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From the Australian Cataract Research Foundation, Department of Chemistry, University of Wollongong, Wollongong, New South Wales, Australia.
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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METHODS. Human lenses were analyzed by high-performance liquid chromatography
(HPLC) after separate incubation with 3H-tryptophan
(3H-Trp), ß-benzoylacrylic acid,
D,L-
-amino-ß-benzoylpropionic acid, or
D,L-3-hydroxykynurenine
O-ß-D-glucoside. The effect of pH on the
model compound D,L--amino-ß-benzoylpropionic acid and
D,L-3-hydroxykynurenine
O-ß-D-glucoside was also investigated.
RESULTS. UV filters were not detected in fetal lenses, despite a 5-month
postnatal lens displaying measurable levels of UV filters. In adults no
radiolabel was incorporated into
4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid
O-ß-D-glucoside after 3H-Trp
incubations. ß-Benzoylacrylic acid was readily reduced in lenses.
D,L--Amino-ß-benzoylpropionic acid and
D,L-3-hydroxykynurenine
O-ß-D-glucoside slowly deaminated at
physiological pH and were converted to ß-benzoylpropionic acid and
4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid
O-ß-D-glucoside, respectively, after lens
incubations.
CONCLUSIONS. UV filter biosynthesis appears to be activated at or near birth. Compounds containing the kynurenine side chain slowly deaminate, and in the lens, the newly formed double bond is rapidly reduced. These findings suggest that 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-ß-D-glucoside is derived from L-3-hydroxykynurenine O-ß-D-glucoside through this deamination-reduction process. The slowness of the deamination presumably accounts for the absence of incorporation of radiolabel from 3H-Trp into 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-ß-D-glucoside.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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More recently, another UV-absorbing glucoside has been identified in
human lenses.7
8
This compound,
4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid
O-ß-D-glucoside (AHBG) is the second
most abundant UV filter compound of the human lens. It is similar to
3OHKG in UV absorbance profile8
and structure, the only
difference being the absence of an -amino group. The biosynthetic
pathway for the formation of AHBG, however, has not been determined.
Although no definite function has been established for these lens-specific compounds, their UV-filtering properties7 8 indicate a role in the protection of ocular tissue from long-wave UV radiation and/or as an aid to visual acuity by decreasing chromatic aberration. It has been proposed that the UV filter compounds may also be involved in the normal age-dependent coloration of the human lens and in crystallin modification during the development of senile nuclear cataract.9 For proper evaluation of the role of the UV filter compounds and their involvement in the cause of aging and cataract, a greater understanding of the endogenous UV protection pathway is needed. In this study, we investigated aspects of UV filter synthesis, in particular the biosynthetic origin of AHBG within the human lens.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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-Amino-ß-benzoylpropionic acid
was synthesized according to the method previously described by Cerani
and Tarzia.10
D,L-3-Hydroxykynurenine
O-ß-D-glucoside and
4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid
O-ß-D-glucoside were synthesized by
the method of Manthey et al.11 Normal lenses were obtained from donor eyes at The Sydney Eye Hospital Lions Eye Bank. Fetal lenses were obtained from abortions performed at Westmead Hospital, Sydney. After removal, lenses were immediately placed into 5-ml artificial aqueous humor (AAH) in 30-ml sterile plastic screw-capped vials. The vials were kept at 35°C until transported to the laboratory, usually within 24 hours. Lenses not incubated in AAH were preserved in liquid nitrogen. The AAH12 consisted of Eagle’s minimum essential medium (EMEM; Auto-Pow version; ICN Biomedicals, Sydney, Australia) supplemented with 50 µM L-Trp, 10 mM HEPES, 2.0 mM L-glutamine, and 200 mg/ml streptomycin sulfate/200 IU penicillin G (Boehringer–Mannheim, Sydney, Australia). The pH was adjusted to 7.4 with 1 M NaOH. Lenses were incubated individually in 35-mm sterile plastic petri dishes (Corning, NY).
