Advanced glycation end product (AGE) modified proteins in tears of diabetic patients

Abstract

Purpose: High glucose level in diabetic patients may lead to advanced glycation end product (AGE) modified proteins. This study investigated AGE modified proteins in tears and compared their levels in diabetic patients (DM) with non-diabetic controls (CTL).

Methods: Basal tears were collected from DM with (DR) or without (DNR) retinopathy and CTL. Total AGE modified proteins were detected quantitatively by a dot immunobinding assay. The AGE modified proteins were separated in 1D- and 2D-SDS gels and detected by western-blotting. The individual AGE modified proteins were also compared between groups using densitometry.

Results: Compared with the CTL group, tear concentrations of AGE modified proteins were significantly elevated in DR and DNR groups. The concentration of AGE modified proteins in diabetic tears were positively correlated with AGE modified hemoglobin (HbA1c) and postprandial blood glucose level (PBG). Western blotting of AGE modified proteins from 1D-SDS gels showed several bands, the major one at around 60 kDa. The intensities of AGE modified protein bands were higher in DM tears than in CTL tears. Western blotting from 2D-SDS gels showed a strongly stained horizontal strip, which corresponded to the major band in 1D-SDS gels. Most of the other AGE modified protein species were within molecular weight of 30-60 kDa, PI 5.2-7.0. Densitometry analysis demonstrated several AGE modified proteins were elevated in DR or DNR tears.

Conclusions: Total and some individual AGE modified proteins were elevated in DM tears. AGE modified proteins in tears may be used as biomarkers to diagnose diabetes and/or diabetic retinopathy.

Figure 1

Images of dot-immunobinding assay of total AGE modified proteins in tears from DR, DNR, and CTL subjects. One dot represented a tear sample from a subject. The left lane was the AGE-BSA standard in various amounts.

Figure 2

Correlation of tear total AGE modified proteins and HbA1c and PBG in DM patients with or without retinopathy.

Figure 3

1D western blotting images of AGE modified tear proteins from DR, DNR, and CTL samples. PS: a Ponceau S stained tear proteins on the blot. A: lactoferrin, B: tear lipocalin, C: lysozyme.

Figure 4

2D western blotting images of AGE modified tear proteins from DR, DNR, and CTL samples and a Sypro ruby stained 2D gel image of a tear sample.

Figure 5

Image analysis to detect expression differences of the individual AGE modified protein spot between the groups. Only the spots showing significant differences were included in the figure table. No significant differences were detected between DNR and DR groups.