Ion analyses of human cataractous lenses

The sodium, potassium, calcium, magnesium and chloride concentrations have been determined in 52 human cataractous lenses. Three main types of association between concentrations and cataract type were found. Group A, mainly nuclear cataracts, had a near normal range of concentrations. Group B, a mixture of cataract types, including mixed cataracts, had a significantly raised sodium and lowered potassium concentration, but particularly a raised calcium concentration. Group C, mainly cortical and mature cataracts, had grossly increased sodium and calcium concentrations, an increased chloride concentration and a very low potassium level. In all groups the magnesium concentration remained relatively constant.The role of calcium is particularly interesting. In pure nuclear cataracts its concentration is relatively normal and below the equilibrium concentration, but when there is cortical involvement very high calcium levels are found. The values, which are above the calculated equilibrium concentration, indicate that binding of calcium occurs. Group B and C cataractous lenses also showed a significant decrease in dry weight.     

Multi-crystallin complexes exist in the water-soluble high molecular weight protein fractions of aging normal and cataractous human lenses

The purpose of the study was to identify non-covalently held complexes that exist in the water-soluble high molecular weight (WS-HMW) protein fractions of normal human lenses of 20-year-old and 60- to 70-year-old, and in the age-matched 60- to 70-year-old cataractous lenses. The WS protein fractions were prepared from five pooled normal lenses of 20-year-old donors or five pooled lenses of 60- to 70-year-old donors or four pooled cataractous lenses (with nuclear opacity) of 60- to 70-year-old donors. Each WS protein fraction was subjected to size-exclusion chromatography using an Agarose A 5m column to recover the void volume WS-HMW protein fraction. A method known as blue-native polyacrylamide gel electrophoresis (BN-PAGE), which allows the isolation of large multi-protein complexes (MPCs) in their native state for further characterization, was used to separate such complexes from individual WS-HMW protein fractions. The protein species that existed as a complex were excised from a gel and trypsin-digested, and the amino acid sequences of the tryptic fragments analyzed by electrospray tandem mass spectrometry (ES-MS/MS). After the second-dimensional sodium dodecyl sulfate-PAGE during BN-PAGE, protein complexes containing a total of 16, 12, and 24 species with M_r between 10 and 90 kDa were identified in the HMW protein fractions of normal lenses of 20-year-old, 60- to 70-year-old and cataractous lenses of 60- to 70-year-old donors, respectively. Based on the amino acid sequences of tryptic peptides of individual protein species in the complexes by the ES-MS/MS method, the presence of α-, β-, and γ-crystallin species along with beaded filament proteins (filensin and phakinin) was observed in the 20-year-old normal lenses. The 60- to 70-year-old normal lenses contained filensin and aldehyde dehydrogenase in addition to the above crystallins. Similarly, the age-matched cataractous lenses also contained the above crystallins and aldehyde dehydrogenase but lacked beaded filament proteins. Protein complexes, held mostly via non-covalent bonding, were seen in the WS-HMW proteins of 20-year-old normal, 60- to 70-year-old normal, and 60- to 70-year-old cataractous lenses. The complexes in the normal lenses were made of α-, β-, and γ-crystallin species, beaded filament proteins (filensin and/or phakinin), and aldehyde dehydrogenase. The complexes in the age-matched cataractous lenses also contained these crystallins, and aldehyde dehydrogenase, but not the beaded filament proteins. Further, the crystallin fragments were greater in number in the cataractous lenses compared to the age-matched normal lenses. During multi-angle light scattering (MALS), the HMW proteins from cataractous lenses exhibited species with lower molecular weight range than age-matched normal lenses. The HMW protein preparations from both normal and cataractous lenses showed spherical structures on electron microscopic analysis.     

Inorganic element concentrations in cataractous human lenses.

