Free and Bound Water in Normal and Cataractous Human Lenses

Abstract

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.

The human lens is composed predominantly of lens fibers that are filled with a highly concentrated protein solution. The lack of organelles in the lens assists its transparency, but this also leads to an inability for regeneration and repair once the cells have differentiated. The lifespan of the components of the lens coupled with a lack of turnover make it a unique structural tissue in the human body and leaves it open to age-related disorders. Numerous investigations have been conducted into protein changes in the human lens, and myriad posttranslational modifications have been reported. ^{1 2 3} Few, however, have analyzed how these alterations affect the interaction of the proteins with other components, such as lenticular water. 

In addition to biochemical changes, pronounced physical changes also take place in the lens with age. Several studies have reported a change in the stiffness of the human lens with age. ^{4 5 6} One mechanism suggested for the increase in lens stiffness with age is compaction of nuclear fibers, and electron microscope data do show an apparent decline in the volume of human nuclear fiber cells with age. ⁷ Compaction is observed in animal lenses in which there are declines in nuclear water content with age. ⁸ In humans, this phenomenon does not appear to take place, and the water content of the lens center remains constant at approximately 65%. ^{8 9 10} A gradient in water content across the lens has been demonstrated using Raman spectroscopy, ⁹ microsectioning, and freeze drying. ¹¹ Refractive index is dependent on protein concentration, and this influences the focusing power of the lens. As fiber cells differentiate and mature, the proteins become more concentrated. We know very little about this process. 

Both aging and cataract are associated with increased insolubility of the proteins in the lens. However, the basis for the precipitation of proteins is not fully understood. Protein aggregation, some of which involves α-crystallin acting as a chaperone, is implicated. ¹² Crystallin insolubility could be influenced by interactions with other components in the lens, such as the cytoskeleton, membranes, and water. The interaction of water with proteins in the lens may well be vital for lens transparency. ¹³ When the balance is altered, as in the case of cortical cataract, ^{14 15} opacification can result. 

The purpose of this study was to investigate water changes in the lens with age. Normal human and cataractous lenses were investigated. Thermogravimetric analysis (TGA) was used to determine lens total water, and both TGA and differential scanning calorimetry (DSC) were used to analyze the ratio of free to bound water. 

Materials and Methods

Normal lenses from persons 15 to 85 years of age were collected from the Lions New South Wales Eye Bank at the Sydney Eye Hospital. These were transferred to Wollongong, Australia, and stored at −80°C. Analysis of lenses by DSC was typically carried out within 1 week of collection. Cataractous human lenses were sourced from an eye clinic in Rajkot, Gujurat, India, and were immediately frozen and transported frozen to Australia. They were then stored at −80°C until analyzed. Porcine eyes (approximately 6 months of age) were obtained from Crownpork (South Strathfield, NSW, Australia) and were typically used the same day. 

The work was approved by the human research ethics committee at the University of Wollongong. Graphs were prepared using SPSS software (Sigmaplot 8.02; SPSS Inc., Chicago, IL). 

Analysis of Nuclei from Normal and Cataractous Lenses

Samples of the nuclear region from normal and cataractous lenses were obtained by coring the lenses with a 4.5-mm trephine. The ends of the cylinders (approximately 1 mm) were removed. This core was then split into two nuclear sections. One sample was used for TGA (total water) and the other for DSC (free water). 

Analysis of Total and Free Water in Separated Lens Regions

For the analysis of free water in human lens regions, human lenses were sectioned using two trephines while frozen. These lenses had been stored at −80°C for less than 1 month. This provided three sections of 4.5-mm diameter (core) and two doughnut-shaped regions, one between 4.5 mm and 8 mm (intermediate) and the other larger than 8 mm (outer). Ends (approximately 1 mm) of the central core were removed, ensuring that it was composed entirely of nuclear material. DSC analyses were begun immediately after dissection to avoid dehydration. 

A second study on total water content separated human lenses into four sections: core (4.5-mm diameter), inner (4.5–6 mm), barrier (6–8 mm), and outer (>8 mm) regions. These samples were used for TGA. The time taken for dissection and the size of the sections in this procedure precluded additional DSC analysis. 

