Free Radicals in Biology and Medicine

Cover
Preface to the fifth edition
Acknowledgements
Contents
Abbreviations
Plates
1 Oxygen: boon yet bane—introducing oxygen toxicity and reactive species
1.1 The history of oxygen: an essential air pollutant
1.1.1 The paradox of photosynthesis
1.1.2 Hyperoxia in history?
1.1.3 Oxygen in solution
1.2 Oxygen and anaerobes
1.2.1 Why does oxygen injure anaerobes?
1.3 Oxygen and aerobes
1.3.1 Oxygen transport in mammals
1.3.2 Oxygen sensing
1.3.3 Mitochondrial electron transport
1.3.4 The evolution of mitochondria
1.3.5 Nicotinamide nucleotide reduction
1.3.6 Bacterial electron transport chains
1.4 Oxidases and oxygenases in aerobes
1.4.1 Cytochromes P450
1.5 Oxygen toxicity in aerobes
1.5.1 Bacteria, plants, insects, and alligators
1.5.2 Mammals
1.5.2.1 Retinopathy of prematurity and brain damage
1.5.2.2 Resuscitation of newborns
1.5.2.3 Factors affecting oxygen toxicity
1.6 What causes the toxic effects of oxygen?
1.7 So free radicals contribute to oxygen toxicity and oxygen is one of them? What then are free radicals?
1.8 Oxygen and its radicals
1.8.1 Singlet oxygen
1.8.2 Superoxide radical
1.9 How to describe them: oxygen radicals, oxygen-derived species, reactive oxygen species, or oxidants?
1.10 Sources of superoxide in aerobes
1.10.1 Enzymes
1.10.2 Auto-oxidation reactions
1.10.3 Haem proteins
1.10.4 Mitochondrial electron transport
1.10.4.1 Mitochondrial DNA (mtDNA)
1.10.5 Uncoupling proteins as antioxidants?
1.10.6 Endoplasmic reticulum (ER)
1.10.7 Nuclear and plasma membranes
1.10.8 Bacterial superoxide production and biofilms
1.11 Thinking about cell culture
1.12 Some numbers
2 Redox chemistry: the essentials
2.1 Introduction
2.2 How do free radicals react?
2.3 Radical chemistry: thermodynamics versus kinetics
2.3.1 Redox chemistry
2.3.1.1 Caveats
2.3.1.2 Thermodynamics of oxygen reduction
2.3.2 Reaction rates and rate constants
2.3.3 Measuring reaction rates and rate constants
2.3.3.1 Pulse radiolysis
2.3.3.2 Stopped-flow methods
2.4 Transition metals: biocatalytic free radicals
2.4.1 Iron
2.4.2 Copper
2.4.3 Manganese
2.4.4 The Fenton reaction
2.4.5 Iron chelators and Fenton chemistry: speed it up or slow it down?
2.4.6 Reaction of copper ions with H2O2
2.5 Chemistry of other biologically important radicals
2.5.1 Hydroxyl radical
2.5.1.1 Generation
2.5.1.2 Chemistry
2.5.2 Carbonate radical
2.5.3 Superoxide radical
2.5.3.1 Making superoxide in the laboratory
2.5.3.2 Reactions of superoxide
2.5.3.3 Superoxide–iron interactions
2.5.3.4 Reductants and Fenton chemistry
2.5.3.5 Semiquinones and quinones
2.5.3.6 Superoxide in hydrophobic environments
2.5.4 Peroxyl and alkoxyl radicals
2.5.4.1 Chemistry
2.5.4.2 Generation of RO2• /RO• radicals
2.5.5 Sulphur radicals
2.5.5.1 Formation
2.5.5.2 Reactions
2.5.5.3 Artefacts involving sulphur compounds
2.5.5.4 The perils of dithiothreitol, thiourea, and N-acetylcysteine
2.5.6 Nitric oxide
2.5.6.1 Basic chemistry
2.5.6.2 Nitric oxide as a free radical scavenger
2.5.6.3 Physiological roles
2.5.6.4 Synthesis of nitric oxide
2.5.6.5 Removal of NO• in vivo
2.5.6.6 Nitrate and nitrite: inert end-products or physiologically important sources of NO•?
2.5.6.7 Nitric oxide donors
2.6 Chemistry of biologically important non-radicals
2.6.1 Peroxynitrite
2.6.1.1 How does peroxynitrite cause damage?
2.6.1.2 Toxicity of nitrotyrosine and nitrated proteins?
2.6.1.3 Nitric oxide, superoxide, peroxynitrite, and nitrated lipids: a balance
2.6.1.4 Can peroxynitrite be antioxidant?
2.6.1.5 More things to beware of
2.6.2 Hydrogen peroxide
2.6.2.1 Production of H2O2
2.6.2.2 Chemistry of H2O2
2.6.3 Hypohalous acids and their derivatives
2.6.3.1 Chlorhydrins, chloramines, and hydroxyl radical from HOCl
2.6.3.2 Atomic chlorine
2.6.4 Singlet oxygen
2.6.4.1 Singlet O2 from photosensitization
2.6.4.2 Type I and II reactions
2.6.4.3 Biological damage by photosensitization
2.6.4.4 Uses of photosensitization
2.6.4.5 Other sources of singlet O2
2.6.4.6 Reactions of singlet oxygen
2.6.5 Ozone, a radical or not?
3 Antioxidant defences synthesized in vivo
3.1 Introduction
3.2 What is an antioxidant?
3.3 Antioxidant defences: general principles
3.4 The simplest antioxidant defence: minimize exposure to oxygen
3.4.1 Protecting nitrogenases
3.4.2 Stem cells
3.5 Antioxidant defence enzymes: superoxide dismutases (SODs)
3.5.1 Copper–zinc SOD
3.5.1.1 CuZnSOD in eukaryotes and prokaryotes
3.5.1.2 Catalytic ability of CuZnSOD
3.5.1.3 CuZnSOD structure
3.5.1.4 Inhibitors of CuZnSOD
3.5.1.5 Isoenzymes of CuZnSOD
3.5.1.6 Pro-oxidant effects of CuZnSOD?
3.5.2 Manganese SOD
3.5.2.1 Where is MnSOD found?
3.5.2.2 Regulation of MnSOD activity
3.5.2.3 Structure of MnSOD
3.5.3 Iron and cambialistic SODs
3.5.3.1 Distribution of FeSODs
3.5.4 Evolution of SODs
3.5.5 Nickel-containing SODs
3.5.6 Assaying SOD
3.5.6.1 Distinguishing between different types of SOD
3.5.7 Using SOD enzymes to implicate superoxide
3.6 Superoxide reductases
3.7 Superoxide dismutases: evidence for their role in vivo?
3.7.1 Gene knockout in bacteria and yeasts
3.7.2 Transgenic animals
3.7.2.1 Caveats about transgenic animals
3.7.3 RNA interference
3.7.4 Induction experiments
