|Year : 1998 | Volume
| Issue : 4 | Page : 233-237
Flavin nucleotides in human lens: Regional distribution in Brunescent cataracts
K Seetharam Bhat, Sujatha Nayak
National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India
K Seetharam Bhat
Department of Biochemistry, National Institute of Nutrition, Jamai Osmania PO, Hyderabad - 500 007
Source of Support: None, Conflict of Interest: None
The biochemical mechanism(s) underlying brunescent cataracts remain unclear. Oxidative stress due to reactive oxygen species may have a role in the pigmentation process in eye lens. We have analysed human cataractous lenses for flavins by high-performance liquid chromatography (HPLC), since flavins are light sensitive and act as endogenous sensitizers generating reactive oxygen species in the eye. The most significant observation in this study is that higher levels of flavin nucleotides occur in brown lens compared to yellow lens. The concentration of flavin nucleotides (flavin monouncleotide, FMN + flavin adenine dinucleotide, FAD) was highest in the nuclear region of the lens followed by the cortical and capsule-epithelial regions. However, the ratio of FAD/FMN was lowest in the nuclear region of the lens followed by other regions. On the other hand, riboflavin was not detected in any of the lens (cataractous) regions. These results suggest that the observed increase in flavin nucleotides in the ocular tissue could contribute towards deepening of lens pigmentation.
Keywords: Flavin nucleotides, brunescent cataract, human lens, lens pigmentation, lens regions
|How to cite this article:|
Bhat K S, Nayak S. Flavin nucleotides in human lens: Regional distribution in Brunescent cataracts. Indian J Ophthalmol 1998;46:233-7
|How to cite this URL:|
Bhat K S, Nayak S. Flavin nucleotides in human lens: Regional distribution in Brunescent cataracts. Indian J Ophthalmol [serial online] 1998 [cited 2019 Jun 18];46:233-7. Available from: http://www.ijo.in/text.asp?1998/46/4/233/24171
Human cataractous lenses vary in colour from pale yellow to brown. The pigmentation of the human lens appears to be due to fluorophores. The events leading to the formation of brunescent cataract which is highly prevalent in India, are still not known. Oxidative insult to lens due to reactive oxygen species (ROS) may play a role in the deepening of the nuclear pigmentation. Our earlier study suggested that during lenticular browning there are changes associated with the glutathione redox cycle defense system against oxidative stress in the ocular tissue. Even though riboflavin (RF) deficiency has been associated with experimental and human cataract, the exact role of flavin in browning of human lens is not known.
While RF through its coenzymatic function helps to generate a reducing agent like reduced glutathione (GSH), flavins per se are light sensitive and rapidly photo-oxidise to generate ROS. Photo-oxidation of lens proteins has been associated with crystallin (structural proteins) modification which is also seen during ageing and cataractogenesis., Endogenous sensitisers like RF (loosely bound to protein) and oxidatively degraded products of tryptophan (Trp) may facilitate such a photo-oxidation process.,
RF, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) have been separated and quantified in biological fluids and ocular tissues by high-performance liquid chromatography (HPLC). Although flavins have been measured in animal eye lens, little or no information is available on their levels in human lens. The present study was therefore undertaken to measure the concentration of flavin nucleotides in different regions of human cataractous lenses and to determine whether pigmentation of lenses is correlated in any way with flavin nucleotide levels.
| Materials and Methods|| |
FAD, FMN and RF were obtained from Sigma Chemical Company (St. Louis, Missouri, USA); the other reagents were of analytical grade.
