Oxidative Stress and Antioxidant Protection: The Science of Free Radical Biology and Disease

List of contributors

Aneela Afzal

Advanced Imaging Research Center Oregon Health and Science University Portland, OR, USA

Mohammad Afzal

Department of Biological Sciences Faculty of Science Kuwait University Safat, Kuwait

Ashok Agarwal

American Center for Reproductive Medicine Cleveland Clinic Cleveland, OH 44195, USA

María José Alcaraz

Department of Pharmacology and IDM University of Valencia Valencia, Spain

Ryyan Alobaidi

Pathology Department King Saud University Riyadh, KSA

Juan A. Ardura

Bone and Mineral Metabolism Laboratory Instituto de Investigación Sanitaria (IIS) – Fundación Jiménez Díaz and UAM Madrid, Spain

Donald Armstrong

Department of Biotechnical and Clinical Laboratory SciencesState University of New York at Buffalo Buffalo, NY, USA

Department of Ophthalmology University of Florida College of Medicine Gainesville, FL, USA

Ines Batinic-Haberle

Department of Radiation Oncology Duke University School of Medicine Durham, NC, USA

Maurizio Battino

Department of Dentistry and Specialized Clinical Sciences Biochemistry Section Università Politecnica delle Marche Ancona, Italy

Bogdan Calenic

Department of Biochemistry Faculty of Dental Medicine University of Medicine and Pharmacy ‘CAROL DAVILA’ Bucharest, Romania

Jing Chen

Department of Pathology, Immunology, and Laboratory Medicine University of Florida College of Medicine Gainesville, FL, USA

Xiwei Chen

Department of Biostatistics School of Public Health and Health Professions State University of New York at Buffalo

Renan C. Chisté

Departamento de Ciências Químicas Faculdade de Farmácia Universidade do Porto REQUIMTE 4050-313 Porto, Portugal

Patrick Colahan

Department of Large Animal Clinical Sciences College of Veterinary Medicine University of Florida Gainesville, FL, USA

D. Scott Covington

Healogics, Inc. Jacksonville, FL, USA

Anna Dietrich-Muszalska

Department of Biological Psychiatry The Chair of Experimental and Clinical Physiology Medical University of Lodz Lodz, Poland

Herminia Domínguez

Departamento de Enxeñería Química Facultad de Ciencias Universidade de Vigo (Campus Ourense) Ourense, Spain

Hassan A. N. El-Fawal

The Pharmaceutical Research Institute and Neurotoxicology Laboratory Albany College of Pharmacy and Health Sciences Albany, NY, USA

Pedro Esbrit

Bone and Mineral Metabolism Laboratory Instituto de Investigación Sanitaria (IIS) – Fundación Jiménez Díaz and UAM Madrid, Spain

Elena Falqué

Departamento de Química Analítica Facultad de Ciencias Universidade de Vigo (Campus Ourense) Ourense, Spain

Eduarda Fernandes

Departamento de Ciências Químicas Faculdade de Farmácia Universidade do Porto REQUIMTE 4050-313 Porto, Portugal

Marisa Freitas

Departamento de Ciências Químicas, Faculdade de Farmácia Universidade do Porto REQUIMTE 4050-313 Porto, Portugal

Natan Gadoth

Department of Neurology Maynei Hayeshua Medical Center and The Sackler Faculty of Medicine Tel Aviv University Tel Aviv, Israel

Reza Ghiasvand

Department of Community Nutrition School of Nutrition and Food Sciences Isfahan University of Medical Sciences Isfahan, Iran

Steve Ghivizzani

Department of Orthopedics and Rehabilitation University of Florida College of Medicine Gainesville, FL, USA

Maria Greabu

Department of Biochemistry Faculty of Dental Medicine University of Medicine and Pharmacy ‘CAROL DAVILA’ Bucharest, Romania

Mitra Hariri

Department of Community Nutrition School of Nutrition and Food Sciences Isfahan University of Medical Sciences Isfahan, Iran

M. Elizabeth Hartnett

Department of Ophthalmology Moran Eye Center University of Utah Salt Lake City, UT, USA

Hanbo Hu

Pulmonary Division Department of Medicine University of Florida Gainesville, FL, USA Malcom Randall Veteran Affairs Medical Center Gainesville, FL, USA

Priyanka M. Jadhav

Department of Pathology, Immunology and Laboratory Medicine UF Diabetes Institute College of Medicine University of Florida College of Medicine Gainesville, FL, USA

Surinder K. Jindal

Jindal Clinics Chandigarh, India

Colleen G. Le Prell

Callier Center for Communication Disorders School of Behavioral and Brain Sciences University of Texas at Dallas Dallas, TX, USA

Judith Lightsey

Department of Radiation Oncology University of Florida College of Medicine Gainesville, FL, USA

Bin Liu

Department of Pharmacodynamics College of Pharmacy University of Florida Gainesville, FL, USA

Chao Liu

Department of Pathology, Immunology, and Laboratory Medicine University of Florida Gainesville, FL, USA

Clayton E. Mathews

Department of Pathology, Immunology, and Laboratory Medicine University of Florida Gainesville, FL, USA

Josef M. Miller

Department of Otolaryngology Kresge Hearing Research Institute University of Michigan Ann Arbor, MI, USA

Christina L. Mitchell

Department of Dermatology University of Florida College of Medicine Gainesville, FL, USA

Arshag D. Mooradian

Department of Medicine University of Florida College of Medicine Jacksonville, FL, USA

Shaker A. Mousa

The Pharmaceutical Research Institute Albany College of Pharmacy and Health Sciences Rensselaer, NY, USA

Carlos Palacio

Department of Medicine University of Florida College of Medicine Jacksonville, FL, USA

Aaron Panicker

Department of Urology Wayne State University Detroit, MI, USA

Sergio Portal-Núñez

Bone and Mineral Metabolism Laboratory Instituto de Investigación Sanitaria (IIS) – Fundación Jiménez Díaz and UAM Madrid, Spain

Robert Rembisz

The Pharmaceutical Research Institute and Neurotoxicology Laboratory Albany College of Pharmacy and Health Sciences Albany, NY, USA

Heinrich Sauer

Department of PhysiologyFaculty of Medicine Justus-Liebig University Giessen, Germany

Fatemeh Sharifpanah

Department of Physiology Faculty of Medicine Justus-Liebig UniversityGiessen, Germany

Bechan Sharma

Department of Biochemistry Faculty of Science University of Allahabad Allahabad, India

Janet H. Silverstein

Department of Pediatrics University of Florida College of Medicine Gainesville, FL, USA

Shweta Singh

Department of Biochemistry Faculty of Science University of Allahabad Allahabad, India

Robert D. Stratton

Department of Ophthamology University of Florida College of Medicine

Department of Small Animal Clinical Sciences College of Veterinary Medicine University of Florida Gainesville, FL, US

Hanna Tadros

Center for Reproductive Medicine Glickman Urological and Kidney Institute Cleveland Clinic Cleveland, OH, USA

Rajiv Tikamdas

Department of Pharmacodynamics College of Pharmacy University of Florida Gainesville, FL, USA

Artak Tovmasyan

Department of Radiation Oncology Duke University School of Medicine Durham, NC, USA

Hirokazu Tsukahara

Department of Pediatrics Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

Eva Tvrdá

Department of Animal Physiology Slovak University of Agriculture Nitra, Slovakia

Albert Vexler

Department of Biostatistics School of Public Health and Health Professions State University of New York at Buffalo Buffalo, NY, USA

Rachael Watson

Department of Orthopedics and Rehabilitation University of Florida College of Medicine Gainesville, FL, USA

James R. Wilcox

Department of Clinical Research & Physician Education Serena GroupCambridge, MA, USA

William E. Winter

Department of Pathology, Immunology and Laboratory Medicine University of Florida College of Medicine Gainesville, FL, USA

Justin Wray

Department of Radiation Oncology University of Florida College of Medicine Gainesville, FL, USA

Masato Yashiro

Department of Pediatrics Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

Ping Zhang

Department of Pharmacodynamics College of Pharmacy University of Florida Gainesville, FL, USA

Special recognition

In memory of Christine M. Armstrong the editors and authors of this textbook unanimously acknowledge her for her dedication to support the concept of oxidative stress and antioxidant therapy.

