Lipids secondary oxidation products

A number of methods are available for following the oxidative behaviour of food samples. The consumption of oxygen and the ESR detection of radicals, either directly or indirectly by spin trapping, can be used to follow the initial steps during oxidation (Andersen and Skibsted, 2002). The formation of primary oxidation products, such as hydroperoxides and conjugated dienes, and secondary oxidation products (carbohydrides, carbonyl compounds and acids) in the case of lipid oxidation, can be quantified by several standard chemical and physical analytical methods (Armstrong, 1998 Horwitz, 2000). 

According to the Cd 18-90 AOCS ° official method, the ANV is 100 times the optical density measured in a 1 cm cell, at 350 nm, of a solution containing 1.00 g of oil in 100 ml of the test solution. The measured absorbance is due to Schiff bases (167) formed when p-anisidine (166) undergoes condensation reaction with carbonyl compounds, according to equation 55. The carbonyl compounds are secondary oxidation products of lipids, such as a, S-unsaturated aldehydes and ketones derived from the hydroperoxides (see Scheme 1 in Section n.A.2.c), and their presence points to advanced oxidation of the oil. 

Alkanals, secondary oxidation products, 665 n-Alkanals, lipid peroxidation, 614 Alkanes... 

The primary products from autoxidation are hydroperoxides, which are often simply referred to as peroxides. Peroxides are odorless and colorless, but are labile species that can undergo both enzymatic and nonenzymatic degradation to produce a complex array of secondary products such as aliphatic aldehydes, alcohols, ketones, and hydrocarbons. Many of these secondary oxidation products are odiferous and impart detrimental sensory attributes to the food product in question. Being able to monitor and semi-quantitate the development of peroxides by objective means (e.g., PV determination) over time is important for food scientists who want to characterize the quality of an oil or a lipid-containing food product, even though the peroxides themselves are not directly related to the actual sensory quality of the product tested. 

The time to reach a certain PV may be used as an index of oxidative stability for food lipids. The effects of antioxidants and food processing on fats are often monitored in this way. Thus, a longer time period to reach a certain PV is generally indicative of a better antioxidant activity for the additive under examination. However, a low PV represents either early or advanced oxidation the breakdown of peroxides to secondary oxidation products will result in a decrease in PVs during the storage period. For determination in foodstuff, a major disadvantage to the classical iodometric PV assay is that a 5-g test portion is required it is sometimes difficult to obtain sufficient quantities of lipid from foods low in fat. Despite its drawbacks, PV determination is one of the most common tests employed to monitor lipid oxidation. 

The results from the TBA test, known in older literature as the TBA number, are usually expressed as mg malonaldehyde/kg sample for methods a to c cited above (note that results have also been reported as nmol malonaldehyge/kg or g sample) and as mg of malonaldehyde per unit of lipid for method d. Since it is known that malonaldehyde is not the only aldehyde present in the sample extract and because other aldehydes are capable of producing the same red pigment with TBA when the conditions are favorable, the TBA number/value is more appropriately expressed as the TBARS value, i.e., mg malonaldehyde equivalents/kg sample. To confuse the matter, the AOCS method, which is based on the protocol reported by Pokomy and Dieffenbacher (1989) and permits the direct determination of TBA value in oils and fats without preliminary isolation of secondary oxidation products, defines the TBA value as the increase of absorbance measured at 532 nm due to the reaction of the equivalent of 1 mg of sample per 1 ml volume with... 

Lipids are susceptible to oxidation and, as such, require analytical protocols to measure their quality. As described in vnitd2.i, autoxi-dation is one of the chief processes by which lipids degrade. The primary products from this reaction are hydroperoxides. These odorless and colorless transient species break down by various means to secondary products, which are generally odoriferous by nature. Being able to measure secondary oxidation products by simple spectrophotometric means is important for the food scientist so that he or she is able to characterize the extent of lipid oxidation. However, the researcher should be cautioned that one assay (e.g., TBA test) does not provide all the answers. To get a better picture of the story, both primary and secondary products of lipid oxidation should be assessed simultaneously by the different methods available (unitdu). 

In recent years, modern instrumental methods have been developed to monitor lipid oxidation in biological samples, including dairy products. These include use of electron spin resonance (ESR) spectrometry, direct measurement of secondary oxidative products such as malonaldehyde, static and dynamic GC/MS methods. ESR spectrometry permits detection of free radicals formed in the very early stages of oxidation prior to the formation of peroxides. The method has been applied successfully to dairy products such as milk powders and processed cheese (Nielsen et al., 1997 Stapelfeldt... 

