The Hydroxyl Radical in Lens Nuclear Cataractogenesis
1998; Elsevier BV; Volume: 273; Issue: 44 Linguagem: Inglês
10.1074/jbc.273.44.28603
ISSN1083-351X
AutoresShanlin Fu, Roger T. Dean, Michael Southan, Roger J.W. Truscott,
Tópico(s)Advanced Glycation End Products research
ResumoCataract is the major cause of blindness; the most common form is age-related, or senile, cataract. The reasons for the development of cataract are unknown. Here we demonstrate that nuclear cataract is associated with the extensive hydroxylation of protein-bound amino acid residues, which increases with the development of cataract by up to 15-fold in the case of DOPA. The relative abundance of the oxidized amino acids in lens protein (assessed per parent amino acid) is DOPA > o- and m-tyrosine > 3-hydroxyvaline, 5-hydroxyleucine > dityrosine. Nigrescent cataracts, in which the normally transparent lens becomes black and opaque, contain the highest level of hydroxylated amino acids yet observed in a biological tissue: for example, per 1000 parent amino acid residues, DOPA, 15; 3-hydroxyvaline, 0.3; compared with dityrosine, 0.05. The products include representatives of the hydroperoxide and DOPA pathways of protein oxidation, which can give rise to secondary reactive species, radical and otherwise. The observed relative abundance corresponds closely with that of products of hydroxyl radical or metal-dependent oxidation of isolated proteins, and not with the patterns resulting from hypochlorite or tyrosyl-radical oxidation. Although very little light in the 300–400-nm range passes the cornea and the filter compounds of the eye, we nevertheless also demonstrate that photoxidation of lens proteins with light of 310 nm, the part of the spectrum in which protein aromatic residues have residual absorbance, does not give rise to the hydroxylated aliphatic amino acids. Thus the post-translational modification of crystallins by hydroxyl radicals/Fenton systems seems to dominate their in vivo oxidation, and it could explain the known features of such nuclear cataractogenesis. Cataract is the major cause of blindness; the most common form is age-related, or senile, cataract. The reasons for the development of cataract are unknown. Here we demonstrate that nuclear cataract is associated with the extensive hydroxylation of protein-bound amino acid residues, which increases with the development of cataract by up to 15-fold in the case of DOPA. The relative abundance of the oxidized amino acids in lens protein (assessed per parent amino acid) is DOPA > o- and m-tyrosine > 3-hydroxyvaline, 5-hydroxyleucine > dityrosine. Nigrescent cataracts, in which the normally transparent lens becomes black and opaque, contain the highest level of hydroxylated amino acids yet observed in a biological tissue: for example, per 1000 parent amino acid residues, DOPA, 15; 3-hydroxyvaline, 0.3; compared with dityrosine, 0.05. The products include representatives of the hydroperoxide and DOPA pathways of protein oxidation, which can give rise to secondary reactive species, radical and otherwise. The observed relative abundance corresponds closely with that of products of hydroxyl radical or metal-dependent oxidation of isolated proteins, and not with the patterns resulting from hypochlorite or tyrosyl-radical oxidation. Although very little light in the 300–400-nm range passes the cornea and the filter compounds of the eye, we nevertheless also demonstrate that photoxidation of lens proteins with light of 310 nm, the part of the spectrum in which protein aromatic residues have residual absorbance, does not give rise to the hydroxylated aliphatic amino acids. Thus the post-translational modification of crystallins by hydroxyl radicals/Fenton systems seems to dominate their in vivo oxidation, and it could explain the known features of such nuclear cataractogenesis. high performance liquid chromatography o-phthaldialdehyde 3-hydroxyvaline 5-hydroxyleucine. Cataract is a major problem worldwide. At present the etiology of the cataract is poorly understood and the only treatment involves surgical removal of the opaque lens. In the developing world, access to ophthalmic facilities is a limiting factor; it is estimated that there are 4 million newly blind each year in India alone (1Minassian D.C. Mehra V. Br. J. Ophthalmol. 