Development of an ELISA for Measurement of Urinary 3-Hydroxypropyl Mercapturic Acid (3-HPMA), the Marker of Stroke

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.

Lipid peroxidation derived reactive carbonyl species in free and conjugated forms as an index of lipid peroxidation: limits and perspectives

“…Moreover, the yield of phase II metabolites (GSH adducts) depends not only on the RCS amount but also on the GSH availability in the site of oxidation which is affected by the same oxidative stress so that the amount of adducts can paradoxically decrease due to the consumption of GSH because of the oxidant reactions. In any case, mercapturic acid of RCS seems a good marker of exposure to exogenous RCS and ELISA methods have been proposed which will permit a greater application of the method [ 37 ].…”

“…Mercapturic acid adducted with the aldehyde in reduced or oxidized forms, such as 3-(s-glutathionyl)-1,4-dihydroxynonane (GSH-DHN) and 3-(s-glutathio-nyl)-4-hydroxynonanoic acid (GS-HNA) for HNE, are the final metabolic products and hence fulfill the first requirements and also have a peculiar structure allowing them to be easily identified by use of a mass analyzer. HPLC-ESI-MS is the main analytical tool which is used for mercapturic acid adduct analysis and most recently an ELISA test has been proposed and validated [ 37 ]. So far, validated analytical methods for mercapturic acid of HNE and ACR analyses in urine have been set-up and also applied as an index of oxidative stress.…”

Inorganic Matrices Assisted Laser Desorption/Ionization Mass Spectrometry for Metabolic Analysis in Biofluids

“…Also, it shows great superiority in diagnostics in terms of sampling invasiveness and low expense. To date, metabolic analysis based on body fluids, mainly including serum, plasma, urine, and cerebrospinal fluid, has been successfully applied to the diagnosis of cancer, [3] stroke, [4] mental diseases, [5] autoimmune diseases, [6] discovering a series of potential biomarkers for accurate diseases classification. Nevertheless, due to the low metabolite abundance and high sample complexity, metabolic analysis from bio‐fluids still faces several challenges: 1) detection methods with high throughput and sensitivity; [7] 2) sample treatment with simple operation and low‐costs; [8] and 3) devices and materials to combine the detection methods and sample treatment [9] .…”