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.

中文翻译：

---

N-(2-羧乙基)脯氨酸作为人类内源代谢物的提议和确认

原理丙二醛是多不饱和脂肪酸的过氧化产物之一，已被广泛报道为许多疾病中的氧化应激生物标志物。然而，丙二醛在生物基质中本质上不稳定，这使得所有报道的分析方法的测量都不可靠。为了找到替代的氧化应激生物标志物，我们设想氮-(2-羧乙基)脯氨酸，一种丙二醛和脯氨酸的修饰缀合物，可能是用于此目的的稳定候选物。方法化学合成了所提出的化合物，并开发了液相色谱-质谱方法并用于在人体中寻找该化合物生物样品。结果发现人类粪便和尿液样品中的内源代谢物与合成的代谢物相匹配氮‐(2-羧乙基)脯氨酸的色谱保留及其分子离子的裂解模式。结论结果证实氮‐(2-羧乙基)脯氨酸是人类粪便和尿液样本中的一种新代谢物。此外，我们的结果证明了通过基于知识的可能代谢物假设，然后用合成标准进行实验确认，成功鉴定出真正的未知代谢物的案例。