The Assay Genie Fatty Acid Oxidation Assay kit is a highly sensitive colorimetric assay for the quantitative measurement of Fatty Acid Oxidation in cells and tissues.
This unique assay utilizes a highly soluble Octanoyl-CoA substrate which enables the accurate measurement of Fatty Acid Oxidation versus longer chain less-soluble substrates such as the 18C unsaturated fatty acid oleate.
Fatty acids provide more ATP per carbon than carbohydrates such as glucose. In tissues with high energy requirements such as heart tissue, up to 50 - 70% of energy comes from fatty acid beta-oxidation, which converts fatty acids to acetyl-CoA with concomitant production of FADH2 and NADH in the mitochondria.
This strictly aerobic oxidative pathway consists of four distinct steps: acyl-CoA dehydrogenation by acyl-CoA dehydrogenase, hydration by enoyl-CoA hydratase, dehydrogenation by 3-hydroxyacyl-CoA dehydrogenase, and thiolytic cleavage by beta-ketothiolase.
Importance of Studying Fatty Acid Oxidation
The importance of the beta-oxidation system in humans is exemplified by the existence of a group of genetic diseases categorized as fatty acid oxidation disorders (FAODs), which are a heterogeneous group of defects in fatty acid transport and oxidation, and are inherited as autosomal recessive disorders, having a wide range of clinical presentations.
In addition, evidence indicates that regulation of activity of FAO during fetal development is not only important for fetal life but also has implications for health and disease in adulthood. The measurement of FAO activity thus provides valuable insights into the pathophysiologic and metabolic basis of many diseases.
The Assay Genie non-radioactive FAO assay is based on oxidation of the substrate octanoyl-CoA. Generation of NADH is coupled to the reduction of the tetrazolium salt INT to formazan. The intensity of the red-colored formazan is proportional to increased FAO activity. The assay solution and substrate should be stored in aliquots at -80°C.
Figure 1: Fatty Acid Oxidation Assay: Tissue lysates were prepared from healthy Hamster hearts and the hearts of the TO2 Hamster model which is associated with heart failure. Lysates were used in the assay and the levels of fatty acid oxidation quantified.
Figure 2: Fatty Acid Oxidation Assay: Octanoyl-CoA is the substrate, not the product of the reaction. Please see the diagram.
Preparation of Cell/Tissue Extracts
Step
Procedure
1.
Wash ~106 cells twice with ice-cold phosphate-buffered saline (PBS), and remove PBS completely from the cell pellet. Cell pellet should be stored at -80ºC. Tissue sample should be washed with PBS thoroughly to remove blood cells, which can cause inconsistent assay result.
2.
Prepare enough 1x Cell Lysis Solution by diluting 10x Cell Lysis Solution with ice-cold dH2O. Add 50 – 100 µl ice-cold 1x Cell Lysis Solution to cell pellet. Extract cells by pipetting up and down (gently but thoroughly). Leave lysate on ice for 5 min with intermittent gentle agitation. If lysate is viscous, add more 1x Cell Lysis Solution and repeat pipetting. Centrifuge lysate in a refrigerated microfuge for 5 min at maximal speed. Recover supernatant for assay. Tissue is homogenized in 1x Cell Lysis Solution, and lysate is clarified by centrifugation. Use ~25 mg of tissue per 0.5 ml of 1x Cell Lysis Solution for tissue homogenization.
3.
Perform protein assay to determine sample protein concentration. Normalize sample protein concentration by diluting with ice-cold 1x Cell Lysis Solution to 1 – 3 mg/ml. Keep protein sample on ice at all times. Freeze thawed crude protein lysate can exhibit reduced enzyme activity. Lysate should be stored at -80ºC. Note: Do not use a buffer containing reducing agents or SDS.
Reagent Thawing:
Keep thawed FAO Assay Solution and 20x FAO Substrate on ice. Gently agitate solution prior to pipetting. It is important to minimize the time the reagents are thawed. Freeze solutions immediately after use.
Preparation of Control Solution and Reaction Solution:
Control solution is prepared by mixing 1 part of dH2O and 20 parts of FAO Assay Solution, e.g. 25 µl dH2O mixed with 500 µl FAO Assay Solution. Keep freshly prepared control solution on ice during assay.