The dimensions of the lens regions taken for analysis yielded sufficient material for measurement and allowed age-related comparisons in zones that corresponded to the following: 4.5 mm is approximately the size of the fetal nucleus, where the simplest Y suture is found ¹⁶ ; 6 mm corresponds approximately to the size of the human lens at birth ^{7 17 18} and 6 to 8 mm encompasses the barrier region and contains within it the first zone of discontinuity. ¹⁹  

Analysis of Total Water Content

Lens total water was calculated by TGA using the mass loss under controlled conditions of heating in a nitrogen atmosphere. Initial studies were conducted at the University of Technology, Sydney (SDT 2960; TA Instruments, New Castle, DE) with platinum crucibles. Later TGA studies for total lens water were conducted (Q500 TGA; TA Instruments) in Wollongong. These were carried out with aluminum sample pans placed in a platinum crucible. The temperature was ramped at 5°C per minute to 300°C. The total water loss was taken as the point where the derivative value reached near zero (around 200°C). “Free water” was calculated from the TGA traces of normal lenses by measuring the loss in mass before a trough in the derivative trace (typically 100°C) and expressing this as a percentage of the total mass loss. 

Analysis of Lens Free Water (DSC)

The instrument used for the measurement of free water (Q100 DSC; TA Instruments) was fitted with refrigerated cooling system to facilitate cooling. Standard aluminum pans were used and were not crimped. During runs the purge gas used was nitrogen. Cycles were reproducible (data not shown). DSC measures the input of energy to the sample that is required to keep it at the same temperature as a standard (sample pan only) under the same conditions. 

Each lens nucleus was placed in a pre-weighed sample pan to determine the mass of the sample and then a lid was placed on it before insertion into the instrument. Samples were generally between 10 and 20 mg. The DSC was equilibrated at −30°C, and then ramped at 3°C per minute to +30°C. 

The data were interpreted using instrumental analysis software (TA Instruments) to obtain a value for the total heat in joules per gram. This value was compared to the value for a sample composed entirely of distilled water to allow quantification of the mass of free water because only free water should have a phase change around 0°C. 

Effect of Lens Freezing

Porcine lenses were frozen at −80°C for periods of 0, 1, 2, 3, and 4 weeks. The lenses were stored in Eppendorf tubes with pieces of moistened Kimwipe placed in the recess of the lid to ensure that the air of the tube was saturated to reduce the risk of possible dehydration. The time 0 lenses were not frozen. Where a sample of lens nuclear material was required for both TGA and DSC, the lens was sectioned using a 4-mm trephine, and the core was cut in half along the anterior-posterior axis to provide two equal sections. 

Results

TGA Analysis

The use of TGA involves heating samples under controlled conditions while the mass is monitored over time and is typically used to calculate total water content. When this technique was used on lens samples, there appeared to be consistent differences in the appearance of the profiles of mass loss, depending on the age of the lenses. 

Figure 1shows typical traces obtained from TGA analysis of two normal human lenses and one type III cataractous lens. Two lines are depicted on these graphs; the solid line shows the change in mass with increasing temperature, and the broken line is the derivative of mass loss (i.e., the rate of mass loss per °C). As can be seen, most of the lens mass was lost by 150°C, and the derivative then plateaued. Beyond 200°C, the variable mass response is likely to be the result of combustion of the remaining dehydrated lens material. Total lens water was taken to be the mass loss below 200°C. 

The data for total water in the lens nucleus, as a function of age, are shown in Figure 2 . These results do not appear to indicate any significant change in lens water content with age. A line of best fit plotted to the data using SPSS software (Sigmaplot) has an equation of y = 0.0232x + 64.4. 

Free Water by TGA

There appeared to be two main zones of mass loss in normal lenses, one centered at approximately 75°C, the other at 125°C (Figs. 1a 1b) . It was considered that these regions may represent free and bound water because free and bound components will presumably be lost from the lens at different temperatures. The nature of the interactions of bound water with proteins should mean that more energy is required for these molecules to be removed. Reducing the rate of heating did not lead to greater separation of the two water populations. 