1990; 74: 341-343Crossref PubMed Scopus (116) Google Scholar). The cost of surgical procedures is now a considerable proportion of the health budget of most developed nations (2Javitt J.C. Wang F. West S.K. Ann. Rev. Public Health. 1996; 17: 159-177Crossref PubMed Scopus (126) Google Scholar). There are two main types of age-related cataract, nuclear and cortical. In cortical cataract the opacity, localized in the outer region of the lens, is associated with an ionic imbalance (3Maraini G. Mangili R. The Human Lens in Relation to Cataract. Elsevier, Amsterdam1973: 79-95Google Scholar). Nuclear cataract is characterized by a high degree of light scattering in the center of the lens. In the vast majority of cases this opacity is colored (4Duncan G. Duncan G. Mechanisms of Cataract Formation in the Human Lens. Academic Press, London1981: 1-5Google Scholar). In most cases the lens in such patients is yellow or brown, but in rare cases the normally transparent and colorless lens can be transformed into a black opaque "nigrescent" cataract. Much of what is known about the biochemical changes associated with age-related cataract has come from studies in which cataractous lenses have been classified as types I–IV on the basis of an increase in nuclear color as first proposed by Pirie (5Pirie A. Invest. Ophthalmol. Vis. Sci. 1968; 7: 634-642Google Scholar). Type I cataract is characterized mainly by cortical changes without significant nuclear alterations. It has been shown that the increase in nuclear color is closely linked with progressive increases in the amount of protein methionine sulfoxide, urea-insoluble protein, and crystallin cross-linking (6Buckingham R.H. Exp. Eye Res. 1972; 14: 123-129Crossref PubMed Scopus (72) Google Scholar, 7Truscott R.J.W. Augusteyn R.C. Biochim. Biophys. Acta. 1977; 492: 43-52Crossref PubMed Scopus (215) Google Scholar, 8Truscott R.J.W. Augusteyn R.C. Exp. Eye Res. 1977; 25: 139-148Crossref PubMed Scopus (152) Google Scholar, 9Truscott R.J.W. Augusteyn R.C. Exp. Eye Res. 1977; 24: 159-170Crossref PubMed Scopus (121) Google Scholar, 10Garner M. Spector A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1274-1277Crossref PubMed Scopus (259) Google Scholar). One of the earliest changes is the loss of protein sulfhydryl groups, and these decrease progressively with the development of cataract such that in type IV nuclear cataract less than 5% of the original sulfhydryl groups remain (8Truscott R.J.W. Augusteyn R.C. Exp. Eye Res. 1977; 25: 139-148Crossref PubMed Scopus (152) Google Scholar). On the basis of these observations it was postulated that the reaction of the lens proteins with H2O2 derived from superoxide may be responsible for the changes associated with nuclear cataract formation (7Truscott R.J.W. Augusteyn R.C. Biochim. Biophys. Acta. 1977; 492: 43-52Crossref PubMed Scopus (215) Google Scholar). Peroxide is known to oxidize cysteine and methionine efficiently (11McNamara M. Augusteyn R. Exp. Eye Res. 1984; 38: 45-56Crossref PubMed Scopus (38) Google Scholar, 12Vogt W. Free Radic. Biol. Med. 1995; 18: 93-105Crossref PubMed Scopus (787) Google Scholar). H2O2 may be derived from the aqueous humor which bathes the anterior segment of the lens (13Spector A. Garner W.H. Exp. Eye Res. 1981; 33: 673-681Crossref PubMed Scopus (449) Google Scholar) and is the primary source of nutrients and oxygen, although the lens is remarkable in its ability to detoxify external peroxide (14Giblin F.J. McCready J.P. Reddan J.R. Dziedzic D.C. Reddy V.N. Exp. Eye Res. 1985; 40: 827-840Crossref PubMed Scopus (56) Google Scholar, 15Zigler J.S. Lucas V.A. Du X. Invest. Ophthalmol. Vis. Sci. 1989; 30: 2195-2199PubMed Google Scholar). Since the human lens grows throughout life by the addition of fiber cells, and since the crystallins in the lens nucleus undergo little turnover (16Harding J.J. Cataract—Biochemistry, Epidemiology and Pharmacology. Chapman & Hall, London1991Google Scholar), the proteins in the central region of the lens are as old as the individual. Thus post-translational modifications to these crystallins may accumulate over a person's lifetime. It is also possible that protein modifications linked with cataract could be the result of a reaction of lens crystallins with other oxidizing agents such as the hydroxyl radical (17Wolff S.P. Garner A. Dean R.T. Trends Biochem. Sci. 1986; 11: 27-31Abstract Full Text PDF Scopus (471) Google Scholar, 18Davies K.J. J. Biol. Chem. 1987; 262: 9895-9901Abstract Full Text PDF PubMed Google Scholar, 19Davies K.J. Delsignore M.E. Lin S.W. J. Biol. Chem. 1987; 262: 9902-9907Abstract Full Text PDF PubMed Google Scholar, 20Dean R.T. Gieseg S. Davies M.J. Trends Biochem. Sci. 1993; 18: 437-441Abstract Full Text PDF PubMed Scopus (235) Google Scholar, 21Stadtman E.R. Annu. Rev. Biochem. 