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Updated: Jun 19, 2026
Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay
Published on: May 12, 2020
Methylglyoxal augments intracellular oxidative stress in human aortic endothelial cells
Noriko Miyazawa1, Michiaki Abe, Tomokazu Souma
1Tohoku University Hospital, Division of Nephrology, Endocrinology and Vascular Medicine, Sendai, Miyagi, 980-8574, Japan.
Abstract:
Methylglyoxal (MGO) is a non-enzymatic metabolite in the glycolytic pathway and its concentration in blood and tissues is elevated in diabetes and renal failure. MGO induces tissue injuries via ROS; however, the mechanism remains to be clarified. The present study examined the harmful actions of MGO. Human aortic endothelial cells were assessed under real-time fluorescent microscopy with continuous superfusion. Increases in intracellular ROS were measured with fluorescent indicator, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (DCFH-DA). The addition of MGO rapidly increased the ROS in a dose-dependent manner. The increment of DCF was entirely abolished by pre-treatment with superoxide anion scavenger and membrane-permeable catalase, indicating that MGO induces superoxide production. The increment was completely inhibited by 2-thenoyltrifluoroacetone or carbonyl cyanide 3-chlorophenylhydrazone and partially inhibited by N-methyl-L-arginine. These data suggest that MGO stimulates superoxide production from mitochondria and partially stimulates nitric oxide synthase in human endothelial cells.
Insights
Methylglyoxal (MGO) increases reactive oxygen species (ROS) in endothelial cells, primarily through mitochondrial superoxide production. This finding clarifies MGO
Area of Science:
- Biochemistry
- Cell Biology
- Pathophysiology
Background:
- Methylglyoxal (MGO) is a metabolite linked to diabetes and renal failure.
- Elevated MGO levels correlate with tissue injury, but the underlying mechanisms are unclear.
- Reactive oxygen species (ROS) are implicated in MGO-induced damage.
Purpose of the Study:
- To investigate the cellular mechanisms by which MGO causes harm.
- To elucidate the role of ROS in MGO-induced endothelial cell injury.
- To identify the cellular sources of ROS stimulated by MGO.
Main Methods:
- Human aortic endothelial cells were cultured and subjected to real-time fluorescent microscopy.
- Intracellular ROS levels were quantified using the fluorescent indicator DCFH-DA.
- Cells were pre-treated with various scavengers and inhibitors, including superoxide anion scavenger, catalase, 2-thenoyltrifluoroacetone, carbonyl cyanide 3-chlorophenylhydrazone, and N-methyl-L-arginine.
Main Results:
- MGO exposure rapidly and dose-dependently increased intracellular ROS.
- The ROS increase was abolished by superoxide anion scavengers and catalase, confirming superoxide production.
- Inhibition studies indicated MGO stimulates mitochondrial superoxide production and partially activates nitric oxide synthase.
Conclusions:
- MGO induces oxidative stress in human aortic endothelial cells.
- Mitochondria are a primary source of MGO-induced superoxide production.
- MGO also partially activates nitric oxide synthase, contributing to cellular dysfunction.