She was instrumental in typing the first International Symposium in 1982 and over many years helped prepare other manuscripts from her knowledge of English grammar and sentence structure into the series on Methods and Protocols and the series on Oxidative Stress in Basic Research and Clinical Practice. She worked laboriously in transcribing numerous symposia.

Anyone who met Chris knew her persona. She showed a gracious and genuine interest in the diverse efforts of many colleagues who wrote in the Protocols and Clinical Practice series. She was always an advocate for natural foods and antioxidant supplements. Chris died on May 27, 2014, from pancreatic cancer and will be sorely missed by everyone who were fortunate to know her on a personal and spiritual level. We dedicate this textbook as a token of our esteem.

For his help in research for this textbook, the editors thank Dennis E. Armstrong.

Foreword

Don Armstrong and I grew up together in Oregon and Colorado, both at the lab bench and during shared family gatherings over several decades. Half a century ago, we coauthored two papers on sulfatase deficiencies responsible for two human neurological diseases. It is both a special privilege and an honor to add a personal observation to my long-esteemed colleague's latest textbook.

In 1963, neither he nor I could have anticipated the remarkable scope and depth of his many scientific interests, research publications, careers as a teacher, and related editorial accomplishments. His efforts over that time span have helped clarify the intricate biochemical and subcellular mechanisms of oxidative damage, in both human disease and animal models. These pivotal mechanisms have a practical application – helping us all defer that condition overly simplified in the old phrase, normal aging.

I hope that as you read the chapters of this extraordinary text written by a cadre of international experts, the diversity of topics reflects the inspired efforts of an exceptionally foresighted person who envisioned long ago how crucial this whole field would become in biology and medicine.

James H. Austin, MD

Professor Emeritus

University of Colorado Health and Science Center

Columbia, Missouri

Preface

This textbook starts with the principles of the oxidative stress process and a historical perspective of oxidative stress pioneers to explain the basic principles related to biochemistry and molecular biology showing pathways and biomarkers. The section also gives an explanation of differential diagnosis and brief descriptions of the use of diagnostic imaging.

The second section covers clinical correlations on acute and chronic disease and discusses our novel approach that bridges the gap between these two concepts. It shows how oxidative damage and inflammation trigger the disease process and how compromised immunity then leads to the proliferation of disease, with a bench-to-bedside approach to clinical application. These discussions focus on immune responses in early and chronic diseases. There is a glossary and explanation on medical terminology and a set of questions for the student. The effects of aging, senescence, and life span are presented. A thematic summary box is included for each chapter so that students know what is required of them.

Chapters on Clinical Correlations cover the most common medical diseases and cover developmental change. The goal is to stimulate new directions for student education in their professional career. The clinical topics are taken from a series of books on specific diseases whose emphasis was on oxidative stress and antioxidants entitled, Oxidative Stress in Applied Basic Research and Clinical Practice. This book represents a compilation of scientific and clinical data summarized from books in the series that emphasize result-oriented findings in (1) neurology, psychiatry, and behavioral data including the eye and ear, (2) pediatrics, (3) disorders of skin and musculoskeletal system, (4) gender-related issues, (5) chronic diseases, (6) pain and inflammation management, (7) wellness issues, and (8) biostatistics.

The book can be used as a college text as well as a graduate level course of pre-professional level, providing an overview covering oxidative damage and antioxidant imbalance. This section links science and medicine at the pre-professional and graduate levels and is appropriate for medical and veterinary residents, providing an overview of disease mechanisms related to oxidative damage and antioxidant imbalance.

Our authors have extensive experience in the laboratory or in medical therapy. Each of the chapters provides an understanding of appropriate mechanistic information and what biomarker(s) and physiological functions may be relevant to the over-riding concept of redox issues leading to critical mass of stress oxidants in early disease to chronic conditions including potential alternative therapy. Areas of research that are not yet fully developed are indicated, helping students in choosing a direction for their careers.

The textbook is the first of its kind and should have wide appeal to students majoring in life science, biology, and allied health professions, as well as to physician assistants and nurse practitioners, with a secondary appeal to those in pharmacy, food and nutrition, exercise, sports medicine, psychology, and public health. This textbook will also be of use to programs in herbalist and holistic schools and could likewise be used for residency training and Continuing Medical Education courses.

Our goal was to present the most authoritative source of current and emerging knowledge on oxidative stress-related disorders. To this end, we are confident we have achieved our aim as editors. We are pleased with the collegial support given to us by these authors. We sincerely hope students who study the information presented herein, will gain from this perspective and it will be helpful in their future professional careers.

Donald Armstrong and Robert D. Stratton

Section I

Introduction

Chapter 1

Introduction to free radicals, inflammation, and recycling

Donald Armstrong¹,²

¹Department of Biotechnical and Clinical Laboratory Sciences, State University of New York at Buffalo, Buffalo, NY, USA

²Department of Ophthalmology, University of Florida School of Medicine, Gainesville, FL, USA

This introductory chapter will give you information to fill in the gaps and understand the complexities reported in Chapters 3–31.

THEMATIC SUMMARY BOX

At the end of this chapter, students should be able to:

Show free radical formation

Show how endogenous and exogenous free radicals stimulate and potentially initiate disease

Define inflammation and the immune response

Differentiate between acute and chronic inflammation

Describe pathways leading to apoptosis, necrosis, cell death, and disease

Define pathogenesis

Show how antioxidants scavenge free radicals and participate in recycling pathways

Describe biomarker measurements using their abbreviations

Historical perspective

In 1993, an International Symposium on Free Radicals in Diagnostic Medicine: a systems approach to laboratory technology, clinical correlations, and antioxidant therapy was organized, and in 1994 it was published as volume 366 in Advances in Experimental Medicine and Biology.¹ This was the first attempt to coordinate the various laboratory findings from research publications that were divided into subsections on pathophysiology, analysis, organ-specific disorders, systemic involvement, and therapeutic intervention. In 2007, another International Symposium on Free Radicals in Biosystems was conducted.² These two conferences set the standard for the present textbook, which is an extension of those meetings in the application and understanding of free radical (FR) methods and protocols, and is once again timely 20 years later. The author of this chapter has multiple books in the field.

In 1990, a new series of books covering laboratory techniques in Methods in Molecular Biology (MiMB) was initiated and later became Advanced Protocols 1–3 including separate volumes on lipidomics and nanotechnology that contain specific sections on oxidative stress and antioxidants. This was followed in 2000 by a new series devoted to clinical studies (Oxidative Stress in Applied Basic Research and Clinical Practice), which increased our oxidative stress library database to over 1300 research collaborators from 34 countries and 20 states in the United States.

