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(Investigative Ophthalmology and Visual Science. 2003;44:2059-2066.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-0345
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Influence of Age, Diabetes, and Cataract on Calcium, Lipid-Calcium, and Protein-Calcium Relationships in Human Lenses

Daxin Tang,1 Douglas Borchman,1 Marta C. Yappert,2 Gijs F. J. M. Vrensen,3 and Vittorio Rasi4

1From the Departments of Ophthalmology and Visual Science and 2Chemistry, University of Louisville, Louisville, Kentucky; the 3Lens and Cornea Research Unit, The Netherlands Ophthalmic Research Institute, Amsterdam, The Netherlands; and 4Udine, Italy.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
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.

It has been almost a century since it was shown that calcium is elevated in cataractous lenses1 and 75 years since total lens calcium was measured in clear lenses.2 More recent work has confirmed a more than 23-fold increase in total lens calcium with cataract (Tables 1 2) .1 2 3 4 5 6 7 8 9 Maintenance of calcium homeostasis is critical to the clarity of the lens.12 13 14 The inward passive diffusion of calcium, perhaps through a nonspecific cation channel,15 is countered by the actions of the plasma and sarco- and endoplasmic reticular Ca2+-adenosine triphosphatase (ATPase) pumps.16 17 18 19 20 21 With age, the increased entry of calcium into clear lenses is offset by an increase in the activity of Ca2+-ATPase pumps.22 With cataract, however, lens membrane permeability increases further,23 24 25 26 total lens calcium is elevated,1 2 3 4 5 6 7 8 9 and Ca2+-ATPase activity is decreased by 50%.27 The Ca2+-ATPase pump is sensitive to lipid order,28 29 which changes with age29 and cataract.27 31 32 Accordingly, the decrease of Ca2+-ATPase activity with cataract27 may be a consequence of lipid structural changes,27 31 32 an increase in Ca2+-ATPase oxidation, or both.33 34 35


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  		TABLE 1. Calcium in Clear Lenses

 

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  		TABLE 2. Calcium in Cataractous Human Lenses

 
Elevated calcium levels are related to numerous processes: activation of proteases,36 37 inhibition of Na,K-ATPase activity,38 39 cell growth,40 protein synthesis,41 disintegrative globulization,42 calcium influx,43 cell death,44 increased membrane permeability, and aggregation of proteins14 45 46 47 and lipids.48 49 50 All these factors could contribute to alterations in lens molecular structure and increased light-scattering by the lens. Hightower and Reddy7 showed that lens calcium content correlates with opacity in cataractous human lenses.

As determined by electrodes, the total calcium concentration (Table 1) is approximately 150 times higher than that of free calcium in human lenses,15 and it also is twice as high as the calcium concentration in the aqueous humor.8 These differences suggest that calcium is sequestered in intracellular compartments or spaces, is bound to components of the lens, or both. Duncan and van Heyningen6 noted that "elevated sodium could arise from Donnan forces alone, whereas elevated calcium must require the action of additional binding or complexing forces."

Intracellular compartments are present only in the outer cortical fibers of the lens. Accordingly, compartmentalization of calcium cannot account for the 150-fold difference between total calcium and free calcium in the cytoplasm of clear lenses. Furthermore, the nuclear region of the human lens, which has no organelles, contains twice as much calcium per dry weight as does the cortical region.6

ß-Crystallin is not likely to bind calcium in the lens, because the dissociation constant (Kd) of 0.38 mM51 is several orders of magnitude higher than the free calcium concentration of the lens.15 Calcium-binding proteins such as calmodulin have Kd values in the lower 3-µM range.52 53 54 Although these proteins can be expected to bind to calcium in the lens, they are present at an insufficient concentrations to account for the 100-fold difference between free and bound calcium.

Calcium oxalate has been found in rare Morgagnian cataracts and in 39% of other cataracts.55 56 57 However, calcium oxalate crystals have never been observed in clear lenses of individuals younger than 50 years55 57 and therefore cannot account for the difference between free and bound calcium in clear, young lenses.

An electron tomographic study has shown that a considerable amount of calcium in the human lens is located in intercellular spaces, where it is bound to the outer leaflet of the bilayer.58 There is evidence that this is the case in rat lens as well.59 It has been shown that calcium can bind to membranes composed of synthetic lipids,48 50 thereby increasing light scattering. Membrane properties, such as permeability, membrane density, and hydration have been related to the intensity of light scattering from lipids.60 61 These studies encouraged us to conduct this in vitro exploration of the relationships between light-scattering and calcium-lipid-binding in human lenses. We also measured age-related changes in the total lens calcium content of human lenses, which has not previously been studied by atomic absorption spectroscopy.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Clear lenses were obtained from deceased human donors within 8 hours after death, from the Kentucky Lions Eye Bank (Louisville, KY) and the University of Kentucky Lions Eye Bank (Lexington, KY), and then frozen in liquid nitrogen. Clear lenses from eyes of donors who had been insulin-dependent for at least 5 years were noted. All human lenses were collected with informed consent. Bovine lenses were obtained from a slaughterhouse within 3 hours after the death of the animals. Reagents, bovine brain sphingomyelin, and {alpha}-crystallin were purchased from Sigma Chemical Co. (St. Louis, MO). With patients’ permission, an author (VR) collected cataractous lenses after performing extracapsular cataract extractions in Udine and Rome, Italy. All experiments were performed in accordance with the Declaration of Helsinki.