1993; 62: 797-821Crossref PubMed Scopus (1266) Google Scholar), which might also partly derive from hydrogen peroxide through the transition-metal ion catalyzed Fenton reactions (22Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. 2nd Ed. Clarendon, Oxford1989Google Scholar). With the recent development of HPLC1 procedures which enable hydroxyl radical damage to proteins to be detected and quantified without any drastic treatment (e.g. derivatization or volatilization for mass spectrometry) after protein hydrolysis (23Gieseg S.P. Simpson J.A. Charlton T.S. Duncan M.W. Dean R.T. Biochemistry. 1993; 32: 4780-4786Crossref PubMed Scopus (172) Google Scholar, 24Fu S. Hick L.A. Sheil M.M. Dean R.T. Free Radic. Biol. Med. 1995; 19: 281-292Crossref PubMed Scopus (71) Google Scholar, 25Davies M.J. Fu S. Dean R.T. Biochem. J. 1995; 305: 643-649Crossref PubMed Scopus (211) Google Scholar, 26Dean R.T. Fu S. Gieseg G. Armstrong S.G. Punchard N.A. Kelly F.J. Free Radicals: A Practical Approach. IRL Press, Oxford1996: 171-183Google Scholar, 27Fu S. Dean R.T. Biochem. J. 1997; 324: 41-48Crossref PubMed Scopus (103) Google Scholar), it has become feasible to use these techniques to examine proteins from biological sources for evidence of modification by this oxidant. Furthermore, we have demonstrated that certain protein oxidation pathways give rise to reactive intermediates, such as hydroperoxides and the reducing moiety DOPA (28Dean R.T. Fu S. Stocker R. Davies M.J. Biochem. J. 1997; 324: 1-18Crossref PubMed Scopus (1463) Google Scholar, 29Davies M.J. Dean R.T. Radical Mediated Protein Oxidation. Oxford University Press, Oxford1997Google Scholar), which can generate secondary reactive species and damage other biological targets such as DNA (30Morin B. Davies M.J. Dean R.T. Biochem. J. 1998; 30: 1059-1067Crossref Scopus (61) Google Scholar), and these methods permit the assessment of the occurrence of these pathways. In this study we analyzed the nuclei and cortices of individual cataractous lenses to assess the involvement of the hydroxyl radical in the etiology of the nuclear cataract. We show that the development of human nuclear cataracts is associated with a pronounced increase in the levels of modified amino acids in the lens proteins, including representatives of the hydroperoxide and DOPA pathways. The observed results are consistent with hydroxyl radical damage, and not with photoxidation. We therefore propose that the hydroxyl radical may play a role in the development of the nuclear cataract in man. o-Phthaldialdehyde (OPA) crystals and OPA diluent (containing 3% potassium hydroxide and 3% boric acid at pH 10.4) were from Pickering Laboratories (Mountain View, CA). 2-Mercaptoethanol was from Sigma. Mercaptoacetic acid was from Merck (VIC, Australia). Other chemicals, solvents, and chromatographic materials were of analytical reagent or HPLC grade. Cataractous lenses were classified on the basis of nuclear color as described by Pirie (5Pirie A. Invest. Ophthalmol. Vis. Sci. 1968; 7: 634-642Google Scholar). Nigrescent lenses were treated as a subcategory of type IV (nigrescent type IV). Lenses were stored at −20 °C and thawed immediately prior to dissection. Some nigrescent lenses, removed from patients attending eye camps in India, were shipped in alcohol and then stored at −20 °C. Analyses we performed on type I and IV cataractous lenses showed that storage in alcohol does not affect the content of the hydroxylated amino acids. Lenses were dissected using a 4-mm cork borer to obtain the central core, and the ends of the core were then removed. The average wet weight of the nucleus thus obtained was 20.3 mg. Cortices were obtained by taking a sample of the outer lens ring