These books have been a major effort to cover subject areas in advanced detail, which are germane to background information that supports perturbations of lipids and proteins in our concept of oxidative stress. In addition, platform technology on metabolomics and transcriptomics using high-pressure liquid chromatography (HPLC) and mass spectroscopy (MS) can be read in the literature by the student separately and integrated with the state-of-the-art coverage. The result of these activities is the basis for the present textbook, which illustrates the concept of educational research in stress-induced disease reactions.

Oxidative stress concept

The concept of oxidative stress in disease means an environmental stimulus is able to create an FR by random chance. The number of hits per day is estimated at 100,000 coming from the mitochondria as molecular oxygen moves through the electron respiratory chain and from environmental radiation. FRs are characterized by loss of an electron-making species highly reactive to other biochemicals resulting in cellular damage (Figure 1.1). We think of FRs as tipping the balance toward disease, so that as FRs increase along the up slope as a function of progressive disease, antioxidants (AOXs) decrease along the down slope as they are being consumed due to oxidative stress, so that homeostasis tips toward disease. Supplementation with AOX nutraceuticals leads to protection and eventual homeostasis when the two processes become equal, and no disease is clinically evident by conventional physiological testing in the patient populations.

Figure 1.1 Homeostasis is a balance between levels of free radicals (FR) and antioxidants (AOX).

The most damaging FRs are the hydroxyl (HO•) and hydrogen radicals produced during ionizing radiation or environmental toxicology reactions. Superoxide FRs ( c01-math-0001 ) are produced in mitochondrial electron transport reactions. Superoxide dismutase rapidly forms hydrogen peroxide (H2O2) from c01-math-0002 . Reduced free iron Fe²+ and copper Cu¹+ participate in Fenton reactions with H2O2 in the millisecond range to produce hydroxyl radicals and hydroxide anions, oxidizing the metals to Fe³+ and Cu²+. Oxidized free iron or copper can then oxidize H2O2 to form hydroperoxyl (HOO•) radicals and protons (H+), reducing the metals to Fe²+ and Cu¹+, so that new peroxidation reactions can occur in a cyclical manner. Thiol (SH•) and lipidperoxide radicals (L•/LO•/LOO•) are other radicals that cause FR damage to cellular components.

Hydrogen peroxide also interacts at Fe²+ metal-binding sites on heme-containing enzymes to generate the reactive hydroxyl radical. The hydroxyl radical damage to proteins has been shown to produce covalently bound protein aggregates, and when disulfide bridges are causing the aggregation, cross-linked protein adducts are formed. These reactive oxygen radicals modify amino acids at metal-binding sites and facilitate proteolytic attack. Other FR damage includes DNA strand scission and lipid hydroperoxidation (LHP).

Polyunsaturated fatty acids (PUFA) contain multiple carbon–carbon double bonds. These fatty acids provide mobility and fluidity to the plasma membrane, properties which are known to be essential for the proper function of biological membranes. The process of lipid peroxidation (LHP) is a step-wise process with the removal of an electron at the initiation step and at subsequent propagation reactions. Iron salts and other iron complexes help initiate the process by forming alkoxy or peroxy radicals upon reaction with oxygen species.

In general, there are three damaging consequences of LHP within the plasma membrane in vivo. The first consequence is a decrease in membrane fluidity. Saturated fatty acids are structurally more rigid than the flexible PUFA. The second consequence of LHP is an increase in the leakiness of the membrane pores to substances, which normally do not pass through the membrane. Finally, membrane-bound proteins are damaged by propagation reactions of LHP. Hydroxyl radicals remove a hydrogen atom from methylene groups in a PUFA resulting in a PUFA lipid radical. The lipid radical then reacts with normal oxygen (triplet O2) to form a lipid peroxyl radical, which then reacts with another PUFA to form another lipid radical. This propagates the production of reactive oxygen species (ROS) in what is called the peroxidative chain reaction (Figure 1.2).

Figure 1.2 Oxidative stress starts a cascade that can lead to chronic disease if not modified by corrective actions.

Reactive nitrogen species (RNS) can also participate in nitrosative oxidative damage. Nitric oxide synthase is the enzyme that drives this reaction. FRs can attack proteins, lipids, carbohydrates, and nucleic acids causing extracellular matrix, cellular, and subcellular damage. Nitrous oxide + superoxide → peroxynitrite (ONOO−), which yields the c01-math-0003 reactive oxidant.

Oxidative stress is highest in the plasma membrane, mitochondria, nucleus, golgi, and lysosomes. AOXs represent about 50% of total Internet citations and cover anti-cancer, anti-inflammatory, and anti-proliferation. FRs can also act in signaling and function as messenger agents.

Inflammation is most often the initial step in a disease process followed by an immune response, but the immune response may occur at nearly the same time. With time, various molecular dysfunctions develop into a chronic disease such as documented in diabetes, cardiovascular disease, organ failure, and cancer. The first step is called initiation, followed by propagation, termination, and/or protection. The AOX pathways are generated by endogenous (internally synthesized) or exogenous sources that come from dietary preferences and lifestyle controlling modifications.

The pathway originates from stimuli that trigger radical formation. These molecular events lead to inflammation and immune responses, provoke neovascularization, and upregulate proinflammatory cytokines. Protective scavenger AOXs are divided into small water-soluble molecules (vitamin C, glutathione (GSH)), lipid-soluble molecules (vitamin E, lipoic acid, carotenoids, and coenzyme Q10 (CoQ10)), and larger enzyme molecules that need to be synthesized internally (superoxide dismutase, catalase, and GSH peroxidase). These detoxify aqueous and lipid-soluble peroxides. Preventive AOXs bind to essential proteins (albumin, metallothionein, transferrin, ceruloplasmin, myoglobin, and ferritin).

These factors can lead to an imbalance – a lack of AOXs because of their underproduction, misdistribution, or environmental stressor depletion. In the clinical realm, these can lead to pre, acute, and chronic disease and may advance to a potentially terminal event.

Biomarkers of oxidative stress can be analyzed in cells; tissues; blood; urine; CSF; synovial fluid; saliva; tears; and many other substances such as botanical nutraceuticals, marine algae, and food samples. Key markers used in most scientific publications are shown by the following oxidative stress metrics (Table 1.1).

Table 1.1 Biomarkers for oxidative stress

Cytokines are immunomodulatory agents that act as intracellular chemical mediators. They activate antigens and carry signals to adjacent cells of the immune system, thus magnifying the response to disease. Chaperones are functionally related groups of proteins synthesized in the endoplasmic reticulum. They are cellular machines that assist in protein folding and protect against degradation. Under physiological stress, cells respond to an increase by less than 5°C of temperature to produce heat shock proteins, which participate in anoxia, inflammation, and oxidant injury. They can be analyzed by electron microscopy.

Oxidative stress plays a major role in many human diseases and may well become the salient feature in most diseases. To date, involvement of oxidative stress has been confirmed in over 100 disorders. Oxidative stress has been previously linked to a plethora of changes induced during aging as well as in specific diseases such as obesity, diabetes, cancer, cardiovascular disease, stroke, neurodegenerative disease, trauma, hypoxia, psychological behavior, pain, chronic fatigue, fibromyalgia, pulmonary disease, hepatic disease, renal disease, gastrointestinal disease, macular degeneration of the eye, disorders of noise-induced hearing loss, fertility, menopause, osteoporosis, endocrine disorders, skin disease, musculoskeletal disorders, bone marrow abnormalities, oral health, nutrition, environmental health, and complications following extended space travel. Genetics may also play a role in the overall pathology of oxidative stress. Many of these topics have extensive publications in peer-reviewed scientific and clinical journals, corroborating the role of oxidative stress. Therefore, oxidative stress should be considered a primary cause of most diseases, or at least the result of several compounded processes that require more data and are currently under investigation. The student should consult the Internet, PubMed, Citation Index, or ISI Web of Science to study the many oxidative stress-related activities present in tissues and organs measured with new appropriate biomarkers.

