Iron and Oxygen Radicals in Brain John M. C. Gutteridge, PhD, DSc, FIBiol ~
~
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The brain has been shown to contain regions with high concentrations of non-heme iron, and some of this iron can be chelated, which suggests that it is in a low molecular mass form. The chelatable iron appears to be responsible for the in vitro peroxidation of homogenized brain tissue lipids. It has been suggested that low molecular mass iron is essential for normal brain functions, and that iron is present in the reduced ferrous state. Recent studies have shown that chelatable iron is often present in micromolar concentrations in apparently normal cerebrospinal fluids (CSF), and that this iron is often increased in certain neurological diseases. Using a molecular recognition assay, based on the in vitro degradation of DNA by bleomycin and chelated ferrous ions, it has been possible to conclude that the low molecufar mass iron present in CSF is also in the reduced ferrous state. Ferrous ions are able to transfer electrons to molecular oxygen to form reactive and damaging intermediates of oxygen, some of which are free radicals. Increased levels of iron in brain tissue or CSF, however, are not necessarily synonymous with increased activation of oxygen. Detailed studies of brain iron deposits in neurological diseases are long overdue, and require new and pioneering methodologies to approach the problem. Gutteridge JMC. Iron and oxygen radicals in brain. Ann Neurol 1992;32:S16-S21
An Introduction to Free Radicals During the chemical and biological evolution of the Earth, its atmosphere changed from a highly reducing state to the current oxygen-rich state. Free molecular oxygen probably appeared on the Earth’s surface some 2 x lo9 years ago as a result of photosynthetic microorganisms acquiring the ability to split water. Oxygen is now the most abundant element in the Earth‘s crust and the second most abundant element in the biosphere. The concentration of molecular oxygen in dry air has risen to 21%; even at this level, oxygen has been shown to be mutagenic [l] and damaging to biological molecules [2f. We use oxygen to “burn” (oxidize) carbon- and hydrogen-rich molecules to obtain the energy and heat necessary for life. During this process, the oxygen molecule is reduced to water. The stepwise reduction of oxygen (addition of electrons) leads to the formation of reactive oxygen species (ROS), some of which are free radicals. A free radical may be defined as any chemical species having one or more unpaired electron. This broad biochemical definition, which does not define exactly where the unpaired electron is, embraces the atom of hydrogen, most transition metals ions, nitric oxide (endotheliumderived relaxing factor), and the oxygen molecule. Oxygen has two unpaired electrons, with parallel spins, and is therefore a hiradical with some unique properties. If oxygen attempts to oxidize another atom or molecule by accepting a pair of electrons from it, both new electrons must be of parallel spin to fit into the vacant From the Oxygen Chemistry Laboratory, Department of Anaesthesia and Intensive Care, Royal Brompton Hospital and National Heart and Lung Institute, London SW3 6NP, United Kingdom.
S16
orbitals. Most biological molecules, however, are covalent bonded nonradicals in which the two electrons forming a covalent bond have opposite spins. Hence, the reaction of oxygen with biomolecules is considerably slowed down, which is a point of considerable importance to aerobic life. To overcome this spin restriction, oxygen tends to accept electrons one at a time, and the sequential addition of electrons (e) to oxygen (0,)leads to the formation of reactive oxygen intermediates, two of which are free radicals.
0,
+ e H+\ HO; 7 PH H+ + 02-
H,O,
‘OH
+e +e
-
Hi
OH-
+ ‘OH
H20
(3) (4)
The unpaired electron of a free radical is represented with a dot (.). The four-electron reduction of oxygen to water, as shown in equations 1 through 4,gives rise to the superoxide anion radical (02-), hydrogen peroxide (H,O,), and the hydroxyl radical (’OH).The superoxide radical, formerly represented as 0,- is now shown as 0,- because it is less of a radical than molecular oxygen (having two unpaired electrons 0;’ that are not normally shown in this way). Superoxide is produced in numerous biological processes, particularly Address correspondence to Prof Gutteridge.
the electron transport chains of mitochondria and the endoplasmic reticulum [3}. Production of superoxide by activated phagocytic cells is one of the most studied radical producing systems [41. When opsonized particles are contacted by neutrophils a “respiratory burst” ensues: Oxygen uptake occurs and superoxide radicals are released into the phagocytic vacuole. Neutrophils in the presence of H202,which is formed by the dismutation of superoxide, can also oxidize chloride ions (C1-) into the powerful oxidant hypochlorous acid (HOCI).
