Proposal and confirmation of N‐(2‐carboxyethyl)proline as a human endogenous metabolite

Abstract

RationaleMalondialdehyde, one of the peroxidation products of polyunsaturated fatty acids, has been widely reported as an oxidative stress biomarker in many diseases. However, malondialdehyde is inherently unstable in biological matrices, which renders its measurement unreliable with all the reported analytical methods. To find an alternative oxidative stress biomarker, we envisioned that N‐(2‐carboxyethyl)proline, a modified conjugate of malondialdehyde and proline, could be a stable candidate for this purpose.MethodsThe proposed compound was chemically synthesized, and liquid chromatography–mass spectrometry methods were developed and used to search for the compound in human biological samples.ResultsAn endogenous metabolite in human feces and urine samples was found to match the synthetic N‐(2‐carboxyethyl)proline by chromatographic retention and the fragmentation pattern of its molecular ion.ConclusionThe results confirmed that N‐(2‐carboxyethyl)proline was a new metabolite in human feces and urine samples. In addition, our results demonstrated a case of successful identification of true unknown metabolite by knowledge‐based hypothesis of possible metabolites followed by experimental confirmation with a synthetic standard.