Figure 5 Hemozoin-mediated lipid peroxidation. Redox cycling of iron in complexes like HZ can initiate lipid peroxidation of fatty acids like arachidonic acid (LH). Abstraction of the bisallylic hydrogen leaves an unpaired electron on the methylene carbon that can rearrange to form a reactive alkyl radical (L ). Oxidation of this radical leads to a peroxyl radical (LOO ), and on reduction, forms a lipid peroxide (LOOM) that can undergo additional reactions to yield a variety of secondary oxidation products.

As oxidation normally proceeds very slowly at the initial stage, the time to reach a sudden increase in oxidation rate is referred to as the induction period (6). Lipid hydroperoxides have been identified as primary products of autoxidation decomposition of hydroperoxides yields aldehydes, ketones, alcohols, hydrocarbons, volatile organic acids, and epoxy compounds, known as secondary oxidation products. These compounds, together with free radicals, constitute the bases for measurement of oxidative deterioration of food lipids. This chapter aims to explore current methods for measuring lipid oxidation in food lipids. 

The primary oxidation products (hydroperoxides) are unstable and susceptible to decomposistion. A complex mixture of volatile, nonvolatile, and polymeric secondary oxidation products is formed through decomposition reactions, providing various indices of lipid oxidation (5). Secondary oxidation products include aldehydes, ketones, alcohols, hydrocarbons, volatile organic acids, and epoxy compounds, among others. Methods for assessing lipid oxidation based on their formation are discussed in this section. 

It has been observed that the content of secondary oxidation products, such as malonaldehyde (MA), decreases with increased lipid oxidation, which can be explained by further reaction of MA with proteins. MA reacts with compounds containing primary amino groups (proteins, amino acids, DNA, phospholipids) to form fluorescent products (Figure 5) (37). A fluorescence assay has been successfully used to assess lipid oxidation in muscle foods and biological tissues. 

Analysis of the Decomposition Products of Hydroperoxides. Some authors have monitored formation of some of the decomposition products of the lipid hydroperoxides. Direct spectrophotometric measurements of the formation of oxo-octadecadienoic acids at 280 nm are possible , as are measurements of secondary oxidation products like a-diketones and unsaturated ketones at 268 nm. The formation of various aldehyde products of lipid peroxide decomposition can be monitored by reacting them with 2,4-dinitrophenylhydrazine and, after HPLC separation, measuring at 360-380 mn the DNPH derivatives formed , althongh the sensitivity of this particular technique makes it very susceptible to interference. 

Carbonyl compounds in oxidized lipids are the secondary oxidation products resulting from the decomposition of the hydroperoxides. They can be quantified by the reaction with 2,4-dinitrophenylhydrazine and the resulting colored hydrazones are measured spectrophotometrically at 430-460 nm. The carbonyl value is directly related to sensory evaluation, because many of the carbonyl molecules are those responsible for off-flavor in oxidized oil. The anisidine value is a measure of carbonyl compounds that have medium molecular weight and are less volatile (Frankel 1998). It can be used to discover something about the prior oxidation or processing history of an oil. 

Many methods have been developed to access the extent of oxidative deterioration, which are related to the measurement of the concentration of primary or secondary oxidation products or of both. The most commonly used are peroxide value (PV) that measures volumetrically the concentration of hydroperoxides, anisidine value (AV), spectrophotometric measurement in the UV region and gas chromatographic (GC) analysis for volatile compounds. Vibrational spectroscopy, because of its high content in molecular structure information, has also been considered to be useful for the fast measurement of lipid oxidation. In contrast to the time consuming chromatographic methods, modem techniques of IR and Raman spectrometry are rapid and do not require any sample preparation steps prior to analysis. These techniques have been used to monitor oil oxidation under moderate and accelerated conditions and the major band changes have been interpreted. ... 

The carotenoid activity during oxidation is strongly influenced by the oxygen pressure (PO2) of the experimental conditions. Kiokias and Oreopoulou have shown that certain natural carotenoid mixtures (paprika, bixin and tomato, and palm-oil preparations) inhibited the azo-initiated oxidation of sunflower oil-in-water emulsions (operated rapidly under low pOj) in terms of both primary and secondary oxidation products. However, other studies " concluded that carotenoids not only did not inhibit aerial lipid autoxidation (high PO2) but even exerted a prooxidant character, a phenomenon also observed at high carotenoid concentrations that could be due mainly to a more increased formation of carotene-peroxyl radicals, promoting the propagation of autoxidation. 