Free radicals

Oxidation reactions cause the formation of a variety of FRs, which are unstable substances, that can initiate chain reactions in microseconds, leading to disease and programmed apoptotic cell death. Cells may recover or may undergo apoptotic autophagy or uncontrolled necrosis. Necrosis is when the tissue cannot regulate the influx of fluids and the loss of electrolytes, most notably in mitochondria and is associated with extensive damage resulting in an inflammatory response.

Apoptosis is by definition a phenomenon of programmed cell death under normal homeostasis control, and consequently no inflammatory response is observed. The cell shows membrane blebbing, shrinkage in size, nuclear condensation, DNA chromatin fragmentation, aggregation of chromatin, nuclear condensation, and partition of cytoplasm into membrane-bound vesicles, which contain ribosomes and nuclear material. These are phagocytized by macrophages, but there is no inflammatory response. Chronic apoptosis may cause widespread atrophy.

Free radicals may occur from a specific stimulus such as ultraviolet radiation, multiple environmental factors, poor nutrition, or sedentary lifestyle. Oxidative stress can stimulate neutrophils in the blood to ingest pathogens, but these are replaced on a daily basis by younger cells. FRs can attack proteins, lipids, carbohydrates, and nucleic acids causing cellular and subcellular damage to cells by ROS or RNS (Figure 1.3).

Figure 1.3 Free radicals (FR) are key to initiation and propagation of the paths that lead to disease. Antioxidants (AOX) are key to protection.

Note that the amount of oxidized protein is proposed as the tipping point in the FR and AOX balance scheme. Oxidized proteins activate the caspase enzyme cascade in the proteasome, form intracellular aggregates, and are predominately nonrepairable because cross-links limit repair mechanisms so that recycling of amino acids for continuous protein synthesis is diminished.

The master AOX for recycling and FR inactivation is reduced GSH, but lutein and phenols with hydroxyl groups can readily take up unpaired electrons together with ascorbate; α-tocopherol/tocotrienol; and enzymes such as superoxide dismutase, catalase, GSH peroxidase, GSH reductase, and CoQ10.

Inflammatory pathways

Inflammation facilitates healing from noxious or foreign stimuli. The initiating event is tissue damage. It involves the formation of nuclear factor kappa B (NFκB) and systemic cytokine by-products such as TNF-α and prostaglandin E2-α. Biomarkers are thiobarbituric acid reactive substances (TBARS), derivatives of reactive oxygen metabolites (dROMS), oxygen radical absorbance capacity (ORAC), hydroxyl radical antioxidant capacity (HORAC), arachidonic acid, thromboxane, lipopolysaccharides, and trolox equivalent antioxidant capacity (TEAC)). Many biomarkers degrade over time, so it is advised to use fresh or freshly frozen samples. For example, in type 2 diabetic patients, oxidative stress is closely associated with chronic inflammation by upregulating key vascular ROS- and RNS-producing enzymes and the corresponding endogenous AOXs. Heme oxygenases (HO-1) utilize NADPH and oxygen to rupture the heme moiety, causing modulation of cellular bioenergetics and leading to apoptosis and inflammation. These are my interpretations of oxidative stress and AOX events in disease to gain a mechanistic summary and thus an approach to therapy.

Mitochondria

Molecular oxygen diffuses into the mitochondrial inner membrane where ROS sources are actively produced through the electron transport chain and nitric oxide synthase reactions. Cytochromes are present in mitochondria and transfer electrons along the respiratory chain that involves oxidation and reduction of iron. CoQ10 is a naturally occurring AOX and a prominent component of mitochondrial electron transport chain. CoQ10 is recognized as an obligatory cofactor for the function of thermogenesis uncoupling proteins and a modulator of the mitochondrial transition pore. It was also observed that CoQ10 is part of an endogenous AOX defense that increases SOD2 and GSH peroxidase. In disease-prone cells, mitochondrial superoxide is exported to adjacent cells, triggering lipid peroxidation, propagation reactions, and inflammation.

Educational redox

The following list of information on products for redox therapy as an experimental therapy is based on results-oriented data. The following list has contact information from prominent companies specializing in pro-oxidant and AOX agents in cells. These also cover alternative and holistic medicine.

1. Life Extension Foundation, 5th edition, 2013 (www.lifeextension.com/track)

2. Integrative Therapeutics, Inc (www.integrativepro.com)

3. (www.naturalmedicines.therapeuticresearch.com)

4. Advanced Bionutritionals (www.advancedbionutritionals.com)

5. Oxford Biomedical Research (http://www.oxfordbiomed.com)

6. OXIS Research International (www.oxisresearch.com)

7. Cayman Chemical Co. (www.caymanchem.com)

8. ALPCO Diagnostics (www.alpko.com)

9. The Japan Institute for the Control of Aging, Nikken SEIL Corp. ([email protected]), Genox is the USA distributor.

10. ALEXIS Biochemicals (www.alexis-biochemicals.com)

11. INOVA Diagnostics (www.inovadx.com)

12. Molecular Probes by Life Technologies (www.lifetechnologies.com)

13. PROBIOX SA, Belgium (www.probiox.com)

14. The National Center for Comparative Alternative Medicine (nccam.nih.gov) is an investigator-initiated project on advanced research covering complementary and alternative medicine.

Great variability in the activities of AOXs is present in over-the-counter nutraceuticals. Disclaimers for food products that are not under FDA regulation are treated as a food, not as a pharmaceutical. The caveat that must be put on the label is These statements have not been evaluated by the FDA and the product is not intended to diagnose, treat, cure, or prevent any disease. Therefore, be careful with sources of AOX treatments.

Stay current with new research initiatives. The student is also encouraged to keep a record of these applications. In searching the Internet, the student should key in on MiMB and Oxidative Stress in Applied Basic Research and Clinical Practice, published by Humana Press, a brand of Springer and part of Springer Science + Business Media.

References

1 Armstrong, D. (1994) Free radicals in diagnostic medicine: a systems approach to laboratory technology. In: Advances in Experimental Medicine and Biology. Vol. 366. Plenum Press.

2 Afzal, M. & Armstrong, D. (eds) (2007) Molecular biotechnology: an international symposium on free radicals in biosystems. In: Molecular Biotechnology. Vol. 37. Humana Press.

3 Armstrong D. 1998. Free radical and antioxidant protocols, Methods in Molecular Biology, 108. Humana Press.

4 Armstrong D. 2002. Oxidative stress biomarkers and antioxidant protocols, Methods in Molecular Biology186. Humana Press.

5 Armstrong D. 2002. Oxidants and antioxidants: ultrastructure and molecular biology: ultrastructure and molecular biology protocols. Methods in Molecular Biology196. Humana Press.

6 Armstrong, D. (ed) (2008) Advanced protocols in oxidative stress I. In: Methods in Molecular Biology. Vol. 477. Humana Press, a part of Springer Science + Business Media, LLC, NY.

7 Armstrong, D. (ed) (2010) Advanced protocols in oxidative stress II. In: Methods in Molecular Biology. Vol. 594. Humana Press, a part of Springer Science + Business Media, LLC, NY.

8 Armstrong D. 2014. Advanced protocols in oxidative stress III Methods in Molecular Biology1208. Humana Press, a part of Springer Science + Business Media, LLC, NY.

