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European Review for Medical and Pharmacological Sciences 2007; 11: 309-342

Oxidative stress tests: overview on


reliability and use
Part I
B. PALMIERI, V. SBLENDORIO
Department of General Surgery and Surgical Specialties, University of Modena and Reggio Emilia
Medical School, Surgical Clinic, Modena, Italy

Abstract. Monitoring oxidative stress can initiate cellular tissue damage by modifying
in humans is achieved by assaying products of lipids, proteins and DNA, which can seriously
oxidative damage or by investigating the po- compromise cell health and viability or induce a
tential of an organism, tissue or body fluids to
withstand further oxidation. Unfortunately, variety of cellular responses through generation
there is little consensus concerning the selec- of secondary reactive species, leading, at last, to
tion of parameters of oxidative stress or an- cell death by necrosis or apoptosis. Oxidative
tioxidant state to be determined in defined pa- damage of any of these biomolecules, if
tients or diseases. This is not only due to the unchecked, is probably responsible of disease de-
uncertainty wheter or not a certain parameter velopment. However, definitive evidence for this
is playing a causative role. Moreover, the
methods of determination described in the lit-
association is often lacking because of recog-
erature represent very different levels of ana- nized shortcomings with methods available to as-
lytical practicability, costs, and quality. Gener- sess oxidative stress status in vivo in humans3.
ally accepted reference ranges and interpreta- There are some exogenous sources of free rad-
tions of pathological situations are lacking as icals such as UV-photolysis, radiation, ozone,
well as control materials. At present, the situa- pollution, pharmacological agents, smoking, al-
tion is changing dramatically and sophisticat- cohol, iron-overload, pesticides and mycotoxins4.
ed methods like HPLC (High Performance Liq-
uid Chromatography) and immunochemical de- Imbalance between production and elimination
terminations have become more and more of free radicals may cause oxidative stress.
common standard. Free radicals can be scavenged by several met-
alloenzymes (e.g., glutathione peroxidase, cata-
Key Words: lase, superoxide dismutase) as well as by the
Oxidative stress, Free radicals, Antioxidants, Reac- non-enzymatic antioxidant defence system (e.g.,
tive oxygen species, Lipid peroxidation. tocopherol, -carotene, ubiquinol, vitamin C,
glutathione, lipoic acid, uric acid, metalloth-
ionein, bilirubin) which quench their activity.
Therefore, much attention of nutritionists is now
focused on the possible role of the enhancement
Introduction of the defences against ROS5 (Table II).
Despite the harmful cellular damaging effects,
The role of free radicals is gaining increasing free radical reactions are also involved into bene-
worldwide attention since so many physiological ficial physiological response when produced in
and pathophysiological phenomena are related to high levels mediating cytotoxicity of polymor-
redox status cell modification. phonuclear leukocytes, macrophages and mono-
A free radical is, by definition, a chemical cytes during the respiratory-burst6.
species containing unpaired electrons and is Moreover, low levels of ROS are involved in
therefore paramagnetic1. Most of the oxygen de- the regulation of the tone of smooth muscle cells7
rived free radicals relevant to cell biology are un- and have been demonstrated to upregulate the re-
stable, short-lived and highly reactive2 (Table I). dox sensitive transcription factors such as nu-
For these reasons, reactive oygen species (ROS) clear factor-B and activator protein-18-10.

Corresponding Author: Beniamino Palmieri, MD; e-mail: palmieri@unimo.it 309


B. Palmieri, V. Sblendorio

Table I. Free radicals. the ROS11 and reactive nitrogen species (RNS)
generated because of excessive production of
Half-lives(s) ROS/RNS, loss of antioxidant defences, or both.
Molecules Symbol at 37C
The localization and effects of oxidative
Molecular oxygen O2 > 102
stress, as well as information regarding the na-
Lipid peroxide ROOH > 102 ture of the ROS/RNS, may be revealed from the
Semiquinone radical Q > 102 analysis of discrete biomarkers of oxidative/ni-
Hydrogen peroxide H2O2 10 trosative stress/damage isolated from tissues and
Peroxyl radical ROO 1 102 biological fluids. Biomarkers are quali-quantita-
Superoxide radical O2 1 106 tive indicators of normal and pathological bio-
Singlet oxygen 1
O2 1 106 chemical processes or of druginduced effect in
Alkoxyl radical RO 1 106 therapeutic protocols. Several in vitro markers of
Hydroxyl radical OH 1 109 oxidative/nitrosative stress are available, includ-
ing ROS/RNS themselves, but most are of limit-
ed value in vivo because they lack sensitivity
Highly specific analytical techniques are re- and/or specificity or require invasive methods.
quired to monitor the biological significance of Although some ROS/RNS have been directly de-
free radicals. tected in vitro by electron spin resonance with or
Increased oxidative/nitrosative stress generally without spin trapping reagents or by chemilumi-
describes a condition in which cellular antioxi- nescence, these methods are not yet applicable in
dant defences are unable to completely inactivate clinical practice because of the instability of
many reactive species and the need for expensive
Table II. Antioxidant defence system.

Preventive antioxidants:
a) Non-radical decomposition of hydroperoxides and hydrogen peroxide:

Catalase: Decomposition of hydrogen peroxide: 2H2O2 2H2O + O2


Glutathione peroxidase (cellular) Decomposition of hydrogen peroxide and free fatty acid
hydroperoxides:
H2O2 + 2 GSH 2 H2O + GSSG
LOOH + 2GS LOH + H2O + GSSG
Glutathione peroxidase (plasma) Decomposition of hydrogen peroxide and phospholipid
hydroperoxides
Phospholipid hydroperoxide PLOOH + 2GSH PLOH + H2O
Glutathione peroxidase GSSG
Glutathione-S-transferase Decomposition of lipid hydroperoxides
Thioredoxin Reduction of peroxides
b) Sequestration of metals by chelation
Transferrin, lactoferrin: Iron
Haptoglobin Haemoglobin
Hemopexin Stabilisation of heme
Ceruloplasmin, albumin Copper
c) Quenching of active oxygens
Superoxide dismutase (SOD) Disproportionation of superoxide: 2O2 + 2H + H2O2 + O2
Carotenoids, vitamin E Quenching of singlet oxygen

Radicals-scavenging antioxidants: scavenge radicals to inhibit chain initiation and break chain propagation
Lipophilic: Vitamin E, ubiquinol, carotenoids
Hydrophilic: Vitamin C, uric acid, bilirubin, albumin

Repair and de novo enzymes: repair the damage and reconstitute membranes:
Lipase, protease, DNA repair enzymes, transferase

Adaptation: generate appropriate antioxidant enzymes and transfer them to appropriate site at the right time and
concentration

310
Oxidative stress tests: overview on reliability and use. Part I

equipment. Furthermore, ROS/RNS are usually free radicals that begat them. The second major
too reactive and/or have a half-life too short problem is that the most commonly available bi-
(even much shorter than seconds) to allow direct ological fluid to be screened are blood, urine and
measurements in cells/tissues or body fluids. Be- expired breath. Clinical biochemistry detects
cause molecular products formed from the reac- usually abnormal metabolic products, recovered
tion of ROS/RNS with biomolecules are usually from these sources, which are related to specific
considered more stable than ROS/RNS them- diseases. On the contrary, reactive free radicals
selves, most commonly ROS/RNS have been as end products of intracellular metabolism from
tracked by measuring stable metabolites (e.g., ni- different tissues have a microseconds-measurable
trate/nitrite) and/or concentrations of their oxida- half life and they are not detectable in the blood
tion target products, including lipid peroxidation stream. In a very few special cases, the actual
end products and oxidized proteins12-16. Tech- site of free radical generation may be the blood
niques for quantification of oxidative damage and direct (or semi-direct) detection of free radi-
markers are often called fingerprinting methods cal species may be possible, but generally speak-
by which specific end products deriving from the ing only secondary free radical products are de-
interaction of the ROS with biomolecules, such tectable in a body fluid. A wide array of analyti-
as DNA, proteins, lipid and LMWA (low-molec- cal techniques has been developed to measure
ular-weight antioxidant) are measured. The pres- these end products though not all of them are
ence of these end products serves as proof of the suitable to detect clinical conditions sampling
prior existence of ROS that left their footprints in blood, urine and expired breath. Lipid peroxida-
the cell. To function as suitable biomarkers of tion is the most intensively studied process and
oxidative modifications in relation to disease, it provides a number of possibilities for assays.
is critical that such oxidation products are stable, Protein and nucleic acid oxidation are presently
can accumulate to detectable concentrations, re- very appealing. The currently available tech-
flect specific oxidation pathways and correlate niques, however, are limited to semi-quantitative
with disease gravity, so that they can be utilised assays of damage to broad classes of biomole-
as diagnostic tools. cules and there is an urgent need for more specif-
To demonstrate a role of ROS in a particular ic and informative methods.
type of tissue injury, evidence should be present-
ed that:

1. ROS are detectable locally and the time- Electron Spin Resonance
course of their formation is such that they and Radical Trapping
could play a role;
2. the chemical production of ROS produces The only analytical technique that directly
similar lesions; measures free radicals is electron spin resonance
3. compounds able to remove ROS protect from (ESR) spectrometry. However, since it is rela-
the injury. tively insensitive and requires steady-state con-
centrations of free radicals in the micromolar
Measuring Free Radicals in Vivo range its of very limited value for use in vivo.
The increasing interest in the role of free radi- Whole-body ESR, analogous to whole-body
cals in the pathogenesis of human disease has led NMR, has been investigated but not yet fully de-
to widespread attempts to develop techniques veloped. Nevertheless, ESR has been used to de-
suitable to measure free radicals and their reac- tect free radicals in human tissue obtained ex vi-
tions in vivo, specifically, in clinical pathology. vo: an example is the detection of a signal be-
The first major problem to be faced is the quick lieved to be that of lipid peroxyl radical in hu-
reactivity of free radicals reaction close to their man uterine cervix19. ESR spectrometry can usu-
biochemical source. Consequently, free radicals ally be applied to analysis of samples in vivo on-
are not amenable to direct assay and free radical ly through the technique of spin trapping. This
activity is usually assessed by indirect methods involves the addition to samples of a compound
such as measurement of the various end products known as spin-trap, which reacts rapidly with the
of reactions with lipids, proteins and DNA17,18. free radicals to form radical-adducts that are very
However, many of these products are themselves much more stable and longer-lived than the origi-
reactive, albeit orders of magnitude less than the nal species and can therefore build up to steady-

