What is the principle of LDH assay for the measurement of cell death?

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LDH assay is one of the most commonly used biochemical methods to measure cell membrane damage and cell death, especially during necrosis or late-stage apoptosis. LDH stands for Lactate Dehydrogenase, which is an intracellular enzyme found in the cytoplasm of almost all cells. Under normal conditions, LDH remains inside the cell. But when the cell membrane becomes damaged or ruptured due to stress, toxin, or cell death, LDH leaks out into the surrounding medium. The LDH assay takes advantage of this leakage to estimate the extent of cell death.

Principle of the LDH Assay

The principle of the LDH assay is based on the ability of LDH enzyme to catalyze a reaction that changes lactate to pyruvate. This reaction also results in the conversion of NAD⁺ to NADH. The NADH produced can then react with specific substrates to create a colored product, which can be measured using a spectrophotometer.

The overall reaction looks like this:

Lactate + NAD⁺ → Pyruvate + NADH + H⁺

The NADH produced is responsible for reducing a tetrazolium salt or INT reagent in the assay. This reduction results in a color change and the intensity of the color correlates with the amount of LDH released.

The more LDH in the culture medium, the more NADH is formed, leading to more intense color production. This color is measured at a specific wavelength (usually 490 nm or 540 nm) and the resulting absorbance value indicates the level of cell death.

Importance and Use of LDH Assay

It is used to measure cytotoxicity in drug testing and cell viability studies.

It helps to distinguish between necrotic and apoptotic cell death.

It is non-radioactive, easy to perform and suitable for high-throughput experiments.

It does not require destruction of the remaining cells, so the same sample can be used for other tests.

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