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Detection of Antioxidants Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies 309
common cellular antioxidants with an emphasis on the principles and major procedures of the assays for
measuring antioxidant enzymes.
2. MAJOR EXPERIMENTAL APPROACHES TO ANALYZING ANTIOXIDANTS
As stated in Chapter 3, mammalian cells and tissues are equipped with a range of antioxidants, including
both protein and non-protein antioxidants synthesized by cells. In addition, mammalian antioxidant
defenses also comprise the antioxidant compounds (e.g., vitamin C, vitamin E, carotenoids, and
polyphenols) derived from the diet. Although the proper functionality of mammalian cells depends on the
coordinated actions of these diverse dietary antioxidants, endogenous antioxidant enzymes seem to play a
more vital role in protecting against oxidative stress. This is evidenced by the essentiality of some
antioxidant enzymes in embryonic development as well as the disease phenotypes caused by deletion of
each of the many cellular antioxidant genes in animal models. Hence, methodologies have been developed
to specifically detect and measure cellular and tissue antioxidant enzymes and proteins. Antioxidant
enzymes and proteins can be assessed by a number of major experimental approaches, as summarized
below (Fig. 15.1).
TF
Protein
mRNA
Gene Transcription
Transcription Factors
Enzyme Activity Assays
Immunoblot Analysis
Immmunohistochemistry
Proteomics
Real‐Time PCR Analysis
RT‐PCR Analysis
Gene Array Profiling
Nuclear Run‐On Assay
Reporter Gene Assays
Immunoblot Analysis
Gel Shift Assay
Fig. (15.1). Major experimental approaches to analyzing cellular and tissue antioxidant defenses. See text (Section 2)
for detailed description. TF, transcription factor; RT-PCR, reverse transcriptase-polymerase chain reaction.
2.1. Measurement of Enzyme Activities
This is frequently done by using biochemical assays based on the chemical reactions catalyzed specifically
by the antioxidant enzymes. The rest of the chapter primarily describes the commonly used assays for
measuring the activities of cellular and tissue antioxidant enzymes that are commonly encountered in
biomedicine.
2.2. Analysis of Protein Expression
In addition to assaying the enzyme activities, the protein levels of cellular antioxidant enzymes can be
determined by immunoblot analysis via using specific antibodies. Immunoblot analysis is considered
semiquantitative. A concomitant standard curve utilizing purified enzymes may be carried out to estimate
the absolute amounts of the antioxidant proteins in the samples. Immunohistochemistry staining with the
use of specific antibodies can be carried out to visualize the protein expression of antioxidant enzymes in
individual cells of tissue slices. The recently developed proteomic techniques are powerful for large-scale
studies of cellular proteins, including antioxidant enzymes and other cytoprotective proteins.
2.3. Determination of mRNA Levels
Increased activities of cellular antioxidants usually result from increased protein expression, which in turn
frequently stems from increased levels of the mRNA. The mRNA levels can be analyzed via various