Lipid oxidation in meat products

mammalian Mbs (sperm whale and horse) (Cashon et al., 1997). Mackerel Mb had low oxygen affinity and high oxygen dissociation rates compared to zebrafish, yellowfin tuna, and Antarctic teleost (Madden et al., 2004). However Antarctic teleost had the highest koxrate among the four fish species indicating factors other than equilibrium oxygen dissociation constants affect autooxidation rates. Mackerel Mb had the most flexible structure based on dynamic simulations. It is interesting to note that aldehydic lipid oxidation products (4-hydroxy-2-nonenal) have been shown to covalently bind to histidine residues of bovine Mb which accelerated metMb formation (Suman et al., 2006).

(^·ÿO2) (reaction 1). This is activation of oxygen. It should be noted that certain chelators will facilitate reaction 1 while other chelators will not. The experimental conditions (e.g., pH, metal to chelator ratio) will also dictate rates of reaction.

Fe + chelator ÿ! Fe (chelate)

Fe²⁺(chelate) + O2ÿ!^·ÿO2+ Fe³⁺(chelate) (reaction 1) At post-mortem pH values, there are appreciable amounts of the conjugate acid form of superoxide (^·OOH, neutral superoxide radical) in equilibrium with the conjugate base form (^·ÿO2, superoxide anion radical) (pKa ~ 5) which leads to rapid production of hydrogen peroxide (reactions 2±4) (Halliwell and Gutteridge, 1999).

·ÿO2‡^·ÿO2‡ 2H^‡ÿ! HOOH ‡ O2(k < 0.3 M^ÿ1s^ÿ1) (reaction 2)

·ÿO2‡^·OOH ‡ H^‡ÿ! HOOH ‡ O2(k = 9:7  10⁵M^ÿ1s^ÿ1)

(reaction 3)

·OOH ‡^·OOH ÿ! HOOH ‡ O₂(k = 8:3  10⁵M^ÿ1s^ÿ1) (reaction 4) Hydrogen peroxide converts oxyHb to metHb (Weiss, 1982) (reaction 5).

HOOH ‡ 2oxyHb(2‡) ‡ 2H^‡ÿ! 2metHb(3‡) ‡ 2HOH ‡ O2

(reaction 5) Hydrogen peroxide is especially efficient in converting deoxyMb to metMb (Wazawa et al., 1992, Yusa and Shikama, 1987) (reactions 6 and 7).

HOOH ‡ deoxyHb(2‡) ÿ! ferrylHb(4‡) ‡ 2OH^ÿ (reaction 6) ferrylHb(4‡) ‡ deoxyHb(2‡) ÿ! 2metHb(3‡) (reaction 7) Hydrogen peroxide reacts with metHb and Fe^2‡(chelates) to form the ferryl heme protein radical and hydroxyl radical, respectively (reactions 8 and 9) (Reeder et al., 2004).

HOOH ‡ metHb(3‡) ÿ! ferrylHb^(·+)(4‡)=O ‡ H₂O (reaction 8) HOOH ‡ Fe^2‡(chelate) ÿ! ^·OH ‡^ÿOH ‡ Fe^3‡(chelate) (reaction 9) Both the hydroxyl radical and ferryl Hb radical can abstract hydrogen atoms from polyunsaturated fatty acids (reactions 10 and 11) resulting in peroxyl radical and lipid hydroperoxide accumulation (reactions 12 and 13). The hypervalent Hb produced in reaction 11 can also abstract hydrogen atoms from polyunsaturated fatty acids (reaction 14) and decompose LOOH to peroxyl radicals (reaction 15) (Kanner and Harel, 1985, Reeder et al., 2004). Note that metHb is regenerated in reactions 14 and 15.

