Measurement of antioxidant activity with the thiobarbituric acid reactive substances assay

Accepted Manuscript Measurement of antioxidant activity with the thiobarbituric acid reactive substances assay Md Ahsan Ghani, Celia Barril, Danny R Bedgood Jr., Paul D Prenzler PII: DOI: Reference:

S0308-8146(17)30339-4 http://dx.doi.org/10.1016/j.foodchem.2017.02.127 FOCH 20680

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

21 December 2015 1 October 2016 26 February 2017

Please cite this article as: Ghani, M.A., Barril, C., Bedgood, D.R. Jr., Prenzler, P.D., Measurement of antioxidant activity with the thiobarbituric acid reactive substances assay, Food Chemistry (2017), doi: http://dx.doi.org/ 10.1016/j.foodchem.2017.02.127

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Measurement of antioxidant activity with the thiobarbituric acid reactive substances assay

Md Ahsan Ghania,b, Celia Barrila, Danny R Bedgood, Jr.a, Paul D Prenzlera,b,* a

School of Agricultural and Wine Sciences, Charles Sturt University, Wagga Wagga, NSW-

2650, Australia b

Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga Wagga, NSW-

2650, Australia.

*Corresponding author: School of Agricultural & Wine Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW 2678, Australia, Tel,:+61 2 6933 2978, Email: [email protected]

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Abstract: The thiobarbituric acid reactive substances (TBARS) assay is widely used to measure lipid oxidation and antioxidant activity in food and physiological systems. However, there has been no review (to our knowledge) that focuses exclusively on this test. This review presents an overview of the current use of the TBARS test in food and physiological systems, before looking at the various ways in which the assay is used in studies on antioxidant activity. As an antioxidant assay, the TBARS test may lack acceptable reproducibility, and long reaction times may preclude its adoption as a rapid screening method. Despite these potential limitations, there are features of the TBARS test that make it useful as a complement to popular screening tests such as Trolox equivalent antioxidant capacity. This review concludes with proposals for development of the TBARS test so that it can be used as a rapid and robust antioxidant assay.

Key words: TBARS assay; methodology; lipid substrate; antioxidant activity; screening.

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1. Introduction: Lipid oxidation is problematic in food systems, where oxidative rancidity causes nutritional loss and development of toxic compounds, and in human physiology, where oxidation of lipids is a major contributor to diseases such as atherosclerosis (Spickett, Fedorova, Hoffmann, & Forman, 2015). The mechanism of lipid oxidation is widely studied and numerous reviews are available to provide the reader with further background (Frankel, 2014). Briefly, lipid oxidation proceeds via a free radical chain mechanism giving many end products, with aldehydes prominent among them. One of the most studied aldehydes is malondialdehyde (MDA) as it is associated with offflavours and aromas in meat products (Fernandez, Perez-Alvarez, & Fernandez-Lopez, 1997), as well as being a marker of oxidative damage in physiological systems (Del Rio, Stewart, & Pellegrini, 2005).

There are numerous methods for measuring MDA, including gas chromatography, high performance liquid chromatography (Del Rio, Stewart, & Pellegrini, 2005) and capillary electrophoresis (Wilson, Metz, Graver, & Rao, 1997) but, by far, the most common method is through reaction of MDA with thiobarbituric acid (TBA) to produce a pink-coloured dimeric compound. The ease of this reaction combined with the simplicity of using visible spectrometry to quantify the pink adduct has contributed to the ongoing widespread use of this method, particularly in the meat industry (Fernandez, Perez-Alvarez, & Fernandez-Lopez, 1997), but also increasingly in studies in physiological systems (Kondo, Masutomi, Noda, Ozawa, Takahashi, Handa, et al., 2014). This is despite recurring criticisms of the method showing poor reproducibility (Janero, 1990) and over-estimation of results (Del Rio, Stewart, & Pellegrini, 2005), which point to its lack of specificity for accurately quantifying MDA. These issues have 1

not deterred researchers from using the method, as evidenced by over 1,300 entries over the last 10 years (using the search terms thiobarbituric acid and malondialdehyde or variants, e.g. TBA, MDA) in the Web of Science database.

The lack of specificity of the reaction involving TBA to quantify MDA has been recognised since the early 1950s with numerous researchers showing that TBA reacts with a variety of aldehydes and the breakdown products of proteins and carbohydrates (Guillen-Sans & GuzmanChozas, 1998). Some of these reactions give pink-coloured compounds, while some produce yellow species the absorbance of which tail into the region around 530 nm and hence still interfere with the measurement of MDA. Nevertheless, early work, particularly with meat products, found correlations between the intensity of the pink colour generated through reaction with TBA, and the sensory properties of the product (Guillen-Sans & Guzman-Chozas, 1998). Thus, the term “TBARS” was coined as thiobarbituric acid reactive substances and, even though not specific, the TBARS assay has a prominent place in food chemistry (Guillen-Sans & Guzman-Chozas, 1998). Similarly, the TBARS test is well-entrenched in studies on human health and diseases, including inter alia: cardiovascular disease (Okauchi, Kishida, Funahashi, Noguchi, Ogawa, Okita, et al., 2011), obesity and diabetes (Furukawa, Fujita, Shimabukuro, Iwaki, Yamada, Nakajima, et al., 2004). Various methods have been developed to increase the specificity of the TBA reaction to measure just MDA, including distillation prior to colour development (Díaz, Linares, Egea, Auqui, & Garrido, 2014), GC (Giera, Lingeman, & Niessen, 2012) and HPLC (Papastergiadis, Mubiru, Van Langenhove, & De Meulenaer, 2012) methods. However, they have not yet gained widespread use, as the vast majority of workers still use the simple colourimetric test. 1

In addition to the well-documented lack of specificity of the TBARS assay, our review has found that there may also be some confusion in the literature as to how to report the results quantitatively. Analysis of the references in Tables 1-5 and Supplementary Materials S1-S2, revealed that of 58 studies, 26 reported results consistent with methodology (i.e., when TBARS were measured, correct units were used, e.g. mg MDA equivalents/per unit sample). In the remaining studies, the most common error was to confuse measuring TBARS with measuring MDA. Wrolstad et al. (2005) are unequivocal that they are not the same thing and that most, if not all methods, are non-specific, i.e., TBARS are measured not MDA. Evidence of confusion is found in studies where TBARS are reported in units of moles as if a pure compound was being determined. In other studies, the incorrect quantitation of TBARS may simply be due to imprecise terminology, i.e., “mg MDA” rather than “mg MDA equivalents”, a subtle, yet important distinction. It is difficult to pinpoint exactly where the notion that TBARS = MDA originated, but Buege and Aust (1978) stated that “Malondialdehyde has been identified as the (emphasis ours) product of lipid peroxidation that reacts with thiobarbituric acid to give a red species absorbing at 535 nm." Perhaps it is not surprising given this influential reference (see below) is in the physiology area, that the incorrect reporting of TBARS occurs more frequently in this field of research.

Along with the measurement of lipid oxidation products, of equal importance has been the search for ways to stop or inhibit lipid oxidation through the use of antioxidants. In this context, antioxidants are defined as “any substance that, when present at low concentration compared to that of an oxidisable substrate, significantly delays or prevents oxidation of that substrate” 1

(Frankel & Meyer, 2000). The last twenty years have seen a surge in research on antioxidants driven by two main factors. One has been increasing interest between diet and health, particularly linked to the so-called “French Paradox” (Yamagata, Tagami, & Yamori, 2015). This dietary effect has led to an interest in understanding the connection between diets rich in fruits, vegetables, nuts, etc. and low incidence of heart and other diseases in certain populations. For many years, this line of inquiry looked particularly at phenolic compounds1 (mainly those with an ortho-diphenol moiety) due to their inherently high reducing capacity (i.e., antioxidant activity). Recently, the dietary phenol-antioxidant hypothesis has been challenged and a wider variety of bioactivities for dietary phenols are now being explored (Tucker & Robards, 2008). The second main driver for research in antioxidants has come from the food industry, where synthetic antioxidants are being replaced by those derived from plant sources. This field of research has arisen as consumer attitudes towards food have changed, with the prevailing view that “natural” ingredients are preferred over synthetic ones (Laguerre, Bayrasy, Panya, Weiss, McClements, Lecomte, et al., 2015). There are also concerns over the safety of some of the synthetic antioxidants, and some of these compounds have been banned for use in foods (Shahidi & Zhong, 2010).

In order to assess the effectiveness of an antioxidant in preventing or delaying lipid oxidation, the activity must be measured. Again, the literature on antioxidant assays has been extensively reviewed by ourselves (Antolovich, Prenzler, Patsalides, McDonald, & Robards, 2002) and others (Prior, Wu, & Schaich, 2005; Tan & Lim, 2015). Yet, despite more than twenty years of research, no antioxidant assay is entirely suitable. Many popular and recommended (Huang, Ou, 1

sometimes referred to as “polyphenols”, although this term is often not strictly chemically correct, or “biophenols”, mainly in the olive literature

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& Prior, 2005) assays such as the Trolox equivalent antioxidant capacity (TEAC) assay simply measure the ability of an antioxidant (or mixture in the case of a plant extract, for example) to transfer an electron to a free radical. This assay is thus not strictly an antioxidant assay because there is no substrate being protected. The oxygen radical absorbance capacity (ORAC) assay, while having a substrate, now most commonly fluorescein (Huang, Ou, & Prior, 2005), can be criticised on the basis that the substrate is not relevant to food or physiological systems. The Folin-Ciocalteu (FC) assay is commonly associated with studies on antioxidant activity and seems to be a mandatory assay in this area. It is often referred to as the total phenols assay, even though it is well known that many non-phenolic compounds will reduce the polyoxomolybdotungstate complex to a blue colour. Although the FC assay has been reported to correlate well with antioxidant assays such as TEAC (Huang, Ou, & Prior, 2005), it may be criticised as an antioxidant assay due to lack of a substrate and lack of biological relevance.

Interestingly, there is a group of antioxidant assays that utilise a lipid substrate, although they do not appear to be among those recommended in the literature (e.g. (Prior, Wu, & Schaich, 2005)). Among them are: the β-carotene bleaching assay; crocin bleaching assay; TBARS assay, and ferric thiocyanate (FTC) assay for hydroperoxides, the latter two using a variety of lipid substrates such as linoleic acid (LA) (Dutta & Maharia, 2012). Numerous criticisms of these assays have arisen (discussed in more detail below for the TBARS assay), yet they do have the advantage in that any antioxidant activity detected occurs according to the definition of an antioxidant, i.e., a substrate is being protected during the assay. Furthermore, the assays continue to be used to study various aspects of antioxidant activity. In the last 10 years, more than 15,000 TBARS, 760 β -carotene bleaching, 30 crocin bleaching, 420 conjugated dienes, and 280 FTC 1

publications have appeared (Web of Science database). Clearly, the TBARS assay is the most widely used lipid-based assay, yet, to the authors’ best knowledge, there has been no review dedicated to the use of this assay.

