High-performance liquid chromatographic quantification of allysine as bis-p-cresol derivative in elastin

The first step in normal cross-linking in elastin is the formation of alpha -aminoadipic-delta -semialdehyde, allysine, through oxidative deamination of specific peptidyl lysine by the enzyme lysyl oxidase (EC 1.4.3.13). For the analysis of allysine, allysine was derivatized with p-cresol. The deriv atization was carried out by acid hydrolysis (6N HCl containing 5% (w/v) p- cresol at 110 degreesC for 48 h) accompanied with the hydrolysis of elastin . A bis-p-cresol derivative of allysine was isolated from bovine ligamentum nuchae elastin hydrolysates, and was characterized by UV, FAB-IMS and NMR. This derivative was identified as 2-amino-6,6-bis(2-hydroxy-5methylphenyl) hexanoic acid. A rapid, sensitive reverse-phase highperformance liquid chro matographic method with UV detection was developed for the quantitative det ermination of allysine as its bis-p-cresol derivative. The lower limit of d etection of the bis-p-cresol derivative was 58 pmol in the standard sample with a 20-mul injection at a signal-to-noise ratio of 3. This method was ap plied to the determination of allysine in bovine ligamentum nuchae, aorta, lung, and rat aorta elastin. The allysine content in rat aorta elastin dram atically increased from 1 week to 2 weeks of age.