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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Oxidative Processes
  5. Reactive Species
  6. Biological Effects of RS
  7. Defense Systems against Oxidative Processes
  8. Other Endogenous Antioxidants
  9. Genetic Effects on the Oxidative Processes in Lamb and Beef
  10. Factors Influencing the Oxidative Processes of Meat
  11. Measurement of Biochemical Components in Tissues of Living Animal or Postmortem Muscles That is Associated with Oxidative Stress
  12. Biological Functions of Isoprostanes
  13. Factors Affecting the Formation of Isoprostanes
  14. Conclusions
  15. Acknowledgments
  16. References

Oxidation of meat occurs under postmortem conditions and is inevitable. This oxidation includes the biochemical changes in meat leading to changes in color pigments and lipids. As a consequence, color deteriorates, and undesirable flavors and rancidity develop in meat thereby impacting on consumer appeal and satisfaction. Across carcasses, there is variation in the rate at which muscle undergoes chemical reactions under postmortem conditions that reflect inherent variation at the biochemical level. It is expected that this underlying biochemical variation will be reflected in living muscle through oxidative processes. The oxidative process of muscle tissues will vary according to an animal's immunity status, temperament, and ability to cope with stress, with all these affected by nutrition, genetics, management practices, and environmental conditions (hot and cold seasons). Identification of biomarkers that indicate the oxidative status levels of animals or muscle tissues in vivo could provide insight as to how the muscle will respond to the anoxic conditions that produce undesirable results in meat. This review outlines the potential use of 1 group of biomarkers, the isoprostanes, in the context of complex biochemical reactions relating to oxidative processes that take place in the biological systems of live animals (in vivo) and subsequently in meat (in vitro).


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Oxidative Processes
  5. Reactive Species
  6. Biological Effects of RS
  7. Defense Systems against Oxidative Processes
  8. Other Endogenous Antioxidants
  9. Genetic Effects on the Oxidative Processes in Lamb and Beef
  10. Factors Influencing the Oxidative Processes of Meat
  11. Measurement of Biochemical Components in Tissues of Living Animal or Postmortem Muscles That is Associated with Oxidative Stress
  12. Biological Functions of Isoprostanes
  13. Factors Affecting the Formation of Isoprostanes
  14. Conclusions
  15. Acknowledgments
  16. References

In nature, the progression of cells toward apoptosis and death is facilitated by oxidative processes that involve the formation of free radicals and their reactions within the tissue systems of animals and humans (Fiers and others 1999). A free radical is a molecule that is able to exist in an independent state (that is, free) and carry unpaired electrons in the valence orbits (that is, radical). This condition is thermodynamically unfavorable and the molecules will attempt to reach a more stable state by reacting with another molecule, cleaving a hydrogen atom from a carbon–hydrogen bond, and donating or accepting electrons from neighboring compounds (Halliwell and Gutteridge 2007). This results in free radicals displaying a more behaviorally reactive chemical species than nonradical molecules. Mitochondria are regarded as the main source of free radical formation (Cadenas and Davis 2000) in the tissue systems of living animals. The mitochondrial electron transport chain and its reactions generate superoxide anion radicals that can be subsequently converted to hydrogen peroxide. This can lead to the formation of hydroxyl radicals that react with muscle and other tissue systems impacting on the well-being or the performance of animals.

Due to their highly reactive nature, free radicals exist in low concentrations (10−4 to 10−9 M) and their effects are initiated locally where they are generated (Southorn and Powis 1988). There are 2 possible outcomes from free radical reactions. The first is chemical modification of the surrounding compounds (such as oxidation of amino acids, lipids, or vitamins). This can promote loss of physiological function in living animals or initiation of undesirable changes in the muscle tissue system leading to deterioration of quality attributes such as color stability, flavor, or nutritive characteristics after death. Second, the affected compound can become a radical causing a series of electron exchanges among several molecules leading to DNA damage or oxidation of lipids and proteins.

The nutrient composition of the diet consumed by animals, including humans, is the major factor that influences the biochemical components and oxidative process of muscle tissue systems (Ames and others 1993; Fang and others 2002). When the oxidative potential of a muscle tissue system is suboptimal, this may cause an imbalance or instability in the functioning of muscle biological systems (Bagchi and Puri 1998). As such, this may affect the reactive capacity of chemical components within muscle systems leading to the formation of free radicals and secondary oxidative substrates in the muscle of living animals as well as in postmortem muscle (Dabbagh and others 1994; Stephens and others 1996; Brigelius-Flohe and Traber 1999). This review will first outline the critical processes controlling oxidation and factors that can impact on oxidation. Following this, it will provide some insights on the biological actions of isoprostanes in circulatory systems of living animals, their relation to the development of secondary components, and their evaluation in live animals to predict the quality of muscle in red meat.

Oxidative Processes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Oxidative Processes
  5. Reactive Species
  6. Biological Effects of RS
  7. Defense Systems against Oxidative Processes
  8. Other Endogenous Antioxidants
  9. Genetic Effects on the Oxidative Processes in Lamb and Beef
  10. Factors Influencing the Oxidative Processes of Meat
  11. Measurement of Biochemical Components in Tissues of Living Animal or Postmortem Muscles That is Associated with Oxidative Stress
  12. Biological Functions of Isoprostanes
  13. Factors Affecting the Formation of Isoprostanes
  14. Conclusions
  15. Acknowledgments
  16. References

Oxidative processes are those chemical/biochemical reactions that involve the transfer of 1 or more electrons from an electron donor (reductant) to an electron acceptor (oxidant) leading to the transformation of both the oxidant and the reductant. These reactions are commonly associated with significant pathological diseases in humans and animals, and also undesirable changes in food systems.

The impact of oxidative stress on human health through propagation of diseases has extensively been reviewed (Southorn and Powis 1996; Cadenas and Davis 2000; Stadtman and Levine 2000; Villamena and Zweier 2004; Andreyev and others 2005; Davies 2005; Trachootham and others 2008; Ilbert and others 2009). Generally speaking, oxidative processes usually involve reactive oxygen and nitrogen species (ROS and RNS, respectively). A detailed account of the chemistry of these reactive species (RS) can be found in the above-mentioned references. Several other modifying compounds are produced by nonenzymatic glycation this is chemical reaction between glucose and protein starts with a Schiff base and undergoing a series of reactions and/or rearrangements ending with the formation of compounds called advanced glycation end products (AGE). These compounds are usually target protein residues and lead to the formation of complex compounds such as alkyl-formyl-diglycoxylpyrrole (a cross-link between arginine and lysine promoted by pentose), pyrroline, carboxymethyllysine, and pentosidine (Bourdon and Blache 2001).

Many of the findings and fundamentals generated in the medical field form the basis for our understanding of oxidative processes and their effects in animal physiology and biochemical processes and postmortem muscle quality. In relation to meat, the effects of lipid and myoglobin oxidation on the quality of meat have been the main focus of several reviews over the past 3 decades (Ladikos and Lougovois 1990; Kanner 1994; Baron and Andersen 2002; Min and Ahn 2005; Faustman and others 2010). The effects of the oxidative processes on the health of farm animals have been recognized, but few reports are available that summarize this relationship (Lykkesfeldt and Svendsen 2007; Pamplona and Costantini 2011). Furthermore, the impact of oxidative stress in live animals and its subsequent effect on the quality of meat are poorly understood. A recent review (Li and Liu 2012) discussed the effects of some farm practices and their influence on lipid oxidation of meat, which is a step toward reducing the gap in our understanding about the relationship between the oxidative stress of farm animals and the quality of meat produced. The relationships between different factors affecting the oxidative processes in meat postmortem are quite complex and regulated by several physical, biochemical, and chemical processes (Figure 1) with the RS being the common factor in many of these processes. The impact of oxidative processes on food components, such as pigments, lipids, protein, carbohydrates, and other nutrients will affect the nutritional value, functional properties, texture, and perceived keeping quality of the food. In addition, due to RS being fundamental to this review, a summary of some important compounds and their biological actions is provided.

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Figure 1. Overview of cellular reactions involving RS. Source: Bekhit (2004).

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Reactive Species

  1. Top of page
  2. Abstract
  3. Introduction
  4. Oxidative Processes
  5. Reactive Species
  6. Biological Effects of RS
  7. Defense Systems against Oxidative Processes
  8. Other Endogenous Antioxidants
  9. Genetic Effects on the Oxidative Processes in Lamb and Beef
  10. Factors Influencing the Oxidative Processes of Meat
  11. Measurement of Biochemical Components in Tissues of Living Animal or Postmortem Muscles That is Associated with Oxidative Stress
  12. Biological Functions of Isoprostanes
  13. Factors Affecting the Formation of Isoprostanes
  14. Conclusions
  15. Acknowledgments
  16. References

The RS in vivo are generated from diverse sources (oxygen species, nitrogen species, lipid and protein oxidative products, and transition metals such as iron and copper). With the loss of antioxidant defenses in living tissues and then in meat postmortem, more complex oxidative processes will emerge. The main RS of interest are shown in Table 1 and comprehensive reviews of these systems have been published by Villamena and Zweier (2004) and Davies (2012). Mitochondria are the major source for the formation of various radicals and oxidants by several enzymatic systems (Table 2).

Table 1. The half-life and rate constants of biological RS
SpeciesSymbolHalf-life (s) at 37 °CRate constant* (M−1s−1)
  1. * = rate constant with methionine.

  2. Compiled from Florence (1991), Klein and Biskupič (1993), Zheng and others (2003), Radi (2004), Bocci and others (2009), and Davies (2005).

Radicals   
Semiquinone radical>102
Peroxyl radicalROO˙>1 × 10−2
Superoxide radicalO2˙>1 × 10−6< 0.3
Alkoxyl radicalRO˙>1 × 10−6
Hydroxyl radicalHO˙>1 × 10−9 
PerhydroxylHOO˙1–30
Nitric oxide radical•NO1–309.1× 109
Carbonate radical anionCO3˙ 1.2 × 108
AzideN3˙10−5—10−6<107
Oxidants   
Molecular oxygenO2>1021.9 × 1010
Lipid peroxideROOH>102
Singlet oxygen1O2>1 × 10−62 × 107
Hydrogen peroxideH2O2101 × 10−2
OzoneO39 × 1035 × 106
PeroxynitriteONOO10–20 × 10−3
Hypochlorous acidHOCl 3.8 × 107
Table 2. Enzymatic systems involved in free radical generation
EnzymeRadical generatedEnzyme functionlocationReference
NADH oxidaseO2˙Unknown functionMuscle sarcoplasmic reticulumXia and others (2003)
NAD(P)H oxidase (EC 1.6.3.1)H2O2  Feillet-Coudray and others (2009)
Dihydroorotate dehydrogenase (EC 1.3.3.1 or EC 1.3.99.11)H2O2, O2˙Catalyzes conversion of dihydroorotate to orotate, a step in the synthesis of pyrimidine nucleotides.MitochondriaAndreyev and others (2005)
Glycerol-3-phosphate dehydrogenase (EC 1.1.99.5)H2O2FAD-containing enzyme catalyzes oxidation of glycerol-3-phosphate to dihydroxyacetone phosphate, utilizing mitochondrial coenzyme Q as an electron acceptor.  
Succinate dehydrogenase (EC 1.3.5.1)ROSOxidizes succinate to fumarate using coenzyme Q as an electron acceptor.Mitochondria complex II 
Aconitase (EC 4.2.1.3)HO˙Catalyzes conversion of citrate to isocitrate as part of the tricarboxylic acid cycle.Mitochondria 
α-Ketoglutarate dehydrogenase complex [multiple copies of 3 enzymes: α-ketoglutarate dehydrogenase (EC 1.2.4.2), dihydrolipoamide succinyltransferase (EC 2.3.1.12), and lipoamide dehydrogenase (EC 1.6.4.3].H2O2, O2˙Catalyzes oxidation of α-ketoglutarate to succinyl-CoA using NAD+ as an electron acceptor.Mitochondria 
Pyruvate dehydrogenase (EC 1.2.4.1)H2O2, O2˙Multiple functions. See Brenda websiteMitochondria 
Cytochrome b5 reductase (EC 1.6.2.2)O2˙∼300 nmol/min /mg protein.It oxidizes cytoplasmic NAD(P)H and reduces cytochrome b5 in the outer membrane.MitochondriaWhatley and others (1998)
Monoamine oxidases (EC 1.4.3.4)H2O2Catalyzes oxidation of biogenic amines and the oxidative deamination of primary aromatic amines along with long-chain diamines and tertiary cyclic amines.Outer mitochondrial membraneOrrenius and others (2007)
Succinate-cytochrome c reductase system (may be EC 1.6.2.1)O2˙ MitochondriaChretien and others (1990)
NADH:ubiquinone reductase (EC 1.6.5.3)O2˙Oxidizes NADH, produced predominantly by the tricarboxylic acid cycle in the mitochondrial matrix, and reduces ubiquinone in the inner mitochondrial membrane.MitochondriaLambert and Brand (2004)
Nitric oxide synthase (EC 1.14.13.39)NOMultiple see Brenda websiteMitochondriaStamler and Meissner (2001)

Superoxide (O2˙)

Mitochondria are the most significant intracellular source of O2˙ with an estimated 5- to 10-fold increase in O2˙ concentration in the mitochondria compared to the nuclear space or the cytosol (Cadenas and Davis 2000; Orrenius and others 2007). About 5% of the oxygen consumed by the living organism can be converted into O2˙ by mitochondria under physiological conditions (Valko and others 2006). The production of O2˙ in mitochondria is estimated to be approximately 2 to 3 nmol of O2˙/min per mg of protein (Inoue and others 2003) underpinning its importance as the major source of this radical in living organisms. O2˙ can be produced enzymatically (cytochrome P450-dependent oxygenases and xanthine oxidase, Table 2) and nonenzymatically by direct electron transfer to an oxygen molecule (Inoue and others 2003).

The amount of O2˙produced in the muscle tissue can increase dramatically as a result of muscle contraction (Reid 2001). The production of O2˙ in mitochondria complex I is stimulated by succinate (an important substrate for complex II) that suggests the existence and involvement of a reverse electron flow mechanism in this stimulation process (Orrenius and others 2007). The main issue associated with O2˙ is the potential production of the extremely reactive hydroxyl radical (HO˙) through dismutation by superoxide dismutase (SOD), which will produce H2O2, and subsequent reactions of the H2O2 or O2˙ with a transition metal such as Fe2+ or Cu2+ (Fenton and Haber–Weiss reactions).

Superoxide is involved in several mechanisms that initiate oxidative damage. For example, O2˙ generates equimolar amounts of H2O2 when O2˙ inactivation of Fe-S-containing enzymes occurs (for example, complex I NADH dehydrogenase and aconitases; Liochev and Fridovich 1999). This subsequently leads to the generation of HO˙ or directly causes the oxidation of neighboring molecules. Also, the reaction of O2˙ with nitric oxide (NO˙) can lead to the production of potent reactive nitrogen species. The formation of a conjugate acid (HO2˙) is possible in Fenton reactions (Carlsen and others 2005). This consequently can lead to more damaging effects since the formed acid has a much higher reaction rate compared to O2˙ (Table 3). Inhibition of O2˙ is facilitated by enzymatic dismutation by SOD or spontaneous dismutation (Loschen and others 1974).

Table 3. Rate constants for reaction of HO˙ with various biological compounds at pH 7
 O2˙* (L/mol−1 s−1)HO2˙# (L/mol−1 s−1)Increase in rate constant of HO2˙/O2˙ (fold)
  1. *= measured at different pHs (ranged 8.3 to 10.9).

  2. #= measured at different pHs (ranged 1.2 to 1.6).