Instrumental Conditions
The high-performance liquid chromatography (HPLC) system
consisted of a pump (model K35D; ICI, GBC, Sydney, Australia),
a sample injector (model 7125; Rheodyne, Cotati, CA) fitted with a
100-µl sample loop and a Knauer (Berlin-Zehlendorf, Germany) variable
wavelength UV detector. Chromatograms were recorded and peak areas
integrated on an integrator (Chromatopac CR6A; Shimadzu, Columbia, MD).
The UV filter compounds from the AAH and protein-free lens extracts and
the D,L-3-hydroxykynurenine
O-ß-D-glucoside pH studies were analyzed
on either a 250 x 4.6-mm C18 reversed-phase column (Spherisorb
S5ODS2; Activon, Sydney, Australia) or on a 250 x 4.6-mm
C18 reversed-phase column (Microsorb; Varian, Sunnyvale, CA), using the
same solvent conditions as previously described.8
A
radiochromatography detector (Flo-One Beta A-100; Radiomatic, Tampa,
FL) with a 500-µl flow cell (TR-LSC; Canberra Packard, Canberra,
Australia) was used to detect and integrate peaks of radioactivity
after tritiation experiments. A fluorescence detector (LC1250; ICI) was
used to detect peaks after the D,L-3-hydroxykynurenine
O-ß-D-glucoside incubation studies.
Analyses of the ß-benzoylacrylic acid and
D,L--amino-ß-benzoylpropionic acid experiments were
performed on the Varian Microsorb C18 reversed-phase column. A mobile
phase of 25 mM phosphate buffer (pH 6.8) in 10% acetonitrile was used,
with a flow rate of 0.6 ml/min for the ß-benzoylacrylic acid lens
incubation experiments and a flow rate of 1.0 ml/min for the
D,L--amino-ß-benzoylpropionic acid experiments. The
peaks were detected at 250 nm. Electrospray ionization mass
spectrometry was performed on a mass spectrometer with a hexapole
collision cell (Quattro; VG Biotech, Altrincham, UK). UV spectra were
recorded on a recording spectrophotometer (UV-265; Shimadzu).
Lens Experiments
Analysis of UV Filters.
Lenses were removed from the AAH or thawed if preserved in liquid
nitrogen, and the protein-free lens extracts were obtained and analyzed
by high-performance liquid chromatography (HPLC), following the
conditions previously described.8
Tritiated Trp Experiments.
Lenses were removed from the AAH in which they had been transported and
stabilized separately overnight in fresh AAH (4.5 ml) at 35°C. The
AAH was changed again at the start of the experiment, and
[5-3H]-L-Trp was added to each
medium to obtain a final activity of 2.0 µCi/ml. After 24 hours,
lenses were removed from the culture and rinsed twice for 10 seconds in
unlabeled AAH to remove any label adhering to the capsule. The
protein-free lens extracts were obtained for each lens and analyzed by
HPLC.8
Lenses not extracted immediately were frozen at
-20°C until needed.
Model Compounds.
The lenses from a 64-year-old donor were incubated separately at 35°C
in AAH (10 ml) containing 1 mM ß-benzoylacrylic acid and 0.05%
ethanol (to aid dissolution of ß-benzoylacrylic acid). One lens was
incubated for 24 hours and the other for 48 hours. The lenses and AAH
were separated, and both the AAH and protein-free extracts of each lens
were examined by HPLC. The peaks at 24 minutes (retention time of
ß-benzoylpropionic acid) for both protein-free lens extracts were
collected, acidified with dilute HCl, extracted with ether, and
evaporated to dryness under argon. As a control, a solution of the AAH
(10 ml) containing 1 mM ß-benzoylacrylic acid and 0.05% ethanol was
incubated at 35°C for 48 hours and analyzed by HPLC.
