Oxidants and Antioxidants, Part A

536 Pages·1999·23.66 MB·English

by  Lester Packer

METHODS IN ENZYMOLOGY
EDITORS-IN-CHIEF
John N. Abelson Melvin I. Simon
DIVISION OF BIOLOGY
CALIFORNIA INSTITUTE OF TECHNOLOGY
PASADENA, CALIFORNIA
FOUNDING EDITORS
Sidney P. Colowick and Nathan O. Kaplan
Methods in Enzymology
Volume 299
Oxidants and Antioxidants
Part A
EDITED BY
Lester Packer
UNIVERSITY OF CALIFORNIA
BERKELEY, CALIFORNIA
Editorial Advisory Board
Bruce .N Ames
Enrique Cadenas
Balz Frei
Matthew Grisham
Barry Halliwell
Pryor William
Catherine Rice-Evans
Helmut Sies
ACADEMIC PRESS
San Diego London Boston New York Sydney Tokyo Toronto
Preface
The importance of reactive oxygen and nitrogen species (ROS and
RNS) and antioxidants in health and disease has now been recognized in
all of the biological sciences and has assumed special importance in the
biomedical sciences. Overwhelming evidence indicates that ROS play a
role in most major health problems, that antioxidants play a critical role
in wellness and health maintenance, and that by inhibiting oxidative damage
to molecules, cells, and tissues prevent chronic and degenerative diseases.
We now know that ROS are essential for many enzyme-catalyzed re-
actions. Low levels of reactive oxygen and reactive nitrogen species are
signaling molecules. At high concentration, these ROS are essential in the
antitumor, antimicrobial, antiparasitic action, etc., of neutrophils and mac-
rophages and contribute to oxidative damage to molecules, cells, and tissues.
In this volume all of the major natural antioxidants with respect to
assays for evaluating their antioxidant activity have been included. There
has been wide usage of methods to access total antioxidant activity, and
some of the new methods in this area have also been included.
Many antioxidant substances have biological activities which may or
may not depend on their antioxidant actions. Although this is of course
relevant to understanding their actions in biological systems, we have cho-
sen not to include such methods. Antioxidant activity can be defined as
the protection against oxidative damage; however, it is becoming eminently
clear that it is difficult to define an antioxidant. Antioxidants have so many
different biological activities, in addition to their direct quenching of radicals
or acting as redox molecules in reducing reactions, that their definition
must surely be very broad.
In bringing this volume to fruition, credit must be given to experts in
various specialized fields of oxidant and antioxidant research. Our apprecia-
tion is to the contributors who, with those who helped select them, have
produced this state-of-the-art volume on oxidant and antioxidant methodol-
ogy. The topics included were chosen on the excellent advice of Bruce N.
Ames, Enrique Cadenas, Balz Frei, Matthew Grisham, Barry Halliwell,
William Pryor, Catherine Rice-Evans, and Helmut Sies. To these col-
leagues, I extend my sincere thanks and most grateful appreciation.
LESTER PACKER
xiii
1 ECNECSENIMULIMEHC SDOHTEM 3
1 Total Antioxidant Activity Measured by
Chemiluminescence Methods
By HANNU ALHO and JANNE LEINONEN
Introduction
Reactive oxygen species (ROS) have been implicated in more than 100
diseases, from malaria and hemorrhagic shock to acquired immunodefi-
ciency syndrome. 1 This wide range of diseases implies that ROS are not
something esoteric, but that their increased formation accompanies tissue
injury in most, if not all, human diseases. 1 Tissue damage by disease, trauma,
toxins, ischemia/repeffusion, and other causes usually leads to the forma-
tion of increased amounts of putative "injury mediators," as well as to
increased ROS formation, 3'e
Four endogenous sources appear to account for most of the oxidants
produced by cells: (i) normal aerobic respiration, i.e., mitochondria, con-
sume 02 by reducing it in sequential steps, thus producing H202; (ii) stimu-
lated polymorphonuclear leukocytes and macrophages release superoxide,
which in turn is a source for H2Oe, HOC1, and NO; (iii) peroxisomes,
organelles responsible for degrading fatty acids and other molecules, pro-
duce HeOe as a by-product; and (iii) induction of P450 enzymes can also
result in oxidant by-products. 4
Hydroxyl radical OH., the fearsomely reactive oxygen species, has been
proposed to be produced in living organisms by at least three separate
mechanisms: (i) by reaction of transition metal ions with H202, the so-
called superoxide-driven Fenton reaction; (ii) by peroxynitrite, a nonradical
product of NO- and O~-, can protonate and decompose to a range of
noxious products, including nitrogen dioxide and nitronium iron; and (iii)
by making -OH in vivo by the reaction of O~- with hypochlorous acid.