3.7.5 SOD and oxygen toxicity in animals
3.7.6 SOD and hibernation
3.8 The superoxide theory of oxygen toxicity: variations and anomalies
3.8.1 Anaerobes with SOD and aerobes without SOD
3.8.2 Manganese can replace SOD
3.9 Why is superoxide cytotoxic?
3.9.1 Direct damage by superoxide or HO2•?
3.9.2 Cytotoxicity of superoxide-derived species
3.9.2.1 Hydrogen peroxide and peroxynitrite
3.9.2.2 Hydroxyl radical
3.10 Glutathione in metabolism and cellular redox state
3.10.1 GSH as a direct antioxidant
3.10.2 Glutathione reductase
3.10.2.1 Sources of NADPH
3.10.3 Glutathione biosynthesis and degradation
3.10.4 Defects in GSH metabolism: humans and other organisms
3.11 Glutathionylation: pathological or protective?
3.12 Protein-disulphide isomerase
3.13 Peroxiredoxins: leaders in peroxide metabolism
3.13.1 Introducing thioredoxins, cofactors of peroxiredoxins
3.13.2 The peroxiredoxins themselves
3.13.2.1 Reaction with peroxynitrite
3.13.2.2 Hyperoxidation
3.13.2.3 Circadian rhythms
3.14 Antioxidant defence enzymes: the glutathione peroxidase family
3.14.1 A family of enzymes
3.14.2 The role of selenium
3.14.3 Watching GPx in action
3.14.4 Consequences of GPx deficiency
3.15 Other enzymes using glutathione
3.15.1 Glyoxalases
3.15.2 The glutathione S-transferase superfamily
3.15.2.1 Subclasses of GST
3.15.2.2 GSTs and lipid peroxidation
3.16 Other sulphur-containing compounds and antioxidant defence
3.16.1 Trypanothione: an antioxidant defence in some parasites
3.16.2 Ergothioneine
3.17 Antioxidant defence enzymes: catalases
3.17.1 Catalase structure
3.17.2 The reaction mechanism of catalase
3.17.3 Catalase inhibitors
3.17.4 Peroxidatic activity of catalase
3.17.5 Subcellular location of catalase: the peroxisome
3.17.6 Manganese-containing catalases
3.17.7 Does catalase matter? Acatalasaemia
3.18 NADH oxidases
3.19 Antioxidant defence enzymes: an assortment of other peroxidases
3.19.1 Cytochrome c peroxidase: another specific peroxidase
3.19.2 ‘Non-specific’ peroxidases
3.19.3 Horseradish peroxidase
3.19.4 Why do plants have so much peroxidase?
3.19.5 Chloroperoxidase and bromoperoxidase
3.19.6 Ascorbate peroxidase
3.19.7 Peroxidase ‘mimetics’
3.20 Making sense of it all. What fits where in peroxide metabolism?
3.20.1 Peroxisomes and mitochondria
3.20.2 Erythrocytes, lung, and yeast
3.20.3 Allowing redox signalling?
3.20.4 Bacteria
3.20.5 Selenium deficiency: reinterpretation of an old paradigm
3.20.5.1 Human selenium deficiency
3.20.5.2 Selenium deficiency and antioxidant defences
3.21 Further co-operation
3.21.1 Superoxide dismutases and peroxide-metabolizing enzymes
3.21.2 Down syndrome
3.22 Antioxidant defence: sequestration of metal ions
3.22.1 Iron metabolism
3.22.1.1 Transferrin
3.22.1.2 Other iron-binding proteins
3.22.1.3 Iron within cells
3.22.1.4 Ferritin
3.22.1.5 Regulation of cellular iron balance
3.22.2 Copper metabolism
3.22.2.1 Caeruloplasmin and copper chaperones
3.22.2.2 A phantom copper pool?
3.22.2.3 Caeruloplasmin as an oxidase
3.22.2.4 Caeruloplasmin as a peroxidase
3.22.3 Haem and haem proteins: powerful pro-oxidants
3.22.4 Metal ion sequestration: why do it?
3.22.4.1 Keeping micro-organisms at bay
3.22.4.2 Diminishing free-radical reactions
3.22.5 Metal ion sequestration: when it goes wrong
3.22.5.1 Iron overload: diet-derived
3.22.5.2 Iron overload: genetic
3.22.5.3 Thalassaemias
3.22.5.4 Non-transferrin-bound iron: is it pro-oxidant?
3.22.5.5 Copper overload
3.23 Metal ions and antioxidant defence: comparing intracellular and extracellular strategies
3.23.1 The intracellular environment: metals and oxidative damage
3.23.2 Metallothioneins
3.23.3 Extracellular antioxidant defence
3.23.3.1 Low antioxidant defence enzymes and limited metal ion availability
3.23.3.2 Extracellular superoxide dismutase
3.23.3.3 Other extracellular SODs
3.23.3.4 Binding haem and haemoglobin
3.23.3.5 Albumin
3.23.3.6 Artefacts with albumin
3.24 Haem oxygenase
3.25 Antioxidant protection by low-molecular-mass agents synthesized in vivo
3.25.1 Bilirubin
3.25.2 α-Keto acids
3.25.3 Melatonin
3.25.4 Lipoic acid
3.25.5 Coenzyme Q
3.25.6 Uric acid
3.25.7 Histidine-containing dipeptides
3.25.8 Trehalose (α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside)
3.25.9 Melanins: hair, skin, corals, fungi, and fish
3.26 Antioxidant defence: a question of sex
4 Antioxidants from the diet
4.1 Introduction
4.2 Ascorbic acid (vitamin C)
4.2.1 Ascorbate as an antioxidant
4.2.2 ‘Recycling’ of ascorbate
4.2.3 Pro-oxidant effects of ascorbate
4.2.4 Taking ascorbate supplements?
4.3 Vitamin E
4.3.1 Its physiological role
4.3.2 What is vitamin E?
4.3.3 Chemistry of vitamin E
4.3.4 Recycling of α-tocopheryl radicals
4.3.5 Pro-oxidant effects of α-tocopherol?
4.3.6 Processing of dietary vitamin E
4.3.7 The fate of γ-tocopherol
4.3.8 α-Tocopherol deficiency
4.3.9 Vitamin E: only an antioxidant, or something else as well?
4.4 Carotenoids
4.4.1 Carotenoid chemistry
4.4.2 Metabolic roles of carotenoids
4.4.3 Carotenoids and vitamin A as antioxidants?
4.4.3.1 Do carotenoids react with radicals?
4.4.3.2 Stability of carotenoids
4.4.3.3 The interesting case of lycopene