Seventeen intact lenses extracted intracapsularly by cryoprobe from human cataract patients without any systemic pathology were collected in screw-capped 5 ml glass vials kept on ice at a local hospital and transported to the laboratory within 2-3 hours. The lenses were classified as yellow (very pale yellow, pale yellow and yellow) and brown ( yellowish brown/dark yellow, brownish yellow/ very dark yellow and brown) through comparison with standard colour photographs of lenses on a white background, as suggested by the American Co-operative Cataract Research Group (CCRG). The lenses were dissected into capsule-epithelium (entire capsule with single-layered epithelium), cortex, and nucleus as described elsewhere. A 5% (W/V) homogenate in water, of each individual lens region was prepared using the potter-Elvehjam homogenizer. An equal volume of acetonitrile was added to the homogenate to precipitate proteins, and the contents were mixed and centrifuged at 10,000 x g (Hitachi, Himac automatic high-speed refrigerated centrifuge, SCR 20 BA) for 15 min at 4° C. Chloroform was added to the clear supernatant (1:6 ratio) to extract fats, and the contents mixed and centrifuged at 1800 x g (clinical centrifuge, IEC) for 15 min. at 22° C. Aqueous phase containing flavin nucleotides was filtered using a 0.45 μm Millipore filter, lyophilized (Virtis Freeze mobile SEL, USA) and reconstituted with water as flavins are present in very low concentration (ng) in the lens regions. All the above procedures were done under dim light to prevent the photo-degradation of flavins in the samples. 20 μ1 of the sample was used for flavin analysis.
The flavins were separated and quantified by HPLC system using a fluorometric detector. The HPLC column was reverse-phase octadecylsilane (C18) column, 250 x 4.6 mm I.D. (Shodex Shoko Co. Ltd., Tokyo, Japan) (particle size 10 μm). The HPLC system consisted of a single piston pump (Shimadzu LC-6A liquid chromatograph, Kyoto, Japan) with a Beckman (Model 210) sample injector (20 μl loop, Fullerton, California, USA) linked to a Shimadzu RF-530 fluorescence detector. Detector wavelengths were set at 450 nm for excitation and 530 nm for emission. The response to the detector was recorded with an integrator (Shimadzu C-R6A chromatopac). The flow rate of the mobile phase (methanol:water, 55:45 and methanol:5mM ammonium acetate buffer PH 6.0, 30:70) was set at 1 ml/min. Protein analysis was done by Lowry's method using bovine serum albumin as the standard.
The results were analysed for statistical significance using student's t-test and modified t-test.
| Results|| |
When RF, FMN and FAD either individually or in combination (FMN+FAD), were processed with acetonitrile and chloroform, the extraction recoveries were 98% (RF) and 100% (FMN, FAD and FMN-FAD). The advantage of extraction of RF with acetonitrile and chloroform is that the sample is undiluted before injection onto the column. As reported by Gatautis and Naito, with methanol:water, 55:45, FMN and FAD were eluted together (FMN+FAD) within the void volume when loaded individually or as mixture (FMN+FAD and RF+FMN+FAD) [Figure - 1]. Flavins were identified based on the retention times of standard RF and FMN+FAD [Figure - 1]A and were calculated based on their peak area. FMN and FAD were eluted together, while RF was well separated [Figure - 1]B. The peaks from the lens samples were eluted at retention times identical to those of standards [Figure - 1]C & [Figure - 1]D. The peak detector response was linearly related to the amount of flavins injected, over a wide range of concentration (50 ng to 1 μg/ml). With a mixture of methanol (30%) and 5 mM ammonium acetate buffer (70%), FMN and FAD which were eluted together earlier [Figure - 1] were now separated with the retention time 4.6 and 2.6 min respectively [Figure - 2].
The data for male and female patients were pooled and presented under yellow and brown lenses respectively, since the ranges for age and all biochemical parameters were found to be similar between male and female patients.
The mean ± SE values for age in years of both groups of subjects (yellow or brown cataract) were similar (yellow lens 57 ± 3.7, brown lens 62 ± 3.5). Whole lens wet weights (yellow 232 ± 12.3 mg/lens, brown 240 ± 10.0 mg/lens) were comparable.
The results on tissue weight, total protein and falvin nucleotides in each region of yellow and brown cataractous lenses are shown in [Table - 1]. Tissue weight and total protein concentration were comparable with our earlier results. RF was not detected in any of the human cataractous lenses (whole lens as well as its different regions). Since normal (non-cataractous) human lenses were difficult to obtain, few normal lenses from rats and rabbits were analysed and RF was detected in these ocular tissues. When samples were spiked with RF, a peak corresponding to standard RF was separated from FMN+FAD peak [Figure - 1]C. The concentration of total flavin nucleotides in whole yellow lens (90 ± 21.2 ng/g) was significantly lower (p<0.0004) than that of brown lens (349 ± 54.5 ng/g). The concentration of flavin nucleotides (FMN+FAD) was comparable in all regions of the yellow lens (Table). However, in the brown lens there were regional differences. The nuclear region had the maximum concentration of flavin nucleotides, followed by the cortex and the capsule-epithelium. The concentration of flavin nucleotides was significantly high in all regions of brown lens compared to yellow lens. In human cataractous lens the relative proportion of decreased from 8.0 (yellow lens) to 1.9 (brown lens) during browning. A similar trend was also observed in different regions, the magnitude of change being greater in brown lens.
