgements
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The authors thank Vibeke Rootwelt Andersen and Thomas Kebely,
Sentralslakteriet Vestfold/Tønsberg, for assistance during collection
of the specimens; Egil Jellum, Institute of Clinical Biochemistry,
National Hospital, for performing the amino acid analyses; and Diana de
Besche, Eli Gulliksen, and Astrid Østerud for technical assistance.
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Footnotes
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Submitted for publication November 15, 1999; revised January 20, 2000; accepted February 15, 2000.
Commercial relationships policy: N.
Corresponding author: Amund Ringvold, Eye Department, National Hospital, University of Oslo, N-0027 Oslo, Norway. a.d.ringvold{at}klinmed.uio.no
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Abstract
Introduction
