Free Fatty Acid Assay Kit (Colorimetric)

Catalog No. ABIN2345128

Product Details

Detection Method: Fluorometric, Colorimetric

Application: Biochemical Assay (BCA)

Sample Type: Serum, Plasma

Characteristics: Free Fatty Acid Assay Kit measures non-esterified fatty acids (NEFA) in serum and plasma by a coupled enzymatic reaction system (ACS-ACOD Method). First, Acyl CoA Synthetase (ACS) catalyzes fatty acid acylation of coenzyme A. Next, the acyl-CoA product is oxidized by Acyl CoA Oxidase (ACOD), producing hydrogen peroxide which reacts with the kit's Colorimetric Probe (absorbance maxima of 570 nm). The Free Fatty Acid Assay Kit is a simple, colorimetric assay that quantitatively measures the free fatty acid concentration (non-esterified) in various samples using a 96-well microtiter plate format. Each kit provides sufficient reagents to perform up to 100 assays, including blanks, standards and unknown samples. The kit contains a palmitic acid standard and has a detection sensitivity limit of ~15 μM. Note: This kit is not suitable for urine or heparin-containing samples. Fatty acids (C8 and longer) can be quantified with this kit.

Components:

1. FFA Standard : One 100 μL vial of 50 mM palmitic acid in ethanol.
2. 20X Assay Buffer : One 1.5 mL vial.
3. 5X Enzyme Mixture A : Four 1 mL vials (containing ACS, Ascorbate Oxidase, and necessary cofactors).
4. 5X Enzyme Mixture B : Four 0.5 mL vials (containing ACOD).
5. NEM Reagent : One 150 μL amber vial.
6. Colorimetric Probe : One 110 μL amber vial.

Material not included:

1. 96-well microtiter plate
2. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
3. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
4. Multichannel micropipette reservoir
5. Microplate reader capable of reading at 570 nm

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Measures non-esterified fatty acids (NEFA) in serum or plasma
• Available with colorimetric or fluorometric detection
• Standard included for quantitative results

Reagent Preparation:

FFA Standard: Thaw at 37 °C for 10 minutes. Once homogeneous and mixed well, maintain the standard at room temperature during assay preparation. The solution is stable for 1 week at room temperature. For longer term storage, the solution should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws. 1X Assay Buffer: 20X Assay Buffer should be thawed/maintained at 4 °C during assay preparation. Dilute the 20X Assay Buffer with deionized water. Stir to homogeneity. The 1X solution is stable for 1 week at 4 °C. For longer term storage, any unused 20X stock material should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws. 1X Enzyme Mixture (A or B): Each 5X Enzyme Mixture should be thawed/maintained at 4 °C during assay preparation. Dilute the 5X Enzyme Mixture with cold, deionized water. Stir to homogeneity. The 1X solution is stable for 1 week at 4 °C. For longer term storage, any unused 5X stock material should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws. Note: These components are provided in multiple tubes to minimize multiple freeze/thaws. NEM Reagent: Thaw and maintain at 4 °C during assay preparation. The solution is stable for 1 week at 4 °C. For longer term storage, the solution should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws. Colorimetric Probe: Thaw and maintain at room temperature during assay preparation. Any unused material should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws.

Sample Preparation:

Urine: This kit is not recommended for urine samples. Plasma: Collect blood with an anticoagulant such as citrate, EDTA or oxalate and mix by inversion. Centrifuge the blood at 1000 x g at 4 °C for 10 minutes. Collect plasma supernatant without disturbing the white buffy layer. Sample should be tested immediately or frozen at -80 °C for storage. Plasma must be diluted before assaying (1:2 to 1:20 in 1X Assay Buffer). Normal FFA levels in human plasma are typically 150-450 μM. Note: Heparin is known to interfere with the assay. Heparin-containing samples including heparinized plasma should be avoided. Serum: Collect blood in a tube with no anticoagulant. Allow the blood to clot at room temperature for 30 minutes. Centrifuge at 2500 x g for 20 minutes. Remove the yellow serum supernatant without disturbing the white buffy layer. Samples should be tested immediately or frozen at -80 °C for storage. Serum must be diluted before assaying (1:2 to 1:20 in 1X Assay Buffer). Normal FFA levels in human serum are typically 100-700 μM.

Assay Procedure:

Each FFA standard and sample should be assayed in duplicate or triplicate. A freshly prepared standard curve should be used each time the assay is performed.

