Isocitrate is a crucial metabolite in intermediary metabolism, particularly within the tricarboxylic acid (TCA) cycle, also known as the Krebs cycle. The enzyme isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate, a key step in cellular energy production and biosynthetic precursor generation. Assay kits designed to measure isocitrate or isocitrate dehydrogenase activity provide valuable tools for studying metabolic flux, enzyme kinetics, and the effects of drugs or environmental factors on metabolism.

Biological Role of Isocitrate and Isocitrate Dehydrogenase

Isocitrate is formed from citrate by the enzyme aconitase and subsequently converted to α-ketoglutarate by isocitrate dehydrogenase (IDH), which exists in multiple isoforms (IDH1, IDH2, and IDH3) located in different cellular compartments such as the cytoplasm, mitochondria, and peroxisomes. This reaction is coupled with the reduction of NADP⁺ or NAD⁺ to NADPH or NADH, respectively, which are essential cofactors in cellular redox balance and biosynthesis. IDH plays a pivotal role in energy metabolism, redox regulation, and biosynthetic processes, including lipid metabolism and response to oxidative stress.

In immune cells like macrophages and dendritic cells, altered IDH activity and isocitrate levels influence metabolic reprogramming during activation, linking metabolism with immune function and inflammation.

Principle of Isocitrate Assay Kits

Modern isocitrate assay kits typically employ enzymatic reactions where isocitrate is oxidized by isocitrate dehydrogenase, producing NADPH. The generated NADPH is then used to reduce a colorimetric dye, resulting in a measurable color change proportional to the isocitrate concentration. The absorbance is commonly read at 565 nm. These assays are sensitive, fast, and adaptable to high-throughput formats, making them suitable for various sample types including food, beverages, cell lysates, tissue homogenates, and serum.

Features of Isocitrate Assay Kits

• Detection Range and Sensitivity: Kits generally detect isocitrate concentrations ranging from 20 to 5000 µM, with some offering detection limits as low as 0.2 mmol/L for IDH activity assays.
• Sample Types: Compatible with diverse biological samples such as tissues, cells, and fluids, as well as food and environmental samples.
• Assay Format: Most kits use a homogeneous "mix-incubate-measure" format, enabling rapid and convenient quantification within 10 minutes.
• Storage and Handling: Components are typically shipped on ice and stored at -20°C to maintain enzyme stability.

Applications in Research and Industry

• Metabolic Studies: Quantification of isocitrate levels and IDH activity helps elucidate metabolic flux through the TCA cycle under physiological and pathological conditions, including cancer metabolism and diabetes.
• Drug Screening: Evaluation of drug effects on isocitrate metabolism and IDH activity supports pharmacological research and development.
• Food and Environmental Analysis: Measurement of isocitrate in food and environmental samples aids in quality control and safety assessment.
• Immunometabolism: Understanding the role of isocitrate in immune cell function and inflammation through metabolic assays contributes to immunological research.

Isocitrate assay kits provide essential tools for the quantitative assessment of isocitrate and isocitrate dehydrogenase activity, facilitating research into intermediary metabolism and its regulation. By enabling sensitive, rapid, and versatile measurements, these assays support a broad range of scientific and industrial applications, from fundamental metabolic studies to clinical diagnostics and food safety.