Here we present a review of the various aspects of the TBARS assay including: TBARS measurement in food systems [see also (Guillen-Sans & Guzman-Chozas, 1998)]; TBARS measurement in physiological systems; TBARS measurement to assess antioxidant activity in food and physiological systems; and the use of TBARS in antioxidant screening studies. The latter will be the focus of this review where we will compare and contrast this assay with the recommended assays (see above).

2. Application of the TBARS assay to measure oxidation in lipid systems. The use of the TBARS assay to measure oxidation in lipid systems originated with the work of Kohn and Liversedge in 1944 (Kohn & Liversedge, 1944). In this study, oxidised brain tissue was shown to generate a metabolite that reacted with TBA to give a red/pink colour. Over the next 70 years, the TBARS assay has developed to become one of the most widely used assays to measure lipid oxidation in food and physiological systems. Despite the many variations of methodology reported in the literature (see below), all TBARS assays share common features. A food, tissue, or fluid that contains oxidised lipidic material is prepared for reaction with TBA by homogenising the sample or extracting MDA (and other TBARS) from the substrate. Often an antioxidant (e.g. BHT) is added at this time to prevent further oxidation. TBA is added, under acidic conditions, and the red/pink colour is developed over time (10-40 min) usually at around 100 °C. Depending on the sample, further extraction of the red/pink chromogen may be required 1

and often n-butanol is used for this purpose. Finally, the absorbance at 530-538 nm is measured: the higher the absorbance, the greater the amount of TBARS formed, and hence the greater the extent of oxidation.

2.1 Food Systems The TBARS assay has been widely used for over 60 years to measure oxidative rancidity in meat products, since the work of Turner et al. in the 1950s (Turner, Paynter, Montie, Bessert, Struck, & Olson, 1954). The original method of Turner et al. (1954) involved direct reaction of a TBA solution with oxidised pork meat and subsequent extraction of the red/pink pigment. The pork meat was ground to a specific size and TBA was added in the presence of phosphoric acid and trichloroacetic acid (TCA). After 30-50 min heating in a boiling water bath, the coloured pigment was extracted with an iso-amyl alcohol-pyridine mixture and absorbance at 538 nm measured. One of the important aspects of the method was that strong acid was used to release MDA from proteins in the sample, a point emphasised by Fernandez et al. (1997) in their review (Fernandez, Perez-Alvarez, & Fernandez-Lopez, 1997). Since this initial work, a plethora of alternate methods have been reported, with a selection of those published in the last five years for different meat products given in Table 1.

{Table 1}

As can be seen in Table 1, every possible reaction parameter has been varied including: the amount of sample 0.1‒10 g; added antioxidant (BHT, BHA, propyl gallate); concentration of TCA (5-15%, v/v); volume and concentration of TBA; heating time and temperature; and 1

absorbance wavelength. Sørensen and Jørgensen (1996) observed “To our knowledge no single version of the TBA test is suitable for application to all sample matrices”, yet, it is unclear why so many different methods are currently in use. Even within a single year and in the same journal, variations on the TBARS method are evident (Supplementary Material, Table S1).

Looking at Tables 1 and S1, some reaction conditions are more commonly used than others. For example, the most commonly used acid is TCA, colour development is most commonly achieved by heating in a boiling water bath, and the wavelength for quantification is usually 532 nm. A recent review on measuring lipid oxidation in foods (Barriuso, Astiasaran, & Ansorena, 2013) noted the many variations on the TBARS measurement, but provided no explanation as to why they have occurred.

Vegetable oils represent another food system where there has been intense interest in oxidation reactions and the measurement of rancidity by various means. The TBARS assay is also utilised in this context, but not as widely as in meat products. Furthermore, to the authors’ best knowledge, there have been no studies where the TBARS test has been used to screen antioxidants in vegetable oils. Because the focus of this review is on antioxidant activity studies, no further discussion of vegetable oils will be provided. However a table has been prepared showing some recent studies on the use of the TBARS assay in vegetable oils and is available in Supplementary Material (Table S2).

2.2 Human physiology including animal model studies

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Lipid oxidation has been implicated in many diseases in humans including atherosclerosis, cancer, rheumatoid arthritis, and neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases (Reed, 2011). As such, a number of studies using the TBARS assay have been performed to assess lipid oxidation in human and related (e.g. animal model) biological samples. The method proposed by Buege and Aust (1978) has been widely cited (over 180 times according to Google Scholar). In their method, Buege and Aust (1978) mixed 1.0 mL of sample with 2.0 mL of 15% (v/v) TCA, 0.375% (w/v) TBA and 0.25 M HCl and heated the mixture for 15 min in a boiling water bath. The solution was cooled, then centrifuged at 1000 g for 10 min, and absorbance was measured at 535 nm.

Table 2 shows variations of the TBARS assay across a range of samples related to the measurement of oxidation in different physiological systems. The samples include exhaled breath condensate, blood/plasma, and tissue homogenates. As has been previously noted, no standard conditions appear to be operative in this sphere of research. One of the main differences with the methods reported in the previous section is the common use of sodium dodecyl sulfate (SDS), as originally proposed by Ohkawa et al. (Ohkawa, Ohishi, & Yagi, 1979).

{Table 2}

In the context of this review, the many variations on measuring TBARS covered in Section 2 have implications for considering its use as an antioxidant assay. One of those is that an antioxidant assay requires standardised conditions such that results may be comparable across laboratories. With an extraordinary number of different reaction conditions used to measure 1

TBARS, finding a set of conditions that will be accepted by researchers as definitive may be a challenge.

3. The use of the TBARS assay to measure antioxidant activity in lipid systems: The production of TBARS occurs at the end of the various stages of lipid oxidation (Figure 1). However as highlighted in Figure 1, an antioxidant may act at any steps of the process prior to the formation of TBARS. Therefore, the measurement of TBARS gives no indication of the mechanism of action of the antioxidant, i.e., whether it is able to interact with oxygen or metal ions, react directly with hydroperoxides, or intercept the free radicals involved in the breakdown of primary to secondary oxidation products. Nevertheless, just as the measurement of TBARS is utilised in thousands of studies to monitor lipid oxidation, so too there are numerous reports in the literature of the use of the TBARS assay to monitor the effectiveness of an antioxidant.

The use of the TBARS assay can be generally classified in two ways depending on the research question. Firstly, and most commonly, it is used to answer the question – does the presence of an antioxidant have an effect on the oxidation of the substrate of interest? In these studies, a substrate (e.g. beef patty) is prepared with and without the antioxidant, oxidation is initiated, and TBARS are measured at some fixed time point. If the TBARS are lower in the sample containing the antioxidant, it is concluded that the substance has indeed behaved as an antioxidant. These types of studies are briefly reviewed in this section.

In the second type of study, a number of antioxidants (e.g. medicinal plant extracts) are screened for antioxidant activity. In these types of studies, the research question is – what is the best 1

antioxidant? Here, quantification of antioxidant activity is essential in order to rank the various antioxidants tested. Section 4 will review this use of the TBARS assay.

{Figure 1}

3.1 Food Systems Table 3 lists some recent studies where antioxidant activity was monitored in meat products through the TBARS assay. The meat products were chosen so that where possible, they match those shown in Tables 1 and S1. This is not an exhaustive list, rather it is illustrative of some of the variations that can be found in these types of studies. The conditions employed to measure TBARS are similar, and as variable, as highlighted above and will not be discussed here. Instead, the discussion will briefly touch upon the issues that must be considered when attempting to measure antioxidant activity in these products.

{Table 3}

Some such considerations include: how the antioxidant is added (solid, dissolved in solvent, concentration, volume, etc.); oxidation time; whether oxidation is stopped prior to measuring TBARS; and whether other measures of oxidation are monitored. The antioxidant must be added to achieve homogenous dispersal through the sample, and therefore the mode of delivery is critical. Not surprisingly, the majority of studies listed in Table 3 utilised minced meat samples in order to aid homogenisation with the antioxidant. Another reason for using minced meat in antioxidant studies is that ground meat is more susceptible to oxidation due to the higher surface 1

area exposed to oxygen. As such, there is much interest in finding ways to protect this particular form of meat from oxidative spoilage.

A variety of delivery modes are evident from Table 3, including addition of antioxidants as solids (Ryu et al. 2014); dissolved/dispersed in water/oil (Naveena, Vaithiyanathan, Muthukumar, Sen, Kumar, Kiran, et al., 2013), as essential oils (Djenane, Aïder, Yangüela, Idir, Gómez, & Roncalés, 2012), or in feeding trials prior to the animal being slaughtered (King, Griffin, & Roslan, 2014). The latter is generating increasing interest as a way of improving meat quality, including altering fatty acid profiles through diet (Biondi, D'Urso, Vasta, Luciano, Scerra, Priolo, et al., 2013) and lowering oxidation status of the meat through antioxidants, as in the study reported here.

Two of the studies listed in Table 3 used antioxidants added as solids. Karwowska and Dolatowski (2014) incorporated ground mustard seed (0.2 and 0.5% (w/w)) into ground pork, and found that the antioxidant activity was dose-dependent. However, Ryu and Shin (2014), using freeze-dried grape skin and seed pomace (GSP) extracts, also in ground pork, found that 0.5% (w/w) GSP extract reduced TBARS more effectively than 1% (w/w) GSP extract. The authors concluded that in a complex system such as a crude botanical extract, there may be prooxidants that, at higher concentration, outweigh the antioxidants in the extract. It is not possible to compare antioxidant activity of GSP extract with that of ground mustard seed because of differences in: pork preparation methods; cooking temperatures; cooling and storage of the cooked meat; and differences in the TBARS assay itself (Table 3). Such differences illustrate the

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importance (and challenges) of developing standardised protocols for undertaking antioxidant studies utilising the TBARS assay.

In terms of mode of delivery, another point to consider is whether the antioxidant is dissolved/dispersed in aqueous solution or oil. The delivery vehicle may impact the antioxidant effectiveness due to differences in polarity and partitioning behaviour of antioxidant compounds. Such differences are likely in crude plant extracts where the chemical complexity may be high. An example of a study that utilised both aqueous and lipid deliveries is that by Naveena et al. (2013). In their study, three commercial preparations of rosemary extract were trialed in minced buffalo patties: a crude extract and a refined extract both dissolved in groundnut oil, and a refined extract dispersed in water. The extracts were standardised to carnosic acid content of 10, 2.5 and 2.5%, respectively; and low (22.5 ppm carnosic acid) and high (130 ppm carnosic acid) doses were tested. In this case, both oil- and water-based preparations had the same antioxidant activity, but the low dose treatments showed higher TBARS. As far as the authors are aware, no other recent study has investigated different modes of delivery.

