Several seconds elapsed between the mixing of the solutions and the initiation of absorbance measurements. Much of the decrease in absorbance would have been lost during the initial time interval, especially for strong redox inhibitors. Their patterns may appear to be similar to those of weak redox inhibitors, based on the slow phase of the reaction curve after rapid substrate consumption. We found that zileuton showed increases in absorbance after the peroxide substrate was fully consumed. On the contrary, the fluorescence assay resolved these issues by only measuring the values at the completion of the reaction. Five of the tested compounds are known to be redox-active. However, according to the absorbance assay, only three appeared to be redox-active. The other two showed non-redox patterns of increasing absorbance. Meanwhile, results from the fluorescence assay showed EC50 values that ranged from 130 nM to over 100 mM. The three compounds with the highest EC50 values matched those with known non-redox mechanisms. The DAPT discrepancies between the absorbance and fluorescence results may be partially explained by the endpoint measurements and high-signal windows. Furthermore, NDGA and CDC showed increases in the absorbance assay, which suggested that they are non-redox compounds. NDGA is a well-known redox compound, and the fluorescence assay revealed that CDC was the strongest redox compound. The low sensitivity of the absorbance assay alone cannot explain the discrepancies between its results and the known mechanisms of action. Different buffer condition was not the reason of disagreement between absorbance and fluorescence assay. The absorbance change is related to the loss of the conjugated system of 13-HpODE. The consumption of 13-HpODE is complex and includes the alkoxide and epoxyallylic radicals. From the unstable radical, several hydroxyl derivatives and cleavage products are produced, some of which can yield absorbance changes at 234 nm. In the given situation, radical scavenging activity may explain the contradictory results of NDGA and CDC. Czapski et al. suggested that strong antioxidants, such as NDGA and baicalein, may work by inhibiting the enzymatic activity of 5-LO and directly scavenging free radicals. Furthermore, they claimed that AA-861 -3,5,6-trimethyl-p-benzoquinone) and zileuton are weak antioxidants that can serve as specific tools to the 5-LO inhibition study. Czubowicz et al. also suggested that the antioxidant effect should be taken into 
consideration when evaluating 5-LO inhibitors. It is not rare for inhibitors of same target to have different mechanisms and to have multiple functions. Caffeic acid and its derivatives, such as CDC, have radical scavenging activities. NDGA is a well-known radical scavenger, and its activity was confirmed in studies by Czapski et al. and Czubowicz et al.. Their radical scavenging activities may have caused the intermediate radicals in the redox assay to produce different products. When these resulting products have UV absorbance, the redox absorbance assay can reflect the incorrect results that we have obtained with NDGA and CDC. The fluorescence assay is not affected by product Rapamycin variation because the dye reacts with substrate. By comparing the known mechanisms with the experimental results, we showed that the fluorescence assay is much more reliable in terms of sensitivity and accuracy. The redox mechanisms of known 5-LO inhibitors were assessed using the absorbance method. We found that the redox absorbance results were easily biased by many factors related to UV absorption, thus leading to inaccurate results. To overcome these limitations, we developed a fluorescence assay, which provides large signal windows and is not easily affected by reaction components, reaction speed, and radical scavenging activities.