To determine whether the first peak in the derivative trace may indeed correspond to free water, porcine lenses were examined. Sectioning one porcine lens and spiking samples of approximately equal mass with varying amounts of distilled water tested this. The distilled water should increase the amounts of free water in the sample; increasing the relative size of the peak centered at approximately 75°C. An example is shown in Figure 3and illustrates that the addition of water did lead to an increase in the proportion of the first peak with little effect on the second peak. 

To investigate the quantities of these two apparent populations of water in human lenses, the trace was divided into two sections based on the minimum in the derivative plot of mass loss. This typically occurred at 100°C (Figs. 1a 1b) . Results are shown in Figure 4 . There is considerable scatter in this graph; however, there appears to be a clear upward trend in the data. If this TGA approach can indeed be used to estimate the content of free and bound water in tissues such as the lens, then the following values were obtained. In the younger lenses, free water represents approximately 35% to 45% of total water, and this figure climbs to approximately 55% to 65% in older normal lenses. 

DSC Analysis

A standard method for the analysis of free water (DSC) in samples was also investigated. DSC is based on the fact that free water and bound water behave differently in terms of their freezing points. Free water is similar to normal water and freezes/melts at approximately 0°C. Bound water has a much lower freezing point. This allows quantification of the free water component by freezing the sample, then monitoring the amount of energy absorbed in the phase change from solid to liquid (at approximately 0°C). Quantification is based on the knowledge of the energy required for pure water: the specific heat of water for this phase change is 340 J/g. 

Figure 5shows a plot of the heat required to maintain the sample pan containing a human lens nucleus at the same temperature as that of the reference pan, which contained no sample. The software enables calculation of the area under the peak and, therefore, quantification of the energy associated with the phase change. This plot shows that 123 J/g was required for this sample to change phase and therefore that 123/340 (36.2%) of this sample is composed of free water. Using a value of nuclear free water of 65% (Fig. 2) , a percentage of free water in total water (55.7%) could be calculated. 

Using these data, a plot was generated (Fig. 6) , for persons between 5 and 88 years of age. The graph shows a clear upward trend with age. Younger lenses (younger than 20) ranged between 49% and 56% free water, but this rose to more than 60% in lenses older than 60. This increase may have significant consequences. In young lenses, the ratio of free to bound water molecules was typically 1:1, whereas in a 70-year-old lens there were, on average, two free water molecules for each bound water molecule. This conversion of bound to free water with age will likely have a dramatic effect on the properties of the lens material. 

Effect of Freezing Lenses on Free/Bound Water

The human lenses examined in this study, unless otherwise stated, had been frozen at −80°C before analysis, sometimes for periods of days or even weeks. We examined whether such prolonged storage may affect the ratio of free to bound water in lenses. For this study porcine lenses were used because they were found to be most similar to human lenses in terms of size and water content. Porcine lenses were analyzed after having been frozen for different times and were compared with lenses that had not been frozen. The results are shown in Figure 7 . 

Based on these results there appeared to be no major changes to the distribution of lens water as a result of freezing for a period of up to 4 weeks (Fig. 7) . Analysis of groups using a Student’s t-test showed that there were no significant differences between the sample groups (P = 0.05). 

Analysis of Cataractous Lenses

Values for the total water content of nuclei dissected from cataractous lenses were revealing (Fig. 8) . When all cataractous lenses were compared against normal lenses, little difference was found between the two. However, when types I and II and types III and IV cataractous lenses were separated into subgroups, the types I and II lenses showed no significant differences (t-test, P = 0.32), whereas the types III and IV lenses were significantly different from normal lenses (t-test P = 3.7 × 10⁻⁶). Types III and IV lenses, therefore, appear to have slightly lower total water content than normal lenses of a similar age. 

The results of free water analysis in normal and cataractous lenses using DSC are shown in Figure 9 . It can be seen that, when compared with normal human lenses, the values for free water in the cataractous lenses were within the range expected for normal lenses of the same age 

Water in Lens Regions

Total water content in lens sections was studied by dissecting human lenses into four parts using three trephines. Values were measured using TGA in the core (4.5-mm diameter), inner (4.5–6 mm), barrier (6–8 mm), and outer (>8 mm) regions. Results of this analysis are shown in Table 1 . As expected, the sections of lens containing epithelial and the newer fiber cells around the lens periphery were more hydrated. As the fibers matured, they became progressively less hydrated. The inner region was not different from the core, indicating a constant protein concentration to at least 6 mm across the adult lens. The barrier region showed slightly higher total water concentration compared with the core and inner regions. 

