Enzymatic assay for quantitative analysis of (D)-2-hydroxyglutarate

Area of Science:

• Biochemistry
• Oncology
• Metabolomics

Background:

• Elevated (D)-2-hydroxyglutarate [D2HG] levels are associated with metabolic diseases and cancers harboring isocitrate dehydrogenase (IDH) mutations.
• Accurate D2HG measurement is vital for disease diagnosis and patient monitoring.
• Current detection methods like chromatography-mass spectrometry are complex and time-consuming.

Purpose of the Study:

• To develop a novel, rapid, cost-effective, and sensitive enzymatic assay for quantifying D2HG.
• To establish the assay's utility in various biological samples, including tumor tissues and serum.

Main Methods:

• An enzymatic assay utilizing (D)-2-hydroxyglutarate dehydrogenase (HGDH) and nicotinamide adenine dinucleotide (NAD+) to convert D2HG to α-ketoglutarate (αKG).
• Detection of stoichiometrically generated NADH to determine D2HG concentration.
• Quantification limits established for tumor tissue (0.44 μM) and serum (2.77 μM).

Main Results:

• The enzymatic assay demonstrated high sensitivity, enabling detection of basal D2HG levels in human tumor tissues and serum.
• Significantly elevated D2HG levels in IDH-mutated tumor tissues and serum from acute myeloid leukemia patients were readily identified.
• The assay successfully differentiated basal from elevated D2HG levels across multiple sample types.

Conclusions:

• The developed enzymatic assay provides a practical and efficient tool for D2HG detection.
• This assay is valuable for clinical applications, aiding in the diagnosis and management of diseases linked to D2HG.
• The assay's applicability extends to tumor tissue, serum, urine, cultured cells, and culture supernatants.