Abstract
We previously observed an inverse correlation between stroke and urinary 3-hydroxypropyl mercapturic acid (3-HPMA), an acrolein-glutathione metabolite, through its measurement by liquid chromatography with tandem mass spectrometry (LC-MS/MS). However, the cost of equipment for LC-MS/MS and its maintenance fee is very expensive and a cost-efficient method is required. In this study, we have developed a sensitive enzyme-linked immunosorbent assay (ELISA) system to measure 3-HPMA using a chicken antibody recognizing 3-HPMA-conjugated chicken albumin as antigen. Linearity to measure 3-HPMA was obtained from 0 to 10 μM, indicating that this ELISA system is useful for measurement of urine 3-HPMA. It was confirmed that 3-HPMA in urine of stroke patients decreased significantly compared with that of control subjects using the ELISA system. Using the ELISA kit, it became possible to evaluate the risk of brain stroke by not only plasma but also by urine. These results confirm that shortage of glutathione to detoxify acrolein is one of the major causes of stroke incidence. Our method contributes to maintenance of quality of life (QOL) of the elderly.
Highlights
Stroke is a serious common pathology and the incidence and severity increase with aging.early detection of the risk of stroke and management of the risk factors are crucial to maintain quality of life (QOL), but there were no reliable biomarkers available for the early phase of stroke.We found that increased plasma levels of protein-conjugated acrolein (PC-Acro) and the enzymes responsible for acrolein (CH2 =CH–CHO) production, polyamine oxidases, were good biomarkers for human stroke [1,2]
We successfully developed a sensitive enzyme-linked immunosorbent assay (ELISA) system to measure 3-hydroxypropyl mercapturic acid (3-HPMA) in urine using antibodies raised against 3-HPMA
In the system using a polyclonal antibody against 3-HPMA, linearity was obtained in the calibration curve
Summary
Stroke is a serious common pathology and the incidence and severity increase with aging. We found that increased plasma levels of protein-conjugated acrolein (PC-Acro) and the enzymes responsible for acrolein (CH2 =CH–CHO) production, polyamine oxidases, were good biomarkers for human stroke [1,2]. It is thought that cell damage in stroke is caused mainly by reactive oxygen species (ROS) [6,7]. Such as superoxide anion radical (O2 − ), hydrogen peroxide (H2 O2 ), and hydroxyl radical (OH). We tested whether 3-HPMA in urine of stroke patients decreased compared with that in urine of control subjects, because PC-Acro increased in plasma of stroke patients [1], probably due to the shortage of glutathione, a detoxifying compound of acrolein. We tried to develop a sensitive enzyme-linked immunosorbent assay (ELISA) system to measure 3-HPMA in urine
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