Hello Fatemeh Maleki

1. What are the recommended ratios (vol/vol or w/v) or typical concentrations of sulfuric acid, phosphotungstic acid, and TBA relative to serum/sample volume? Please specify typical volumes per µL or per mL of serum if possible.

1. For sulfuric acid, say for 20µL of serum, you will need with 500µL of 42 mM sulfuric acid. This is a 1:25 ratio (500/20=25).

2. For phosphotungstic acid, for 20µL of serum, you will need 125µL of phosphotungstic acid. This is a 1:6.25 ratio (125/20=6.25).

3. After the acid precipitation steps and resuspending the pellet in 200µL of water, a standard protocol calls for 600µL of TBA reagent for the colorimetric reaction. This is a 1:3 ratio (600/200=3). The specific concentration of TBA reagent can vary, but it is often prepared as a 0.67% (w/v) solution.

2. Can TBA be dissolved using a boiling water bath (bain-marie)? Are there recommended temperatures, times, or precautions to avoid decomposition or contamination?

Yes, a boiling water bath (bain-marie) can be used to dissolve TBA. As TBA is not very soluble in water alone, it often requires heat for proper dissolution. The melting point of TBA is around 245°C. It is also noted that the compound decomposes upon melting though some sources may report slightly different values like 235°C. This value can vary depending on the specific grade and purity of the chemical. So, always check the manufacturer's specifications for the most accurate information on the melting point of a specific thiobarbituric acid product.

A standard procedure for preparing a TBA reagent involves the below steps:

3. Do you have practical tips to enhance color intensity and improve assay sensitivity (e.g., incubation time/temperature, pH adjustments, additives, extraction steps, or reference protocols)?

To enhance color intensity and sensitivity in MDA assay,

a) use acidic conditions (around pH 2.5) as the optimal pH for the reaction between MDA and TBA is around 2.5.

b) optimize incubation temperature and time. A 60-70°C incubation for 30 to 40 minutes is common.

c) after incubation, rapidly cooling the reaction mixture, for example on ice for 5-10 minutes, can improve stability and assay performance.

d) use high-purity water for sample preparation.

e) include an anti-foaming agent, such as butylated hydroxytoluene to prevent oxidation and minimize interference.

References are provided below.

Article Determination of the Optimal Method for Measuring Malondiald...

Article Effects of Analytical Procedures on the Repeatability of Mal...

Best,