Table 3 also shows other types of oxidation products that may be monitored along with TBARS. For example, Naveena et al. (2013) monitored peroxides using the FTC assay, and found that both low and high doses of carnosic acid showed effectively the same activity in preventing peroxide formation, in contrast to their effect on TBARS (see above). In the study reported by Contini et al. (2012), hexanal was monitored in cooked turkey meat to “validate [the] TBARS results”. As hexanal is also a secondary oxidation product, it might be expected to follow a similar trend to that of TBARS, as was found by Contini et al. (2012). However, each product is 1

formed via a different mechanism and inconsistent results may thus be an indication of a more specific antioxidant interaction in a particular reaction pathway. Finally, Karwowska and Dolatowski (2014) monitored cholesterol oxidation products due to “their toxicological risk [...] more directly connected to the development of atherosclerosis and coronary heart disease.” After one day storage, there was a significant difference among the treatments (0, 0.2 and 0.5% (w/w) mustard seed) with the highest concentration of mustard seed exhibiting the highest antioxidant activity. However, after 12 days storage, no significant differences were found, and in fact the cholesterol oxidation products had decreased to low levels, which the authors attributed to possible interactions with other components in the meat. This behaviour was contrary to that reported for TBARS in this study: TBARS increased over the 12 days of storage, but more slowly for samples containing mustard seed. It appears evident that while the TBARS assay provides an assessment of antioxidant effectiveness, such assessment is dependent on the mode of action of the antioxidant and its effectiveness towards specific types of oxidation products.

3.2 Human physiology including animal model studies There is currently much interest in reducing the oxidation status of human tissues, fluids, etc. through dietary intervention in order to prevent or treat diseases such as atherosclerosis, diabetes, etc. (Toaldo, Cruz, de Lima Alves, de Gois, Borges, Cunha, et al., 2015). Therefore, knowledge of how diet affects markers of oxidation is required, where possible in human feeding trials, or alternatively in animal models. Table 4 lists some illustrative examples of both types of studies. Similar to Table 3, the intention is not to present an exhaustive list, but rather to give an overview of the different approaches used to measure antioxidant effectiveness with the TBARS assay, as well as trying to match the sample types with those shown in Table 2. 1

{Table 4}

As with the studies in meat products, when conducting research on antioxidant activity in physiological systems, various issues need attention including: how the antioxidant is added (through normal diet, intraperitoneal (IP) injection, etc.); length of time to administer the antioxidant; when to take the sample; what sample to take (blood, organs, other); and whether other measures of oxidation are monitored. Some of these considerations are only relevant to animal studies, e.g. IP injection has not been reported as a means of delivering antioxidants to humans (as far as the authors are aware).

Two of the studies listed in Table 4 involved feeding trials with humans. One investigated the acute effect of a single “dose” of grape juice on TBARS and lipid hydroperoxides in plasma taken 1 h after consumption (Toaldo, Cruz, de Lima Alves, de Gois, Borges, Cunha, et al., 2015). In contrast, Li et al (2015) conducted a 56 day trial with subjects consuming tomato juice. In this case, TBARS were measured in serum along with the [2,2′-azinobis-(3-ethylbenzothiazoline-6sulfonic acid)] (ABTS) radical scavenging capacity of the serum. These studies highlight the diversity of approaches to understanding the role of antioxidants in the diet. Further information on nutritional methodologies commonly used can be found in a review by Kendall et al. (2008).

Animal studies exhibit a greater flexibility in the mode of delivery of antioxidants. As well as dietary consumption (da Silva, Cazarin, Batista, & Maróstica, 2014), pure compounds and extracts may be delivered orally through an intragastric tube (Silambarasan, Manivannan, Priya, 1

Suganya, Chatterjee, & Raja, 2014), or via IP injection (Yang, et al., 2014). Antioxidants may be administered in aqueous solution, emulsion, or oil matrices. The matrix undoubtedly affects absorption of compounds from the gut, yet this was not commented on by Yang et al. (2014), who used saline solution to deliver quercetin; nor by Silambarasan et al. (2014), who used corn oil to deliver sinapic acid. Furthermore, compounds or extracts with low water solubility, such as quercetin, need to be dissolved in a solvent such as DMSO prior to dilution with aqueous solution (Yang, et al., 2014).

In both human and animal studies, it is noticeable that TBARS are but one of multiple measures of oxidation/antioxidant activity, which is in contrast to studies on meat products above. Typically, antioxidant enzymes such as superoxide dismutase (SOD), catalase (Cat), and glutathione peroxidase (GSH-Px) are assayed. Lipid hydroperoxides, as primary oxidation products, may also be measured, and in animal studies, organ morphology may be monitored. Together, these multiple measurements provide a comprehensive picture of the effect of dietary or other interventions on an organism’s oxidative status. However, as has been noted previously, since there is no standardised TBARS method, it would not be possible to compare results across studies on the effects of a particular treatment on lipid oxidation.

4. TBARS assay in antioxidant screening studies: 4.1 Current applications In contrast to research reported in Section 3, antioxidant screening studies are seeking to find the “best” antioxidant from a range of candidates, be they plant extracts from different species (Xu, Yuan, & Chang, 2007); different extracts from the same species (Céspedes, Valdez-Morales, 1

Avila, El-Hafidi, Alarcón, & Paredes-López, 2010); protein hydrolysates (Hogan, Zhang, Li, Wang, & Zhou, 2009); or pure compounds (Alamed, Chaiyasit, McClements, & Decker, 2009). These studies typically employ a number of different assays designed to assess various types of antioxidant activities (single electron transfer, hydrogen atom transfer, etc). Not only is it important to identify whether the extract/compound is having an antioxidant effect (and what that effect might be), but quantification of that effect so as to rank the samples is also important. Ideally, standardised assays should be developed so that results from one laboratory can be compared to those from another (Frankel & Finley, 2008).

Compared to the number of studies that utilise assays such as 2,2’-diphenyl-1-picrylhydrazyl (DPPH), ABTS, or ORAC, few screening studies incorporate an assay that has an oxidisable substrate, such as a lipid. Of those that do, the TBARS test is a very common method for quantifying oxidation. Two main types of antioxidant screening studies incorporating the TBARS assay can be found (see Table 5 for a selection of studies). The first are those that use rapid radical-scavenging assays to screen for antioxidant activity, then having found the most effective antioxidants, undertake further testing with a “real” substrate and measure antioxidant activity using the TBARS assay. For example, Yu et al (2005a) first screened methanolic extracts of cold-pressed seed oils using DPPH, ABTS, ORAC and FC assays, and based on the results for ABTS and DPPH, extracts from cranberry and black caraway seed oils were further tested for antioxidant activity by oxidising LDL and monitoring TBARS formation. Although both extracts showed significantly different (p < 0.05) ABTS activity and total phenols, they exhibited the same (p > 0.05) antioxidant activity in the TBARS assay. Similarly, Hogan et al. (2009) prepared a series of hydrolysates from milk protein and assessed antioxidant activity via ORAC and 1

DPPH assays. Three protein hydrolysate fractions were selected for further antioxidant testing by incorporation into ground beef, and TBARS were measured in the cooked samples. Again, only one fraction was observed to have antioxidant activity in the cooked ground beef, despite all three fractions having antioxidant activity as measured by ORAC and DPPH assays.

{Table 5}

These results highlight some of the challenges when trying to select the most effective antioxidant. For example, there may be very little correlation between some measures of antioxidant activity and total phenols, as was observed in Yu et al. (2005b), and also noted by McDonald et al. (2001). In both cases, total phenols did not correlate with antioxidant activity in the TBARS assay. Further challenges may arise where one type of antioxidant assay (e.g. DPPH) is used to select an antioxidant for further testing using a different type of assay (e.g. TBARS). For example, in the study conducted by Hogan et al. (2009), using DPPH or ORAC assays may not necessarily have revealed the most effective antioxidant in a different system where the TBARS assay was employed. It is entirely possible that one of the other nine protein fractions not tested by the TBARS assay may have in fact been more effective as an antioxidant in ground beef.

The second type of screening studies involving the TBARS assay is that where this assay is used along with all other assays to assess antioxidant activity. In other words, all potential antioxidants are screened by all assays, and any correlations (or contradictions) among assays may be revealed. For example, Xu et al. (2007) found significant correlations between TBARS 1

and DPPH (r = 0.97, p < 0.01) and TBARS and ORAC (r = 0.81, p < 0.001) for acetone/water extracts of various legumes (Table 5). On the other hand, McDonald et al. (2001) found that compounds that were the most effective in the TBARS assay (caffeic acid, tyrosol, and naringin) had minimal activity in protecting phycoerythrin from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-initiated oxidative damage. Similarly, Yu et al. (2005b) reported “No correlation […] among the three antioxidative activities on per mg of TPC [total phenol content] basis”. In Table 5, 11 studies are reported where TBARS is used to screen, but of those, only 6 show a correlation between TBARS and the other assays. The lack of consistency between assays does raise concerns about studies where results from one type of assay are used to select antioxidants for further testing based on TBARS. As noted above, there is a chance that suboptimal antioxidants are selected.

The potential lack of correlation between results of antioxidant activity from free radical scavenging assays and real life systems such as foods was explicitly investigated by Alamed et al. (2009). In this study, 9 compounds were evaluated for antioxidant activity, either as polar compounds in a modified ORAC assay, or as non-polar compounds in the DPPH assay. A statistically significant (p ≤ 0.05) rank for the polar antioxidants was achieved: ascorbic acid < gallic acid < propyl gallate < coumaric acid < ferulic acid. Similarly, the non-polar antioxidants were also ranked: α-tocopherol < tert-butylhydroquinone (TBHQ) ≈ butylated hydroxytoluene < rosmarinic acid. However, in cooked ground beef, only propyl gallate and TBHQ were effective in inhibiting oxidation as measured by the TBARS assay. Various explanations were offered by Alamed et al. (2009) in order to explain the results. One is that in complex food systems, the 1

ability to chelate iron (a pro-oxidant) becomes as important as, or more important than, the ability to scavenge free radicals. This may explain why possible chelators such as propyl gallate and TBHQ are more effective than compounds such as ferulic acid, which is not able to chelate metal ions. Another possible explanation is that some compounds, e.g. ascorbic acid, may reduce ferric ions to ferrous ions, which are more effective as pro-oxidants. A further explanation may relate to the physical properties of the antioxidants where those that are more hydrophobic may partition more effectively into the lipid phase of heterogeneous systems such as ground beef, and therefore offer more protection at the site of oxidation. Regardless of the explanation, the results of Alamed et al. (2009) suggest that care must be exercised when using one type of assay to select antioxidants for use in a different system. It appears from a citation search of the work of Alamed et al. (2009) that this issue has not been addressed in the literature. Given the current interest in finding alternatives to synthetic antioxidants, there appears to be quite an opportunity to develop screening assays better suited to real world applications.

