In order to provide a sufficient supply of iron many foods, and especially cereals, are fortified with iron in the form of ferrous sulfate or ferrous gluconate.
However, generally, iron is an undesirable element in food processing because it catalyzes oxidation of lipids and also others compounds such as antioxidant vitamins, polyphenols and many other reducing compounds found in food.
Dehydrated foods (cereals snacks, biscuits, milk powder, eggs powder, etc.), fried products, oils, muscle foods, and many others such tea, coffee or orange juice are susceptible to oxidation by metal ions.
The amount of total iron (g/g fresh weight) for different red muscle foods such as chicken, pork, turkey, lamb, beef, are: 5,10, 12, 16, 26, respectively from those heme-iron 3, 5, 8, 9, 16 and non-heme-iron 2, 5, 4, 7, 10, respectively. The amount of iron in fruits and vegetables, which is mostly non-heme iron is about 5±10 g/g fresh weight. However, in cereals which are dehydrated the amount of iron mostly non-heme is about 20±30 g/g dry weight.
2.3 Mechanism of metal oxidation in biological systems and foods
2.3.1 Free metal ions electron configuration
The `free iron' ion pool seems to be chelated to small molecules. The exact chemical nature of this pool is not clear, but it may represent iron ions attached to phosphate esters (ATP, ADP, phospholipids) organic acids (citrate, fatty acids), membrane lipids, nucleic acids, amino acids, and reducing sugars (Spiro, 1969; Kakhlon and Cabantchik, 2002). Most recently it was suggested that saturated fatty acids are the pathologic mediators of iron translocation in vivo (Yao et al., 2005).
Elementary iron has the following electronic configuration, 1s22s22p63s23p63d8. The outermost valence shell is the 3d orbital. In ferric iron, there are five valence electrons and in ferrous there are six. Ligands binding to the iron, due to their electronegativity and special arrangement, alter the energies of the electrons in the 3d orbital. This is termed field splitting.
In most biological complexes, the orbitals occupy an octahedral orientation in which the d orbitals are split into two levels: a high energy pair of electron orbitals (eg) and a lower energy trio (+2g). The presence and magnitude of the energy differences between these orbitals are due exclusively to the presence of the ligand in the gas phase. The free 3d orbitals are all of equal energy. For the 38 Oxidation in foods and beverages and antioxidant applications
most stable state of non-liganded iron, the valence electrons fill all possible orbitals with predominantly parallel spins, due to the orbital degeneracy and the Pauli exclusion principle. Ligand bonding, however, alters the energy of the orbitals and consequently, the lowest energy distribution of spin states is altered as well. This affect the high and low spin states of ferric and ferrous ions. The electrons in the 3d orbital of ferrous ion are in a high spin, however during the formation of a ligand, they adopt a low spin arrangement in the t2g level in such a way that the eg orbital remain free for occupation by the electrons of the ligand which form with iron coordinative bonding (Kanner et al., 1987).
Transition metals, iron and copper, with their labile d-electron system, are well suited to catalyzed redox reactions. Stable paramagnetic states, resulting from the presence of impaired electrons, are common for transition metals and facilitate their reaction with free radicals or molecules at the triplet state such as oxygen.
2.3.2 Oxygen activation by metal ions
Oxygen is a vital component in oxidation of foods and biological matter. The electronic structure of oxygen has two unpaired electrons at energy level of antibonding, in triplet state, 3g. The reaction of oxygen, therefore, is spin forbidden with ground state molecules of singlet multiplicity, which are more than 99% of the molecules in biological matter. This barrier does not apply to reactions with single electrons, hydrogen atoms, molecules in triplet state or molecules containing unpaired electrons, such as free radicals or transition metals. Oxygen is a bi-radical and paramagnetic compound. Thus, transition metals are able to remove the spin restriction of oxygen and be a bridge between oxygen and other molecules such reducing compounds, poly-unsaturated fatty acids, proteins and sugars.
Transition metals may initiate oxidation in foods and other biological systems by several mechanisms:
1. They are able to interact directly with triplet oxygen to generate reactive oxygen species (ROS) such superoxide radical (O2)ÿ, which by interaction with other O2to form O2, and by addition of two protons to form hydrogen peroxide (H2O2).
