Folin–Ciocalteu reagent

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The Folin–Ciocalteu reagent (FCR) or Folin's phenol reagent or Folin–Denis reagent, also called the Gallic Acid Equivalence method (GAE), is a mixture of phosphomolybdate and phosphotungstate used for the colorimetric in vitro assay of phenolic and polyphenolic antioxidants.[1] It is named after Otto Folin, Vintilă Ciocâlteu, and Willey Glover Denis.

The reagent does not only measure phenols, and will react with any reducing substance. It therefore measures the total reducing capacity of a sample, not just phenolic compounds. This reagent is part of the Lowry protein assay, and will also react with some nitrogen-containing compounds such as hydroxylamine and guanidine.[2] The reagent has also been shown to be reactive towards thiols, many vitamins, the nucleotide base guanine, the trioses glyceraldehyde and dihydroxyacetone, and some inorganic ions. Copper complexation increases the reactivity of phenols towards this reagent.[3]

This reagent is distinct from Folin's reagent, which is used to detect amines and sulfur-containing compounds.

A 1951 paper entitled "Protein measurement with the Folin phenol reagent"[4] was the most cited paper in the 1945–1988 Science Citation Index, with 187,652 citations[5]

Physiologic significance

Because it measures anti-oxidant capacity in vitro, the reagent has been used to assay foods and supplements in food science. The oxygen radical absorbance capacity (ORAC) used to be the industry standard for antioxidant strength of whole foods, juices and food additives.[6][7] However, the United States Department of Agriculture (USDA) withdrew these ratings in 2012 as biologically invalid, stating that no physiological proof in vivo existed to support the free-radical theory.[8] Consequently, the ORAC method, derived only in in vitro experiments, is no longer considered relevant human diets or biology.

The Trolox equivalent antioxidant capacity assay is an alternative in vitro measurements of antioxidant capacity.[9]

References

  1. ↑ Singleton, Vernon L.; Orthofer, Rudolf; Lamuela-Raventós, Rosa M. (1999). [14] Analysis of total phenols and other oxidation substrates and antioxidants by means of folin–ciocalteu reagent 299. p. 152. doi:10.1016/S0076-6879(99)99017-1. 
  2. ↑ Ikawa M, Schaper TD, Dollard CA, Sasner JJ (2003). "Utilization of Folin–Ciocalteu phenol reagent for the detection of certain nitrogen compounds". J. Agric. Food Chem. 51 (7): 1811–5. doi:10.1021/jf021099r. PMID 12643635. 
  3. ↑ Everette, Jace D.; Bryant, Quinton M.; Green, Ashlee M.; Abbey, Yvonne A.; Wangila, Grant W.; Walker, Richard B. (2010). "Thorough Study of Reactivity of Various Compound Classes toward the Folin−Ciocalteu Reagent". J. Agric. Food Chem. 58 (14): 8139. doi:10.1021/jf1005935. PMID 20583841. 
  4. ↑ Oliver H. Lowry,Nira J. Rosebrough,A. Lewis Farr, and Rose J. Randall (1951). "Protein Measurement with the Folin Phenol Reagent". J. Biol. Chem. 193 (1): 265–275. PMID 14907713. 
  5. ↑
  6. ↑ Cao G, Alessio H, Cutler R (1993). "Oxygen-radical absorbance capacity assay for antioxidants". Free Radic Biol Med 14 (3): 303–11. doi:10.1016/0891-5849(93)90027-R. PMID 8458588. 
  7. ↑ Ou B, Hampsch-Woodill M, Prior R (2001). "Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe". J Agric Food Chem 49 (10): 4619–26. doi:10.1021/jf010586o. PMID 11599998. 
  8. ↑ "Withdrawn: Oxygen Radical Absorbance Capacity (ORAC) of Selected Foods, Release 2 (2010)". United States Department of Agriculture, Agricultural Research Service. 16 May 2012. Retrieved 13 June 2012. 
  9. ↑ Prior R, Wu X, Schaich K (2005). "Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements". J Agric Food Chem 53 (10): 4290–302. doi:10.1021/jf0502698. PMID 15884874. 