9 Armstrong, D. (2009) Lipidomics: methods and protocols. In: Methods in Molecular Biology. Vol. 579–580. Humana Press, a part of Springer Science + Business Media, LLC, NY.

10 Armstrong, D. & Bharali, D.J. (2013) Oxidative stress and nanotechnology. In: Methods and Protocols. Vol. 1028. Humana Press, a part of Springer Science + Business Media, LLC, NY.

Chapter 2

Diagnostic imaging and differential diagnosis

Robert D. Stratton¹,²

¹Department of Ophthalmology, College of Medicine, University of Florida, Gainesville, FL, USA

²Department of Small Animal Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA

THEMATIC SUMMARY BOX

At the end of this chapter, students should be able to:

Describe the main diagnostic methods

Describe the main methods of measuring bioelectrical signals

Describe the main methods of imaging using electromagnetic energy

Describe the main methods of imaging using radioisotopes

Diagnostic method in clinical practice

History

Clinicians are trained to employ the differential diagnostic method using a wide range of diagnostic methods and tests to eliminate diagnostic possibilities and narrow the diagnostic list. The history and physical exam are the most important first steps in the differential diagnosis. The history is a conversation with the patient to ascertain the chief complaint, which may be more generally described as the presenting problem so as to include unresponsive or uncooperative patients. A history of the present illness is then taken to find the symptom locations, the character of pain the problem may be causing, including location of any radiating discomfort, the time of onset of the symptom and duration, the severity of discomfort, and aggravating or relieving factors. Other complaints are investigated in a similar manner. To elicit other complaints, a past medical and surgical history is taken, including major past and present illnesses, hospitalizations, treatments, procedures and surgeries, allergic reactions, and present medications. A review of systems is next taken, which is a systematic review of the major organ systems, including the head, eyes, ears, nose, throat, teeth, tongue, cardiovascular system, respiratory system, gastrointestinal system, hematological system, neurological system, musculoskeletal system, genitourinary system, endocrine system, skin, psychiatric problems, and orientation. A family history elicits diseases that may be passed on to progeny, and a social history elicits workplace and home dynamics.

Physical examination

Equipped with data from the history, a focused physical exam is then performed. A standard physical examination includes vital signs, alertness assessment, general body assessment, and a systematic organ systems examination guided by the history. Many of the organ system examinations are performed in much greater detail by medical and surgical specialists, but all clinicians evaluate head, eyes, ears, nose, and throat (HEENT), respiratory system, cardiovascular system, abdomen, neurological system, genitourinary system, integument, and musculoskeletal system.

Specialized examinations are performed when indicated. Neurological examination may include coma assessment, detailed motor and sensory testing of somatic and cranial nerves including reflexes, gait evaluation, and cognitive evaluation. Ophthalmological examination may include detailed vision testing, visual field examination (perimetry and color perimetry), contrast sensitivity, extraocular muscle evaluation, slit lamp examination of the anterior and posterior segments of the eye including optic nerve head evaluation, and indirect ophthalmoscopy with scleral indentation to examine the peripheral retina and ciliary body. Otologic examination may include otoscopy with the manipulation of the tympanic membrane, evaluation of hearing acuity and tuning fork, and evaluation of the vestibulo-ocular reflex. Laryngoscopy, with the familiar ENT head mirror being replaced with direct light sources, is performed to evaluate the larynx and vocal cords. Auscultation, percussion, and palpation are performed to evaluate the chest and abdomen. Musculoskeletal examination can be one of the most complex of the general physical exam but, focused by the history, evaluates the part in question.

Differential diagnosis

Once the history and physical exams are complete, the clinician must then consider all the possible compatible diagnoses drawn from the clinician's fund of knowledge. Diagnostic testing is then considered if the diagnosis is in doubt. In diseases of either systemic or organ-specific damage owing to oxidative stress, diagnostic testing usually depends on biomarker measurements and imaging techniques. A biomarker is almost any measurement reflecting an interaction between a biological system and its environment, which may be chemical, physical, or biological, and the measured response may be a functional, physiological, biochemical, or molecular interaction.¹ These measurements are used to detect a current state.

Biomarkers in biological investigation

Biochemical biomarkers

Clinical chemistry laboratories provide measurements of chemicals, proteins, DNA, RNA, and many organic macromolecules in any biological fluid, including plasma, serum, and cerebrospinal fluid. Measurements include enzymes, antibodies, hormones, cytokines, antigens, cytosol proteins, and levels of pharmaceuticals. Trace amounts of DNA or RNA can be detected using polymerase chain reaction (PCR) technology, making early diagnosis of some infections and cancers possible. An increasing array of PCR techniques are used in medical, forensic, and genetic investigation.²

Electromagnetic biomarkers

Biomarkers from the whole range of electromagnetic radiation (photons) are used for imaging and detection, from a Hertz of 0 (standing potentials), to a Hertz of up to 10²⁰ (γ radiation).

Electrooculography (EOG) measures the electrical potential across the eye from the cornea to the choroid, and is mostly a measurement of the electrical potential generated by the monolayer of retinal pigment epithelial (RPE) cells, called the standing potential because of its nontransient character (near 0 Hz). This potential can be directly measured accurately with invasive placement of measuring electrodes, but in order to find this value noninvasively, measurements of electrical potential are made external to the eye and compared to a reference electrode. The potentials measured with the eye gazing far right is compared to the potential with the eye gazing far left, and a calculation of the vector of electrical potential of the RPE is made. Although the resulting calculated standing potential is not very accurate, a change in this standing potential in response to dark and light adaptation accurately reflects the function of the RPE in normal and dysfunctional states.

Electroretinography (ERG) measures the transient electrical potential generated by the neurosensory retina in response to a brief whole field simulation with light (Figure 2.1). This can be done in a dark adapted state to assess the scotopic retinal function (mainly the rod-based neural network) or in the light adapted state to assess the photopic retinal function (mainly the cone-based neural network). An electrode placed on the anesthetized cornea is compared to a reference electrode (usually on the forehead). The initial negative response, called the A wave, is generated by the photoreceptor cell that has the first response to the light stimulus. The following positive B wave is a combined response from the photoreceptor, bipolar, and Mueller cells. The A wave in ERG done during dark adaptation is mostly from the rod photoreceptor cells. The A wave done during light adaptation is mostly from the cone photoreceptor cells. A more definitive way to differentiate between slow recovering rods and more quickly recovering cones is to use a flickering light stimulus of 30 Hz so that the rods cannot recover before the next flash, whereas normal cones recover in time to give a measurable electrical impulse. Differentiation among various retinal degenerations is greatly aided by results of EOG and ERG testings. Progression and prevention of retinal degeneration can be measured by serial testing. Multifocal electroretinography measures the same transient electrical signal from the retina, but a carefully planned and patterned stimulus rather than a whole field light stimulus is used and the ERG pattern from focal retinal areas are computed from the corneal electrode recorded signals. Dark adaptometry measures subjective light sensitivity thresholds at a focal spot in the retina as the eye adapts from bright light to dim light levels. Several subjective color vision tests are available including Ishihara pseudochromatic plates, Farnsworth-Munsell D-15 panel, and a more detailed F-M 100 hue test.

Figure 2.1 Electroretinogram waveform. t0 is the time of light stimulus, ta is the time to the peak of the negative deflection and tb is the time to the peak of the positive deflection. A wave is the amplitude of the negative deflection, mainly from the photoreceptor cell depolarization; B wave is the amplitude of the positive deflection from the neurosensory retinal response to the photoreceptor cell depolarization. Times vary with species, but are generally in the 10–300 ms range. Amplitudes also vary widely, but are in the 20–500 mV range.