When considering the TBARS assay as a potential candidate for a standardised screening assay, a review of Table 5 highlights some of the challenges that need to be overcome for development. One is the choice of substrate: a substrate that is chemically consistent is essential so that results from one research group can be compared to those of another. In Table 5, the substrates used are: LA; SDS emulsion of LA (or another polyunsaturated fatty acid, e.g. arachidonic acid), rat brain homogenate; LDL; rat liver microsome homogenate; liposomes; human blood plasma; tilapia muscle; ground beef; and ground poultry meat. Many of these substrates, e.g. LDL, liver/brain homogenates, or beef or poultry, cannot be guaranteed to be chemically similar from one sample to the next. For example, increases in polyunsaturated fatty acids across these substrates may 1

result in significant differences in susceptibility to oxidation. Of the substrates listed in Table 5, only LA has the potential to be chemically consistent from one study to the next, since it is a pure compound (available in ≥ 99% purity). The use of LA in antioxidant assays has been both advocated (Roginsky & Lissi, 2005) and criticised (Frankel, 2014). In our view, the advantages of using LA as a substrate outweigh its disadvantages vis.: it is a fatty acid of relevance to both food and physiological systems; it is readily available commercially in high purity; and it is at least as relevant, if not more so, than other popular lipid substrates such as crocin or β-carotene.

Another challenge to be addressed for TBARS to be used as a standardised assay is that of reaction conditions. As highlighted previously in Section 2, the reaction conditions for the TBARS reaction are many and varied. To be utilised as an antioxidant assay, the reaction conditions for both the oxidation step and the colour development step need to be consistent across studies. Looking at studies utilising LDL, concentrations of LDL and reaction media varied from 50 µg/mL in PBS (Xu, Yuan, & Chang, 2007), to 100 µg/mL in PBS (Yu, Zhou, & Parry, 2005a), to 200 µg/mL in HEPES (Roginsky & Lissi, 2005). In the studies reported in Table 5, oxidation times varied from 30 min (M. Domínguez, Nieto, Marin, Keck, Jeffery, & Céspedes, 2005) to 20 h (McDonald, Prenzler, Antolovich, & Robards, 2001). Also, some authors used n-butanol to extract TBARS (McDonald, Prenzler, Antolovich, & Robards, 2001), whereas others did not (these tend to be studies where the substrate is a solid). Clearly, there has been a wide variety of reaction conditions utilised in the TBARS assay in screening studies, making it very difficult to compare results from one study to the other.

1

Even when conditions are standardised, there is still no guarantee that an assay will be sufficiently robust to give comparable results across different laboratories as shown by Buenger et al. (2006). In this study, six laboratories evaluated four antioxidant assays, vis. DPPH, TEAC, “lipid assay” (i.e., conjugated diene assay, LA as substrate), and “TBA” (i.e., TBARS assay, liposomes from alpha-lecithin as substrate). Variability differed according to the assay, within laboratory: TEAC (5.7–7.7%), DPPH (2–18%), conjugated dienes (2.6–14%) and TBARS (18– 102%); and among laboratories: TEAC (9–40%); DPPH (6–57%); conjugated dienes (10–67%); TBARS (78–154%). It was noted that the TBARS assay was especially poor in terms of reproducibility, but the cause was not investigated further. Interestingly, assays were found to be more or less variable according to the antioxidant tested. For example, BHT exhibited a CV of 18% in the DPPH test, whereas other antioxidants showed CVs of 2-8%. In the TBARS assay, tocopherol was found to be particularly problematic with a CV of 102%. These results would again suggest that caution is needed in interpreting data from other studies where the TBARS assay has been used. And indeed, given the lack of standard conditions in general (see above), there appears to be an urgent need to standardise the operation of this assay across all its uses.

4.2 Towards a standardised TBARS assay for use in rapid screening of antioxidant activity According to Prior et al. (2005), a standardised method to measure antioxidant capacity should meet the following criteria: “(1) measures chemistry actually occurring in potential application(s); (2) utilizes a biologically relevant radical source; (3) simple; (4) uses a method with a defined endpoint and chemical mechanism; 1

(5) instrumentation is readily available; (6) good within-run and between-day reproducibility; (7) adaptable for assay of both hydrophilic and lipophilic antioxidants and use of different radical sources; (8) adaptable to “high-throughput” analysis for routine quality control analyses.”

In this section, we evaluate the TBARS assay in light of these eight criteria and with reference to the literature as summarised throughout this review.

(1) Measures chemistry actually occurring in potential application(s): This is perhaps the hardest criteria for any antioxidant assay to fulfil. Inevitably, in a real system, the chemistry is complex and multiple reaction pathways are occurring that are not possible to model. Assays such as FC and TEAC are utilised due to their ability to model some aspects of the chemistry taking place in a potential application, e.g. electron transfer, but they are not able to account for all the chemistry that occurs in a real system. On the other hand, the TBARS assay would appear to be well-placed with respect to this criterion. The chemistry underlying the TBARS assay is lipid oxidation and this is of interest in a wide range of potential applications. Moreover, a substrate is present and measuring the decrease in TBARS formation in the presence of an antioxidant is a good indication that the substrate is being protected. Furthermore, in some applications, TBARS correlate with other measures of oxidative damage (e.g. off-odours in meat), and hence reduction in TBARS by an antioxidant is of practical significance. A challenge with the TBARS assay is finding a substrate that is suitable, but it may be argued that having a substrate, even if not optimal, is advantageous over an assay that has none. 1

(2) Utilises a biologically relevant radical source: The radicals generated in a TBARS assay may be considered to be essentially the same as those generated during “normal” lipid oxidation, i.e., alkyl, peroxyl, alkoxyl, etc. However, the use of a catalyst (see (8), below) such as Cu2+ to speed up lipid oxidation may alter the proportion of the different radicals (Frankel, 1993), which may lead to differences with a real system. Nevertheless, the TBARS assay compares favourably with other more widely used assays, all of which utilise radicals with limited biological relevance.

(3) Simple: Assays such as TEAC and FC are two-component systems – the antioxidant and the colourimetric “reporter” ‒ and are therefore about as chemically “simple” as possible. In comparison, the TBARS assay is more complicated due to the inclusion of a substrate. Further complications arise when considering the choice of substrate, e.g. triglyceride, fatty acid, LDL etc., and the means by which the antioxidant is added, e.g. as a separate phase, dispersed in the lipid etc. The development of the pigment also requires additional steps, increasing complexity, as well as introducing inconsistencies among reported methods. Nevertheless, if a standardised procedure were adopted, the basic reaction conditions, chemicals, and instrumentation, could be implemented as a routine procedure in most laboratories.

(4) Uses a method with a defined endpoint and chemical mechanism: The formation of TBARS, as secondary oxidation products, could be viewed as a defined endpoint. However, the rate of TBARS formation is dependent inter alia on the substrate, whether or not a catalyst is used, the presence of other substances that can generate TBARS, etc. This points again to the need for a well-defined substrate and standardised reaction conditions. With respect to a defined chemical 1

mechanism, that is the subject of ongoing research. Lipid oxidation is a widely studied phenomenon, yet there is much that remains to be learned. The fact that the products of lipid oxidation are very dependent on reaction conditions (Frankel, 1993) suggests complex chemistry that is yet to be fully understood.

(5) Instrumentation is readily available: By far the most common method of measuring TBARS is with a UV-Vis spectrophotometer. Other equipment that may be required would be a constant temperature environment for the oxidation step, a centrifuge for phase separation (if using nbutanol to extract the pigment), and a water bath for the colour development step. All equipment should be readily available in most laboratories.

(6) Good within-run and between-day reproducibility: The widespread use of the TBARS assay would suggest that the reproducibility is acceptable. Yet Buenger et al. (2006) found that the TBARS assay was the least reproducible among those they evaluated with large coefficients of variation, some over 100%. In addition, the authors are aware of considerable anecdotal evidence that underlines the unreliability of the assay in biological systems and some of the issues that may contribute to variability in the TBARS assay have been addressed in a separate publication (Ghani, et al., 2016 accepted for publication). It is likely that an analytically robust assay can be developed providing that all reaction conditions are well controlled, as would be the case for a standardised assay.

(7) Adaptable for assay of both hydrophilic and lipophilic antioxidants and use of different radical sources: Again, the TBARS assay compares favourably with more common assays such 1

as FC with regards to adaptability. Whereas the FC assay (and TEAC, and others) cannot be used with a highly lipophilic antioxidant sample, the TBARS assay can accommodate samples of virtually any polarity. For example, highly hydrophilic substances may be dissolved in ethanol, added to a lipid substrate (including bulk oil), and then the ethanol removed under vacuum, leaving the antioxidant in the lipid (Haiyan, Bedgood, Bishop, Prenzler, & Robards, 2006). On the other hand, lipophilic antioxidants can be added directly to a lipid substrate and the assay run “normally”, unlike assays such as ORAC, where alternate reaction conditions are required for antioxidants of low polarity (Prior, Wu, & Schaich, 2005).

(8) Adaptable to “high-throughput” analysis for routine quality control analyses: This criterion has two parts, one relating to the ability of an assay to be used in a high-throughput environment, and the second relating to the goal of the analysis, i.e., routine quality control. With respect to the former, the main consideration is the time required for oxidation of a substrate to occur. In all of the studies listed in Table 5, where TBARS has been used in a screening capacity, the time required to oxidise a substrate can be considerable (up to 20 h). This is clearly much longer than in assays such as TEAC, which can be performed in around an hour. The rate of lipid oxidation can be increased by raising the temperature, through the use of a catalyst, or through the use of an oxidation initiator such as AAPH. However, all of these strategies lead to a change in the reaction mechanism of lipid oxidation, which may then give results unrelated to the original (slower) reaction conditions of the assay (Frankel, 1993). This is particularly problematic in studies that investigate bioactive compounds in plants for antioxidant activity relevant to human diet or physiology, where the temperature of the system should be around 37 °C (body temperature). Many of the studies in Table 5 were conducted at 37 °C and hence long oxidation 1

times were often used. An exception seems to be studies in which LDL is used as a substrate; however, LDL is a complex chemical system, not amenable to a robust, analytically reproducible assay.

The second aspect of criterion (8) relates to the goal of the analysis – as specified by Prior et al. (2005) “routine quality control analyses”. Presumably, this might relate to the production of a health supplement standardised to a particular antioxidant capacity, which would then require a rapid and robust assay to be used for QC purposes. It is not envisaged that the TBARS assay would be employed in such a way. Rather, if it could be made faster, TBARS could be a valuable assay to use in large scale screening studies. As it is an assay where protection of a substrate from oxidative damage is measured, it could provide useful and complementary information to other, more widely used assays such as TEAC and FC.