External links

Food antioxidants
Fuel antioxidants
Measurements

47641 Folin-Ciocalteu's phenol reagent Product Description: Appearance: Clear bright yellow solution. Acid concentration: 2 N based on sodium hydroxide titration. Folin & Ciocalteu’s phenol reagent should be stored tightly capped at room temperature. The reagent can be diluted with deionized water. Method of Preparation: Dissolve 10 g sodium tungstate and 2.5 g sodium molybdate in 70 ml water. Add 5 ml 85% phosphoric acid and 10 ml concentrated hydrochloric acid. Reflux for 10 hr. Add 15 g lithium sulfate, 5 ml water and 1 drop bromine. Reflux for 15 min. Cool to room temperature and bring to 100 ml with water.1 Hexavalent phosphomolybdic/phosphotungstic acid complexes with the following structures are formed in solution.2 3H2O•P2O5•13WO3•5MoO3•10H2O 3H2OxP2O5•14WO3•4MoO3•10H2O Applications: Folin & Ciocalteu’s phenol reagent does not contain phenol. Rather, the reagent will react with phenols and nonphenolic reducing substances to form chromogens that can be detected spectrophotometrically. It can also be used as a spray reagent in chromatographic procedures. The color development is due to the transfer of electrons at basic pH to reduce the phosphomolybdic/phosphotungstic acid complexes to form chromogens in which the metals have lower valence.3 The most common usage of this reagent is in the Lowry method for determining protein concentration.4 In this method, protein is pretreated with copper(II) in a modified biuret reagent (alkaline copper solution stabilized with sodium potassium tartrate). Addition of Folin & Ciocalteu’s phenol reagent generates chromogens that give increasing absorbance between 550 nm and 750 nm. Normally, absorbance at the peak (750 nm) or shoulder (660 nm) are used to quantify protein concentrations between 1-100 μg/ml while absorbance at 550 nm is used to quantitate higher protein concentrations. In the absence of copper, color intensity would be determined primarily by the tyrosine and tryptophan content of the protein, and to a lesser extent by cysteine, and histidine. Copper(II) enhances color formation by chelation with the peptide backbone, thus facilitating the transfer of electrons to the chromogens. Copper(II) has no effect on color formation by tyrosine, tryptophan, or histidine, but reduces that due to cysteine.2,4,5,6 Many modifications of the original assay procedure have been published2 including methods for enhancing the color development,5,7 for determining the content of insoluble proteins,4,8 and for automating the procedure.9 A list of compounds that interfere with the Lowry protein assay, including many buffers, chelating agents, detergents, and cyclic organic compounds, has been published.2 To control for the effect of these compounds on color development and, thus, on the calculated protein concentration of the sample, it is essential that the blank and standards be made up in the same medium as the samples. Precautions: For Laboratory Use Only. Not for drug, household or other uses. References 1. Krebs, K.G., et al. (1969) In E. Stahl (ed.), Thin Layer Chromatography. New York: Springer- 1. Verlag, p. 878. 2. Peterson, G.L., Anal. Biochem. 100, 201-220 (1979). 3. Bray, H.G., and Thorpe, W.V., Meth. Biochem. Anal. 1, 27-52 (1954). 4. Lowry, O.H., et al., Protein measurement with the Folin phenol reagent J. Biol. Chem. 193, 265-275 (1951) 5. Onishi, S.T., and Barr, J.K., Anal. Biochem. 86. 193-200 (1978). 6. Chattopadhyay, M.K.J., Pharm. Pharmacol. 45, 80 (1993). 7. Larson, E., et al., Anal. Biochem. 155, 243-248 (1986). 8. Peterson, G.L., Anal. Biochem. 83, 346-356 (1977). 9. Fryer, H.J.L., et al., Anal. Biochem. 153, 262-266 (1986).

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