  3. From Bielski and Shiue (1978); Bocci and others (2009); and Davies (2005).

DL-Alanine0.144.0733.3
L-Arginine0.163.0484.6
DL-Asparagine0.253.0331.3
DL-Aspartic acid0.212.066.7
L-Cysteine15.0601.040.1
L-Cystine0.4
Glycine0.448.6115.7
L-Glutamic acid0.430.076.9
L-Glutamine0.323.092.0
L-Histidine1.095.095.0
DL-Isoleucine2.038.919.5
L-Leucine0.223.0109.5
DL-Lysine3.313.34.0
DL-Methionine0.348.8147.9
L-Phenylalanine0.4180.0500.0
D-Proline0.217.3108.1
DL-Serine0.554.6103.0
DL-Threonine0.212.559.5
L-Tryptophan24.0
L-Tyrosine10.0
DL-Valine0.210.558.3

Hydroxyl radical (HO˙)

The hydroxyl radical HO˙ is the most reactive (Table 4) and is regarded as the most damaging free species since it is capable of attacking any neighboring molecules. All mitochondrial enzyme proteins are susceptible to inactivation by HO˙, but are rather resistant to the effects of H2O2 (Cadenas and Davis 2000). It is important to note that the Fenton reaction is a major route for the formation of HO˙. This reaction is dependent on the availability of metal ions that are tightly bound and well regulated in the circulatory system and tissues of living animals. Upon slaughter, the integrity of cells is compromised by the drop in muscle pH, loss of cofactors, and actions of proteases, and therefore, mechanisms controlling the metal ions will no longer operate effectively. This suggests the contribution of HO˙ will be eventually high in postmortem muscles and unless endogenous antioxidant systems can inhibit this radical, fast deterioration in the quality of meat will occur through oxidation processes. The Fenton reaction (reactions 1 and 2) is the most reported pathway for the generation of HO˙ as shown below.

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Table 4. Rate constants for reaction of HO˙ with various biological compounds at pH 7
CompoundRate constant (L/mol s)
  1. Cadenas and Davies (2000) and Valko and others (2006).

DNA8 × 108
RNA1 × 109
Hyaluronan7 × 108
Linoleic acid9 × 109
Collagen4 × 1011
Albumin8 × 1010
Ascorbate1 × 1010
GSH1.4 × 1010
Trolox6.9 × 109
Alanine7.7 × 107
Arginine3.5 × 109
Asparagine4.9 × 107
Aspartic acid7.5 × 107
Cysteine3.4 × 1010
Cystine2.1 × 109
Glutamine5.4 × 108
Glutamic acid2.3 × 108
Glycine1.7 × 107
Histidine1.3 × 1010
Isoleucine1.8 × 109
Leucine1.7 × 109
Lysine3.4 × 108
Methionine8.3 × 109
Phenylalanine6.5 × 109
Proline4.8 × 108
Serine3.2 × 108
Threonine5.1 × 108
Tryptophan1.3 × 1010
Tyrosine1.3 × 1010
Valine7.6 × 108
N-Ac-Gly–Gly(backbone attack)7.8 × 108

The reaction is not limited to iron and several other ions (Cu2+, Fe3+, Ti4+, and Co3+) can be involved in the reaction (Valko and others 2006).

Perhydroxyl radical (HOO˙)

The perhydroxyl radical molecule is the simplest peroxyl radical. Garrison (1987) described reactions where the availability of electrons, suggested to be generated by pulsed radiolysis, are favorably scavenged to produce HO2˙ through reactions 3 to 5.

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In mitochondria, e and O2 are available which can give rise to the conditions required for the formation of HO2˙. There is an evidence that HO2˙ is a far more powerful radical than for O2˙ (Table 3). The reaction rate constants for HO2˙ are 6 to 9 orders of magnitude higher than O2˙ (Bielski and Shiue 1978). This is due to its ability to extract hydrogen atoms from linoleic, linolenic, and arachidonic fatty acids with rate constants of 1 to 3 × 103 L/mol s (Eq. (6) to (8), suggesting a role in the initiation of lipid oxidation (Bielski and others 1983). The perhydroxyl radical can also extract hydrogen atoms from NADH or glyceraldehyde-3-phosphate dehydrogenase–NADH at reaction constants of 2 × 105 and 2 × 107 L/mol s, respectively (Land and Swallow 1971; Chan and Bielski 1980) leading to the formation of H2O2.

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It has been suggested that HO2˙ is very important compared to O2˙ (De Grey 2002). This is despite the concentration of O2˙ being 2- to 3-fold higher than HO2˙ at pH 6.8 to 7.8, due to its high reactivity (2 to 3 orders of magnitude) with biological material, for example, muscle tissues, and its ability to diffuse through membranes (De Grey 2002). Meat oxidation by perhydroxyl radicals is not likely except in irradiated meat, since high-energy input (exposure to radioactive material or use of a high-energy electron beam) is required to initiate the reactions leading to formation of this radical.

Nitric oxide (NO˙)

Nitric oxide is mainly produced by the different forms of NO synthase, but the formation has also been suggested through H2O2 reaction with arginine (Nagase and others 1997) and the decomposition of S-nitrosothiols in the presence of metal ions (Singh and others 1996). Muscle expresses the different forms of NO (Stamler and Meissner 2001). Nitric oxide exerts both beneficial and damaging effects in vivo (Rubbo and others 1994). For example, the presence of NO˙ can provide some protection to tissue when excess production of reactive oxygen species (ROS) is encountered (Rubbo and others 1994). At low concentrations, NO˙ can alleviate the oxidative activation of the calcium release channel caused by ROS actions, but at high concentrations of NO, the oxidative processes are stimulated (Reid 2001). The damaging facet is probably brought about by formation of the potent oxidant ONOO through the reaction of NO˙ and O2˙. This reaction is 3 times faster than the enzymatic dismutation of O2˙ by SOD (Rubbo and others 1994), and therefore, it is the likely event for NO˙ reactions. Once formed, ONOO- can exert a direct oxidation reaction against biomolecules and be involved in metal-catalyzed reactions. Unlike O2˙, NO is able to oxidize metal-containing enzymes and proteins such as myoglobin and its derivatives (Laverman and others 2001) and can regulate the activity of calpains (Michetti and others 1995). Nitric oxide is a known inhibitor for glutathione peroxidase (GPx) (Asahi and others 1995), lipoxygenase (O'Donnell and others 1999), and several oxidases such as NADPH oxidase and cytochrome c oxidase (Clancy and others 1992; Cleeter and others 1994), all of which will contribute positively or negatively to postmortem muscle quality. Comprehensive reviews of the role of NO in the manipulation of meat quality (Warner and others 2005) and the nitrosylation of muscle structural proteins (Lonergan and others 2010) are available.

Peroxynitrite (ONOO)

The formation of ONOO is afforded by the reaction between nitric oxide and superoxide and could play a significant prooxidant role in vivo and in muscle foods. ONOO can cause inactivation of complex I of mitochondria that may involve S-nitrosation or Fe-nitrosylation (Orrenius and others 2007). This eventually increases the production of H2O2. A rapid oxidation of liposomes and muscle homogenates was triggered by ONOO that was suppressed by CO2, low pH, and metal chelators (Brannan and Decker 2001). The peroxynitrite in that study was prepared by the reaction of isoamyl nitrite with 9 M hydrogen peroxide at pH 13. The role of ONOO as prooxidant in live animals can be significant since higher levels of thiobarbituric-acid-reactive substances (TBARSs) were found in muscle homogenates at pH 7.2 compared with pH 5.2 (Brannan and Decker 2001). Peroxynitrite generates sulfur-centered radicals through a 1-electron oxidation step reaction with thiols and, as a result, the formation of thioyl radicals from cysteine, glutathione, methionine, and albumin have been reported (Pryor and others 1994; Gatti and others 1994; Augusto and others 1994). These reactions are stimulated in the presence of CO2 and ascorbate (Scorza and Minetti 1998). A feature that increases the damaging effects of ONOO in an aqueous environment is the potential of peroxynitrous acid formation that has the ability to pass through lipid bilayers and can initiate lipid peroxidation and also cause nitration of lipids (Radi and others 1991).

Heme proteins such as cytochrome c oxidase, cytochrome c peroxidase, myoglobin, and catalase react with peroxynitrite (Keng and others 2000). Myoglobin is a good target for peroxynitrite as it is abundant in muscles and oxidation of myoglobin can lead to deleterious effects on meat quality (Connolly and others 2002). Giving the exceptionally high half-life of NO˙ (stated to be up to 3 min, Brannan and Decker 2001), the ability to react with O2˙ and generation of peroxynitrite in meat is high.

Transition metals

The production of free radicals by transition metals mediated reactions (such as Fenton reactions) has been well established (Stadtman 1990; Min and Ahn 2005; Halliwell and Gutteridge 2007). The role of transition metals in generating free radicals in vivo was suggested to be insignificant since the metals are bound to proteins and have limited availability for reactions (Chen and others 2000). However, after slaughter and during retail display or post farm storage, significant biochemical changes (such as pH, protein/fat denaturation in muscles, and O2 availability) take place and these metals will become freely available for reactions in meat that may lead to quality deterioration.

Biological Effects of RS

  1. Top of page
  2. Abstract
  3. Introduction
  4. Oxidative Processes
  5. Reactive Species
  6. Biological Effects of RS
  7. Defense Systems against Oxidative Processes
  8. Other Endogenous Antioxidants
  9. Genetic Effects on the Oxidative Processes in Lamb and Beef
  10. Factors Influencing the Oxidative Processes of Meat
  11. Measurement of Biochemical Components in Tissues of Living Animal or Postmortem Muscles That is Associated with Oxidative Stress
  12. Biological Functions of Isoprostanes
  13. Factors Affecting the Formation of Isoprostanes
  14. Conclusions
  15. Acknowledgments
  16. References

The damaging effects of oxidative processes are well recognized in the medical field and the food industry, for example, the involvement of lipid oxidation in the generation of reactive compounds, which causes damage to cellular materials. These effects have been implicated in a number of physiological disorders and diseases (Montine and others 2004) and negative changes in meat quality (Kanner 1994). Having noted these effects, it is important to understand that low levels of RS like H2O2 can in some circumstances enable cells to survive otherwise lethal oxidative stress (Niki 2012). Three significant changes take place due to increased oxidative changes; namely, changes in DNA, lipid oxidation, and protein oxidation. These processes will be further explained.

Changes in DNA

Oxidative processes cause modification to the various DNA components (such as purine and pyrimidine bases, the deoxyribose backbone), damage to the DNA strands, and cross-linking of several molecules. These reactions are causative in the development of several diseases, but have limited influence on the quality of meat. A possible positive effect, however, is the increase of the lag phase of bacterial growth (Jamaludin and others 2010) during meat storage and thus an increase in meat retail display shelf life.

Lipid oxidation

The main targets for oxidation in the lipids are the polyunsaturated fatty acids (PUFAs) and phospholipids that are vulnerable to the action of HO˙ (Esterbauer and others 1991). The presence of carbon double bonds in PUFAs weakens the C-H bonds and makes the H+ vulnerable for oxidation reactions triggered by HO˙, HO2˙, RO˙, and ROO˙ that are generated in mitochondrial and microsomal membranes. These radicals (HO˙, HO2˙, RO˙, and ROO˙) are able to abstract hydrogen atoms from methylene groups of fatty acids (Götz and others 1994) that can initiate lipid oxidation. The progression of lipid oxidation will promote several changes (such as alteration of membrane properties, loss of physiological function, inactivation of enzymes; changes in membrane fluidity and permeability of the membrane increases; denaturation and subsequent rupture causing leakage of cellular components) and further biochemical changes will have both desirable and undesirable effects. For example, a low level of oxidation can be desirable for flavor development and proteolysis (Estévez 2011). This can occur when lipid peroxidation products cause damage to Ca2+–ATPase and Ca+2 regulation systems leading to a build-up of Ca2+ and activation of calpains and lipoxygenase. However, the undesirable outcomes are often outnumbered the desirable outcomes.

Undesirable effects result from the decomposition of lipid oxidation products when further reactions with metal ions occur (Chapple 1997), which will promote the formation of off-flavors (4-hydroxy-2-nonenal, malondialdehyde, alkanes, ethane, pentane, and isoprene). The by-products generated by lipid oxidation can be regarded as 2 groups; 1) primary end products such as hydroperoxides and conjugated dienes; 2) secondary end products such as isoprostanes, prostaglandin (PG) F2-like compounds (discussed later), carbonyls (ketones and aldehydes), alcohols, hydrocarbons (including pentane, ethane), isofurans, and malondialdehyde that cause the formation of rancidity and off-flavors (Min and Ahn 2005). In particular, the oxidation of n-3 and n-6 fatty acids have been reported to generate off-flavors by the formation of 4-hydroxy-2-nonenal (HNE), hexanal, 1-octen-3-ol, and 2-nonenal (from n-6 fatty acids) and 2,4-heptadienal, 4-heptenal, 2-hexenal, propanal, 2,4,7-decatrienal, 1,5-octadien-3-ol, 2,5-octadien-1-ol, 1,5-octadien-3-one, and 2,6-nonadienal (from n-3 fatty acids) (Min and Ahn 2005). The mechanisms involved have been reviewed extensively (Gutteridge 1984; Ladikos and Lougovois 1990; Kanner 1994; Grandemer 1998; Morrissey and others 1998; Baron and Andersen 2002; Carlsen and others 2005; Min and Ahn 2005; Chaijan 2008; Faustman and others 2010; Li and Liu 2012) and therefore will not be covered in this review.

Although many reports indicate that n-3 fatty acids generate off-flavors in foods, this area warrants further investigation. Recent research has shown that n-3 fatty acids do not enhance lipid oxidation per se, as this is dependent on the concentration of antioxidants or heme iron availability in muscles. To explore this further, Najafi and others (2012) have shown that canola oil supplementation of feed significantly increased linolenic acid content in blood, liver, and muscle tissues. Their findings on TBARSs from these samples as an assessment of lipid oxidation indicated that the increase in the n-3 fatty acid (linolenic acid) did not increase lipid oxidation, instead it significantly lowered the TBARS levels. Related work by this group on lambs provides evidence that the level of lipid oxidation in postmortem muscles will be determined by the vit E, total omega-6 fatty acids, and heme iron concentration in the muscle (Ponnampalam and others 2012a), but not the long-chain omega-3 fatty acid levels per se. Another study has shown no evidence of a reduction in meat color as assessed by redness associated with the level of long-chain omega-3 fatty acid content in muscles (Ponnampalam and others 2012b). In light of these recent findings, one would expect that if n-3 fatty acids cause adverse effects in the muscle systems postmortem, it would also lead to detrimental effects in living animals and humans when consuming it as food or used in medicine. However, there is mounting evidence that omega-3 fatty acids improve immunity systems and health by lowering the oxidation process in the body and tissues of living animals and humans (Simopoulos 2002; Benatti and others 2004). The main message from the above cited reviews indicates those oxidants and metals, individually and combined, are capable of initiating and propagating lipid oxidation. The oxidation of PUFA by HO˙, HO2˙, O2˙, RO˙, RO2˙, and NO2˙ can be initiated via 1 electron reduction (Buettner 1993). The involvement of metal ions through the Fenton and Haber–Weiss reactions has been well documented (Stadtman 1990; Kanner 1994; Grandemer 1998; Morrissey and others 1998; Baron and Andersen 2002; Min and Ahn 2005; Halliwell and Gutteridge 2007). Lipid peroxidation in mitochondria caused by ROS can severely damage mitochondrial metabolism. Lipid peroxides modify essential functions of mitochondria (such as respiration and oxidative phosphorylation, inner membrane barrier properties, the uptake of Ca2+, and the overall integrity of mitochondria) by interacting with the protein and lipid moieties in the membrane (Orrenius and others 2007). The retention of Ca2+ in mitochondria causes acute changes in the morphology and functionality of mitochondria (Haworth and Hunter 1979). During the postmortem phase, these modifications will greatly affect the oxygen consumption rate (OCR) of muscle tissues, and hence, the availability of Ca2+ that will have a potential impact on the color and tenderness properties of the meat.