·OH + LHÿ! L^·+ HOH (reaction 10)

ferrylHb^(·‡)(4‡)=O ‡ LH ÿ! L^·‡ ferrylHb(4‡)=O (reaction 11)

L^·‡ O2ÿ! LOO^· (reaction 12)

Heme proteins and oxidation in fresh and processed meats 87

LOO^·‡ LH ÿ! LOOH ‡ L^· (reaction 13) ferrylHb(4‡)=O ‡ LH ÿ! L^·‡ metHb(3‡) ‡ HOH (reaction 14) ferrylHb(4‡)=O ‡ LOOH ÿ! LOO^·‡ metHb(3‡) (reaction 15) Lipid hydroperoxides react with metHb, certain Fe2‡(chelates), and certain Fe3‡(chelates) to form ferryl Hb radical, alkoxyl, and peroxyl radicals, respectively (reactions 16±18). Alkoxyl radicals can also form due to reaction of metHb with LOOH (reaction 19) (Reeder et al., 2004). Alkoxyl radicals more readily abstract hydrogen atoms from polyunsaturated fatty acids compared to peroxyl radicals based on their one-electron reduction potentials (Buettner, 1993). It should be noted that the ferryl cation radical in reactions 8 and 16 reacts with O2to form peroxyl radical (Reeder et al., 2004).

metHb(3‡) ‡ LOOHÿ! ferrylHb^(·‡)(4‡) =O ‡ LOH (reaction 16) Fe^2‡(chelate) ‡ LOOHÿ! LO^·‡ OH^ÿ‡ Fe^3‡(chelate) (reaction 17) Fe^3‡(chelate) ‡ LOOHÿ! LOO^·‡ H^‡Fe^2‡(chelate) (reaction 18) metHb(3‡) ‡ LOOHÿ! ferrylHb(4‡)±OH ‡ LO^· (reaction 19) Lipid hydroperoxides also accelerate met formation (Nagy et al., 2005) as does hydrogen peroxide (reactions 5±7). Porphyin release occurs around 60-fold faster from metMb compared to ferrous forms of Mb (e.g., oxyMb and deoxyMb) (Tang et al., 1998). Released hemin porphyrin from metHb reacts with LOOH to form alkoxyl and peroxyl radicals (reactions 20 and 21) that stimulate lipid oxidation.

Hemin^(3‡)‡ LOOH ÿ! LO^·‡ hemin^(4‡)±OH (reaction 20) Hemin^(4‡)±OH + LOOH ÿ! LOO^·‡ hemin^(3‡)‡ HOH (reaction 21) The reaction schemes above suggest a continuous cycle of free radical and lipid hydroperoxide production yet often a small amount of the lipid substrate becomes oxidized in even rancid muscle (Xing et al., 1993). The reason only a small fraction of the total lipids become oxidized can be due to termination reactions of free radicals with other free radicals when their concentration becomes high (reaction 22).

LO^·‡ LO^·ÿ! LOOL (reaction 22)

It should also be noted that lipophilic free radicals (LO^· and LOO^·) (see reactions 12, 15 and 17±21) can randomly attack the carbon methene bridges of the tetrapyrrole rings, producing various pyrrole products in addition to releasing iron (Nagababu and Rifkind, 2004). This destruction of the porphyrin will prevent hemin and ferryl Mb-mediated lipid oxidation. The ability of low molecular weight metals, heme protein autooxidation, released hemin, and ferryl heme proteins to promote lipid oxidation and termination reactions are illustrated in a schematic representation (Fig. 4.4).

88 Oxidation in foods and beverages and antioxidant applications

4.6.2 Relative ability of Hb and Mb to autooxidize and promote lipid oxidation

Human Hb is known to release its ferric hemin moiety much more rapidly compared to human Mb (Bunn and Jandl, 1968; Gattoni et al., 1998). In fact apoMb can completely extract hemin from Hb (Banerjee, 1962).This is relevant because released hemin is capable of stimulating extensive lipid oxidation through decomposition of pre-formed lipid hydroperoxides (Tappel, 1955, Van der Zee et al., 1996) (reactions 20 and 21). Release of hemin from trout Hb occurred much more rapidly compared to trout Mb (Richards et al., 2005). On the other hand, trout Mb autooxidized to metMb much more rapidly compared to trout Hb (Richards et al., 2005). Ferrous, trout Hb was a more effective promoter of lipid oxidation in washed fish muscle at pH 6.3 compared to ferrous, trout Mb (Richards et al., 2005). This suggested that hemin loss from metHb was the primary factor that promoted lipid oxidation in washed fish muscle at pH 6.3 while metMb and the rapid burst of superoxide that formed during Mb autooxidation were relatively weak reactants. Hb promoted lipid oxidation more effectively compared to Mb in lipoproteins which was also attributed to the lower hemin affinity of Hb (Grinshtein et al., 2003).