HOCl and H202are called reactive oxygens, but they are not free radicals because they do not contain unpaired electrons. The enzymatic generation of reactive oxygens by activated phagocytic cells has evolved as a purposeful contribution to host defense [ S } . The reaction of iron salts with H,O, to generate a powerful oxidant was first described by Fenton in the 1890s [6}. We now know that the simple sequence represented in equation 6, known as the “Fenton reaction,” involves higher oxidation states of iron and is considerably more complex than shown [71. Fe2+
+ H202-
Fe3+
+ OH- + ’OH
(6)
Hydroxyl radicals are a major product of the radiolysis of water that can attack most biological molecules at almost diffusion-controlled rates but have little or no specificity in the damage they cause. However, when ‘OH radicals are formed by the Fenton reaction, they have considerable site-specificity because ‘OH will be formed close to where the metal ion is located [8}. The reactive oxygens and free radicals discussed are low molecular mass inorganic molecules; however, organic oxygen radicals are equally important in biological systems. Radicals tend to beget radicals, so that when ’OH attacks a biological molecule (RH) and steals an electron from it, an unpaired electron is left behind on the biological molecule (K).R’ is an organic free radical, and when the unpaired electron is on a carbon atom, the carbon-centered radical reacts rapidly with oxygen to form an organic oxygen radical called a peroxyl radical (RO,’). RO,’ is still able to cause damage, although it is less reactive than ’OH. For example, it can remove a hydrogen atom (one electron) from another biological molecule (RH) to form an organic hydroperoxide (ROOH) plus another radical (R’) to continue the sequence.
+ ‘OH-R’ K + 0,-RO,’
RH
R 0 , ’ t RH-ROOH
+ H,O
+ R’
(9)
When the biological molecule (RH) is a polyunsaturated fatty acid, the process results in a radical chain reaction, known as lipid peroxidation, leading ultimately to the oxidative destruction of the lipid. Radical chain reactions can be prevented or delayed by the presence of chain-breaking antioxidants; one of the most studied antioxidants is the lipid-soluble membrane antioxidant vitamin E (a-tocopherol). Lipid peroxides and their aldehydic breakdown products are not free radicals, because they have no unpaired electrons, but they are important cytotoxic components of oxidative stress and damage (91. The conversion of poorly reactive reduction intermediates of oxygen, such as 0,- and H , 0 2 , into highly reactive and damaging intermediates requires, as mentioned, the participation of transition metal ions. Iron is by far the most abundant and therefore the most likely biological participant. For this reason, the body takes great care to sequester iron in safe storage (ferritin) and transport (transferrin) forms. Detection and measurement of reactive low molecular mass iron in disease states is of fundamental importance to understanding how oxygen radical reactions occur [7}, and implies that site-specific metal chelation is a logical approach to intervention therapy [lo}.