We attempted to determine free water values in regions of the lens using DSC (Fig. 10) . There was considerable scatter in the data, which may reflect the time taken to perform the dissections and measurements compared with simple coring of the lens. There did not appear to be statistically significant differences in the three lens regions, though the outer and core regions tended to show the highest values for free water content. 

Discussion

This study sought to examine the state of water in normal human and cataractous lenses as a function of age. In particular, we focused mostly on the oldest, central part of the lens, which we refer to as the core (4.5-mm diameter) and which corresponds in size approximately to that of the fetal nucleus. This region of the lens contains the simplest (Y) suture pattern. ¹⁶  

Total Water

The results of TGA showed that the water content of the human lens center was approximately 65% and that this value did not change with age. This finding agrees with previous findings. ^{8 9 10} Based on these data, compaction of the center of human lenses does not occur with age. There is a marked difference between human lenses and those of most animals, especially rodents, in which there is a pronounced age-related decline in the water content in the lens center. ⁸ Interestingly, the appearance of the TGA profiles for nuclear cataractous lenses was typically different from that of normal lenses (Fig. 1) , but the reason for this was unclear. 

Previous research from our group ^{20 21} has demonstrated the importance of the lens barrier for the later onset of nuclear cataract. The barrier develops in normal lenses at middle age and has dimensions of 7 mm equatorial and 3 mm axial. To localize water profiles to sites in the lens, regions were dissected to yield a core (4.5-mm diameter) and “doughnut”-shaped inner (4.5–6 mm), barrier (6–8 mm), and outer (>8 mm) regions. Analysis of these lens sections showed that a gradient in water content exists in the human lens. In our study (Table 1) , the core region had the same water content as the inner region, indicating that in human lenses there is a zone in which water is constant, extends at least 6 mm across the lens, and does not change with age. The finding that most of the human lens has a constant protein content is in agreement with the data of Fagerholm et al. ⁹  

The barrier region of the human lens seems to correspond to the region with incomplete compaction of crystallins (Table 1) . Outside this barrier, the lens has a lower protein content, and inside the barrier is the region of constant protein concentration. ^{8 9 10} The lens barrier, therefore, seems to correspond to a region in which the protein concentration has increased compared with the outer part of the lens but has not reached the maximum values. Little is known about the way in which cytosolic protein is concentrated to such high levels in the mature fiber cells (reaching 35% in humans and up to 60% in rodents ⁸ ) or how this process is controlled. 

Cortical and early-stage nuclear cataractous lenses (types I and II) were found to have levels of total water that were similar to those of age-matched normal lenses, whereas types III and IV cataractous lenses showed lower total water contents (normal lenses, 65.7% ± 1.8%; types III and IV, 62.1% ± 0.8%). Hence, it appears that for earlier stages of cataract, no change in lens hydration is observed in the nucleus, but for dark brown lenses, which are the most advanced type (type IV) ²² of nuclear cataracts, there is a decline in total water. This would indicate that the major oxidative changes known to be associated with the formation of age-related nuclear cataract ²³ in human lenses may, at later stages of development, be linked with dehydration. We cannot rule out the possibility that some dehydration might have resulted from the transportation of lenses from India to Australia. If this were the case, however, similar changes should have been observed in the types I and II lenses that were collected and transported at the same time. 

Free Water

It is important to recognize that the terms free water and bound water in biological terms are not absolute. For example, there are likely to be populations of tightly bound and less tightly bound water molecules. ^{24 25} Nevertheless, methods such as those used in this study can provide information on changes that take place over time. TGA and DSC data indicated that the content of free water in the center of the lenses increases linearly with age. 