Exogenous sources of free radicals include tobacco smoke, ionizing
radiation, certain pollutants, organic solvents, anesthetics, hyperoxic envi-
ronment, and pesticides. Some of these compounds, as well as certain
medications, are metabolized to free radical intermediates that have been
I B. Halliwell, Haernostasis 23, 118 (1992).
2 B. Halliwell and J. M. C. Gutteridge, eds., in "Free Radicals in Biology and Medicine,"
2nd ed., p. 253 Clarendon Press, Oxford, 1989.
3 S. Toyokuni, K. Okamoto, J. Yodoi, and H. Hiai, FEBS Lett. 358, 1 (1995).
4 B. N. Ames, M. K. Shigenaga, and T. M. Hagen, Proc. Natl. Acad. Sci. U.S.A. 90, 7915 (1993).
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4 TOTAL ANTIOXIDANT ACTIVITY [ 1]
shown to cause oxidative damage to the target tissues. Exposure to radiation
results in the formation of free radicals within the exposed tissues.
To protect itself against the deleterious effects of free radicals, the
human body has developed an antioxidant defense system that consists
of enzymatic, metal-chelating, and free radical-scavenging properties. In
addition to the protective effects of endogenous enzymatic antioxidant
defenses, consumption of dietary antioxidants appears to be important. 4
The concentration of antioxidants in human blood plasma is important in
investigating and understanding the relationship among diet, oxidative
stress, and human disease. The measurement of the total antioxidant activity
of biological fluids, especially plasma, serum, or serum lipoprotein fractions,
is of value in estimating the capability to resist oxidative stress. Different
methods applicable to this task have been reviewed previously in this series. 5
The principle of practically all of these methods is to by some means
produce free radicals at a known rate and to study the capability of a
sample to inhibit this radical production by a certain end point. In this
study, chemiluminescence-based methods are evaluated for measuring the
peroxyl radical-scavenging capacity of human plasma, low-density lipopro-
tein (LDL), and cerebrospinal fluid (CSF).
Methods of assessing antioxidant activity vary greatly with regard to
the radical species that is generated (Table I), the reproductivity of the
generation process, and the end point that is used (Table II). One of the
most widely used end points is chemiluminescence. Earlier methods 7'6
were based on the inhibition of spontaneous tissue autoxidation, but
Wayner 8 took advantage of their discovery that the thermal decomposition
of water-soluble azo compound 2,2'-azobis([2-amidinopropane])hydro-
chloride (ABAP) yields peroxyl radicals at a known constant rate. The
decomposition of ABAP has been shown to induce the following temporal
order of consumption of plasma antioxidants: ascorbate > thiols > bili-
rubin > urate > a-tocopherol.
It has been shown that chemiluminescence as an end point offers a
sensitive way to observe antioxidant-consuming free radical reactions
against either whole plasma or LDL, and several modifications utilizing
chemiluminescence have been developed. Hirayama et aL 9 have used a
5 C. Rice-Evans and N. J. Miller, Methods Enzymol. 234, 279 (1994).
6 T. Ogasawara and M. Kan, Tohoku J. Exp. Med. 144, 9 (1984).
7 j. Stocks, J. M. C. Gutteridge, R. J. Sharp, and T, L. Dormandy, Clin. Sci. Mol. Med. 47,
215 (1974).
8 D. D. M. Wayner, G. W. Burton, K. U. Ingold, and S. Locke, FEBS Lett. 187, 33 (1985).
90. Hirayarna, M. Tagaki, K. Hukumoto, and S. Katoh, Anal Biochem. 247, 237
(1997).
1 ECNECSENIMULIMEHC SDOHTEM 5
TABLE I
SDOHTEM ROF GNITARENEG LACIDAR SEICEPS a
CuZ+/cumene hydroperoxide (5)
Cu2+/H202 (5)
HRP/H202 (11)
OPD/H202 (5)
Ferrimyoglobin radicals and ABTS (5)
Peroxyl radicals from ABAP (8, )31
AAPH (31)
AMVN (17)
Lipoperoxides in brain homogenates (5)
Superoxide (6)
Photoinduction (12)
"HRP, Horseradish peroxidase; OPD, o-phenyl-
enediamine; ABTS, 2,2'-azinobis(3-ethylben-
zothiazoline 6-sulfonate); ABAP, 2,2'-azobis
(2-amidinopropane hydrochloride); AAPH,
2,2'-azobis(2-amidinopropane) dihydrochlo-
ride; AMVN, 2,2'-azobis(2,4-dimethylvaleroni-
trile). Numbers in parentheses refer to litera-
ture cited in the text.
mixture of lipid hydroperoxides and microperoxidase to produce oxyradi-
cals and further light emission by luminol oxidation to study the antioxidant
activity of plasma and saliva. Because cumene hydroperoxide induces a
rapid chemiluminescence that is followed for only 3 min, half-inhibition
values of the initial chemiluminescence are determined for various anti-
TABLE II
SDOHTEM ROF DNE TNIOP SNOITAVRESBO a
Fluorescence inhibition (5)
Chemiluminescence (8-11, ,31 24, 32)
Oxygen uptake (32)
Absorbance change (5)
TBA-RS (5)
CO production (5)
Cell morphology (6)
"TBA-RS, Thiobarbituric acid-reactive sub-
stances. Numbers in parentheses refer to litera-
ture cited in the text.