Ledward and others (1977) observed that mincing meat more than once caused the meat to form MetMb more readily, which led the authors to conclude that mincing destroys the reducing ability of the meat. They also observed that the rate of Mb oxidation in mince was about 5 times as fast as that reported for OxyMb in solution. This would allow a complete destruction of a large proportion of cells and direct interaction between different reactants involved in various oxidative processes. According to Gray and Pearson (1994), lipid oxidation in meat is affected by species, anatomical location (muscle type), temperature, antioxidants like vit E influenced by diet, age, sex, and the lipid composition (PUFAs, phospholipids) of the meat. The development of a MetMb layer under the surface of meat depends on the partial O2 pressure, and therefore one may expect that a high O2 pressure should stabilize the color since MetMb formation requires a low O2 pressure. According to Wang (1962), the rate of OxyMb oxidation is slower than the corresponding process for free heme by a factor of about 108. Hence, maintaining the Mb in the oxygenated form reduces the autoxidation rates dramatically. However, high O2 pressure increases the rate of lipid oxidation and may counteract the beneficial effects on color gained by that treatment (Jayasingh and others 2002).

The shelf life of meat and the rate of discoloration are closely related to the content of n-3 PUFAs in beef. The inclusion of fish oil (3%) in the feed significantly increased lipid oxidation of beef (Wood and others 2008). Increased n-3 PUFA also led to more intense flavor and volatiles upon cooking (Elmore and others 2000), but this is dependent on inclusion rates (Najafi and others 2012).

Protein oxidation

The effects of oxidative processes on the oxidation of protein in tissue systems have been an important part in human medical investigations and several excellent reviews have outlined the various mechanisms involved in pathological diseases and protein oxidation (Stadtman 1990; Florence 1991; Dean and others 1997; Stadtman and Berlett 1999; Stadtman and Levine 2000; Dunlop and others 2002; Davies 2005; Bocci and others 2009; Ilbert and others 2009). However, this research area is less developed in meat. The issue of protein oxidation in meat was raised by the studies of Xiong and Decker (1995) and Mercier and others (1995) and the topic has been recently reviewed (Estévez 2011; Lund and others 2011).

The damaging effects of protein oxidation are seen in the oxidation of sulfhydryl groups, formation of oxidative adducts on amino acids, reactions with aldehydes, establishment of cross-links between proteins, and protein fragmentations (Starke-Reed and Oliver 1989; Stadtman and Oliver 1991). Several products can be generated from the oxidation of amino acids and the formation of protein carbonyls, or RNS can be the outcome of the oxidation of the majority of the amino acids (Table 5). Stadtman (2006) described the mechanisms that can lead to protein–protein cross-linking by RS as

  1. Formation of disulfide cross-links due to the oxidation of cysteine sulfhydryl groups on the proteins.
  2. Interaction between active groups on different proteins such as the reaction of an aldehyde group of an HNE protein adduct and the -NH2 group of a lysine residue in 2 separate proteins.
  3. The interaction between a carbonyl group of a glycated protein and a -NH2 group of lysine residue in another protein.
  4. Reaction of malondialdehyde with the reactive -NH2 groups of lysine residues in 2 different protein molecules.
  5. The formation of covalent linkages between carbon-centered radicals in 2 different protein molecules.
Table 5. Amino acid oxidation products caused by RS
Amino acidsOxidation products
  1. Source: Bourdon and Blache (2001) and Davies (2005).

AlanineGlutamic semialdehyde
ArginineHydroperoxides (unstable); 5-hydroxy-2-aminovaleric acid; carbonyl compounds; chloramines (unstable—from HOCl)
CysteineCystine (disulfide); oxyacids (RSO2H, RSO3H); sulfonamides; sulfenyl chloride (unstable—from HOCl)
Glutamic acidHydroperoxides (unstable)
GlycineAminomalonic acid
Histidine2-Oxohistidine; asparatate; asparagines; 2-oxohistidine; chlorinated materials (unstable—from HOCl); hydroperoxides (unstable—from 1O2); alcohol and carbonyl products (from 1O2)
IsoleucineHydroperoxides (unstable); alcohols; carbonyl compounds
LeucineHydroxyleucine; alcohols; a-ketoisocaproic acid; isovaleric acid; isovaleraldehyde; isovaleraldehyde oxime; carbonyls
Lysine2-Aminoadipic semialdehyde; hydroperoxides (unstable); alcohols chloramines (unstable—from HOCl); carbonyl compounds
MethionineMethionine sulfoxide; methionine sulfone
PhenylalanineOrtho-tyrosine (2-hydroxyphenylalanine); meta-tyrosine (3-hydroxyphenylalanine); tyrosine
ProlineGlutamate, pyroglutamate; cis/trans-4-hydroxyproline; 2-pyrrolidone; glutamic semialdehyde; g-aminobutyric acid; hydroperoxides (unstable); alcohols; 5-hydroxy-2-aminovaleric acid; carbonyl compounds
Threonine2-Amino-3-ketobutyric acid
TryptophanN-Formylkynurenine; kynurenine; 5-hydroxytryptophan; 7-hydroxytryptophan; hydroperoxides (unstable—from 1O2); alcohol and cyclized products (from 1O2)
Tyrosine3,4-Dihydroxyphenylalanine (DOPA) (unstable); di-tyrosine (carbon-carbon dimer and carbon-oxygen dimer, and higher species); 3-chlorotyrosine; 3,5-dichlorotyrosine; 3-nitrotyrosine; hydroperoxides (unstable—from 1O2); alcohols and cyclized products (from 1O2)
ValineHydroperoxides (unstable); alcohols; carbonyl compounds

Oxidation of amino acids can be used as a marker for protein oxidation and modification (Orrenius and others 2007). As mentioned above, the oxidation of protein can lead to conformational changes in the protein structure that will affect the biological functions of the protein. The impact of the oxidation site can affect the degree of modification. For example, the oxidation of amino acids buried in the inner part of protein will have a more significant effect on the protein conformation than the oxidation of the surface amino acids (Rostovtseva and others 2005). The oxidation of aliphatic (hydrophobic) side chains can produce carbonyl groups, peroxide, or alcohol (Table 5) that can lead to new hydrogen bonds with the surrounding solvent due to their high dipole moments (Orrenius and others 2007). The formation of protein carbonyl can alter the tertiary structure of the protein and lead to various degrees of irreversible and irreparable protein unfolding (Aldini and others 2007). The unfolded structure promotes the hydrophobicity of the polypeptide and supports protein–protein interactions. Protein oxidation causes the loss of their normal functions, such as enzymatic activity, channel forming properties, and so on, and the proteins become more susceptible to proteolytic degradation. The susceptibility of protein to degradation by proteases will depend on the level of the protein modification. For example, the rate of degradation of oxidized protein by 20S proteasome is enhanced with the modification of Met residues and decreased hydrophobicity in the secondary structure (Orrenius and others 2007). However, when the tertiary structure is altered, and increased hydrophobicity is attained by oxidation, the rate of protein degradation is reduced (Matsuishi and Okitani 1997). An interesting observation is the inactivation of proteasome by HNE, the secondary product of lipid oxidation (see Szweda and others 2002). It does not seem, however, to explain why a reduction in proteolysis can be found at high oxidation levels as proteasome does not have a significant influence on degradation that leads to improvements in tenderness (Hopkins and Geesink 2009).

There are some reports indicating the inhibition of the proteolytic activity of calpains in the presence of H2O2 that can be reversed upon the addition of a reducing agent (Guttmann and others 1997; Carlin and others 2006). Rowe and others (2004b) demonstrated that oxidation induced by irradiation can negatively affect μ-calpain activity in beef and result in less tender meat. In that study, meat from vit E-supplemented animals had higher degradation of troponin-T compared with meat from nonsupplemented animals at 2 d postmortem only, but it was not clear whether this supplementation alleviated any negative impact of irradiation or not. Similarly, high oxidation of myosin reduced its susceptibility to degradation by cathepsin B (Kristensen and others 1997), pepsin, trypsin, and chymotrypsin (Liu and Xiong 2000). The negative influence of high oxidation on the proteolysis of myofibrillar proteins was also confirmed in a study using papain (Morzel and others 2006). In contrast to these reports, a recent study examined the susceptibility of myofibrillar proteins to degradation by μ-calpain, m-calpain, or caspase-3 under 3 levels of oxidation (low, medium, and high). It was found that proteolysis by these enzymes increased with an increase in protein oxidation (Smuder and others 2010).

It is clear that different proteases may have different effects on oxidized proteins. A potential role of vit E is possible in downregulating protein oxidation to a level that renders these proteins susceptible to the actions of proteases rather than eliminating oxidation (Santé-Lhoutellier and others 2008a), and this warrants further investigation. It is important to state that the literature available in the public domain clearly shows that the oxidation process in tissues of living animals and postmortem muscles is mostly interrelated to lipids, minerals (Fe, Ca, Se), and antioxidants (vit E, vit C, and carotenoids), not the proteins that are associated with DNA damage.

Defense Systems against Oxidative Processes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Oxidative Processes
  5. Reactive Species
  6. Biological Effects of RS
  7. Defense Systems against Oxidative Processes
  8. Other Endogenous Antioxidants
  9. Genetic Effects on the Oxidative Processes in Lamb and Beef
  10. Factors Influencing the Oxidative Processes of Meat
  11. Measurement of Biochemical Components in Tissues of Living Animal or Postmortem Muscles That is Associated with Oxidative Stress
  12. Biological Functions of Isoprostanes
  13. Factors Affecting the Formation of Isoprostanes
  14. Conclusions
  15. Acknowledgments
  16. References

In living organisms, there are several synchronized defense systems (Table 6) that work on inactivating RS and repairing the damage induced by these species. Defense systems can generally be classified as enzymes, metal chelators, RS scavengers, and several other compounds that have demonstrated in vivo and in vitro antioxidant activity (Table 6). Another group of cellular materials has been suggested to act as sacrificial antioxidants such as albumin (Halliwell 1988), myoglobin (Yang and de Bono 1993), and amino acids (Stadtman and Berlett 1991). These are normally regenerated in the living organism through enzymatic processes (for example, oxidized myoglobin MetMb is reduced by cytochrome b5 reductase).

Table 6. Endogenous antioxidants in muscles
AntioxidantsTarget ROSMechanism
  1. Source: Wayner and others (1987); Halliwell (1988); Stadtman and Berlett (1991); Yang and de Bono (1993); Woodford and Whitehead (1998); Rãisãnen and others (1999); Fujii and Ikeda (2002); Arora and others (2003); Jongberg and others (2009); Faustman and others (2010).

Enzyme systems  
Catalase (EC 1.11.1.6)H2O2Decomposes H2O2 into O2 and H2O.
Glutathione peroxidase (EC 1.11.1.9)H2O2, LOOHUses GSH for the reduction of H2O2 to H2O
Glutathione S-transferase (EC 2.5.1.18)LOOHProtects mitochondria from various insults by adding a GSH molecule (such as lipid peroxidation such as 4-hydroxynonenal)
Thioredoxin reductase (EC 1.11.1.15)LOOHDecomposes peroxides
Phospholipid hydroperoxide glutathione peroxidase (EC 1.11.1.12)H2O2, LOOH, Cholestrol peroxidesBroad selectivity
Glutathione reductase (EC 1.8.1.7)O2˙, HO˙Reduces oxidized glutathione that can either scavenge O2˙ and HO˙ nonenzymatically or by serving as an electron donating substrate to several enzymes involved in ROS detoxification
SOD (EC 1.15.1.1)O2˙Dismutation of superoxide radical to H2O2
Carbonic anhydrases (EC 4.2.1.1)H2O2Zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide.
Metal chelators  
TransferrinIron 
HaptoglobinHemoglobin 
HemepexinHeme 
CeruloplasminCopper 
AlbuminCopper 
Quenching ROS radical scavenaging  
Lipophilic:1O2, ROSVit E is a ubiquitous lipid-soluble free-radical-scavenging
Carotenoids, vit E, vit D, Ubiquinol (reduced coenzyme Q) antioxidant present in mitochondrial membranes.
Hydrophilic:ROS 
Vit C, uric acid, bilirubin, albumin, Estrogens  
Sacrificial compounds  
Cytochrome c.O2˙The reduced cytochrome c is regenerated (oxidized) by its natural electron acceptor, cytochrome c oxidase.
GluthathioneH2O2Regenerated by glutathione reductase
Amino acidsMetal ions, H2O2 
MyoglobinO2˙Regenerated by cytochrome b5 reductase
Vit C, Vit E, bilirubin, uric acid, glutathioneRS 
Other muscle components demonstrated antioxidants activity  
Anserine, Carnitine, Carnosine, Lactate, NADH, NADPH, Putrescine, Pyruvate, Spermine, Spermidine, Urate, α-lipoic acid, and peptides.ROSIn vitro activity under optimal conditions. Activity in postmortem meat may differ.

Antioxidant enzymes

Physiological importance of antioxidant enzymes

The majority of the enzymes catalyze reactions that decompose peroxides (H2O2 or lipid peroxides), with the exception of SOD that catalyzes the 1-electron dismutation of O2˙ to H2O2. The availability of several enzymes that target H2O2 may indicate the importance of eliminating this oxidant. H2O2 is considered a relatively stable compound and it has high membrane permeability. Therefore, it can diffuse within the cell and cause major damage to the cells and muscle systems of living organisms. The availability of cytosolic or mitochondrial antioxidant enzymes (including catalase, GPx, and thioredoxin peroxidase) are therefore an important defense system in eliminating H2O2 (Marchi and others 2012).

SOD does not require a cofactor for reaction, and therefore, its activity is not associated with an energy cost. SOD in mammalian cells exists in 2 forms: cytosol SOD (Cu/Zn-SOD) and mitochondrial SOD (Mn-SOD). The intracellular concentrations of Cu/Zn-SOD range between 10−6 and 10−5 M and they has a very high rate constant of 2 × 109 M−1 s−1 (Chaudieáre and Ferrari-Iliou 1999). The ion (Cu, Zn, Mn, Fe, Se, or S) available in the active side of these antioxidant enzymes can potentially be of significance to the rate constant of the reactions, the affinity to target certain RS, and the location of the enzyme (Chaudieáre and Ferrari-Iliou 1999).

Antioxidant enzymes in meat

There are few studies investigating the effects of nutrition or dietary background on the activities of antioxidant enzymes (Table 7). Diet appears to modify the level of antioxidant enzymes in the circulatory and muscle systems, although no consistent effects have been observed (Table 7). For example, while Petron and others (2007) found that the pasture type can affect the activity of GPx, but not SOD and catalase, Descalzo and others (2005, 2007) reported higher catalase only in meat from cattle raised on pasture compared with meat from grain-fed cattle. However, this effect was not in agreement with other studies conducted by the same group (Gatellier and others 2004; Mercier and others 2004). While the background of the animals used in these studies varied greatly and could be a contributing factor in the reported findings, the effects seem to be dependent on the sex and age of the animals (Gatellier and others 2004; Mercier and others 2004) and the postslaughter processing conditions (Pastsart and others 2010). These factors are expected to dictate the carcass cooling rate postmortem and muscle quality (Pastsart and others 2010). The influence of vitamin E supplementation on the antioxidant enzyme activities in meat, however, is inconclusive (Table 7).

Table 7. Effect of diet, muscle, and supplementation of vitamin E on antioxidant enzymes
TreatmentMuscleSOD (Ug−1)CAT (Ug−1)GPX (Ug−1)α-tocopherol (μg/g)ß-carotene (μg/g)Ascorbic acid (μg/g)Reference
  1. – = no information provided.

  2. a-b = Means of different diet treatments, within each publication, with different letters are significantly different.

  3. # SOD = One unit was defined as homogenate volume per mg protein that inhibited epinephrine autoxidation by 50%. Cat = Units were calculated as pmol of disproportionate H2O2 min−1 mg−1 protein using a constant value for pure catalase of 3.4 × 107 l/mol s. Activity was expressed as units of enzyme per mg protein. GPX = Units were expressed as nmol of oxidized NADPH mg−1 protein.