We evaluated the presence of calcium, potassium, zinc, copper, and selenium in human lenses (53 cataractous and 10 clear lenses). The determinations of these elements were done using atomic absorption spectrometry techniques, namely flame and flameless methods, after acidic digestion of the samples. Compared with the results obtained from samples of normal lenses (zinc, 16.5 +/- 2.5 mg/kg dry weight; copper, 0.53 +/- 0.08 mg/kg dry weight; selenium, 0.83 +/- 0.18mg/kg dry weight; potassium, 10,306 +/- 1232mg/kg dry weight, and calcium, 9.9 +/- 2.7mg/kg dry weight), the mean concentration values of the cataractous lenses showed some significant changes. Increases were found for zinc, copper, and calcium; the potassium concentration decreased. No significant changes occurred in selenium values. A positive correlation was found between zinc and copper concentrations (y = 0.030x + 0.007, r = .79). An inverse correlation was evident between calcium and potassium values (y = -0.097x + 1141, r = -.65).     

On the presence and mechanism of formation of heavy molecular weight aggregates in human normal and cataractous lenses

It has been suggested that the presence of high molecular weight protein aggregates in the lens can lead to light scattering and a consequent loss of transparency. We have measured the concentration of aggregates having a molecular weight greater than approximately 150 × 10⁶ g/mole present in the soluble fraction of both aging normal and cataractous human lenses. This protein population is approximately 5% of the total soluble protein in lenses up to age 75 and increases to 10–15% in lenses aged greater than 75 years and in cataracts. The amino acid composition of the heavy molecular weight soluble aggregates of normal lenses is different in leucine content from alpha, beta and gamma crystallins and different in tyrosine content from beta and gamma crystallins. The aggregates from cataractous lenses show a greater increase in leucine and greater decrease in tyrosine. An unidentified component is present in the cataractous aggregates. Furthermore, calcium, an ion which increases in concentration in cataractogenesis, induces alpha crystallin to aggregate and induces the heavy molecular weight fraction to aggregate. This aggregation is irreversible by dialysis or chelation.     

Cadmium, lead and copper concentrations in normal and senile cataractous human lenses

Cadmium, lead and copper concentrations were measured in normal and cataractous human lenses by atomic absorption spectrophotometry. The concentrations of all three elements were relatively higher in cataractous lenses compared with normals, and the ratios of cataract/normal concentrations were in the order Pb greater than Cd greater than Cu. The concentration of these trace elements also varied according to lense age. It is likely that the major source of cadmium is tobacco smoke while that of lead is the exhaust gases of motor cars.     

Changes in the state of water in senile cataractous lenses as studied by nuclear magnetic resonance

Measurements of NMR relaxation times T₁ and T_1? are presented for water protons in normal human, porcine and senile cataractous lenses, together with determinations of both total water content and the fraction of bound water. Relaxation times measured in completely opaque cortical cataractous lenses are longer than for normal lenses at temperatures above the freezing point of the free water, the difference being most marked in the case of spin-lattice relaxation in the rotating frame (T_1?). The changes reflect a reduction in the proportion of bound water. No such changes were observed in lenses affected only by nuclear cataract, which appear to retain a normal water balance.     

Pyridine nucleotides in normal and cataractous human lenses

The levels of NAD⁺, NADH, NADP⁺ and NADPH have been determined in each cortex and nucleus of about 80 normal and cataractous human lenses and in a small number of calf, rabbit and rat lenses. Cataractous lenses had lower levels of all nucleotides than the normal lenses but no progressive decreases were associated with the development of nuclear colour, or with cortical, diabetic, traumatic and steroid cataracts. It was concluded that a lack of reduced pyridine nucleotides was not responsible for a decrease in the ability of the lens to protect itself against oxidative damage.     

Amino acid transport and protein synthesis in human normal and cataractous lenses

The ability of normal and cataractous human lenses to accumulate amino acids and synthesize proteins was studied under organ culture conditions. As expected, normal lenses regulate their internal ion levels, accumulate amino acids against a concentration gradient, and continue to synthesize proteins even in advanced age (greater than 60 yrs). The cataractous lenses fell basically into two groups. Those with a low internal sodium and calcium content behaved in a similar manner to normal lenses, but cataractous lenses with high sodium and calcium contents showed a markedly reduced ability to accumulate amino acid and synthesized less low molecular weight protein. They incorporated, however, a much higher proportion of labelled amino acid into high molecular weight protein. While sodium appears to be the ion involved in changes in amino acid accumulation, calcium seems to play a critical role in the disturbance of lens protein synthesis and also protein-protein interaction. Both loss of protein and accumulation of high molecular weight aggregates may be due to modifications induced by the calcium-activated protease, calpain. None of the changes appeared to be correlated with increasing nuclear brunescence.     