5. Conclusions In this review, various aspects of the TBARS assay have been reported, including its use in food and physiological systems to monitor lipid oxidation. A recurring theme is that there are many variations in the reaction conditions used to measure TBARS. Such differences make it difficult to compare results among studies. These issues are compounded when the TBARS assay is used to monitor lipid oxidation in the presence of an antioxidant, as further variations in methodology arise, including: antioxidant mode of delivery, time of oxidation, choice of substrate, etc. Despite the challenges, the TBARS assay has potential to be further developed for antioxidant screening studies. Most importantly, it meets a number of criteria suggested by Prior et al. (2005) to be considered a standard assay, and has a particular advantage over most assays in that there is a 1

substrate being protected from oxidation. Future research is required to find ways to increase the rate of lipid oxidation so that the overall assay can be made faster – a key prerequisite for use in screening studies ‒ while maintaining the intrinsic lipid oxidation pathways. Following on from the work of Buenger et al. (2006), another key area of research should be to thoroughly investigate the sources of variability in the TBARS assay, such that low coefficients of variation can be consistently obtained. Due to its widespread use, the TBARS assay is well accepted in many fields of study, and if a rapid and robust antioxidant assay based on TBARS was developed, it would likely be utilised in diverse areas where lipid oxidation is an issue.

Acknowledgements: Md Ahsan Ghani acknowledges receipt of a Faculty of Science (Compact) scholarship, Charles Sturt University. The authors would also like to acknowledge the Graham Centre for Agriculture Innovation for supporting this research.

References: Aksu, T., Aksu, M., Yoruk, M., & Karaoglu, M. (2011). Effects of organically-complexed minerals on meat quality in chickens. British poultry science, 52(5), 558-563. Alamed, J., Chaiyasit, W., McClements, D. J., & Decker, E. A. (2009). Relationships between free radical scavenging and antioxidant activity in foods. Journal of Agricultural and Food Chemistry, 57(7), 2969-2976. American Oil Chemists’ Society (2013) In: Firestone D (ed), Official methods and recommended practices of the American Oil Chemists’ Society, 6th edn. AOCS, Champaign. Ansari, M. A., Roberts, K. N., & Scheff, S. W. (2008). A time course of contusion-induced oxidative stress and synaptic proteins in cortex in a rat model of TBI. Journal of neurotrauma, 25(5), 513-526. Antolovich, M., Prenzler, P. D., Patsalides, E., McDonald, S., & Robards, K. (2002). Methods for testing antioxidant activity. Analyst, 127(1), 183-198. Barriuso, B., Astiasaran, I., & Ansorena, D. (2013). A review of analytical methods measuring lipid oxidation status in foods: a challenging task. European Food Research and Technology, 236(1), 1-15. 1

Biondi, L., D'Urso, M., Vasta, V., Luciano, G., Scerra, M., Priolo, A., Ziller, L., Bontempo, L., Caparra, P., & Camin, F. (2013). Stable isotope ratios of blood components and muscle to trace dietary changes in lambs. animal, 7(09), 1559-1566. Buege, J. A., & Aust, S. D. (1978). Microsomal lipid peroxidation. Methods in Enzymology, 52, 302. Buenger, J., Ackermann, H., Jentzsch, A., Mehling, A., Pfitzner, I., Reiffen, K. A., Schroeder, K. R., & Wollenweber, U. (2006). An interlaboratory comparison of methods used to assess antioxidant potentials. International Journal of Cosmetic Science, 28(2), 135-146. Capitani, C. D., Carvalho, A. C. L., Rivelli, D. P., Barros, S., & Castro, I. A. (2009). Evaluation of natural and synthetic compounds according to their antioxidant activity using a multivariate approach. European Journal of Lipid Science and Technology, 111(11), 1090-1099. Carpi, A., Menabò, R., Kaludercic, N., Pelicci, P., Di Lisa, F., & Giorgio, M. (2009). The cardioprotective effects elicited by p66 Shc ablation demonstrate the crucial role of mitochondrial ROS formation in ischemia/reperfusion injury. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1787(7), 774-780. Céspedes, C. L., Valdez-Morales, M., Avila, J. G., El-Hafidi, M., Alarcón, J., & Paredes-López, O. (2010). Phytochemical profile and the antioxidant activity of Chilean wild black-berry fruits, Aristotelia chilensis (Mol) Stuntz (Elaeocarpaceae). Food Chemistry, 119(3), 886895. Contini, C., Katsikogianni, M., O'Neill, F., O'Sullivan, M., Dowling, D., & Monahan, F. (2012). PET trays coated with Citrus extract exhibit antioxidant activity with cooked turkey meat. LWT-Food Science and Technology, 47(2), 471-477. Costa, P., Gonçalves, S., Valentão, P., Andrade, P. B., Almeida, C., Nogueira, J. M., & Romano, A. (2013). Metabolic profile and biological activities of Lavandula pedunculata subsp. lusitanica (Chaytor) Franco: Studies on the essential oil and polar extracts. Food Chemistry, 141(3), 2501-2506. da Silva, J. K., Cazarin, C. B. B., Batista, Â. G., & Maróstica, M. (2014). Effects of passion fruit (Passiflora edulis) byproduct intake in antioxidant status of Wistar rats tissues. LWTFood Science and Technology, 59(2), 1213-1219. Del Rio, D., Stewart, A. J., & Pellegrini, N. (2005). A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutrition Metabolism and Cardiovascular Diseases, 15(4), 316-328. Díaz, P., Linares, M., Egea, M., Auqui, S., & Garrido, M. (2014). TBARs distillation method: Revision to minimize the interference from yellow pigments in meat products. Meat science, 98(4), 569-573. Djenane, D., Aïder, M., Yangüela, J., Idir, L., Gómez, D., & Roncalés, P. (2012). Antioxidant and antibacterial effects of Lavandula and Mentha essential oils in minced beef inoculated with E. coli O157: H7 and S. aureus during storage at abuse refrigeration temperature. Meat science, 92(4), 667-674. Domínguez, M., Nieto, A., Marin, J. C., Keck, A.-S., Jeffery, E., & Céspedes, C. L. (2005). Antioxidant activities of extracts from Barkleyanthus salicifolius (Asteraceae) and Penstemon gentianoides (Scrophulariaceae). Journal of Agricultural and Food Chemistry, 53(15), 5889-5895. 1

Dutta, R. K., & Maharia, R. S. (2012). Antioxidant responses of some common medicinal plants grown in copper mining areas. Food Chemistry, 131(1), 259-265. Fernandez, J., Perez-Alvarez, J. A., & Fernandez-Lopez, J. A. (1997). Thiobarbituric acid test for monitoring lipid oxidation in meat. Food Chemistry, 59(3), 345-353. Frankel, E. (1993). In search of better methods to evaluate natural antioxidants and oxidative stability in food lipids. Trends in Food Science & Technology, 4(7), 220-225. Frankel, E. N. (2014). Lipid oxidation: Elsevier. Frankel, E. N., & Finley, J. W. (2008). How to standardize the multiplicity of methods to evaluate natural antioxidants. Journal of Agricultural and Food Chemistry, 56(13), 49014908. Frankel, E. N., & Meyer, A. S. (2000). The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants. Journal of the Science of Food and Agriculture, 80(13), 1925-1941. Furukawa, S., Fujita, T., Shimabukuro, M., Iwaki, M., Yamada, Y., Nakajima, Y., Nakayama, O., Makishima, M., Matsuda, M., & Shimomura, I. (2004). Increased oxidative stress in obesity and its impact on metabolic syndrome. Journal of Clinical Investigation, 114(12), 1752. Ghani, M. A., Barril, C., Bedgood, D. R., & Prenzler, P. D. (2016) Substrate and TBARS variability in a multi-phase oxidation system. European Journal of Lipid Science and Technology. Accepted for publication. doi : 10.1002/ejlt.201500500 Giera, M., Lingeman, H., & Niessen, W. M. A. (2012). Recent Advancements in the LC- and GC-Based Analysis of Malondialdehyde (MDA): A Brief Overview. Chromatographia, 75(9-10), 433-440. Guillen-Sans, R., & Guzman-Chozas, M. (1998). The thiobarbituric acid (TBA) reaction in foods: A review. Critical Reviews in Food Science and Nutrition, 38(4), 315-330. Haimeur, A., Messaouri, H., Ulmann, L., Mimouni, V., Masrar, A., Chraibi, A., Tremblin, G., & Meskini, N. (2013). Argan oil prevents prothrombotic complications by lowering lipid levels and platelet aggregation, enhancing oxidative status in dyslipidemic patients from the area of Rabat (Morocco). Lipids in health and disease, 12(107), 1-9. Haiyan, Z., Bedgood, D. R., Bishop, A. G., Prenzler, P. D., & Robards, K. (2006). Effect of added caffeic acid and tyrosol on the fatty acid and volatile profiles of camellia oil following heating. Journal of Agricultural and Food Chemistry, 54(25), 9551-9558. Hogan, S., Zhang, L., Li, J., Wang, H., & Zhou, K. (2009). Development of antioxidant rich peptides from milk protein by microbial proteases and analysis of their effects on lipid peroxidation in cooked beef. Food Chemistry, 117(3), 438-443. Huang, D., Ou, B., & Prior, R. L. (2005). The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry, 53(6), 1841-1856. Janero, D. R. (1990). Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indexes of lipid-peroxidation and peroxidative tissue-injury. Free Radical Biology and Medicine, 9(6), 515-540. Juárez, M., Failla, S., Ficco, A., Peña, F., Avilés, C., & Polvillo, O. (2010). Buffalo meat composition as affected by different cooking methods. Food and Bioproducts Processing, 88(2), 145-148. Karwowska, M., & Dolatowski, Z. J. (2014). Effect of mustard on lipid oxidation in model pork meat product. European Journal of Lipid Science and Technology, 116(3), 311-318. 1