Fig. 4.4 Heme protein (HP) oxidation and HP-mediated lipid oxidation can occur by multiple pathways. Metals (M), oxyHP, and deoxyHP are sources of^·ÿO2and H2O2that

accelerate HP oxidation. MetHP readily releases hemin. Hemin decomposes LOOH stimulating free radical-mediated lipid oxidation. H2O2and LOOH produce ferryl(+4)HP forms that can initiate lipid oxidation. Reactions involving M are facilitated or prevented

depending on the type of chelator bound to the metal.

Heme proteins and oxidation in fresh and processed meats 89

Ser(F7) and His(FG3)in Mbs interact with the heme-7-propionate while Leu(F7) and Leu(FG3) in Hb do not so that the porphyrin group is more

`anchored' in Mbs. Evidence for this can be seen when examining Mb mutants in which the native residues at F7 and FG3 were replaced with residues identical or similar to those found in Hbs at the same site. For example, the Ser(F7)Leu Mb mutant released its hemin moiety 20-fold faster compared to WT Mb (Smerdon et al., 1993). The His(FG3)Val mutant released its hemin moiety 23-fold more rapidly compared to WT Mb (Hargrove et al., 1996).

It should be noted that the tendency of Hb tetramers to dissociate to monomers and dimers accelerates hemin loss and Hb oxidation (Griffon et al., 1998; Hargrove et al., 1997). Enhanced ionic strength and dilution of Hb promotes dissociation of Hb tetramers to subunits (Antoni and Brunoni, 1971;

Manning et al., 1998). There have been reports that decreasing pH (in the pH range of 7 to 5) enhances Hb subunit formation but this may be due to effects from using acetate buffer. An Hb mutant (rHb 0.1) with decreased ability to form subunits was a weaker promoter of lipid oxidation compared to wild type Hb (Grunwald and Richards, 2006a).

The reaction of nitrite with oxyMb resulted in the formation of a ferryl Mb radical while reaction of nitrite with oxyHb did not (Keszler et al., 2006).

Formation of the Mb radical was inhibited by catalase indicating involvement of hydrogen peroxide in Mb radical formation. The ferryl heme protein radical is capable of abstracting hydrogen atoms from polyunsaturated fatty acids which can initiate lipid oxidation (reaction 11).

4.6.3 Role of released hemin compared to other oxidative forms of Hb and MbThe challenge in understanding the pathway by which heme proteins promote lipid oxidation is that heme protein autooxidation, ferryl radical formation, hemin release, heme protein crosslinking, hemichrome formation, and iron release can all occur in a very short time sequence (and simultaneously) so that the most relevant step related to lipid oxidation is obscured. However, amino acid substitutions of native Hb and Mb can be used to manipulate various properties of the heme proteins. For example, the ability of the hemin porphyrin to remain attached to the globin can be varied 975-fold by comparing wild-type sperm whale Mb with V68T and H97A (Hargrove et al., 1996). Substitution of valine at site E11 with threonine increases hemin affinity 25-fold. Thr(E11) will hydrogen bond with liganded water increasing hemin affinity while the native valine cannot hydrogen bond with liganded water (Fig. 4.5). Substituting histidine at site FG3 with alanine decreases hemin affinity 39-fold. The smaller alanine at FG3 allows water to rapidly enter into the heme crevice which hydrates the proximal histidine lowering hemin affinity; the native histidine at FG3 sterically blocks water from the proximal side of the heme crevice and chemically bonds with the heme-7-propionate (Fig. 4.6). The different mutants are separately added to washed fish muscle at post-mortem pH to assess the 90 Oxidation in foods and beverages and antioxidant applications

ability of each Mb mutants to promote lipid oxidation. H97A readily promoted lipid oxidation in washed cod at pH 5.7 while WT Mb was intermediate and V68T was the weakest promoter of lipid oxidation (Grunwald and Richards, 2006b). These results suggest that hemin release is the primary mechanism by which heme proteins promote lipid oxidation in washed fish muscle at pH 5.7.

Hemin readily decomposes preformed lipid hydroperoxides producing alkoxyl Fig. 4.5 Valine at site E11 (68th residue) in wild-type sperm whale metMb cannot hydrogen bond with water that is liganded to the iron atom of the porphyrin. Threonine at E11 does form a hydrogen bond with liganded water increasing hemin affinity 25-fold.

Adapted from Hargrove et al., 1996.