  4. *SOD = One unit was defined as the activity that inhibits the pyrogallol autoxidation by 50%. Cat = One unit was defined as nmol of decomposed H2O2 min−1 mg−1 protein.

  5. GPX = One unit was defined as nmol of oxidized NADPH min−1mg−1 protein.

  6. @ SOD = A unit of enzyme activity was defined as the amount of sample needed to inhibit the reaction (pyrogallol autoxidation) by 50%. The activity was expressed per gram sample.

  7. Cat = One unit of Cat activity was defined as the amount of sample required to decompose 1 μmol of H2O2 per minute at room temperature. The activity was expressed per gram sample.

  8. GPX = One unit of GPx activity was defined as the amount of extract required to oxidize 1 μmol of NADPH per minute at 25 °C. The activity was expressed per gram sample.

Intensive ryegrassLongissimus0.090a40.864.41.72Petron and others (2007)@
Botanically diverse 0.082a30.868.51.24  
Leguminosa-rich 0.181b30.172.41.09 
GrainPsoas major16.0319.6a40.21.50a0.06a15.92aDescalzo and others (2007) and Descalzo and others (2005) #
Grain + Vit E 18.8119.7a36.91.76a0.05a17.39a 
Pasture 15.8427.2b42.93.08b0.45b25.3b 
Pasture + Vit E 16.5123.7b443.91b0.63b21.98b 
Mixed-diet steersLongissimus0.7a2100ab195b2.8aGatellier and others (2004)*
    Heifers 0.6a2800b195b2.8a   
    Cows 0.5a3000b190b3.7ab   
Pasture steers 2.8b2200ab75a4.2b 
    Heifers 4.0b1800a70a4.2b   
    Cows 3.8b320065a3.9ab   
Mixed-dietLongissimus0.58a2678197.83.6Mercier and others (2004)*
Pasture 3.65b352763.64.2 
 Longissimus1.57b2056b132.58aPastsart and others (2010)*
 Outer biceps femoris1.51b2203b152.80b 
Inner biceps femoris1.33a1650a137.20a 

Results from Renerre and others (1996) reported significant differences in the antioxidant enzymes among different muscles [psoas major (PM), diaphragm (D), longissimus lumborum (LL), and tensor fasciae latae (TFL)]. Higher catalase and GPx activities were found in D muscle only, whereas SOD activity was higher in PM and D muscles than in LL and TFL. Higher oxidation was reported in the oxidative-glycolytic (PM) and oxidative (D) muscles compared with the glycolytic TFL muscle due to higher mitochondria and myoglobin contents in the oxidative muscles. While these results support increased antioxidant enzyme expression in high-oxidative environments of living tissues, it does not substantiate a role for these enzymes in postmortem muscle tissues or meat. The activities of catalase and GPx were not different over 8 d of postmortem storage, while significant oxidative changes were observed. Similarly, Pradhan and others (2000) found that catalase activity was not affected by postmortem storage time at 4 °C, frozen storage at −20 °C for 2 mo, or freeze/thaw cycles.

The stability of catalase during postmortem storage was also reported in other studies (Descalzo and others 2007; Petron and others 2007). These observations negate the contribution of these endogenous enzymes to the stability of meat during postmortem storage, since oxidation processes are known to increase with storage time in postmortem meat from the time of slaughter. However, the addition of exogenous catalase to beef mince at high levels (1600 and 4000 units/g mince) significantly reduced lipid oxidation (measured as TBARS) by 10% and 6%, respectively (Pradhan and others 2000). The authors suggested that catalase plays an important role in stabilizing lipids in postmortem meat due to higher lipid oxidation in meat treated with sodium azide. However, sodium azide, a potent antimicrobial compound, may interfere with the measured oxidation products. It is worth noting that the approach used in examining the effects of antioxidant enzymes may not represent their real function in meat. For example, most of the assays used to measure the enzyme activities are performed at pH > 7.0, a condition that does not reflect the actual pH of normal meat (5.4 to 5.8).

Furthermore, the activities of the antioxidant enzymes in large beef muscles including the biceps femoris were shown to be negatively affected by the location on the muscle (Pastsart and others 2010). In the study by Pastsart and others (2010), the oxidation of myoglobin (as measured by rate of change in a*-value or MetMb%) was in the following order: outside biceps femoris < longissimus < inner biceps femoris. The oxidation of lipids (measured by TBARS) was in the following order: longissimus < inner biceps femoris < outside biceps femoris. The orders of oxidation were different from the order found with the GPx and catalase (inner biceps femoris < outside biceps femoris = longissimus) or SOD (inner biceps femoris = longissimus < outside biceps femoris), suggesting a lack of association between antioxidant enzymes and oxidative processes in postmortem muscles or meat.

The addition of catalase and SOD in a model system (containing microsomes, OxyMb, FeCl3, and ascorbic acid in acetate buffer, pH 6.5) at 400 units/mL produced different outcomes. While SOD and catalase decreased the oxidation of OxyMb by 10% and 40%, respectively, these enzymes had no effect on lipid oxidation (Gorelik and Kanner 2001). The addition of catalase in a model system (containing liposomes, OxyMb, xanthine, and xanthine oxidase, pH 5.6) decreased the oxidation of OxyMb and lipid by 22.1% and 31.7%, respectively (Chan and others 1997). In another model system, meat homogenates from cattle with different feeding backgrounds (pasture and mixed-feed) showed higher lipid oxidation in samples that had low SOD and high GPx when challenged with FeSO4 and H2O2, but no effect was found on protein oxidation (Mercier and others 2004). It is worth mentioning that the meat from both treatments did not differ in vit E and catalase contents, but no information on the fatty acid composition/content was provided, which more than likely would have assisted in explaining the results and the difference observed. The outcomes from these model systems appear to be dependent on the radical/oxidant generator used in the assay and it would be interesting to compare these assays using the same meat sample. Similar studies that measured the antioxidant activity of meat demonstrated that the activity varied with the assay used (Gatellier and others 2004; Descalzo and others 2007).

The systems implicated in the reduction of metmyoglobin (MetMb) and hemoglobin are reviewed by Bekhit and Faustman (2005). The role of metmyoglobin-reducing activity in human and animal health, mostly exhibited by metmyoglobin cytochrome b5 reductase, is certainty important in regenerating myoglobin and hemoglobin (suggested as sacrificial antioxidants) (Herold and others 2002). However, the significance of metmyoglobin-reducing activity in postmortem meat is questionable. Mitochondrial cytochrome b5 metmyoglobin reductase (Cyt b5 met r) is located in the outer mitochondrial membrane and the enzyme is found in many mammalian tissues. The enzyme is suspected to be involved in the production of O2˙ at a very high rate of 300 nmoL/min/mg protein (Marchi and others 2012). Several studies using meat, rather than mitochondria, found that increased myoglobin and lipid oxidation was strongly linked to increased metmyoglobin-reducing activity of the meat (Mikkelsen and others 1999). For example,

  1. Partially purified Cyt b5 met r and cytochrome b5 from cow liver in the presence of NADH have been shown to increase lipid peroxidation in frozen beef patties (Mikkelsen and Skibsted 1992). NADH is a crucial cofactor for metmyoglobin reduction (Bekhit and Faustman 2005).
  2. Increases of MetMb percentage and TBARS values were associated with an increase of myofibrillar metmyoglobin-reducing activity and total metmyoglobin-reducing activity (Bekhit 2004).
  3. Earlier immunochemical studies by Hirokata and others (1978) investigating a NADH-dependent lipid oxidation in microsomes demonstrated that Fe3+ ions supported NADH lipid peroxidation of liver microsomes. The authors pointed out the involvement of NADH-Cyt b5 r and cytochrome b5 in the transfer of electrons from NADH to the lipid peroxidation reaction because NADH-dependent lipid peroxidation was inhibited by antibodies for these proteins.
  4. Low color stability across 19 bovine muscles had high MetMb reducing activity, OCR, and lipid oxidation (McKenna and others 2005), which provides evidence for increased oxidation processes with higher MetMb-reducing activity.
  5. A recent study using lamb cores (Bekhit and others 2007b) demonstrated a 3-fold increase in superoxide anion production in the presence of NADH relative to its absence (Figure 2A). The prooxidant-mediated oxidation of dichlorofluorescein diacetate was increased 22-fold in the presence of NADH and meat cores, compared with the absence of NADH (Figure 2B). Because NADH is required by metmyoglobin-reducing activity for the reduction of metmyoglobin in meat (see Bekhit and Faustman 2005), this indicates the involvement of the system in ROS generation and lipid oxidation. The production of ROS as a by-product of the reaction should be expected due to the loss of cell integrity. The production of ROS was inhibited by resveratrol, a potent plant antioxidant (Bekhit and others 2003), and was stimulated by the addition of carnosine, a muscle antioxidant that exhibits prooxidant activity at pH 5.6 (Bekhit and others 2004) supporting the production of ROS when meat is stimulated with NADH (Figure 3). The produced ROS will lead to increased lipid oxidation. That study confirmed a hypothetical mechanism for the production of ROS earlier suggested by Bekhit (2004) as a result of increased metmyoglobin-reducing activity in meat (Figure 4) where infusing meat with Zinc chloride, a powerful antioxidant and strong inhibitor for calpains, reduced the oxidative processes in postmortem meat.
  6. Even in some cases where the reducing activity was measured by nitric oxide metmyoglobin reduction assay, the inhibition of MetMb-reducing activity produced significantly reduced lipid oxidation (Ramanathan and others 2012).
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Figure 2. Prooxidant activities in fresh lamb (3 d postmortem) as detected using 2`,7`-dichlorofluorescein diacetate (DCFH-DA) assay (A) and cytochrome c reduction assay (B) from Bekhit and others (2007b).

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Figure 3. Increase in carnosine and decrease in resveratrol in DCFH oxidation compared with control (DCFH + meat core + NADH) in a dose-dependent fashion reaction from Bekhit and others (2007b).

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Figure 4. Effect of Zn++ induction on the oxidative pathway of meat as proposed by Bekhit (2004). Cycle A represents the oxidative processes in meat due to free radicals the production of superoxide as proposed by Hansen and others (1996). Cycle B represents the reduction pathway of MetMb as proposed by Kuma and others (1976).

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Collectively, this information suggests an increased lipid oxidation in meat with the increase in metmyoglobin reducing activity and therefore any beneficial role for this activity in postmortem meat maybe questionable.

Other Endogenous Antioxidants

  1. Top of page
  2. Abstract
  3. Introduction
  4. Oxidative Processes
  5. Reactive Species
  6. Biological Effects of RS
  7. Defense Systems against Oxidative Processes
  8. Other Endogenous Antioxidants
  9. Genetic Effects on the Oxidative Processes in Lamb and Beef
  10. Factors Influencing the Oxidative Processes of Meat
  11. Measurement of Biochemical Components in Tissues of Living Animal or Postmortem Muscles That is Associated with Oxidative Stress
  12. Biological Functions of Isoprostanes
  13. Factors Affecting the Formation of Isoprostanes
  14. Conclusions
  15. Acknowledgments
  16. References

Physiological importance of other endogenous antioxidants

Several compounds have demonstrated antioxidant activities that play important roles in living organisms and in postmortem meat (Table 6). Ascorbate (vitamin C) and glutathione (γ-glutamylcysteinylglycine) are probably the major scavengers of free radicals as they are available in the tissue at higher concentrations than reducing nucleophilic ones and biomolecules (Gérard-Monnier and Chaudiere 1996). Vitamin C is a water-soluble antioxidant that has broad scavenging activities against ROS (O2˙, HO˙, and peroxides), but it exhibits a strong prooxidant activity in the presence of Fe3+ or Cu2+. Vitamin C plays a major role in restoring the antioxidant activity of oxidized vit E through a reduction step. Α-lipoic acid supports the antioxidant activity of vitamines C and E. A similar protective role against the oxidation of vit C is provided by uric acid. Uric acid, a by-product from the degradation of purine catalyzed by xanthine dehydrogenase, has strong scavenging activities against O2˙, HO˙, and peroxides, and metal-binding capacity. Bilirubin, a by-product from the catabolism of hemeprotein, has chain-breaking antioxidant activity and inhibits peroxides. Muscle contains several metal binding compounds. Transferrin is a protein that is responsible for the movement of iron in the body and displays extremely high capacity to bind iron (1 molecule can bind up to 2 g atomic iron). Similarly, albumin provides a protective role by binding metal ions. Ceruloplasmin, a copper-containing protein, supports the binding of iron to transferrin and interferes with the oxidation of Fe2+ by H2O2 (Kosman 2002). Coenzyme Q is an important antioxidant present in lipoproteins and protects cell membranes from oxidation.

Hydrophobic antioxidants are found in lipoproteins and membranes where they provide an important role in interrupting or delaying lipid oxidation. ß-Carotene is an important antioxidant that protects lipid from oxidation. It can scavenge O2˙ and inhibit peroxyl radicals. It acts synergistically with α-tocopherol (vit E), but it can exhibit prooxidant activity under high oxygen pressure (Burton and Ingold 1984). Vitamin E is preferentially incorporated in phospholipid bilayers and is the most effective scavenger of peroxyl radicals (Traber and Kayden 1989). Vitamin E is the main lipid-soluble antioxidant in the cellular system despite its concentration being as low as 1 molecule per 2000 to 3000 lipid molecules. Vitamin E prevents the formation of peroxides and modulates the metabolism of arachidonic acid initiated by lipooxygenase and/or cyclooxygenase. Vitamin E works in coordination with GPx to protect the cells from peroxides by hydrogen atom transfer to ROO˙ that results in the hydroperoxide equivalent ROOH, which then is reduced by the peroxidase. The oxidized tocopherol will be regenerated by ascorbate. However, vit E does not scavenge hydrogen peroxide or superoxide (Niki 2012).

The muscle and other mammalian tissues/fluids contain many compounds that exhibit antioxidant activity (Table 6) some which are considered more powerful than vitamins C and E. For example, the Trolox equivalent antioxidant capacity (TEAC) of various compounds in plasma was in the following order; bilirubin > uric acid > vit C > vit E > glutathione > albumin > tyrosine > cysteine > transferrin (Rice-Evans and Miller 1997).

Other endogenous antioxidants in meat

Vitamins C and E are the endogenous antioxidants most investigated in meat studies. Vitamin C is regarded as a safe antioxidant. However, the ability of vitamin C to maintain the color or improve the color stability of raw meat depends on the method of incorporation into meat. Hood (1975) reported that dipping or spraying meat with 2.5% or 5% ascorbate solution inhibited the formation of MetMb but impaired blooming (the oxygenation process that results in the desirable bright-cherry red color of meat). Preslaughter injection of ascorbate improved the color stability and the shelf life of psoas major (PM), gluteus medius (GM), and semimembranosus (SM), but not longissimus (LL) (Hood 1975). Harbers and others (1981) confirmed that ascorbic acid dipping retarded the formation of MetMb. However, beef steaks dipped in ethanolic (70%) vitamin C solutions exhibited higher MetMb% than controls during cold storage of 3 and 6 d. The opposite effect was noted after 9 d of storage (Okayama and others 1987). Jugular infusion of sodium ascorbate before slaughter extended the shelf life of PM, GM, and LL by reducing the rate of discoloration (Schaefer and others 1995). In conclusion, some researchers have found that the formation of MetMb is retarded in beef treated with vitamin C (Shivas and others 1984; Mitsumoto and others 1991). Others (Benedict and others 1975) have reported a prooxidant effect of ascorbic acid when added to raw ground beef with higher lipid oxidation evidenced. This effect seems to be dependent on concentration, the method of administration, and the shape of the meat. The use of ascorbic acid supplementation as part of the animal diet to improve the oxidative stability of the meat produced has been questioned (Morrissey and others 1998). Direct application of ascorbate to fresh meat is not permitted in some countries.