Kendall, M., Batterham, M., Prenzler, P. D., Ryan, D., & Robards, K. (2008). Nutritional methodologies and their use in inter-disciplinary antioxidant research. Food Chemistry, 108(2), 425-438. Kim, I.-S., Jin, S.-K., Mandal, P. K., & Kang, S.-N. (2011). Quality of low-fat pork sausages with tomato powder as colour and functional additive during refrigerated storage. Journal of food science and technology, 48(5), 591-597. King, A. J., Griffin, J. K., & Roslan, F. (2014). In Vivo and in Vitro Addition of Dried Olive Extract in Poultry. Journal of Agricultural and Food Chemistry, 62(31), 7915-7919. Kohn, H. I., & Liversedge, M. (1944). On a new aerobic metabolite whose production by brain is inhibited by apomorphine, emetine, ergotamine, epinephrine, and menadione. Journal of Pharmacology and Experimental Therapeutics, 82(3), 292-300. Kondo, Y., Masutomi, H., Noda, Y., Ozawa, Y., Takahashi, K., Handa, S., Maruyama, N., Shimizu, T., & Ishigami, A. (2014). Senescence marker protein-30/superoxide dismutase 1 double knockout mice exhibit increased oxidative stress and hepatic steatosis. FEBS open bio, 4, 522-532. Kosar, M., Goger, F., & Baser, K. H. C. (2008). In vitro antioxidant properties and phenolic composition of Salvia virgata Jacq. from Turkey. Journal of Agricultural and Food Chemistry, 56(7), 2369-2374. Laguerre, M., Bayrasy, C., Panya, A., Weiss, J., McClements, D. J., Lecomte, J., Decker, E. A., & Villeneuve, P. (2015). What makes good antioxidants in lipid-based systems? The next theories beyond the polar paradox. Critical Reviews in Food Science and Nutrition, 55(2), 183-201. Li, X., Li, J., Zhu, J., Wang, Y., Fu, L., & Xuan, W. (2011). Postmortem changes in yellow grouper (Epinephelus awoara) fillets stored under vacuum packaging at 0 C. Food Chemistry, 126(3), 896-901. Li, Y.-F., Chang, Y.-Y., Huang, H.-C., Wu, Y.-C., Yang, M.-D., & Chao, P.-M. (2015). Tomato juice supplementation in young women reduces inflammatory adipokine levels independently of body fat reduction. Nutrition, 31(5), 691-696. Margonis, K., Fatouros, I. G., Jamurtas, A. Z., Nikolaidis, M. G., Douroudos, I., Chatzinikolaou, A., Mitrakou, A., Mastorakos, G., Papassotiriou, I., & Taxildaris, K. (2007). Oxidative stress biomarkers responses to physical overtraining: implications for diagnosis. Free Radical Biology and Medicine, 43(6), 901-910. McDonald, S., Prenzler, P. D., Antolovich, M., & Robards, K. (2001). Phenolic content and antioxidant activity of olive extracts. Food Chemistry, 73(1), 73-84. Naveena, B., Vaithiyanathan, S., Muthukumar, M., Sen, A., Kumar, Y. P., Kiran, M., Shaju, V., & Chandran, K. R. (2013). Relationship between the solubility, dosage and antioxidant capacity of carnosic acid in raw and cooked ground buffalo meat patties and chicken patties. Meat science, 95(2), 195-202. Nowak, D., Kałucka, S., Białasiewicz, P., & Król, M. (2001). Exhalation of H2O2 and thiobarbituric acid reactive substances (TBARs) by healthy subjects. Free Radical Biology and Medicine, 30(2), 178-186. Ohkawa, H., Ohishi, N., & Yagi, K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry, 95(2), 351-358. Okauchi, Y., Kishida, K., Funahashi, T., Noguchi, M., Ogawa, T., Okita, K., Iwahashi, H., Ohira, T., Imagawa, A., Nakamura, T., & Shimomura, I. (2011). Cross-sectional and 1

longitudinal study of association between circulating thiobarbituric acid-reacting substance levels and clinicobiochemical parameters in 1,178 middle-aged Japanese men the Amagasaki Visceral Fat Study. Nutr Metab (Lond), 8, 82. Papastergiadis, A., Mubiru, E., Van Langenhove, H., & De Meulenaer, B. (2012). Malondialdehyde measurement in oxidized foods: evaluation of the spectrophotometric thiobarbituric acid reactive substances (TBARS) test in various foods. Journal of Agricultural and Food Chemistry, 60(38), 9589-9594. Penko, A., Polak, T., Polak, M. L., Požrl, T., Kakovič, D., Žlender, B., & Demšar, L. (2015). Oxidative stability of n-3-enriched chicken patties under different package-atmosphere conditions. Food Chemistry, 168, 372-382. Pereira, C. M., Marques, M. F., Hatano, M. K., Castro, I. A. (2010). Effect of the Partial Substitution of Soy Proteins by Highly Methyl-esterified Pectin on Chemical and Sensory Characteristics of Sausages. Food Science and Technology International, 16, 401-407. Prior, R. L., Wu, X. L., & Schaich, K. (2005). Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. Journal of Agricultural and Food Chemistry, 53(10), 4290-4302. Quevedo, R., Valencia, E., Cuevas, G., Ronceros, B., Pedreschi, F., & Bastias, J. (2013). Color changes in the surface of fresh cut meat: A fractal kinetic application. Food Research International, 54(2), 1430-1436. Reed, T. T. (2011). Lipid peroxidation and neurodegenerative disease. Free Radical Biology and Medicine, 51(7), 1302-1319. Roginsky, V., & Lissi, E. A. (2005). Review of methods to determine chain-breaking antioxidant activity in food. Food Chemistry, 92(2), 235-254. Ryu, K. S., & Shin, D. K. (2014). ARTICLES: Effect of Grape Pomace Powder Addition on TBARS and Color of Cooked Pork Sausages during Storage. KOREAN JOURNAL FOR FOOD SCIENCE OF ANIMAL RESOURCES, 34(2), 200-206. Saluk, J., Bijak, M., Nowak, P., & Wachowicz, B. (2013). Evaluating the antioxidative activity of diselenide containing compounds in human blood. Bioorganic Chemistry, 50, 26-33. Shahidi, F., & Zhong, Y. (2010). Novel antioxidants in food quality preservation and health promotion. European Journal of Lipid Science and Technology, 112(9), 930-940. Silambarasan, T., Manivannan, J., Priya, M. K., Suganya, N., Chatterjee, S., & Raja, B. (2014). Sinapic Acid Prevents Hypertension and Cardiovascular Remodeling in Pharmacological Model of Nitric Oxide Inhibited Rats. PloS one, 9(12), e115682. Sørensen, G., & Jørgensen, S. S. (1996). A critical examination of some experimental variables in the 2-thiobarbituric acid (TBA) test for lipid oxidation in meat products. Zeitschrift für Lebensmittel-Untersuchung und Forschung, 202(3), 205-210. Spickett, C. M., Fedorova, M., Hoffmann, R., & Forman, H. J. (2015). An Introduction to Redox Balance and Lipid Oxidation. Lipid Oxidation in Health and Disease, 1. Sun, W., Meng, P., & Ma, L. (2014). Relationship between N-nitrosodiethylamine formation and protein oxidation in pork protein extracts. European Food Research and Technology, 239(4), 679-686. Tan, J. B. L., & Lim, Y. Y. (2015). Critical analysis of current methods for assessing the in vitro antioxidant and antibacterial activity of plant extracts. Food Chemistry, 172, 814-822.

1

Tarladgis, B. G., Watts, B. M., Younathan, M. T., & Dugan, L. (1960). A distillation method for the quantitative determination of malonaldehyde in rancid foods. Journal of the American Oil Chemists' Society, 37(1), 44-48. Toaldo, I. M., Cruz, F. A., de Lima Alves, T., de Gois, J. S., Borges, D. L., Cunha, H. P., da Silva, E. L., & Bordignon-Luiz, M. T. (2015). Bioactive potential of Vitis labrusca L. grape juices from the Southern Region of Brazil: Phenolic and elemental composition and effect on lipid peroxidation in healthy subjects. Food Chemistry, 173, 527-535. Tucker, G., & Robards, K. (2008). Bioactivity and structure of biophenols as mediators of chronic diseases. Critical Reviews in Food Science and Nutrition, 48(10), 929-966. Turner, E., Paynter, W., Montie, E. J., Bessert, M., Struck, G., & Olson, F. (1954). Use of the 2thiobarbituric acid reagent to measure rancidity in frozen pork. Food Technology, 8(7), 326-330. Utrilla, M., Ruiz, A. G., & Soriano, A. (2014). Effect of partial reduction of pork meat on the physicochemical and sensory quality of dry ripened sausages: Development of a healthy venison salchichon. Meat science, 98(4), 785-791. Varamenti, E. I., Kyparos, A., Veskoukis, A. S., Bakou, M., Kalaboka, S., Jamurtas, A. Z., Koutedakis, Y., & Kouretas, D. (2013). Oxidative stress, inflammation and angiogenesis markers in elite female water polo athletes throughout a season. Food and Chemical Toxicology, 61, 3-8. Wang, J., Jin, G., Zhang, W., Ahn, D., & Zhang, J. (2012). Effect of curing salt content on lipid oxidation and volatile flavour compounds of dry-cured turkey ham. LWT-Food Science and Technology, 48(1), 102-106. Wilson, D. W., Metz, H. N., Graver, L. M., & Rao, P. S. (1997). Direct method for quantification of free malondialdehyde with high-performance capillary electrophoresis in biological samples. Clinical Chemistry, 43(10), 1982-1984. Wrolstad, R. E., Acree, T. E., Decker, E. A., Penner, M. H., Reid, D. S., Schwartz, S. J., Sporns, P. (2005). Lipid Composition Handbook of Food Analytical Chemistry (pp. 423-511): John Wiley & Sons, Inc. Xu, B., Yuan, S., & Chang, S. (2007). Comparative Studies on the Antioxidant Activities of Nine Common Food Legumes Against Copper‐Induced Human Low‐Density Lipoprotein Oxidation In Vitro. Journal of Food Science, 72(7), S522-S527. Xu, G., Tang, X., Tang, S., You, H., Shi, H., & Gu, R. (2014). Combined effect of electrolyzed oxidizing water and chitosan on the microbiological, physicochemical, and sensory attributes of American shad (Alosa sapidissima) during refrigerated storage. Food Control, 46, 397-402. Yamagata, K., Tagami, M., & Yamori, Y. (2015). Dietary polyphenols regulate endothelial function and prevent cardiovascular disease. Nutrition, 31(1), 28-37. Yang, T., Kong, B., Gu, J.-W., Kuang, Y.-Q., Cheng, L., Yang, W.-T., Xia, X., & Shu, H.-F. (2014). Anti-apoptotic and anti-oxidative roles of quercetin after traumatic brain injury. Cellular and Molecular Neurobiology, 34(6), 797-804. Yu, L. L., Zhou, K. K., & Parry, J. (2005a). Antioxidant properties of cold-pressed black caraway, carrot, cranberry, and hemp seed oils. Food Chemistry, 91(4), 723-729. Yu, L. L., Zhou, K., & Parry, J. W. (2005b). Inhibitory effects of wheat bran extracts on human LDL oxidation and free radicals. LWT-Food Science and Technology, 38(5), 463-470. 1

Table 1. Selected publications (last 5 years) using the TBARS assay to measure oxidation in meat products. Sample

Sample preparation

Buffalo

5 g meat + 15 mL 0.38 M HClO4 ; hom.a 3 min in ice bath; + 0.5 mL 0.19 M BHT; 3000 g, 5 min, 5 °C; filtered meat hom. 18000 rpm; 5 g + 45.8 mL 10% TCA in H3PO4 + 1 mL 0.02 mg/mL BHT; hom., 18000 rpm, 30 s; filtered 5 g + 50 uL 7.2% BHA (in EtOH) + 15 mL H2O; hom. 1 g ground meat + 6 mL (7.5% TCA, 0.1% EDTA, 0.1% 1-propyl gallate); hom., 20-30 s; filtered 5 g minced ham + 50 uL BHA (7.2 g/100 g) + 15 mL H2O; hom., 15 s