remaining after coring and removing the lens capsule. No reagents were added to the cortices, and no dialysis step was necessary. Lens samples were frozen individually in liquid nitrogen and powdered in an Eppendorf tube by using a Teflon hand homogenizer. After freeze-drying, the samples were hydrolyzed individually for analysis of oxidized amino acids, as follows. The issue of artifactual formation of oxidized amino acids during tissue homogenization has been studied extensively with human plaque tissue (31Fu S. Davies M.J. Stocker R. Dean R.T. Biochem. J. 1998; 333: 519-525Crossref PubMed Scopus (228) Google Scholar). No formation of oxidized amino acids was observed when standards of tyrosine, phenylalanine, valine, and leucine were added to the sample prior to mechanical homogenization. A gas phase and acid- catalyzed method described previously (23Gieseg S.P. Simpson J.A. Charlton T.S. Duncan M.W. Dean R.T. Biochemistry. 1993; 32: 4780-4786Crossref PubMed Scopus (172) Google Scholar) was adopted, in which mercaptoacetic acid (5% v/v) and phenol (1% w/v) were added to the HCl (6m) solution as reductant and antioxidant, respectively, to permit the optimal recovery of DOPA (95–100% as judged by the recovery of free DOPA). We also achieved 95–100% recovery of freeo- and m-tyrosine and dityrosine. The recovery of 3-hydroxyvaline (Val.OH1) was c.80% and of 5-hydroxyleucine (Leu.OH2) c.70%, as detailed previously (24Fu S. Hick L.A. Sheil M.M. Dean R.T. Free Radic. Biol. Med. 1995; 19: 281-292Crossref PubMed Scopus (71) Google Scholar, 26Dean R.T. Fu S. Gieseg G. Armstrong S.G. Punchard N.A. Kelly F.J. Free Radicals: A Practical Approach. IRL Press, Oxford1996: 171-183Google Scholar, 27Fu S. Dean R.T. Biochem. J. 1997; 324: 41-48Crossref PubMed Scopus (103) Google Scholar). The known quantity (approximately 4 mg) of dried lens powder was used for each hydrolysis. The hydrolysate was lyophilized and dissolved in about 200 μl water (such that the final sample concentration was 20 mg/ml hydrolysate) for HPLC analysis. Artifactual formation of m- and o-tyrosine (from free phenylalanine), and DOPA (from free tyrosine) during protein hydrolysis, in the presence or absence of protein, has also been investigated in detail in the study relating to human atherosclerotic plaques (31Fu S. Davies M.J. Stocker R. Dean R.T. Biochem. J. 1998; 333: 519-525Crossref PubMed Scopus (228) Google Scholar). The maximum artifactual formation ranges from 10 μmol of m-tyrosine/mol of phenylalanine, 20 μmol of o-tyrosine/mol of phenylalanine, to 100 μmol of DOPA/mol of tyrosine, which are low values compared with those reported in this study for either the normal or cataractous lenses. Artifactual formation of hydroxyvaline and hydroxyleucine was not observed during such protein hydrolysis. In comparable experiments in which these four amino acids were added to lens samples prior to freezing and powdering, and then taken through the hydrolysis procedure, indistinguishable results were obtained, confirming that artifactual oxidation was very limited. All HPLC analyses were carried out on a LC-10A HPLC system (Shimadzu) consisting of two pumps, a high pressure mixer, an autosampler, and a sample cooler. System operation and peak integration was driven by Class LC-10 software (Shimadzu) operated under a PC-based Windows environment. A column oven (Waters) set at 30 °C was also used for each analysis. Detection of Val.OH1 and Leu.OH2 requires two steps of HPLC. In the first step, the hydrolysate was fractionated on a LC-NH2column (25 cm × 4.6 mm, 5-μm particle size, Supelco). The purified fraction was then analyzed by a second HPLC step on a Zorbax ODS column (25 cm × 4.6 mm, 5-μm particle size, Rockland Technologies) following pre-column derivatization with OPA reagent. The detailed experimental conditions have been described previously (24Fu S. Hick L.A. Sheil M.M. Dean R.T. Free Radic. Biol. Med. 1995; 19: 281-292Crossref PubMed Scopus (71) Google Scholar,27Fu S. Dean R.T. Biochem. J. 1997; 324: 41-48Crossref PubMed Scopus (103) Google Scholar). This second HPLC procedure was also used to measure unmodified valine, leucine, and phenylalanine in the hydrolysate (without any prior fractionation). For detection of DOPA, m-tyrosine,o-tyrosine, and dityrosine, the hydrolysate was separated on a Zorbax ODS column with a Pelliguard column (2 cm, Supelco). Elution at 1 ml/min was performed with a gradient of solvent A (100 mm sodium perchlorate in 10 mm sodium phosphate buffer, pH 2.5) and solvent B (80% methanol in water) as described previously (32Fu S. Fu M.