General Antioxidant Protection in t h e Body The term antioxidant can be used to describe any substance that inhibits or delays an oxidative sequence when present at concentrations considerably lower than the oxidizable substrate. Oxygen is metabolized inside cells, and it is inside cells where protein antioxidants have evolved to deal specifically and speedily (catalytically)with products of reduced oxygen. Enzymes such as the superoxide dismutases (SOD), catalase, and glutathione peroxidase (selenium enzyme) function in a coordinated way to eliminate reduction intermediates of oxygen. The removal of oxygen intermediates should allow a low molecular mass pool of iron, required for the synthesis of iron-containing proteins, to exist safely inside the cell 1111. Different types of radicals are formed in the lipid interior of membranes, and special antioxidants are required to deal with them. Lipid-soluble molecules such as a-tocopherol (vitamin E), beta carotene, and possibly cholesterol fulfil some of these functions in animal and plant membranes. An extremely important feature of membrane protection appears to be the structural integrity of the membrane, which requires that the correct ratios of phospholipid and cholesterol are present as well as the correct phospholipids and their fatty acid side chains [121. We do not find enzymes like the intracellular SODS, catalase, and glutathione peroxidase in extracellular fluids, and the concentration of reduced glutathione Gutteridge: Iron and Oxygen Radicals in Brain
S17
(GSH) is very low. Nevertheless, extracellular Adds are often subjected to fluxes of superoxide and H202 by “activated” phagocytic cells and by substrate autooxidations. As a result of studies in our own laboratories over the last 18 years, we have proposed that extracellular antioxidant defenses substantially depend on mechanisms that remove or inactivate reactive transition metal complexes, particularly iron, before they can form more aggressive and damaging oxidants [12}. In this way, it may be possible to carefully control the levels of 02-and H 2 0 2in extracellular fluids so they can act as signal or trigger molecules between cells { l l , 131. Numerous low molecular mass chemicals exist in extracellular fluids such as ascorbate, urate, glucose, and bilirubin, which have the potential to act as scavengers and chain-breaking antioxidants. Fractionation studies on human serum, however, have shown that ceruloplasmin and transferrin, which represent only 4% of the total proteins present in serum, account for almost all of the antioxidant activity of human serum (when tested for its ability to inhibit iron-dependent peroxidation of phospholipid) C 12). It appears that a main function of extracellular antioxidant activity is the efficient inactivation or removal of metal catalysts likely to stimulate the formation of damaging and reactive forms of oxygen.
Iron and Free Radicals in Brain Brain tissue contains non-heme iron at levels estimated at approximately 0.074 pg/mg protein C14, 151 and has regions with high iron concentrations, particularly the globus pallidus, the substantia nigra, and the red nucleus { 16-18}. Slices of brain tissue peroxidize slowly, if at all, at room temperature in air, but when the tissue is homogenized, rapid peroxidation is observed [17, 201. The peroxidation can be inhibited by iron chelators, by enzymatically removing ascorbate, or by adding chain-breaking antioxidants 1201. Elegant studies by Zaleska and Floyd [ 2 l} showed that the rate of brain tissue homogenate peroxidation was dependent on the intrinsic iron content of different regions. The process of disrupting structural integrity by homogenizing tissue brings into contact reactants, such as polyunsaturated fatty acids, ascorbate, oxygen, and various metal complexes (particularly iron), normally safely held in discrete compartments. Trauma to the brain is known to promote lipid peroxidation in vivo, and antioxidant intervention therapy has recently been successfully targeted toward these events by using drugs with metal chelation and chain-breaking properties [22}. Several reports suggest that part of the non-heme iron (Table 1) present in the brain is low molecular
Table I . Biological 1ron Complexes and Their Possible Participation in Oxygen Radical Reactions
Type of Iron Complex Loosely bound iron Iron ions attached to: Phosphate esters (e.g., ATP) Carbohydrates and organic acids (e.g., citrate, picolinic acid, deoxyribose) DNA Membrane lipids Iron tightly bound to proteins: Non-heme iron Ferritin (4,5001mol Fe/mol protein) Hemosiderin Lactoferrin (iron saturated, 2 mol Fe3+/molprotein) Transferrin (iron saturated, 2 mol Fe3+/molprotein) Heme iron Hemoglobin Leghemoglobin Myoglobin Cytochrome c Catalase
Decomposition of Lipid Peroxides to Form Alkoxyl and Peroxyl Radicals
Reaction with H,O, to Form Hydroxyl Radicals by Fenton Chemistry
Yes Yes
Yes Yes
Yes Yes
Yes Yes