2. Reduced metals reduce H2O2 to hydroxyl radical (HO·), the most reactive radical generated in biological systems. The redox potential of hydroxyl radical at pH7.0 (HO·/H2O) is a +2.3V (Koppenol and Liebman, 1984), high enough to oxidize all bio-molecules. However, if the concentration of reduced metal is high enough, hydroxyl radical will oxidize the metal to form a hydroxyl anion by the following reactions 2.1±2.4:
Fe2 O2ÿ! Fe3 O2·ÿ 2.1
Fe2 H2O2ÿ! Fe3 HO· HOÿ 2.2
Fe2 HO·ÿ! Fe3 HOÿ 2.3
Metals and food oxidation 39
net 4Fe2 O2ÿ! 4Fe3 2HOÿ 2H 2.4 Fe2 O2·ÿÿÿÿ!2H Fe3 H2O2
Our work (Harel, 1994) showed that 100 M of ferrous sulfate was oxidized during interaction of 50 M of H2O2with 200 M of FeSO4at pH7.0 buffer acetate. This could be explained only if ferrous ions react by equations 2.3 and 2.4. The same stoichiometric ratio of Fe2/H2O2 of 2:1 was shown by Qian and Buettner (1999) to prevent oxidation of target molecules by HO·. 3. The most important reason to consider the oxidative chemistry initiated by Fe2 O2 as a significant route to biological oxidation is that the overall steady state concentration of oxygen is much greater, about 103higher than pre-existing H2O2 in living systems (O2, 10 M and H2O2, 10 nM). Data demonstrated by Qian and Buettner (1999) showed that when [O2]/[H2O2]
>100, Fe2 O2chemistry is an important route to initiation of detrimental biological free radical oxidation, much more than Fe2 with pre-existing H2O2(Fenton reaction). This chemistry leads to the formation of ferryl ion from loosely bound iron by the following reactions:
Reaction 2.1 will generate Fe3 O2·ÿwhich further generate a complex between Fe2 and O2.
Fe3 O2·ÿ ÿ! Fe2-O2 2.5
Fe2-O Fe2 ÿ! Fe2-O2±Fe2 2.6
Fe2-O2±Fe2 ÿ! 2Fe4=O oxo-ferryl ion 2.7 However, fresh muscle tissue after slaughtering and grounding at 37 ëC generates H2O2at a very significant amount of about 0.9 nmole/g min and after 60 min produce a steady state concentration of abut 50 M (Kanner and Harel, 1985b; Jorgenson and Skibsted, 1998; Harel and Kanner, 1985a).
Aging muscle tissues at 4 ëC for a period of 5 days increases H2O2production almost 2.3 fold. It seems that in muscle foods, endogenous generation of H2O2 plays an important role in the formation of the primary pool of biological catalysts.
4. Reducing agents are the most important co-factors turning transition metals such as Fe or Cu ions into significant catalysts of non-enzymic oxidation in biological and food systems. The most active reducing compounds involved in such reaction are ascorbic acid, cysteine, polyphenols, protein-SH, NADPH, NADH and dopa, dopamine and other minor reducing agents. We could assume that nearly all `loosely bound' or catalytic iron is present as Fe2 (Keyer and Imlay, 1996).
The interaction between ascorbic acid and transition metals could be described by the following reaction:
Fe3/Cu2 2AH2 ÿ! Fe2/Cu1 2AH· 2H2 2.8
AH· AH· ÿ! AH2 A 2.9
40 Oxidation in foods and beverages and antioxidant applications
The reduction of the transition metal generates ascorbyl radical which by disproportion form ascorbic acid and dehydroascorbic acid (DHAA). DHAA is not a toxic compound; however, in many foods it could increase non-enzymatic browning through Maillard reaction and Strecker degradation.
Reduction of transition metal ions by some polyphenols generate free radicals with the potential to produce further oxidizing compounds by the following reactions:
Fe3/Cu2 PhOH ÿ! Fe2/Cu1 PhO· Fe2/Cu 2.10
PhO· O2 ÿ! Ph=O O2·ÿ 2.11
PhO· PhO· ÿ! Ph±OH Ph=O 2.12
Equations 2.11 and 2.12 demonstrate that some polyphenols generate through reduction of transition metals, superoxide, H2O2and oxidized polyphenols, which are well-known oxidizing and cytotoxic compounds (Galati et al., 2006). Redox compounds are the ultimate driving force in obtaining high concentration of ferrous or cuprous ions, which are essential for the generation of oxygen reactive species such as superoxide, perhydroxyl radical, hydrogen peroxide and hydroxyl radicals, and the initiation of peroxidation of lipids, but also of proteins and carbohydrates. This reaction is also known as the `metal-redox cycle' (Winterbourn, 1979; Kanner et al., 1986; Harel, 1994).