-X. Baynes J.W. Thorpe S.R. Dean R.T. Biochem. J. 1998; 330: 233-239Crossref PubMed Scopus (68) Google Scholar). The eluent was monitored by both UV (Shimadzu, at 280 nm) and fluorescence (Hitachi F-1080) detectors in series. The excitation wavelength was set at 280 nm for all components, while the emission wavelength was set at 320 nm for DOPA, m-tyrosine,o-tyrosine, and 410 nm for dityrosine through the built-in time program of the detector. Quantitation of (unmodified)p-tyrosine was based on UV detection due to its off-scale response in the fluorescence channel. Pooled lens protein samples (10 mg) were hydrolyzed and then redissolved in water (500 μl). The samples were then fractionated using the standard HPLC methodology described above. DOPA and o-Tyr fractions were collected and combined from 25 injections of the hydrolysate (20 μl for each injection). The collected samples were freeze-dried and rechromatographed on the same HPLC column with 0.1% trifluoroacetic acid in water, replacing the standard mobile phase solvent A (which contains 100 mmsodium perchlorate and 10 mm sodium phosphate). The desalted fractions were dissolved in 80 μl of 1% aqueous formic acid and acetonitrile (1:1 v/v) and subjected to mass spectroscopic analysis. The electrospray mass spectrometry was conducted on a VG Platform mass spectrometer (Fisons, Homebush, NSW, Australia). The solvent (50% acetonitrile in water) was delivered by a Phoenix (Fisons) syringe pump at a flow rate of 100 μl/min; 10 μl of each sample solution were injected for analysis. Dry nitrogen gas at atmospheric pressure was employed to assist evaporation of the electrospray droplets. A positive electrospray mode was used with electrospray probe tip potential at 2.19 kV, counter-electrode potential at 0.5 kV, and cone potential at 20 V. Two human lenses (normal and nigrescent type IV) were used for determination of the proportion of free native and oxidized amino acids in lens compared with their population in lens proteins. Lens samples were powdered with the aid of liquid nitrogen and solubilized in water by sonicating. Proteins were precipitated out by means of trichloroacetic acid. Both the protein portion after amino acid hydrolysis and the supernatant portion were analyzed by HPLC for the presence of tyrosine, phenylalanine, valine, and leucine. As summarized in Table I, the free amino acids comprised less than 1% of the total amino acids in proteins in lens, in agreement with published literature (7Truscott R.J.W. Augusteyn R.C. Biochim. Biophys. Acta. 1977; 492: 43-52Crossref PubMed Scopus (215) Google Scholar, 8Truscott R.J.W. Augusteyn R.C. Exp. Eye Res. 1977; 25: 139-148Crossref PubMed Scopus (152) Google Scholar, 9Truscott R.J.W. Augusteyn R.C. Exp. Eye Res. 1977; 24: 159-170Crossref PubMed Scopus (121) Google Scholar). There were no detectable oxidized amino acids in the supernatant portion of the samples, confirming that any oxidized amino acids found in the lens are protein-bound.Table IFree amino acids as percentages of total amino acids in human lensesAmino acidNormal lensNigrescent type IV lensTyr0.210.30Phe0.370.52Val0.830.89Leu0.400.47Dried and powdered human lens samples (4 mg, one normal and one nigrescent type IV) were dissolved in water (200 μl) by sonicating at 4 °C for 5 min. Into the solutions were added 0.3% sodium desoxycholate (25 μl) and 50% (v/v) trichloroacetic acid (50 μl). After mixing, the mixture was centrifuged at 16,000 ×g for 2 min. After separation from the supernatant the protein pellet was hydrolyzed with the conditions described under "Materials and Methods." HPLC analyses for oxidized as well as native amino acids were carried out on both the supernatant and protein hydrolysate fractions. Free amino acids are expressed as percentages of total amino acids (free plus protein-bound) in the lenses and are means of duplicate measurements. Open table in a new tab Dried and powdered human lens samples (4 mg, one normal and one nigrescent