Yes
Yes (when iron is released)
Weakly Weakly (not if
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The brain has been shown to contain regions with high concentrations of non-heme iron, and some of this iron can be chelated, which suggests that it is in a low molecular mass form. The chelatable iron appears to be responsible for the in vitro peroxidation of homogenized brain tissue lipids. It has been suggested that low molecular mass iron is essential for normal brain functions, and that iron is present in the reduced ferrous state. Recent studies have shown that chelatable iron is often present in micromolar concentrations in apparently normal cerebrospinal fluids (CSF), and that this iron is often increased in certain neurological diseases. Using a molecular recognition assay, based on the in vitro degradation of DNA by bleomycin and chelated ferrous ions, it has been possible to conclude that the low molecufar mass iron present in CSF is also in the reduced ferrous state. Ferrous ions are able to transfer electrons to molecular oxygen to form reactive and damaging intermediates of oxygen, some of which are free radicals. Increased levels of iron in brain tissue or CSF, however, are not necessarily synonymous with increased activation of oxygen. Detailed studies of brain iron deposits in neurological diseases are long overdue, and require new and pioneering methodologies to approach the problem. Gutteridge JMC. Iron and oxygen radicals in brain. Ann Neurol 1992;32:S16-S21
An Introduction to Free Radicals During the chemical and biological evolution of the Earth, its atmosphere changed from a highly reducing state to the current oxygen-rich state. Free molecular oxygen probably appeared on the Earth’s surface some 2 x lo9 years ago as a result of photosynthetic microorganisms acquiring the ability to split water. Oxygen is now the most abundant element in the Earth‘s crust and the second most abundant element in the biosphere. The concentration of molecular oxygen in dry air has risen to 21%; even at this level, oxygen has been shown to be mutagenic [l] and damaging to biological molecules [2f. We use oxygen to “burn” (oxidize) carbon- and hydrogen-rich molecules to obtain the energy and heat necessary for life. During this process, the oxygen molecule is reduced to water. The stepwise reduction of oxygen (addition of electrons) leads to the formation of reactive oxygen species (ROS), some of which are free radicals. A free radical may be defined as any chemical species having one or more unpaired electron. This broad biochemical definition, which does not define exactly where the unpaired electron is, embraces the atom of hydrogen, most transition metals ions, nitric oxide (endotheliumderived relaxing factor), and the oxygen molecule. Oxygen has two unpaired electrons, with parallel spins, and is therefore a hiradical with some unique properties. If oxygen attempts to oxidize another atom or molecule by accepting a pair of electrons from it, both new electrons must be of parallel spin to fit into the vacant From the Oxygen Chemistry Laboratory, Department of Anaesthesia and Intensive Care, Royal Brompton Hospital and National Heart and Lung Institute, London SW3 6NP, United Kingdom.
S16
orbitals. Most biological molecules, however, are covalent bonded nonradicals in which the two electrons forming a covalent bond have opposite spins. Hence, the reaction of oxygen with biomolecules is considerably slowed down, which is a point of considerable importance to aerobic life. To overcome this spin restriction, oxygen tends to accept electrons one at a time, and the sequential addition of electrons (e) to oxygen (0,)leads to the formation of reactive oxygen intermediates, two of which are free radicals.
0,
+ e H+\ HO; 7 PH H+ + 02-
H,O,
‘OH
+e +e
-
Hi
OH-
+ ‘OH
H20
(3) (4)
The unpaired electron of a free radical is represented with a dot (.). The four-electron reduction of oxygen to water, as shown in equations 1 through 4,gives rise to the superoxide anion radical (02-), hydrogen peroxide (H,O,), and the hydroxyl radical (’OH).The superoxide radical, formerly represented as 0,- is now shown as 0,- because it is less of a radical than molecular oxygen (having two unpaired electrons 0;’ that are not normally shown in this way). Superoxide is produced in numerous biological processes, particularly Address correspondence to Prof Gutteridge.
the electron transport chains of mitochondria and the endoplasmic reticulum [3}. Production of superoxide by activated phagocytic cells is one of the most studied radical producing systems [41. When opsonized particles are contacted by neutrophils a “respiratory burst” ensues: Oxygen uptake occurs and superoxide radicals are released into the phagocytic vacuole. Neutrophils in the presence of H202,which is formed by the dismutation of superoxide, can also oxidize chloride ions (C1-) into the powerful oxidant hypochlorous acid (HOCI).