1. Add 10 μL of each FFA standard, sample and blank to the 96-well microtiter plate.
2. Add 200 μL of 1X Enzyme Mixture A (see Preparation of Reagents) to each well.
3. Cover the plate wells to protect the reaction from light.
4. Incubate at 37 °C for 30 minutes.
5. During the step 4 incubation, separately prepare the desired volume of Detection Enzyme Mixture according to Table 2 below, based on the number of tests to be performed. Maintaining all components and mixtures at 4 °C throughout this step, add components in the following sequence: a. In a tube, add the appropriate volume of 1X Enzyme Mixture B (see Preparation of Reagents). b. To the 1X Enzyme Mixture B add the corresponding volume of NEM Reagent. Mix well. c. Finally, add the corresponding volume of Colorimetric Probe. Mix well and use immediately. Note: Detection Enzyme Mixture will appear slightly pink in color. This is normal background and should be subtracted from all absorbance values. 1X Enzyme NEM Reagent Colorimetric Total Volume of # of Tests in Mixture B (μL) Probe (μL) Detection 96-well Plate (mL) Enzyme Mixture (100 μL/test) (mL) 10 100 100 10.2 100 5 50 50 5.1 50 2.5 25 25 2.55 25 Table
6. Preparation of Detection Enzyme Mixture 5
7. Transfer 100 μL of the above Detection Enzyme Mixture to each well (from step 4).
8. Cover the plate wells to protect the reaction from light.
9. Incubate at 37 °C for 10 minutes.
10. Read absorbance in the 540-570 nm range on a microplate reader.
11. Calculate the concentration of free fatty acid within samples by comparing the sample absorbance to the standard curve. Negative controls (without FFA) should be subtracted.

Restrictions: For Research Use only

Handling

Storage: -80 °C

Storage Comment: Store the entire kit at -80°C. Avoid multiple freeze/thaws by aliquoting. The NEM Reagent and Colorimetric Probe are light sensitive and should be maintained in amber tubes. 3

References

Ditzel, Nguyen, Parker, Camenisch: "Effects of Arsenite Exposure during Fetal Development on Energy Metabolism and Susceptibility to Diet-Induced Fatty Liver Disease in Male Mice." in: Environmental health perspectives, Vol. 124, Issue 2, pp. 201-9, (2016) (PubMed).

Healy, Lahiri, Hargett, Chow, Byrne, Breen, Kenwood, Taddeo, Lackner, Caldwell, Hoehn: "Dietary sugar intake increases liver tumor incidence in female mice." in: Scientific reports, Vol. 6, pp. 22292, (2016) (PubMed).

Miller, Snow, Henriquez, Schladweiler, Ledbetter, Richards, Andrews, Kodavanti: "Systemic metabolic derangement, pulmonary effects, and insulin insufficiency following subchronic ozone exposure in rats." in: Toxicology and applied pharmacology, Vol. 306, pp. 47-57, (2016) (PubMed).

Nicholas, Salto, Boston, Kim, Larios, Beeson, Firek, Casiano, Langridge, Cordero-MacIntyre, De Leon: "Identification of Anti-Long Chain Saturated Fatty Acid IgG Antibodies in Serum of Patients with Type 2 Diabetes." in: Mediators of inflammation, Vol. 2015, pp. 196297, (2015) (PubMed).

Diané, Nikolic, Rudecki, King, Bowie, Gray: "PACAP is essential for the adaptive thermogenic response of brown adipose tissue to cold exposure." in: The Journal of endocrinology, Vol. 222, Issue 3, pp. 327-39, (2014) (PubMed).

Nagajyothi, Weiss, Zhao, Koba, Jelicks, Cui, Factor, Scherer, Tanowitz: "High fat diet modulates Trypanosoma cruzi infection associated myocarditis." in: PLoS neglected tropical diseases, Vol. 8, Issue 10, pp. e3118, (2014) (PubMed).

Target Details

Background: Triglycerides (TAG) are a type of lipid in the blood, serving as an energy source and playing a key role in metabolism. Triglycerides are the digestive end product of breaking down dietary fats. Any extra carbohydrates and fats that are not immediately used are chemically converted into triglycerides. In the intestines, secreted enzyme lipases hydrolyze the triglyceride ester bond, yielding glycerol and free fatty acids (FFA) in a process called lipolysis. Additionally, hormones induce and regulate lipase activity in adipose tissue, resulting in changes to blood FFA levels. Free fatty acids then bind plasma albumin for circulation in the body, serving as a readily absorbed energy source for muscle, brain and other organ tissues. Measurement of free fatty acids has become useful in monitoring and diagnosis of several diseases and metabolic disorders (e.g. obesity, insulin resistance, diabetes, cancer).