A 27-year-old lens was incubated at 35°C in a 1-mM solution of
D,L--amino-ß-benzoylpropionic acid in AAH (5 ml) for
48 hours, and the AAH and protein-free lens extract were examined by
HPLC. The peak at 17 minutes (retention time of ß-benzoylpropionic
acid) in the lens extract and peaks at 17 and 20 minutes (retention
time of ß-benzoylacrylic acid) in the AAH were collected and
extracted as described. As a control, a 1-mM solution of
D,L--amino-ß-benzoylpropionic acid in AAH (5 ml) was
incubated for 48 hours at 37°C and analyzed by HPLC.
D,L-3-Hydroxykynurenine
O-ß-D-Glucoside Lens Incubation.
A 26-year-old lens was incubated at 35°C in AAH (6 ml), and aliquots
(100 µl) were taken every 2 hours and snap frozen in liquid nitrogen.
After 8 hours, D,L-3-hydroxykynurenine
O-ß-D-glucoside was added to the AAH
(final concentration, 5 mM), and the incubation was continued for
another 40 hours with aliquots collected as described approximately
every 2 to 4 hours. The lens was separated from the AAH and the
protein-free extract obtained. The protein-free extract and AAH
aliquots were analyzed by HPLC for the presence of
D,L-3-hydroxykynurenine
O-ß-D-glucoside and
4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid
O-ß-D-glucoside. As a control, the
contralateral lens was incubated at 35°C in AAH (6 ml) for 48 hours
and the protein-free lens extract analyzed by HPLC.
pH Stability of D,L--Amino-ß-benzoylpropionic Acid
D,L--Amino-ß-benzoylpropionic acid (2 mg/ml) was
incubated at 37°C in 25 mM phosphate buffer (pH 6, 7, and 8) and 25
mM carbonate buffer (pH 9 and 10). Duplicate aliquots (100 µl) were
taken at various intervals over a period of 25 hours. The aliquots were
diluted (1:1) with 25 mM phosphate buffer (pH 6.8) and analyzed by
HPLC. For confirmation of structure, the peak at approximately 20
minutes (retention time of ß-benzoylacrylic acid) was collected from
the pH 7 experiment, extracted, and dried as described.
pH Stability of D,L-3-Hydroxykynurenine
O-ß-D-Glucoside
D,L-3-Hydroxykynurenine
O-ß-D-glucoside (0.22 mg/ml) was
incubated at 37°C in 25 mM carbonate buffer (pH 9) for 24 hours.
Duplicate aliquots (100 µl) were taken at t = 0
minutes, 2 hours, and 24 hours, diluted (1:1) with 25 mM phosphate
buffer (pH 6.8) and analyzed by HPLC. The peak at 47 minutes’
retention was collected from the 24-hour aliquot, freeze dried,
desalted with a C18 Sep-Pak (Waters, Milford, MA), and
lyophilized. D,L-3-Hydroxykynurenine
O-ß-D-glucoside (1 mg/ml) was also
incubated at 37°C with 25 mM phosphate buffer (pH 7) for 7 days.
Duplicate aliquots (100 µl) were taken once a day and analyzed by
HPLC as described.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Radiolabeled Trp incubation experiments performed over 24 hours on individual fetal lenses also showed no incorporation of radiolabel into UV filters. Because there was concern that the apparent absence of incorporation may have been due to the small size of fetal lenses, three fetal lenses aged 12, 13, and 17 weeks were incubated together in AAH containing tritiated Trp (3H-Trp). The incubation was maintained for 72 hours to maximize incorporation of label. Analysis of the protein-free extract of the lenses showed that, although radiolabeled Trp entered the lenses, it was not metabolized into the UV filter compounds in any detectable level. Integration of the 3H-Trp peak produced a value of 16,000 disintegrations per minute, a quantity of radiolabel more than adequate to detect metabolism to 3OHKG at typical incorporation levels found in adult lenses.
