The Free Fatty Acids Half-Micro Test Kit from Roche is a colorimetric assay designed to quantify free fatty acids (FFA) in plasma and serum samples. Metabolically, FFAs are converted into acyl-CoA by ATP and CoA in a reaction catalyzed by Acyl CS, yielding AMP and pyrophosphate. Further, acyl-CoA reacts with oxygen in a reaction catalyzed by acyl-CoA oxidase (ACOD), producing 2,3-enoyl-CoA. The resulting hydrogen peroxide converts 2,4,6-tribromo-3-hydroxy-benzoic acid (TBHB) and 4-aminoantipyrine (4-AA) into a red dye in the presence of peroxidase (POD). The dye is then measured in the visible range light at 546 nm.
I used this kit to measure the FFA levels in frozen conserved serum samples from rats. I was looking for differences in serum levels of FFAs between control and test animals in my studies. This method for measuring FFAs is a little bit complicated because of both the large number of solutions to be prepared and steps involved. However, in my opinion, it is one of the best and most accurate methods available. The protocol has to be followed precisely in order to avoid problems related to the preparation of the numerous solutions. The kit contains almost all of the required solutions for the protocol. Palmitic acid is required (as a fat standard) and recommended by the company, but oleic acid may be used in its place with satisfactory results. Other solutions not contained in the kit are commonplace in most labs (i.e. Absolute ethanol and Triton X-100), thus obtaining them is not a problem.
Before starting the procedure itself, a few working solutions have to be prepared. This is a potential concern with this kit, as the prepared solutions do not remain stable for long thus, it is better to measure numerous samples simultaneously to prevent waste. A standard solution, such as oleate or palmitate, has to be prepared first (described in kit’s protocol) to generate a standard curve and ensure accurate measurement. Next, working solution #1 is made by dissolving Triton® X-100 in redistilled water and warming, cooling and diluting the mixture. Palmitic acid (or oleic acid) is then dissolved in warm ethanol, sealed and allowed to cool to make working solution #2. The two working solutions are combined as directed within the instructions to create the standard solution which is stable for only 3 days in a refrigerator.
The next step is the preparation of the kit itself by combining the solutions (1-5) as described within the instruction manual to make reaction mixtures A and B (use forceps when handling tablets included in the kit), which are good for 10 assays. Both reaction mixtures A and B are stable for 5 days at 2-8°C or 8 h at 15-25°C and are light sensitive. Either fresh or frozen serum may be used for the assay. In my studies I used frozen rat serum and a standard curve prepared with oleic acid. I used small cuvettes to measure the light absorbance for the samples and standard curve. For best results, the blank, standard curve and samples should be prepared as described in the kit. This may become complicated, especially when analyzing a large number of samples, as the user has a limited amount of time to pipette the required solutions in many cuvettes and mix the reagents. Light absorbance (546 nm) is measured at two steps of the reaction (termed A1 and A2) to calculate FFA in the sample relative to both the standard and the blank. This method shows linearity up to a concentration of 1.5 mmol FFA, an important consideration during sample dilution and analysis.
As a summary for using this kit, the method involved is relatively simple for a person with some experience in lab techniques. Most of the critical solutions for the protocol are part of the kit and some are ready to use while others require preparation (a potential source of human error). The full protocol is described in detail in the kit instructions, though the timing for each step and the proportions/preparation of solutions may be additional sources of error. A major advantage of the kit is the accuracy obtained if the whole procedure is followed carefully. In my rat serum samples, I was content with the degree of accuracy with which I accomplished my research goals.
Andrei I. Oprescu
Medical Science Graduate Student
University of Toronto