6 LATOT TNADIXOITNA ACTIVITY [ 11
oxidants and biological samples. Maxwell et aL lo have measured the total
antioxidant activity of LDL, HDL, and VLDL and Whitehead et al.ll that
of serum by measuring luminol-based chemiluminescence catalyzed by
horseradish peroxidase (HRP) by the addition of phenolic enhancer
compounds. One method for testing and quantification of nonenzymatic
antioxidants is based on a photoinduced, chemiluminescence-accom-
panied, and antioxidant-inhibitable autoxidation of luminol. 21 The mean
values of an integral antioxidant capacity (AC) of human blood plasma
showed age-dependent patterns with maximal values with newborns. The
AC of six tested animal species was lower than that of humans, with max-
imal values with guinea pigs and spontaneously hypertensive rats (see
Ref. 12).
However, the most often used chemiluminescence-based methods are
modifications of the original total peroxyl radical-trapping potential
(TRAP) method of Wayner et al. 8 The problem with the original TRAP
assay method lies in the oxygen electrode used to measure the end point,
aist will not maintain its stability over the period of time required. However,
the TRAP assay measured with a chemiluminescence modification devel-
oped by Mets~i-Ketel~i 3~ produces an assay of considerably better precision
than the original TRAP assay. This chemiluminescence-enhanced TRAP
utilizes water- or lipid-soluble azo initiators as sources of a constant flux
of carbon-centered peroxyl radicals. Using this approach, the ability of
plasma antioxidants to inhibit the artificial propagation phase of membrane
lipid peroxidation can be tested. It also lends itself to a higher degree of
automation and significant numbers of samples can be processed.
We have previously reported changes of total antioxidant capacity of
plasma, CSF, or LDL in control materials and various clinical situations
by using this TRAP method. ~2-41 In addition to the methodological corn-
01 .S R. J. Maxwell, O. Wiklund, and G. Bondjers, Atherosclerosis 11, 79 (1994).
n T. P. Whitehead, G. H. G. Thorpe, and .S R. J. Maxwell, Anal Chim. Acta 266, 265
(1992).
lz I. N. Popov and G. Lewin, Free Radio. Biol. Med. 17, 267 (1994).
31 T. Mets~i-Ketel~i, in "Bioluminescence and Chemiluminescence Current Status" (P. E.
Stanley and L. J. Kricka, eds.), Wiley, Chirehester, 1991.
41 R. Aejmelaeus, T. Mets~i-Ketela, P. Laippala, and H. Alho, FEBS Lett. 384, 128 (1996).
51 R. Aejmelaeus, T. Mets~i-Ketel~i, T. Pirttil~i, A. Hervonen, and H. Alho, Free Radic. Res,
26, 335 (1996).
61 R. Aejmelaeus, P. Holm, U. Kaukinen, T. Mets~i-Ketela, P. Laippala, A. Hervonen, and
H. E. R. Alho, Free Radio. Biol. Med. 23, (1996).
71 R. Aejmelaeus, T. Mets~i-Ketela, P. Laippala, T. Solakivi, and H. Alho, Mol. Asp. Med.
18, 113 (1997).
] 1 [ CHEMILUMINESCENCE METHODS 7
ments on TRAP developed by Mets~i-Ketelfi, 31 this chapter presents de-
tailed protocols for the measurement of chemiluminescence-enhanced
TRAP of both plasma and LDL.
Total Peroxyl Radical-Trapping Potential
General Principle
Thermal decomposition of the water-soluble azo compound ABAP or
the lipid-soluble (AMVN) generates peroxyl radicals at a known constant
rate. Their reaction with the chemiluminescent substrate luminol leads to
the formation of luminol radicals that emit light that can be detected by a
luminometer. Antioxidants in the sample inhibit this chemiluminescence
for a time that is directly proportional to the total antioxidant potential of
the sample. This potential of the sample is compared to that of either water-
or lipid-soluble tocopherol analogs, capable of trapping 2 moles of peroxyl
radicals per 1 mole of Trolox (6-hydroxy-2,-5,7,8-tetramethylchroman-2-
carboxylic acid, Aldrich, Germany).