Incorporation of Radiolabel from 3H-Trp into UV
Filters
Eleven adult human lenses were incubated separately with
3H-Trp for 24 hours at 35°C, and the UV filters
present in the protein-free extracts were analyzed by HPLC. A typical
HPLC chromatogram is shown in Figures 1A
and 1B
for a 21-year-old lens. Both online radiochemical detection and
UV absorption were used and the profiles plotted simultaneously. At the
commencement of the analysis, the detection wavelength was set at 365
nm. The sharp line at 30 minutes on the UV trace of Figure 1A
(top)
indicates the alteration of the detection wavelength to 278 nm, to
detect Trp, which eluted between 60 and 70 minutes. Using elution with
acetate buffer, three 365-nm absorbing peaks were detected. Each of
these UV peaks also corresponded to peaks of radioactivity (Fig. 1A
,
bottom). The small peaks running at 13 minutes and 27 minutes have been
previously identified as 3OHKyn and Kyn, respectively, whereas the
large peak at 15 minutes has been identified as 3OHKG.5
Electrospray mass spectroscopy was used to confirm the identities of
these peaks.
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After elution of Trp (Fig. 1A) , a return in the detection wavelength to 365 nm and replacement of the acetate buffer mobile phase with 20% methanol resulted in the detection of two additional long-wave UV-absorbing peaks, with retention times of 21 and 25 minutes (Fig. 1B) . The earlier eluting compound has previously been identified as AHBG,8 whereas the structure of the latter peak is not yet known. No radiolabel coincided with the AHBG peak (or the unknown peak) in the analysis shown in Figure 1B , nor was any label observed in the other 10 lens experiments performed over 24 hours, or after longer periods of incubation with 3H-Trp (up to 65 hours) in two further lens experiments. A peak with radiolabel was observed at 19 minutes (Fig. 1B) , which did not correspond to the elution position of AHBG. This peak was not consistently observed in the other lens experiments. Incubation of lenses with tritiated 3OHKyn, as described previously,6 also demonstrated incorporation of label into 3OHKG, but not into AHBG.
The integrated UV peak areas of 3OHKG and AHBG from the lenses used in the radiolabeling studies and from 18 other lenses were used to estimate quantities of these two UV filter compounds. The lenses varied in age ranging from 21 to 84 years. Lens pairs showed little variation in 3OHKG and AHBG levels and in any given lens, the quantity of 3OHKG was always greater than that of AHBG (Fig. 2) . Four lenses with very low levels of 3OHKG (one each 71- and 83-year-old lenses and a pair of 84-year-old lenses), also had the lowest levels of AHBG. One 64-year-old lens, with an exceptionally high level of 3OHKG, also possessed the highest quantity of AHBG. No correlation was observed between the levels of AHBG and age.
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Because the structures of AHBG and 3OHKG are very similar and there was
a correlation between the levels of these UV filters in the human lens,
it seemed feasible that AHBG could in fact be derived from 3OHKG
despite the absence of 3H-Trp incorporation. A
possible pathway for this involves the elimination of ammonia
(deamination) from 3OHKG to produce the corresponding
,ß-ketoalkene, followed by reduction, possibly enzymatically, of
the ,ß-ketoalkene to AHBG (Fig. 3)
. The feasibility of this mechanism was therefore investigated using
the model compounds D,L--amino-ß-benzoylpropionic
acid, ß-benzoylacrylic acid, and ß-benzoylpropionic acid (Fig. 4)
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Model Studies for Deamination
D,L--Amino-ß-benzoylpropionic acid was incubated
at pH 6, 7, 8, 9, and 10 at 37°C and monitored by HPLC over a period
of 25 hours for the formation of ß-benzoylacrylic acid. The identity
of ß-benzoylacrylic acid was confirmed by mass spectrometry of the
HPLC peak. The deamination was optimal at pH 9, with approximately 74%
conversion of the amino acid to ß-benzoylacrylic acid, whereas a 12%
conversion was observed at pH 7 and 33% conversion at pH 8 (Fig. 6)
.