Vitamin E is considered as a safe supplement. The incorporation of vit E into animal diets is an effective mean to increase the level in the muscles and subsequently in meat (Faustman and others 1989; Table 7 and 8). A proper duration of vitamin E supplementation via diets and the concentration used are very important for maximizing the color stability of fresh meat (Mitsumoto and others 1993). Dietary supplementation with α-tocopheryl acetate is an effective mean to improve the stability of the meat postmortem without exogenous intervention (Faustman and others 1998). Significant improvements in beef color have also been achieved through dietary supplementation with vit E (Faustman and others 1989; Lanari and others 1993, O'Grady and others 2001; Mitsumoto and others 1993). For example, steaks from cattle supplemented with dietary vitamin E exhibited better appearances than controls and were preferred over controls during display by 91% of surveyed Japanese consumers (n = 10941) (Sanders and others 1997). It was found that vit E influenced the length of the MetMb induction period (Sanders and others 1997) and diminished the adverse effect of temperature abuse on color stability (Chan and others 1995). This effect was dependent on dose (the higher the vitamin E supplement the longer the induction period) and muscle type (LL had a longer induction period than the color-labile GM). Faustman and others (1989) and Arnold and others (1993) reported that to gain the maximum protection against discoloration, 3 to 3.5 mg vit E/kg muscle was needed depending on the muscle type. Ponnampalam and others (2012a) recently showed that lipid oxidation was significantly impaired when vit E levels were maintained above 2.95 mg/kg muscle in lamb LL stored at 2 °C for 4 wk before retail display.

Table 8. Effect of diet and vitamin E supplementation on the concentration of vitamin E (α-tocopherol) in the meat
Feed typeMuscleα-tocopherol (μg/g)Source
GrassLongissimus3.42Ponnampalam and others (2012b)
Grain 1.69 
GrassLongissimus4.07bDe la Fuente and
Grain 1.50aothers (2009)
GrassPsoas major2.1bInsani and others
Grain 0.8a(2008)
Grass silageLongissimus1.3Warren and others
Concentrate 3.4(2008b)
GrassLongissimus3.91bRealini and others
Grain 2.92a(2004)
GrainLongissimus1.8aYang and others
Grain + Vit E 4.3b(2002)
Pasture 4.5b 
Pasture + Vit E 4.6b 
GrainSemimembranosus2.0a 
Grain + Vit E 5.3b 
Pasture 4.4b 
Pasture + Vit E 4.3b 
GrainGluteus medius2.4a 
Grain + Vit E 6.0b 
Pasture 5.8b 
Pasture + Vit E 6.1b 
GrainSemimembranosus3.4Formanek and
Grain + Vit E 2.1others. (1998)
GrainLongissimus2.1Eikelenboom and
Grain + Vit E 4.4others (2000)
GrainPsoas major3.2 
Grain + Vit E 8.3 
Mixed-feedLongissimus2.3Lynch and others
Mixed-feed+Vit E 5.4(1999)
Mixed-feedGluteus medius3.1 
Mixed-feed+Vit E 4.4 
Mixed-feedPsoas major4.5 
Mixed-feed+Vit E 6.9 
GrainLongissimus0.8O′ Grady and
Grain + Vit E 2.3others (2001)

The impact of animal feed (pasture, grain, or mixed diet) on the level of vit E in meat has been extensively investigated and has been reviewed by Descalzo and Sancho (2008). Pasture is a rich source of vit E, and meat from animals grazed on pasture contains more vit E than animals fed on grain (Table 8). Vitamin E supplementation successfully increases the level of this antioxidant in the meat, but the level of increase is dependent on the concentration used, the type of basal diet used, and the muscle (Table 8). The level of change in vitamin E is generally higher in animals fed grain + vitamin E (the difference from basal level and the level obtained after treatment).

Other endogenous antioxidants such as carnosine and anserine have been suggested to improve the stability of meat during postmortem storage. For example, Chan and Decker (1994) suggested that the better stability of white fiber muscles against oxidative processes could be due to the higher concentrations of anserine and carnosine in these muscles compared to red muscles. The presence of several muscle compounds such as anserine, carnosine, lactate, NADH, and others (Table 6) have been shown to have various physiological roles including antioxidant activity (Halliwell 1988; Faustman and others 2010). However, their role in postmortem muscle is controversial due to changes in muscle pH that can affect the activity of the compounds (as for carnosine) (Bekhit and others 2003; 2004), and hasten the depletion of others (such as NADH and NADPH) (Bekhit and others 2005).

The antioxidant activity of carnosine has been reported to vary across research laboratories. Carnosine is a dipeptide found in skeletal muscle of most vertebrates at concentrations of 1 to 20 mM (Kohen and others 1988). Several reports demonstrated its antioxidant activity in various model systems (Decker and Faraji 1990; Decker and Crum 1991, 1993; Decker and others 1995; Lee and Hendricks 1997; Lee and others 1998). Carnosine inhibits lipid oxidation in different model systems; however, its activity depends on the catalyst used for oxidation (Kansci and others 1997). Since carnosine is naturally available in the muscle, it was proposed as a beneficial natural food antioxidant (Decker and Faraji 1990; Decker and Crum 1991). Lee and Hendricks (1997) found that the pH range of a meat homogenate should be 7.0 to 8.0 for carnosine to exert antioxidant activity. Carnosine was not effective in protecting beef patties from oxidation (Bekhit and others 2003, 2005) and was not capable of delaying the lipid oxidation in liposomes and an oxygen model system, even at a concentration of 100-fold the effective Trolox concentration (Jongberg and others 2009). Therefore, carnosine is unlikely to exert any protective function in meat.

Pyruvate inhibited lipid oxidation in a microsome model system, but had no effect on the oxidation of oxymyoglobin in a model system (Ramanathan and others 2012), and the addition of pyruvate to minced beef decreased lipid oxidation and improved color stability over 4 d of storage at 2 °C (Ramanathan and others 2012). The authors concluded that pyruvate exerted its protective effects on color through its antioxidant effect on lipids.

Antioxidant elements

Metal ions such as Cu, Zn, Mn, Fe, and Se are cofactors and contribute to active sites of many enzymes. Postmortem protein denaturation (through processing and RS) and the actions of proteases can lead to the release of some of these metals to be freely available for reactions. The oxidative roles of Fe and Cu have been mentioned previously. Zinc and Se can exhibit several biological effects. For example, Zn is a strong inhibitor of calpains and its availability would maintain the cell structure and delay oxidative processes. Zinc can be involved in several biochemical reactions that will affect meat quality. For instance, zinc can bind to Mb and increase the O2 affinity of Mb (oxygenation) (Rifkind and others 1977). Zn2+ also inhibits mitochondrial respiration (Saris and Niva 1994), which diminishes the mitochondrial OCR assisting the maintenance of meat color. Furthermore, zinc has been reported to prevent the formation of ROS through a mechanism that may involve protection of sulfhydryl groups against oxidation (Bettger and O'Dell 1981) and/or displacement of redox transition metals from site-specific loci. Essentially, this means that zinc exerts its antioxidant action by occupying the binding sites of iron and copper available in lipids and proteins (Stohs and Bagchi 1995). In addition, it has been reported that zinc has a synergistic action with a lipid-soluble antioxidant (α-tocopherol) that prevents lipid oxidation (Zago and Oteiza 2001).

Genetic Effects on the Oxidative Processes in Lamb and Beef

  1. Top of page
  2. Abstract
  3. Introduction
  4. Oxidative Processes
  5. Reactive Species
  6. Biological Effects of RS
  7. Defense Systems against Oxidative Processes
  8. Other Endogenous Antioxidants
  9. Genetic Effects on the Oxidative Processes in Lamb and Beef
  10. Factors Influencing the Oxidative Processes of Meat
  11. Measurement of Biochemical Components in Tissues of Living Animal or Postmortem Muscles That is Associated with Oxidative Stress
  12. Biological Functions of Isoprostanes
  13. Factors Affecting the Formation of Isoprostanes
  14. Conclusions
  15. Acknowledgments
  16. References

There is no direct information for cattle and sheep for the impact of genetic background on oxidative stress. The meat industry, seedstock producers, and animal breeders have practiced animal selection for more than a century to improve the animals’ performance targeting certain traits (higher growth rates, leaner meat and better tenderness attributes). In many cases, the selection for these traits influenced the muscle characteristics and thus the quality of meat (for example Hopkins and others 2005). For example, higher proportions of type I myosin and slow-twitch red fibers (high oxidative capacity) were associated with higher growth rates (Renand and others 1995). The selection for leaner animals also leads to a higher proportion of fast-twitch and glycolytic (type IIb) fibers (Renand and others 1995). These changes will definitely have an impact on the meat retail color, tenderization rate, and the oxidative processes of the meat. A recent review by Hopkins and others (2011) provided plausible evidence that supports genotype effects on the meat pH and the amount of unsaturated fatty acids and intramuscular fat, all of which suggest potential different oxidative events. Changes in the IMF and fatty acid composition could result in higher oxidative processes in meat since a strong association (P < 0.001, n = 2052) between the rate of meat discoloration (Oxy/Met) and intramuscular fat (IMF) was reported by Warner and others (2010). Furthermore, genotype effects have been reported for some important nutritional elements that may influence the oxidative changes of the meat postmortem such as iron (prooxidant) and zinc (antioxidant) although further research is needed to confirm the reported observations (Hopkins and others 2011).

Warner and others (2007a) reported significant differences in the color stability of loins obtained from lambs with different genotypes and Border Leicester × Merino] having the same age groups (8, 14, and 22 mo) confirming other reports on these genotypes (Hopkins and Fogarty 1998; Hopkins and others 2007). Knuckles (quadriceps femoris) from Merino sheep exhibited lower color stability compared with the other genotypes (Warner and others 2007a), but this was not the case for loins. The reported genotype effects on different elements, muscle type, and color stability may provide indirect evidence for genetic influence on oxidative processes.

Factors Influencing the Oxidative Processes of Meat

  1. Top of page
  2. Abstract
  3. Introduction
  4. Oxidative Processes
  5. Reactive Species
  6. Biological Effects of RS
  7. Defense Systems against Oxidative Processes
  8. Other Endogenous Antioxidants
  9. Genetic Effects on the Oxidative Processes in Lamb and Beef
  10. Factors Influencing the Oxidative Processes of Meat
  11. Measurement of Biochemical Components in Tissues of Living Animal or Postmortem Muscles That is Associated with Oxidative Stress
  12. Biological Functions of Isoprostanes
  13. Factors Affecting the Formation of Isoprostanes
  14. Conclusions
  15. Acknowledgments
  16. References

Animal nutrition

Animal diet (pasture, grain, or mixed feed) can affect the composition of fat and the amount of vit E deposited in muscle (Table 7 and 8). Cattle finished on a grass diet had higher levels of long-chain omega-3 fatty acid and conjugated linoleic acid (CLA) compared with cattle finished on short-term or long-term grain-based diets (Ponnampalam and others 2006). Generally, higher (CLA) (C18 : 2) isomers, trans-vaccenic acid (C18:1 t11), and n-3 PUFA (g/g fat basis) are found in meat from grass-fed animals compared with other diets (Daley and others 2010). Similar observations were found for precursors for vitamins A and E (Daley and others 2010; Table 7 and 8) and infrequently reported for antioxidant enzymes (Table 7). Increasing the level of n-3 PUFA is commonly perceived as a nutritional advantage for meat (Simopoulos 2002). The inclusion of good sources of fatty acids (such as flaxseed and algae) in a diet can achieve significant increases in the meat (Wood and others 2008; Ponnampalam and others 2012a). The use of different sources of fatty acids can have different effects on the oxidative stability of the meat. For example, the inclusion of fish oil increased lipid oxidation compared to flaxseed (Nute and others 2007) and increased rancid and abnormal flavor (Elmore and others 2000). Ponnampalam and others (2001, 2002) reported that elevated PUFA levels in the meat can be achieved through different feeding regimes (such as the inclusion of 7% fish meal in the feed) without compromising the color or lipid oxidative stability. The high phenolic content (powerful plant antioxidants) in flaxseed may provide some protective effects, but this needs to be confirmed by experimental work. To add to this, a recent study indicated that supplementing lambs, grazing on annual pastures with flaxseed or flaxmeal resulted in a higher blood total antioxidant status than in lambs fed on annual pasture and supplemented with oat grain (Burnett and others 2012).

Protecting the unsaturated fatty acids in the feed from the rumen biohydrogenation, by the addition of formaldehyde, which decrease the protein breakdown by the rumen microorganisms and thus any emulsified PUFA will be protected, can significantly improve the level of PUFAs in muscle tissue (Cooper and others 2004). Grass-feeding not only improves the level n-3 PUFAs, but also provides higher vit E and β-Carotene that protect these PUFAs in the meat postmortem (Descalzo and Sancho 2008; Garcia and others 2008; Warren and others 2008a,b). The influence of diet (pasture compared with concentrate) on the balance of oxidation and antioxidative capacities was evaluated in lamb LL muscle (Santé-Lhoutellier and others 2008b). In this study, samples from pasture-fed lambs had higher vit E content than their concentrate-fed lamb counterparts with a diet displaying no effect on antioxidant enzymes (Santé-Lhoutellier and others 2008b). Meat from pasture-fed lambs has higher MUFA, PUFA, n-3 PUFA, and CLA contents compared to concentrate-fed lambs. Despite the high level of PUFA, lipid oxidation during postmortem storage was inhibited and was significantly lower in pasture-fed lambs compared with concentrate-fed samples due to the protective effects of vitamin E (6.42 compared with 1.61 mg/kg tissue in pasture and concentrate treatments, respectively). Similar results were reported for beef (Maughan and others 2012). Interestingly, this protective effect did not extend to the protein or meat pigments as the carbonyls and a*-values were not different in both treatments over 7 d of storage. Related to this, Najafi and others (2012) have reported that goats fed with a 3% fish oil supplement in the diet compared to palm oil or soybean oil had significantly higher levels of long chain omega-3 fatty acid in muscles. However, in the latter study, the meat color, assessed by L*, a*, and b* values or sensory properties evaluated by flavor intensity, was not affected, although the vit E concentration in meat was not reported. Karamia and others (2013) have recently shown that the inclusion of canola oil into the diet of goats increased blood, muscle, and liver omega-3 fatty acid contents, but lipid oxidation assessed by the levels of TBARS was significantly lowered in blood and muscle.

Rowe and others (2004a) investigated the impact of vitamin E supplementation in concentrate-fed steers (1000 IU per animal/day for 120 d) on the protein oxidation and color stability of LL over 14 d of postmortem storage after irradiation treatment (average of 6.4 kGy). The supplementation increased vit E content in the muscle (4.19 compared with 1.22 mg/kg tissue in supplemented and control treatments, respectively), and this increase in vit E had an effect on protein oxidation as determined by carbonyl assay. The authors provided qualitative evidence using western blots of sarcoplasmic proteins that showed some protective effects against irradiation exerted by vit E. In this study, a*-values of vit E-irradiated samples were significantly lower than a*-values of control-irradiated samples on 3 (out of 5) measurement times, suggesting increased myoglobin oxidation. These results collectively indicate that vit E does not provide enough protection to avoid protein oxidation in meat that is associated with redness of meat during postmortem storage. From a meat color perspective, vit E and heme iron accounted for 70% of the changes in lamb color and the contribution of PUFA to the rate of discoloration was minimal (Ponnampalam and others 2012b).

Age

Age effects on the total fat content and its composition are well established. The total lipids, phospholipid, neutral lipids, and 18:1cis-9 contents of the meat increase with an animal's age (Wood and others 2008). However, the phospholipids and PUFA% decreased in IMF with an increase in age (Warren and others 2008b). Age can also alter the muscle fiber composition. Warner and others (2007b) reported that as an animal age increased (4 to 22 mo), the muscle fibers become more oxidative and the myoglobin content, redness, and fiber cross-section area of LL and semitendinosus (ST) muscles increases, whereas tenderness decreases.