Membrane water permeability in normal and cataractous human lenses

The permeability of lens membranes to water has been studied in human cataractous lenses and in transparent post-mortem lenses. Both the exchange of radioactive water and the flow of water under an osmotic pressure gradient have been measured. The experimental evidence obtained suggests that the osmotic behaviour of a lens with early senile cataract approximates that of a perfect osmometer and that no significant difference exists between normal and cataractous lenses in the percentage of exchangeable water.When the in vitro uptake of Na²² by cataractous lenses is investigated the results indicate a progressive deterioration of the mechanisms maintaining the impermeability of lens membranes to sodium.     

Membrane permeability characteristics of perfused human senile cataractous lenses

Human cataractous lenses were removed by the cryoprobe technique and were maintained for up to 24 hr in a solution of similar ionic composition to human aqueous humour. The bimodal distribution of internal sodium concentrations was similar to that previously reported for unincubated human lenses. Lenses with lower total and free sodium contents had relatively higher membrane potentials and they lost 86Rb at a slower rate than lenses with high internal sodium. The 86Rb efflux in these lenses was stimulated four-fold by removing external calcium. The efflux was reduced by increasing external calcium, but was increased during a small (60 mosmol) hyperosmotic shock. A similar hyperosmotic shock also surprisingly increased 86Rb efflux. Lenses with increasing internal sodium (and calcium) levels showed an increasing rate of loss of 86Rb and the stimulation by calcium removal was progressively diminished. The efflux from lenses with disturbed ion levels was also relatively insensitive to changes in external osmolarity and to increasing external potassium. Lenses with raised sodium concentrations also had an increased inulin space. Frog, rat and rabbit lenses were also exposed to the same range of stimuli and only frog lenses responded to the low calcium solution with more than a four-fold increase in efflux rate. Although only a two- to four-fold increase in efflux rate was obtained from rabbit lenses exposed to Ca-free conditions, this was the only type of animal lens so far tested that, like the human lens, responded to both hyperosmotic and isosmotic shocks with an increase in efflux rate. All three species of mammalian lenses responded with an increase in efflux rate when exposed to a hyperosmotic test solution while in the frog, the efflux rate from the lens decreased. The glucose efflux from human cataractous lenses was inhibited by cytochalasin B in a similar manner to the efflux from rat and frog lenses. It was concluded, therefore, that the cryoprobed human lens can be kept for a limited period in a relative simple artificial aqueous humour solution. The potassium permeability characteristics of low sodium cataracts remained relatively intact and showed a unique response (relative to lenses from other animals) when exposed to various stimuli that are known to be potentially cataractogenic.     

Glutathione and glutathione-related enzymes in human cataractous lenses

Glutathione and its related enzymes were measured for normal and cataractous human lenses. Glutathione decreased progressively with the development of cataracts. This decrease was more pronounced in the nucleus than in the capsule-epithelia of cataractous lenses. Glutathione reductase in nuclear extracts was relatively unchanged during cataract progress, while glutathione synthetase was significantly low in the advanced stages of cataracts. gamma-Glutamylcysteine synthetase was not measurable in the nuclei of cataractous lenses.     