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B. Palmieri, V. Sblendorio

state concentrations in the detactable range. spectral information about the trapped radical is
Spin-traps have been used in experimental ani- the major drawback of this class of spin trap.
mals to demonstrate the generation of free radi- Three commonly used spin traps will be dis-
cals in vivo, but as no effective spin traps cussed: phenyl-t-butyl nitrone (PBN), (4-
presently exist that can be administered to hu- pyridyl-1-oxide)-N-t-butyl nitrone (POBN), and
mans, the technique is currently limited to sam- 5,5-dimethyl-1-pyrroline N-oxide (DMPO).
ples of blood mixed with the spin trap as soon as The technique has been associated with vari-
possible after taking them. Despite the obvious ous cases of incorrect interpretations; these gen-
shortcomings of this approach, valuable data has erally can be attributed to:
been obtained, for example, relating to free radi-
cal production during angioplasty20. 1. changes in the spin trap (nonradical, chemical,
Other trapping procedure allow a radical to re- photochemical, enzymatic reactions);
act with a detector molecule to yield a stable 2. perturbation of the biological system by the
product that can be evaluated using a variety of probe;
techniques, such as hydroxylation of salicyclic 3. artifactual reporting associated with intrinsic
acid21, the deoxyribose assay22, 23, the cytochrome properties of the probe.
c reduction assay for detection of superoxide rad-
icals24, and detection of nitric oxide radicals by Spin trapping is a good example of the com-
colored end-product compounds25. plex interaction between the model system and
Thus, the attack of hydroxyl radicals on sali- the artificial addition of a probe, with a high pos-
cylic acid produces 2,3-dihydroxybenzoate sibility of recording artifactual results.
(DHB) and on phenylalanine produces o-and m- The first that has proposed the term spin trap-
tyrosines. These products are not produced enzy- ping has been Janzen27. Spin trapping in biology
matically in humans. Thus, the method can be is covered by various reviews28,29. An extensive
used in vivo and detection of 2,3-DHB or the ty- literature survey has been carried out by Dodd30.
rosines in body fluids can be taken as evidence Specifically devoted to examining the problems
of hydroxyl radical generation26. As the trapping associated with the spin trapping of oxygen-cen-
compound has to complete with all other biomol- tered free radicals are the reviews of Finkelstein
ecules for reaction with the radicals, this tech- et al1, Rosen and Rauckman31, Rosen et al32, and
nique, like ESR-spin-trapping, is unlikely to pro- Pou and Rosen33.
vide more than semi-quantitative data. Invaluable help in disentangling the number of
Spin trapping is a powerful method that facili- spectra and attributions is given in the database
tates the visualization of free radicals, including for spin-trapping by Li and Chignell34, which has
those formed in complex biological systems. The been made feely available to all those interested
spin trap is a diamagnetic compound that reacts in the field.
with a reactive free radical to form a more stable
radical adduct. Although detection through ESR
spectroscopy offers some distinct advantages in
its high sensitivity, and in some cases its speci- Electron Paramagnetic Resonance
ficity toward some radical species, there are also (EPR)
several drawbacks to using this technique.
The technique was developed in the lates Another technique for the measurement of the
1960s by several laboratories27. Two groups of oxidative stress status in biological systems is
compounds are commonly utilized as spin-trap- based on the X-band EPR (electron paramagnetic
ping agents: nitroso and nitrone compounds. The resonance) detection of a persistent nitroxide
nitrogen atom of the nitroso spin trap reacts di- generated under physiological or pseudo-physio-
rectly with the free radical species, giving dis- logical conditions by oxidation of a highly
tinctive spectral features. Two nitroso com- lipophylic hydroxylamine probe. The probe em-
pounds are currently used in biological investiga- ployed is bis(1-hydroxy-2,2,6,6-tetramethyl-4-
tion: 2-methyl-2-nitroso propane (MNP) and 3,5- piperidinyl)-decandioate which is administrated
dibromo-4-nitrosobenzenesulfonate (DBNBS). as hydrochloride salt. This way of making OS
The nitrone spin traps each have a radical status detectable involves the use of exogenous
added to the carbon; the radical will be with re- nitroxides as probe of the redox balance in a giv-
spect to the nitroxide radical center. The lack of en enviroment. This probe is able to give a fast

312
Oxidative stress tests: overview on reliability and use. Part I

reaction with the most of radical species in- the spectrometer response it was possible to ob-
volved in the oxidative stress. The rate at which tain quantitative values for the oxidative stress.
the nitroxide is reduced to the diamagnetic hy- These results indicate that the OS level in dis-
droxylamine, which can be evaluated by EPR, is eased liver is several orders of magnitude higher
related to the reducing capacity of the organism than in healthy controls and the differences were
and hence to its oxidative status35. Furthermore, highly significant.
it crosses cell membranes and distributes in a bi- An endogenous molecule might also function
ological environment without the need to alter or as a trap, although it can be argued that measur-
destroy compartmentation. The method is there- ing specific end products of the trapping of RS
fore suitable for quantitative measurements of (reactive species) by endogenous molecules is
ROS and can be applied to human tissues in real the same as measuring biomarkers. Ascorbate
clinical settings36. It has been successfully em- reaction with free radicals is one example; anoth-
ployed in systems of growing complexity and in- er is urate, which is readily oxidized by a range
terest, ranging from subcellular fractions to of RS39, including proxynitrite40. Several groups
whole animals and human liver. Liver disease have used urate as a selective scavenger of
was chosen as the prototype of a patology in ONOO- in animal studies, neglecting the fact that
which the involvement of inflammatory process- it reacts with many species41. One of urates oxi-
es has a relevant role in the evaluation of the dis- dation products, allantoin, can be measured in
ease37. Thirty-two subjects, including 10 healthy human body fluids and its plasma levels are ele-
controls, were enrolled after giving informed vated in conditions associated with oxidative
consent. Ten of the 22 patients had hepatitis C, 3 stress, such as chronic inflammation, diabetes,
had hepatitis B, while the remainder had a vari- premature birth, iron overload, chronic hearth
ety of diseases characterized by an autoimmune failure and exercise42-45. Allantoin can also be
nature which, for statistical purpose, were clus- measured in urine46 and cerebral microdialysis
tered in a group called nonviral liver diseases fluid 47. Levels of allantoin rise in the human
(NVLD). The method developed by Valgimigli et muscle during exhaustive exercise, presumibly
al38 was enough simple and only moderately in- due to oxidation of urate by RS generated during
vasive: 2-3 mg of liver biopsy (obtained by the exercise48. Allantoin measurement may be one of
fine needle technique) were weighted and incu- the more promising techniques for human use,
bated for 5 minutes at 37C with a physiological since human urate levels in vivo are high and
solution of the hydroxylamine I (1 mM) contain- urate reacts with a wide range of RS3.
ing a metal chelating agent. After incubation, the
sample was quickly frozen in liquid nitrogen to
denaturate enzymes and stop any reaction, and
subsequently warmed at room temperature prior Nuclear Magnetic Resonance (Nmr)
to the EPR measurement. For practical reasons, Based Metabolomics/Metabonomics
these researchers monitored the maximum con- Analysis of Biofluids
centration of nitroxide instead of the full time
evolution. Diseased tissue provided a more oxi- Metabolomics (also called metabonomics) is
dizing enviroment than healthy liver. Further- defined as the quantitative measurement of the
more, the nature of the disease affected the ox- dynamic multiparametric metabolic response of
idative status. living systems to pathophysiological stimuli or
The effect of the various experimental condi- genetic modification49. High resolution nuclear
1
tions on the final result, including lenght of incu- H-magnetic resonance (NMR) spectroscopic
bation, time from tissue extraction to addition of analysis of biofluids allows simultaneous detec-
the probe and time from incubation to EPR mea- tion of hundreds of low molecular weight species
surement, were sistematically investigated in or- within a sample of body fluid, resulting in the
der to set the optimal standardized experimental generation of a metabolic profile or NMR fin-
conditions. Interestingly, these results revealed gerprint that is altered characteristically in re-
that omogenization of the tissue is unnecessary sponse to physiological status 50. Once NMR
since the signal measured immediately after spectra are obtained, the highly complex spectra
omogenization in the presence of the probe was are analyzed using pattern recognition and multi-
very close to that obtained after 5 minutes incu- variate statistical methods to produce models for
bation with the whole biopsy. After calibration of samples classification 51. This technology has

313
B. Palmieri, V. Sblendorio

been widely applied to toxicology studies with a or pre-treatment of exhaled breath. The develop-
range of biological fluids, such as urine and plas- ment and introduction of this biosensor technique
ma, in both experimental animals and for immediate analysis of EBC (exhaled breath
humans52,53. Statistical analysis of urine samples condensate) has potential for undertaking real-
has been shown to result in inherent clustering time EBC monitoring of oxidative stress in ani-
behaviour for drugs and toxins acting on differ- mal research and clinical practice. Newer tech-
ent organs, such as liver or kidney, or having dif- niques, such as online masurements using sensi-
ferent toxic mechanisms. These cluster analyses tive biosensors, are being developed for more re-
are similar to those currently being developed for producible measurement of hydrogen peroxide.
gene array expression analysis and proteomics, For example, it is possible to detect hydrogen
and have been demonstrated to classify toxins in peroxide online (real-time) using a silver elec-
test samples correctly. Metabolomic analysis, trode or polymer with horseradish peroxidase56,57.
therefore, seems particularly suited to the analy- A similar enzyme detector system also may be
sis of biofluids from clinical/laboratory studies developed for real-time monitoring of 8-iso-
with the potential to measure simultaneously a prostane.
range of oxidative stress products and other in-
flammatory markers. Analysis of such samples
may lead to the identification of novel single bio-
markers of interest for wider study in patient Lipid Peroxidation
populations. Brindle and colleagues 54 studied
serum metabolome obtained from coronary heart Lipid peroxidation is a complex process
disease and healthy individuals. Moreover, appli- whereby polyunsaturated fatty acids (PUFAs) in
cation of metabolomics allows the simultaneous the phospholipids of cellular membranes undergo
analysis of multiple end products and it may be reaction with oxygen to yield lipid hydroperox-
that these fingerprints characteristic of disease ides (LOOH). The reaction occurs through a free
are a more powerful and robust means by which radical chain mechanism initiated by the abstrac-
to stratify disease severity, progression and to as- tion of a hydrogen atom from a PUFA by a reac-
sess drug efficacy than the analysis of any single tive free radical, followed by a complex se-
marker over a patient population. quence of propagative reactions.
The LOOH and conjugated dienes that are
formed can decompose to form numerous other
products including alkanals, alkenals, hydrox-
Online Measurements of yalkenals, malondialdehyde (MDA) and volatile
Oxidative Stress Biomarkers hydrocarbons58. Lipid peroxidation is often the
first parameter to which researchers turn when
Infrared laser spectroscopy is a promising they wish to prove the involvement of free radi-
method for free radical research, enabling online cals in cell damage. There are several reasons
measurement of oxidative stress biomarkers, for this. First, lipid peroxidation is an extremely
such as lipid peroxidation products, with high likely consequence if a reactive free radical is
sensitivity and efficiency. Murtz et al55 have de- formed in a biological tissue where PUFAs are
veloped real-time analysis of volatile ethane frac- generally abundant. Second, lipid peroxidation
tions in exhaled breath (gaseous molecular is a very important process in free radical
species) using laser absorption spectroscopy. The pathology as its so damaging to cells. Finally, a
group monitored the ethane fraction exhaled by a vast array of analytical techniques has been de-
smoker after smoking a cigarette every 30 min veloped to measure lipid peroxidation, though
over a period of 4 h, and observed a strong in- not all of them are applicable to the situation in
crease and subsequent decay of the ethane frac- vivo59.
tion after smoking. This method is unique, with For all assays its important that artifactual
very sensitivity and specificity for rapid and pre- changes in lipid peroxidation products are min-
cise breath testing. The detection limit is 300 imised both during and after sampling. Radical-
volume parts per trillion ethane in exhaled breath scavenging antioxidants and metal-chelating
with an integration time of 5 s. Another major agents are added to prevent the further formation
advantage of this method is that it allows the of lipid hydroperoxides and the breakdown of
analysis of biomarkers without pre-concentration existing lipid hydroperoxides. Enzymic reactions