The oxidation of protein is increased with age, and several mechanisms were proposed for the age-related increase in protein oxidation (Stadtman and others 2005; Stadtman 2006):

  1. With the increase of age, a significant reduction in the muscle mass occurs, and the ability to degrade oxidized proteins decreases. This will lead to accumulation of oxidized proteins and promote further oxidative reactions.
  2. Increased RS generation due to changes in the mitochondrial metabolic processes (such as mitochondrial electron transport activity) and/or environmental factors.
  3. Decreased antioxidant capacity.

Xiong and others (2007) investigated the effects of cow age on the oxidative stability of meat during postmortem storage. The authors found no effect of age group (2 to 4, 6 to 8, or 10 to 12 y) on the color stability of patties; however, the effect was muscle-dependent. The authors noted that the animal age-associated susceptibility to lipid oxidation was not dependent on the lipid content of the samples since no significant differences were found among all the muscle samples.

Emerging technologies

Producing meat with consistent tenderness is one of the major objectives for the meat industry's profitability and international competitiveness, and to achieve that goal various technologies are employed (Hopkins 2004; Taylor and Hopkins 2011). Many of these technologies rely on manipulating the shape (through compression and stretching), applying pressure (hydrodyne/shock wave, ultrasound and high hydrostatic pressure), applying force (blade/needle tenderization, flaking, mincing) or physical stimulus (electrical stimulation of carcasses, accelerated conditioning (temperature), freeze-thaw cycles, all of which will affect the cellular system in different ways in postmortem muscles. For example, the structural changes occurring during freeze-thaw, needling/blade treatment will lead to direct contact between enzymes, catalyst (such as iron), and substrates (for example, phospholipids, PUFA, myoglobin) all of which can lead to increased oxidative reactions. Freeze-thawing of muscles increases lipid oxidation (Benjakul and Bauer 2001) and promotes protein oxidation (Xia and others 2009).

A recent review on high-pressure treatment of meat (Simonin and others 2012) concluded that this treatment at >300 MPa increases lipid and protein oxidation. The authors suggested the release of iron as the mechanism for increased oxidation. Another possible mechanism is the biological action of Ca2+ that is released during the process as a result of the physical disruption of the sarcoplasmic reticulum (Okamoto and others 1995). Ca2+ stimulates the activity of lipoxygenase (St. Angelo and others 1991) and stimulates the mitochondrial respiration process (Carafoli and Gazzotti 1970) that will lead to an increase in the oxidative processes. A rapid increase in Ca2+ can lead to loss of glutathione (Anderson and Sims 2002) and inactivation of GPx and glutathione reductase (Zoccarato and others 2004) that will lead to increased ROS emission. Interestingly, high-pressure treatment up to 300 MPa increases the redness of meat (a*-values), and the redness is affected negatively at pressure above that level (Simonin and others 2012). Other technologies that rely on cellular disintegration (such as pulsed electric field and ultrasound) may cause similar effects, but optimization of the process can minimize any deleterious side effects. For example, ultrasound treatment of beef can significantly affect (Pohlman and others 1997), slightly affect (Stadnik and Dolatowski 2011), or have no effect (Jayasooriya and others 2007) on the oxidative processes of meat depending on the treatment intensity.

Measurement of Biochemical Components in Tissues of Living Animal or Postmortem Muscles That is Associated with Oxidative Stress

  1. Top of page
  2. Abstract
  3. Introduction
  4. Oxidative Processes
  5. Reactive Species
  6. Biological Effects of RS
  7. Defense Systems against Oxidative Processes
  8. Other Endogenous Antioxidants
  9. Genetic Effects on the Oxidative Processes in Lamb and Beef
  10. Factors Influencing the Oxidative Processes of Meat
  11. Measurement of Biochemical Components in Tissues of Living Animal or Postmortem Muscles That is Associated with Oxidative Stress
  12. Biological Functions of Isoprostanes
  13. Factors Affecting the Formation of Isoprostanes
  14. Conclusions
  15. Acknowledgments
  16. References

Antioxidant measurements

To determine the biological efficacy of antioxidant compounds, 3 main test systems (test with subcellular fractions/chemical reactions; tests with intact cells; and human trials) have been used in fields of medicine and nutrition (Hoelzl and others 2005). These systems can provide different levels of information such as actual effects on intracellular tissues (including DNA) and circulatory (plasma) components, time kinetics, and distribution in tissue. Given the wide range of antioxidants either available in vivo or by supplementation to improve the antioxidant capacity of the muscle/meat, a wide range of methods have been used to quantify the antioxidants (Table 9). Generally speaking, the methods can be classified as those that measure dietary antioxidants (such as Vit E and Vit C); endogenous antioxidants (antioxidant enzymes, bilirubin, GSH, and uric acid), or total antioxidant activity/capacity. The latter are groups of assays that determine the net balance of antioxidants and prooxidants present in an animal system or tissues that is presented as the total antioxidant/reducing capacity. Many of these methods have critically been evaluated and several reviews on the topic are available that describe the mechanisms of action, and the advantages and disadvantages of the assays (Ghisellia and others 2000; Sánchez-Moreno 2002; Huang and others 2005; Prior and others 2005; Roginsky and Lissi 2005; Somogyi and others 2007; Karadag and others 2009). Most of these methods challenge the antioxidant's availability in the system by generating a free radical which will vary depending on the assay. While all the assays can provide some useful information, the nature of the free-radical and the mechanism of action have been criticized since in many cases, there is no physiological relevance (Sánchez-Moreno 2002; Huang and others 2005; Roginsky and Lissi 2005) and, in many cases, the lack of standardization of the assays does not allow proper evaluation of studies from various investigators (Prior and others 2005).

Table 9. Measurements of antioxidants
SystemAntioxidantsMethod of measurementReference
Dietary antioxidantsVitamin EHPLC, Spectrophotometric, ELISABarua and others (1993); Nilsson and others (1978); Tütem and others (1997)
 Vitamin CHPLC; Titration; SpectrophotometricOmaye and others (1979)
 Carotenoids (such as α- and β-carotene, lycopene, lutein, zeaxanthin, and β-cryptoxanthin)HPLC; Resonance Raman SpectroscopyBarua and others (1993); Darvin and others (2005)
 Flavonoids (such as quercetin, myristin, phloridzin, and catechins)HPLC; SpectrophotometricErlund and others (1999); Oomah and others (1996)
Endogenous antioxidantsSODHPLC; SpectrophotometricAbbe (1986); Kakkar and others (1984)
 Catalase (CAT)SpectrophotometricAebi (1984) Slaughter and O'Brien (2000)
 Glutathione peroxidase (GSHPx) enzymesspectrophotometricWendel (1981)
 Glutathione (GSH),Spectrophotometric; HPLCCereser and others (2001); Akerboom and Sies (1981); Ellman (1959)
 Glutathione-S-transferase (GST)SpectrophotometricHabig and others (1974)
 Plasma proteins such as albumin, ceruloplasmin, transferrin and bilirubinSpectrophotomertic; HPLC; RIAHeirwegh and Blanckaert (1981); Arnaud and others (1988); Tonik and Sussman (1987) Duly and others (2003) Infusino and others (2010)
 Uric acidColorometric, fluorometric;HPLCElsayed and others (1993); Hochstein and others (1984)
Total antioxidant capacityRandox Koracevic and others (2001)
 PLC (phytochemoluminescence) assay Wood and others (2006)
 TEAC (2,2′-azino-bis/3-ehylbenzthioazoline-6-sulfonic acid radical ABTS+/Metmyoglobin) Robak and Gryglewski (1988)
 TOSCA (total antioxidant scavenging assay)  
 Oxygen radical absorbance capacity assay (ORAC)  
 2,4,6-tri(2-pyridyl)-s-triazine  
 (TPTZ) and 1,1-diphenyl-2-picrylhydrazyl (DPPH˙) scavenging activity 
 Peroxyl radical Scavenging (trichlormethyl peroxyl or alkoxyl peroxyl radical  
 Ferric reducing ability (FRAP)  
 Superoxide anion (O2˙) scavenging activity  
 TBARSs  
 Total radical-trapping antioxidant parameter (TRAP) Pavelkova and Kubala (2004)

The technical procedures to perform the methods, including high-performance liquid chromatography (HPLC), gas chromatography (GC), spectrophotometry, and commercial kits, all have several advantages and disadvantages. HPLC and GC techniques are very accurate, rapid and sensitive techniques that can be customized to operate at high throughput. However, the equipment is expensive to purchase and to run, and it requires some expertise and technical skills which makes these techniques unaffordable to small and medium-size companies or laboratories. The analysis involves time-consuming extraction procedures that render the extracts very unstable ex vivo and special protective steps during the processing, extraction, handling, and storage of the extracts need to be in place. Information generated from extracts, especially those targeting a single molecule, normally do not provide the actual in vivo kinetics of antioxidants and their real effects in tissues. Spectrophotometer measurements and commercial kits are relatively inexpensive and do not require the skills needed in HPLC and GC techniques, but the same level of care for handling the samples is required. Many of these assays have been used in meat research (Savary-Auzeloux and others 2008; Bekhit and others 2007a; Wu and others 2008; Tansawat and others 2013; Table 7a and 8) and various conclusions have been reached. The sample preparation method influences the measured antioxidant activity very much (Wu and others 2008), and the use of intact meat tissue, such as meat cores, is recommended (Bekhit and others 2007a) since the integrity of the cells remains uncompromised.

Prooxidant measurements

The prooxidants, also known as oxidative stress, assays are procedures that quantify the generation rates of RS (Table 10) or the effect of RS actions (hydroperoxide, volatiles, carbonyls, and isoprostane). The generation of RS or oxidation products is higher when the animal's antioxidant defense system is low or fails to cope with damage caused by oxidation and the production of RS. This is translated into altered or damaged biological materials such as DNA, lipids, and protein, which all can be used as indicators of oxidative stress. Many assays are available that cover a wide range of biological materials in their different forms (extract, homogenate, or tissue) and these methods vary in their technical skills requirement and cost associated with subsequent analysis (Table 10). In some instances, the assay can provide an indication of the antioxidant capacity of animals or tissues (for example, the lag phase in the fluorescence production in DCFH assay) that can be useful when comparing tissues of animals have been under different conditions. Clearly, any steps used in extracting or homogenizing the samples can lead to loss of information or can alter the actual measurements. Therefore, sample handling and processing need to be evaluated and critical control procedures (CCPs) need to be in place in order to eliminate possible sources of interference.

Table 10. Different methods for the measurements of prooxidant activity and oxidative processes
AssaySystem usedMaterialUnits of measurementSampleReference
2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA)FluorescentBrainresults expressed as pmol DCFHomogenateDriver and others (2000)
 Ex485 nm, Em 530 nmMuscleformed/mg protein/minIntact tissueBekhit and others (2007b)
TBARSSpectrophotometric (532nm) HPLCVarious biologicalnmol MDA/g tissueHomogenateSingh and others (2009)
 • Wakosil, C18 column and an SGE C18, or Phenomenex Gemini columnPlasmanmol MDA/min/mg protein Jardine and others (2002)
 • Fluorescence Ex 525 nm, Em 560 nm    
Lucigenin enhanced chemiluminescenceN/AOrgansChemiluminescence intensity (Arb. Units)HomogenateIntact tissueSeljeskog and others (2006)Matveeva and others (2007)
CrocinUV/Vis (443 nm, 40 °C)Various biologicalThe number of crocin moles consumedExtractsManzocco and others (2002)
OH˙(PMS-NADPH)UV/Vis (224 nm)Liverng/mg proteinMicrosomesCederbaum and Cohen (1984)
Autofluoresence spectroscopy400–700 nmTissueTissueWold and Mielnik (2000)
Electron paramagnetic resonance spin trappingVarious  Villamena and Zweier (2004)
Hydroperoxide measurements     
FOX (ferrous ion oxidation xylenol orange)UV/Vis (560 nm)VariousΔLOOH (μM)/180 minExtractsNourooz-Zadeh (1999)
     Kanner and Lapidot (2001)
HPLC (Eurospher-100 Si column)UV/Vis (234 nm)VariousLOOH (μM)Extracts 
Conjugated dienesUV/Vis (233 nm)VariousmL/mg sampleExtractsAndreo and others (2003)
Peroxide valueEnd point reactionVariousMilliequivalents (meq) of peroxide per kilogram for sampleExtracts 
GC-MSRtx MS fused-silica capillary column (5% phenyl, 95% dimethyl polysiloxane); 5% SE 30Plasmaμg/mLPlasmaBertucci and others (2002)
 on gas-chrom Q (80 to 100 mesh) column m/z 155 and 169 m/z 2255, 295, and 335Livernmol/gExtractHughes and others (1986)
HPLC436 nm (Nacalai Tesque Cosmosil5 C18 column)Variousng/μLFatty acids in model systemWu and Lin (1995)
 254 nm (technopak C18 reversed-phase column) nmol/mLHuman serumKaratas and others (2002)
Lipid Volatiles (GC-MS, LC-MS or PTRMS)VariousVarious ExtractsFrank and others (2012)
     Ross and Smith (2006)
Comet assay (DNA damage)Horizontal gel electrophoresis Basic DNA damage (without FPG treatment) and total DNA damageBloodWeisel and others (2006)
Cupric ion reducing ability450 nmVariousTrolox equivalent (μg/mL)ExtractGǘlçįn (2008).
DMPD˙+ radical scavenging activity505 nmVariousTrolox equivalent (μg/mL)Extract 
Oxidation isElectrochemical detectorDNA from tissue8-OHdG (ng/mL)ExtractLoft and others (1992)
8-hydroxydeoxyguanosineHPLC (Spherisorb ODS2)   Subash and others (2010)
 ELISA kit    
Modified tyrosinesELISAExhaled breath condensate  Jones and others (2000)
 GC-MS a DB5-MS column m/z 582 and 576   Lärstad, and others (2005)
Protein carbonylsSpectrophotometric 370 nm Nanomoles of carbonyl per milligram of proteinExtractEstévez and others (2009)
DNPHScan range of m/z 100 to 700. Luna reverse-phase columnVarious   
LC ESI- MS     
IsoprostanesGC-MS, HPLC-MS, ELISA, RIAVarious ExtractSee below

Protein and lipid modification in tissues caused by RS generates aldehyde and ketone derivatives and the measurement of the carbonyl groups is regarded as an important marker for protein or lipid oxidation. There are several spectrophotometric, immunochemical (Rimbach and others 1999), and LC-MS (Estévez and others 2009) methods that vary in their sensitivity and ability to identify individual carbonylated proteins. In meat quality research, fluorescent light produced during storage (Renerre and others 1996; Morzel and others 2006; Chelh and others 2007) and during cooking (Gatellier and others 2009) can be used as a good indicator for meat oxidative reactions. To measure this fluorescence, the samples are homogenized and separated into aqueous phase (phosphate buffer 20 mM, NaCl 100 mM buffer solution at pH 6) and organic solvent phase (dichloromethane : ethanol containing butylated hydroxytoluene added as antioxidant). The fluorescence of these 2 phases is then measured using a spectrofluorometer at excitation wavelength of 360 nm and emission wavelength 390 to 600 nm (Gatellier and others 2009). This fluorescence is produced as a result of the interactions between proteins and aldehydes or sugars and has been proposed as a marker for intermolecular cross-linking and polymerization of proteins. Volatiles produced as end products of lipid oxidation [for example, 4-hydroxy-2-nonenal (4-HNE)] are determined using various methods (HPLC, GC, and ELISA), but rapid and highly accurate methods such as proton-transfer-reaction mass spectrometry (PTR-MS) are currently used for the measurements of lipid oxidation products (Frank and others 2012). The extremely high cost of these instruments makes them unsuitable for commercial analysis.

Currently, there is no methodology available to measure the oxidative status of animals on a farm and to relate this to the quality of muscles or muscle foods. Prediction of oxidative status in live animals may provide an opportunity to improve animal productivity and profitability through on-farm nutritional management. Therefore, the development of nondestructive techniques is an important priority (Wold and Mielnik 2000). Techniques using tissues or blood (for example, chemiluminescence and DCFH-DA) rather than extracts or homogenates would be beneficial (Bekhit and others 2007b; Matveeva and others 2007). One of the most important markers used for oxidative stress in human and laboratory animals is isoprostanes. The use of isoprotanes and its biological functions will be discussed herein.