Fig. 4.6 Histidine at site FG3 (97th residue) in wild type sperm whale Mb excludes solvent from the heme crevice and also interacts with the heme-7-propionate. An alanine substitution at site FG3 allows water to hydrate the proximal histidine decreasing hemin

affinity. Ala(FG3) cannot interact with the heme-7-propionate group which also decreases hemin affinity. The proximal histidine is shown below the heme group. The distal histidine is shown above the heme group. The PDB structure 1A6K was used to

prepare the image shown using PyMOL software.

Heme proteins and oxidation in fresh and processed meats 91

and peroxyl radicals that propagate lipid oxidation (Van der Zee et al., 1996) (reactions 20 and 21).

L29F/H64Q is susceptible to porphyrin destruction in the presence of hydrogen peroxide compared to WT Mb (Alayash et al., 1999). Porphyrin destruction releases the iron in the heme ring and produces biliverdin. L29F/

H64Q was a weaker promoter of lipid oxidation in washed cod muscle compared to WT Mb (Grunwald and Richards, 2006b). This may be partly due to the antioxidant action of biliverdin (Baranano et al., 2002). It also indicates that liberated iron atoms from the porphyrin were not pro-oxidative. It should be noted hydrogen peroxide is water soluble so that heme destruction likely occurred in the aqueous phase. Iron atoms in the aqueous phase may be less reactive than iron atoms that incorporate into the membrane. Hemin is noted as a molecule that transports reactive iron atoms to lipid sites (Balla et al., 1991;

Grinshtein et al., 2003).

Ferrous V68T and ferrous WT Mb were compared to assess the ability of autooxidation relative to hemin affinity to promote lipid oxidation. V68T autooxidizes rapidly compared to WT while WT has lower hemin affinity.

Ferrous WT Mb was a better promoter of lipid oxidation in washed cod compared to ferrous V68T (Grunwald and Richards, 2006a). This suggested that hemin affinity was more critical in promoting lipid oxidation compared to Mb autooxidation rate in washed cod.

The fact that animal tissues contain hemopexin and heme oxygenase further implicates hemin as an oxidant that must be removed for cells to avoid oxidative effects associated with unbound hemin. Hemopexin binds hemin and is then detoxified in the liver (Paoli et al., 1999). Heme oxygenase degrades the heme ring releasing iron atoms and forming biliverdin that is reduced by biliverdin reductase to bilirubin, a potent antioxidant (Halliwell and Gutteridge, 1990;

Kumar and Bandyopadhyay, 2005)

Tyrosine at site G4 is considered a critical residue that facilitates ferryl Mb formation from hydrogen peroxide and metMb. Ferryl Mb can stimulate lipid oxidation (Baron et al., 1997). Substituting Tyr with Phe decreased ferryl Mb radical formation 1.4-fold (Witting et al., 2002). The Tyr(G4)Phe human Mb mutant promoted lipid oxidation about half as effectively as wild-type Mb when examining linoleic acid with added hydrogen peroxide at pH 7.4 (Rayner et al., 2004). This suggested that decreasing ferrylMb radical formation through mutagenesis decreased the ability of Mb to promote lipid oxidation in linoleic acid.

Hydrogen peroxide caused the heme moiety of horse heart Mb to be covalently cross-linked to the globin at pH values near 7 (Vuletich et al., 2000).

This cross-linked Mb promoted lipid oxidation in low density lipoproteins more readily compared to native Mb. Thus under certain conditions, cross-linked Mb promotes lipid oxidation more effectively than Mb that is not cross-linked.

There is also evidence that cross-linked Mb formation is favored at acidic pH values (Reeder et al., 2002). Hydrogen peroxide was also found to degrade the heme ring of Hb (Nagababu and Rifkind, 1998). This indicates hydrogen 92 Oxidation in foods and beverages and antioxidant applications

peroxide can have varying effects depending on whether Mb or Hb is examined.

More studies are needed to compare the relative ability of dissociated hemin and cross-linked Mb (and Hb) to promote lipid oxidation.