Extraction of Human Lens Lipid
Lipid was extracted from human lenses by using a monophasic extraction protocol.62 To remove oxygen, all solvents were bubbled with argon gas. The extraction was performed in an argon atmosphere. Glass centrifuge tubes were used throughout the extraction. Each pair of lenses was put in a glass centrifuge tube containing 10 mL of methanol. Lenses were cut with a metal spatula and sonicated with a microprobe sonicator (Branson; Ultrasonics Co., Danbury, CT) three to four times for 15 seconds, with a 30-second pause between sonication bursts to ensure that the samples were not heated. The solution was centrifuged at 5000 rpm for 1 hour and the supernatant decanted into another centrifuge tube, leaving a small amount of the upper layer to avoid disrupting the pellet. The methanol in the supernatant was evaporated with a rotary evaporator (Buchi Rotavapor 011; Brinkman Instruments, Inc., Westbury, NY). Hexane and isopropanol (2:1 vol/vol, 10 mL) were added to the dry lipid film and gently sonicated with a microprobe for 15 seconds. The solution was transferred to a centrifuge tube and centrifuged at 5000 rpm for 1 hour. The lipid-containing supernatant was decanted into another tube, taking care not to disturb the pellet, and the hexane and isopropanol were then evaporated with the rotary evaporator (Buchi Rotavapor 011; Brinkman Instruments, Inc.).

Preparation of Large Unilamellar Vesicles of Human Lens Lipid
Human lens lipids (milligram range) dissolved in chloroform and methanol (7/3, vol/vol) were dried under nitrogen in glass test tubes, then lyophilized for 6 hours to remove remaining organic molecules. The dried lipid was suspended in 5 mM HEPES and 100 mM KCl buffer (pH 6.9). The dispersion was mixed with a vortex mixer (Scientific Industries Inc., Bohemia, NY) for 5 minutes at 45°C to make multilamellar vesicles. The vesicles were cooled to 4°C for 30 minutes and heated to 45°C for 30 minutes. We repeated the heating and cooling cycle three times to ensure that vesicles were completely formed. We prepared large unilamellar vesicles (LUVs) from the multilamellar vesicles at 45°C, using a lipid extruder (Lipex Biomembranes, Vancouver, British Columbia, Canada) and polycarbonate membranes (Whatman Nuclepore, Pleasanton, CA) according to the method of Hope et al.63

Measurement of Light-Scattering Intensity
Each sample contained 1.25 mg of LUVs in 1.5 mL 5 mM HEPES buffer and 100 mM KCl buffer (pH 6.9). To determine the effect of calcium on the light-scattering intensity of LUVs, we added 1.0 M CaCl2 stock solution to the LUV solution to give the appropriate final calcium concentration, ranging from 0 to 8 mM, and then incubated the samples for 16 hours. Light-scattering was measured with a photon-counting spectrofluorometer (PC1; ISS, Champaign, IL) at 90° with respect to the direction of the incident light. Radiation at 360 nm was used for excitation. During the measurement of light-scattering, samples were stirred with a magnetic stirrer. Data were averaged from at least 200 scans.

Measurement of Calcium Binding
We measured light-scattering and then binding of calcium to lipid. After centrifuging the lipids at 150,000g for 1 hour, we decanted and diluted the supernatant and measured its calcium content by atomic absorption spectroscopy (Analyst 100 spectrometer; Perkin Elmer, Wellesley, MA). We compared the calcium content of the supernatant, representing unbound calcium, with the total calcium content of samples prepared in the same way but with no lipid. Bound calcium was calculated as the difference between the calcium content of samples containing lipid and those without lipid. The composition of bovine lens phospholipids was estimated from 31P-NMR and chemical composition studies.64 We estimated that the average lipid molecular mass of bovine phospholipids and cholesterol is 575 g/mole.

Measurement of Human Lens Calcium Content
For the determination of total calcium in pooled samples, we used six clear human lenses and eight human lenses with cataracts (four exclusively cortical and four with posterior subcapsular opacity). Lenses were fragmented with a metal spatula and sonicated with a microprobe sonicator (Branson; Ultrasonics Co.) in an argon-bubbled methanol solution. We evaporated the methanol solution under a stream of nitrogen gas and then lyophilized the samples for 16 hours to remove water and methanol. After weighing the samples, we added approximately 2 mL deionized water to each of them. Calcium content was measured by atomic absorption spectroscopy.

To determine the total amount of calcium in individual lenses, we lyophilized lenses for 18 hours to remove all water and weighed them three times to 0.01 mg with a relative SD of 0.8%. Each lens was mixed with and brought to a volume of 10 mL with concentrated nitric acid containing 0.1% LaCl3 (7H2O). The lenses were sonicated in a bath-type sonicator (Model FS-9; Fisher Scientific, Pittsburgh, PA) for 45 minutes. Eighteen hours later, the lenses were completely solubilized.

For atomic absorption spectroscopy, a 100-mM stock solution of CaCl2 was prepared by mixing CaCO3 with 75 mL of concentrated HCl. CaCO3 was used because it is not hydroscopic, as is CaCl2. Ten calcium standards ranging from 0.5 to 10 µM were prepared from the stock solution of calcium. Each standard solution contained 0.1% LaCl3 (H2O) and 10 mL concentrated nitric acid in a 200-mL volume. Standards were measured before and after samples, and samples were measured three times with a relative SD of 3.8%.