4.5 Flavonoids and other phenols
4.5.1 Phenols in the diet
4.5.1.1 Do humans and other animals absorb phenols?
4.5.2 Are phenols antioxidants in vivo?
4.5.2.1 More than antioxidants
4.5.3 Pro-oxidant effects of phenols?
4.5.4 Herbal medicine
4.6 Dietary antioxidants: insights from epidemiology
4.6.1 Problems of interpretation
4.6.2 The gold standard of intervention trials: hope unfulfilled
4.6.3 The need for biomarkers
4.6.3.1 Do fruits and vegetables decrease the risk of disease by lowering oxidative damage?
4.6.4 Cardiovascular intervention trials
4.6.5 Cancer prevention by antioxidants?
4.6.5.1 The Finnish study
(α-tocopherol/β-carotene [ATBC] cancer
prevention study) and CARET
4.6.6 Some rays of hope and a gender bias
4.6.7 Lycopene, other carotenoids, and human disease
4.6.8 Antioxidants and neuroprotection; insights from epidemiology?
4.7 Other dietary constituents and oxidative damage
4.8 What does it all mean? What should we poor mortals eat?
5 Oxidative stress and redox regulation: adaptation, damage, repair, senescence, and death
5.1 Introduction
5.1.1 Defining oxidative stress and oxidative damage
5.2 Consequences of oxidative stress
5.2.1 Proliferation
5.2.2 Adaptation
5.2.3 Migration and adhesion
5.2.4 Cell injury and senescence
5.2.5 Poly(ADP–ribose)polymerase
5.3 Oxidative stress causes changes in cellular ion metabolism
5.3.1 Basic principles
5.3.1.1 Cell volume changes
5.3.2 Calcium
5.3.2.1 Keeping it low
5.3.2.2 Oxidative stress raises Ca2+ levels
5.3.2.3 Ca2+ and mitochondria
5.3.3 Oxidative stress and transition metal ion mobilization
5.3.3.1 Demonstrating iron mobilization
5.3.4 Copper
5.4 Consequences of oxidative stress: cell death
5.4.1 Basic definitions
5.4.2 Apoptosis
5.4.2.1 Molecular mechanisms of apoptosis
5.4.2.2 Reactive species and apoptosis
5.5 Redox regulation
5.5.1 What is it and how does it work?
5.5.2 Bacterial redox regulation: oxyR, soxRS and HOCl-sensitive transcription factors
5.5.3 Redox regulation in yeast
5.5.4 Redox regulation in animals: kinases and phosphatases
5.5.4.1 What is it about?
5.5.4.2 Protein kinases
5.5.4.3 How do RS modulate signalling?
5.5.4.4 Reactive species as mediators of the actions of signalling molecules?
5.5.5 Mitochondrial communication by ROS?
5.5.6 NF-κB
5.5.6.1 ROS or no ROS?
5.5.7 AP-1
5.5.8 The antioxidant response element and Nrf2
5.5.9 Co-operation and combination
5.5.10 Physiological significance of redox regulation in animals
5.5.11 Lessons from an amoeba
5.6 Heat-shock and related ‘stress-induced’ proteins; cross-talk with ROS
5.7 Cytokines, hormones, and redox-regulation of the organism
5.7.1 TNF-α
5.7.2 Interleukins
5.7.3 Transforming growth factors β
5.7.4 The acute-phase response
5.8 Mechanisms of damage to cellular targets by oxidative stress: DNA
5.8.1 DNA structure
5.8.2 Damage to DNA by reactive species
5.8.2.1 Hydroxyl radical
5.8.2.2 Hydrogen peroxide and the role of transition metals
5.8.2.3 Use of iron and hydrogen peroxide for oxidative ‘footprinting’
5.8.2.4 Singlet oxygen
5.8.2.5 Carbonate radical anion
5.8.2.6 Peroxyl and alkoxyl radicals
5.8.2.7 Hypohalous acids
5.8.2.8 Ozone
5.8.2.9 Reactive nitrogen species
5.8.2.10 Ultraviolet light
5.8.2.11 Oxidation of oxidation products
5.8.3 Damage to mitochondrial and chloroplast DNA
5.9 Consequences of damage to DNA and RNA by reactive species
5.9.1 Mutation
5.9.2 Slowing protein synthesis
5.9.3 Misincorporation
5.9.4 Changes in gene expression
5.9.5 Having sex
5.10 Repair of oxidative DNA damage
5.10.1 Reversing the chemical change
5.10.2 Don’t let it in: sanitization of the nucleotide pool
5.10.3 Cut it out: excision repair
5.10.4 Mismatch repair
5.10.5 Repair of 8-hydroxyguanine (8OHG)
5.10.6 Repair of double-strand breaks
5.10.7 Mitochondrial DNA repair
5.10.8 Is DNA repair important?
5.10.8.1 Bacteria to mice
5.10.8.2 Mice to men
5.10.9 Polymorphisms in genes encoding antioxidant and repair enzymes
5.10.10 Dealing with oxidative RNA damage
5.11 Mechanisms of damage to cellular targets by oxidative stress: lipid peroxidation
5.11.1 A history of peroxidation: from oils and textiles to breast implants, fish meal, and plastic wrapping
5.11.2 Targets of attack: membrane lipids and proteins
5.11.2.1 What’s in a membrane?
5.11.2.2 Membrane structure
5.11.3 Targets of attack: dietary lipids and lipoproteins
5.11.4 How does lipid peroxidation begin?
5.11.5 Propagation of lipid peroxidation
5.11.6 Transition metals and lipid peroxidation
5.11.6.1 Iron
5.11.6.2 Copper
5.11.6.3 Other metals
5.11.7 Microsomal lipid peroxidation
5.11.8 Acceleration of lipid peroxidation by species other than oxygen radicals
5.11.8.1 Singlet oxygen
5.11.8.2 Reactive halogen species
5.11.8.3 Adding organic peroxides or azo initiators
5.12 Lipid peroxidation products: bad, good, or indifferent?
5.12.1 General effects
5.12.2 Lipid hydroperoxides (ROOH)
5.12.3 Isoprostanes, isoketals, and cyclopentenone compounds
5.12.4 Cholesterol oxidation products (COPs)
5.12.5 Decomposition products of lipid peroxides: yet more bioactive products
5.12.5.1 Ethane and pentane
5.12.5.2 Malondialdehyde
5.12.5.3 4-Hydroxy-2-trans-nonenal (HNE), acrolein, and other unsaturated aldehydes
