The TBARS method is an assay for measuring the end products of lipid peroxidation or more specifically malondialdehyde (MDA) need not cause controversy but it was worth noting how attempts to improve its application have fared.
The TBARS assay as it is known (the thiobarbituric acid reactive substances assay) has been used for many years as a method for detecting end products of lipid peroxidation. It was first discovered when the reaction between MDA and 2-thiobarbituric acid (TBA) was analysed and the level of MDA could be correlated with lipid oxidation (Bernheim et al., 1948). It was then used as the standard test for rancidity in milk (Dunkley and Jennings, 1951; Dunkley and Franke, 1967) as well as meat which was more accurate than a direct peroxide measure (Zipser et al., 1964; Fernandez et al., 1997). It is now widely employed because there are so few other methods covering this aspect of food quality which are as simple to use, inexpensive and with its rapidity suited to a large number of samples, which can be assessed with little effort (Armstrong and Browne, 1994).
The Reactions In The TBARS Method
The assay relies on the reaction of 2-thiobarbituric acid (TBA) with malondialdehyde to yield a red chromogenic fluorescent substance measured spectrophotometrically between 530 and 537nm and is usually expressed as mgs of malondialdehyde per kg of substance.
Malondialdehyde (MDA) itself is the major carbonyl oxidation product derived mainly from polyunsaturated fatty acids with at least two conjugated double bonds. To improve upon the measurement of MDA, modifications using HPLC methods have also been devised (Agarwal and Chase, 2002).
Unfortunately, the specificity of the method for measuring MDA leaves something to be desired because other substances can generate the same end product with TBA, so it is not entirely specific a measure for lipid peroxidation. Attempts to understand the errors began with investigation of the chemistry behind the reaction (Tarladgis et al., 1964). MDA also binds to other substances such as proteins, amino acids and glycogen which can then lead to under reporting of values (Kwon et al., 1965). Hence the need to report results as TBAR values.
One reason for non-specificity is a result of the acid-heating step which attempts to release bound MDA from protein causing the formation of TBA/MDA-type derivatives (Liu et al., 1997). Other interfering substances include caseinate in milk (Caprioli et al., 2010). It also suffers from over-inflation of lipid peroxidation values when other interfering substances take part in the reaction too. One method to overcome this problem has been to subtract the absorbance at 532 nm of an unreacted control where other coloured substances such as anthocyanins in plant extracts were present (Hodges et al., 1998). Other attempts to standardise the assay have been posited when assessing in vitro lipid peroxidation (Rael et al., 2004). When a variety of lipids are available, individual fatty acids have differing degrees of unsaturation which leads to an unspecific but general measure. Their findings suggested standardisation using multiple fatty acid concentrations.
Markers In Food
Researchers making assessments on how diets rich in dietary oils or in other additives affect meat quality have used the TBARS method to make judgements on lipid quality in animal muscle (Rey et al., 2001). It is also a useful and simple screen for peroxides from lipid breakdown in blood plasma and serum (Yagi, 1998). In fact, the technique has long been the subject of assessment for measuring oxidative stress in the etiology of chronic conditions such as cardiovascular disease (Valenzuela, 1991; Trevisan et al., 2001). It was recognised early on that the standard assay was not suitable for measuring MDA in human plasma which was essential if oxidative stress was to be measured accurately (Largilliere and Melancon, 1988). Markers of oxidative stress, besides plasma derived thiobarbituric acid substances also include measures for erythrocyte glutathione and glutathione peroxidase. The non-specificity of the TBARS assay is problematic especially for assessing lipid damage in human plasma. It has been a condition in some EFSA opinions that a more specific method be found. Isoprostane might be more suitable but is much more difficult to measure unless HPLC or ELISA is available.
With time, the TBARS method may well become superseded by others but it still remains the method of choice for assessing lipid oxidation. It’s limitations are understood and it provides a general assay for screening and monitoring lipid peroxidation.
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