Measurement of the Influence of Calcium on Light-Scattering by {alpha}-Crystallin
We used 11 samples, each containing 500 mg {alpha}-crystallin in 2 mL of 5 mM HEPES and 100 mM KCl buffer (pH 6.9). Calcium chloride stock solution was added to a final calcium concentration ranging from 0 to 220 mM. After 16 hours of incubation at 37°C, the light-scattering intensity was measured at 21°C.

Measurement of the Effect of Calcium on the Protein of Young, Old, and Cataractous Lenses
Delipidated proteins were obtained from the pellets after lipid extraction. Each pellet was dried and weighed, then resuspended in 5 mM HEPES and 100 mM KCl buffer (pH 6.9). The resultant stock solution was stored at 4°C. Each protein stock solution was diluted to 1 mg protein in 2 mL HEPES buffer. The final calcium concentration was adjusted to 0, 2, 4, 5, and 6 mM with CaCl2. The protein suspension was incubated at 37°C for 16 hours under argon. Light-scattering intensities were measured at 21°C, as described earlier.

Measurement of LUV Size
The size of lipid vesicles was estimated by cryoelectron microscope. We dipped bare grids into the samples, blotted them with filter paper, and vitrified the fluid film remaining on the grids in liquid ethane at -193°C. The samples were examined with the electron microscope at 100 kV and -174°C, in a vacuum of more than 10-8 mbar. For freeze-fracture microscopy, the samples were vitrified, fractured at a temperature of -120°C in a vacuum of more than 5 x 10-7 mbar, replicated with 2-nm Pt at an angle of 45°, and strengthened with 20-nm carbon. The replicas were cleansed with household bleach, rinsed with distilled water, and collected on bare 300-mesh grids.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
The total calcium content decreased significantly (P = 0.003) in clear human lenses aged between 18 and 55 years. It then increased significantly (P =0.002) with age between 55 and 75 years (Fig. 1A) . The relative SD of total calcium in paired lenses from the same individual was 10%.



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  		FIGURE 1. (A) Total calcium in clear human lenses. Solid line: linear regression fit of data using a line order of 5. Dotted line: 95% confidence limits. Large open circles encompass overlapping data points. (B) Data correspond to lenses in (A). No postmortem-time-dependent changes in calcium were observed. Solid lines: linear regression fit of data using a line order of 1. (C) Data are for the lenses in (A). The time from death to dissection was not significantly different in the different age groups.

 
The time between death and lens dissection, which ranged from 3 to 9 hours, did not influence the level of total calcium (Fig. 1B) . The average time of death to the time of lens dissection was similar in at all ages (Fig. 1C) . Total calcium increasedsignificantly with cataract. In clear lenses from eyes of insulin-dependent donors, total calcium levels did not significantly differ from the levels in age-matched control lenses (Fig. 2) .



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  		FIGURE 2. Cataractous lenses were from a pool of lenses having four cortical and three posterior subcapsular cataracts, but no nuclear opacities. The control group of clear lenses was the same as that shown in Figure 1 . Donors had been insulin dependent for (at least) 5 years. Data are presented as average ± SEM in individual lenses, except for the cataractous lens data, in which ± represents the experimental deviation. Ten diabetic and 13 control lenses were examined.

 
The equivalent of either two bovine brain sphingomyelin molecules or two bovine lens lipids (phospholipid and cholesterol) had the capacity to bind approximately one calcium molecule with half-maximal binding at 2.79 ± 0.02 mM (SEM; Fig. 3 ).



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  		FIGURE 3. Calcium binding to LUVs formed in extracted and extruded bovine lens lipids ({circ}) and bovine brain sphingomyelin (•). The experiment was performed at 37.5°C. Data are expressed as ± SEM. An average molecular mass of 545 g/mol was used to calculate moles of bovine lens lipid.

 
Figure 4A shows a cryoscanning electron micrograph of human lens lipid LUVs. Two extrusions of the same lipid were made on different days. In young lenses (34 ± 16 years), the average diameter of LUVs was 260.8 ± 16 nm (n = 2568). Vesicle aggregation was evident after 1 hour of incubation with 1 mM free calcium (Fig. 4B) .



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  		FIGURE 4. Cryoelectron micrograph of LUVs extruded from older human lenses. (A) No calcium; (B) 1 hour of incubation with 1 mM free calcium at 36.5°C.

 
With increasing age and cataract, the calcium-lipid-binding capacity (Fig. 5) and calcium-induced light-scattering (Fig. 6B) of human lens lipids decreased above a free calcium concentration of 2 mM. The level of half-maximum binding of calcium to lens lipids varied much more (2.6–5 mM) than that measured in bovine lens lipids or sphingomyelin (Fig. 3) . This is a consequence of the increase in the relative experimental error in assessing the 100-fold lower binding capacity of human lens lipids.



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  		FIGURE 5. Calcium-binding capacity in three pools of human lenses. A saturating calcium concentration of 6 mM was used. The average age of the young, older, and cataractous lens tissue is 30.3 ± 8.4, 75.6 ± 5.7, and 69.3 ± 8 years, respectively.