5. Numerous iron (copper, zinc, manganese, molybdenum) containing enzymes and other iron-containing non-enzymatic compounds are important in ROS chemistry and biochemistry, in particular, lipoxygenase, cyclooxygenase, xanthine-oxidase, peroxidases, myoglobin and hemoglobin. They can directly or indirectly initiate ROS and lipid peroxidation. The enzymatic activity in foods is significant only in fresh products and is very important in the generation of `preformed' H2O2and hydroperoxides (ROOH). A great part of our food is processed by heating, which destroys the enzymatic activity and only catalysts such as free metal ions, myoglobin, hemoglobin or other hemeproteins still remain active for catalysis of ROS formation and lipid peroxidation (Kanner, 1994).
2.3.3 Hemeproteins Activation of hemeproteins
Hemoglobin and myoglobin play an essential role in maintaining aerobic metabolism in animal tissues. Iron-hemeproteins, especially myoglobin, are very abundant in muscle tissues. After slaughtering, autooxidation of oxyhemoglobin and oxymyoglobin (P±Fe2±O2) results in the formation of methemeproteins (P±Fe3) and superoxide and hydrogen peroxide. This process is accelerated by low pH, anions, low oxygen pressure, high temperature and is very much affected by the type of the myoglobin or hemoglobin (Kanner and Harel, 1985a;
Kanner, 1994; Aranda et al., 2009) by the following reactions: Auto-oxidation and oxygen activation:
Metals and food oxidation 41
P±Fe2ÿ O2ÿÿÿ!H P±Fe2 O2 2.13
Anions
P±Fe2 O2ÿÿÿ!H P±Fe3 O2·ÿ 2.14
Anions
O2·ÿ O2·ÿÿÿÿ!2H H2O2 O2 2.15
where P porphyrin±protein ligand.
Ferrous hemeproteins are oxidized by two-electron transfer when mixed by preformed H2O2forming an oxo-ferryl specie by the following reaction:
P±Fe2 H2O2ÿ! P±Fe4=O H2O 2.16
Ferrous hemeproteins should be regenerated from ferryl hemeproteins if a catalytic cycle reaction is expected. However, such regeneration is not expected as the ferryl state is preferentially reduced to ferric state in one electron reactions.
The active form for the cycle reaction remains methemeproteins, P±Fe3. Catalyst activation I
Hj
P±Fe3 H2O2 ÿ! P±Fe3±O±O±H 2.17
Hj
P±Fe3±O±O±H ÿ! P±Fe4=O· H2O 2.18
P±Fe4=O· ÿ! ·P±Fe4=O 2.19
Catalyst activation I is formed by the interaction of ferri-hemeproteins with H2O2generating a porphyrin cation radical, also called perferryl or oxo-ferryl radical. The Fe3 states of peroxidases, such as horseradish peroxidase, lactoperoxidase or myeloperoxidase form the oxo-ferryl state by a two-electron transfer. However, myoglobin and hemoglobin could form by two-electron transfer a pseudo-perferryl state generating a ·P±Fe4=O, the radical on the tyrosine and not on the porphyrin (Egawa et al., 2000). Myoglobin and hemoglobin interact with H2O2~105slower than peroxidases, ~102Mÿ1sÿ1and
~107Mÿ1sÿ1, respectively. Both myoglobin and hemoglobin could by one-electron transfer to form a ferryl and a HO· by the following reaction:
P±Fe3 H2O2 ÿ! P±Fe4 HO· HOÿ 2.20 Hydroxyl radical forming near the porphyrin will most probably oxidize the heme ring or the protein formed a oxo-ferryl radical (Kanner and Harel, 1985a).
Hemeproteins, like myoglobin and hemoglobin, could be activated by hydroperoxides (Kanner and Harel, 1985a; Adachi et al., 1993; Matsui et al., 1999; Carlsen et al., 2005). Reaction could activate P±Fe3 by two-electron or one-electron transfer (Fig. 2.1). The two-electron transfer will generate the following reactions:
42 Oxidation in foods and beverages and antioxidant applications
Hj
P±Fe3 LOOH ÿ! P±Fe3±O±O±L 2.21
Hj
P±Fe3±O±O±L ÿ! P±Fe4±O· LOH 2.22
P±Fe4±O· ÿ! ·P±Fe4=O 2.23
where·P is a protein radical and LOH is an alcohol such as cumyl alcohol.
