The TOSC assay was developed for SIFT-MS analysis of aqueous systems from the GC-based method by Winston et. al., and recently has been adapted further by Senthilmohan & McEwan(19) to measure oil-in-water emulsions.
Peroxyl radicals are produced by thermal decomposition of AAPH at 37°C.
Reaction of these radicals with KMBA releases ethene (figure 7.2)(15). The other two oxidising species from the TOSC assay(3), OH● and peroxynitrite, are produced in the same way in the SIFT-MS-TOSC assay as in the original TOSC assay, i.e. via a Fenton reaction and decomposition of 3-morpholinosydnonimine N-ethylcarbamide(3). The peroxyl and hydroxyl radicals only were used in the present research.
NH2 AAPH forms ethene along with several other products.
The reaction of hydroxyl radicals with KMBA is thought to proceed via single electron transfer from the sulfide group(17), however no reaction mechanism has been proposed for the reaction of peroxyl radicals with KMBA. The measured products are the same for both reactions, therefore the mechanisms are expected to be similar. It should be noted however that the hydroxyl radical reacts via electron abstraction, while the peroxyl radical generally reacts via hydrogen atom transfer.
Whatever the mechanism, radical attack causes the KMBA molecule to cleave, giving two molecules of carbon dioxide, one of ethene and a methylthio radical (two of which may combine to produce dimethyl disulfide)(17). The production of acetone (the concentration of which is also observed to increase during the reaction)
ANTIOXIDANT INTRODUCTION 169
was not described by Winston et. al.(15), and is thought to be due solely to AAPH break-down products. The ethene concentration in the reaction vial headspace is monitored by SIFT-MS from its reaction with O2+
(reaction 7.12).
k = 1.0 x 10-9 cm3s-1
O
2++ C
2H
4→ C
2H
4++ O
2(7.12)
The important reactions in the TOSC assay occur in the aqueous phase, as both the KMBA substrate and the radicals derived from AAPH are highly soluble in water. To include olive oil, an oil-in-water emulsion is used. The hydrophilic components of olive oil dissolve into the aqueous phase from the emulsion droplets, while the hydrophobic components remain. These hydrophobic compounds are still able to protect the substrate, as they can scavenge radicals at the oil-water interface(20).
The method details are described in section 8.1. In brief, several different concentrations of oil are used in different bottles, giving a different inhibition value for each concentration. Using these inhibition values, it is possible to obtain the oil concentration which will cause 50 % inhibition of ethene production compared to the blank solution without olive oil. This value is called the Inhibition Concentration at 50 % (IC50). Due to the variability in composition of olive oil, a reliable measure of its concentration in mol L-1 is difficult to obtain. In the present research, the volume necessary to produce 50 % inhibition of ethene was found as it
is much easier to calculate and is considered to be proportional to the concentration to within experimental error.
The concentrations of AAPH and KMBA chosen are appropriate to give a good head space ethene concentration over the hour that the reaction is allowed to proceed. The range of concentrations of emulsified oil in the system is currently based on an estimate. A series of oil volumes are chosen which are then used to estimate the volume at which 50 % inhibition is achieved. This volume is termed the VI50 value. If the VI50 value is outside the chosen concentration range of the oils, the assay must be repeated, as the line of best fit is not always linear and extrapolation may be unreliable.
The SIFT-MS-TOSC assay has potential as a model for in vivo radical scavenging, where oil is emulsified and reactions occur in the aqueous phase, so the relative contributions of different antioxidant species in the SIFT-MS-TOSC assay are as close as currently possible to those in vivo. There are also different radical species which may be used, approximating this aspect of biological systems.
However, the SIFT-MS-TOSC assay, although much better suited to rapid analysis than the TOSC assay, does have some difficulty with high-throughput analysis.
Also, while one of the best substrates currently available is used (KMBA, which is similar to the naturally occurring amino acid methionine, the suspected source of ethene in plants(17)), the biological relevance of an assay which measures the ability to protect only KMBA is limited.
ANTIOXIDANT INTRODUCTION 171
7.4.5 Concluding Remarks
Many different assays exist for assessing antioxidant capacity, but only a selection of the most widely used assays have been mentioned above. In 2004 the first of a series of annual meetings on antioxidant methods was held with the purpose of choosing an analytical method or methods to best measure antioxidant capacity in foods and dietary supplements(21). A number of good review articles have also been written on the subject(9;11). As no single assay adequately recreates the in vivo situation, Frankel and Meyer(9) suggest the use of several standardised assays, as each assay has its own potential for interference and bias towards measuring certain types of antioxidants. Analysing the same sample with several different assays gives a better appreciation of the true in vivo antioxidant capacity.
