X-ray fluorescence study of Ca, Fe and Zn distribution in human RPE/Bruch’s membrane/choroid with and without early AMD

Authors: Marta Ugarte (University of Manchester; Manchester University NHS Foundation Trust) , Kalotina Geraki (Diamond Light Source) , Glen Jeffery (UCL, Institute of Ophthalmology) , Paul Bishop (University of Manchester; Manchester University NHS Foundation Trust)
Co-authored by industrial partner: No

Type: Conference Paper
Conference: ARVO 2020
Peer Reviewed: No

State: Published (Approved)
Published: June 2020

Open Access Open Access

Abstract: Purpose : The purpose of this study was to investigate the distribution of Ca, Fe and Zn using X-ray fluorescence in human RPE/Bruch’s membrane/choroid with and without early AMD. Methods : We used a set of unfixed frozen human retinal pigment epithelium (RPE)/choroid samples from young (n=1, female aged 34) and aged donors with (n=4, males, aged 73-78) and without (n=3, one female, aged 75-77) early AMD from Manchester Eye Tissue Repository. Using X-ray fluorescence microscopy (I18 Diamond light source, UK) with a 2 um beam, we obtained high-resolution Ca, Zn, Fe, sulphur, potassium, chloride and phosphorus maps covering areas up to 100 x 600 um2 of 30 um thick sections placed on quartz holders and scanned at room temperature Results : Calcification was observed in the 3 groups. In the 34-year old sample, sparse small Ca spherules (2x2 um2) at the RPE/Bruch’s membrane interface not colocalising with Zn or Fe. In aged samples, with and without AMD, calcified nodules within RPE cells, at RPE/Bruch’s membrane interface and within druse in AMD. Every calcified nodule colocalised with Zn. Quantification revealed two types (high Zn content, low Zn content). In aged donors without AMD, high-Zn calcified nodules with Ca concentration of 3083+1679 ppm (mean+SD) (maximum 12809+9311) and Zn 66+55 ppm (max 125+75). The average size was 27+15 um2. In aged samples with AMD, high-Zn calcified nodules with average Ca concentration of 4316+1723 ppm (max 13105+7563) and Zn 97+57 ppm (max 201+143). Size 26x10 um2. In the aged non-AMD group, the low zinc-calcification nodules contained an average Ca ppm of 915+259 (max 1475+380) and Zn 33+18 ppm (max 40+21). The average size 28x16 um2. In the AMD group, the low Zn calcified nodules average Ca ppm 1544+1450 (max 3526+5289) and average Zn ppm 47+23 (max 64+37). Average size 19x10 um2. Calcified plaques in Bruch’s membrane from aged donors with and without AMD. Some of these plaques colocalised with Zn and also Fe. Fe-loaded structures in the choriocapillaris underlying calcified nodules and plaques. Conclusions : Calcific nodules contain zinc in older eyes with and without AMD. Calcified nodules with lower amounts of Ca contained lower amounts of Zn, so the accumulation of Ca may occur in parallel to Zn. It is possible that iron-loaded structures in the choriocapillaris are macrophages.

Subject Areas: Biology and Bio-materials

Instruments: I18-Microfocus Spectroscopy