L-Ascorbic acid

 

Principle

 

L-Ascorbic acid and related reducing compounds reduce the tetrazolium salt MTT [3-(4.5-DimethylthiazoIyl-2)-2,5-diphenyltetrazolium bromide] in presence of the catalyzing electron transfer molecule PMS (5-Methylphenaziniummethyl sulfate) at pH 3,5 forming a Formazane.

 

The test is suited to determine the sum of all reducing molecule in the sample.

 

 

 

L-Ascorbic acid  + MTT (+ PMS)  --> Dehydroascorbic acid + MTT-Formazane^- + H⁺

 

 

To obtain a specific signal for ascorbic acid the acid is oxidized by Ascorbate-Oxidase (AAO) using molecular oxygen.

 

The dehydroascorbate formed will not reduce MTT.

 

 

 

L-Ascorbate + 1/2 O₂ (+ AAO)  -->  Dehydroascorbate + H₂O

 

 

The difference of an assay with and without pretreatment will give the specific signal for ascorbic acid.

 

MTT-Formazane is quantified photometrically at 578 nm.