Malondialdehyde TBARS assay

In the UVB study we also measured malondialdehyde, a marker of lipid peroxidation, but were unable to detect any increase in response to UV light, in contrast to other reports. There are a number of possible explanations. In our experiment, a-tocopherol levels remained high after irradiation, and it is known that lipid peroxidation does not begin in in vitro membrane systems until a-tocopherol is completely depleted [29], Several studies have shown a time lag of one to several hours after irradiation to occur before a measurable increase in cutaneous lipid peroxidation can be detected [26-28] since skin was processed immediately in our experiments, lipid peroxidation may not have reached detectable limits. Finally, the TBARS assay for malondialdehyde is notoriously fraught with artifact and has a relatively high background [30], and noise levels may simply have been too high to detect peroxidation. Results... 

This test is used for both in vitro and in vivo determinations. It involves reacting thiobarbituric acid (TBA) with malondialdehyde (MDA), produced by lipid hydroperoxide decomposition, to form a red chromophore with peak absorbance at 532 nm (Fig. 10.1). The TBARS reaction is not specific. Many other substances, including other alkanals, proteins, sucrose, or urea, may react with TBA to form colored species that can interfere with this assay. 

Two bioassays were used to evaluate the potency of Sal B and SM-EW-1 fraction to inhibit human LDL oxidation in vitro. The first assay was based on inhibition of malondialdehyde (MDA) formation, determined as TBARS by reacting with thiobaibituric acid (TBA), at fixed tin (2-6 hr). The ICso values indicated that Sal B (1.1 pM) was 4.0 times more potent than probucol (4.5 pM). The time course study of Sal B to inhibit conjugated diene formation in a standard LDL oxidation assay (LDL at 50 pg/mL was reacted with 5 pM Cu was also carried out in a microtiter plate (quartz) (44,45). Sal B prolonged the lag phase (ATiag) of LDL oxidation 7.5 times longer than probucol both at 1.0 pM (Table 1) (Figure 4). Sal B accounted for up to 75% of antioxidant activity in SM-EW-1 fraction. 

---