The concentrations (ng/g tissue) of FMN+FAD and RF in rat lenses (n=4) were 29.3 ± 7.38 (mean ± SE) and 2.1 ± 0.68 respectively. A similar trend was observed for total flavin nucleotides (80.7 ng/g) and RF (4.4 ng/g) concentrations in rabbit lenses (n=2). The relative proportion of FAD/FMN was 4.6 in normal (transparent) rat lens.
| Discussion|| |
To the best of our knowledge, this is the first report on flavin nucleotide levels in human eye lens. The most significant observation in the present study is the markedly higher concentration of flavin nucleotides in the brown cataractous lens compared to the yellow lens. When flavins were separated in a few lenses, the ratio of FAD/FMN decreased during lens pigmentation. In our earlier studies we have observed greater proportions of insoluble proteins of high molecular weight (HMW) in the brown lens compared to the yellow lens.
Trp metabolities have been etiologically implicated in the causation of cataract on near-ultraviolet (near-UV) light exposure.,,,, In a few human cataractous lenses we had measured the fluorescence of Trp and N-formylkynurenine (NFK) in insoluble protein fractions and found increased generation of NFK as a result of Trp oxidation in brown lens (Padmini Rao and K. Seetharam Bhat, unpublished data). Flavins are light sensitive and rapidly photo-oxidize to generate ROS.
It is interesting to note that the reported photochemical quantum yields of singlet oxygen (1O2) from RF and FMN were of much higher magnitude compared to NFK, the principal oxidative metabolite of Trp, observed in the lens in vitro. It seems likely that the observed increase in flavin nucleotides could also contribute to increased generation of ROS, during browning in addition to the damage due to Trp metabolites.
As to the source of increased nucleotides in the lens and nature of their influence on lens pigmentation, one can only speculate at this stage. The accumulation of flavin esters in the brown lens could be due to lower utilisation of the coenzymes as a result of the reduced activity of glutathione reductase, GSH-R (expressed/unit tissue). The accumulation of fluorogenes has been associated with increased yellowing of human lens during ageing.
FMN, a coloured fluorogen, is a very efficient photo sensitiser. It also generates ROS, which in turn may influence HMW aggregation and pigmentation. Lens pigmentation could also be due to the binding of flavins to crystallins. In the human cataractous lens, RF was not detected. However, using the same methodology, we found that while 93% of RF in normal rat lens occurs as nucleotides, 7% is present as free (unbound to nucleotide) RF. Similar observations have been made by other investigators. Batey and Eckhert showed that in rabbits, the highest concentration of FAD, FMN and RF occurred in the cortex followed by the nucleus. Whether lenses from normal subjects also show a similar pattern of flavin distribution is not known at present and needs to be studied.
It has been shown that where the etiology of cataract involves oxidative stress, cataract formation begins in the nuclear region of the lens. The higher concentration of flavin nucleotides, especially FMN, found in the nuclear region of brown lens, is in accordance with this oxidative etiology.
Earlier, we had observed that the riboflavin status of cataract patients as judged by the erythrocyte GSH-R activation coefficient is lower than matched controls., Also, GSH content in cataractous lens has been reported to be lower than the non-cataractous lens. Earlier, we had also observed decreased GSH levels in brown cataractous lenses compared to yellow lenses. Both suboptimal riboflavin status and lenticular accumulation of flavins may contribute to cataract formation, the brown type being more advanced.
| Acknowledgment|| |
The authors wish to thank Dr. P. Ranga Reddy, Superintendent, Sarojini Devi Eye Hospital, Hyderabad (India) for the human lenses. This investigation was supported in part under a contract awarded to Dr. V.N. Reddy by the National Eye Institute, USA for collaborative research on brunescent cataract with Sarojini Devi Eye Hospital, Hyderabad, India.
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[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5]
[Table - 1]