HOCl and H202are called reactive oxygens, but they are not free radicals because they do not contain unpaired electrons. The enzymatic generation of reactive oxygens by activated phagocytic cells has evolved as a purposeful contribution to host defense [ S } . The reaction of iron salts with H,O, to generate a powerful oxidant was first described by Fenton in the 1890s [6}. We now know that the simple sequence represented in equation 6, known as the “Fenton reaction,” involves higher oxidation states of iron and is considerably more complex than shown [71. Fe2+
+ H202-
Fe3+
+ OH- + ’OH
(6)
Hydroxyl radicals are a major product of the radiolysis of water that can attack most biological molecules at almost diffusion-controlled rates but have little or no specificity in the damage they cause. However, when ‘OH radicals are formed by the Fenton reaction, they have considerable site-specificity because ‘OH will be formed close to where the metal ion is located [8}. The reactive oxygens and free radicals discussed are low molecular mass inorganic molecules; however, organic oxygen radicals are equally important in biological systems. Radicals tend to beget radicals, so that when ’OH attacks a biological molecule (RH) and steals an electron from it, an unpaired electron is left behind on the biological molecule (K).R’ is an organic free radical, and when the unpaired electron is on a carbon atom, the carbon-centered radical reacts rapidly with oxygen to form an organic oxygen radical called a peroxyl radical (RO,’). RO,’ is still able to cause damage, although it is less reactive than ’OH. For example, it can remove a hydrogen atom (one electron) from another biological molecule (RH) to form an organic hydroperoxide (ROOH) plus another radical (R’) to continue the sequence.
+ ‘OH-R’ K + 0,-RO,’
RH
R 0 , ’ t RH-ROOH
+ H,O
+ R’
(9)
When the biological molecule (RH) is a polyunsaturated fatty acid, the process results in a radical chain reaction, known as lipid peroxidation, leading ultimately to the oxidative destruction of the lipid. Radical chain reactions can be prevented or delayed by the presence of chain-breaking antioxidants; one of the most studied antioxidants is the lipid-soluble membrane antioxidant vitamin E (a-tocopherol). Lipid peroxides and their aldehydic breakdown products are not free radicals, because they have no unpaired electrons, but they are important cytotoxic components of oxidative stress and damage (91. The conversion of poorly reactive reduction intermediates of oxygen, such as 0,- and H , 0 2 , into highly reactive and damaging intermediates requires, as mentioned, the participation of transition metal ions. Iron is by far the most abundant and therefore the most likely biological participant. For this reason, the body takes great care to sequester iron in safe storage (ferritin) and transport (transferrin) forms. Detection and measurement of reactive low molecular mass iron in disease states is of fundamental importance to understanding how oxygen radical reactions occur [7}, and implies that site-specific metal chelation is a logical approach to intervention therapy [lo}.