type IV) were dissolved in water (200 μl) by sonicating at 4 °C for 5 min. Into the solutions were added 0.3% sodium desoxycholate (25 μl) and 50% (v/v) trichloroacetic acid (50 μl). After mixing, the mixture was centrifuged at 16,000 ×g for 2 min. After separation from the supernatant the protein pellet was hydrolyzed with the conditions described under "Materials and Methods." HPLC analyses for oxidized as well as native amino acids were carried out on both the supernatant and protein hydrolysate fractions. Free amino acids are expressed as percentages of total amino acids (free plus protein-bound) in the lenses and are means of duplicate measurements. The HPLC methods employed in this study have been used successfully in several other studies investigating protein oxidation by free radicals in biological and pathological samples, for example, native human plasma, 2S. Fu and R. Dean, unpublished results.cerebral malaria-affected mice brain homogenates, 3L. Sanni, S. Fu, R. Dean, G. Bloomfield, R. Stocker, G. Chaudhri, M. Dinauer, and N. Hunt, unpublished results. and rat tail collagen incubated with glucose (32Fu S. Fu M.-X. Baynes J.W. Thorpe S.R. Dean R.T. Biochem. J. 1998; 330: 233-239Crossref PubMed Scopus (68) Google Scholar). Fig. 1 shows typical HPLC traces of hydrolysates of normal as well as cataractous lens proteins. Oxidized amino acids were identified on the basis of their coelution with spiked standards. The identification of the hydroxy-aliphatic amino acids is very strong, since they undergo two successive HPLC chromatography steps, in each of which they behave characteristically. Indeed, in Fig. 1 B, Leu.OH2 (peak 6) is essentially base-line resolved; similarly, Val.OH1 is well resolved from a smaller preceding peak, although the spiking material used in Fig. 1 B, ii, contains very large proportions of such an adjacent molecule, which gives a slightly misleading impression. Again the identification of the individual oxidized species is very strong, with dityrosine distinguished not only by its elution position, but also by its characteristic fluorescence spectrum. o-Tyrosine (peak 3) is closely followed by a smaller peak, not present in the spiking material (Fig. 1 A, ii versus i). All these individual chromatographic peaks could be readily integrated and well resolved, giving precise measurements of the oxidized species, which were quite reproducible (see below). Nevertheless, we used mass spectrometry of some such peaks isolated from pooled normal or pooled diseased lens proteins, to confirm their identity. Fig. 1 Cshows for the cases of DOPA and o-tyrosine that the relative abundance of the appropriate molecular ion in the two samples was similar to that observed by HPLC in the same samples, and in the later data for individual lenses. Mass spectrometry thus confirmed the identity of the species under study, although it cannot readily distinguish o-, m-, or p-tyrosine from each other. To obtain information on interassay variation for each oxidized amino acids, repeat analyses of the same lens sample were performed on different days. As summarized in Table II, the interassay variation for oxidized amino acids was generally larger (around 10%) than that for native amino acids (around 5%), which might be due to either the modest artifactual formation of oxidized moieties during hydrolysis (in the case of DOPA, dityrosine, o-tyrosine, and m-tyrosine) or to the need for two steps of HPLC with the extra inherent variability this entails (in the case of hydroxyvaline and hydroxyleucine).Table IIInterassay variation in the determination of oxidized and parent amino acidsOxidized amino acidsVariationnParent amino acidsVariationn%%DOPA14.54Tyr4.44Dityrosine10.14m-Tyr9.44Phe2.74o-Tyr9.44Val.OH110.03Val4.13Leu.OH214.33Leu4.73Repeat analysis of a type III lens sample was performed on different occasions using the same analytical procedure described under "Materials and Methods." The interassay coefficients of variation were calculated according to the mean values and standard deviations as percentages (S.D. × 100/mean). Open table in a new tab Repeat analysis of a type III lens sample was performed on different occasions using the same analytical procedure described under "Materials and Methods." The interassay