High sensitivity HPLC method for determination of the allysine concentration in tissue by use of a naphthol derivative

- Improved method to quantify the allysine concentration in tissue samples using HPLC.
- Assay sensitive to 0.02 pmol of allysine from milligram quantities of tissue.
- Method provides a means to assess the effects of novel anti-fibrotic therapeutics.

Common to all fibrotic and metastatic diseases is the uncontrollable remodeling of tissue that leads to the accumulation of fibrous connective tissue components such as collagen and elastin. Build-up of fibrous tissue occurs through the cross-linking of collagen or elastin monomers, which is initiated through the oxidation of lysine residues to form α-aminoadipic-δ-semialdehyde (allysine). To provide a measure of the extent of collagen oxidation in disease models of fibrosis or metastasis, a rapid, sensitive HPLC method was developed to quantify the amount of allysine present in tissue. Allysine was reacted with sodium 2-naphthol-7-sulfonate under conditions typically applied for acid hydrolysis of tissues (6 M HCl, 110 °C, 24 h) to prepare AL-NP, a fluorescent bis-naphthol derivative of allysine. High performance liquid chromatography was applied for analysis of allysine content. Under optimal reaction and detection conditions, successful separation of AL-NP was achieved with excellent analytical performance attained. Good linear relationship (R2 = 0.994) between peak area and concentration for AL-NP was attained for 0.35-175 pmol of analyte. A detection limit of 0.02 pmol in the standard sample with a 20 μL injection was achieved for AL-NP, with satisfactory recovery from 88 to 100% determined. The method was applied in the quantification of allysine in healthy and fibrotic mouse lung tissue, with the fibrotic tissue showing a 2.5 fold increase in the content of allysine.