5.12.6 Peroxidation of other molecules
5.12.7 Repairing oxidized lipids?
5.12.8 Lipids as antioxidants?
5.12.8.1 The plasmalogens
5.13 Mechanisms of damage to cellular targets by oxidative stress: protein damage
5.13.1 Does protein damage matter?
5.13.2 How does protein damage occur?
5.13.3 Chemistry of protein damage
5.13.4 Damage to specific amino acid residues
5.13.4.1 Cysteine and methionine
5.13.4.2 Histidine
5.13.4.3 Proline, lysine, and arginine
5.13.4.4 Tryptophan
5.13.4.5 Tyrosine and phenylalanine
5.13.4.6 Valine, leucine, and other aliphatic amino acids
5.13.4.7 Hydroxy-amino acids (serine and threonine)
5.14 Dealing with oxidative protein damage
5.14.1 Repair of methionine residues
5.14.1.1 A methionine cycle?
5.14.2 Removal: spatial segregation
5.14.3 Removal: proteolysis
5.14.3.1 Autophagy
5.14.3.2 Lon proteinase and the proteasome
5.14.3.3 Any role for ubiquitin?
5.14.3.4 Clogging up the proteasome
5.15 Summary: oxidative stress and cell injury
6 Measurement of reactive species
6.1 Introduction
6.1.1 Trapping
6.1.2 Fingerprinting: the biomarker concept
6.2 ESR and spin trapping
6.2.1 What is ESR?
6.2.2 Measurement of oxygen
6.2.3 Spin trapping
6.2.4 DMPO, DEPMO, and PBN
6.2.5 Ex vivo trapping in humans
6.2.6 Cautions in the use of spin traps
6.2.7 Trapping thiyl radicals
6.2.8 Spin trapping without ESR?
6.3 Other trapping methods, as exemplified by hydroxyl radical trapping
6.3.1 Aromatic hydroxylation
6.3.1.1 Aromatic hydroxylation
6.3.2 Use of hydroxyl radical scavengers
6.3.3 The deoxyribose assay
6.3.4 Measurement of rate constants for OH• reactions
6.3.5 Other trapping methods for hydroxyl radical
6.4 Detection of superoxide
6.4.1 The aconitase assay for superoxide
6.4.2 Rate constants for reactions with O•–2
6.4.3 Triphenyl radical-based probes
6.4.4 Histochemical detection
6.5 Detection of nitric oxide
6.5.1 Calibration
6.6 Detection of peroxynitrite
6.6.1 Probes for peroxynitrite
6.6.2 Nitration assays
6.6.2.1 Specificity for peroxynitrite?
6.6.2.2 Accuracy of nitration assays?
6.7 Detection of reactive halogen species
6.8 Detection of singlet oxygen
6.8.1 Direct detection
6.8.2 Use of scavengers and traps
6.8.3 Deuterium oxide (D2O)
6.9 Studies of ‘generalized’ light emission (luminescence/fluorescence)
6.10 Changes in gene expression: ROS biosensors?
6.11 Detection of hydrogen peroxide
6.11.1 Fluorescent ‘probes’ for H2O2
6.12 Other methods to measure reactive species in cultured cells: be wary of DCFHDA!
6.12.1 2’,7’-Dichlorodihydrofluorescein diacetate
6.12.2 Dihydrorhodamine 123 (DHR)
6.12.3 Dihydroethidium
6.12.4 Luminol, lucigenin, and L-012
6.12.5 Alternative luminescent probes for superoxide
6.12.6 Effects of reactive species on other probes
6.13 Biomarkers: oxidation of bilirubin and of urate
6.14 Biomarkers: oxidative DNA damage
6.14.1 DNA damage: why measure it?
6.14.2 Characterizing DNA damage: what to measure?
6.14.3 Characterizing DNA damage: how to measure it
6.14.4 Steady-state damage: the artefact problem
6.14.5 Overcoming the artefact
6.14.5.1 The comet assay
6.14.6 Interpreting the results: measure DNA levels or urinary excretion? What do the levels mean?
6.14.7 Reactive nitrogen and chlorine species
6.14.8 Gene-specific oxidative damage
6.14.9 RNA oxidation
6.14.10 DNA–aldehyde adducts
6.15 Biomarkers of lipid peroxidation
6.15.1 Why measure lipid peroxidation?
6.15.2 Measurement of peroxidation and peroxidizability
6.15.3 Loss of substrates
6.15.4 Measurement of intermediates
6.15.4.1 Radicals
6.15.4.2 Diene conjugates
6.15.5 Measurement of end-products: peroxides
6.15.6 Measurement of end-products: isoprostanes (IsoPs), isofurans (IsoFs), and isoketals (IsoKs)
6.15.7 Measurement of end products: aldehydes
6.15.8 The thiobarbituric acid (TBA) assay
6.15.8.1 Problem 1: most TBARS (TBA-reactive substances) are generated during the assay
6.15.8.2 Problem 2: false chromogens
6.15.8.3 Problem 3: real chromogens but not from lipids
6.15.8.4 Urinary TBARS
6.15.8.5 Should the TBA assay be abandoned?
6.15.9 Measurement of end-products: breath analysis
6.15.10 Measuring lipid peroxidation: light emission
6.15.11 What is the best method to measure lipid peroxidation in tissues, cells, and body fluids?
6.15.12 Visualizing lipid peroxidation
6.16 Biomarkers of protein damage by reactive species
6.16.1 Damage by reactive oxygen species
6.16.2 Damage by reactive halogen and nitrogen species
6.16.3 The carbonyl assay
6.16.4 Glutathione oxidation and synthesis
6.16.5 γ-Glutamyltranspeptidase
6.16.6 The ‘ thiol-ome’
6.16.7 Advanced oxidation products and modified albumin
6.17 ‘Indirect’ approaches
6.17.1 Erythrocyte and plasma enzymes
6.17.2 Vascular reactivity
6.17.3 Assays of total (‘non-enzymic’) antioxidant capacity
6.17.3.1 What do changes in total antioxidant capacity mean?
6.18 Is there a single biomarker of oxidative stress or oxidative damage?
7 Reactive species can pose special problems needing special solutions: some examples