4.6.4 Ability of mammalian and fish Hbs to promote lipid oxidation Perch Hb was found to promote lipid oxidation in washed cod muscle rapidly compared to trout Hb at pH 6.3 (Richards and Dettmann, 2003). Bovine Hb was a remarkably poor promoter of lipid oxidation in washed cod compared to trout Hb at pH 6.3 (Richards et al., 2002). These findings can be partly attributed to the rapid autooxidation of the fish Hbs compared to the bovine Hb (Aranda et al., 2009). The hemin affinity of the met forms of fish and mammalian Hbs should also be considered based on the ability of released hemin to promote lipid oxidation (Tappel, 1955).

Perch Hb and trout IV Hb released hemin 55-fold and 26-fold faster com-pared to bovine Hb, respectively at pH 6.3 (Aranda et al., 2009). These dramatic differences can be attributed to steric and amino acid differences around the heme moiety when comparing fish and mammalian hemoglobins. The gap for solvent entry (water and protons) into the heme crevice at CD3 was 8 AÊ in perch Hb, around 6 AÊ in trout IV Hb, and around 4 AÊ in bovine Hb (Aranda et al., 2009). Hydration of the proximal histidine will decrease hemin affinity (Hargrove et al., 1996). Lysine at site E10 in bovine Hb formed favorable electrostatic and hydrogen bond interactions with the heme-7-propionate group while the smaller threonine at site E10 in the fish Hbs did not (Aranda et al., 2009). The interaction of Lys(E10) with the heme-7-propionate group increases hemin affinity in the bovine Hb. Perch Hb and trout IV Hb have glycine at site E14 while bovine Hb has the larger alanine ( chains) and serine ( chains) at this site. We have found that the Ala(E14)Gly Mb mutant rapidly autooxidized and had lower hemin affinity compared to WT Mb (unpublished observation).

Gly(E14) creates a channel for solvent entry into the heme crevice and may affect stability of the E-helix.

4.6.5 Effect of pH on Mb and Hb-mediated lipid oxidation

There are numerous reasons that decreasing pH increases the ability of Hb and Mb to promote lipid oxidation. First, protonation of the heme propionates at low pH will decrease hydrogen bonding and electrostatic interactions of the heme propionates with neighboring amino acids of the globin. Loss of these inter-actions decreases hemin affinity which promotes lipid oxidation. Second, protonation of the proximal histidine at low pH weakens the covalent linkage between the proximal histidine and the iron atom of the porphyrin. Hemin affinity of sperm whale Mb decreased 200-fold when decreasing the pH from 6.0 to 5.0 (Hargrove et al., 1994). Third, protonation of the distal histidine prevents hydrogen bonding with liganded water in metMb (Fig. 4.5) which decreases hemin affinity (Hargrove et al., 1996). Fourth, formation of hydrogen peroxide Heme proteins and oxidation in fresh and processed meats 93

(e.g., from superoxide that is released from Hb and Mb during autooxidation) occurs more rapidly at reduced pH (reactions 2±4). Hydrogen peroxide converts reduced Hb and Mb to more oxidative forms (reactions 5±8). Fifth and sixth, protonation of liganded O2and the distal histidine as it relates to autooxidation (Fig. 4.2) and solvent exposure to the heme crevice (Fig. 4.3) has been addressed (see Section 4.5.1). Seventh, low molecular weight metals also have enhanced solubility and thus enhanced reactivity at low pH (see Section 4.6.1).

4.6.6 Protein oxidation

Protein oxidation due to Hb and Mb can negatively impact textural attributes of muscle foods (see Section 4.2). Addition of hydrogen peroxide to metMb in the presence of albumin resulted in formation of ferryl forms of Mb that caused cross-linking of Mb to albumin and formation of dityrosine (éstdal et al., 2001).

MetMb and hydrogen peroxide caused the formation of thiyl, tyrosyl, and unidentified radical species on myosin as well as cross-links between myosin molecules (Lund et al., 2008). Blocking of sulfhydryls with N-acetylmaleimide prevented formation of myosin radicals and myosin cross-links when exposing porcine myosin to metMb and hydrogen peroxide (Frederiksen et al., 2008). This demonstrated that thiol groups of myosin were important in the formation of cross-links due to protein oxidation. Protein oxidation in a myofibrillar protein isolate was generally highest when using a hydroxyl radical generating system (FeCl3and ascorbate) compared to a metMb oxidizing system (0.05±0.5 mM metMb) and a lipid oxidizing system (linoleic acid and lipoxidase) (Park et al., 2006).

4.7 Inhibition of Hb- and Mb-mediated quality deterioration