314
Oxidative stress tests: overview on reliability and use. Part I

that may affect levels of products are inhibited Rises in intracellular free Ca2+, with conse-
by mixing the sample with acid or organic sol- quent activation of proteases and nucleases and
vents. It is generally advisable to assay samples formation of membrane blebs, oxidation of
as quickly as possible after taking them, since a critical SH groups and DNA damage are often
tendency to increased lipid peroxidation on stor- more relevant toxic events than is the bulk perox-
age has been reported60,61. Conversely, lipid hy- idation of membrane lipids68.
droperoxides can deteriorate on storage62. Lipid peroxidation is often (but by no means
The lipid peroxidations reaction in biological always) a late event, accompanying rather than
membranes causes impairment of membrane causing final cell death69. Indeed, cell and tissue
functioning63,64, decreases fluibility, inactivation destruction (whether mediated by radicals or
of membrane-bound receptors and enzymes and otherwise) can often lead to more lipid peroxi-
increases non-specific permeability to ions such dation because antioxidants are diluted out and
as Ca2+. Additionally, lipid hydroperoxides de- transition metal ions that can stimulate the per-
compose upon exposure to iron or copper ions, oxidation process are released from disrupted
simple chelates of these metal ions (e.g. with cells.
phosphate esters), haem, and some iron proteins, This stimulation of lipid peroxidation as a con-
including haemoglobin and myoglobin. Products sequence of tissue injury can sometimes make a
of these complex decomposition reactions in- relevant contribution to worsening the injury. For
clude hydrocarbon gases (such as ethane and example, in atherosclerosis there is good evi-
pentane), radicals that can abstract further hydro- dence that lipid peroxidation occurs within the
gen atoms from fatty acid side chains and cyto- atherosclerotic lesion and leads to foam cell gen-
toxic carbonyl molecules, of which the most eration and hence lesion growth70. In traumatic
harmful are the unsaturated aldehydes such as 4- injury to the brain and spinal cord, good evi-
hydroxy-2-trans-nonenal. Indeed, a major con- dence again exists that iron ion release into the
tributor to extracellular antioxidant defence in surrounding area, and consequent iron-stimulated
mammals is the existence in body fluids of pro- free radical reactions, worsen the injury71.
teins that bind copper ions (caeruloplasmin and It is equally likely that in some other dis-
albumin), iron ions (transferrin), haem eases, the increased rates of free radical reac-
(haemopexin) or haem proteins (haptoglobins) tions induced as a result of tissue injury make
and stop them from accelerating lipid peroxida- no significant contribution to the disease pathol-
tion and other free radical reactions65,66. ogy. Each proposal that free radicals in general,
or lipid peroxidation in particular, are important
Biomedical Lipid Peroxidation contributors to the pathology of a given disease
The measurement of putative elevated end must be carefully evaluated on its merits. This
products of lipid peroxidation in human samples obviously requires accurate methodology for
is probably the evidence most frequently quoted measuring these processes in cells, tissues and
in support of the involvement of free radical re- whole organisms.
actions in tissue damage by disease or toxins.
Studies beginning in the 1950s provided good Detection and Measurement of Lipid
evidence that several halogenated hydrocarbons Peroxidation: General Principles
exert some, or all, of their toxic effects by stimu-
lating lipid peroxidation in vivo. This is particu- Oxidation of lipids can be measured at differ-
larly true of carbon tetrachloride and probably ent stages, including:
true of bromobenzene.
This early choice of halogenated hydrocarbons 1. losses of unsaturated fatty acids;
for study was both casual (in that it gave early 2. measurement of primary peroxidation prod-
emphasis to the important biological role of free ucts;
radical reactions) but also unfortunate, since later 3. measurement of secondary carbonyls and hy-
studies have shown that most toxins stimulating drocarbon gases.
oxidative damage to cells do not appear to act by
accelerating the bulk peroxidation of cell mem- Between phases 1, 2 and 3 it is possible detect
brane lipids67: carbon-and oxygen-centred radicals (by ESR
combined with the use of spin traps) and iden-
toxin lipid peroxidation cell damage tify these radicals by their ESR spectra72.

315
B. Palmieri, V. Sblendorio

It should be noted that the chemical composi- cals during reoxygenation in perfused rat hepato-
tion of the end products of peroxidation will de- cytes is related to lipid peroxidation. Superoxide
pend on the fatty acid composition of the lipid anion was detected by lucigenin-enhanced
substrate used and upon what metal ions (if any) chemiluminescence. Lipid peroxidation and cell
are present. Thus, copper and iron ions give dif- injury were assessed by the release of malondi-
ferent end-product distributions and so the selec- aldehyde and lactic dehydrogenase. Upon reoxy-
tion of only a single test to monitor peroxidation genation following 2.5 h of anoxia, isolated he-
can give misleading results. Copper salts effi- patocytes generated considerable amount of O2.
ciently decompose peroxides, leading to low Following O2 formation, a significant increase
concentrations of detectable peroxides but high in malondialdehyde release was measured. Cell
amounts of some carbonyl molecules containing injury was temporally delayed relative to O 2
amino groups to form fluorescent products. The generation, but preceded the occurrence of a sig-
most accurate assays of lipid peroxidation are the nificant lipid peroxidation. Treatment with Vita-
most chemically sophisticated ones. They also min E abolished lipid peroxidation but had no ef-
require the most sample preparation and great fect upon superoxide anion formation and cell in-
care (e.g. by working under nitrogen) has to be jury. These results suggest that in perfused rat
taken to ensure that peroxidation does not occur hepatocytes non-peroxidative mechanisms are
during the handling of lipid material. more important than peroxidative mechanisms in
the pathogenesis of the early phases of reoxy-
Measurement of Lipid Hydroperoxides genation injury.
LOOH are the major initial molecular prod- Gasbarrini et al79 wanted to determine whether
ucts of lipid peroxidation and can be measured in the formation of oxygen free radicals occurs in
plasma by a lot of techniques. A sensitive and murine osteoblast-like cells (MC3T3-E1) exposed
specific assay is based on the capacity of LOOH to anoxia and reoxygenation and to explore its re-
to initiate the cyclooxygenase reaction catalysed lation to the reoxygenation injury. Cells were cast
by activation of prostaglandin endoperoxide syn- in agarose and perfused with oxygenated Krebs-
thase and uses an oxygen electrode73. Another Henseleit bicarbonate buffer. Anoxia was ob-
sensitive, even if expensive, method to measure tained by shifting the gas phase of the media to
plasma LOOH uses gas chromatography-mass 95% N2-5% CO2. Oxygen free radicals were de-
spectrometry (GC/MS) and involves reduction of tected by enhanced chemiluminescence: anion su-
LOOH to the hydroxy acids with triphenylphos- peroxide or hydrogen peroxide was measured by
phine74. adding lucigenin or luminol plus horseradish per-
Plasma LOOH can be measured using com- oxidase to the media, respectively. Cell injury
mercially-available assay kits. One such kit relies was assessed by the rate of lactate dehydrogenase
upon the reaction of LOOH with a haem com- release. During the control period, lucigenin and
pound, concomitantly oxidising a precursor to luminol plus horseradish chemiluminescences
produce methylene blue which is measured spec- were 15 1 nA per chamber and 20 2 nA per
trophotometrically. Although very simple and chamber, respectively. and lactate dehydrogenase
quick, only total hydroperoxide concentrations release was 10 1 mU per minute. During anox-
are measured and results do not relate well with ia, both chemiluminescences dropped to back-
some other measures of lipid peroxidation75. ground levels, although lactate dehydrogenase re-
Some methods have been developed that can lease increased progressively to 38 7 mU per
distinguish specific or different classes of minute. During reoxygenation, O2 formation in-
LOOH. These are based on separation according creased sharply to 45 6 nA and decreased to
to lipid class of the various hydroperoxides in a control levels; H2O2 production increased slowly,
Folch lipid extract of plasma by high perfor- reaching 42 7 nA at the end of the reoxygena-
mance liquid chromatography (HPLC) and mea- tion period; lactate dehydrogenase declined pro-
surement of the chemiluminescence produced gressively to control values. These data show that
during their breakdown in the presence of either osteoblastlike cells produce measurable amounts
luminol76 or isoluminol77. of superoxide and hydrogen peroxide radicals
The pathogenic role of lipid peroxidation in during reoxygenation. Because lactate dehydroge-
the reperfusion injury of the liver is still contro- nase release did not appear to relate to chemilu-
versial. Caraceni et al 78 wanted to determine minescence, oxyradical flux may serve as a signal
whether the damage caused by oxygen free radi- for other events that eventually lead to cell injury.

316
Oxidative stress tests: overview on reliability and use. Part I

Ojetti et al80 evaluated the combined use of Measurement of Conjugated Dienes


chemiluminescence and gastroendoscopy tech- LOOH possess a conjugated diene structure
niques and to assess the real-time production of having a characteristic UV absorption around
free radicals during ischemic damage of the gas- 234 nm. Measurement of this absorbance has
tric wall in an animal model. For the experiment, been extremely useful as an index of peroxida-
an optical junction was set up between a fibroen- tion in pure lipid systems and in tissue prepara-
doscope and a luminograph apparatus. Three tions from experimental animals. There are, how-
pigs were submitted to gastrofibroendoscopy be- ever, difficulties in measuring conjugated dienes
fore, during and after 30 min of clamping of the in biological materials because many of the other
coeliac artery. Under basal conditions, at the end substances present (e.g. haem proteins) absorb
of the ischemic phase and at the beginning of strongly in the UV and create a high background.
reperfusion, 1 mM of lucigenin, a specific super- This is partly eliminated by extraction of conju-
oxide enhancer, was injected in the left gastric gated dienes into an organic solvent such as chlo-
artery of the animal. The endoscopic live images roform/metahnol. However, PUFA, themselves,
and chemiluminescence emission were recorded and carbonyl compounds produced from the
and successively superimposed to measure rate breakdown of LOOH absorb UV light strongly at
and spatial distribution of photon emission (pho- about 210 nm so that the conjugated diene ab-
tons/s). Free radical production was not observed sorbance appears as a shoulder on the PUFA ab-
under basal conditions or during the ischemic sorbance spectrum82.
phase, but significantly increased during reperfu- Measurement is also complicated by the rela-
sion reaching a maximum peak after 15 min (0.6 tively low levels of conjugated dienes normally
0.2 photons 10(5)/s) and decreased progres- present in human plasma. A second derivative
sively thereafter. The superimposition of live and spectroscopy method83 allows greater sensitivity,
chemiluminescence images allowed the determi- since the conjugated diene shoulder that appears
nation of the regional production rate and distrib- in the ordinary spectrum translates into a sharp
ution of photons. minimum peak that is more easily measurable.
The LOOH are identified by comparison with The increased resolution of this technique may
authentic standards: although hydroperoxides of allow discrimination between the different conju-
free fatty acids are commercially available, those gated diene structures present. However, most
of others lipids (e.g. phospholipids, cholesterol es- (90%) of the conugated diene in human plasma is
ters) must be synthesised, which is both time-con- a non-oxygen-containing isomer of linoleic acid
suming and inconvenient. The assays have pico- (9, 11-octadecadienoic acid) that can be assayed
molar sensitivity and additionally, LOOH is specifically by HPLC 84 . This product is not
achieved by monitoring conjugated dienes either at found in the plasma of animals subjected to ox-
234 nm or by measuring the complete UV absorp- idative stress and may be of dietary origin or pro-
tion spectrum of the sample with a diode-array de- duced by the metabolism of gut bacteria. Appli-
tector. Alternatively, treatment with the reducing cation of conjugated diene methods to human
agent sodium borohydride will eliminate the body fluids is thus probably not measuring lipid
chemiluminescent signal. The assay is relatively peroxidation and is not recommended for human
specific for hydroperoxides although ubiquinols in studies.
human plasma produce a positive response.
Accurate measurement of LOOH is difficult Measurement of Thiobarbituric Acid
due to their rapid degradation in vitro. It is ex- Reacting Substances (TBARS) and
tremely important to minimise this by the addi- Malondialdehyde (MDA)
tion of antioxidants and quick processing of sam-
ples at 4C which is often not possible in a clini- The thiobarbituric acid (TBA) assay is the
cal situation. Also, these HPLC methods, al- most common and easiest method used as an in-
though specific and sensitive, are time-consum- dicator of lipid peroxidation and free radical ac-
ing in their analysis and preparation of standards tivity in biological samples. The assay is based
and are best used only when information on indi- upon the reaction of two molecules of TBA with
vidual hydroperoxides is required. Free fatty one of MDA, a physiologic ketoaldehyde pro-
acid- and cholesterol-hydroperoxides have been duced by peroxidative decomposition of unsatu-
detected in patients with adult respiratory distress rated lipids as a byproduct of arachidonate me-
syndrome81 or undergoing angioplasty. tabolism. The excess MDA produced as a result