7.1 Introduction
7.2 The gastrointestinal tract
7.2.1 The threats it faces
7.2.2 Defence systems
7.2.2.1 Saliva
7.2.2.2 Antioxidants from diet?
7.3 The respiratory tract
7.3.1 The challenges
7.3.2 Defending the respiratory tract
7.3.3 Asthma and antioxidants
7.4 Erythrocytes
7.4.1 What problems do erythrocytes face?
7.4.2 Solutions: antioxidant defence enzymes
7.4.3 Solutions: diet-derived antioxidants
7.4.4 Erythrocyte peroxidation in health and disease
7.4.4.1 Problems in blood transfusion
7.4.5 Glucose-6-phosphate dehydrogenase (G6PDH) deficiency
7.4.6 Solutions: destruction
7.5 Erythrocytes as targets for toxins
7.5.1 Hydrazines
7.5.2 Sulphur-containing haemolytic drugs
7.5.3 Favism
7.5.4 Erythrocyte apoptosis
7.6 Bloodthirsty parasites: problems for them and for us
7.6.1 Malaria, oxidative stress, and an ancient Chinese herb
7.7 The problems of plants
7.8 The key to life: photosynthetic oxygen production
7.8.1 Trapping of light energy
7.8.2 The water splitting mechanism: a radical process and the reason for this book
7.8.3 What problems do green leaves face?
7.8.4 Solutions: minimizing the problem
7.8.5 The xanthophyll cycle
7.8.6 Solutions: antioxidant defence enzymes control, but do not eliminate, reactive species
7.8.6.1 Superoxide dismutases
7.8.6.2 Removal of hydrogen peroxide
7.8.6.3 Redox signalling in plants
7.8.7 Ascorbate and glutathione
7.8.8 Plant tocopherols
7.8.9 Solutions: sequestering transition metal ions
7.8.10 Solutions: repair and replacement
7.8.11 The special case of the root nodule
7.9 Plants as targets for stress and toxins
7.9.1 Inhibition of electron transport and carotenoid synthesis
7.9.2 Bipyridyl herbicides
7.9.2.1 Redox cycling
7.9.2.2 Evidence that ROS are important in paraquat toxicity
7.9.3 Environmental stress: air pollutants (ozone, sulphur dioxide, nitrogen dioxide)
7.9.4 Environmental stress: heat, cold, and drought
7.9.5 Coral reef bleaching and toxic algal blooms: examples of plant-dependent oxidative stress?
7.10 The eye
7.10.1 What problems does the eye face?
7.10.1.1 Macular degeneration, lipofuscin, and singlet oxygen
7.10.2 Protecting the eye
7.10.2.1 Screening, prevention, and crying
7.10.2.2 Antioxidants in the eye
7.10.2.3 Sequestration of metal ions
7.10.2.4 Repair of damage
7.10.3 Toxins, inflammation and the eye
7.10.4 Ocular carotenoids: a Chinese herb good for the eyes?
7.10.5 Antioxidants, cataract and macular degeneration
7.11 Reproduction and oxidative stress
7.11.1 Pre-conception: spermatozoa face problems
7.11.2 Spermatozoa: the solutions
7.11.3 Spermatozoa as targets for toxins
7.11.4 The female story
7.11.5 Problems of the embryo
7.11.6 Problems of pregnancy: normal and abnormal O2 levels
7.11.6.1 Endometriosis
7.11.7 The embryo/foetus as a target for toxins
7.11.8 Birth
7.11.8.1 A cold hyperoxic shock
7.11.8.2 Prematurity
7.11.8.3 Antioxidants and babies
7.11.8.4 Iron metabolism in the newborn
7.11.8.5 Parenteral nutrition
7.11.8.6 Antioxidants, PUFAs and iron
7.12 The ear
7.13 The skin
7.13.1 What problems does the skin face?
7.13.1.1 Photosensitization
7.13.1.2 Ultraviolet light
7.13.1.3 Inflammation
7.13.1.4 Air pollutants
7.13.2 Protecting the skin
7.13.3 Wounds and burns
7.14 Skeletal muscle: is exercise a cause of or a protection against oxidative stress?
7.14.1 Exercise, lack of exercise and oxidative damage
7.14.1.1 Antioxidant supplements and exercise
7.14.2 Exercise, health and free radicals
8 Reactive species can be useful: some more examples
8.1 Introduction
8.2 Radical enzymes: ribonucleotide reductase and its colleagues
8.2.1 The enzyme mechanism
8.2.2 Inhibitors of RNRs
8.2.3 Class III ribonucleotide reductases and other ‘sons of SAM’ enzymes
8.2.4 Class II ribonucleotide reductases and other cobalamin radical enzymes
8.3 Pyruvate–formate lyase: a similar mechanism
8.3.1 Pyruvate–ferredoxin oxidoreductase
8.4 Assorted oxidases
8.4.1 Galactose oxidase
8.4.2 Indoleamine and tryptophan dioxygenases
8.5 Useful peroxidases
8.5.1 An ‘antimolestation’ spray
8.5.2 Sea urchins and brine shrimp
8.5.3 Making and degrading lignin
8.5.3.1 Making lignin
8.5.3.2 Breaking lignin down
8.5.3.3 A role for hydroxyl radical?
8.6 Light production
8.6.1 Green fluorescent protein: another example of autocatalytic oxidation
8.7 Phagocytosis
8.7.1 Setting the scene
8.7.2 Neutrophils, monocytes, and macrophages
8.7.3 Phagocyte recruitment, adhesion, activation, and disappearance
8.7.3.1 Getting to the right place
8.7.3.2 What must neutrophils do?
8.7.4 How do phagocytes kill?
8.7.4.1 Phagocytes show a respiratory burst
8.7.4.2 Priming of the respiratory burst
8.7.4.3 The respiratory burst makes superoxide
8.7.4.4 Superoxide is required to kill some bacteria
8.7.4.5 So how does superoxide kill? Via H2O2?
8.7.4.6 Via hydroxyl radical?
8.7.4.7 Via singlet O2?
8.7.4.8 Via peroxynitrite?
8.7.4.9 By facilitating the action of other microbicidal agents?
8.7.4.10 Interference with quorum sensing