The electrocardiogram is a recording of the electrical signals of myocardial contraction from electrodes on the skin, familiar to most as an ECG. The more invasive processes of introducing electrodes behind the heart through a transesophageal ECG yield additional information. An ECG of an electrode attached to the needle used to drain fluid from the pericardial sac can indicate contact with the myocardium by an abrupt change in the ECG and guide the placement of the aspirating needle.

Electrocorticography (ECOG) is the direct recording of cerebral cortical electrical potentials during craniotomy using electrodes. Single neuron recording is achieved using a wide variety of microelectrodes and microelectrode arrays. The electroencephalogram (EEG) records mass activity of the brain and is, therefore, very useful in detecting seizures where there is mass recruitment of neuronal activity or in confirming brain death where there is no activity. A strong stimulus can be used to evoke an EEG response, called sensory evoked potential (SEP). Visual evoked potential (VEP) or visual evoked response (VER) is an EEG recording of the scalp overlying the visual cortex. Flash whole field stimuli evoke strong signals with inconsistent timing. Pattern stimulus evoked responses are weaker but are much more consistent. Delays in the evoked signal show pathology in the neural pathways. Optic nerve lesions most often cause unilateral delays. The auditory system is assessed by auditory brainstem response (ABR) with a click auditory stimulus and scalp EEG electrodes. The initial response (Wave I) is predominantly the auditory nerve, and later responses (i.e., Wave V) are predominantly from the brainstem. Magnetoencephalography (MEG) also records mass electrical signals from the cerebral cortex by detecting the magnetic fields generated by the mass neuronal activity. The changes in signals over time give a way to measure functional brain activity.

Electromyography (EMG) is the clinical recording of the mass signal of muscle from skin electrodes overlying a muscle or transcutaneous needle electrode in a muscle. Nerve conduction velocity (NCV) is the measurement of the speed and strength of a peripheral nerve from an electrical stimulus. EMG and NCV together help to differentiate between myopathic and neuropathic diseases and also measure responses to drugs or treatment.

Spectrographic biomarkers

Chromatography is the separation of mixtures based on some differing characteristics of the populations in the mixture, typically molecular size, charge, and shape. Gas chromatography, gel column chromatography, affinity chromatography, and high performance liquid chromatography separate by elution. Gel electrophoresis separation of DNA is a common technique in modern laboratories. Many specialized forms of chromatography have been devised and are used extensively in research laboratories.

Mass spectrometry measures the mass-to-charge ratio of ions produced from a sample by using the deflection of accelerated charged particle by an electromagnetic field. The measurement is the relative abundance of ions of a particular m/z ratio so that the data are presented as a graph or image (see Figure 2.2). Different ionization and fragmentation techniques have been developed to help analyze samples. Separation procedures with gas chromatography or with tandem mass spectrometers and quadrupole filtering allow for the identification of complex samples. Rasterizing tissue samples with laser allows for imaging by mass spectrometry.³

Figure 2.2 Simple mass spectrogram of methane. The less abundant ¹³C is hidden in the ¹²C peaks except for ¹³C¹H4 visible right of ¹²C¹H4 peak.

Biological imaging

When the amount of biomarker information increases to the point of being hard to conceptualize, the data are presented as an image. Imaging can be a way of presenting a large amount of data so that trends and relationships can be seen. This is used not only with electromagnetic data but also in other large data sets as in genomics, proteomics, and brain mapping.

Acoustic imaging

Ultrasonography or echography is a technique that uses acoustic reflections to make measurements and construct images. There is a trade-off between higher frequency giving better resolution and lower frequency giving better penetration. Sound frequencies of 2–20 MHz are typically used. A-scan is recording of the echo of a series of pings along a single vector for the purpose of measuring distance or sound attenuation. B-scan is recording of a series of pings along a series of adjacent vectors for the purpose of constructing a 2D image. Multiple B-scan images can be computationally constructed into a 3D image, sometimes called a C-scan. Doppler scan identifies moving media and is useful in visualizing blood flow velocity and direction. Doppler information is often displayed as a color palette overlying a B-scan image. Ultrasound has become a widely used technology due to the rapid and dynamic assessment, noninvasiveness, and low cost. Computer enhancement of images and Doppler data have made evaluation of blood vessels with ultrasound a good alternative to angiography, and intravascular ultrasound (IVUS) has enhanced angiography.

Light imaging

Photography has been expanded to a vast array of technologies. Fundus photography and retinal fluorescein angiography capture images of the retina, optic nerve head, and blood vessels in the posterior segment of the eye. Photomicroscopy can be enhanced in many different ways. Stains can enhance the structural details of tissue on histologic sections, with hematoxylin staining the nucleus and eosin staining the cytoplasm in the most common H&E stain. Special stains can identify various cellular and extracellular components in tissue sections and cell preparations. Antibody-linked immunofluorescent staining has become a very powerful investigative tool with advances in new techniques for antibody production.

Phase contrast microscopy can provide details of living cells by the enhancement of the changes in phase as light passes through transparent cellular structures. Optical coherence tomography (OCT) is an imaging technique that uses interference patterns from reflected light to construct an image, somewhat analogous to ultrasonography, but with a much higher resolution. OCT is most easily used in investigating surfaces or the optically clear structures of the eye, but investigation into using lower frequency light to penetrate tissue and higher frequency light to give higher resolution will continue. Intravascular OCT has increased the optical resolution of vascular imaging. Diffuse optical imaging is an infrared light technique using computer reconstruction of images similar to the familiar computed tomography (CT) scan imaging technique. This is able to detect a range of metabolic biomarkers including oxyhemoglobin, deoxyhemoglobin, and cytochrome C oxidase redox status.⁴ Transmission electron microscopy (TEM) uses magnetic fields to accelerate and focus electrons to produce a 2D absorbance image on a fluorescent screen recorded by a charge-coupled device. Resolution of TEM depends on the wavelength of the electrons, giving resolutions that approach 1 nm and below. Tissue samples are stained with heavy metals to increase absorption contrast. Osmium, uranium, and gold are often used. Specific antibody stains including colloidal gold–antibody complexes are used to stain specific antigens. Scanning electron microscopy (SEM) produces an image of electrons from a conductive surface, usually sputter coated with a thin metal layer, stimulated by a raster scanning narrow electron beam. Scanning a specimen with a physical probe rather than a beam of light or electrons is called scanning probe microscopy (SPM). Many variations of SPM have been developed that give very high resolution imaging of atomic structures. Scanning tunneling microscopy and atomic force microscopy are two of the more familiar techniques used in oxidative stress research.

Endoscopy has transformed diagnosis and surgery, with an endoscopic instrument available for almost every organ system, even the eye. Diagnostic ultrasound and OCT catheters are becoming available, along with numerous tissue sampling techniques and treatment techniques. Moore's law is at work, with steady improvements in imaging systems and miniaturization.

X-ray projection (plain) radiology was the only imaging system for internal organs for many years, and is still widely used in clinical medicine, primarily in instances when there is good contrast, such as bone lesions, lung tissue lesions, and fluid levels. Contrast material is use to increase the range of usefulness of plain films. Barium is used to visualize the gastrointestinal tract, and iodinated compounds are used to visualize arteries (angiography), veins (venography), lymph vessels and nodes (lymphography), spinal cord (myelography), brain (ventriculography), bile and pancreatic ducts (cholangiography), and joints (arthrography). X-ray computed tomography (CT) allows for more detailed imaging by using computer analysis of X-ray images along multiple vectors. CT scan images may be reconstructed through computer processing to give 2D and 3D images. Iodinated contrast material is used intravenously with CT in a technique called computed tomography angiography (CTA). X-ray diffraction techniques (crystallography) allow determination of molecular structures of a wide variety of complex molecules, including DNA. Dual-energy X-ray absorptiometry (DEXA scan) measures a bone density assessment to evaluate osteopenia and osteoporosis.