317
B. Palmieri, V. Sblendorio

of tissue injury can combine with free amino Direct assessment of free MDA is most reli-
groups of proteins (MDA reacts mainly with Lys ably done by HPLC but the technique requires
residues by Michael addition), producing MDA- very careful handling of the sample. However,
modified protein adducts. Modification of pro- MDA is a minor product of lipid peroxidation
teins by MDA could conceivably alter their bio- and is readily metabolised; it is therefore not a
logical properties. Moreover, MDA-modified promising subject for the analysis of lipid peroxi-
proteins are immunogenic, and autoantibodies dation in vivo.
against MDA-modified Lys residues have been Plasma MDA concentrations are increased in
detected in the sera of rabbits and humans. Some diabetes mellitus and MDA can be found in the
studies have reported that the titer of these au- atherosclerotic plaques promoted by diabetes88.
toantibodies is related to the burden of, and may Increasead MDA concentrations have been found
predict progression of, atherosclerosis and my- in samples from women with preeclampsia89, in
ocardial infarction. Higher titers of autoantibod- plasma and breath condensates from asthmatics90
ies have also been correlated to coronary artery and in the brains of patients suffering from
disease11. Parkinson disease (PD), whereas increased
There are a lot of variations85 but basically the TBARS have been observed in plasma of pa-
sample is heated with TBA under acidic condi- tients with amyotrophic lateral sclerosis (ALS) as
tions and the amount of pink-coloured MDA- well as in Alzheimers patients91.
TBA adduct produced is measured at 532 nm.
For increased sensitivity, the complex can be ex- Measurement of Aldehydes Other
tracted into an organic solvent such as butanol Than MDA
and measured fluorometrically86. In a few experi- A great number of various aldehydes are pro-
mental systems the TBA test has been demon- duced during lipid peroxidation and they differ
strated actually to be measuring MDA itself. In greatly in their biological activity and capacity to
uncharacterised systems it is usual to refer to the cause further damage. The different classes of
assay of TBA-reactive substances (TBARS) as aldehydic peroxidation products in biological
the test is not specific for MDA. samples can be quantified by a method devel-
The test itself is very simple and quick but its oped by Esterbauer and Cheeseman86. The alde-
application to biological samples can be prob- hydes are derivatised with dinitrophenylhy-
lematic. The exact conditions of the test are drazine (DNPH), the various classes of different
very important. Biological samples normally polarity (e.g. alkanals, hydroxyalkenals, alke-
contain only a small amount of free MDA and nals) separated by TLC (Thin Layer Chromatog-
in tests where the unseparated sample is incu- raphy) and the individual aldehydes then re-
bated for a prolonged time the majority of the solved by HPLC with UV detection. An alterna-
MDA measured is formed by the decomposition tive procedure involves HPLC separation of the
of LOOH and further peroxidation during the fluorescent cyclohexanedione (CHD) derivatives
heating stage of the assay itself. The widely of the aldehydes92. These techniques are general-
used Yagi test that utilise TBA87, are probably ly extremely time-consuming and quite expen-
assays of lipid hydroperoxides. Various biologi- sive and are unlikely at present to be used as rou-
cal compounds react with TBA and the fluores- tine measures of lipid peroxidation. They are on-
cence method may be more selective than spec- ly likely to be used where it is necessary to know
trophotometry. Other factors that can markedly the full range of aldehydes produced in a particu-
affect the apparent concentration of TBARS in lar condition.
plasma include the iron content of reagents used Hydroalkenals, such as 4-hydroxynonenal
in the analysis and the storage of samples at (HNE), are probably the most important end
70C, although the addition of EDTA to products of the lipid peroxidation process in
chelate iron may reduce variability. To further terms of cytotoxicity. HNE is a major and toxic
minimise the problems related with the TBA aldehyde produced by free radical attack on -6
test, the MDA-TBA adduct may be measured polyunsaturated fatty acids (arachidonic, linoleic
by HPLC and GC although this is time-consum- and linolenic acids)93 and is considered a second
ing, involving complex sample preparation to toxic messenger of oxygen free radicals94,95. HNE
remove contaminants or sample extraction into undergoes many reactions with proteins, pep-
organic solvents to improve sensitivity and peak tides, phospholipides and nucleic acids; it there-
separation. fore has a high biological activity and exhibits

318
Oxidative stress tests: overview on reliability and use. Part I

various cytotoxic, mutagenic, genotoxic and sig- dothelium and, particularly, neutrophils of COPD
nal effects, including inhibition of protein and patients were found to be inversely related to
DNA synthesis, inactivation of enzymes, stimu- lung function104. COPD patients also had higher
lation of phospholipase C, reduction of gap-junc- diaphragm concentrations of both protein car-
tion communication, stimulation of neutrophil bonyls and HNE-protein adducts. Furthermore, a
chemotaxis, modulation of platelet aggregation negative correlation was found between carbonyl
and modulation of the expression of some groups and airway obstruction (i.e., concentra-
genes96. Additionally, HNE may be an important tions of reactive carbonyls related to disease
mediator of oxidative stress-induced apoptosis, severity) and between HNE-protein adducts and
cellular proliferation and signaling pathways97. respiratory muscle strength (i.e., HNE-protein
HNE is permanently formed at basal concentra- adduct formation associated to respiratory mus-
tions under physiologic conditions, but its pro- cle function)105.
duction is greatly enhanced in pathologic condi- Because HNE is such an important, biological-
tions associated to lipid peroxidation. Under ly active product it may be of interest to measure
physiologic conditions, the cellular concentra- specifically. HNE can be measured by HPLC
tions of HNE ranges from 0.1 to 3 mol/L. Un- with UV detection or GC-MS which is more sen-
der conditions of oxidative stress, HNE concen- sitive but expensive.
trations are significantly increased in plasma,
some organs and cell types 98 . During heavy Isoprostanes
stress, e.g., in patients with severe rheumatologic F2-Isoprostanes (F2-IsoPs), isoprostanes con-
diseases such as rheumatoid arthritis, systemic taining an F-type prostane ring, are a family of,
sclerosis, lupus erythematosus, chronic lym- theoretically, 64 prostaglandin F2-like molecules
phedema or chronic renal failure, serum HNE is produced in vivo, primarily in situ, by nonenzy-
increased to concentrations up to 3- to 10-fold matic free-radical-catalyzed peroxidation of es-
higher than physiologic concentrations99. HNE terified arachidonic acid and then cleaved and re-
and acrolein, compound present in some environ- leased into the circulation by phospholipases(s)
mental sources like cigarette smoke, are highly before excretion in the urine as free isoprostanes.
reactive toward proteins (particularly, HNE is Reports have shown that F2-IsoPs are authentic,
much more reactive to proteins than to DNA), reliable biomarkers of lipid peroxidation and are
forming stable covalent adducts with His, Lys useful in vivo indicators of oxidative stress in
and Cys residues through Michael addition; these various clinical conditions, such as acute and
adducts are known as advanced lipoxidation end chronic inflammation, ischemia/reperfusion in-
products (ALEs)100,101. This process introduces jury, diabetes and atherosclerosis106-110. F2-IsoPs
carbonyl groups into proteins. have also been used to assess in vivo oxidative
Furthermore, concentrations of acrolein- and response to some drugs, antioxidants or dietary
HNE-protein adducts are increased in cardiovas- interventions for their free-radical-scavenging
cular disease 102 . Acrolein reacts with Lys properties. Various techniques for F2-IsoP quan-
residues of apolipoprotein A-I (apoA-I), the ma- tification are available (GC-MS)111. Additionally,
jor protein of HDL, which plays a relevant role to being markers of oxidative stress and antago-
in mobilizing cholesterol from artery wall nists of the action of prostaglandins, they may al-
macrophages. Acrolein adducts colocalize with so exert unique biological effects.
apoA-I in human atherosclerotic lesions. More- A tissue that does not contain isoprostanes is
over, the capacity of acrolein-modified apoA-I to yet to be reported. Isoprostanes have also been
remove cholesterol from cultured cells is im- found in measurable quantities in most of the bi-
paired, suggesting that carbonylation might inter- ological fluids analyzed, including plasma, urine,
fere with the normal function of apoA-I in pro- synovial fluid, bronchoalveolar fluid, bile,
moting cholesterol removal from artery wall lymph, microdialysis fluid from various organs,
cells, thus playing a critical role in atherogene- and amniotic, pericardial, and seminal fluid, even
sis103. if plasma and urine are the sample types that are
Increased concentrations of HNE-protein commonly analyzed, being the most convenient
adducts have been reported in the lungs of smok- to obtain and the least invasive112.
ers with and without chronic obstructive pul- At present, measurement of F2-IsoPs is regard-
monary disease (COPD). Notably, HNE concen- ed as one of the most reliable approaches for the
trations in the pulmonary epithelium, airway en- assessment of oxidative status or free-radical-

319
B. Palmieri, V. Sblendorio

mediated lipid peroxidation in vivo. Available da- says for MDA, TBARS, lipid hydroperoxides or
ta indicate that quantification of F2-isoprostanes conjugated dienes, which are hampered by some
in either plasma or urine gives a highly precise methodologic limits.
and accurate index of oxidative stress113. Where- F2-IsoPs are very well suited as biomarkers of
as the biological validity of F2-IsoPs as biomark- oxidative stress for the following reasons:
ers of oxidative status is well established, it is
technically quite complicated to measure F 2- 1. The in vivo formation of isoprostanes increas-
IsoPs and their metabolites in body fluids and es as a function of oxidative stress115,116.
some limits with respect to their measurement 2. They can be measured accurately down to pi-
must be taken into account. F2-IsoPs are chemi- comolar concentrations with analytical meth-
cally stable in vivo and ex vivo, but once they ods such as GC-MS, GC-MS/MS, LC-MS,
produced and released into the circulation, they LC-MS/MS or RIA. The first 4 methods can
are fastly metabolized (even if not as quickly or easily differentiate among the various types of
as extensively as prostaglandins) and eliminated. isoprostanes, but they require extensive prepa-
Their fast disappearance from plasma may pre- ration of the material (e.g., phospholipid ex-
vent practical application. Current techniques: traction and alkaline hydrolysis) and/or expen-
GC-MS, GC-tandem MS (GC-MS/MS), liquid sive instrumentation. RIAs are somewhat easi-
chromatography (LC)-MS, LC-MS/MS, enzyme er to perform and are widely available com-
immunoassays and radioimmuno assays (RIAs), mercially. However, many of these are not
are able to detect the steady-state concentrations able (or have not been shown to be able) to
of F2-IsoPs in many tissues and body fluids, even distinguish between the prostanoids and the
in the basal state, concentrations after any degree isoprostanes, much less between the different
of oxidant stress or lipid peroxidation in vivo. types of isoprostanes.
Different internal standards (18O- or 2H-labeled 3. They are stable in isolated samples of body
analogs of specific isoprostane isomers) are fluids, including urine and exhaled breath con-
available from commercial sources to quantify densates, providing an exceedingly noninva-
the isoprostanes by MS techniques. sive route for their measurement.
Alternative methods have also been developed 4. Their measured values do not exhibit diurnal
to quantify F2-IsoPs by immunologic techniques variations and are not affected by lipid content
(RIAs and enzyme immunoassays) and a few im- in the diet 117,118 . However, they do vary
munoassay reagent sets are commercially avail- markedly in clinical and experimental condi-
able. A potential drawback of these techniques is tions characterized by oxidative stress and
that limited information is currently available re- closely parallel disease severity. Some diurnal
garding their precision and accuracy. Moreover, variation in urinary F2-IsoP excretion does oc-
few data exist comparing F2-IsoP concentrations cur within individual humans, even if this
measured by immunoassays with MS results. variation is not present when F 2-IsoPs are
Futhermore, the sensitivity and/or specificity of evaluated on a group level. Furthermore, even
these assays may vary substantially among man- if pooled urine samples are likely preferable,
ufacturers. However, even if MS techniques of F2-IsoPs determined in urine collected in the
F2-IsoP quantification are considered the gold morning or in several spot urine samples ade-
standard, immunoassays have expanded re- quately represent the daily F2-IsoP excretion.
search in this area because of their low cost and 5. They are specific products of peroxidation.
relative ease of use. Additionally to commercial 6. They are present in detectable amounts in all
immunoassays, some researchers have generated healthy tissues and biological fluids, thus al-
polyclonal antibodies and have developed assays lowing definition of a reference interval.
for F2-IsoP114. It appears that there is good corre-
lation between these techniques and MS. Because of free-radical-catalyzed conversion
Various analytical methods are available for of arachidonic acid to isoprostanes, precautions
the analysis of isoprostanes, the most sensitive, must be taken to avoid artifactual formation dur-
highly specific and reliable technique being GC ing sample storage and processing. Blood plasma
with negative-ion chemical ionization (NICI) samples contain considerable amounts of arachi-
MS. For quantification of lipid peroxidation, donic acid, mainly esterified to membrane phos-
measurements of F2-IsoP have a clear advantage pholipids. Storage of these samples at 80C and
over currently available techniques such as as- addition of antioxidants (e.g., butylated hydroxy-