8.7.4.11 By NETs formation
8.7.4.12 Fitting it together
8.7.5 Myeloperoxidase (MPO)
8.7.5.1 Hypochlorous acid production
8.7.5.2 The MPO reaction mechanism
8.7.5.3 Singlet O2 from MPO?
8.7.5.4 The enigma of MPO
8.7.5.5 Nitration by MPO
8.7.5.6 Peroxidasins
8.7.5.7 Other defensive peroxidases
8.7.5.8 Fitting it together (part 2)
8.8 Other phagocytes: similar but different
8.9 What do phagocyte-derived reactive species (RS) do to the host?
8.9.1 Extracellular RS: what can they do?
8.9.2 Signalling
8.9.3 Damage to the phagocyte
8.9.4 RS: promoters or suppressors of chronic inflammation?
8.9.5 What does it all mean? Are RS both pro- and anti-inflammatory?
8.9.6 Defeating the defences: bacterial and fungal avoidance strategies
8.10 NADPH oxidases in other cell types
8.10.1 The gastrointestinal and respiratory tracts
8.10.2 Thyroid hormone synthesis
8.10.2.1 Iodide as an antioxidant
8.10.3 C. elegans
8.10.4 Blood vessel walls and the regulation of blood pressure
8.10.5 Lymphocytes
8.10.6 Renal function and oxygen sensing
8.10.7 Platelets
8.10.8 Bone formation and degradation
8.10.9 Other redox systems
8.11 Plants use reactive species for defence and regulation
8.11.1 Plant NOXes
8.11.2 The hypersensitive response
8.11.3 Plant lipoxygenases
8.11.4 The injury response and oxylipin signalling
8.11.5 Germination and senescence
8.12 Animal lipoxygenases and cyclooxygenases: stereospecific lipid peroxidation
8.12.1 Oxidation of PUFAs by enzymes
8.12.2 Eicosanoids: prostaglandins and leukotrienes
8.12.3 Prostaglandins and thromboxanes
8.12.4 Prostaglandin synthesis
8.12.5 Regulation by ‘peroxide tone’
8.12.6 Prostaglandins from isoprostanes? Cross-talk of the systems
8.12.7 Levuglandins
8.12.8 Prostacyclins and thromboxanes
8.12.9 Leukotrienes and other lipoxygenase products
9 Reactive species can be poisonous: their role in toxicology
9.1 Introduction
9.1.1 What is toxicology?
9.1.2 Principles of toxin metabolism
9.1.3 How can reactive species contribute to toxicity?
9.2 Carbon tetrachloride
9.2.1 Carbon tetrachloride synthesis: a free-radical chain reaction
9.2.2 Toxicity of CCl4
9.2.3 How does CCl4 cause damage?
9.3 Other halogenated hydrocarbons
9.3.1 Chloroform and bromotrichloromethane
9.3.2 Pentachlorophenol and related environmental pollutants
9.4 Redox-cycling toxins: bipyridyl herbicides
9.4.1 Toxicity to bacteria
9.4.2 Toxicity to animals
9.4.3 Why is paraquat toxic to the lung?
9.5 Redox-cycling toxins: diphenols, quinones, and related molecules
9.5.1 Interaction with O2 and superoxide
9.5.2 Interaction with metals
9.5.3 Mechanisms of toxicity
9.5.4 Quinone reductase
9.5.5 Catechol oestrogens
9.5.6 Substituted dihydroxyphenylalanines and ‘manganese madness’
9.5.7 Neurotoxicity of 6-hydroxydopamine
9.5.8 Benzene and its derivatives
9.5.9 Toxic-oil syndrome and a new Society
9.6 Redox-cycling agents: toxins derived from Pseudomonas aeruginosa
9.7 Diabetogenic drugs
9.7.1 Alloxan
9.7.2 Streptozotocin
9.8 Alcohols
9.8.1 Ethanol
9.8.1.1 Ethanol metabolism and CYP2E1
9.8.1.2 Ethanol toxicity
9.8.1.3 Does ethanol increase RS formation?
9.8.1.4 How does ethanol cause oxidative stress?
9.8.1.5 Does oxidative damage explain ethanol toxicity?
9.8.1.6 Other liver diseases
9.8.1.7 Therapeutic options?
9.8.2 Allyl alcohol and acrolein
9.9 Other recreational drugs
9.10 Paracetamol (acetaminophen) and naphthalene, glutathione-depleting toxins
9.11 Chlorine gas
9.12 Air pollutants
9.12.1 Ozone
9.12.2 Nitrogen dioxide
9.12.2.1 Nitrogen dioxide as a free radical
9.12.2.2 Antioxidants and nitrogen dioxide
9.12.3 Sulphur dioxide
9.13 Toxicity of mixtures: ‘real’ air pollution, cigarette smoke, and other toxic smokes
9.13.1 Chemistry of tobacco smoke
9.13.2 Mechanisms of damage by cigarette smoke
9.13.3 How does the respiratory tract defend itself?
9.13.4 Adaptation
9.13.5 Environmental tobacco smoke (ETS)
9.13.6 Other tobacco usage
9.13.7 Other smokes, fumes, and dusts
9.14 Diesel exhaust and airborne particulates
9.14.1 Nanoparticles
9.15 Toxicity of asbestos and silica
9.16 Toxicity of metals
9.16.1 Cause or consequence?
9.16.2 Arsenic
9.16.3 Nickel
9.16.4 Chromium
9.16.5 Cobalt
9.16.6 Cadmium
9.16.7 Mercury
9.16.8 Lead
9.16.9 Vanadium
9.16.10 Titanium
9.16.11 Aluminium
9.16.12 Zinc
9.17 Antibiotics
9.17.1 Tetracyclines as pro- and antioxidants
9.17.2 Quinone antibiotics
9.17.3 Aminoglycoside nephrotoxicity
9.18 Stress
9.19 Nitro and azo compounds
9.19.1 Nitro radicals and redox cycling
9.19.2 Further reduction of nitro radicals
9.19.3 Azo compounds
9.20 Ionizing radiation
9.20.1 The oxygen effect
9.20.1.1 A role for superoxide?
9.20.2 Antioxidants and radiotherapy
9.20.3 Hypoxic cell sensitizers
9.20.4 Food irradiation
9.21 Summary and conclusion
10 Reactive species in disease: friends or foes?
10.1 Setting the scene
10.2 Does oxidative stress matter?
10.2.1 Establishing importance
10.3 Atherosclerosis
10.3.1 What is atherosclerosis?