Analysis of changes in free water by TGA has not, to our knowledge, been previously attempted. This method was complicated by the fact that it was sometimes difficult to clearly separate the two apparent populations of water in the trace. Based on this method, younger lenses (younger than 40 years) had lower free water values (approximately 35%–45% of total water), and this value increased with age in the older lenses (older than 60 years) to approximately 55% to 65% free water. 

Although these values were a little lower than those found using a standard method for determination of free water (DSC), the same trend with age was observed. Using DSC, free water content in the lens nucleus also showed a progressive increase with age. In younger lenses, approximately 50% of the water in the lens was composed of free water. By age of 70 to 80, however, free water content had increased to approximately 65%. This appears to be a relatively small increase, but it equates to a shift from a ratio of 1:1 (free/bound) in young lenses to approximately 2:1 in older lenses. Because the total water content does not change, this significant shift in water ratios would indicate that less of the total water hydrates proteins in older lenses. 

Our DSC results are in broad agreement with other DSC data ^{26 27} but differ quantitatively from those using nuclear magnetic resonance (NMR). ²⁸ As suggested, ²⁶ one reason for this difference is that the DSC measurements are based on using a value for pure water to calculate the free water content. ^{13 26 27} It is likely that the value for 1 g free water undergoing a phase change in tissues will be lower than that of pure water because ions are present. Taking this factor into consideration will make them agree more closely with those gained by NMR analysis. Most studies in the literature have used the value for pure water. ^{13 26 27} We evaluated this factor by comparing DSC measurements of distilled water with those of PBS. The following data were obtained: for distilled water, 332.7 ± 1.0 J/g (n = 3); for PBS, 289.7 ± 2.9 J/g (n = 3). If the value for PBS is substituted for that of pure water, all the values shown in the graph (Fig. 6)will be moved upward by 14.9%. 

Most lenses analyzed for DSC had been frozen for a short period (less than 2 weeks). Freezing was necessary to allow easier sectioning of the lenses and to facilitate access to instruments. Freezing did not appear to affect the ratio of free and bound water because no significant changes to the free water content of porcine lenses, which had been stored for a period of up to 4 weeks at −80°C, was observed (Fig. 7) . 

Initially, we hypothesized that the changes we observed in lens free water with age might simply have reflected the major protein changes demonstrated in the human lens during this time period. For example, large-scale aggregation of proteins in lenses with age ^{12 29 30} and an increase in insoluble protein ^{12 31 32} could reduce the amount of water that can bind because of a reduction in the net surface area of proteins exposed to the solvent. We examined this with a model system involving porcine lenses. When intact lenses were incubated at 50°C, there was marked conversion of soluble protein into high molecular weight aggregates and insoluble protein. However, when such lenses were analyzed for the content of free and bound water, there were no detectable changes (Fig. 11) . On this basis we conclude that the age-dependent conversion of bound to free water in the human lens is unlikely to be simply a consequence of protein aggregation. 

Given that the increase in insoluble protein with age in humans, and in model systems in which lenses are exposed to heat, does correlate with increased lens stiffness, ¹² we conclude that the conversion of bound to free water may not be directly responsible for the marked changes in lens stiffness that appear to be responsible for presbyopia. 

There may be a circulation pathway within the lens to aid the movement of small molecules, though the model ³³ needs significant revision because experimental data have indicated that molecules enter the lens at the germinative zone ³⁴ and move inward toward the lens center in the direction opposite that predicted by the model. Such equatorial movement is consistent with the distribution of square arrays, which contain aquaporin 0 and which facilitate water diffusion, and with gap junctions, which permit passage of small molecules such as glutathione. Both types of cellular pores are concentrated in the lens equator. ^{35 36} Each of these membrane pores permits the flow of water molecules within the lens, so any impairment of their function in a particular region may lead to changes in the state of water in parts of the lens that are internal to that zone. We have postulated that the lens barrier, which develops in human lenses at middle age, ^{20 21} involves a decrease in the permeability of the gap junctions and aquaporins. Such a process could, therefore, affect the state of water in the inner and core regions, as we have documented in this article. In addition, the progressive increase in posttranslational modification of the long-lived crystallins with age, the most abundant of which are deamidation of Gln and Asn residues, ^{37 38} may also lead to a decrease in hydration of these macromolecules. At present, the reason for the age-related decrease in bound water in the center of the human lens is unknown. 