320
Oxidative stress tests: overview on reliability and use. Part I

toluene and triphenylphosphine) during sample hypercholesterolemia and atherosclerosis. Uri-


preparation is therefore recommended. More- nary 8-iso-PGF2, measured by GC-MS/MS, was
over, isoprostanes in blood samples may occur as found to be a novel, sensitive and independent
free fatty acids or esterified to phospholipids or risk marker in patients with coronary heart dis-
lipoproteins. Thus, one has to distinguish be- ease, additionally to know risk factor of this
tween the two fractions of isoprostanes in human pathology, i.e., diabetes mellitus, hypercholes-
blood, i.e., free and total (free plus esterified). terolemia, hypertension, obesity and smoking122.
Analysis of the esterified molecules requires hy- Increased concentrations of 8-iso-PGF2 have al-
drolysis to yield the free derivatives. Because so been found in plasma or urine samples from
urine samples have a very low lipid content, au- patients with type 2 diabetes.
tooxidation is not a problem. Nevertheless, as a
precaution, samples should be supplemented Lipid peroxidation in Human Material:
with EDTA and 4-hydroxy-2,2,6,6-tetram- Past and Future
ethylpiperidine 1-oyl (4-hydroxy-TEMPO) and Despite the problems that can occur with as-
stored at 20C. says such as the TBA test, diene conjugation and
Different diseases and experimental conditions light emission, they usually work adequately
have been shown to be related to marked increas- when applied to measurements of peroxidation in
es in urinary, plasma and tissue concentrations of liposomes, microsomes or other isolated mem-
F2-IsoPs. However, it has been suggested they brane fractions, provided that one is alert to the
should be considered not just mere markers, but various artefacts that can arise123. Much more se-
also mediators of disease, as they evoke impor- rious problems occur when these assays are ap-
tant biological responses in virtually every cell plied to human body fluids or to tissue extracts.
type found within the lung. Infact, the iso- By far the most misleading assay to use on hu-
prostanes may mediate many of the features of man material, especially plasma, is the TBA test.
the disease states for which they are used as indi- Plasma contains many compounds that react in
cators. 8-iso-prostaglandinF2, the biologically the TBA assay, including bile pigments, amino
active component, is produced in great amount in acids and carbohydrates. Some of these sub-
otherwise normal individuals exposed to ciga- stances (e.g. bile salts) produce a different chro-
rette smoke, allergenes, ozone or hyperoxia and mogen, and this interference can be overcome by
during ventilated ischemia. It is also markedly separating out the authentic (TBA)2-MDA adduct
increased, serving as a biomarker, in the bron- (e.g. by HPLC) before measurement. However,
choalveolar lavage (BAL) fluid, plasma, urine or this solves only part of the problem because
exhaled breath condensate (a noninvasive tech- some molecules (especially amino acids and sug-
nique for direct measurement of oxidative stress ars) react in the assay to form an authentic
in the lungs) in some pulmonary diseases such as (TBA)2-MDA adduct. The lack of specificity of
asthma, COPD, interstitial lung disease, cystic fi- the TBA assay when applied to plasma is dra-
brosis, pulmonary hypertension, acute chest syn- matically illustred by a simple experiment per-
drome, sickle cell disease, acute lung injury (in- formed by Lands et al124. Using the cyclooxyge-
cluding acute respiratory distress syndrome, nase assay these researchers measured the lipid
ARDS) and severe respiratory failure in infants peroxide amount of some human plasma samples
as well as in healthy chronic smokers119,120. Sys- as about 0.5 M. Expressing the results of a TBA
temic and synovial fluid concentrations of 8-iso- test on the same samples as peroxide equiva-
PGF2 are higher in patients with rheumatoid lentsgave a value of 38 M. When specific
arthritis, psoriatic arthritis, reactive arthritis and chemical assays are used, the authors and oth-
osteoarthritis than in healthy controls. Plasma ers125 find that human plasma, freshly taken from
concentrations are increased in patients with car- healthy subjects, has less than 0.1 M lipid per-
diovascular disease and it has been suggested oxide. This is perhaps not surprising, since even
that this may be a useful biomarker of risk121. if peroxides do form in vivo and enter the circu-
Similarly, some cardiovascular conditions feature lation, they can be quickly cleared. For example,
marked increases in F2-IsoP concentrations, in- although lipid peroxidation is thought to be rele-
cluding during and after cardiopulmonary by- vant in atherosclerosis, it seems to be peroxida-
pass, renal, cerebral and myocardial ischemia- tion in the arterial wall that matters, not peroxi-
reperfusion injury, unstable angina, heart failure, dation in the bulk plasma. Thus, some of the ear-
coronary heart disease, acute ischemic stroke, lier suggestions that circulating lipid peroxides

321
B. Palmieri, V. Sblendorio

kill vascular endothelial cells and initiate athero- of whole-body GSH status and a useful indicator
sclerosis need to be re-evaluated. of oxidative stress status in humans. Different
In order to know as much as possible about the techniques have been optimized to identify and
real occurrence of lipid peroxidation in human quantify glutathione forms in human samples, in-
material, it is important to use methods that give cluding spectrophotometric, fluorometric and bi-
specific chemical information about what is pre- oluminometric assays, often applied to HPLC
sent. Indeed, food scientists have followed this analysis, as well as the more recently developed
principle for years. Thus various groups are sepa- GC-MS and HPLC-electrospray ionization-MS
rating the different peroxidation products before techniques131,132. Futhermore, a wide variety of
measuring them. This is often done by HPLC; techniques have been introduced for the determi-
for example, HPLC methods for measuring cyto- nation of GSH and GSSG in human blood, the
toxic aldehydes are available126. However, con- measurement of which, particularly that of
version of material into volatile derivatives, sep- GSSG, could be overstimated if samples are not
aration by gas chromatography and identification properly processed133,134. A specific warning has
by mass spectrometry is likely to give more pre- to be addressed to correct sample manipulation
cise chemical information when complex mix- and prevention of artifactual GSH oxidation. The
tures are being studied127,128. Thus, derivatization authors have shown that the main artifact results
and mass spectrometry have been used to charac- from sample acidification (for protein separation)
terize peroxidized fatty acids and cholesterol oxi- without prevention of artificial oxidation of SH
dation products in human atherosclerotic le- groups by blocking with alkylating agents. Actu-
sions129. ally, many published articles reporting concentra-
Specificity can also be achieved by the use of tions of GSH, GSSG and S-glutathionylated pro-
antibody techniques, particularly monoclonal an- teins in blood, both from healthy controls and pa-
tibodies. Thus, antibodies directed against low- tients affected by various pathologies, are not ar-
density lipoprotein that has undergone peroxida- tifact free, which makes the conclusions reached
tion or has been treated with 4-hydroxynonenal in these articles meaningless. Consequently, the
bind to rabbit atherosclerotic lesions. Additional- notion that some pathophysiologic conditions
ly, low-density lipoproteins eluted from such le- can alter and/or be influenced by the GSH/GSSG
sions can bind to antibody specific for MDA- homeostasis of blood still needs to be confirmed.
treated low-density lipoproteins. Antibody-based It is well known that a decrease in GSH con-
methods can also be applied to plasma samples. centration may be associated with ageing135 and
Using such specific techniques, the exact role the pathogenesis of many diseases, including
played by lipid peroxidation in cell injury and rheumatoid arthritis, amyotrophic lateral sclero-
death mediated by toxins and in human disease sis, acquired immune deficiency syndrome,
should at last become clearer130. Alzheimers disease, alcoholic liver disease,
cataract genesis, respiratory distress syndrome,
cardiovascular disease and Werner syndrome.
Furthermore, there is a drastic depletion in cyto-
Measurement of Protein Damage plasmic concentrations of GSH within the sub-
stantia nigra of Parkinsons disease patients136.
Reactive free radicals can modify amino acid Depletion of total GSH (GSH + 2 GSSG + pro-
residues of proteins and lead to cross-linking, tein-bound glutathione) and a decreased
changes in conformation and loss of function. GSH:GSSG ratio are indicators of oxidative/ni-
Oxidatively damaged proteins are likely to be re- trosative stress in ischemic brain disease137, car-
moved rapidly by proteases rather than accumu- diovascular diseases 138 and cancer 139, and de-
late to readily-detectable levels. creased concentrations of GSH are consistently
observed in both types of diabetes mellitus. Low
Glutathione and GSH concentrations and a high GSSG:GSH ratio
S-Glutathionylated Proteins have been measured in blood of patients with
Because blood glutathione concentrations may various diseases, including breast and lung can-
reflect glutathione status in other, less accessible cer, coronary heart surgery and preeclampsia140.
tissues, measurement of both reduced glutathione The GSH system is also altered in lung inflam-
(GSH) and glutathione disulfide (GSSG) in matory conditions. For example, GSH concentra-
blood has been considered relevant as an index tions are increased in the epithelial lining fluid of

322
Oxidative stress tests: overview on reliability and use. Part I

chronic smokers, whereas they decrease fastly in Increased concentrations of stable halogenated
patients with mild asthma during an asthma ex- Tyr residues have been detected in proteins iso-
acerbation. Similarly, GSH concentrations in the lated from atherosclerotic plaques as well as in
epithelial lining fluid are decreased in idiopathic plasma and airway secretions of patients with
pulmonary fibrosis, asbestosis, acute respiratory asthma, ARDS and cystic fibrosis, and halo-
distress syndrome and in HIV-positive genated Tyr residues are widely used as markers
patients141. Total GSH was markedly decreased for damage mediated by hypohalous acids (HO-
in older patients with chronic diseases 142, the CL and HOBr) in these diseases153-156. The major
deficit being attributable to lower GSH concen- products are Cl-Tyr and 3-bromotyrosine, but di-
trations and not to higher GSSG. These results halogenated compounds (3,5-dichlorotyrosine
suggested that the decrease in GSH might be and 3,5-dibromotyrosine) are formed with high
used to monitor the severity and progress of the excesses of HOCL and HOBr. Dramatic selective
diseases. Conversely, total GSH concentrations enrichment in protein-bound NO2-Tyr and Cl-Tyr
are high in the blood of elderly persons who are amount within ApoA-I, the major protein con-
in excellent physical and mental breath143. stituent within HDL, recovered from human
plasma and atherosclerotic lesions has been
Tyrosine Oxidation, demonstrated by proteomic and MS methods.
Nitration and Halogenation Analysis of serum also showed that protein-
The toxicity of NO is enhanced by its reaction bound NO 2-Tyr and Cl-Tyr concentrations in
with a superoxide to form ONOO144. It or sec- ApoA-I are markedly higher in individuals with
ondary metabolites can cause tyrosine nitration established coronary heart disease157,158. These re-
in protein, creating nitrotyrosine, a footprint de- sults suggest that increased concentrations of Cl-
tectable in vivo. Tyr and NO2-Tyr in circulating HDL might rep-
Analysis of 3-nitrotyrosine (NO2-Tyr), a stable resent specific markers for clinically significant
marker for NO derived oxidants, and halo- atherosclerosis.
genated Tyr products such as 3-chlorotyrosine Increased concentrations of nitrated plasma
(Cl-Tyr) or 3-bromotyrosine has been performed proteins have been associated with predisposition
in some diseases and different techniques have to develop lung injury in premature infants as
been developed for such measurements145-149. The well as with unfavorable outcome on develop-
quantitative measurement of NO2-Tyr is hindered ment of lung injury159. The clinical relevance of
by severe methodologic problems. The most of protein Tyr nitration has been emphasized by the
the data available on NO2-Tyr in tissues and flu- observation of a strong association between pro-
ids have been derived from antibody-based tech- tein bound NO2-Tyr concentrations and coronary
niques, which however, are often not rigorously artery disease risk. Circulating concentrations of
validated. Therefore, such immunologic tech- protein-bound NO2-Tyr may serve as an indepen-
niques should be considered semiquantitative dent biomarker to assess atherosclerosis risk,
and the results interpreted accordingly. HPLC burden and incident cardiac events, as well as to
with ultraviolet detection does not provide ade- monitor the vasculoprotective action of drugs
quate sensitivity or specificity for biological ma- such as statins (hydroxymethylglutaryl-CoA re-
terials. In contrast, HPLC with electrochemical ductase inhibitors)160.
detection, LC-MS/MS, electron capture-negative Patients with lung cancer have significantly
chemical ionization (EC-NCI) GC-MS and GC- higher serum concentrations of nitrated proteins,
MS/MS are able to quantify NO2-Tyr in biologi- supporting the presence of oxidative and ni-
cal materials and human plasma150-152. trosative stress161,162. Specific locations and tar-
At present, only MS/MS-based techniques, gets of Tyr nitration in lung cancer have, recent-
both GC-MS/MS and LC-MS, provide reliable ly, been detailed163. Increased nitrotyrosine im-
values for circulating and excreted NO2-Tyr, with munostaining is limited mainly to the tumor and
LC-MS/MS being at present considerably less not to surrounding healthy tissue or is weakly re-
sensitive than GC-MS/MS and that the basal active in different regions of the lung from the
concentrations obtained by this analytical ap- same patients with cancer, suggesting a unique
proach may serve as reference values. enviroment inside the tumor that may contribute
Another methodologic problem is consider- to the disease process. This was noted in squa-
able interference by coeluting molecules, which mous cell carcinoma as in the well-differentiated
can eliminated only by use of MS/MS. adenocarcinoma. Using proteomic and genomic