Free and bound water in normal and cataractous human lenses

PURPOSE: To analyze free and total water in human normal and cataractous lenses. METHODS: Thermogravimetric analysis was used to determine total water, and differential scanning calorimetry was used for free water. RESULTS: In normal human lenses, the total water content of the nucleus remained unchanged with age, but the state of the water altered. The ratio of free to bound water increased steadily throughout adult life. In a 20-year-old person, there was approximately one bound water molecule for each free water molecule in the lens center, whereas in a 70- to 80-year-old person, there were two free water molecules for each bound water molecule. This conversion of bound to free water does not appear to be simply a consequence of the aggregation of soluble crystallins into high molecular weight aggregates because studies with intact pig lenses, in which such processes were facilitated by heat, did not show similar changes. The region of the lens in which the barrier to diffusion develops at middle age corresponds to a transition zone in which the protein concentration is intermediate between that of the cortex and the nucleus. In cataractous lenses, the free-to-bound water ratio was not significantly different from that of age-matched normal lenses; however, total water content in the center of advanced nuclear cataractous lenses was slightly lower than in normal lenses. CONCLUSIONS: As the human lens ages, bound water is progressively changed to free water. Advanced nuclear cataract may be associated with lower total hydration of the lens nucleus.     

Nickel, chromium, manganese, iron and aluminum levels in human cataractous and normal lenses.

Atomic absorption spectrophotometry was used to detect lenticular nickel, chromium, manganese, iron and aluminum levels in 37 senile cataractous and 9 normal human lenses. The nuclear parts of the lenses were used for the study in both groups. It was found that the concentrations of nickel and iron were significantly higher, and those of chromium, manganese and aluminum were significantly lower in human cataractous lenses than those in normal human lenses. None of the elements showed a significant difference according to sex in both groups. As remarkable differences in chromium, manganese, iron, aluminum and nickel levels exist in cataractous lenses, all the five elements may play some roles in cataractogenesis.     

Argpyrimidine, a blue fluorophore in human lens proteins: high levels in brunescent cataractous lenses

PURPOSE. To determine whether the human lens contains argpyrimidine, a modification of arginine by methylglyoxal, to establish how argpyrimidine content relates to lens aging and cataract formation. METHODS. A monoclonal antibody was used to measure argpyrimidine by a competitive ELISA in water soluble (WS) and insoluble (WI) lens fractions from young, aged, nuclear cataractous, and brunescent cataractous lenses. Brunescent cataractous lens proteins were digested by enzymes, the digest was subjected to HPLC, and the eluate was analyzed for argpyrimidine. Lens proteins from aged lenses (from donors 65 to 80 years of age) were fractionated on a Sephadex G-200 column, and the crystallins were tested for argpyrimidine. RESULTS. The competitive ELISA showed two to three times as much argpyrimidine in water-insoluble proteins as in water-soluble proteins. Although no clear cut increase with the age of the lens donors in either the water-soluble or the insoluble protein fractions was found, the argpyrimidine levels in brunescent cataractous lenses were significantly higher (254.0 +/- 155 pmol/mg protein, P < 0.005) than in age-matched, aged (16.1 +/- 8 pmol/mg) or nuclear cataractous lenses (49.0 +/- 26 pmol/mg). Lenses from diabetic individuals showed a modest increase (50.3 pmol/mg) compared with age-matched normal lenses. HPLC results provided additional evidence that human lenses contain argpyrimidine. Western blotting experiments showed consistently stronger reactions with cataractous lens proteins than those from noncataractous lenses, and argpyrimidine was found in both crystallin monomers and polymers. All crystallins and several cross-linked high-molecular-weight aggregates reacted with the antibody to argpyrimidine, but a protein of approximately 28 kDa in the alpha-crystallin fraction displayed the greatest immunoreactivity. CONCLUSIONS. Methylglyoxal modifies arginine within the human lens, and the changes occur at a much higher rate in brunescent lens proteins than in either nuclear cataractous or normal lenses. All crystallins contained argpyrimidine and covalently cross-linked aggregates. This is the first report of immunologic evidence for an arginine modification in the human lens by a physiologically important alpha-dicarbonyl compound.     