Nuclear magnetic resonance spectroscopy (NMRS) aids in identification of organic molecules, with techniques developed for protein and DNA analyses. Magnetic resonance imaging (MRI) uses computer analysis of large data sets of NMRS relaxation energy measurements along multiple vectors to construct images using a process similar to the one used in CT scanning. Techniques to improve image contrast have been found using differences in relaxation times of various tissue types. Contrast material is also used to enhance images in MRI. Chelated gadolinium, iron oxide nanoparticles, chemical exchange saturation transfer (CEST) compounds, and mangafodipir (containing paramagnetic Mn²+) can be used. To obtain images of vessels, a technique called magnetic resonance angiography (MRA) is used with a contrast agent to make blood vessels stand out.

Electron paramagnetic resonance spectroscopy (electron spin resonance) detects unpaired electrons in a way much similar to NMRS. A free radical has an unpaired electron, so EPRS is used extensively in free radical chemistry research. In a strong magnetic field, a paramagnetic unpaired electron will align its magnetic spin moment in either a positive parallel or negative (anti-) parallel direction, creating an energy state difference between each alignment, the strength of which is proportional to the strength of the magnetic field. Absorption or emission of photons is measured to detect energy transformations between the two energy states.

Carbon-13 (¹³C) isotopes are commercially available in a wide variety of compounds for a nonradioactive method of labeling carbon atoms in reactions. Because ¹²C is more abundant (99%) and ¹³C is less abundant (1%), mass spectrometry can be used to identify where a labeled carbon (¹³C) is located in a reaction product. Unlike the abundant ¹²C that has a spin quantum number of 0 and cannot be detected with NMRS, ¹³C is paramagnetic with a net spin of 1/2. Both NMRS and NMI (2D and 3D) can be done.

Radioactive isotope imaging (scintigraphy)

Radioactive isotopes are used in laboratory investigation in a wide range of applications to label molecules in cellular structures and biochemical reactions. This allows for imaging of specific metabolic activities rather than just tissue anatomy. Nuclear medicine uses radioactive isotopes to detect, measure, or image by measuring α, β, or γ emissions from the radioactive decays. Radioactive thyroid uptake scans image the amount of iodine the thyroid gland concentrates by measuring γ radiation emission after intravenous radioactive iodine injection. Uptake of iodine in thyroid nodules, which are detected by ultrasound, can be quantified, helping to differentiate between benign and cancerous nodules. The technetium isotope ⁹⁹mTc is a metastable isotope that emits γ radiation with a half-life of 6 h. ⁹⁹mTc is used to label a variety of compounds for scintigraphy of various organs. An increase in the uptake of phosphate labeled with ⁹⁹mTc indicates an area of increased osteoblast metabolism in bone scans, allowing detection of fractures, inflammation, and metastatic cancers. ⁹⁹mTc-labeled compounds that are taken up by the liver and excreted into the biliary tract are used for imaging the liver, biliary tract, and gall bladder in cholescintigraphy, helping in the diagnosis of cholecystitis and congenital biliary anatomical anomalies. Parathyroid scintigraphy with ⁹⁹mTc-labeled complexes can identify an active parathyroid adenoma, allowing selective excision and retaining uninvolved glands. ⁹⁹mTc or γ-emitting ¹³³Xe are used in pulmonary scintigraphy to evaluate ventilation and perfusion defects, helping to diagnose pulmonary embolism and other anomalous pulmonary perfusion defects. ⁹⁹mTc-labeled dimercaptosuccinic acid or l,l-ethylenedicysteine is concentrated in the renal cortex, allowing renal scintigraphy to identify anomalous or diseased areas of the kidney, ureters, and bladder.

Single-photon emission computed tomography (SPECT)

Rotating the γ detector around the patient to acquire images from varying vectors allows computer analysis similar to X-ray CT scan and MRI technology, and can produce a 3D image. SPECT bone scan produces a 3D image, allowing better localization of a defect. Cardiac SPECT scans use ²⁰¹Tl-(thallium) or ⁹⁹mTc-labeled compounds to detect perfusion defects and help to differentiate between infarction and ischemia. Brain SPECT scans use ⁹⁹mTc-labeled compounds to measure perfusion defects in the brain, helping in differentiation of stroke and dementia. Gallium ⁶⁷Ga³+ is concentrated in areas of inflammation and rapid cell growth, so gallium scans using either plain scintigraphy or SPECT technology can identify areas of inflammation, infection, and tumor growth.

Current SPECT scanners do not have very high spatial resolution, but are widely available and relatively inexpensive. The problem with resolution is the lack of a technology to focus these high-energy γ photons. Collimators are used which, by blocking all photons not traveling parallel, do give a type of focus that greatly reduces the signal and therefore sensitivity. In research investigation on animal models, higher levels of radiation are tolerated, so pinhole collimators that reduce signal strength dramatically but allow very good resolution are available. Positron emission radionuclides can be detected without collimation in positron emission tomography (PET). The γ-emitting radioisotopes used in scintigraphy are, in general, heavier elements, relatively inexpensive, and have half-lives that are long enough to be commercially available, except for ⁹⁹mTc, which is generated from the more stable isotope ⁹⁹Mo. Positron-emitting isotopes are, in general, lighter, more expensive, and short lived. No collimator is needed in PET, for focusing the image because γ radiation is detected, not from the tissue, but from the position of the positron when positron–electron annihilation occurs, giving a very small localization error but high sensitivity. With annihilation, two photons of γ energy are released in approximately opposite directions, which give vector information, in contrast to single photon emission. This allows for a greater sensitivity of 100–1000×, with better signal-to-noise ratio and higher resolution. There are positron-emitting isotopes of oxygen, carbon, nitrogen, and fluorine, allowing for easier labeling of specific biological molecules. The shorter half-lives of PET isotopes allow a higher initial dose, shorter time sequence intervals for dynamic studies, and shorter intervals for repeat studies. Glucose, labeled with ¹⁸F as fluorodeoxyglucose, is able to pass freely through the blood–brain barrier and concentrates at areas of metabolic activity, allowing brain imaging and cancer detection. Brain PET is useful in evaluating stroke, tumor, and neurodegenerations, and has been used extensively in brain research because of the ability to measure functional changes and to identify various neuroreceptors using specific ¹⁸F-labeled neurotransmitters. An Oncology PET is widely used in cancer diagnosis, staging and evaluating treatment effects. The fluorodeoxyglucose is rapidly taken up by growing cancer cells, enzymatically phosphated, but not metabolized because of the fluorine substitution for an oxygen, so that the radioactive label is highly concentrated in the cancer cell, allowing sensitive detection of metastatic tumors. The 110 min half-life of ¹⁸F allows time for transport from a cyclotron facility to the PET facility, but the shorter half-lives of ¹¹C, ¹³N, and ¹⁵O require that the PET scanner be located close to the cyclotron. Rubidium is metabolized as if it were potassium, allowing rapid uptake of intravenously injected positron emitter ⁸²Rb by myocardium, indicating areas of normal and deficient perfusion. The problem with the short halve-life of ⁸²Rb (75 s) has been overcome by an ⁸²Rb generator using ⁸²Sr (strontium), which decays to ⁸²Rb by electron capture with a half-life of 25 days. Cardiac PET scans using ⁸²Rb chloride are more sensitive than are cardiac SPECT scans, but are more expensive and not as widely available.

