1978 Volume 6 Issue 1 Pages 73-81
It has been widely recognized that the determination of serum malondialdehyde (MDA), a product of lipid peroxidation, is one of the useful measure for the clinical study on the role of lipoperoxide in atherosclerotic disorders. Determination of serum MDA has been carried out mainly by the colorimetry of compound formed by coupling of MDA with thiobarbituric acid (TBA). Recently, however, it has been pointed out that sialic acids have the great influence on MDA determination in these methods. In this study, an attempt was made to establish the specific colorimetric method for the estimation of serum MDA eliminating such an influence.
The most important point in our modification is to use the TBA reagent dissolved in 2M sodium sulfate instead of the reagent in water used in conventional methods. Using this TBA reagent, the optimum conditions for the specific serum MDA determination was searched and established.
The procedure elaborated is as follows; MDA in serum specimen was precipitated with protein by adding 5 volumes of 20% trichloracetic acid to 0.5ml of serum in the test tube covered with aluminum foil to shut off the light. After centrifugation at 3, 500r.p.m. for 10 minutes the supernatant was decanted and the precipitate was washed once with 0.1N sulfuric acid. Then 2.5ml of 0.1N sulfuric acid and 3.0ml of TBA reagent were added to this precipitate and the coupling of MDA with TBA was carried out heating in boiling water bath for 30 minutes. After cooling in cold water the resulting chromogen was extracted with 4.0ml of n-butanol by vigorous shaking. This was followed by centrifugation at 3, 000 r.p.m. for 10 minutes and optical density of the organic phase was determined at the wavelength of 530nm.
In the absorption spectra obtained by conventional methods, exogenously added sialic acid greatly raised the optical density at 530nm, which is specific for MDA. On the contrary, in our method, sialic acid gave little color and had no absorbance at 530nm, and addition of sialic acid did not result in any changes in serum MDA value. Heated in trichloracetic acid, sialic acids may give rise to formation of aldehyde that reacts with TBA in the conventional method. Since coupling of MDA with TBA is performed by heating in 0.1N sulfuric acid, there seems to be no aldehyde formation from sialic acids in our method. This, with the effect of sodium sulfate to facilitate the transfer of chromogen into organic phase, may enable us to eliminate the interference from sialic acids.
The new method is specific for MDA and suitable for clinical application.