323
B. Palmieri, V. Sblendorio

approaches, authors have identified the protein Assays of general oxidative damage to pro-
targets. Most of the nitrated proteins fall into 4 teins, while an important index of oxidative
categories: oxidant defense (such as manganese stress occuring in vivo, need to be replaced with
superoxide dismutase and carbonic anhydrase), assays of oxidative damage to specific proteins
energy production (many glycolitic enzymes), having relevance to the lesion under considera-
structure (such as -actin, - and -tubulin and tion.
vimentin) and those involved in apoptosis (an- As a marker of oxidative damage to proteins,
nexins). carbonyls have been shown to accumulate during
Tyrosine nitration is one of the earliest mark- aging, ischemia/reperfusion injury, chronic in-
ers found in brains from persons affected by flammation, cystic fibrosis and many of age-re-
Alzheimers disease, in the plaques of brains lated diseases in some organisms171.
from persons with multiple sclerosis and in de- Specific carbonylated proteins have been de-
generating upper and lower motor neurons in tected in both the brain tissue and plasma of
ALS patients164. Nitrated -synuclein selectively Alzheimers disease patients172. The observation
accumulates in Lewy bodies and protein inclu- of carbonylated proteins in plasma suggests that
sions in many pathologies (Alzheimers disease, these oxidized species may be useful as diagnos-
Parkinsons disease, synucleinopathies and tic biomarkers for (possibly early) Alzheimers
tauopathies). Nitrated proteins have been evi- disease.
denced in some inflammatory disease, chronic The carbonyl content in plasma proteins
renal failure, rheumatoid arthritis, type 1 and (mainly albumin and -globulins) from children
type 2 diabetes and cystic fybrosis. On the other with different forms of juvenile chronic arthri-
hand, basal protein nitration has been detected tis was significantly higher than in healthy chil-
under physiologic conditions in most tissues, in- dren, and more importantly, the carbonyls in-
cluding plasma and the human pituitary and creased in parallel with the activity of the dis-
some of the nitrated proteins have been identi- ease.
fied. Two-dimensional Western blotting and LC- Correlation between the carbonyl concentra-
MS/MS analyses have been used to detect and tion and the activity or the type of chronic juve-
characterize 4 nitrated proteins, including actin, nile arthritis indicates that plasma protein car-
in the healthy human pituitary, which partecipate bonyl groups are a good marker of inflammatory
in neurotransmission, cellular immunity, and cel- process activity and may allow the use of car-
lular structure and motility165. These results are bonyls as a clinical marker of antioxidant barrier
consistent with the emerging perspective that impairment in this group of patients as well as
low-level Tyr nitration may be a physiologic reg- for monitoring possible pharmacologic treat-
ulator of a signaling pathway166. ments173.
Plasma concentrations of protein carbonyls, as
Carbonylated Proteins well as free F2-IsoPs and protein reduced thiols,
Protein carbonyls may be produced by the oxi- differ significantly between chronic kidney dis-
dation of some amino acid side chains (e.g., in ease patients and healthy people. Furthermore,
Lys, Arg, Pro and Thr); by the formation of such biomarkers of oxidative/nitrosative stress
Michael adducts between Lys, His and Cys are significantly higher in patients with diabetes
residues and ,-unsaturated aldehydes, forming and hypercholesterolemia174.
ALEs (Advanced Lipooxidation End Products); Winterbourn et al175 determined that protein
and by glycation/glycoxidation of Lys amino carbonyl concentrations were increased in both
groups, forming advanced glycation end AGE plasma and BAL (bronchoalveolar lavage) fluid
products167-169. The generation of carbonyl mole- of patients with severe sepsis or major trauma,
cules is the most general and widely used marker which correlated well with measured concentra-
of severe protein oxidation both in vitro and in tions of ALEs and with indices of neutrophilia
vivo, with different assays developed for the and neutrophil activation. Moreover, patients
quantification of these species (170). The chemi- with acute pancreatitis had significantly in-
cal stability of protein carbonyls makes them creased plasma concentrations of protein car-
suitable targets for laboratory measurements and bonyls, which were related to disease severity,
is also useful for their storage: their stability dur- thus confirming that this protein modification
ing storage for 10 years at 80C has been could be a useful plasma marker of oxidative
demonstrated. damage.

324
Oxidative stress tests: overview on reliability and use. Part I

Measurement of DNA Damage 8-hydroxydeoxyguanosine (8-OHdG), an oxi-


dized form of guanine, is a major oxidative
Cellular DNA damage can be caused by ROS DNA-damage product that can produce mutation.
produced under several conditions and different This compound causes A:T to C:C or G:C to T:A
methods have been developed to measure the transversion mutations because of its base pair-
oxidatively modified nucleobases in DNA176,177. ing with adenine as well as cytosine. Measure-
Oxidative DNA damage seems to relate to an in- ment of 8-OHdG in urine has been used to assess
creased risk of cancer development later in whole-body oxidative DNA damage. This can
life178. DNA subjected to attack by hydroxyl rad- be achieved by HPLC and MS methods. Howev-
ical produces a wide range of base and sugar er, 8-OHdG can arise from degradation of oxi-
modification products. Amongst these, the major dized dGTP in the DNA precursor pool, not just
reaction product of .OH with thymine is thymine from removal of oxidized guanine residues from
glycol and with guanine, 8-hydroxy-guanine. DNA by repair processes. Furthermore, there are
These DNA products are eliminated by repair many other products of oxidative DNA damage.
enzymes (excision enzyme and glycolases) and Hence, urinary 8-OHdG is a partial measure of
are excreted in the urine either as the free base damage to guanine residues in DNA and its nu-
products or as the nucleoside derivatives, thymi- cleotide precursor pool, and 8-OHdG concentra-
dine glycol (Tg) and 8-hydroxydeoxyguanosine. tions may not truly reflect rates of oxidative
The latter products can be used as an index of damage to DNA.
radical attack upon DNA in vivo and Cathcart179 Papa et al181 wanted to assess the production of
haved calculated from such measurements that ROS and 8-OHdG in gastric mucosa, according to
oxidative damage to mammalian DNA may total H. pylori status and cytotoxic associated gene
about 105 oxidative hits per cell per day179. product A (CagA) and to determine the relation-
Measurement of urine samples is based on chro- ship between ROS production and amount of 8-
matographic pre-purification by normal, reverse- OHdG. Gastric biopsy specimens were obtained
phase or immuno-affinity columns to prevent in- from 60 consecutive patients. ROS generation was
terference by many urinary compounds, fol- measured by luminol enhanced chemilumines-
lowed by derivatization and analysis. Originally, cence. 8-OHdG detection was performed by an
HPLC combined with UV detection was used to immunoperoxidase method, using a specific anti
measure Tg31 but great concentrations of sam- 8-OHdG monoclonal antibody. 40/60 patients
ples were required to obtain sufficient sensitivi- (67%) were H. pylori-positive. ROS generation
ty. Most current procedures are based on HPLC, was significantly higher in patients positive for H.
GC-MS, LC-MS and antibody-based methods180. pylori infection as compared to negative. 8-OHdG
The advantages of artifacts produced during detection was performed in 30 patients in which
measurement of 8OHdG are useful for visualiza- CagA presence was also investigated. High ex-
tion of damage, but they seem likely to be only pression of 8-OHdG was detected in 14/20 (70%)
semiquantitative. H. pylori-positive patients (13 CagA + and 1
DNA can also be damaged by RNS, undergo- CagA) and in 2/10 (20%) H. pylori-negative pa-
ing mainly nitration and deamination of purines. tients. A significant correlation was found be-
Techniques for the measurement of DNA base tween ROS production and 8-OHdG content.
nitration and deamination products have been de- However, the recently completed Biomarkers
veloped but may need more refinement and vali- of Oxidative Stress Study (BOSS), using acute
dation before they can routinely applied to hu- CCl4 poisoning in rodents as a model for oxida-
man materials. tive stress, has demonstrated that 8-OHdG in
None of the analytical techniques mentioned urine is a potential candidate general biomarker
above identifies where the oxidative damage is of oxidative stress, whereas neither leukocyte
located. Another problem in studying damage DNA-MDA adducts nor DNA-strand breaks re-
to DNA by ROS/RNS is the limited availability sulted from CCl4 treatment.
of human tissues from which to obtain DNA. Immunohistochemical accumulation of high
Most studies are performed on DNA isolated levels of 8-OHdG was reported to occur in vari-
from lymphocytes or total leukocytes from hu- ous human tumors, like high-grade glioma, com-
man blood and it is assumed (possibly erro- pared to adiacent, normal tissue or low-grade
neously) that changes here are reflected in oth- glioma182. These studies suggested that oxidative
er tissues. stress may play a role in tumor progression.

325
B. Palmieri, V. Sblendorio

As with other indices of whole-body oxidative serve as a stimulating species for induction of an-
stress, the measurement of products of oxidative tioxidant enzymes on the one hand and, on the
DNA damage is limited by some problems, in- other, may themselves damage the proteins; for
cluding the obscurity of the tissue of origin of the example, O2 might inactivate catalase. Determi-
products. nation of the fate of LMWA may serve as a better
Another technique utilized to detect DNA indicator for ROS, because the adduct is specific
adducts is the comet assay183. Other methods ex- to these molecules.
ist to determine single- and double-strand Determination of the ratio between oxidant
breaks184. Different oxidized adducts of DNA can and reductant (e.g., ascorbate/dehydroascorbic
be determined. Examples are DNA-aldehyde acid or GSH/GSSG) may therefore serve as in-
adducts, such as deoxyguanosine-malondialde- dicator of oxidative damage. One of the ap-
hyde adducts185, or the end product of the reac- proaches most commonly used is the measure-
tion between DNA and 4-hydroxynonenal, the ment of the total anti-oxidant activity of a bio-
aldehyde formed following exposure to ROS186 logical site. Depletion of one anti-oxidant mol-
to produce N2-ethenodeoxyguanosine. ecules causes changes in the level of overall
anti-oxidant molecules and may be evaluated
using a variety of techniques including bio-
chemical, immunohistochemical, spectroscopi-
Measurement of Antioxidants cal and electrochemical188.
The total-antioxidant-activity assay offers
Different animal studies have shown that an- many advantages and is considered a useful
tioxidants delay or protect against the oxidative tool for detecting oxidative stress phenomena
damage produced by free radical reactions. Radi- in bodily fluids and tissues. It may serve as an
cal-scavenging antioxidants are consumed during appropriate tool for the evaluation of anti-oxi-
this process and antioxidant status is sometimes dant therapy. Determinations of total LMWA
used indirectly to assess free radical activity. The rather than individual anti-oxidants are impor-
commonly used TRAP assay (Total [peroxyl] tant, because LMWAs work in concert189, and
Radical-trapping Antioxidant Parameter) is, in it measurement of only one or a few compounds
basic form, an empirical measurement of antioxi- out of many present at a specific biological lo-
dant activity in plasma187. Assessment of the rela- cation might be misleading. Moreover, mea-
tive contribution of individual antioxidants surement of the total LMWA ensures a reliable
(ascorbate, urate, -tocopherol, protein sul- picture of the physiological situation. Now its
phydryls) to the total antioxidant capacity re- dont know the concentration of a specific
quires separate specific assays. Measurements of compound at a specific location at a given mo-
either TRAP or the individual antioxidants are ment. Sometimes the researchers try to detect
not likely to be useful indices of free radical gen- compounds that are not present in the site un-
eration as the latter would need to be extensive der investigation.
for the steady-state concentrations of the antioxi- A few dozen LMWA exist, and usually only a
dants to be disturbed in vivo. However, antioxi- few of them, such as vitamin E and ascorbic and
dant amounts are interesting parameters in them- uric acids, are revealed; thus, many compounds
selves, indicative of the propensity of the indi- that can be present at the biological site are ne-
vidual to oxidative stress. glected. The measurement of the total LMWA is
Many methods exist for evaluating the activity designed to overcome these problems. Numer-
and composition of the anti-oxidant enzymes, ous procedures allow measurement of the total
which, along with the LMWA, constitute the two LMWA activity. These include indirect and di-
major components of the anti-oxidative system. rect methods for measuring total anti-oxidant
Some techniques that directly evaluate enzymatic activity originating from the LMWA. Indirect
activity utilize spectroscopic measurements or methods are those that measure consequential
gel-activity procedures; other methods employ factors of redox capacity, such as oxidation
immunocytochemistry. Assays of anti-oxidant products formed or concentrations of major re-
enzymes may indicate prior exposure of the cell dox couples in the biological enviroment, by
to oxidative stress, even if these enzymes are un- fluorescent or spectrophotometric techniques. In
der regulation, and one might detect an increase, this approach one assumes that a biological re-
rather than a decrease, in their activity. ROS may dox buffer exists in the form of a redox couple