Reaction solution is prepared by mixing 1 part of 20x FAO substrate and 20 parts of FAO Assay Solution, e.g. 25 µl 20x FAO Substrate mixed with 500 µl FAO Assay Solution. Keep solution on ice and use immediately. Each protein sample is treated with 50 µl control solution and 50 µl reaction solution in two separate sets.
Step
Procedure
1.
Add 10 µl of each protein sample to a 96-well plate in duplicate. Note: For drug discovery applications, add 1 µl of a drug inhibitor to both wells, and mix with sample by pipetting up and down.
2.
After all samples have been pipetted to the plate, swiftly add 50 µl control solution to one set of wells and 50 µl reaction solution to another set of wells. Mix contents by gentle agitation for 30 sec. Cover plate and incubate in a humidified 37°C incubator for 1 − 2 hr. FAO activity generates cherry red color in wells. Do not use a CO2 incubator. Note: Increase sample protein concentration and/or incubation time if little red color is detected.
3.
Stop assay by adding 50 µl 3% Acetic acid (not included in the kit) to each control solution well and reaction solution well followed by brief gentle agitation. Measure O.D.492 nm using a plate reader.
4.
Subtract control well reading from reaction well reading for each sample. The subtracted O.D. reading is proportional to fatty acid oxidation activity of the sample.
Pham et al.
Neutrophil trafficking to the site of infection requires Cpt1a-dependent fatty acid β-oxidation
1) Have you tested with cells? If not, how do you know 10^6 is the right number? Could you use less?
Since FAO activity is driven by mitochondrial contents, most cultured cells exhibit low, but detectable, FAO activity. 10^6 cells are required to make a concentrated protein extract for FAO detection.
2) Will the assay work with frozen tissue? Does it not depend on active enzymes for assay to work?
The assay works with frozen tissue. It is an enzyme activity assay.
3) How do you generate a standard curve to determine your results?
There is no standard curve for the assay. Optical density can be converted to enzyme units.
4) What inhibitor can you use for FAO?
We have not tested an FAO inhibitor.
5) What controls do you recommend for the assay especially with cells in culture?
Cells/tissues with high mitochondrial contents can be used as positive control.
6) Would this assay potentially work in whole blood samples or purified PBMCs?
The red colour of the sample will interfere with optical detection at 492nm. PBMC, if free of blood cells, can be processed using the standard protocol.
7) Can this measure Fatty Acid Oxidation in cells and tissues in plant material?
The assay has not been tested with plant cells, but I would expect it to work because this is a chemical rather than immunological assay.
8) Does this assay work with extracellular fluids?
The assay does not work with extracellular fluids.
9) What about long chain FA?
We have tested long chain fatty acid as substrate for the assay. However, the poor water solubility of long chain fatty acid poses a problem that renders the assay result unreliable.
10) Is measuring Long-chain FA is a more accurate determination of FAO? As short and medium do not need mitochondrial CPTs, while long ones do.
No, CPT is only relevant in an intact organ/cell system wherein the mitochondrial structure is maintained. The FAO assay however is based on the use of tissue/cell homogenates, and the activity of CPT therefore cannot be measured in the in vitro assay system.
11) How does the kit distinguish NADH production from the octanoyl-CoA from NADH production from endogenous tissue substrates (glycogen, triglycerides, etc.)?
The control solution prepared according to the protocol will measure NADH production from endogenous tissue substrates. The optical density obtained with the control solution is then subtracted out in final calculation of FAO activity.
12) It doesn’t look like there are standards. The FAO activity is calculated. How do I generate a ΔOD at 30 minutes if I’m only to measure at 30 and 60? Do I measure at time 0 as well?
You can measure OD without stopping the reaction at 30, 60, 90, or 120 min.
13. The 10x cell lysis solution has a lot of white sediment on the bottom that doesn’t seem to be dissolving. Should I warm the solution to resolubilize things?
Yes, it is caused by dry ice freezing. Just warm the solution with agitation.
A 3% Acetic acid solution needs to be prepared for reaction termination. The assay solution contains DMSO and iodonitrotetrazolium violet. Please refer to the product page of our website or contact us for MSDS information.
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