TRAP Assay for Plasma and LDL
Prepare plasma samples by drawing venous (fasting or nonfasting, see
Observations) blood into EDTA-containing Vacutainer tubes on ice, pro-
tected from light. Separate plasma by centrifugation using a temperature-
controlled centrifuge at ° 4 after which plasma can be stored at -80 ° for up
to 6 months without a significant change in the TRAP value. Mix 475/xl
of oxygen-saturated 100/xM sodium phosphate buffer, pH 7.4, in a plastic
cuvette with 50/xl of 400 mM ABAP (Polysciences, Warrington, PA) in
the same buffer and with 50/xl of 10 mM luminol (5-amino-2,3-dihydro-
1,4-phthalazinedione, Sigma Chemical Co., St. Louis, MO) in 20 mM boric
acid-borax buffer, pH adjusted to 9.5 with 10 N HC1. After a 15-rain
is M. Erhola, M. Nieminen, A. Ojala, T. Mets~i-Ketel~i, P. Kellokumpu-Lehtinen, and H. Alho,
J. Exp. Clin. Cancer Res. 17(1), 1 (1998).
~'~ M. Erhola, M. Nieminen, P. Kellokumpu-Lehtinen, T. Mets~i-Ketelfi, T. Poussa, and H.
Alho, Free Radic. Res. 26, 439 (1997).
o2 M. Erhola, P. Kellokumpu-Lehtinen, T. Mets~i-Ketel~, K. Alanko, and M. Nieminen, Free
Radic. Biol. Med. 21, 383 (1996).
l2 K. L6nnrot, T, Metsa-Ketel~i, G. Molnar, J.-P. Ahonen. M. Latvala, J. Peltola, T. Pietil~,
H. Alho, Free Radic. Biol. Med. 21, 211 (1996).
[1]
8 TOTAL ANTIOXIDANT ACTIVITY
incubation at 37 ° the rate of synthesis of peroxyl radicals is constant; dis-
pense 25/zl of plasma into the cuvette. Using LKB Wallac Luminometer
1251, a PC, and software from TriStar Enterprise (Tampere, Finland),
detect chemiluminescence readings at 36-sec intervals for 90 min. The
linear regression line for Trolox in our laboratory is y = 131.7x + 43.2,
where y is the inhibition time in seconds and x is the concentration of
Trolox in riM.
For thorough evaluation of the antioxidant capacity, it is essential to
also measure the concentrations of main chain-breaking antioxidants from
the same sample (see Discussion). For the measurement of ascorbic acid,
we use a final concentration of 5% of metaphosphoric acid as an additive
in the plasma samples. Ascorbic acid and uric acid are then measured by
HPLC according to Frei et al. 22 a-Tocopherol is measured by the modified
HPLC method of Catignani et al.23 and ubiquinol-10 from a heparin-citrate-
precipitated LDL fraction according to Lang et al. 42 Protein sulfhydryl
groups (-SH) are measured according to Ellman. 52 From the individual
concentrations omfe asured antioxidants it is possible to derive the theoreti-
cal TRAP value (TRAPca~c) of the sample by using the stoichiometric
peroxyl radical-scavenging factors (see also Observations and Discussion)
that have been establishedS'13'21: TRAPcalc = 2.0 [sample concentration of
uric acid] + 2.0 [a-tocopherol] + 0.7 [ascorbic acid] + 0.4 [-SH]. It is
also possible to calculate the difference between measured TRAP and
TRAPcalc, i.e., the TRAPunid, which is composed of actions of unmeasured
and partly uncharacterized antioxidants of the sample. TRAP values are
presented as micromoles of peroxyl radicals trapped by 1 liter of the
sample.
For measurement of LDL TRAP (TRAPLDL), heparin-citrate-precipi-
tated LDL is extracted from plasma with chloroform/methanol (1, v/v).
TRAPLDL is measured analogically to plasma TRAP by replacing water-
soluble ABAP with 50/xl of 25 mM lipid-soluble AMVN (Polyscience,
Warrington, PA). D-a-Tocopherol is used asa n internal standard. TRAPLoL
is expressed as picomoles of peroxyl radicals. If the concentrations of
ot-tocopherol and ubiquinol-10 are measured separately from the LDL
extract, the TRAPca~c can be derived using stoichiometric factors 2.0 for
22 B. Frei, L. England, and B. Ames, Proc. Natl. Acado Sci. U.S.A. 86, 6377 (1989).
32 G. Catignani and J. Bieri, Clin. Chem. 29, 708 (1993).
42 j. K. Lang, K. Gohil, and L. Packer, Anal. Biochem. 157, 106 (1986).
52 G. EUman, Arch. Biochem. Biophys. 82, 70 (1959).