Isoprostanes, their biochemistry, and potential use as markers in live animals

The medical need to evaluate oxidative stress in vivo without the use of invasive procedures has led to the discovery of the marker isoprostane (Morrow and others 1990; Morrow 2000). Isoprostanes are prostaglandin F-like compounds (formed via actions of cyclooxygenases, COXs) that are produced by direct free radical development of lipid oxidation and other disorders, and their formation is not dependent on COXs activity (Roberts and Milne 2009). Many of the methods that are traditionally used for the evaluation of oxidative stress in vivo are deficient in several important aspects, such as specificity and/or sensitivity, which make them unreliable (Wood and others 2006). Isoprostanes are regarded as the “gold standard” and “the most reliable” marker of oxidative stress in humans (Morrow 2005; Lykkesfeldt and Svendsen 2007). The formation of isoprostane (specifically 8-iso-PGF, also known as 15-F2t- isoprostane) was suggested as an indirect marker for in vivo lipid oxidation (Morrow 2000; Roberts and Milne 2009; Halliwell and Lee 2010) due to its abundant production. These compounds have the merit of being specific products of lipid oxidation and are relatively stable compounds that provide information about the oxidative status of an individual. This compound is found in body fluids such as blood and urine at detectable levels, and therefore, invasive procedures are not required for measurement (Morrow 2000 2005).

An extensive list of disorders where isoprostanes were used as a marker for oxidative stress was reported by Dalle-Donne and others (2006) and the authors reported that 41 disorders were found to produce high F2-isoprostane (F2-isop) levels. Deficiency in important substances in the body due to an unbalanced diet (for example, α-tocopherol, selenium), aging, or exposure to toxins can increase the levels of F2-isop, see Nikolaidis and others (2011) for a summary of the literature. The production of isoprostane in various organs or biological fluids varies with the clearance of this compound from the tissue system and migration to other body parts in the circulatory system. The fact that higher than normal levels of isoprostane are found along with a wide range of human diseases (diabetes, obesity, smoking, and atherosclerosis among others, Table 11) and that treatments with antioxidants can reduce the levels of F2-isoprostane supports their use as a biomarker for oxidative stress in animals in vivo (Praticó and others 1998b; Cracowski and Ormezzano 2004; Roberts and Milne 2009). F2-isops are widely distributed in the body and their measurements have been reported in several biological fluids (plasma, urine, exhaled breath condensate, saliva, bile, cerebrospinal, seminal, and pericardial fluids) and tissues (including skeletal muscle) (Nikolaidis and others 2011; Table 12). Urine is the most common biological fluid of humans used for the detection of isoprostanes (Roberts and Milne 2009). However, collection of urine on-farm or at slaughter in from live animals is not feasible. In this manner, the collection of blood for the determination of F2-isoprostane, as a marker for oxidative stress, is a viable pathway. While most studies have reported a significant relationship between the detection of F2-isoprostane and pathological damage/disorders, some studies have reported no such relationship. This is attributed to various experimental (handling of the sample, method used for measurements, or level/type of intervention used) and biological factors (material used for the detection) (Table 12 and 13).

Table 11. Model systems used for the evaluation of isoprostane as a biomarker
Disorder/modelUrinePlasmaTissueCerebrospinal fluidModelRemarksReference
  1.  ✗ = relationship was not established. ✓ = relationship was established.

Alzheimer's disease (AD) or Parkinson's disease (PD) Mouse[DOWNWARDS ARROW] with Vit EPraticó and others (1998a)
   Human Mufson and Leurgans (2010)
    Human Montine and others (2011)
   Human Connolly and others (2008)
    HumanTotal NeuroP but not total IsoP, levels are greater in AD patients than controls.Reich and others (2001)
    Human Montine and others (2007)
    Human Feillet-Coudray and others (1999)
   Human Connolly and others (2008)
   Human Praticó and others (2000).
   Human Montine and others (2000)
    Human Montine and others (2002)
Chronic obstructive pulmonary disease (COPD)   Human Praticó and others (1998b)
Antiphospholipid syndrome (APS)   Human[DOWNWARDS ARROW] with Vit E and CPraticó and others (1999)
Oxidative stress with CCl4  Rats Kadiiska and others (2005a,b)
Atherosclerosis   Human Gniwotta and others (1997)
Coronary heart disease   Human Schwedhelm and others (2004)
Exercise   Human Rietjens and others (2007)
    Human McAnulty and others (2007)
   muscle✓ Human Karamouzis and others (2004)
    Human Campbell and others (2010); Schmitz and others (2008), Wang and others (2000), Galassetti and others (2006)
  No change  Human Mice Kelly and others (2007), Moien-Afshari and others (2008), Watson and others (2005)
Asthma   breath✓Human Carraro and others (2010)
Table 12. Effect of antioxidants on F2-isoprostane levels in human body fluids
SubjectsInterventionPeriod of treatmentChangeSampleSystemReference
  1. Enzyme-linked immunosorbent assays = ELISA; enzyme immunoassay kit = EIA; radioimmunoassay = RIA.

  2. Dissociation Enhanced Lanthanide Fluoro Immuno Assay = DELFIA.

Healthy subjects4 g/day Vit C2 wk[DOWNWARDS ARROW]37% in esterified F2-IsopPlasmaGC-MSMorrow and others (1999)
 3200 IU/day Vit E     
 300 mg/d β-carotene     
 400 IU/day α-tocopherol2 wk[DOWNWARDS ARROW]35% in unesterified F2-Isop   
 800 IU/day α-tocopherol2 wk[DOWNWARDS ARROW]37% in unesterified F2-Isop   
 400 IU/d α-tocopherol8 wk[DOWNWARDS ARROW] F2-Isop. The combination was no better than individual compoundsUrineEIAMarangon and others (1999)
 600 mg/d lipoic acid8 wk    
 366 mg/kg phenolics3 wkNo effectPlasmaHPLC-ESI-MS-MSCovas and others (2006a)
 164 mg/kg     
 2.7 mg/kg     
 366 mg/kg phenolicsMeasured after 2, 4, and 6 h[UPWARDS ARROW]4.9%, 11.6%, and 16.5% after 2, 4, and 6 hPlasmaHPLC-ESI-MS-MSCovas and others (2006b)
 164 mg/kg [UPWARDS ARROW]11.9%, 14.2%, and 23.8% after 2, 4, and 6 h   
 2.7 mg/kg [UPWARDS ARROW]5.6%, 21.3%, and 29.9% after 2, 4, and 6 h   
 High-vegetable diet2 wk[DOWNWARDS ARROW] 33% (pooled from 3 d)UrineELISAThompson and others (2005)
 Twice daily6 wkNo effect over 48 h measurementUrineDELFIABailey and others (2011)
 400 mg Vit C + 268 mg Vit E + 2 mg vitamin B6 + 200 μg vitamin B9 + 5 μg zinc sulfate monohydrate + 1 μg Vitamin B12     
 500 mg Vit C + 400 IU Vit E/day4 wk[DOWNWARDS ARROW] 60% (after 3 and 4 h of exercise only)PlasmaRIAFischer and others (2004)
 Fruit and Veg diet12 wk[DOWNWARDS ARROW] 6.8% (between <2 and 2)PlasmaGC-MSRoot and others (2012)
 < 2 serving [DOWNWARDS ARROW] 10% (between 2 and > 2)   
 2 serving     
 > 2 serving     
 Normal diet3 wkNormal diet = low-antioxidant diet,PlasmaEIACornelli and others (2011)
 [DOWNWARDS ARROW] antioxidant diet but [DOWNWARDS ARROW] ≈ 58% in high-antioxidant   
 [UPWARDS ARROW] antioxidant diet diet   
 Phenolics in olive oil (50 mL serve)Consumption and[DOWNWARDS ARROW] 16% with B over AUrineEIAVisioli and others (2000)
 D (1950 mg/L)C (1462 mg/L)B (975 mg/L)A (487.5 mg/L)sample collected 24 hRepeated 4 times after 1 mo wash-out period[DOWNWARDS ARROW] 34% with C over A[DOWNWARDS ARROW] 33% with D over A   
 Anthocyanin/polyphenolic-rich fruit juice (700 ml/day)4 wk[DOWNWARDS ARROW] but not significantUrineGC-MSWeisel and others (2006)
 High flavonoid2 wkNo effectPlasmaGC-MSO'Reilly and others (2001)
 Low flavonoids     
 Low-fat (15% energy from fat)12 mo[DOWNWARDS ARROW] ≈22% in low-fat onlyPlasmaEIAChen and others (2004)
 High fruit and vegetables 9 serves/day     
 Low-fat/high fruit and vegetables (control)     
 Decaffeinated green tea2 wkNo effectUrineGC-MSDonovan and others (2005)
 Light beer4 wk[DOWNWARDS ARROW] 14% in plasma andUrineGC-MSBarden and others (2007)
 Normal beer [DOWNWARDS ARROW] 16% in urine with low alcohol. Only significant in plasma.Plasma  
 Garlic pearls (hypertensive patients compared with healthy control)2 with each meal8 wk[DOWNWARDS ARROW] 31% in plasma and[DOWNWARDS ARROW] 38% in urine in Hyper. No change in control.UrinePlasmaELISADhawan and Jain (2004)
 Vitamin C (2 g single oral dose)Measurement over 8 hNo effectUrineGC-MSKelly and others (2008)
  after consumption Plasma  
 Dark chocolateMeasurement over several stages of exercise.[DOWNWARDS ARROW] in plasma at exhaustion and after 1 h of recoveryPlasmaELISAAllgrove and others (2011); Davison and others (2012)
 Dark soy sauce (30 mL single oral dose)Measurement over 4 h after consumption[DOWNWARDS ARROW] but significant only after 3 hUrineGC-MSLee and others (2006)
    Plasma  
 Cyclosporin A6 wkNo effectUrineGC-MSBarany and others (2001)
 Vitamin E (800 IU/d)  Plasma  
 Tomato sauce (150 g single oral dose)Measurements over 48 h[DOWNWARDS ARROW] 38% in urine at 48 h onlyUrineGC-MSLee and others (2009)
    Plasma  
 Vitamin C (30 to 2500 mg/d)186 ±28 dNo effectUrinePlasmaGC-MSLevine and others (2001)
 Quercetin (200 mg, single oral dose)Plasma (2 h post-treat)No effectUrinePlasmaGC-MSLoke and others (2008)
 Epicatechin (200 mg single oral dose)Urine (5 h post-treat)    
 Epigallocatechin gallate (200 mg single oral dose)     
Diabetic patients600 mg/d Vit E2 wk[DOWNWARDS ARROW] 37%UrineRIADavì and others (1999)
Smokers100 or 800 U/d Vit E,5 d[DOWNWARDS ARROW] 29% with C,UrineGC-MSReilly and others (1996)
 2 g/ Vit C [DOWNWARDS ARROW] 22% with the combination,   
 Combinations of both No effect of E alone   
 1 g/day Vit C2 mo[DOWNWARDS ARROW] 10.6 Vit CPlasmaGC-MSBlock and others (2008)
 800 IU/d Vit E [DOWNWARDS ARROW] 3.9% Vit E   
 Control [UPWARDS ARROW] control   
 300 mg/d Vit E3 wkNo effectUrineRIAPatrignani and others (2000)
 600 mg/d Vit E     
 1200 mg/d Vit E     
 A) 375 mL (13.3% alc/vol,1200 mg/l polyphenols)2 wk[DOWNWARDS ARROW] 20% with C (plasma)[DOWNWARDS ARROW] 6% with C (urine)PlasmaUrineGC-MSAbu–Amsha and others (2001)
 B) 375 mL (13.7% alc/vol, 345 mg/L polyphenols)     
 C) 500 mL (<2% alc/vol, 905 mg/L polyphenols)     
 Aged garlic extract (5 mL/d)2 wk[DOWNWARDS ARROW] 35% and 48% with smokers in plasma and urinePlasmaUrineEIA kitDillon and others (2002)
   [DOWNWARDS ARROW] 29% and 37% with nonsmokers plasma and urine   
Pregnant atopic womenFish oil20 wkFish oil compared with olive oilPlasmaGC-MSBarden and others (2004)
 Olive oil [DOWNWARDS ARROW] 25 plasmaUrine of offspring  
   [DOWNWARDS ARROW] 13 in urine (not significant)   
 515 mg/d Vit C2 mo[DOWNWARDS ARROW] 11%PlasmaGC-MSDietrich and others (2002)
 515 mg Vit C+ 95 mg Lipoic acid+ 794 mg Vit E/day [DOWNWARDS ARROW] 3%   
Hypercholesterolemic100 mg/day Vit E2 wk[DOWNWARDS ARROW] 34% to 36%UrineRIADavi and others (1997)
patients600 mg/d Vit E [DOWNWARDS ARROW] 47% to 58%   
 Soy isoflavones (50 mg/1000 kcal)42 dInconclusiveUrineEIAVega-López and others (2005)
 Phenolics 400 mg/kgSamples obtained[DOWNWARDS ARROW] 30% with high phenolicsPlasmaEIARuano and others (2005)
 80 mg/kgafter consumption over 4 h    
Patients with900 IU/d Vit E4 wk[DOWNWARDS ARROW]25% to 83%UrineGC-MSPraticò and others (1999)
antiphospholipid2 g/d Vit C     
antibodiesBoth     
Patients with liver cirrhosis300 mg Vit E twice/day30 d[DOWNWARDS ARROW] 51%UrineGC-MSFerro and others (1999)
Patients with chronic alcoholic liver disease2.5 g Vit C10 d[DOWNWARDS ARROW] 50%UrineGC-MSMeagher and others (1999)
Breast cancer survivorsFresh carrot juice3 wkNo changeUrineELISAButalla and others (2012)
Table 13. Measurements of isoprostane as a biomarker in animals
SubjectsInterventionPeriodChangeSampleSystemReference
  1. *Acceptable agreement but for some species GC-MS is recommended.

Sprague– Dawley ratsZn-adequate (50 mg/kg of diet)Zn-deficient (<0.05 mg/kg of diet)3 wkNo change in Vit E and C[UPWARDS ARROW] isopro in Zn-deficient[DOWNWARDS ARROW] 50% uric acidPlasma, LiverHPLCBruno and others (2007)
 Vitamin E (20/kg diet)3 wk[DOWNWARDS ARROW] 40% in urine, [DOWNWARDS ARROW]60% in liver. No change in plasmaUrine, Plasma, LiverRIASodergren and others (2000; 2001)
 Wine6 h[DOWNWARDS ARROW] 100% in urine for wine group compared to control. No change in PlasmaPlasma UrineEIARodrigo and others (2004)
SheepSelenium without or with 2.5 mg selenium/kg as sodium selenite (high and low GSH)8 wk[DOWNWARDS ARROW] 37% in High GSH animals onlyPlasmaGC-MSLiu and others (2010)
CatsObese compared with leanNatural weightsOver weight cats had twice the level of lean catsUrineEIAJeusette and others (2009)
DogsHealthy compared with ill[UPWARDS ARROW] in ill dogsUrineELISAMcMichael and others (2006)
Various animalsHealthy compared with ill[UPWARDS ARROW] in animals exposed to adverse conditions*UrineELISA and GC-MSSoffler and others (2010)
HorsesColic compared with healthyColic > healthyUrineGC-MSNoschka and others (2011)

Biological Functions of Isoprostanes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Oxidative Processes
  5. Reactive Species
  6. Biological Effects of RS
  7. Defense Systems against Oxidative Processes
  8. Other Endogenous Antioxidants
  9. Genetic Effects on the Oxidative Processes in Lamb and Beef
  10. Factors Influencing the Oxidative Processes of Meat
  11. Measurement of Biochemical Components in Tissues of Living Animal or Postmortem Muscles That is Associated with Oxidative Stress
  12. Biological Functions of Isoprostanes
  13. Factors Affecting the Formation of Isoprostanes
  14. Conclusions
  15. Acknowledgments
  16. References

Several biological activities have been assigned to isoprostane (15-F2t-isop). For example, it has been demonstrated that vasoconstrictor activity, enhanced count of monocytes, and polymorphonuclear adhesion to endothelial cells, stimulated mutagenesis and induced endothelial cell necrosis (reviewed by Roberts and Milne 2009).