326
Oxidative stress tests: overview on reliability and use. Part I

that is sensitive to changes in the redox enviro- anti-oxidants in vivo could make the measure-
ment. Thus, it reflects changes in the reducing ment of any individual anti-oxidant unrepre-
power of the measured sample, which is in cor- sentative of the overall anti-oxidant status.
relation with all of the LMWAs. Other indirect Moreover, because the measured TAC of a bi-
techniques are inibition methods that involve ological samples depends on which procedure
adding a radical species to the sample together is used in the measurement199, the comparison
with a scavenger that can be monitored with of different analytical methods represents a
laboratory instruments. The LMWAs present in crucial factor in helping researchers to choose
the sample under investigation can quench the and to understand the results obtained using a
radical and, therefore, interfere in its reaction specific method, due to the different princi-
with the added scavenger. Examples of indirect ples on which they are based200. For example,
methods are: studies evaluating the single contribution of
pure plasma component to the total anti-oxi-
1. measurement of electrochemical couples, such dant activities of blood samples, indicate that
as GSH/GSSG (glutathione/oxidized glu- the main contribution in the FRAP assay, but
tathione190); not in others, is the acid uric. On the contrary,
2. NADH/NAD + (reduced nicotinamide dinu- the anti-oxidant capacity of reduced glu-
cleotide/nicotinamide dinucleotide), and tathione is not detected by using the FRAP as-
ascorbic acid/ascorbate191,192; say but significantly contributes to the anti-
3. the Trolox equivalent-antioxidant capacity oxidant capacity measured by utilizing other
(TEAC) assay193; tests. Recent data showed differences between
4. the total radical-trapping potential (TRAP), an the FRAP assay and other methods in TAC
assay to define, for example, the stage of ath- measured in the same sample from normal in-
erosclerosis194,195; dividuals. Thus, each method is sensitive to
5. the chemiluminescence method for superoxide various anti-oxidants in a different manner
detection; and consequently may also evidence, as com-
6. the oxygen-radical absorbance capacity pared to another method, different levels of
(ORAC) methodology. anti-oxidant capacity in the same sample.
Moreover, the interaction between anti-oxi-
Direct methods for measuring total LMWA are dant components may complicate the evalua-
those that utilize an external probe to measure tion of in vivo results.
the reducing or oxidizing capacity of a system. Horoz et al 201 aimed to measure total anti-
An example is an electrode, in which the current oxidant response (TAR) using a novel auto-
is proportional to the concentrations of the scav- mated method in nonalcoholic steatohepatitis
enger or the redox couple under investigation. (NASH) subjects. As a reciprocal measure,
These direct methods can be classified into 2 they also aimed to determine total peroxide
groups: chemical and electrochemical. The levels in the same plasma samples. The ratio
chemical methods measure a known redox active percentage of the total plasma peroxide level
couple whose reduced and measured as a func- to the plasma TAR value was regarded as ox-
tion of concentration. For example, the ferric-re- idative stress index (OSI)202. Twenty-two sub-
ducing antioxidant power (FRAP) assay is based jects with biopsy proven NASH (19 male, 3 fe-
on the reaction of the redox couple ferric/ferrous male; mean age 37.7 8.8) and 22 healthy
with anti-oxidants in the sample and results in controls (17 male, 5 female; mean age 34.6
the creation of a blue color that can be measured 9.3) were enrolled. The most important indica-
at 593 nm196. tions for liver biopsy in those 22 subjects were
Several methods have been developed to ultrasonographically diagnosed fatty liver and
assess the total antioxidant capacity (TAC); elevation in alanine aminotransferase (ALT).
the molecules measured using these assays are The total anti-oxidant status of the plasma was
reducants, able to reduce oxidant species and measured using a novel automated colorimet-
protect oxidizable compounds197,198. However, ric measurement method for TAR developed
the number of different anti-oxidants in serum by Erel203. In this method, the hydroxyl radical,
or other biological samples makes it difficult the most potent biological radical, is produced
to measure each element separately. Addition- by the Fenton reaction, and reacts with the
ally, the possible interaction among different colourless substrate O-dianisidine to produce

327
B. Palmieri, V. Sblendorio

the dianisyl radical, which is brigth yellowish- dase-labelled), calibrators and controls were
brown in colour. Upon the addition of a plasma obtained from Dakocytomation (Glostrop, Den-
sample, the oxidative reactions initiated by the mark) and o-phenylenediamine (Sigma-
hydroxyl radicals present in the reaction mix Aldrich, Poole, Dorset) was used to detect the
are suppressed by the anti-oxidant components amount of bound analyte. Serum aspartate
of the plasma, preventing the colour change transaminase (ASAT), alanine transaminase
and thereby providing an effective measure of (ALAT), alkaline phosphatase (ALP), -glu-
the total anti-oxidant capacity of the plasma. tamyl transferase (-GT), total protein, albu-
The assay results are expressed as mmol min, bilirubin and urate were determined by
Trolox eq./L, and the precision of this assay is standard automated techniques.
excellent, being lower than 3%. The total plas- Markers of lipid peroxidation, antioxidant
ma peroxide concentrations were determined status, hepatic fibrogenesis, inflammation and
using the FOX2 method80 with minor modifi- liver function were measured in blood and
cations. The FOX2 test sistem is based on the urine from 24 patients with established alco-
oxidation of ferrous iron to ferric iron by the holic cirrhosis and in 49 age- and sex-matched
various types of peroxides contained in the controls. In the ALD group, lipid peroxidation
plasma samples, in the presence of xylenol or- markers 8-isoprostane and malondialdehyde
ange which produces a coloured ferric-xylenol were significantly increased (p < 0.001), as
orange complex whose absorbance can be was the ratio of oxidized to reduced glu-
measured. Total antioxidant response of sub- tathione (p = 0.027). The antioxidants seleni-
jects with NASH was significantly lower than um, glutathione (whole blood and plasma) and
controls (p < 0.05), while mean total peroxide vitamins A, C and E were all significantly de-
level and mean oxidative stress index were creased (p < 0.001); median plasma glutathione
higher (all p < 0.05). In subjects with NASH, levels were only 19% of control levels. PIIINP,
fibrosis score was significantly correlated with a serum marker of hepatic fibrogenesis, and
total peroxide level, total antioxidant response CRP were both increased (p < 0.001). Urinary
and oxidative stress index (p < 0.05, r = 0.607; 8-isoprostane correlated positively with PII-
p < 0.05, r = 0.506; p < 0.05, r = 0.728, re- INP, CRP and markers of cholestasis (alkaline
spectively). However, no correlation was ob- phosphatase and bilirubin) and negatively with
served between necroinflammatory grade and glutathione (whole blood), vitamins A and E
those oxidative status parameters (all p > and albumin.
0.05). NASH is associated with increased oxi- The electrochemical methods include a lot
dant capacity, especially in the presence of liv- of techniques, such as potentiometry, electro-
er fibrosis. The novel automated assay is a re- chemical titration and voltammetry 210. Mea-
liable and easily applicable method for total surement of the reducing power by voltammet-
plasma antioxidant response measurement in ric methods offers several advantages. Such
NASH. measurements can be performed easily and and
Total anti-oxidant capacity was measured in rapidly, allow the evaluation of numerous sam-
whole and protein-free serum by an enhanced ples without sophisticated extraction and treat-
chemiluminescence technique 204 . Total glu- ment, and thus are most suitable for screening
tathione in fresh whole blood, GSH and GSSG a large number of samples. Information de-
in plasma were determined using 5,5-dithio- rived from these measurements cannot be ob-
bis(2-nitrobenzoic acid)205. Selenium was deter- tained by other methods. The evaluations pro-
mined using a simple single-tube fluorimetric vide information about all LMWA of both
assay206. Vitamin A207, vitamin C208 and vitamin lipophilic and hydrophilic nature and can be
E209 were determined by established laboratory conducted in cells, biological fluids and tis-
techniques. sues. A unique characteristic, the reducing-
Hepatic fibrogenic activity was measured in power profile can supply information concern-
serum using the Type III procollagen intact PII- ing the type and concentration of LMWA. The
INP radioimmunoassay (Orion Diagnostica, Es- profile is specific to the tissue and each bio-
poo, Finland). C-reactive protein (CRP) was logical site possesses its own characteristic set
assayed using an in-house, antibody sandwich of data. Changes in the profile can immediate-
ELISA technique. Rabbit anti-human CRP anti- ly indicate the occurrence of oxidative stress to
bodies (unlabelled and horse-radish peroxi- system.