10.3.2 Predictors of atherosclerosis
10.3.3 What initiates atherosclerosis?
10.3.4 LDL oxidation and the foam cell
10.3.5 Mechanisms of LDL oxidation
10.3.5.1 Reactive nitrogen and chlorine species
10.3.5.2 Metal ions
10.3.5.3 Lipoxygenases
10.3.5.4 Summing it up: which pro-oxidant(s) oxidize LDL in vivo?
10.3.6 Other aspects of the involvement of RS in atherosclerosis
10.3.7 Does evidence support the ‘oxidative modification hypothesis’ of atherosclerosis?
10.3.8 Chemistry of LDL oxidation: is in vitro LDL oxidation a relevant model?
10.3.8.1 The role of ‘seeding peroxides’
10.3.8.2 Antioxidants and LDL oxidation
10.3.8.3 Pro-oxidant effects of antioxidants
10.3.8.4 Relevance of the model
10.3.8.5 An artefact of eating?
10.3.8.6 Subclasses of LDL
10.3.9 The role of high-density lipoproteins (HDL)
10.3.10 Lipoprotein(a)
10.3.11 Unanswered questions
10.4 Obesity and its opposite
10.5 Diabetes
10.5.1 Can oxidative stress cause diabetes?
10.5.2 ROS in normal insulin function and insulin resistance
10.5.3 Oxidative stress in diabetic patients
10.5.4 How does the oxidative stress originate?
10.5.5 Non-enzymatic glycation and glycoxidation
10.5.5.1 Reversing AGEing?
10.5.6 Other mechanisms of glucose toxicity
10.5.7 A summary: how important is oxidative stress in diabetes?
10.5.7.1 Do antioxidant supplements help diabetic patients?
10.6 Ischaemia–reperfusion
10.6.1 Reoxygenation injury
10.6.2 A role for xanthine oxidase?
10.6.3 Intestinal ischaemia–reoxygenation
10.6.4 Cardiac ischaemia–reoxygenation
10.6.4.1 The phenomenon
10.6.4.2 Importance of the model used
10.6.4.3 The relevance of xanthine oxidase
10.6.4.4 The relevance of transition metals
10.6.4.5 Nitric oxide: good or bad?
10.6.4.6 Heart failure
10.6.4.7 Clinical relevance
10.6.4.8 Cardiopulmonary bypass
10.6.5 Angioplasty, restenosis, and bypass grafting
10.6.6 Ischaemic preconditioning
10.6.7 Shock- and sepsis-related ischaemia–reoxygenation
10.6.7.1 Aneurysm
10.6.8 The eye
10.6.9 Chemical ischaemia–reperfusion: carbon monoxide poisoning
10.6.10 Cold and freezing injury: the enigma of biopsies
10.6.11 Sleep apnoea
10.7 Organ preservation, transplantation, and reattachment of severed tissues
10.7.1 Heart
10.7.2 Kidney
10.7.3 Liver and pancreas
10.7.4 Limbs, digits, and sex organs
10.7.5 Organ preservation fluids
10.7.6 Other examples
10.8 Lung transplants, shock, and ARDS
10.8.1 Oxidative stress in ARDS: does it occur and does it matter?
10.9 Cystic fibrosis
10.9.1 Cystic fibrosis and carotenoids
10.10 Some autoimmune diseases
10.10.1 Adverse drug reactions
10.10.2 Are RS important mediators of autoimmune diseases?
10.10.2.1 Artefacts to watch for: contamination of commercial antioxidants and oxidation on sample storage
10.10.2.2 Periodontal disease: a missed opportunity?
10.11 Rheumatoid arthritis
10.11.1 The normal joint
10.11.2 The RA joint
10.11.3 How does increased oxidative damage arise in RA?
10.11.4 Does oxidative damage matter in RA?
10.11.5 Drugs to treat RA: antioxidant, pro-oxidant, or neither?
10.11.6 Iron and rheumatoid arthritis
10.12 Inflammatory bowel disease
10.12.1 The salazines
10.12.2 Coeliac disease
10.13 Inflammation of other parts of the gastrointestinal tract
10.13.1 Pancreas
10.13.2 Oesophagus and stomach
10.13.3 Liver
10.14 Oxidative stress and cancer: a complex relationship
10.14.1 The cell cycle
10.14.2 Tumours
10.14.3 Carcinogenesis
10.14.3.1 Initiation
10.14.3.2 Tumour promoters
10.14.3.3 Progression
10.14.4 Genes and cancer
10.14.4.1 Oncogenes
10.14.4.2 Tumour suppressor genes
10.14.4.3 Stability genes
10.14.4.4 Angiogenesis and cancer
10.14.5 Reactive species and carcinogenesis: basic concepts
10.14.6 p53 and ROS
10.14.7 Changes in antioxidant defences in cancer
10.14.8 ROS and cancer
10.14.8.1 DNA damage by RS
10.14.8.2 Is there increased oxidative DNA damage in cancer?
10.14.8.3 A role for reactive nitrogen and chlorine species
10.14.8.4 Epigenetics, cell proliferation, and HIF-1α
10.14.8.5 Intercellular communication
10.14.8.6 Suppressing apoptosis
10.14.8.7 Metastasis and angiogenesis
10.14.8.8 Affecting stem cells
10.14.9 Cancer and cachexia
10.14.10 Are malignant cells truly under oxidative stress?
10.14.11 Chronic inflammation and cancer: a close link but is it due to reactive species?
10.14.12 Transition metals and cancer
10.15 Carcinogens: oxygen and others
10.15.1 Carcinogen metabolism
10.15.1.1 Carcinogens can make RS
10.15.2 Carcinogens and oxidative DNA damage
10.15.2.1 Peroxisome proliferators
10.15.3 Carcinogenic reactive nitrogen species?
10.16 Cancer chemotherapy and reactive oxygen species
10.16.1 Oxidative stress and chemotherapy
10.16.2 The anthracyclines and other quinones
10.16.2.1 Mechanisms of cardiotoxicity: redox cycling and others
10.16.2.2 Iron and anthracyclines
10.16.3 Bleomycin
10.16.3.1 Side-effects of bleomycin
10.16.4 Should cancer patients consume antioxidants?
10.17 Oxidative stress and disorders of the nervous system: setting the scene