Formation of isoprostanes

The formation of isoprostanes is initiated by the oxidation of arachidonic acid mediated by free radical attack followed by several steps of oxygen insertion and cyclization (Roberts and Milne 2009). This will generate a complex mixture of 64 enantiomers that are grouped into 4 regioisomeric families of prostanes (see Morrow 2000, 2005) that are commonly referred to as F2-isop 5-, 8-, 12-, or 15-series (see Morrow 2000, 2005; Roberts and Milne 2009). The series-naming refers to the carbon atom position where the hydroxyl side chain is attached with series F2-isop 5-, 8-, and 12-, or 15- derived from the attack at C7, C10, and C13, respectively. The isoprostane 8-epi-PGF2α (aka 8-iso-PGF2α or 15-F2t-isoprostane) is the most studied member of the isoprostanes.

The degradation of components such as lipids, protein, or DNA in vivo can lead to the formation of several compounds that can be used as markers for oxidative stress. For example, the formation of isoprostanes and isofurans from arachidonic acid (F2-isoprostanes), and neuroprostanes (F4-neuroIsop) from docosahexaenoic acid (DHA) that can be present in different isoforms (Montine and others 2004; Roberts and Milne 2009). Isoprostanes can be generated from peroxidation of linolenic acid (F1-isop) and eicosapentaenoic acid (F3-isop) also (Roberts and Milne 2009); however, this is dependent on the antioxidant status of the animal. The majority of the isoprostanes detected in plasma and urine samples are the product of nonenzymatic oxidation of arachidonic acid via RS attack (Roberts and Milne 2009; Halliwell and Lee 2010). The oxidation products are designated as A, E, D, or J that will have a number reflecting the origin of the compounds. For example, those from arachidonic (F2-isop) will be A2-, E2-, D2, and J2-isop. Roberts and Milne (2009) suggested that derived oxidation products from different fatty acids can potentially have different functions. These authors cited a study where EPA-derived isop (15-F3t-isop) demonstrated different activity from the equivalent AA-derived (15-F2t-isop) and the supplementation of EPA reduced F2-isop in mouse heart tissues by 60%. Similarly, the supplementation of EPA and DHA to subjects reduced levels of 8-iso-PGF to their baseline levels (Higdon and others 2000; Barden and others 2004; Mas and others 2010). The latter suggests that the supplementation of EPA and/or DHA may reduce the oxidative stress in animals by lowering the F2-isoprostane levels in the circulatory systems or body. Research indicated that plasma isop concentrations were not affected by the consumption of high-fat meals (Richelle and others 1999; Gopaul and others 2000); however, the type of fatty acid may be more important in modulating isoprostanes in plasma (Higdon and others 2000; Barden and others 2004; Iannone and others 2009; Mas and others 2010).

Measurement of F2-Isoprostane

As mentioned above, several biological fluids and tissues can be used for the measurement of F2-Isop (Table 11 to 13). The most common methods for the quantification of isoprostanes are: GC-MS, LC-MS, enzyme immunoassay (EIA), and ELISA (Table 13). The commercial kits are simple, cheap, and could be used for the rapid screening of animals under realistic commercial conditions. On the other hand, GC-MS and LC-MS methods are very sensitive (picogram range) and can detect several isomeric forms of isoprostanes, but they are laborious, very expensive, require high technical skills, and are time-consuming that will be a significant hurdle for application to the commercial screening of animals. There are several modified techniques that use various GC-MS methodologies. The different methods have been reviewed elsewhere (Morrow 2000; 2005; Roberts and Milne 2009; Halliwell and Lee 2010) and will not be covered here. Modifications of the GC-MS measurement system by Morrow and others (1992) (namely, separation of isops via solid-phase extraction or affinity chromatography with or without the aid of a thin-layer chromatography followed by final quantification of the compounds of interest with spectrometric techniques such as GC–MS, HPLC-MS, or tandem MS) have been reported to cause the comigration of various types of isoprostanes and can potentially produce different results (Montine and others 2007). Therefore, even with the same technique, there is a need for validation of methods. A clear advantage of LCMS-based methods is that they are regarded as a simpler analysis technique due to sample preparation not requiring derivatization of the molecule before analysis unlike the GC-MS technique. There is a strong correlation between EIA and the GC-MS results (Wang and others 1995; Devaraj and others 2001; Wood and others 2005, 2006; Carro and others 2010). A comparison between EIA and gas chromatography (electron capture)/negative ion mass spectrometry (GC-ECNI-MS) showed that both assays provide good relative values (r = 0.91) among samples, but the absolute values were different with higher values found using EIA (Yeoh-Ellerton and Stacey 2003). However, other studies found no or low correlation coefficients (Proudfoot and others 1999; Bessard and others 2001; Saenger and others 2007; Soffler and others 2010) that suggest a cross-validation may be required for the same set of samples (Morrow 2000).

The measurement of F2-isop in several animals has been investigated with the aim of verifying the accuracy of the compound as a biomarker (Table 13). Generally, the measurements of isoprostane were able to distinguish between animals that had been exposed to toxic prooxidants, underwent surgery, were obese, or were deficient in important nutrients (Table 13). Therefore, some authors advocate the use of F2-isop as a prognostic indicator (Noschka and others 2011). The majority of these studies used commercial kits that are promising for the effective screening of large numbers of farm animals. However, caution should be exercised as a recent study (Soffler and others 2010) compared the determination of F2-isop across a range of farm animals using GC/NICI-MS and glucuronidase (GL)-ELISA and found various outcomes. These authors found, since there was acceptable agreement between the 2 methods, that the use of GC-MS for some species is recommended.

Precautions for the measurement of isoprostanes

Several precautions should be considered when measurements of F2-isop are used. Some of these precautions are very relevant to the meat industry where some practices are now regarded as standard processing steps (such as the use of electrical stimulation of carcasses).

  • Prevention of sample auto-oxidation and creation of artificially higher isoprostanes is very important. All body fluids contain lipids at different concentrations, with the exception of urine, and therefore there are requirements for handling samples such as the addition of an antioxidant (such as BHT) during the processing of the sample, rapid freezing in liquid nitrogen, and storage at −80 °C.
  • Blood samples need to be processed very quickly and hemolysis should be avoided as it can catalyze lipid oxidation (Dreibigacker and others 2010).
  • The rate of hydrolysis of F2-isoprostane is different in urine and plasma and cannot be used interchangeably. The use of the biological material should be verified (Halliwell and Lee 2010).

Practices seen in the meat industry that may lead to isoprostane production

  • Mixing of animals and lengthy travel that are common during transportation of animals from farm to processing facility could have an effect on the formation of isoprostane. This is due to increased production of RS during muscle contraction (Nikolaidis and others 2011). F2-isop increased by 193% during exercise (Karamouzis and others 2004). In humans, the formation of F2-isop was delayed and appeared in plasma 1 and 3 d after exercise activity, which was suppressed by presupplementation with α-tocopherol (12 wk with 1000 mg/d) (Sacheck and others 2003). The use of antioxidants can reduce the levels of F2-isop when a stimulus occurs that triggers increased production of F2-isoprostane. For example, antioxidants appear to favor a reduction in isoprostane formation when exercise takes place, but not at rest (Mastaloudis and others 2004; Watson and others 2005). However, chronic exercise appears to decrease the level of F2-isoprostane (Wang and others 2000) suggesting the development of a control system under systematic stress conditions. This was recently demonstrated by Schmitz and others (2008) who showed that regular aerobic exercise can reduce F2-isop in urine samples by 34%.
  • Fasting, another practice that occurs before the processing of animals at an abattoir, can potentially induce the formation of F2-isop. Fasting by humans was found to increase the plasma F2-isop after 24 h (Richelle and others 1999; Lee and others 2004), which appeared to be related to nutrition. The activity of feeding, regardless of the presence of antioxidants or not, appears to reduce the F2-isop to its baseline level (Lee and others 2004; Halliwell and Lee 2010), but whether this effect is detectable in ruminant species is unknown.
  • Handling of tissue can be extremely important. A 7-fold increase in isoprostanes was observed in rat kidneys after 48 h of storage at 4 °C (Salahudeen and others 1999).
  • Increased isoprostane formation (56% or 158%) in rat muscles during electrical stimulation was reported by Delliaux and others (2009a,b). Therefore, the level of isoprostanes determined in the muscles may not be related to the levels found in the blood collected earlier.

Factors Affecting the Formation of Isoprostanes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Oxidative Processes
  5. Reactive Species
  6. Biological Effects of RS
  7. Defense Systems against Oxidative Processes
  8. Other Endogenous Antioxidants
  9. Genetic Effects on the Oxidative Processes in Lamb and Beef
  10. Factors Influencing the Oxidative Processes of Meat
  11. Measurement of Biochemical Components in Tissues of Living Animal or Postmortem Muscles That is Associated with Oxidative Stress
  12. Biological Functions of Isoprostanes
  13. Factors Affecting the Formation of Isoprostanes
  14. Conclusions
  15. Acknowledgments
  16. References

Physiological factors

Generally, positive correlations were found between the level of isoprostane (8-iso-PGF) and human age, with some exception where high levels were found in children at age >7 y which declined with age. Higher levels of Isop were found in females compared with males (Basu and Helmersson 2005). In fact, the effect of dietary intervention can be dependent on gender where male patients with hypercholesterolemia responded positively to vitamin E supplements (8-iso-PGF was decreased with supplementation), whereas female patients were not affected (Salonen and others 2003).

Diet

High-antioxidant diets can exhibit different effects on F2-isoprostane (Table 12). The type of antioxidant can play a very important role with the most effective antioxidant suppressing the generation of F2-isop, as seen with high doses of vitamins E and C (Reilly and others 1996; Davi and others 1997; Praticó and others 1998a; Morrow and others 1999; Abu-Amsha and others 2001). However, the effect appears to be dependent on the model used, the dose used during measurement time point, and the length of the trials (Patrignani and others 2000; Barany and others 2001; Levine and others 2001; Bailey and others 2011). Other antioxidants such as polyphenols, flavones, and carotenoids seem to have no effect (Donovan and others 2005; Vega-López and others 2005; Covas and others 2006a; Loke and others 2008; Butalla and others 2012) and in some cases can increase the level of isop (Covas and others 2006b). The source of polyphenol (composition) may be important, since phenolics from olive oil (Visioli and others 2000) when compared with other fruit and vegetable phenolics (Table 12), did reduce isoprostane levels in plasma. The effects of different antioxidants on the production of F2-isop were investigated by Rabovsky and others (2006). The plasma 8-iso PG F was measured following incubation with linsidomin (SIN-1, an inducer of O2˙ and NO˙ generation) or 2,​2′-​azobis-​2-​methyl-​propanimidamide​ dihydrochloride (AAPH, a free radical generator) using an ELISA kit (Cayman Chemical, Ann Arbor, Mich., U.S.A.). The results demonstrated that vitamin C was more effective in reducing isoprostane formation compared with polyphenols from different sources.

The efficacy of antioxidants in reducing the formation of F2-isop appears to be also dependent on the subjects used for the study (Halliwell and Lee 2010). For example, vitamin E supplementation appeared to be more effective at decreasing F2-isop in patients with type 2 diabetes, hyperhomocysteinemia, and hypercholesterolemia and high doses or long-term intervention (Halliwell and Lee 2010). Deficiency in some elements can lead to increased oxidative stress, particularly those involved with antioxidant enzymes. Zinc deficiency contributed to reductions in hepatic α-tocopherol and γ-tocopherol and in plasma uric acid (Bruno and others 2007).

It is true that, like humans, farm animals can undergo several environmental challenges such as drought, malnutrition, imbalance in feed nutritive values, and heat/cold shock and these can all influence the oxidative status of individual animals. Any variation in the oxidative status can induce the formation of free radicals in the body. This can cause further damage to the living animal system including DNA damage, lipid damage, and protein damage. The above-mentioned biological actions of isoprostanes in circulatory and tissue systems of humans and small animals show that isoprostanes have potential as biomarkers in farm animals to assess the oxidative status in vivo and with the possibilty to relate this to biochemical components in muscle tissue postmortem and, therefore, meat quality. Developing a methodology to identify the oxidative status of farm animals, especially meat-producing livestock, may be a challenge, but this will benefit the meat industry and producers through improved performance and muscle quality using early on-farm detection and nutritional management.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Oxidative Processes
  5. Reactive Species
  6. Biological Effects of RS
  7. Defense Systems against Oxidative Processes
  8. Other Endogenous Antioxidants
  9. Genetic Effects on the Oxidative Processes in Lamb and Beef
  10. Factors Influencing the Oxidative Processes of Meat
  11. Measurement of Biochemical Components in Tissues of Living Animal or Postmortem Muscles That is Associated with Oxidative Stress
  12. Biological Functions of Isoprostanes
  13. Factors Affecting the Formation of Isoprostanes
  14. Conclusions
  15. Acknowledgments
  16. References

This review outlines the critical processes controlling oxidation, the biological actions of isoprostanes in the circulatory systems of humans and small animals, and their relationship with development of secondary components. Farm animals can undergo several challenges, such as poor immune system, malnutrition, imbalance in feed nutritive values, and heat/cold shock, and these can all influence the oxidative status of individuals. Any variation in oxidative status can induce the formation of free radicals in the body, which can lead to further damage to the living animal system including DNA damage, lipid damage, and protein damage. The above-mentioned biological actions of isoprostanes in circulatory and tissue systems show that isoprostanes have potential as biomarkers in farm animals to assess the oxidative status in vivo and with the objective to relate this to biochemical components in muscle tissue postmortem and, therefore, meat quality. Under extensive production systems, it is not always possible to ensure adequate feeds with good nutritive characteristics so as to maintain the antioxidant status in the body (such as vitamin E). In this context, a method to detect animals at “risk” of producing meat with undesirable characteristics is needed. Therefore, identification of the antioxidant status of live animals prior to slaughter using a marker would be a valuable strategy to increase the meat industry revenue through extension of shelf life (retail display) by maintaining the redness of meat or reducing meat discoloration.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Oxidative Processes
  5. Reactive Species
  6. Biological Effects of RS
  7. Defense Systems against Oxidative Processes
  8. Other Endogenous Antioxidants
  9. Genetic Effects on the Oxidative Processes in Lamb and Beef
  10. Factors Influencing the Oxidative Processes of Meat
  11. Measurement of Biochemical Components in Tissues of Living Animal or Postmortem Muscles That is Associated with Oxidative Stress
  12. Biological Functions of Isoprostanes
  13. Factors Affecting the Formation of Isoprostanes
  14. Conclusions
  15. Acknowledgments
  16. References

This work was in part supported by the Australian Meat Processor Corp. (AMPC) and Meat and Livestock Australia (MLA). The in-kind contribution toward this project was provided by The Dept. of Primary Industries, Victoria, Australia; The New South Wales Dept. of Primary Industries, Australia; and The Univ. of Otago, Dunedin, New Zealand.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Oxidative Processes
  5. Reactive Species
  6. Biological Effects of RS
  7. Defense Systems against Oxidative Processes
  8. Other Endogenous Antioxidants
  9. Genetic Effects on the Oxidative Processes in Lamb and Beef
  10. Factors Influencing the Oxidative Processes of Meat
  11. Measurement of Biochemical Components in Tissues of Living Animal or Postmortem Muscles That is Associated with Oxidative Stress
  12. Biological Functions of Isoprostanes
  13. Factors Affecting the Formation of Isoprostanes
  14. Conclusions
  15. Acknowledgments
  16. References
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