Although less common than in adults, pediatric cataracts are the leading cause of childhood blindness worldwide (1, 2). While they may result from a variety of different primary etiologies, cataracts in children share several pathologic features. Cataractous lenses exhibit damage to lens cell proteins and lipids. Biochemically, this damage can include oxidation, crosslinking, denaturation, aggregation, proteolysis/cleavage, misfolding, and other kinds of modifications (3, 4). Disturbances of ionic homeostasis, including large elevations of calcium concentrations, are also observed in many cataractous lenses (5, 6). These biochemical changes are not necessarily identical in all patients and their relative importance for cataract severity is not well established.

Recent studies performed in our laboratory and others using microscopy and micro-computed tomography to study the lenses of mice that develop cataracts due to different genetic abnormalities have shown that the cataracts contain calcium precipitates that localize to the same region as the cataract (7–10). In connexin mutant lenses and the lenses of a mouse with a mutation of γC-crystallin, we used infrared microspectroscopy to identify the mineral in these precipitates as calcium phosphate in the form of apatite (10, 11). Radiographic studies have detected calcium-containing mineral also in canine cataracts (12). Several previous papers have suggested that different types of cataracts in people contain insoluble calcium salts (13–17). These findings suggest that precipitation of calcium ions (biomineralization) might be a general phenomenon in cataracts (18).

We undertook the present study to test the generality of calcified particle formation in human cataracts of different etiologies. We performed microscopic examination of the insoluble material from cataract extractions after staining it with Alizarin red, a dye commonly used to identify and localize calcium deposits in tissues, including bone and teeth.

We studied three pediatric patients with cataracts of different etiologies (Table 1).

We found Alizarin red-stained insoluble particles in all cases within this series of three pediatric patients with cataracts of diverse etiologies, suggesting that biomineralization (calcification of biological material) may be a general occurrence in pediatric cataracts. Many of the particles were discrete with limited background staining of most cellular material and debris in the samples, implying the specificity of our staining. We have not performed “control” experiments to study insoluble material from the clear lenses of normal children for comparison with the cataractous material. However, based on extrapolating our previous findings that Alizarin red-stained particles are found in the lenses of mice with cataracts but not in the lenses of their wild type littermates (10, 11), we suspect that such stained particles are uniquely associated with cataractous lenses.

Alizarin red has been widely used as a histochemical stain to identify calcified material in tissues (20, 21) or in body fluids (22). This dye is not absolutely specific for calcium, since it can also react with salts of other cations (23). However, calcium ions are more abundant in tissues than the other cations (e.g., copper, barium, zinc, lead, iron, nickel and cobalt). In mouse models that develop cataracts, an increase in the intracellular concentration of Ca²⁺ > 1 μM is a common finding (6), making it potentially possible to exceed the K_sp for some calcium salts in these lenses or to favor the deposition of calcium on a scaffold of macromolecules containing negative charges. In our studies of mutant mice, we found mineral in their cataractous lenses and identified it as calcium phosphate in the form of apatite (10, 11). Therefore, it is likely that calcium ions comprise the cation in the Alizarin red-stained insoluble material that we found in pediatric cataracts.

Maintenance of lens cell homeostasis is facilitated by an internal circulation of ions, small molecules and water, which is driven by the regional distribution and activities of ion channels, transporters, and exchangers (24). Ions enter the lens at the anterior and posterior poles and move towards the center through the extracellular spaces. Ions are driven into fiber cells by their electrochemical gradients. Ions move outward through the cytoplasm of fiber cells and across cell boundaries through gap junction channels. Once ions reach the epithelial cells on the lens surface, they are transported out of the lens by epithelial ion pumps, transporters and exchangers. The movement of water is coupled to the circulation of ions.

Cellular Ca²⁺ is highly regulated in the lens. In the epithelial cells, the intracellular Ca²⁺ concentration is maintained at ~100 nM (25, 26). In fiber cells, it is somewhat higher, reflecting a gradient of calcium ions that increases approaching the center of the lens. Regardless of cell type, the intracellular concentration of Ca²⁺ is thousands of times less than the extracellular Ca²⁺ concentration of ~1.3 mM in the aqueous humor (5, 27). Thus, the electrochemical gradient will drive extracellular Ca²⁺ that enter the lens into fiber cells. Impairment of the lens circulation will lead to increases in the intracellular concentration of Ca²⁺, which can interact with anions in the cells (including free phosphate) and form insoluble particles.

The sequence of events leading to biomineralization may differ in subjects that develop cataracts due to different primary etiologies. The initial event may be precipitation of insoluble calcium-containing salts resulting from disturbances in ionic homeostasis or it might be damage to lens components that act as a matrix for the deposition of calcium ions. Each of these sequences may explain the formation of mineralized particles in the different cases studied in this report.

The lens circulation of ions and water is disrupted in many different kinds of cataracts (28). Lens calcification has been observed in mouse cataracts caused by mutations of channel proteins with varied functions (7–9, 11, 29) and cataracts caused by mutations of other lens proteins associated with secondary impairment of the lens circulation (10). We do not know the gene(s) responsible for the congenital cataracts in our series, but a primary disturbance of lens ionic homeostasis might be possible. In the traumatic cataract, direct impact on the globe causes lens damage and cellular disruption (30), exposing negative charges in proteins and other molecules that could readily bind calcium ions. Cataract formation in patients with uveitis is usually attributed to uncontrolled and sustained inflammation (or prolonged use of steroids which was not the case in our patient) (31). It is likely that pathologic calcification followed the inflammatory damage to lens cells.

Formation of calcified material in the lens requires interaction of Ca²⁺ with negatively charged ions or molecules. The extracellular pool would provide the main source for Ca²⁺ accumulation inside lens cells and subsequent mineralization in the many kinds of cataracts that develop when the lens circulation is disrupted (28). Mitochondria and the endoplasmic reticulum hold substantial stores of calcium ions that may contribute to mineralization in some forms of cataracts. Although these organelles are normally only present in epithelial and differentiating fiber cells, damage to cortical cells might result in release of their intraorganellar calcium. The anions (including phosphate and possibly proteins or lipids) are all major constituents of cells and their cytoplasm that may become available for interaction with Ca²⁺ in lenses subjected to insults or damage.

Regardless of the mechanistic sequence of events, biomineralization within the lens would have a major impact on vision (beyond protein damage and aggregation). The calcium-containing particles would scatter or block transmission of light onto the retina. It is not yet clear how the abundance of calcified material relates to the volume or severity of cataracts. All of the patients in our series had dense white cataracts of unknown duration. In the two older patients, the parents had only noticed the white pupil for about one month prior to presentation, while the congenital cataract may have been significant for as little as three months. These results suggest that mineralization may occur relatively early in some (or perhaps all) cataracts.

Taken together, our data suggest that development of a treatment that prevented biomineralization in the lens could be beneficial by interfering with the development or progression of cataracts.

The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by University of Chicago Institutional Review Board. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the minor(s)’ legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.

PM, SR, VB, and EB conceived the project. SR conducted the clinical evaluations of all patients and performed the surgeries. PM performed the microscopy experiments. PM and VB analyzed the data and prepared the figures. All authors contributed to the article and its writing, and approved the submitted version.