Multiple choice questions

1 SPECT scan technology using γ emission has resolution limited mainly by

a. The dose of the radionuclide given

b. The quality of the γ detectors used

c. The lack of technology to focus γ radiation

d. The duration of the scan

2 In evaluating a patient for metastatic cancer, the most important first step is

a. History and physical exam

b. PET scan using ¹⁸F-labeled fluorodeoxyglucose

c. Gallium (⁶⁷Ga³+) SPECT scan

d. SPECT bone scan

3 The stable isotope ¹³C can be detected by

a. OCT

b. Scintigraphy

c. Nuclear magnetic resonance spectroscopy

d. Transmission electron microscopy

4 The short half-lives of ¹¹C, ¹³N, and ¹⁵O require PET scans using these radionuclides be

a. Very fast scanners

b. Near the cyclotron used to make these isotopes

c. Used only early in the week

d. Collimated

References

1 Marmor, M.F., Fulton, A.B., Holder, G.E. et al. (2009) ISCEV Standard for full-field clinical electroretinography (2008 update). Documenta Ophthalmologica, 118, 69–77.

2 Rahman, M.T., Uddin, M.S., Sultana, R. et al. (2013) Polymerase chain reaction (PCR): a short review. AKMMC Journal, 4(1), 30–36.

3 Garrett, T.J., Menger, R.F., Dawson, W.W. et al. (2011) Lipid analysis of flat-mounted eye tissue by imaging mass spectrometry with identification of contaminants in preservation. Analytical and Bioanalytical Chemistry, 401(1), 103–113.

4 Lee, J., Kim, J.G., Mahon, S.B. et al. (2014) Noninvasive optical cytochrome c oxidase redox state measurements using diffuse optical spectroscopy. Journal of Biomedical Optics, 19(5), 055001.

Section II

Clinical correlations on acute and chronic diseases

Chapter 3

Free radicals: their role in brain function and dysfunction

Natan Gadoth¹,²

¹Department of Neurology, Mayanei HaYeshua Medical Center, Bnei Brak, Israel

²The Sackler Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel

THEMATIC SUMMARY BOX

At the end of this chapter, students should be able to:

Describe the main features of the disease entities covered in the chapter including possible etiology and pathophysiology

Recognize and summarize the contribution of oxidative stress to nerve cell function

Outline the similarities in the mechanisms responsible for OS damage in various brain disorders described in the chapter

Review the role of age in OS

Review the therapeutic and practical opportunities of antioxidant treatment of the described CNS disorders

Introduction

The central nervous system (CNS), which is depended on oxygen for its continuous normal function, is protected from damaging oxidation by an endogenous antioxidant defense system, that is, enzymatic and nonenzymatic antioxidants. Glutathione, uric acid, and nicotinamide adenine dinucleotide phosphate (NADP) are endogenous while vitamin C (ascorbic acid) and vitamin E (α-tocopherol) are the major exogenous antioxidants. When an imbalance between prooxidant and antioxidant factors is present, a state of oxidative stress (OS) is formed. This can lead to generation of reactive oxygen species (ROS) and other electrophiles that are capable of either supporting or damaging cellular functions.

The brain, which requires 4 × 10¹² molecules of adenosine triphosphate (ATP) each minute for its normal function, consumes therefore 20% of the inspired oxygen while its weight is only 2% of the total body weight. This vital high turnover of oxygen necessary for normal brain function makes it vulnerable to the interruption of oxygen supply and/or inhibition of mitochondrial production of ATP. About 5% of the oxygen consumed by cells is reduced to form ROS; however, the high oxygen consumption of the brain relatively to other organs may result in an increased amount of ROS produced. Considering the potential damage that ROS may cause by its deleterious effect on apoptosis and on neural membranes, which are particularly rich in polyunsaturated fatty acids (PUFAs), may suggest that they play a role in normal aging and disease-related neurodegeneration. In addition, the antioxidant defense of the brain is relatively reduced due to relatively low levels of the antioxidants such as catalase, glutathione peroxidase (GPX), and vitamin E.

The beneficial role of oxidative stress in the brain

There are "good and bad" ROS. All good ROS are by-products of the turnover in the mitochondrial respiratory chain. In brain tissue, ROS are generated by microglia and astrocytes and modulate synaptic and nonsynaptic communication between neurons and glial cells. ROS also interfere with increased neuronal activity by modifying the myelin basic protein, thus altering its conduction ability and possible induction of synaptic long-term potentiation. Furthermore, results from animal models suggest that the role of O2 in modulating synaptic plasticity and potentiation is altered by age. ROS are also involved in central control of food intake and are required for hypothalamic osmoregulation.

The harmful role of oxidative stress in the brain

The deleterious effects on the brain associated with ROS appear when those reactive compounds are in excess and overcome the relatively feeble antioxidant protective mechanism of the brain. The high concentration of PUFAs (which are rich in double bonds and are responsible for their susceptibility to peroxidation) and the high concentration of brain iron, which may act as a prooxidant, are the main reasons for the vulnerability of the brain to assault by ROS. As a result of lipid peroxidation, further potential damage can be caused by the formation of toxic by-products such as reactive aldehydes, which may enhance neuronal apoptosis (programmed cell death). An additional damaging effect of ROS on brain is their potential ability to cause oxidative modification of DNA with secondary endonuclease-mediated DNA fragmentation, which leads to DNA damage.

Brain mitochondria, the energy source for essential brain activity, are targeted by ROS. Following brain ischemia, the mitochondria will take up calcium in the form of Ca²+, resulting in increased production of ROS by a poorly understood mechanism(s). The increase ROS production results in increased permeability of the mitochondrial membrane followed by a cascade of events starting with Ca²+ which is followed by further influx of K+. The end result is an increase in the osmolarity inside the mitochondria resulting in entry of water (osmotic swelling) and finally a bioenergetic neural tissue failure.

The role of oxidative stress (OS) in programmed neuronal death (apoptosis)

Programmed cell (neuronal) death is an actively regulated mechanism of cell death, which plays a major role in maintenance of normal tissue growth. The intracellular machinery responsible for apoptosis depends on a family of proteases that have cysteine at their active site and cleave their target proteins at specific aspartic acid site, thus they are called caspases (Cysteine-dependent ASPartate-directed proteASEs). Caspases, once activated, amplify a proteolytic cascade that leads to apoptotic cell death. A variety of ROS such as the prooxidants H2O2, among others, induce apoptosis while antioxidants such as N-acetylcysteine (NAC) suppress apoptosis. Thus, apoptosis is strongly associated with normal mitochondrial function, in particular changes in electron transport and altered mitochondrial oxidation–reduction. Neuronal firing is highly dependent on normal mitochondrial function; thus, the mitochondrion is highly susceptible to OS via increased apoptosis.

Oxidative stress in neonatal hypoxic-ischemic encephalopathy (HIE)

Asphyxia is a term frequently used to describe the state of severe lack of oxygen due to abnormal breathing. This state is most frequent in newborns. A total of 0.5–3% of newborns worldwide will suffer from birth asphyxia severe enough to require resuscitation. Out of those, about a million will die and a similar number will be left with significant neurobehavioral disability. Many of those infants will suffer from anoxic brain damage