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-amino-ß-benzoylpropionic acid (1 mM) in AAH for
48 hours at 35°C. HPLC analysis of the AAH displayed peaks at
approximately 10, 17, and 20 minutes, consistent with the retention
times of D,L--amino-ß-benzoylpropionic acid,
ß-benzoylpropionic acid, and ß-benzoylacrylic acid, respectively
(Fig. 7A
). Analysis of the protein-free lens extract displayed peaks consistent
with D,L--amino-ß-benzoylpropionic acid and the
reduced compound ß-benzoylpropionic acid (Fig. 7B)
. No
ß-benzoylacrylic acid was detected. Electrospray mass spectrometry of
the collected peaks confirmed the identity of ß-benzoylpropionic acid
in the AAH and protein-free lens extract and the identity of
ß-benzoylacrylic acid in the lens media. A control in which a 1-mM
solution of D,L--amino-ß-benzoylpropionic acid in AAH
was incubated at 35°C for 48 hours also displayed a peak for
ß-benzoylacrylic acid; however, no peak was observed for the reduced
compound ß-benzoylpropionic acid (Fig. 7C)
. Quantification studies
showed that in both the lens AAH and the control AAH approximately 35%
of D,L--amino-ß-benzoylpropionic acid had undergone
deamination and that in the lens AAH approximately 85% of
ß-benzoylacrylic acid was reduced after 48 hours’ incubation,
whereas the amount of reduction within the lens itself was virtually
100%.
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-amino-ß-benzoylpropionic acid,
deamination of 3OHKG was significantly slower.
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,ß-ketoalkenes, the lenticular
conversion of 3OHKG to AHBG was examined directly. A 26-year-old lens
was incubated in AAH at 35°C. After 8 hours the AAH was spiked with
D,L-3OHKG to achieve a final concentration of 5 mM, and the
incubation was continued for another 40 hours. Aliquots from the AAH
were taken at regular time intervals and examined by HPLC. Previous
research has shown that UV filters accumulate in the AAH when lenses
are incubated.5
Analysis of human vitreous indicates that
this efflux also occurs in vivo.5
During the first 8
hours, 3OHKG was observed at very low levels in the AAH, whereas none
of the other UV filters, including AHBG or the deaminated product, was
detected. AHBG was observed 2 hours after the spiking, with an
integrated peak area approximately 7% that of 3OHKG. A corresponding
decrease in 3OHKG was also observed. The level of AHBG appeared to
increase slowly over the remainder of the experiment, as did the level
of 3OHKG (Fig. 9) . As a control, the contralateral lens was incubated in AAH in the
absence of added 3OHKG at 35°C for 48 hours. Analysis of the
protein-free lens extract of both the spiked lens and the contralateral
lens showed that the spiked lens had significantly higher levels of
3OHKG (approximately 1.6 times higher), whereas AHBG levels were
comparable.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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-interferon, as
described for other cells.13
The second most abundant UV filter compound, AHBG, differs from 3OHKG
only in the absence of an -amino group. This similarity suggests
that AHBG may be a further metabolic product of 3OHKG, yet no
incorporation of radiolabel into AHBG using either radiolabeled Trp or
3OHKyn6
could be shown. In these experiments significant
amounts of radiolabel were incorporated into 3OHKG, thus providing
evidence that AHBG was not formed artifactually during isolation or
extraction. AHBG is almost certainly the unidentified glucoside
isolated by Van Heyningen,4
who also found no
incorporation of radiolabel from Trp into the unknown glucoside,
although label was detected in Kyn and 3OHKG.
Indole-3-propionic acid was not detected in a lens extract, whereas the
precursors of 3OHKG (i.e., Trp, Kyn, and 3OHKyn) were all detected.