10.17.1 Introduction to the brain
10.17.2 Energy metabolism in the brain
10.17.3 Glutamate, calcium, and nitric oxide
10.17.4 Excitotoxicity
10.17.5 Why should the brain be prone to oxidative stress? ROS are both useful and deleterious
10.17.6 Antioxidant defences in the brain
10.17.6.1 Keeping oxygen low
10.17.6.2 Superoxide dismutases and peroxide-removing enzymes
10.17.6.3 Glutathione and ergothioneine
10.17.6.4 Protecting brain mitochondria
10.17.6.5 Ascorbate
10.17.6.6 Vitamin E
10.17.6.7 Coenzyme Q
10.17.6.8 Histidine-containing dipeptides
10.17.6.9 Plasmalogens
10.17.6.10 Carotenoids and flavonoids
10.17.6.11 Metal-binding and related protective proteins
10.17.6.12 Repair of oxidative damage
10.17.6.13 Defence of the blood–brain barrier
10.18 Oxidative stress in ischaemia, inflammation, and trauma in the nervous system
10.18.1 Inflammation: a common feature
10.18.2 Multiple sclerosis
10.18.3 Brain injury: stroke
10.18.3.1 Mediators of damage
10.18.3.2 Therapeutic interventions?
10.18.4 Traumatic injury
10.19 Oxidative stress and neurodegenerative diseases: some general concepts
10.19.1 The role of iron
10.19.2 Are aggregates toxic?
10.20 Parkinson disease
10.20.1 Genetics or environment?
10.20.2 Treatment
10.20.3 Environmental toxins and PD
10.20.4 The vicious cycle: proteasomal dysfunction, oxidative stress, and mitochondrial defects in PD
10.20.4.1 Early or late?
10.20.5 Summing it up; insights from PINK1 and DJ-1
10.21 Alzheimer disease
10.21.1 Definition and pathology
10.21.2 Genetics of AD
10.21.3 Mechanisms of neurodegeneration
10.21.4 Oxidative damage in AD: cause or consequence?
10.21.5 Impairment of proteolysis
10.21.6 An old red herring: aluminium in AD
10.21.7 Diet, lifestyle, and AD
10.21.8 Other amyloid diseases
10.21.9 Prion diseases
10.22 Amyotrophic lateral sclerosis (ALS)
10.22.1 Familial ALS (FALS) and superoxide dismutase
10.22.2 Oxidative damage and excitotoxicity in ALS
10.22.2.1 Therapies
10.23 Other diseases of the brain and nervous system
10.23.1 Friedreich ataxia
10.23.2 Huntington disease
10.23.3 Neuronal ceroid lipofuscinoses
10.24 Pain
10.25 Oxidative stress and viral infections
10.25.1 Reactive species, antioxidants, and HIV
10.25.1.1 Changes in glutathione?
10.25.2 Redox regulation of viral expression
10.25.3 Side-effects of therapy
11 Ageing, nutrition, disease, and therapy: a role for antioxidants?
11.1 Introduction
11.2 Theories of ageing; the basics
11.2.1 General principles
11.2.2 What features of ageing must theories explain?
11.2.2.1 Caloric restriction (CR)
11.2.2.2 Obesity, oxidative stress, and CR
11.3 What theories of ageing exist?
11.3.1 Do genes influence ageing? The story of C. elegans
11.3.1.1 What about mammals?
11.3.2 Genes and human longevity
11.3.3 Premature human ageing
11.3.4 Mechanisms of caloric restriction; learning from yeast
11.3.5 Telomeres and cellular senescence
11.3.5.1 An artefact of cell culture?
11.4 Oxidative damage: a link between the theories of ageing?
11.4.1 Introduction to the free-radical theory of ageing
11.4.2 Do ROS production and oxidative damage increase with age?
11.4.2.1 Be cautious with global biomarkers
11.4.3 Is the rise in oxidative damage due to failure of antioxidant protection with age?
11.4.4 Is there a failure to repair oxidative damage with age?
11.4.5 Testing the free-radical theory of ageing: altering antioxidant levels
11.4.5.1 Transgenic organisms: a confusing picture
11.4.6 ‘Rapidly ageing’ rodents
11.4.7 Lipofuscin and ceroid; fluorescent ‘red herrings’?
11.4.8 Is the oxidative damage theory of ageing ageing badly?
11.4.9 How to live a long time
11.4.10 Iron, ageing, and disease: another gender gap
11.5 Antioxidants to treat disease
11.5.1 Therapeutic antioxidants
11.5.2 Approaches to antioxidant characterization
11.5.3 Superoxide dismutases, catalases, and nanoparticles
11.5.3.1 Viral vectors
11.5.4 SOD mimetics and related redox-active molecules
11.5.5 Spin traps/nitroxides
11.5.6 Vitamins C and E, carnosine, and their derivatives
11.5.7 Coenzyme Q and synthetic chain-breaking antioxidants
11.5.8 Dual action molecules and edaravone
11.5.9 Thiol compounds
11.5.9.1 Glutathione
11.5.9.2 N-Acetylcysteine
11.5.9.3 Other thiols
11.5.9.4 Thiols as radioprotectors
11.5.10 Glutathione peroxidase ‘mimetics’
11.5.11 ‘Pro-oxidants’ and Nrf2 activators
11.5.12 Mitochondrially targeted antioxidants
11.6 Iron and copper ion chelators
11.6.1 Desferrioxamine
11.6.2 Other iron-chelating agents
11.7 Inhibitors of the generation of reactive species
11.7.1 Xanthine oxidase (XO) inhibitors
11.7.2 Myeloperoxidase inhibitors
11.7.3 Inhibitors of phagocyte action
11.7.4 NADPH oxidase inhibitors
11.8 Agents to watch
Appendix: Some basic chemistry
A1 Atomic structure
A2 Bonding between atoms
A2.1 Ionic bonding
A2.2 Covalent bonding
A2.3 Non-ideal character of bonds
A2.4 Hydrocarbons and electron delocalization
A3 Moles and molarity
A4 pH and pKa
References
Index