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Bennett. J Transl Genet Genom 2020;4:36-49 I https://doi.org/10.20517/jtgg.2020.17 Page 37

in distinct oxidation states with environmental dependencies that include (1) the redox environment of the
mitochondrion and the cell; (2) the oxidative stress burden and history; (3) the integrity of the mitochondrial
membrane; and (4) the functionality of the individual MRC components and the electron transfer chain
overall. Other non-MRC metalloproteins, particularly aconitase and catalase, provide distinct and specific
biomarkers for oxidative stress.

[1,2]
Mitochondrial diseases (MD) can arise where depletion of mitochondrial DNA (mtDNA) , or mutations
in mtDNA and/or nuclear DNA lead to altered mitochondrial function [3-7] . Altered catalytic and electron
transferring activities of mitochondrial complexes I-V have been associated with MD, and physiological
consequences of MRC defects include reduced metabolic capacity, reduced ATP synthesis, and increased
oxidative and nitrosative stress [8-18] . Symptoms are manifold and include weakness (from central nervous
system, peripheral nerve, and/or skeletal muscle disease), pain, intolerance of some general anesthetics and
anti-epileptic drugs, gastrointestinal disorders, ophthalmoplegia and/or visual failure, failure to thrive, cardiac
and respiratory disease, liver disease, diabetes, seizures, sensorineural hearing loss, mental retardation,
dementia, movement disorders, increased susceptibility to infection, and pregnancy loss [5,6,19-41] . The primary
manifestation of mitochondrial dysfunction is an inability to generate enough energy from metabolism to
maintain the functional or structural integrity of the associated tissues. Thus, MD is particularly debilitating
when dysfunctional mitochondria are present in tissues with high energy requirements, such as the
cerebrum, nerves and muscle. The other major underlying pathology is oxidative stress i.e. the production
of reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS and RNS are free radicals and
related compounds, some of which exhibit high reactivity and can damage proteins, lipids and nucleic acids.
Depolarization of the mitochondrial membrane can occur due to physical damage. Damage to individual
MRC components can exacerbate oxidative stress and an oxidative stress cascade can occur. Identification
of low ATP production or oxidative stress to be primarily responsible for symptoms may have profound
consequences for subsequent care and therapy.

Traditional diagnosis of MD includes clinical presentation of symptoms, family history, pathology, metabolic
profiling, enzyme activity levels, electrophysiology, magnetic resonance imaging of brain and magnetic
resonance spectroscopy of metabolites, and mtDNA analysis [7,10,34,42-55] . Diagnosis can be challenging, given
that (1) mitochondrial metabolism can be affected in non-mitochondrial diseases; (2) there can be extensive
variability in the distribution of abnormal mitochondria within an individual patient, resulting in “false
negative” testing to occur when tissues containing the abnormal mitochondria are not tested; and (3) there
are no uniform, clear cut pathological abnormalities to distinguish all MD patients from patients with other
disorders, to the extent that some biopsy specimens look structurally normal. MD can also present with an
extraordinary range of clinical symptoms, and laboratory testing abnormalities are common. MD is often
suspected clinically as part of the differential diagnosis in patients with diseases involving the brain, muscle,
or liver and MD-like symptoms are often exhibited in early childhood. Attempts to improve MD diagnosis
have included the use of diagnostic algorithms to predict the likelihood of MD, DNA sequencing, and omics
methods, each with associated advantages and challenges of their own [49,56-62] .

Herein, the application of cryogenic electron paramagnetic resonance spectroscopy (EPR) of intact, flash-
frozen tissue is described for the diagnosis and characterization of metabolic dysfunction in general, and MD
in particular. The article describes (1) the principles of EPR; (2) the EPR signals exhibited by mammalian
and human tissue; (3) sample preparation considerations; (4) analysis methods; and (5) the relevance of EPR
to MD and integration of EPR results with other, complementary investigative methods.

ELECTRON PARAMAGNETIC RESONANCE
EPR, in the present context, is the measurement of the magnetic field-dependence of absorption of a photon
by a paramagnetic substance i.e. containing unpaired electrons in one or more atomic, ionic, or molecular