Hemoglobin is Present in Avascular Pseudoexfoliation Human Lens Capsule Epithelium, Aqueous Humor and Pseudoexfoliation Material

Abstract

Purpose: : To demonstrate the presence of hemoglobin (Hb) in avascular human lens capsule epithelium, aqueous humor, and pseudoexfoliation material (PXFM).

Methods: : Lens capsules (LC) and aqueous humor (AH) were obtained from normal and pseudoexfoliation (PXF) eyes during cataract extraction surgery through avascular cornea and without contamination by hemorrhage. Proteomic analysis of AH and LC was performed using electrospray tandem mass spectrometry. Immunofluorescence (IF) was used to localize Hb within lens capsules and PXFM on LC, which is pathognomonic for PXF. Western blotting of pooled solubilized LC (minimum 7 specimens/sample) and AH (minimum 2 specimens/sample) was used to compare Hb expression levels between normal and PXF eyes. Len capsules were also stained with hematoxylin/eosin and Prussian blue (to detect iron).

Results: : Proteomic analysis of LC and AH differentially identified the presence of Hb in eyes with PXF compared to normal eyes. Immunofluorescence localized Hb to PXF material and lens capsule epithelium. Western blot analysis demonstrated a ~16 kDa Hb protein band in both normal and PXF LC and AH that was significantly greater in PXF eyes. Prussian blue staining did not detect iron suggesting the lack of iron-chelated Hb.

Conclusions: : Using proteomic analysis, immunofluorescence, and immunoblotting, non-iron-bound hemoglobin was observed to be highly expressed in avascular human LC, PXFM, and AH. Prussian blue negative staining of Hb suggests no contamination from bleeding during surgical specimen extraction. Over a decade ago, several groups suggested a low molecular weight protein was associated with PXF glaucoma. We have identified this molecule to be Hb, which is specifically present within PXF material.

Keywords: protein structure/function • proteomics • pathology: human