During lipid oxidation, the primary oxidation products that are formed by the autoxidation of unsaturated lipids are hydroperoxides, which have little or no direct impact on the sensory properties of foods. However, hydroperoxides are degraded to produce additional radicals which further accelerates the oxidation process and produce secondary oxidation products such as aldehydes, ketones, acids and alcohols, of which some are volatiles with very low sensory thresholds and have potentially significant impact on the sensory properties namely odor and flavor [2, 3]. Sensory analysis of food samples are performed by a panel of semi to highly trained personnel under specific quarantined conditions. Any chemical method used to determine lipid oxidation in food must be closely correlated with a sensory panel because the human nose is the most appropriate detector to monitor the odorants resulting from oxidative and non-oxidative degradation processes. The results obtained from sensory analyses provide the closest approximation to the consumers approach. Sensory analyses of smell and taste has been developed in many studies of edible fats and oils and for fatty food quality estimation [1, 4, 5]. 

Autoxidation and photo-oxygenation are two aspects of the non-enzymic reaction between oxygen and unsaturated fatty acids. The enzymic reactions are discussed in Section 10.3. Oxidation of lipids during storage and handling, involving complex substrates and ill-defined reaction conditions, proved difficult to understand. This difficulty is enhanced by the fact that the primary oxidation products are labile and readily converted to secondary oxidation products of several kinds. Understanding of these processes has come from studies of simpler substrates such as methyl oleate or methyl linoleate under clearly defined reaction conditions. 

The complex mixture of regioisomers and stereoisomers structurally related to the endoperoxides and thromboxanes (prostaglandin-like compounds) produced from arachidonic acid and esterified to phospholipids and cholesterol are classified as isoprostanes. These compounds are used as markers of in vivo oxidative injury in biological systems and considered reliable because they are specific, stable and not affected by the lipid content of the diet (Chapter 13. E.4). However, these secondary oxidation products are produced in relatively small concentrations because they are derived from arachidonic acid, which is a minor polyunsaturated lipid compared to linoleic acid in biological samples. Very sensitive gas chromatography-mass spectrometric (GC-MS) and liquid chromatography-MS techniques are required to analyse isoprostanes in biological samples. 

Schiff base compounds formed by the interaction of oxidation products with proteins, phospholipids and nucleic acids produce chromophores showing characteristic fluorescence spectra. The Schiff base formed between malonaldehyde and amino acids is attributed to the conjugated structure -NH=CH-CH=CH-NH-. Lipid-soluble fluorescence chromophores are produced from oxidized phospholipids and from oxidized fatty acid esters in the presence of phospholipids. These chromophores have fluorescence emission maxima at 435-440 nm and excitation maxima at 365 nm. The Schiff base of malonaldehyde and phospholipids has a higher wavelength maximum for emission (475 nm) and excitation (400 nm). The interaction between oxidized arachidonic acid and dipalmityl phosphatidylethanolamine produce similar fluorescence spectra (maximum excitation at 360-90 nm and maximum emission at 430-460 nm). The products from oxidized arachidonic acid and DNA have characteristic fluorescence spectra, with excitation maximum at 315 nm and emission maximum at 325 nm. Similar fluorescence spectra, with excitation maximum at 320 mn and emission maximum at420 nm, are obtained from the interactions of either lipid hydroperoxides or secondary oxidation products with DNA in the presence of metals and reducing agents, or different aldehydes, ketones and dimeric compounds from oxidized linolenate. Therefore, the Schiff base produced from various oxidized lipids and phospholipids and DNA may be considered to be due to a mixture of closely related chromophores. 

The large number of precursors of volatile decomposition products affecting the flavor of oils has been discussed in Chapter 4. Only qualitative information is available on the relative oxidative stability of hydroperoxides, aldehydes and secondary oxidation products. As observed with the unsaturated fatty ester precursors, the stability of hydroperoxides and unsaturated aldehydes decreases with higher unsaturation. Different hydroperoxides of unsaturated lipids, acting as precursors of volatile flavor compounds, decompose at different temperatures. Hydroperoxides of linolenate and long-chain n-3 PUFA decompose more readily and at lower temperatures than hydroperoxides of linoleate and oleate. Similarly, the alkadienals are less stable than alkenals, which in turn are less stable than alkanals. The short-chain fatty acids produced by oxidation of unsaturated aldehydes will further decrease the oxidative stability of polyunsaturated oils. For secondary products, dimers are less stable than dihydroperoxides, which are less stable than cyclic peroxides. 

Many secondary oxidation products of fatty acids have been shown to be more toxic to experimental animals. This is partly due to the fact that the low molecular weight products have shorter carbon chain lengths, and are more easily absorbed into the intestinal wall than lipid hydroperoxides or their polymeric materials. Oarada et al. (1986), for example, have shown that over 73% of radioactivity was found in the urine and CO2 of rats 12 h after receiving " C-labeled low molecular compounds, compared to less than 25% for methyl linoleate hydroperoxides and the polymeric fraction. 

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