Mixed chicken/pork sausages Pork sausages Poultry Turkey

Conditions for addition of TBA (vol. aliquot, vol. & [TBA], etc.) 0.7 mL + 0.7 mL 0.02 M TBA

Incubation temperature & time

Measurement of colour

Reference

100 °C, 30 min

cooled; 3000 g, 15 min, 5 °C; 532 nm

(Juárez, Failla, Ficco, Peña, Avilés, & Polvillo, 2010) (Pereira, Marques, Hatano, & Castro, 2010)

5 mL + 5 mL 0.02 M RTb, 20 h in dark TBA

530 nm

2 mL + 4 mL 20 mM 90 °C, 15 min TBA in 15% TCA 1 mL + 1 mL 0.02 M boiling water bath, 40 TBA min

2000 rpm, 15 min; 532 nm cooled; 2000 rpm, 5 min; 532 nm

1 mL + 20 uL 1% sulphanilamide + 2 mL 15 mM TBA in 15% TCA

boiling water bath, 15 min

95 °C water bath, 10 min

Beef (surface)

0.5 g

+ 2.5 mL 0.375% TBA (made up in 15% TCA, 0.25% HCl)

Pork protein

10 g + 30 mL 7.5% TCA (with 0.1% propylgallate & 0.1%

5 mL + 5 mL 0.02 M 100 °C water bath, 40 TBA min

(Kim, Jin, Mandal, & Kang, 2011) (Aksu, Aksu, Yoruk, & Karaoglu, 2011) cooled in iced (Wang, Jin, Zhang, water, 10 min; Ahn, & Zhang, mixed; 2500 g, 15 2012) min, 4 °C; 531 nm cooled (tap (Quevedo, water); 5500 rpm, Valencia, Cuevas, 25 min; 532 nm Ronceros, Pedreschi, & Bastias, 2013) cooled RT; 532 (Sun, Meng, & Ma, nm 2014) 1

American shad Pork/venison sausage Chicken patties

EDTA); hom., 30 s, 9500 rpm; filtered 2 g + 8 mL 5% TCA + 5 mL 0.8% BHT in hexane; hom.; 3000 g, 30 s; bottom layer made to 10 mL with 5% TCA 2 g + 8 mL 5% TCA; hom., 90 s; filtered; made to 10 mL with 5% TCA

2.5 mL + 1.5 mL of 0.8% TBA

70 °C, 30 min

5 mL + 5 mL 20 mM mix, 20 °C, 20 hr in TBA dark

0.1 g pre-hom. sample + 1 mL 35% TCA + 2 mL 0.36% TBA in 0.1 M Na2SO3 a hom. = homogenised; b RT = room temperature

cooled tap water; 3rd derivative spectroscopy at 521.5 nm 532 nm

mixed 5 min; + 1 mL 1500 g, 6 min; 0.9% TBA in hexane; supernatant 532 90 °C, 30 min; cooled; nm + 1 mL TCA + 2 mL CHCl3

(Xu, Tang, Tang, You, Shi, & Gu, 2014) (Utrilla, Ruiz, & Soriano, 2014) (Penko, Polak, Polak, Požrl, Kakovič, Žlender, et al., 2015)

1

Table 2. Selection of articles showing TBARS conditions for different physiological samples. Sample

Sample preparation

Exhaled breath condensate (EBC)

Conditions for addition of TBA (Vol. aliquot, vol. & [TBA], etc.) 200 µL EBC + 2 mL TBA (0.67 g in 100 mL H2O, diluted 1:1 with glacial acetic acid)

Incubation temperature & time boiling water bath, 30 min

Measurement of colour

Reference

extracted into 2.5 mL BuOH; c’fugeda, 1500 g, 10 min, 25 °C; fluorescence excitation 515 nm, emission 546 nm on ice, 5 min; + 1 mL 70% TCA; vortexed; c’fuged, 15000 g, 3 min; 530 nm

(Nowak, Kałucka, Białasiewicz, & Król, 2001)

(Margonis, Fatouros, Jamurtas, Nikolaidis, Douroudos, Chatzinikolaou, et al., 2007) c’fuged, 800 g, 10 min; (Carpi, Menabò, 535 nm Kaludercic, Pelicci, Di Lisa, & Giorgio, 2009) cooled; + 100 µL H2O + (Kondo, Masutomi, 500 µL n-BuOH-pyridine Noda, Ozawa, Takahashi, (15:1); c’fuged 21000 g, Handa, et al., 2014) 5 min, 4 °C; fluorescence excitation 515 nm, emission .553 nm cool on ice, 500 µL H2O, (Haimeur, Messaouri, + 2.5 mL n-BuOHUlmann, Mimouni, pyridine (15:1); shaken; Masrar, Chraibi, et al.,

Serum

100 µL + 500µL TCA 35% + 500 µL Tris-HCl (200 mM, pH 7.4); mixed; RT, 10 min

+ 1 mL 2 M Na2SO4 + 55 mM TBA

95 °C, 45 min

Rat heart

tissues + 1 mL 0.15 M KCl + 0.024% BHT; hom.b; c’fuged 10000 g tissues hom. in 10 vol icecold 0.1% SDS; c’fuged 21000 g, 15 min, 4 °C

supernatant + 0.5 mL 30% TCA + 0.5 mL 0.8% TBA

100 °C, 1h

10 µL supernatant + 175 µL stock solution (0.023% BHT, 0.926% SDS, 20% acetic acid, pH 3.5) + 150 µL 0.8% TBA + 70 µL H2O; ice, 60 min 500 µL platelet solution + 100 µL 8.1% SDS + 750 µL 20% acetic acid (pH

boiling water bath, 60 min

Rat liver

Platelets

95 °C 60 min

1

Plasma

3.5) + 750 µL TBA 0.8% + H2O to 2.5 mL + 1 mL 2 M Na2SO4 & 55 mM TBA

100 µL + 500µL TCA 35% + 500 µL Tris-HCl (200 mM, pH 7.4); RT, 10 min a c’fuged = centrifuged; b hom. = homogenised

95 °C, 45 min

c’fuged, 1000 g, 10 min; 532 nm cooled on ice, 5 min; + 1 mL 70% TCA; vortexed; c’fuged, 15000 g, 3 min; 530 nm

2013) (Varamenti, Kyparos, Veskoukis, Bakou, Kalaboka, Jamurtas, et al., 2013)

1

Table 3. Selection of articles showing TBARS conditions for antioxidant studies in meat – matching those meats in Table 1 (last 5 years). Sample

Sample preparation & oxidation conditions

Reaction conditions for TBARS

Conditions for addition of TBA (vol. aliquot, vol. & [TBA], etc.)

Beef

essential oils of Lavandula angustifolia & Mentha piperita sprayed onto 70 g minced beef; TBARS measured over 8 days at 9 °C proprietary extracts of rosemary leaves with carnosic acid quantified; extracts dispersed/dissolved in oil or water added to minced buffalo; 100 g patties stored raw or after cooking at 180 °C; TBARS measured over 9 days at 4 °C grape skin & seed pomace extract added as freeze-dried solid to ground pork meat; sausages cooked to internal temperature of 74 °C; TBARS measured over 10 days at 4 °C DOE (olive extract) &

10 g sample + 20 mL 10% TCA; Ultra-Turrax

c’fugeda 2300 g, 30 min, 5 °C; filtered through paper; 2 mL filtrate + 2 mL 20 mM TBA c’fuged 3000 g, 10 min ; 2 mL supernatant + 2 mL 0.1% TBA

Buffalo

Pork sausage

Poultry

Incubation temperature & time, measurement of colour 97 °C, 20 min; 532 nm

Other measures of oxidation

Reference

none

(Djenane, Aïder, Yangüela, Idir, Gómez, & Roncalés, 2012)

100 °C water bath, 30 min; cooled under tap; 532 nm

peroxides

(Naveena, et al., 2013)

5 g sample + 15 1 mL homogenate + 2 mL H2O + 0.1 mL mL of TCA & TBA BHA/BHT; mixture b hom. ; 15 min in dark

boiled 15 min; cooled; c’fuged; 530 nm (with 7.8 x conversion factor)

none

(Ryu & Shin, 2014)

0.05 g sample + 1

100 °C for 60 min in

none

(King, Griffin,

4 g sample + 20 mL 20% TCA

+ 1.5 mL 0.8% TBA in

1

Turkey

myricetin fed to chickens through water, & then added to chicken meat post-mortem in H2O; fresh, heated, & heated-stored samples were prepared; storage at ‒12 °C for 72 h

mL H20 + 1.5 mL 1.1% SDS; vortexed 20% “acidic acid (sic)” pH adjusted to 2

water bath; cooled; + 5 mL n-BuOH; c’fuged, 10 min, 10000 rpm; 532 nm

& Roslan, 2014)

citrus extract & α-tocopherol

3 g sample + 25 mL H2O; hom., 1 min, 8000 rpm, Ultra-Turrax

5 mL distillate + 5 mL hexanal TBA (0.02 M in 90% glacial acetic acid); boiling water bath, 35 min; cooled, tap water, 10 min; 538 nm

(Contini, Katsikogianni, O'Neill, O'Sullivan, Dowling, & Monahan, 2012)

RTc for 15 h or boiling water bath for 1 h; cooled, 10 min, tap water; 532 nm

(Karwowska & Dolatowski, 2014)

added HCl to pH 1.5; distilled (Kjeldahl), collected 50 mL

in MeOH sprayed onto trays, cooked (to internal temperature of 73 °C) turkey slices laid on top; TBARS measured over 4 days at 4 °C Pork ground mustard seed added 10 g sample; filtered through paper; to minced pork; sausages hom. with 34.25 washed with 5 mL H2O; cooked to internal mL 4% HClO4 + adjusted to 50 mL; 5 temperature of 72 °C; 0.75 mL BHT in mL aliquot + 5 mL 0.02 TBARS measured over 12 EtOH, 4 °C, 4000 M TBA in H2O days at 4 °C rpm, 2 min a b c’fuged = centrifuged; hom. = homogenised; c RT = room temperature

cholesterol oxidation products

1

Table 4. Selection of articles showing TBARS conditions for antioxidant studies in different physiological samples ‒ matching those samples in Table 2 (last 5 years). Sample

Sample preparation & oxidation conditions

Exhaled breath condensate Plasma

no studies found

Serum

Rat brain

Vitis labrusca L. grape juices; subjects consumed 400 mL of different juices; blood collected after 1 h 100% pure tomato juice containing 11.6 mg of lycopene/100 mL; 280 mL/day for 56 days; bood collected before study & at day 56 traumatic brain injury induced in rats; quercetin administered by IP; TBARS measured in hippocampus

Formation of TBARS (in vivo, ex vivo, in vitro)

Conditions for addition of TBA (vol. aliquot, vol. & [TBA], etc.)

