What is the principle of LDH assay?

The LDH assay protocol is based on an enzymatic coupling reaction: LDH released from the cell oxidizes lactate to generate NADH, which then reacts with WST to generate a yellow color. The intensity of the generated color correlates directly with the number of lyzed cells.

What is the calculation of LDH?

LDH activity is reported as nmole/min/mL = milliunit/mL One unit of LDH activity is defined as the amount of enzyme that catalyzes the conversion of lactate into pyruvate to generate 1.0 μmole of NADH per minute at 37 °C.

How does LDH mechanism work?

LDH produces lactic acid as an end product through a fermentation reaction. In the process, LDH removes electrons from NADH and makes NAD+, which is channelized in the glycolysis pathway to create ATP.

What is the use of LDH assay in toxicity study?

In the assay, the level of extracellular LDH released from damaged cells is measured as an indicator of cytotoxicity.

What is WST in LDH assay?

Cytotoxicity LDH Assay Kit-WST can be used to measure the released LDH according to the following scheme. LDH catalyzes dehydrogenation of lactate to pyruvate thereby reducing NAD to NADH. NADH reduces a water-soluble tetrazolium salt (WST) in the presence of an electron mediator to produce an orange formazan dye.

What is the specific activity of LDH?

The specific activity of purified LDH was 145.45 U/mg protein.

How do you normalize LDH?

You can normalise your results to total cellular LDH content to thus calculate relative LDH release; To do so use an agent that will cause total cell lysis ( such as 0.2 % Triton X-100) as your positive control, this should result in complete loss of membrane integrity and consequently total ‘leakage’ of LDH content.

How does LDH convert pyruvate to lactate?

LDH is an ubiquitous enzyme having a tetrameric structure. Subunits that can be of two types: M (muscle type) or H (heart type) encoded by two genes LDH-A and LDH-B, respectively. LDH catalyzes the conversion of pyruvate to lactate with the regeneration of NADH to NAD+.

How do you calculate percent cytotoxicity?

Cell cytotoxicity assays

  1. Average the duplicate reading for each sample.
  2. Subtract the culture medium background from your assay readings. This is the corrected absorbance.
  3. Calculate percentage cytotoxicity with the following equation, using corrected absorbance: % cytoxicity = (100 x (control – sample))

What are cytotoxicity assays?

Cytotoxicity assays measure parameters associated with loss of membrane integrity upon cell death. Cytotoxicity assays may detect cytosolic proteins that should not be released by the cell, unless the cell has lost membrane integrity and the proteins have leaked out of the cell.

How does pH affect LDH?

At pH values below 7.2 (pH 5.0-6.8) the enzyme showed high substrate inhibition, which was highly dependent on pH, NADH concentration and temperature. By way of substrate inhibition pH regulates, primarily, lactate dehydrogenase activity towards pyruvate, since the homotropic effects appear not to be dependent on pH.