Analysis of age-related carbonylation of human vitreous humor proteins as a tool for forensic diagnosis

Age-related changes of protein carbonylation due to oxidative damage in human vitreous humors were analyzed. The vitreous samples were collected from 51 autopsied bodies (male: 27, 13–87 years-old, female: 24, 18–80 years-old) whose postmortem interval was less than 4 days. Total protein carbonyl content was assessed by colorimetry using 2,4-dinitrophenylhydrazine and particular carbonylated proteins were identified by immunostaining of vitreous proteins resolved by SDS–PAGE and MALDI-TOF MS with peptide mass fingerprinting. No significant correlation was found between total protein carbonyl content and biological age. However, two major proteins, albumin and transferrin, were found to be heavily carbonylated in the samples from individuals age 40 or over. Furthermore, an immunostained cluster of proteins at 50–60 kDa was discernible in the samples of aged individuals, and it was revealed to contain alpha enolase, pigment epithelial differentiating factor, type 2 keratin subunit protein, and S-arrestin. The results of this study suggest that some retinal proteins detected in the vitreous humor sample would be markers of age-related oxidative stress and biological age.