However, the one-electron transfer of P±Fe3 to the hydroperoxide will produce two possible compounds by the following reactions:
P±Fe3 LOOH ÿ! P±Fe4 LO· H2O 2.24
P±Fe3 LOOH ÿ! P±Fe4 HO· LOH 2.25
Both the alkoxyl radical or hydroxyl radical could oxidize the protein amino acids and be reduced to alcohol and H2O.
Using a simple system activated met-myoglobin by cumene hydroperoxide, Adachi et al. (1993) and Matsui et al. (1999) found that ~70% of MbFe3reacted in two-electron transfer producing cumyl alcohol and oxo-ferryl radical,·P±Fe4=O.
Both pathways generate active species which could initiate lipid peroxidation and co-oxidation of many other compounds such as carotenoids, cholesterol, other lipids, proteins and carbohydrates. The 1-e-transfer and 2-e-transfer pathways generate alkoxy, hydroxyl and oxoferryl radicals with redox potential high enough to initiate oxidation of unsaturated fatty acids (LH) to allyl (L·) radicals and further to propagate lipid peroxidation.
The redox cycle of oxo-ferryl radical and oxo-ferryl
The redox cycle of oxo-ferryl radical by two-electron transfer will regenerate MbFe3(Fig. 2.2). If these donors are lipids or peroxides, the catalytic cycle will
Fig. 2.1 Activation of metmyoglobin by one- or two-electron transfer.
Metals and food oxidation 43
initiate and propagate oxidation. However, if the donors are antioxidants, such as ascorbic acid, tocopherols, polyphenols, or proteins containing amino acids such as tyrosine, tryptophan or SH group, relative unreactive radicals are formed, and the overall effect of the catalytic cycle will be antioxidative, removing the peroxides and hydroperoxides from the system (Lapidot et al., 2005a).
A wide range of compounds including plant polyphenols and other antioxidants efficiently react with oxo-ferryl and regenerate metmyoglogin (Lapidot et al., 2005a, 2005b; Carlsen et al., 2000).
The interaction of MbFe3with hydroperoxides leads to the generation by 1-e- or 2-1-e-transfer of alkoxyl, peroxyl and oxo-ferryl radicals. All of those species could be auto-reduced by the porphyrin ring and amino acids as electron donors of the hemeproteins. Such auto-reduction would produce free radical proteins and cause intra-molecular rearrangement and cross-linkage of the protein (Lewis and Wills, 1963; Kendrick and Watts, 1969; Harel and Kanner, 1989; Carlsen et al., 2000; Mikkelsen and Skibsted, 1992). If the auto-reduction process is very efficient (in the presence of low concentration of hydroperoxides and relatively high concentration of hemeproteins) the antioxidant tone of hemeproteins will dominate in the system. The antioxidant effect has been found to be strongly supported by phenolic antioxidants such as catechin (Lapidot et al., 2005a, 2005b).
The redox cycle of oxo-ferryl radical and oxo-ferryl was found to be dependent on pH (Mikkelsen and Skibsted, 1992; Reeder and Wilson, 1998;
Kanner and Lapidot, 2001). At pH 3.0 metmyoglobin at low concentration (1:30), as compared to hydroperoxides in the lipid system, act pro-oxidatively almost 7 104times as effective at pH 7.0. However, at a high concentration (~1:3), metmyoglobin acted antioxidatively, decomposing hydroperoxides whose concentration then remained at zero for a long period of time.
Polyphenols support the inversion of metmyoglobin catalysis, from pro-oxidation to antipro-oxidation. During this reaction, polyphenols not only donate reducing equivalents to prevent lipid peroxidation, but also prevent the destruction and polymerization of metmyoglobin. The results of our research
Fig. 2.2 The redox cycle of oxo-ferryl radical.
44 Oxidation in foods and beverages and antioxidant applications
highlighted the important and possible reaction of hemeproteins and poly-phenols as couple antioxidants working as hydroperoxidases or as pseudo-peroxidases (Lapidot et al., 2005b).