 


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  		FIGURE 6. Light-scattering (360 nm, 90°) at 36°C. (A) Change in intensity after adding 1 mM calcium to human lens lipid LUVs (B) bovine lens ({blacksquare}) lipid LUVs; human lens ({circ}) lipid LUVs. The average age of the younger, older, and cataractous pool of lens tissue is 30.3 ± 8.4, 75.6 ± 5.7, and 69.3 ± 8.5 years, respectively. (C) Human lens homogenates containing protein from the same three pools of human lenses used in Figures 1 and 4 . (D) Bovine lens {alpha}-crystallin.

 
Addition of 1 mM free calcium to LUVs from human lens lipids increased light-scattering in all 12 samples examined (Fig. 6A) . Calcium-induced light scattering for human lens proteins increased with increasing age and cataract (Fig. 6C) . Scattering from bovine lens {alpha}-crystallin also increased with increasing calcium content, reaching a maximum value near 60 mM of calcium (Fig. 6D) . A nonphysiological range of calcium was used to demonstrate that the Kb for calcium binding to {alpha}-crystallin was above physiological levels (Fig. 6D) . Clear human lenses contained 6 mg/g phospholipid of lens tissue and 9.6 mg/g cholesterol of lens tissue.62


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Total Lens Calcium and Age
Relative to the dry weight of the lens, the total calcium in clear lenses decreased from 1.2 nanomoles/mg near the age of 20 years to 0.98 nanomoles/mg at 60 years of age, then increased to 2 nanomoles/mg near 75 years of age (Fig. 1A) . For total lens calcium to decrease between the ages of 20 and 60 years (Fig. 1A) , membrane permeability must decrease, Ca2+-ATPase activity must increase, or both must occur. Previous studies have shown that Ca2+-ATPase pump activity increases with age22 and that there is little change in cation permeability between 20 and 60 years.15 In clear human lenses, the increase in total calcium after 60 years of age (Fig. 1A) is a consequence of increased membrane permeability,15 which begins after the age of 50 years and is not offset by an age-related increase in Ca2+-ATPase pump activity.22 The levels of free calcium measured electrochemically in clear human lenses increased from approximately 10 µM in younger lenses to 12 to 20 µM in lenses of eyes from donors more than 50 years of age.15 There was a similar increase in total calcium in lenses near 55 years of age (Fig. 1A) .

In clear lenses, the nonlinear changes in total calcium (Fig. 1A) and free calcium15 do not correlate with the linear increase in lens opacity observed with increasing age.65 However, our data do not address possible changes in the regional distribution of calcium within clear lenses that could correlate with lens opacity. The data suggest that it is possible that as total calcium surpasses threshold value of 8 millimoles/kg lens water (Fig. 1A) , cataracts may develop (discussed in the next section). The level of 3.2 millimoles/kg total calcium of lens water is similar to the average value of 2.9 millimoles of total calcium per kilogram of lens water measured by others (Table 1) .

Eckhert66 noted a high degree of variability between individuals with respect to the number of cations in all eye tissues, reporting a relative SD of 43% among clear lenses of unreported ages. The large changes in calcium level with age (Fig. 1A) may have contributed to the variability in data in Eckhert,66 as well as the wide range of total lens calcium content reported by many investigators (Table 1) . We found that the variation in total calcium within individuals (paired lenses) was only 10% compared with the 38% that Eckhert reported.66

Postmortem changes in calcium probably did not contribute to the changes we observed in total calcium with increasing age. As noted earlier, differences in time between death and lens dissection did not influence total calcium levels (Figs. 1B 1C) . That there was no change in the lens after death is not surprising, considering that the energy status and integrity of human lenses persist to 72 hours after death. Between 72 and 86 hours after death, the ATP/inorganic phosphate (Pi) ratio decreases by only 8%.67 Furthermore, a decline in pH is not observed until 144 hours after death.67

Where Is the Calcium in Clear Lenses? Intracellular Compartmentalization, Calcium-Protein, Calcium-Oxalate, and Calcium-Lipid Binding
The total calcium concentration in the human lens (Table 1) is approximately 150 times higher than that of free calcium, as determined by electrodes,15 and twice as high as the calcium concentration of the aqueous humor.8 As discussed in the introduction, neither calcium bound to proteins, nor oxalate, nor calcium sequestered in intracellular compartments can account for the difference between bound and free calcium in the lens. Our data show that only at nonphysiological calcium concentrations did calcium bind to {alpha}-crystallin (Fig. 6D) , confirming that there is minimal binding of calcium to the crystallins in clear human lenses at the free calcium concentration of 20 µM.51

The present study also shows that lens lipids have a high capacity for binding calcium (Fig. 5) . Indeed, they can bind approximately 25 nanomoles of calcium, which represents between 40% and 100% of the total calcium reported in clear human lenses (Table 1) . Because the Kb for calcium binding to lipids is 2.6 mM (Fig. 3) and because free intracellular calcium is in the micromolar range, little calcium is expected to bind to the lipids facing the cytoplasm. However, if most of the diffusible calcium is in the intercellular spaces,58 59 68 and if these spaces are 15 nm wide69 based on the relative area of the intracellular-to-intercellular space, we calculate the total amount of calcium in the intracellular spaces of a young lens would be 6 moles/kg of water. The lens lipids in the outer leaflet of the bilayer would bind to calcium at these concentrations.