This suggests that a parallel pathway to the kynurenine pathway, using
a precursor without the -amino group, was not operating. There did,
however, appear to be a correlation between the levels of 3OHKG and
AHBG in the lens. All this evidence led us to speculate that AHBG was
derived from 3OHKG.
Deamination of 3OHKG to produce an ,ß-ketoalkene, followed
by reduction, was proposed as a possible route to AHBG. Deaminations
from systems similar to that of 3OHKG have been reported to occur in a
facile manner at high pH,14
whereas enzymatic elimination
of ammonia from phenylalanine to produce trans-cinnamic acid
is known to occur in higher plants.15
No previous studies
have examined the reduction of ,ß-ketoalkenes by lenses. Studies
on bovine ocular tissues, however, have confirmed the presence of
reductases capable of reducing the ,ß-ketoalkene
trans-phenyl-1-propenyl ketone in the presence of reduced
nicotinamide adenine dinucleotide phosphate (NADPH) or reduced
nicotinamide adenine dinucleotide (NADH).16
Alkene
reductase enzymes of this type are also present in humans. For example,
the transformation of 7-dehydrocholesterol to cholesterol is catalyzed
by 7-dehydrocholesterol-delta7-reductase.17
The model compound ß-benzoylacrylic acid was readily reduced to ß-benzoylpropionic acid when incubated with lenses in AAH. Because reduction was not observed in the absence of a lens, this reduction was clearly a result of lens activity and not due to any species within the AAH. Given the similarity of ß-benzoylacrylic acid with the proposed elimination product of 3OHKG, it can be concluded that the reduction of this alkene to form AHBG within the lens is feasible. The details of the reduction were not examined further.
Because only small amounts of 3OHKG are available, we initially
investigated deamination of the model compound
D,L--amino-ß-benzoylpropionic acid, which has the same
amino acid side chain as 3OHKG. The model compound was found to
deaminate over a range of pHs, albeit slowly at physiological pH and
maximally at pH 9. Lens incubation studies with the amino acid showed
that the rate of deamination at physiological pH in the presence and
absence of the lens was similar, confirming the nonenzymatic nature of
the deamination. Furthermore, extension of these studies to
D,L-3OHKG showed that the proposed deamination can occur at
physiological pH, although this process was slower than that for
D,L--amino-ß-benzoylpropionic acid. The rate of
reduction was found to be significantly faster than the rate of
deamination, suggesting that the elimination of ammonia from 3OHKG may
be the rate-limiting step in the formation of AHBG.
D,L-3OHKG was found to yield AHBG after incubation of a lens with D,L-3OHKG; however, the amount of conversion was low. This presumably reflects a low rate of deamination as well as other processes that may be occurring in the lens, such as conjugation of the double bond with glutathione.18 It may well be that AHBG formation occurs largely in the lens nucleus where glutathione is low and the effective residence time of 3OHKG is longer, allowing deamination to take place. If so, these factors make lens experiments of AHBG formation difficult to undertake.
The 3OHKG lens incubation study and the model studies provide strong
support for the notion that AHBG is derived from the major UV filter
compound, 3OHKG, through a slow nonenzymatic elimination of ammonia to
produce the corresponding ,ß-ketoalkene, followed by reduction of
the side-chain double bond. The slowness of the deamination presumably
accounts for the absence of radiolabeling of AHBG after the lens
incubations with 3H-Trp. These studies also show
the intrinsic instability of compounds with the kynurenine side chain,
which may have important implications for the human lens.
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Acknowledgements |
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Footnotes |
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Submitted for publication May 14, 1999; accepted July 12, 1999.
Commercial relationships policy: N.
Corresponding author: Joanne F. Jamie, Australian Cataract Research Foundation, Department of Chemistry, University of Wollongong, Wollongong, N.S.W., Australia 2522. E-mail: joanne_jamie{at}uow.edu.au
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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-oxobenzenebutanoic acid J Org Chem. 64,3930-3933
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