The aim of this research was to develop a SIFT-MS-TOSC assay that could be used for a wide range of non-aqueous samples and to evaluate its use in the analysis of olive oil antioxidants.
7.5 References
(1) Huang, D.; Ou, B.; Prior, R. The Chemistry Behind Antioxidant Capacity Assays.
J. Agric. Food Chem. 2005, 53, 1841-1856.
(2) Halliwell, B.; Gutteridge, J. Free Radicals in Biology and Medicine, 3rd ed.;
Oxford University Press: Chennai, India, 1999.
(3) Regoli, F.; Winston, G. W. Quantification of Total Oxidant Scavenging Capacity of Antioxidants for Peroxynitrite, Peroxyl Radicals, and Hydroxyl Radicals.
Toxicol. Appl. Pharm. 1999, 156, 96-105.
(4) Lichtenthäler, R.; Marx, F.; Kind, O. M. Determination of Antioxidative Capacities Using an Enhanced Total Oxidant Scavenging Capacity (TOSC) Assay. Eur. Food Res. Technol. 2003, 216, 166-173.
(5) Belitz, H.-D.; Grosch, W. Food Chemistry, 2nd English ed.; Springer-Verlag:
Berlin, Germany, 1999.
(6) Uri, N. Inorganic Free Radicals in Solution. Chem. Rev. 1951, 50, 375-454.
(7) Knowles, R. G.; Moncada, S. Nitric Oxide Synthases in Mammals. Biochem. J.
1994, 298, 249-258.
(8) Halliwell, B. What Nitrates Tyrosine? Is Nitrotyrosine Specific as a Biomarker of Peroxynitrite Formation In Vivo? FEBS Letters 1997, 411, 157-160.
(9) Frankel, E. N.; Meyer, A. S. The Problems of Using One-Dimensional Methods to Evaluate Multifunctional Food and Biological Antioxidants. J. Sci. Food Agric.
2000, 80, 1925-1941.
(10) Mathews, C. K.; van Holde, K. Biochemistry; The Benjamin/Cummings Publishing Company: Redwood City, CA, USA, 1990.
(11) Prior, R.; Wu, X.; Schaich, K. Standardized Methods for the Determination of Antioxidant Capacity and Phenolics in Foods and Dietary Supplements. J. Agric.
Food Chem. 2005, 53, 4290-4302.
(12) Denisov, E. T.; Khudyakov, I. V. Mechanisms of Action and Reactivities of the Free Radicals of Inhibitors. Chem. Rev. 1987, 87, 1313-1357.
(13) Folin, O.; Ciocalteu, V. On Tyrosine and Tryptophane Determinations in Proteins.
J. Biol. Chem. 1927, 73, 627-650.
(14) Roginsky, V.; Lissi, E. A. Review of Methods to Determine Chain-Breaking Antioxidant Activity in Food. Food Chem. 2005, 92, 235-254.
(15) Winston, G.; Regoli, F.; Dugas, A. J.; Fong, J.; Blanchard, K. A Rapid Gas Chromatographic Assay for Determining Oxyradical Scavenging Capacity of Antioxidants and Biological Fluids. Free Rad. Biol. Med. 1998, 24, 480-493.
(16) Lieberman, M.; Kunishi, A.; Mapson, L.; Wardale, D. Ethylene Production from Methionine. Biochem. J. 1965, 97, 449-459.
(17) Yang, S. Further Studies on Ethylene Formation from
α-Keto-γ-Methylthiobutyric Acid or β-Methylthiopropionaldehyde by Peroxidase in the Presence of Sulfite and Oxygen. J. Biol. Chem. 1969, 244, 4360-4365.
(18) Beauchamp, C.; Fridovich, I. A Mechanism for the Production of Ethylene from Methional. J. Biol. Chem. 1970, 245, 4641-4646.
(19) Senthilmohan, S.; McEwan, M. A Method of Assaying the Antioxidant Activity of Pure Compounds, Extracts and Biological Fluids. New Zealand, 2005.
ANTIOXIDANT INTRODUCTION 173
(20) Frankel, E.; Huang, S.-W.; Kanner, J.; German, J. Interfacial Phenomena in the Evaluation of Antioxidants: Bulk Oils vs Emulsions. J. Agric. Food Chem. 1994, 42.
(21) Conference Targets Uniform Antioxidant Measurements.
http://www.chemistry.org/portal/a/c/s/1/feature_acs.html?id=c373e9fcee851a518f 6a4fd8fe800100 (accessed 15 Aug, 2005)