We found that the binding capacity of human lens lipids was approximately 100 times less than that of bovine lens lipids and bovine brain sphingomyelin (compare Figs. 3 and 5 ). Compositional differences account for these differences in binding. Regarding phospholipid content, the human lens contains approximately 3 times more cholesterol64 70 than does the bovine lens. Our preliminary studies indicate that cholesterol greatly reduces the capacity of sphingomyelin membranes to bind calcium (Borchman D, unpublished results, 2000). In addition, the major lipid in the adult human lens is dihydrosphingomyelin (~52%), whereas the bovine lens contains little dihydrosphingomyelin but, depending on the region, has a relatively large amount of sphingomyelin.64 Molecular interactions may also explain the difference between the calcium-binding capacities of bovine and human lens lipids. Dihydrosphingolipids, which, as noted, are predominant in the human lens, pack together more strongly and more closely71 than do sphingomyelin molecules.72 As a consequence of their tighter packing, calcium appears to interact with sphingomyelin much more effectively than it does with dihydrosphingomyelin.73

Clear Lens Total Calcium and Diabetes
Calcium homeostasis was impaired in at least 15 tissues from humans with diabetes74 and in lenses from diabetic rats75 and rabbits.76 Calcium-phosphate deposits have been identified in human lenses with diabetic cataracts.77 Because of the link between diabetic cataracts and calcium, we examined clear lenses from donors who had been insulin-dependent for at least 5 years. The absence of any statistical difference between the total calcium of these lenses and those from age-matched control subjects (Fig. 2) indicates that factors other than elevated total calcium must contribute to the predisposition toward formation of cataract in those with diabetes. Our findings do not eliminate the possibility that changes in free calcium or alterations in the local distribution of total calcium contribute to diabetic cataracts.

Lens Calcium and Cataract
We found that the total calcium in human lenses with cortical cataracts was four times higher than that in clear lenses (Table 2 ; Fig. 2 ). Our value of 12 millimoles calcium per kilogram of lens water is consistent with the values reported by three other investigators, which ranged from 4 to 65 millimoles calcium per kilogram of lens water (Table 2 ; Fig. 2 ). On the basis of the results in 10 studies that compared clear and cataractous lenses (Table 2) , it can be concluded that total calcium is elevated in almost all cataracts by an average of 2300%. Even pure nuclear cataracts have 2 to 26 times more total calcium than do clear lenses (Table 2) . The average level of 22 millimoles calcium per kilogram of lens water in cataractous lenses is remarkable (Table 2) . It is also 18 times higher than the amount of calcium in the aqueous humor.8 The increase in total calcium with development of cataract cannot be attributed to lipid-calcium binding, because cataractous lens lipids have a diminished capacity to bind calcium (Fig. 5) .

As total calcium in the lens increases, we hypothesize that higher intercellular calcium concentrations, coupled with decreased Ca2+-ATPase activity27 and greater membrane permeability15 22 23 24 25 26 could lead to elevated free intracellular calcium levels. This could, in turn, induce the formation of calcium oxylate crystals55 56 and contribute to trigger a cascade of events that culminate in increased light-scattering from proteins (Fig. 6C) and, to a lesser extent, from lipids (Fig. 6B) .


   Footnotes
 
Supported by National Eye Institute Grant EY07975 (DB), the Kentucky Lions Eye Foundation, and an unrestricted grant from Research to Prevent Blindness.