Incubation temperature & time, measurement of colour

Other measures of oxidation

Reference

in vivo

250 µL blood samples + 0.5 mL 20% TCA + 50 µL 10 mM BHT + 500 µL 1% TBA

lipid hydroperoxides in serum

in vivo

2 g hom.b sample + 10 mL 10% TCA + 1 mL 500 ppm BHT; boiling water bath, 30 min; c’fugedc, 2500 g, 10 min; 2 mL supernatant + 2 mL saturated aqueous TBA

100 °C, 60 min; cooled to RTa (21 °C); + 1.5 mL n-BuOH; c’fuged, 5 min, 1000 g; 532 nm boiling water bath, 30 min; cooled; 532 nm

(Toaldo, Cruz, de Lima Alves, de Gois, Borges, Cunha, et al., 2015) (Li, Chang, Huang, Wu, Yang, & Chao, 2015)

in vivo, post injury

OxiSelectTM TBARS kit: 50-100 mg/mL tissue in PBS & 5% BHT (in MeOH); hom. on ice; c’fuged, 10000 g, 5 min; 100 µL supernatant + 100 µL SDS lysis solution; 5 min, RT; + 250 µL TBA reagent (aqueous pH 3.5)

95 °C, 45-60 min; cooled in ice bath, 5 min; c’fuged, 3000 rpm, 15 min; 300 µL supernatant + 300 µL n-BuOH; vortexed, 12 min; c’fuged, 5 min, 10000 g; 200 µL nBuOH extracted sample; 532 nm.

total antioxidant assay (Cayman kit, based on the ABTS assay)

antioxidant enzymes SOD, GSH-Px, CAT

(Yang, et al., 2014)

1

Rat heart

sinapic acid in corn in vivo oil administered orally (intragastric tube) for 4 weeks

20% heart tissue hom. prepared in cold 0.1 M TrisHCl buffer; 0.5 mL tissue homogenate + 0.5 mL H2O + 2 mL TBA-TCA-HCl reagent

Incubated boiling water bath, 15 min; cooled; c’fuged, 10 min; 535 nm

lipid hydroperoxides & antioxidant enzymes, SOD, GSH-Px, CAT, inter alia.

(Silambarasan, Manivannan, Priya, Suganya, Chatterjee, & Raja, 2014)

Rat liver

Passion fruit peel flour fed to rats for 15 days

liver organs macerated in liquid N2 mixed with 8.1% SDS & TBA reagent (5% acetic acid + 20% NaOH)

95 °C, 60 min; cooled, ice bath, 10 min; c’fuged, 10 min, 10000 g; supernatant at 532 nm boiling water bath, 10 min; cooled; c’fuged; 535 nm

ORAC, FRAP, antioxidant enzymes SOD, GSH-Px

(da Silva, Cazarin, Batista, & Maróstica, 2014) (Saluk, Bijak, Nowak, & Wachowicz, 2013)

Platelets

in vivo

platelets suspended in in vitro prepared platelets + equal buffer; pre-incubated volume 15% cold TCA in (37 °C, 20 min) with 0.25 M HCl + equal volume & without 0.37% TBA in 0.25 M HCl selenorganic compounds; oxidation induced by peroxynitrite (0.1 mM, 2 min, 37 °C) or Fe2+ (37 °C, 20 min) or thrombin (6 U/mL, 37 °C, 5 min) a RT = room temperature; bhom. = homogenised; c c’fuged = centrifuged

none

1

Table 5. Selected studies where the TBARS assay has been used to screen for antioxidant activity along with other antioxidant assays. Antioxidants

Pure compounds & olive fruit extracts

Extracts of Penstemon. gentianoides & Barkleyanthus salicifolius, BHT, quercetin, tocopherol Methanol extracts of black caraway, cranberry, carrot & hemp seed oils

TBARS used as primary (1°) or secondary (2°) screen; other antioxidant assays 1° FC, phycoerythrin assay (AAPH mediated or Cu2+, ascorbate)

Substrate

Reaction conditions & quantification method

Results

Reference

LA

TBARS results did not match those from phycoerythrin assay

(McDonald, Prenzler, Antolovich, & Robards, 2001)

1° ORAC, FRAP, DPPH, FC

rat brain homogenate

in crude fractions, generally a good link between DPPH & TBARS

(Dominguez, Nieto, Marin, Keck, Jeffery, & Cespedes, 2005)

2° FC, DPPH, ABTS, ORAC

LDL

2 phases, Cu2+ catalyst, 37 °C, 20 h, BHT added to quench oxidation, 10 min colour development, extract pink layer into n-butanol, 532 nm; % inhibition at 20 h 1 phase, Fe2+ catalyst, 37 °C, 1 h, 30 min colour development in SDS emulsion, 532 nm; standard curve from tetramethoxypropane (TMP) expressed as nanomoles TBARS/mg protein; % inhibition; EC50 emulsion, Cu2+ catalyst, ambient temperature, 60 min, 30 min colour development, 532 nm; standard curve from TMP

cold-pressed cranberry & black caraway seed oil extracts tested for antiox. activity using TBARS; these extracts had highest antioxidant activity in ABTS & DPPH assays, but not highest total

(Yu, Zhou, & Parry, 2005a)

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Wheat bran extracts from 2 varieties, Akron & Trego, grown in 3 locations in Colorado

1° DPPH, ABTS, ORAC, superoxide scavenging

LDL

same as Yu et al. (2005a)

Pure compounds (12) – data aggregated from various other studies

1° fragmentation of apolipoprotein B-100

various

Ascorbic acid, BHT, 4-MBC (4methylbenzylidene camphor), tocopherol, Trolox, Lipochroman-6 Various pulses

1° DPPH, TEAC, “lipid” (conjugated dienes assay A236)

liposomes; rat liver microsome homogenate; human blood plasma; LDL + VLDL liposome prepared from L-αlecithin

Extracts of Salvia virgata Jacq.

1° conjugated dienes; results compared with another study where FC, DPPH, & ORAC done 1° FRAP, DPPH, FTC

LDL

LA (in ethanol)

liposome (emulsion), Fe2+/ascorbate catalyst, 37 °C, 1 h, BHT added, 15 min colour development, absorbance at 540 nm; not mentioned, but presumably % inhibition LDL “solution”, Cu2+ catalyst, 37 °C, 3 h, EDTA added, 30 min colour development, 532 nm; % inhibition converted to Trolox equivalents 1 phase, AAPH catalyst/initiator, 50 °C, 10 h, 20 min colour development, extraction into n-

phenol content “No correlation exhibited among the three antioxidative activities on per mg of TPC basis” (“TPC” = total phenol content) quercetin: better antioxidant in liposomes, myricetin: better antioxidant in microsomes

no correlation of TBARS with other antioxidant tests; %CV for TBA assay variable with antioxidant & all %CV high correlations observed between TBARS & other antioxidant assays & between TBARS & FC hexane & ethyl acetate fractions active in lipid assays, lower activity in

(Yu, Zhou, & Parry, 2005b)

(Roginsky & Lissi, 2005)

(Buenger, et al., 2006)

(Xu, Yuan, & Chang, 2007)

(Kosar, Goger, & Baser, 2008) 1

Pure compounds polar: ferulic acid, coumaric acid, propyl gallate, gallic acid, ascorbic acid; non-polar: BHT, rosmarinic acid, tertbutylhydroquinone (TBHQ), tocopherol Pure compounds (22)

2° modified ORAC for polar compounds & DPPH for non-polar compounds; peroxide & hexanal for antioxidant activity in oil-in-water emulsion

cooked ground beef

1° LAOX, DPPH, ORAC, FRAP

cooked, ground, poultry meat

Milk protein hydrolysates

2° ORAC, DPPH, metal chelation

cooked ground beef

Chilean wild blackberry fruits, Aristotelia chilensis

1° DPPH, ORAC, FRAP, superoxide & hydroxyl

rat brain homogenate

butanol, 532 nm; IC50 values 2 phases, no catalyst, heating until 77 °C internal temperature, propyl gallate added, colour development 15 min, 533 nm; extinction coefficient of TBA-MDA2 used to convert absorbance measurements to mg TBARS/kg muscle

aqueous assays free radical scavenging assays tested have limited value in predicting antioxidant activity in complex foods

2 phases, no catalyst, heating until 70 °C internal temperature, BHT added, 20 h colour development room temperature, 530 nm; Trolox equivalents 2 phases, no catalyst, heating until 71 °C internal temperature, then stored at 4 °C for 1, 8 & 15 days post cooking, propyl gallate & EDTA added, 60 min colour development in emulsion, extraction into pyridine/n-butanol, 532 nm; “standard curve” presumably TMP see Dominguez et al. (2005)

5 different antioxidant methods gave 5 different orders of ranking

(Capitani, Carvalho, Rivelli, Barros, & Castro, 2009)

based on ORAC, DPPH & metal chelation, 3 fractions tested by TBARS; only one had antioxidant activity;

(Hogan, Zhang, Li, Wang, & Zhou, 2009)

DPPH gave approximately the same ranking order as TBARS

(Céspedes, Valdez-Morales, Avila, El-Hafidi,

(Alamed, Chaiyasit, McClements, & Decker, 2009)

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(Mol) Stuntz radical scavenging (Elaeocarpaceae), EtOH & water extracts & fractions Methanol extracts of 1° 8 medicinal plants DPPH, metal chelation grown on a mine site

Extracts of Lavandula pedunculata subsp. lusitanica (Chaytor) Franco

1° ORAC, TEAC (ABTS)

Alarcón, & Paredes-López, 2010) linolenic acid in SDS emulsion

emulsion, Fe2+ catalyst, 37 °C, 16 h, BHT added, 60 min colour development, 532 nm; IC50 values calculated from % inhibition

mouse brain homogenate

2 phases, Fe2+ catalyst, 37 °C, 1 h, colour development in emulsion, 532 nm; standard curve from tetramethoxypropane

while not strictly a screening study (compared antioxidant activities of plants grown on mine site vs controls not grown at a mine) paper is significant as a recent example of a linolenic acid emulsion system good correlation among assays

(Dutta & Maharia, 2012)

(Costa, Gonçalves, Valentão, Andrade, Almeida, Nogueira, et al., 2013)

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Figure Caption Figure 1. Stages of lipid oxidation and antioxidant action in the TBARS antioxidant activity assay

Figure 1. Stages of lipid oxidation and antioxidant action in the TBARS antioxidant activity assay

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Highlights

• • • •

First review exclusively on the TBARS assay to measure antioxidant activity; Literature surveyed on the TBARS test in food and physiological systems; Methodology of the TBARS assay critically reviewed; Steps needed to enhance use of the TBARS assay in screening studies outlined.

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