328
Oxidative stress tests: overview on reliability and use. Part I

Principles and Methodologies of Examples of Evaluation of


Cyclic Voltammetry (CV) and Total Reducing Power in Some
Examples of Evaluation of Clinical and Pathological Cases
Biological Reducing Power
Since this methodology for quantification of
Voltammetric measurements have been con- the overall LMWA was first introduced 217, it
ducted for many years to measure electron has been used in a variety of clinical situations
transfer between molecules and evaluate oxida- and pathological disorder, including dia-
tion/reduction potentials of various redox-ac- betes218,219 ulcerative colitis220, brain degenera-
tive compounds 211. These methodologies can tive diseases and head trauma 221, skin status
provide information concerning thermodynam- and pathologies 222 and irradiation therapy as
ic, kinetic and analytical features of the tested well as study of the aging process and stages of
compounds212. This technique is useful to the embryonic development223. Biological fluids224
evaluation of the overall reducing power of a such as seminal fluid, cerebrospinal fluid, sali-
biological sample213. Following preparation of va, sweat, urine, plasma and gastric juice pos-
the sample for measurement, the sample is in- sess reducing power derived from their LMWA
troduced into the tested well. A potentiostat content.
with a 3-electrode system, required to conduct
the measurement, consists of a working elec-
trode (e.g., glassy carbon, mercury film or plat-
inum electrode), a reference electrode (e.g., sil- Immunohistochemical Markers Used
ver/silver chloride or calomel electrode) and an in Toxicology Pathology in
auxiliary electrode (e.g., a platinum wire). Fol- Visualization of
lowing introduction of the sample, the voltage Oxidative-Stress Phenomena
is linearly applied to the working electrode and
changed from a start to stop potential and im- Oxidative-stress markers have been divided
mediately swept back at the same swept rate to into 3 categories. First, molecules modified by
the start. This potential is aimed to oxidize or free radicals, such as 4-hydroxy-2-nonenal,
reduce a species present in the solution in the malondialdehyde and 8-oxo-2-deoxyguanosine
voltammetric cell. The resulting current vs po- (8-oxo-dG). The concentrations of these prod-
tential is recorded to produce a cyclic voltam- ucts are proportional to dose and they are de-
mogram214,215 that can supply thermodynamic, tected at the sites where free-radical attacks oc-
kinetic and analytical information concerning cur. Second, antioxidant enzymes and mole-
the electrochemical couple under investiga- cules are associated with the metabolism of
tion216. The position of the current wave (e.g., radicals, such as GSH and catalase. Finally,
anodic wave) on the voltage axis (x-axis of the transcriptional factors are included, such as nu-
voltammogram) can be determined and is re- clear factor- (NF-) and c-myc which are
ferred to as the potential at which the peak cur- modulated by these radicals. Because tissue
rent occurs. This potential can be defined as the collected during toxicity studies are fixed
oxidation potential of a compound for a given chiefly in formalin, researchers must focus on
set of conditions. Analitically, it is used to well-defined products that are stable in this fix-
monitor concentration. When several com- ative and unlikely to share homology with for-
pounds have the same or close oxidation poten- malin-induced modifications225. Reviewing his-
tials, the anodic wave obtained is composed of tochemical and immunohistochemical ap-
all of these compounds and the peak potential proaches to the study of oxidative stress, Raina
is evaluated for the whole group. In this case et al 225 stated that the importance of in situ
the anodic current describes the concentrations methods over bulk analysis cannot be overstat-
of these molecules. This pattern is usually seen ed when considering the structural and cellular
in voltammetric measurements of biological complexity of tissues and the effects of dis-
samples. Although the voltammogram cannot eases thereof. Indeed, in situ detection allows
provide specific information on the exact na- detection of specific cell types affected or spe-
ture of the LMWA, it can supply data concern- cific localization such that a process affecting
ing the reducing power of the sample under in- only a small fraction of the tissue or cells can
vestigation. be readily visualized.

329
B. Palmieri, V. Sblendorio

Nuclear Factor-B ide)236. Although the role of NO in tumor biology


Nuclear factor-B (NF-B) is a transcriptional remains controversial, most data indicate that it
factor implicated in inflammation and immune promotes tumor progression237. Increased iNOS
activation and activated by oxidants and cy- expression may play a role in human tumorigen-
tokines226. This factor normally resides in an in- esis, as, for example, in the prostate where high-
active form in the cytoplasm and has been shown grade prostatic intraepithelial neoplasia (PIN)
to enhance iNOS gene expression in different and carcinoma display more intense iNOS im-
types of cells, like macrophages227. munoreactivity than benign prostatic hyperplasia
and low-grade PIN samples238.
Cyclooxygenase-2 Extensive iNOS immunoexpression (average
Cyclooxygenase catalyzes the formation of grade, severe) has been noted within infiltrating
prostaglandins and other eicosanoids from macrophages at sites of chronic active inflamma-
arachidonic acid. Cyclooxygenase-2 (COX-2) is tion, the major lesion in rats exposed by inhala-
induced at the site of inflammation following tion for 3 months and 2 years to the lung carcino-
stimulation with pro-inflammatory agents, such gen IP239. Lesion progression suggested that the
as interleukin-1 (IL-1), tumor necrosis factor- foreign bodies (IP) introduced into the lungs at-
and lipopolysaccharides. Researchers have sug- tracted macrophages for their digestion and re-
gested that the release from inflammatory cells moval and induced severe inflammation, which
of NO, increases COX-2 activity228. COX-2 over- further enhanced NO generation. The change in
expression is involved in cellular proliferation this and other markers analyzed in this research
and carcinogenesis in different organs229,230 and support the hypothesis that oxidative stress paly
COX-2-specific inhibitors prevent lung carcino- a relevant role in the development of lung cancer
genesis231. from IP inhalation.

Glutathione S-Transferase-pi Haem Oxygenase I


Drug-metabolizing enzymes, such as glu- Haem oxygenase (HO)-1, a heat shock pro-
tathione S-transferase (GST)s and antioxidant tein, is the inducible form of the rate-limiting en-
systems, such as glutathione, vitamins, catalase zyme of haem degradation240. It is induced by a
and superoxide dismutase function concertedly lot of stimuli, including heat shock, hyperoxia
as the two most important inducible defense sys- and oxidative stress and represents a powerful
tems against electrophiles and xenobiotic endogenous protective mechanism against free
toxicity232. The expression of these 2 systems oc- radicals in a variety of pathological conditions.
curs through a common regulatory region named Liu et al241, studying in frozen tissues the im-
the antioxidant responsive element (ARE)233. Nu- munohistochemical localization of HO-1 in ex-
clear factor 2 (Nrf2) has been show to be a key perimental autoimmune encephalomyelitis,
molecule that responds to reactive electrophiles which serves as a model for multiple sclerosis
by activating ARE-mediated gene expression. (MS) in human, noted a high expression in scat-
Glutathione S-transferase-pi (GST-pi), a member tered macrophage-like and perivascular cells in
of this family of phase II detoxification enzymes, inflammed lesions of the spinal cord. Repeated
catalyzes intracellular detoxification reactions, intraperitoneal injection of the HO-1 inducer,
including the inactivation of electrophilic car- hemin, was associated with attenuation of spinal
cinogens by catalyzing their conjugation with cord inflammation and reduced HO-1 immuno-
glutathione234. Additionally, GSTs have endoge- expression. The latter was probably attributable
nous substrates, such as lipid and nucleic acid to fewer macrophages, known to be the main
hydroperoxides and alkenals, which result from source of ROS generation. These results suggest
the decomposition of lipid hydroxyperoxides235. that pharmacological modulation of HO-1 ex-
pression may serve as a novel approach to thera-
Inducible Nitric Oxide Synthase peutic intervention in MS.
Nitric oxide is synthesized in a variety of tis-
sues via the catalytic activity of nitric oxide syn- Uric Acid and Oxidative Stress
thase (NOS). The inducible form, iNOS, is found Uric acid (UA) is the final product of purine
mainly in mononuclear phagocytes where it may metabolism in humans. The final two reactions
be incuced by endotoxins and/or cytokines; it is of its production catalyzing the conversion of hy-
able of producing high levels of NO (nitric ox- poxanthine to xanthine and the latter to uric acid

330
Oxidative stress tests: overview on reliability and use. Part I

are catalysed by the enzyme xanthine oxidore- acid are a risk factor for cardiovascular disease
ductase (XOR), which may exist into two inter- where oxidative stress plays an important patho-
convertible forms, namely xanthine dehydroge- physiological role. Also, allopurinol, a xanthine
nase or xanthine oxidase242. In most species, UA oxidoreductase inhibitor that lowers serum levels
is metabolised to allantoin by the enzyme urate of uric acid exerts protective effects in situations
oxidase: allantoin is then conversed to allantoate associated with oxidative stress (e.g. ischaemia-
and finally glyoxylate plus urea. All of these reperfusion injury, cardiovascular disease). How-
products are much more soluble than UA in wa- ever, there is increasing experimental and clinical
ter. Humans lack the enzyme urate oxidase due evidence showing that uric acid has an important
to a defective gene that is not transcribed243 thus, role in vivo as an antioxidant.
the plasma levels of UA in humans are higher Factors such as hypoxia, cytokines and gluco-
(200-400 mol/l; 3.4-6.8 mg/dl) in comparison corticoids lead also to the strong expression of
to most other mammals. Most of the serum UA is XOR. Harrison 252 suggested that the XHD-
excreted in urine as long as renal function is not NADH oxidase pathway can also lead to the pro-
impaired, while low-sodium ducts have the effect duction of the superoxide anion and contribute to
of raising the net re-absorption of UA in the I/R oxidative stress. Recent evidence suggests
proximal tubule and thus, increase serum UA that XOR can produce NO under hypoxic condi-
concentration244,245. UA is present intracellularly tions through the reduction of inorganic nitrate to
and in all body fluids but usually at lower levels NO253. Allopurinol is a hypoxanthine analogue
than in plasma. At physiological pH, almost all that reacts with XOR to yield alloxanthine (oxy-
UA is ionized to urate and has a single negative purinol), which binds to XOR and inhibits its ac-
charge. Serum levels of UA are correlated with tion, therefore, its use for the managment of
changes in the amounts of dietary purine con- arthritis due to hyperuricaemia254.
sumed246. Due to urates limited solubility in wa-
ter, excess production in vivo can lead to its crys- Antioxidant Properties of UA
tallization out of solution (e.g. in gout, where It has been proposed that UA may represent
urate accumulates in joints causing arthritis). UA one of the most important low-molecular-mass
is also produced in conditions of ischaemia- anti-oxidants in the human biological fluids255-258.
reperfusion (I/R) during the oxidation of hypox- Ames et al255 proposed in the early eighties that
anthine dehydrogenase (XDH) and xanthine oxi- UA can have biological significance as an anti-
dase (XO). In physiological conditions, it is oxidant and showed, by in vitro experiments, that
mainly found in the dehydrogenase form, with it is a powerful scavenger of peroxyl radical
highest levels found in liver and intestine. XHD (RO2), hydroxyl radicals (.OH) and singlet oxy-
has greater affinity for oxidised nicotinamide gen. The researchers speculated that UA may
adenine dinucleotide (NAD+) compared to oxy- contribute to increased life-span in humans by
gen, as the electron acceptor, when catalysing the providing protection against oxidative stress-pro-
oxidation of hypoxanthine and xanthine to urate. voked ageing and cancer. UA is an oxidizable
Under ischaemic conditions, ATP is degraded to substrate from haem protein/H2O2 systems and is
adenine and xanthine, while at the same time able to protect against oxidative damage by act-
there is increased conversion of XDH to XO. ing as an electron donor. Apart from its action as
Consequently, XO uses molecular oxygen in- radical scavenger, UA can also chelate metal
stead of NAD+ during reperfusion and leads to ions, like iron and copper, converting them to
formation of the free radical superoxide anion poorly reactive forms unable to catalyse free-rad-
(O2)247-249. Superoxide anion can form hydrogen ical reactions259,260.
peroxide through superoxide dismutase activity Upon reactions with ROS and other oxidizing
and, in presence of iron, the extremely reactive agents, UA can be oxidized to allantoin and sev-
hydroxyl radical by Fenton-type reactions250,251. eral other oxidation compounds. Thus, the deter-
The latter uses molecular oxygen as electron ac- mination of UA and/or allantoin is a useful tool
ceptor and generates superoxide anion and other in the assessment of the level of oxidative stress
reactive oxygen products. in humans.
The role of uric acid in conditions associated Squadrito261 suggests that UA is a specific in-
with oxidative stress is not completely defined. hibitor of radicals produced by the decomposi-
Evidence mainly based on epidemiological stud- tion of peroxynitrite (ONOO.), the product of in-
ies suggests that increased serum levels of uric teraction of NO with the superoxide anion. Per-

331
B. Palmieri, V. Sblendorio

oxynitrite is a strong oxidizing agent able to in- appropriate for the putative mechanism of dam-
teract with almost all important cell components age: for example, if DNA damage is implicated,
inducing cell injury262. It can also induced nitra- measurement of lipid peroxidation may be irrele-
tion of tyrosine residues in proteins influencing vant. Assays of oxidative damage should be cou-
their structures and functions263. Squadrito et al261 pled with careful observations of the progress of
studied the kinetics of the reaction of UA with the clinical symptoms and the effects of any an-
peroxynitrite using stopped-flow spectroscopy. tioxidant intervention therapies be studied from
They found that peroxynitrite reacts wiyh carbon both aspects. Only in this way can the clinical
dioxide (CO2) in human blood plasma nearly 920 symptoms be reliably correlated with the indices
times faster than with UA. Thus, UA is not a di- of free radical generation and an understanding
rect scavenger of peroxynitrite since it cannot of their association be reached.
compete with CO2. The researchers postulated
that the therapeutic effects of UA may be related
to the scavenging of the radicals such as CO3
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Acknowledgements
The Authors thanks to Dr. Carla Torri from Callegari
Spa-Catellani group, Parma, for a permission of re-
viewing their bibliography archives.

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