Higher phosphate concentrations as in aqueous humor of diabetic patients increase intraocular lens calcification

Abstract

Background: Clinical evidence suggests an association between phosphate concentrations in aqueous humor and the risk of intraocular lens (IOL) calcification. To test this hypothesis the influence of different phosphate concentrations on IOL calcification was evaluated in an in vitro electrophoresis model.

Methods: 20 IOLs of two hydrophilic IOL models (CT Spheris 204, Zeiss; Lentis L-313, Oculentis) and one hydrophobic control IOL model (Clareon CNA0T0, Alcon) were exposed to physiologic and elevated phosphate concentrations, similar to diabetic aqueous humor. IOL calcification was analyzed by alizarin red staining, von Kossa staining, scanning electron microscopy, energy dispersive x-ray spectroscopy and transmission electron microscopy with electron diffraction.

Results: Higher phosphate concentrations were associated with IOL calcification. Analyses of IOL surfaces and cross-sections documented calcification in no CT Spheris and 4 Lentis IOLs following exposure to 10 mM Na₂HPO₄, compared with 7 and 11 positive analyses following exposure to 14 mM Na₂HPO₄, respectively. Furthermore, a clear association between IOL calcification and the duration of electrophoresis was demonstrated, confirming increased phosphate concentrations and duration of exposure as risk factors of IOL calcification.

Conclusions: Findings suggest that higher phosphate concentrations in aqueous humor, as seen in diabetic patients, contribute to an increased IOL calcification risk, potentially explaining clinical observations showing an increased risk of IOL calcification in patients with diabetes.

Keywords: Aqueous humor; Diabetes; IOL Calcification; Phosphate concentration; Proliferative diabetic retinopathy.

Fig. 1

Electrophoresis model and IOL analysis protocol. a. Electrophoresis model with the cathode (left) and anode (right) chamber and the IOL holding plate above. b. Schematic demonstration of the electrophoresis model with the Na₂HPO₄ solution at the cathode (left) and CaCl₂ solution at the anode (right). The phosphate and calcium ions diffuse to the other side respectively, passing through the IOLs in the IOL holding plate, causing crystal formation and therefore IOL calcification. CaCl₂: Calcium chloride, Na₂HPO₄: Disodium hydrogen phosphate. c. Following electrophoresis, the IOLs underwent different analyses as indicated. Gross light microscopy was performed to obtain overview images. Alizarin red staining was used to detect superficial calcium phosphate deposits. Subsequently, the IOL was cut in half and the von Kossa method was used to identify deposits below the IOL polymer surface in cross-sections of one IOL half. SEM and EDX were performed at the Max Planck Institute for polymer research in Mainz (Germany) to detect crystal growth in IOL cross-sections. TEM and ED were performed following EDX, allowing definitive identification of the specific calcium phosphate crystal present. SEM: Scanning electron microscopy, ED: Electron diffraction, EDX: Energy-dispersive X-ray spectroscopy, TEM: Transmission electron microscopy with electron diffraction

Fig. 2

CT Spheris (top row) and Lentis IOL (bottom row) after 20 h exposure to 10 mM Na₂HPO₄. Native images show a general overview. Alizarin red staining detects superficial calcium phosphate deposits. Once the IOL has subsequently been halved, von Kossa staining identifies deposits below the IOL polymer surface in cross-sections of the IOL. Similarly to von Kossa staining, SEM identifies crystals below the IOL surface in cross-sections obtained following halving of the IOL (Fig. 4A1). EDX analysis confirmed that the crystals contain calcium and phosphorus. The bottom left scale bars show 0.5 mm. Na₂HPO₄: Disodium hydrogen phosphate, EDX: Energy-dispersive X-ray spectroscopy, SEM: Scanning electron microscopy

Fig. 3

CT Spheris (top row), Lentis (middle row) and Clareon IOL (bottom row) after 20 h exposure to 14 mM Na₂HPO₄. Native images show a general overview. Alizarin red staining detects superficial calcium phosphate deposits. Once the IOL has subsequently been halved, von Kossa staining identifies deposits below the IOL polymer surface in cross-sections of the IOL. Similarly to von Kossa staining, SEM identifies crystals below the IOL surface in cross-sections obtained following halving of the IOL (Fig. 4A1). EDX analysis confirmed that the crystals contain calcium and phosphorus. Compared to the CT Spheris (top row) and Lentis (middle row) IOLs, no positive histologic staining occurred in the Clareon control IOL and SEM images obtained of both surfaces and cross-sections of the IOL remained free of any crystal growth. The bottom left scale bars show 0.5 mm. Na₂HPO₄: Disodium hydrogen phosphate, EDX: Energy-dispersive X-ray spectroscopy, SEM: Scanning electron microscopy

Fig. 4

Both SEM overview and magnification as well as EDX microanalysis of crystal growth in the Lentis IOL following 20 h exposure to 14 mM Na₂HPO₄. a₁. SEM overview of the investigated IOL cross-section. a_2,3. SEM magnification of the investigated IOL cross-section documenting calcium phosphate crystal growth. a₄. EDX analysis of the precise area indicated. b. Analysis of the elements present shows distinctive peaks for calcium (Ca) and phosphorus  (P) and confirms that the crystal contains both elements. The silicon peak (Si) is an artefact created because a silicon wafer is used in the analysis. The peaks for carbon (C) and oxygen (O) are also expected as these are elements contained in hydroxyapatite, the present calcium phosphate salt. Na₂HPO₄: Disodium hydrogen phosphate, SEM: Scanning electron microscopy, EDX: Energy-dispersive X-ray spectroscopy

Fig. 5

Electron crystallography of crystal growth in the Lentis IOL polymer following 20 h exposure to 14 mM Na₂HPO₄. a. Transmission electron microscopy demonstrating crystal growth. b. Crystal electron diffraction pattern, used to identify the specific chemical composition and crystal structure in the calcium phosphate crystal present. c. The very high match between the crystals’ electron diffraction pattern (gross, blue line) and the database electron diffraction pattern for hydroxyapatite (marker 1, green lines) confirms that the crystal is made of hydroxyapatite. Na₂HPO₄: Disodium hydrogen phosphate

Fig. 6

Qualitative analysis of IOL calcification based on SEM analyses. Both surfaces and sides of the IOL’s cross-sections were analyzed for crystal growth (present/not present) after every duration of exposure (5, 10, 15 and 20 h). C: control IOL, h: hours, mM: millimolar

Fig. 7

Total number of documented IOL calcifications in SEM analyses (present/not present) for every IOL model and concentration tested (Fig. 6), illustrating the clear association between Na₂HPO₄ concentration and IOL calcification. Na₂HPO₄: Disodium hydrogen phosphate

Fig. 8

Lentis IOLs following 5 (a), 10 (b) and 20 h (c) of exposure to 14 mM Na₂HPO₄, illustrating the association between degree of calcification and duration of exposure. Na₂HPO₄: Disodium hydrogen phosphate

Fig. 9

Comparison of crystal growth in SEM magnification of the CT Spheris IOL following 20 h exposure to 10 mM and 14 mM Na₂HPO₄. a. Magnification of CT Spheris IOL cross-section following 20 h exposure to 10 mM Na₂HPO₄, documenting no calcium phosphate crystal growth. b. Magnification of CT Spheris IOL cross-section following 20 h exposure to 14 mM Na₂HPO₄, documenting calcium phosphate crystal growth and demonstrating that crystal growth only occurred following exposure to the elevated Na₂HPO₄ concentration. SEM: Scanning electron microscopy, Na₂HPO₄: Disodium hydrogen phosphate