Influence of age,diabetes, and cataract on calcium,lipid-calcium,and protein-calcium relationships in human lenses

PURPOSE: Calcium is elevated in most cataractous human lenses and may contribute to cataractogenesis. In this study, age-related changes were examined in the total calcium content of clear human lenses and the binding of calcium to lens lipids and proteins. METHODS: Total lens calcium was determined by atomic absorption spectroscopy. Calcium binding was measured by light scattering and measurement of calcium by atomic absorption spectroscopy in bound and unbound fractions. RESULTS: The calcium content of clear human lenses decreased between 18 and 55 years of age and increased between 55 and 75 years, as well as in the presence of cataract. Total calcium levels in clear lenses from subjects with insulin-dependent diabetes did not differ from that in lenses of age-matched control subjects. In vitro binding studies have shown that lens lipids can bind nearly all the calcium present in the human lens. Age and cataract diminished the capacity of lens lipids to bind calcium. Calcium-induced light-scattering, measured in vitro for lens proteins, correlated with increasing age and cataract. CONCLUSIONS: The data support the hypothesis that increased intracellular calcium concentrations and a diminished capacity of lens lipids to bind to calcium initiate a cascade of events that culminates in increased light-scattering from lipids and especially proteins. Calcium binding to lipid membranes cannot directly contribute to light-scattering in cataractous lenses. It has been suggested that most of the diffusible calcium in the lens is in the intercellular spaces and that lens lipids in the outer leaflet of the bilayer bind to that calcium. If so, this could account for the 150-fold difference between free and bound calcium levels in the lens.     

Deaminated UV filter 3-hydroxykynurenine O-beta-D-glucoside is found in cataractous human lenses

Analysis of UV filter levels in 48 cataractous human lenses was performed with the use of HPLC. A new chromophore with the absorption maximum at 410nm and molecular mass of 369Da was detected and assigned as deaminated 3-hydroxykynurenine O-beta-D-glucoside (3OHCKAG). Cataractous lenses are characterized by the wide range of the UV filter concentrations and remarkably lower levels of UV filters and glutathione than published for the normal lenses. No correlation between the lens age and the level of UV filters has been found in cataractous lenses.     

Aminopeptidase III activity in normal and cataractous lenses

Aminopeptidase III activity was demonstrated in extracts from several different mammalian lenses by the hydrolysis of Arg-MCA at pH 6.0. No more than a two-fold difference was seen in overall specific activity. Sections of bovine lenses were removed from the periphery to the center and assayed. A sharp decline in activity was observed in the inner cortical region, and little or no activity was observed in the lens nucleus. This correlated with an increase in the presence of low molecular weight peptides as determined by SDS polyacrylamide gel electrophoresis. The properties of the aminopeptidase from human lens tissue were the same as those previously reported for the purified enzyme from bovine lens. The aminopeptidase activity of normal and cataractous lenses was compared using 4 different substrates. The cataractous lenses had significantly less total aminopeptidase activity. However, little difference in specific activity was observed based on soluble lens protein content. Similarly, electrophoretic separations of normal and cataractous soluble proteins showed little or no differences in the content of low molecular weight peptides. Therefore, this major human lens aminopeptidase remains functional in the cataractous state.     

Analysis of glucose-6-phosphate dehydrogenase in anterior subcapsular and mixed cataractous lenses.

The study was undertaken to determine the different concentrations of glucose-6-phosphate dehydrogenase (G-6-PD) in anterior subcapsular and mixed cataractous lenses. In both types of cataractous lenses an increased concentration of G-6-PD was observed in 41-50 year age groups and a lower concentration of G-6-PD was noted in 61 year and above age groups. Cataractous lenses of females have a higher content of G-6-PD than males. In anterior subcapsular cataractous lenses, a lower concentration of G-6-PD was noted than in mixed lenses.     

Spectral studies on normal and cataractous intact human lenses

Spectral characteristics are presented for intact normal and cataractous human lenses. By comparison with healthy intact lenses, no new spectral features (and therefore pigmentous species) are observed in brunescent cataract. However, in this cataract, a buildup in the components giving rise to high wavelength absorption (400–500 nm) is observed relative to that at lower wavelengths (～ 300 nm). A model of protein tryptophan depletion via oxidation, resulting in protein pigmentation is considered, based on spectral evidence.