In summary, although the total water content of the human lens nucleus does not change with age, the state of the water does alter significantly. Over time there appears to be a steady transformation of bound water molecules to free water. Such changes do not appear to result simply from protein aggregation/insolubilization. Age-related nuclear cataractous lenses do not differ substantially from normal aged lenses in terms of free water content; however, the centers of advanced nuclear cataractous lenses appear to be more dehydrated than comparable age-matched normal lenses. 

 Supported by the Australian Research Council (Grant DP0666847), the National Health and Medical Research Council (NHMRC), and the Ophthalmic Research Institute of Australia. RJWT is an NHMRC fellow.

 Submitted for publication September 5, 2007; revised November 12, 2007; accepted March 6, 2008.

 Disclosure: K.R. Heys, None; M.G. Friedrich, None; R.J.W. Truscott, None

 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.

 

Figure 1.

 

TGA traces of human lenses. (a) Normal lens, age 13. (b) Normal lens, age 73. (c) Type III cataractous lens, age 75. Solid line: change in mass with increasing temperature. Broken line: derivative of this mass loss.

Figure 2.

 

Total water in the human lens nucleus as a function of age, measured using TGA (n = 47).

Figure 3.

 

TGA traces of porcine lens nuclei. (a) Porcine lens; mass = 15 mg, no water added. (b) Lens sample spiked with distilled water (17 mg lens sample with 7 mg distilled water).

Figure 4.

 

Free water in the human lens nucleus as a function of age calculated from TGA traces (n = 35). A value for free water was calculated using the minimum of the derivative trace at approximately 100°C (see Fig. 1 ). The mass of water lost by this stage was calculated and expressed as a percentage of total water loss. The line shown is a line of best fit calculated using SPSS software.

Figure 5.

 

A typical DSC trace of a young human lens.

Figure 6.

 

Free water in the human lens nucleus as a function of age as determined by DSC (n = 39). Values were calculated using distilled water as reference. The line shown is a line of best fit calculated using SPSS software.

Figure 7.

 

Effect of freezing of lenses on measured free water values. Porcine lenses were stored for differing times at −80°C before analysis, from 0 (nonfrozen) to 4 weeks, then analyzed using DSC (n = 10, for t = 0 and 3; n = 5 for t = 1, 2, and 4; values shown are mean ± SD).

Figure 8.

 

Total water in the nucleus of cataractous lenses measured by TGA. Cataractous lenses were divided into two groups, types I and II (•; n = 5) and types III and IV (▾; n = 7). Normal lenses (○) are shown for comparison (from Fig. 2 ); n = 47.

Figure 9.

 

Free water in the nucleus of cataractous lenses measured using DSC. Cataractous lenses were divided into two groups, types I and II (•; n = 5) and types III and IV (▾; n = 7). Normal lenses (○) are shown for comparison (from Fig. 6 ); n =39.

Table 1.

 

Total Water Content in Regions of the Human Lens Using TGA

Lens Region	Dimensions (diameter, mm)	Total Water (%)
Outer	>8	70.6 ± 3.2*
Barrier	6–8	67.2 ± 0.7
Inner	4.5–6	65.9 ± 0.9, †
Core	4.5	65.6 ± 1.0, ‡

 For all sections, mean ± SD (n = 4). Average age, 74.8 ± 10.0 years.

 Results of t-test between groups:

*   P = 0.0260 compared with the core;

†   P = 0.064 and

‡   P = 0.057 compared with the barrier.

Figure 10.

 

Free water in regions of the human lens using DSC, (•) outer (>8 mm), (○) intermediate (4.5–8 mm), and (▾) core (4.5 mm) for all sections (n = 13.)

Figure 11.

 

Incubation of intact porcine lenses at 50°C. Lens nuclei were analyzed for insoluble protein and for free water by DSC. Four lenses were analyzed at each time point, three for insoluble protein and one for DSC. Values shown are mean ± SD.

The authors thank Raj Devasahayam (Sydney Lions Eye Bank) for providing the normal human lenses and Sudha Awasthi Patney for enabling the collection of cataractous lenses.