General Antioxidant Protection in t h e Body The term antioxidant can be used to describe any substance that inhibits or delays an oxidative sequence when present at concentrations considerably lower than the oxidizable substrate. Oxygen is metabolized inside cells, and it is inside cells where protein antioxidants have evolved to deal specifically and speedily (catalytically)with products of reduced oxygen. Enzymes such as the superoxide dismutases (SOD), catalase, and glutathione peroxidase (selenium enzyme) function in a coordinated way to eliminate reduction intermediates of oxygen. The removal of oxygen intermediates should allow a low molecular mass pool of iron, required for the synthesis of iron-containing proteins, to exist safely inside the cell 1111. Different types of radicals are formed in the lipid interior of membranes, and special antioxidants are required to deal with them. Lipid-soluble molecules such as a-tocopherol (vitamin E), beta carotene, and possibly cholesterol fulfil some of these functions in animal and plant membranes. An extremely important feature of membrane protection appears to be the structural integrity of the membrane, which requires that the correct ratios of phospholipid and cholesterol are present as well as the correct phospholipids and their fatty acid side chains [121. We do not find enzymes like the intracellular SODS, catalase, and glutathione peroxidase in extracellular fluids, and the concentration of reduced glutathione Gutteridge: Iron and Oxygen Radicals in Brain
S17
(GSH) is very low. Nevertheless, extracellular Adds are often subjected to fluxes of superoxide and H202 by “activated” phagocytic cells and by substrate autooxidations. As a result of studies in our own laboratories over the last 18 years, we have proposed that extracellular antioxidant defenses substantially depend on mechanisms that remove or inactivate reactive transition metal complexes, particularly iron, before they can form more aggressive and damaging oxidants [12}. In this way, it may be possible to carefully control the levels of 02-and H 2 0 2in extracellular fluids so they can act as signal or trigger molecules between cells { l l , 131. Numerous low molecular mass chemicals exist in extracellular fluids such as ascorbate, urate, glucose, and bilirubin, which have the potential to act as scavengers and chain-breaking antioxidants. Fractionation studies on human serum, however, have shown that ceruloplasmin and transferrin, which represent only 4% of the total proteins present in serum, account for almost all of the antioxidant activity of human serum (when tested for its ability to inhibit iron-dependent peroxidation of phospholipid) C 12). It appears that a main function of extracellular antioxidant activity is the efficient inactivation or removal of metal catalysts likely to stimulate the formation of damaging and reactive forms of oxygen.
Iron and Free Radicals in Brain Brain tissue contains non-heme iron at levels estimated at approximately 0.074 pg/mg protein C14, 151 and has regions with high iron concentrations, particularly the globus pallidus, the substantia nigra, and the red nucleus { 16-18}. Slices of brain tissue peroxidize slowly, if at all, at room temperature in air, but when the tissue is homogenized, rapid peroxidation is observed [17, 201. The peroxidation can be inhibited by iron chelators, by enzymatically removing ascorbate, or by adding chain-breaking antioxidants 1201. Elegant studies by Zaleska and Floyd [ 2 l} showed that the rate of brain tissue homogenate peroxidation was dependent on the intrinsic iron content of different regions. The process of disrupting structural integrity by homogenizing tissue brings into contact reactants, such as polyunsaturated fatty acids, ascorbate, oxygen, and various metal complexes (particularly iron), normally safely held in discrete compartments. Trauma to the brain is known to promote lipid peroxidation in vivo, and antioxidant intervention therapy has recently been successfully targeted toward these events by using drugs with metal chelation and chain-breaking properties [22}. Several reports suggest that part of the non-heme iron (Table 1) present in the brain is low molecular
Table I . Biological 1ron Complexes and Their Possible Participation in Oxygen Radical Reactions
Type of Iron Complex Loosely bound iron Iron ions attached to: Phosphate esters (e.g., ATP) Carbohydrates and organic acids (e.g., citrate, picolinic acid, deoxyribose) DNA Membrane lipids Iron tightly bound to proteins: Non-heme iron Ferritin (4,5001mol Fe/mol protein) Hemosiderin Lactoferrin (iron saturated, 2 mol Fe3+/molprotein) Transferrin (iron saturated, 2 mol Fe3+/molprotein) Heme iron Hemoglobin Leghemoglobin Myoglobin Cytochrome c Catalase
Decomposition of Lipid Peroxides to Form Alkoxyl and Peroxyl Radicals
Reaction with H,O, to Form Hydroxyl Radicals by Fenton Chemistry
Yes Yes
Yes Yes
Yes Yes
Yes Yes
Yes
Yes (when iron is released)
Weakly Weakly (not if
