A novel colorimetric assay for the detection of urinary N1, N12-diacetylspermine, a known biomarker for colorectal cancer

Abstract

Urinary N¹, N¹²-diacetylspermine (DAS) is a known biomarker for colorectal cancer (CRC). However, DAS levels in both healthy and CRC patients’ urine samples are extremely low and often challenging to quantify. Complex and expensive methods do exist to detect DAS in urine, but simpler, less expensive methods to detect DAS are needed, especially in low resource settings. Here we describe a highly efficient, fast, precise, and inexpensive colorimetric assay to detect low levels of DAS in human urine samples. We used recombinant diacetylspermine oxidase (rDAS Ox), expressed and extracted from E. coli, to oxidize DAS, producing three products including hydrogen peroxide (H₂O₂). The level of DAS present, which correlates with H₂O₂ levels, was measured using horseradish peroxidase (HRP), which together with H₂O₂, oxidized Amplex™ Red to produce the pink-colored resorufin. The concentration of resorufin is directly proportional to H₂O₂ (and DAS) levels. As urine contains metabolites which interfere with these oxidation reactions, we developed a simple two column-based protocol using ion exchange resins to remove these compounds and concentrate the DAS. With this novel cleaning and concentrating method, DAS was concentrated 15 times (confirmed by nuclear magnetic resonance (NMR) spectroscopy) and <1 μM DAS could be detected. Correlation graphs of urine samples spiked with known DAS concentrations versus assay-determined DAS concentrations had high coefficients of determination (R²) for 0–10 μM DAS (0.94) and for 0–1 μM DAS (0.91), clearly demonstrating the excellent performance of the two-column protocol with the rDAS Ox reaction mixture. To the best of our knowledge, this is first reported colorimetric enzymatic assay that quantitates DAS in urine.