Submitted for publication April 5, 2002; revised September 10 and December 20, 2002; accepted January 16, 2003.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Douglas Borchman, Kentucky Lions Eye Center, University of Louisville, 301 E. Muhammad Ali Blvd., Louisville, KY 40202; borchman{at}louisville.edu.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Burge, WE. (1909) Analysis of the ash of the normal and the cataractous lens Arch Ophthalmol 38,435-450
  2. Adams, DR. (1929) The role of calcium in senile cataract Biochem J 23,902-912
  3. Duncan, G, Bushell, AR. (1975) Ion analysis of human cataractous lenses Exp Eye Res 20,223-230[CrossRef][Medline][Order article via Infotrieve]
  4. Rasi, V, Costantini, S, Moramarco, A, Giordano, R, Giustolisi, R, Balacco Gabrieli, C. (1992) Inorganic element concentrations in cataractous human lenses Ann Ophthalmol 24,459-464[Medline][Order article via Infotrieve]
  5. Jedziniak, JL. (1976) On the calcium concentration of cataractous and normal human lenses and protein fractions of cataractous lenses Exp Eye Res 23,325-332[CrossRef][Medline][Order article via Infotrieve]
  6. Duncan, G, van Heyningen, R. (1977) Distribution of non-diffusible calcium and sodium in normal and cataractous human lenses Exp Eye Res 25,183-193[CrossRef][Medline][Order article via Infotrieve]
  7. Hightower, KR, Reddy, VN. (1982) Calcium content and distribution in human cataract Exp Eye Res 34,413-421[CrossRef][Medline][Order article via Infotrieve]
  8. Ringvold, A, Sagen, E, Bjerve, KS, Folling, I. (1988) The calcium and magnesium content of the human lens and aqueous humour Acta Ophthalmologica 66,153-156[Medline][Order article via Infotrieve]
  9. Dilsiz, N, Olcucu, A, Atas, M. (2000) Determination of calcium, sodium, potassium and magnesium concentrations in human senile cataractous lenses Cell Biochem Funct 18,259-262[CrossRef][Medline][Order article via Infotrieve]
  10. van Heyningen, R. (1972) The human lens. 3. Some observations on the post-mortem lens Exp Eye Res 13,155-160[CrossRef][Medline][Order article via Infotrieve]
  11. van Heyningen, R. (1972) The human lens. II. Some observations on cataracts removed in Oxford, England Exp Eye Res 13,148-154[CrossRef][Medline][Order article via Infotrieve]
  12. Hightower, KR. (1985) Cytotoxic effects of internal calcium on lens physiology: a review Curr Eye Res 4,453-459[Medline][Order article via Infotrieve]
  13. Duncan, G, Williams, MR, Riach, RA. (1994) Calcium, cell signaling and cataract Prog Retinal Eye Res 12,623-651[CrossRef]
  14. Clark, JI, Mengel, L, Bagg, A, Benedik, GB. (1980) Cortical opacity, Ca2+ concentration and fiber membrane structure Exp Eye Res 31,399-410[CrossRef][Medline][Order article via Infotrieve]
  15. Duncan, G, Hightower, KR, Gandolfi, SA, Tomlinson, J, Maraini, G. (1989) Human lens membrane cation permeability increases with age Invest Ophthalmol Vis Sci 30,1855-1859[Abstract/Free Full Text]
  16. Chiesa, R, Sredy, J, Spector, A. (1985) Phosphorylated intermediates of two Ca++-ATPases in membrane preparations from lens epithelial cells Curr Eye Res 4,897-903[Medline][Order article via Infotrieve]
  17. Zeng, J, Borchman, D, Paterson, CA. (1995) ATPase activities of rabbit and bovine lens epithelial microsomes: a continuous fluorimetic assay study Curr Eye Res 14,87-93[Medline][Order article via Infotrieve]
  18. Duncan, G, Webb, SF, Dawson, AP, Bootman, MD, Elliott, AJ. (1993) Calcium regulation in tissue cultured human and bovine lens epithelial cells Invest Ophthalmol Vis Sci 34,2835-2842[Abstract/Free Full Text]
  19. Bian, L, Zeng, J, Borchman, D, Paterson, CA. (2000) Plasma membrane calcium ATPase gene expression in bovine lens epithelium Ophthalmic Res 32,100-105[CrossRef][Medline][Order article via Infotrieve]
  20. Liu, L, Bian, L, Borchman, D, Paterson, CA. (1999) Expression of sarco/endoplasmic reticular Ca2+-ATPase in human lens epithelial cells and cultured human lens epithelial B-3 cells Curr Eye Res 19,389-394[CrossRef][Medline][Order article via Infotrieve]
  21. Churchhill, GC, Louis, CF. (1999) Imaging of intracellular calcium stores in single permeabilized lens cells Am J Physiol 276,C426-C431
  22. Borchman, D, Paterson, CA, Delamere, NA. (1989) Ca2+-ATPase activity in the human lens Curr Eye Res 8,1049-1054[Medline][Order article via Infotrieve]
  23. Pasino, M, Maraini, G. (1982) Cation pump activity and membrane permeability in human senile cataractous lenses Exp Eye Res 34,887-893[CrossRef][Medline][Order article via Infotrieve]
  24. Lucas, VA, Duncan, G, Davies, PD. (1986) Membrane permeability characteristics of perfused human senile cataractous lenses Exp Eye Res 42,151-165[CrossRef][Medline][Order article via Infotrieve]
  25. Duncan, G, Jacob, TJ. (1984) Human cataract formation CIBA Found Symp 106,132-148[Medline][Order article via Infotrieve]
  26. Gandolfi, SA, Tomba, CM, Maraini, G. (1985) 86-Rb Efflux in normal and cataractous human lenses Curr Eye Res 4,753-758[Medline][Order article via Infotrieve]