coefficients of variation were calculated according to the mean values and standard deviations as percentages (S.D. × 100/mean). The levels of all six amino acid derivatives have been quantified in the separated nuclear and cortical proteins of normal and cataractous human lenses (Fig. 2). Statistical analysis by analysis of variance indicated no significant variation between nuclear and cortical material in any of the lens samples. But there was a marked dependence on cataract type for every oxidized amino acid (p < 0.001 in every case). When unpaired t tests were undertaken, nigrescent type IV and type III values were significantly different from normal lens values for each oxidized amino acid (p < 0.05 in every case). Statistical differences at p < 0.05 were observed in most cases for type II and in one case for type I versusnormal lens cortices and nuclei. The contents of the modified amino acids present in the normal lens are similar to those found in apoprotein B of freshly prepared (33Kritharides L. Jessup W. Gifford J. Dean R.T. Anal. Biochem. 1993; 213: 79-89Crossref PubMed Scopus (178) Google Scholar, 34Kritharides L. Jessup W. Mander E.L. Dean R.T. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 276-289Crossref PubMed Scopus (89) Google Scholar) normal human plasma low density lipoprotein (Table III). Since type I lenses are characterized by cortical cataract, this finding indicates that the development of such cortical opacity is not associated with a general increase in the levels of such modified amino acids, suggesting that the hydroxyl radical is not involved in the etiology of this form of cataract.Table IIIOxidized amino acids in fresh human plasma low density lipoproteinDOPA630 μmol/mol TyrDityrosine2 μmol/mol Tyrm Tyr260 μmol/mol Pheo-Tyr470 μmol/mol PheVal.OH125 μmol/mol ValLeu.OH213 μmol/mol LeuApoprotein B of freshly isolated human plasma low density lipoprotein was prepared as previously described (33Kritharides L. Jessup W. Gifford J. Dean R.T. Anal. Biochem. 1993; 213: 79-89Crossref PubMed Scopus (178) Google Scholar, 34Kritharides L. Jessup W. Mander E.L. Dean R.T. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 276-289Crossref PubMed Scopus (89) Google Scholar). 0.7 ml of low density lipoprotein (2 mg of apoB/ml) was delipidated using trichloroacetic acid/sodium desoxycholate method (26Dean R.T. Fu S. Gieseg G. Armstrong S.G. Punchard N.A. Kelly F.J. Free Radicals: A Practical Approach. IRL Press, Oxford1996: 171-183Google Scholar) (n = 3). The precipitated protein was hydrolyzed and analyzed by HPLC as described under "Materials and Methods." Data are mean values from three donors. We find comparable values in total plasma protein. Open table in a new tab Apoprotein B of freshly isolated human plasma low density lipoprotein was prepared as previously described (33Kritharides L. Jessup W. Gifford J. Dean R.T. Anal. Biochem. 1993; 213: 79-89Crossref PubMed Scopus (178) Google Scholar, 34Kritharides L. Jessup W. Mander E.L. Dean R.T. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 276-289Crossref PubMed Scopus (89) Google Scholar). 0.7 ml of low density lipoprotein (2 mg of apoB/ml) was delipidated using trichloroacetic acid/sodium desoxycholate method (26Dean R.T. Fu S. Gieseg G. Armstrong S.G. Punchard N.A. Kelly F.J. Free Radicals: A Practical Approach. IRL Press, Oxford1996: 171-183Google Scholar) (n = 3). The precipitated protein was hydrolyzed and analyzed by HPLC as described under "Materials and Methods." Data are mean values from three donors. We find comparable values in total plasma protein. The earliest stage of nuclear cataract is the type II lens, characterized by an increase in the protein fraction which is insoluble in 8 m urea and a substantial loss in protein thiol content, on average to 30% of those seen in normal lenses (7Truscott R.J.W. Augusteyn R.C. Biochim. Biophys. Acta. 1977; 492: 43-52Crossref PubMed Scopus (215) Google Scholar, 8Truscott R.J.W. Augusteyn R.C. Exp. Eye Res. 1977; 25: 139-148Crossref PubMed Scopus (152) Google Scholar, 9Truscott R.J.W. Augusteyn R.C. Exp. Eye Res. 1977; 24: 159-170Crossref PubMed Scopus (121) Google Scholar). Interestingly, for DOPA, the hydroxylated amino acid produced at
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