  27. Paterson, CA, Zeng, J, Hussenini, Z, et al (1997) Calcium ATPase activity and membrane structure in clear and cataractous human lenses Curr Eye Res 16,333-338[CrossRef][Medline][Order article via Infotrieve]
  28. Delamere, NA, Paterson, CA, Borchman, D, King, KL, Cawood, SC. (1991) Calcium transport, Ca-ATPase and lipid order in rabbit ocular lens membranes Am J Physiol 260,C731-C737
  29. Zeng, J, Zhang, Z, Paterson, CA, Ferguson-Yankee, S, Yappert, MC, Borchman, D. (1999) Ca2+-ATPase activity and lens lipid composition in reconstituted systems Exp Eye Res 69,323-330[CrossRef][Medline][Order article via Infotrieve]
  30. Borchman, D, Ozaki, Y, Lamba, OP, Byrdwell, WC, Yappert, MC. (1996) Age and regional structural characterization of clear human lens lipid membranes by infrared and near-infrared Raman spectroscopes Biospectroscopy 2,113-123
  31. Borchman, D, Lamba, OP, Yappert, MC. (1993) Structural characterization of lipid membranes from clear and cataractous human lenses Exp Eye Res 57,199-208[CrossRef][Medline][Order article via Infotrieve]
  32. Gooden, M, Takemoto, L, Rintoul, D. (1983) Evidence for reduced lipid order in plasma membranes from cataractous human lenses Curr Eye Res 2,367-375
  33. Ahuja, RP, Borchman, D, Dean, WL, et al (1999) Ca2+-ATPase kinetics and membrane oxidation relationships in lens epithelial microsomes Free Radic Biol Med 27,177-185[CrossRef][Medline][Order article via Infotrieve]
  34. Borchman, D, Delamere, NA, Paterson, CA. (1988) Selective inhibitions of membrane ATPases by hydrogen peroxide in the lens of the eye Basic Life Sci 49,1029-1033[Medline][Order article via Infotrieve]
  35. Borchman, D, Paterson, CA, Delamere, NA. (1989) Oxidative inhibition of Ca2+-ATPase in the rabbit lens Invest Ophthalmol Vis Sci 30,1633-1637[Abstract/Free Full Text]
  36. Yoshida, H, Murachi, T, Tsukahara, J. (1984) Limited proteolysis of bovine lens alpha crystallin by calpain, a Ca2+ dependent cysteine proteinase, isolated from the same tissue Biochim Biophys Acta 798,252-259[Medline][Order article via Infotrieve]
  37. Ireland, M, Maisel, H. (1984) Evidence for a Ca activated protease specific for lens intermediate filaments Curr Eye Res 3,423-429[Medline][Order article via Infotrieve]
  38. Delamere, NA, Borchman, D, Paterson, CA, Manning, RE. (1992) The influence of calcium upon rabbit lens sodium pump Invest Ophthalmol Vis Sci 34,405-412
  39. Hightower, KR, Hind, D. (1982) Cytotoxic effects of calcium on sodium-potassium transport in the mammalian lens Curr Eye Res 2,239-245[Medline][Order article via Infotrieve]
  40. Duncan, G, Riach, RA, Williams, MR, Webb, SF, Dawson, JR, Reddan, JR. (1996) Calcium mobilization modulates growth of lens cells Cell Calcium 19,83-89[CrossRef][Medline][Order article via Infotrieve]
  41. Hightower, KR. (1983) The influence of calcium on protein synthesis in the rabbit lens Invest Ophthalmol Vis Sci 24,1422-1426[Abstract/Free Full Text]
  42. Bhatnager, A, Dhir, P, Wang, LF, Ansari, NH, Lo, W, Srizastava, SK. (1997) Alterations in light transmission through single lens fibers during calcium-mediated disintegrative globulization Invest Ophthalmol Vis Sci 38,586-592[Abstract/Free Full Text]
  43. Thomas, GR, Sanderson, J, Duncan, G. (1999) Thapsigargin inhibits a potassium conductance and stimulates calcium influx in the intact rat lens J Physiol 516,191-199[Abstract/Free Full Text]
  44. Duncan, G, Wormstone, IM, Liu, CSC, Marcantonio, JM, Davies, PD. (1997) Thapsigargin-coated intraocular lenses inhibit human lens cell growth Nat Med 3,1026-1028[CrossRef][Medline][Order article via Infotrieve]
  45. Jedziniak, JA, Kinoshita, JH, Yates, EM, Hocker, LO, Bender, LG. (1972) Ca induced aggregation of bovine lens alpha crystallin Invest Ophthalmol Vis Sci 11,905-915[Abstract/Free Full Text]
  46. Spector, A, Rothschild, C. (1973) The effect of Ca upon the reaggregation of bovine alpha crystallin Invest Ophthalmol Vis Sci 12,225-231[Abstract/Free Full Text]
  47. Giblin, FJ, Hightower, KR, Ragatzki, PA, Reddy, VN. (1984) Calcium-induced high molecular weight proteins in the intact rabbit lens Exp Eye Res 39,9-17[CrossRef][Medline][Order article via Infotrieve]
  48. Brown, K. (1986) Atomic and Raman spectroscopy of the phosphatidic acid-calcium complex and bilayer phase Biochim Biophys Acta 862,134-140[Medline][Order article via Infotrieve]
  49. Bakas, LS, Disalvo, EA. (1991) Effect of encapsulated Ca2+ on the surface properties of curved phosphatidylcholine bilayers Biochim Biophys Acta 1065,114-120[Medline][Order article via Infotrieve]
  50. Bakas, LS, Disalvo, EA. (1989) Effect of asymmetric Ca2+ distribution on the bilayer properties of phosphatidylcholine-sonicated vesicles Biochim Biophys Acta 979,352-360[Medline][Order article via Infotrieve]
  51. Sharma, Y, Rao, CM, Narasu, L, Rao, NSC. (1989) Calcium ion binding to {alpha}- and to ß-crystallins J Biol Chem 264,12794-12799[Abstract/Free Full Text]
  52. van den Eijnden-van Raaij, AJM, de Leeuw, ALM, Broekhuyse, RM. (1986) Calcium-binding lens membrane proteins Doc Ophthalmol 61,255-265[CrossRef][Medline][Order article via Infotrieve]
  53. Broekhuyse, RM, van den Eynden-van Raay., (1986) Calcium binding proteins in lens and other tissues Lens Res 3,17-33
  54. Louis, CF, Johnson, R, Turnquist, J. (1985) Identification of the calmodulin-binding components in bovine lens plasma membranes Eur J Biochem 150,271-278[Medline][Order article via Infotrieve]
  55. Zimmerman, LE, Johnson, FB. (1958) Calcium oxalate crystals within ocular tissues Arch Ophthalmol 60,372-383[Abstract/Free Full Text]
  56. van Heyningen, R. (1972) The human lens I. A comparison of cataracts extracted in Oxford (England) and Shikarpur (W. Pakistan) Exp Eye Res 13,136-147[CrossRef][Medline][Order article via Infotrieve]
  57. Bron, AJ, Brown, NAP. (1987) Perinuclear lens retrodots: a role for ascorbate in cataractogenesis Br J Ophthalmol 71,86-95[Abstract/Free Full Text]
  58. van Marle, J, Jonges, R, Vrensen, GF, Dewolf, A. (1997) Calcium and its localization in human lens fibres: an electron tomographic study Exp Eye Res 65,83-88[CrossRef][Medline][Order article via Infotrieve]
  59. Vrensen, GFJM, Sanderson, J, Wilkekens, B, Duncan, G. (1995) Calcium localization and ultrastructure of clear and CMPS-treated rat lenses Invest Ophthalmol Vis Sci 36,2287-2295[Abstract/Free Full Text]
  60. Disalvo, EA. (1991) Optical properties of lipid dispersions induced by permeant molecules Chem Phys Lipids 59,199-206[CrossRef]
  61. Disalvo, EA. (1985) Thermodynamic factors determining the permeability barrier properties of lipid bilayers Chem Phys Lipids 37,385-397[CrossRef]
  62. Byrdwell, WC, Sato, H, Schwarz, A, Borchman, D, Yappert, MC, Tang, D. (2002) 31P-NMR Quantification and monophasic solvent purification of human and bovine lens phospholipids Lipids 37,1087-1092[CrossRef][Medline][Order article via Infotrieve]
  63. Hope, MJ, Bally, MB, Webb, G, Cullis, PR. (1985) Production of large unilamellar vesicles by a rapid extrusion procedure: characterization of size distribution, trapped volume and ability to maintain a membrane potential Biochim Biophys Acta 812,55-65[CrossRef]
  64. Borchman, D, Cenedella, RJ, Lamba, OP. (1996) Role of cholesterol in the structural order of lens lipids Exp Eye Res 62,191-197[CrossRef][Medline][Order article via Infotrieve]
  65. Bettelheim, FA, Ali, S. (1985) Light scattering of normal human lens. III. Relationship between forward and backscatter of whole excised lenses Exp Eye Res 41,1-9[CrossRef][Medline][Order article via Infotrieve]
  66. Eckhert, CD. (1983) Elemental concentrations in ocular tissues of various species Exp Eye Res 37,639-647[CrossRef][Medline][Order article via Infotrieve]
  67. Cheng, HM, Aguayo, JB. (1988) The energy status of lenses from human donor eyes Exp Eye Res 46,451-456[CrossRef][Medline][Order article via Infotrieve]
  68. Paterson, CA, Delamere, NA. (1982/1983) An analysis of 45Ca fluxes in rabbit lens Curr Eye Res 2,727-734[Medline][Order article via Infotrieve]
  69. Zampighi, G. (November 2001) Presented at Cooperative Cataract Research Meeting Hawaii.
  70. Borchman, D, Delamere, NA, McCauley, LA, Paterson, CA. (1989) Studies on the distribution of cholesterol, phospholipid, and protein in the human bovine lens Lens Eye Tox Res 6,703-724
  71. Ferguson-Yankey, S, Borchman, D, Taylor, KG, DuPre, DB, Yappert, MC. (2000) Conformational studies of sphingolipids by NMR spectroscopy. I. Dihydrosphingomyelin Biochim Biophys Acta 1467,307-325[Medline][Order article via Infotrieve]
  72. Talbott, CM, Vorobyov, I, Borchman, D, Taylor, KG, DuPre, DB, Yappert, MC. (2000) Conformational Studies of sphingolipids by NMR spectroscopy. II. Sphingomyelin Biochim Biophys Acta 1467,326-337[Medline][Order article via Infotrieve]
  73. Rujoi, M, Borchman, D, Taylor, KG, DuPre, DB, Yappert, MC. (2002) Interactions of Ca2+ with sphingomyelin and dihydrosphingomyelin Biophys J 82,3096-3104[Medline][Order article via Infotrieve]
  74. Levy, J. (1999) Abnormal cell calcium homeostasis in type 2 diabetes mellitus: a new look on old disease Endocrine 10,1-6[CrossRef][Medline][Order article via Infotrieve]
  75. Cekic, O, Bardak, Y. (1998) Lenticular calcium, magnesium, and iron levels in diabetic rats and verapamil effect Ophthalmic Res 30,107-112[CrossRef][Medline][Order article via Infotrieve]
  76. Hightower, KR, Riley, MV, McCready, J. (1989) Regional distribution of calcium in alloxan diabetic rabbit lens Curr Eye Res 8,517-521[Medline][Order article via Infotrieve]
  77. Lee do, H, Seo, Y, Joo, CK. (2002) Progressive opacification of hydrophilic acrylic intraocular lenses in diabetic patients (1) J Cataract Refract Surg 28,1271-1275[CrossRef][Medline][Order article via Infotrieve]



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