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Plasma Antioxidant




The PAT test is an innovative test that measures the antioxidant capacity of plasma.
Antioxidants (AOs) represent the main barrier of defense against damaging aggression due to reactive
species, in particular by reactive oxygen species (ROS).
The plasma AO capacity is a measure of physiological, environmental, and nutritional factors (exposure
to ROS and antioxidant supplementation) determining the redox status in humans, and can underline the
oxidative stress conditions (OS) in the progression/development of many diseases. Moreover, changes in
AOs plasma content after supplementation may provide information on the absorption and
bioavailability of nutritional compounds and efficacy of AOs therapy.
The simplicity of execution, the fast evaluation (1 min) and the low cost are key features of PAT test for
the prevention and control of the systemic chronic diseases, or to monitor the efficacy of any
pharmacological treatment.

The report is divided into 5 parts.

Part I is a brief discussion on oxidative stress (OS), from its formation to the control exercised by enzymatic
and no-enzymatic systems till its propagation. The main mode of production of reactive species (RS) and
the molecular structures that are subjected to their oxidizing action are considered. The mechanisms
operated by the cells to defend themselves against such excessive propagation of RS will be also described.
Part II is a brief discussion on plasma composition and functions. The main component of AOs for the
control of the OS.
Part III concerns a brief description of methods to determine the OS balance.
Part IV is dedicated to PAT test characteristics (chemical composition, linearity, repeatability, robustness,
accuracy) and report.
Part V reports in summary the preliminary clinical application addressed to humans (not reported the work
done in pharmacology or veterinary field) and some of future possible fields of application of PAT test.



Part Subject page Part Subject Page

Part I Oxidative stress characteristics 5 Part II Human whole blood and plasma 18
Introduction 5 Blood composition/characteristics 18

Reactive species 5 Blood main functions 19

Reactive Oxygen Species (ROS) 6 The importance of plasma AOs 19

Modalities of ROS formation 7 Soluble antioxidants reserve 20

The oxidative paradigm 10 Kinetic of antioxidants 21

The antioxidant potential 11 Part III The methods for OS balance 25

Main endogenous AO system 11 OS evaluation 25

Thioredoxin 12 AOs evaluation 27

Peroxyredoxin 12 Some critical variables 28

Glutaredoxin 13 Part IV The PAT test 33

Glutathione 13 Principle and validation 33

Catalase 14 Analytical performance 34

Paraoxonase 14 PAT test in normal subject 35

Non-enzymatic antioxidants 15 Part V Clinical application and Possible 37


Chapter I: Clinical applications 37

Chapter II: Possible applications 43


Part I.
Oxidative stress characteristics
Prof Umberto Cornelli
Loyola University School of Medicine-Chicago

In the aerobic organisms the use of oxygen (O2) is vital for the cellular functionality, which is characterized
by a continuous alternation of oxidation and reduction processes.
The oxidation processes can occur also without the O2 since they consist of the removal of one or more
electrons (e-) from a given molecule.
Since hydrogen (H) formed by 1e- + H+, by definition the removal of one H corresponds to oxidation, while
the addition of one H corresponds to a reduction.
The process of reduction and oxidation are usually combined, since an e-(or an hydrogen atom H) passes
from a donor (oxidized element) to an acceptor (reduced element), and donor/acceptor can be defined as a
"redox couple".
In the context of single elements constituting the living matter, such as, O2 (even 1/2 O), carbon (C),
nitrogen (N), hydrogen (H), or metals such as iron (Fe), zinc (Zn), copper (Cu), the oxidation process is
substantially reversible, since all these elements can return to their original condition regaining an e- in a
reduction process. These characteristics allow them to be used to transfer an e- from one element to
another in a process defined "redox exchange".
For more complex molecules such as amino acids, lipids, carbohydrates, or other compounds derived from
their combination (i.e. proteins, lipids, DNA), the oxidation process can lead to a structural modification,
which can prelude to an activation (capacity to perform a specific activity) or to a deactivation
(interruption of a specific activity). In the last case the process could prelude to their elimination also.
Thus for complex molecules of any living organism the processes of oxidation and reduction are part of a
dynamic equilibrium defined as "allostasis" that must be maintained for the purposes of the continuity of
vital processes. One cannot escape that allostasis is based essentially on the exchange is the smallest
chemical entity existing, the e- .

Reactive species
Reactive species (RS) are a wide number of elements or compounds characterized by the capacity to attract
electrons (e-) or hydrogen (H). RS can be based upon different elements and despite the chemical nature
(single element, simple molecule or macromolecule such as a lipid, protein or DNA etc) they are classified


according to the element finally attracting the e- or H. Consequently, the different types of RS are described
as follows:
ROS (reactive oxygen species) because the "attractor" is oxygen (O2 or O); RNS (reactive nitrogen species) in
case the attractor is nitrogen; RSS in case of sulfur (S), RCS for carbon, RClS in case of chloride. In other
terms, the second letter or letters after R indicate the attractor element.
RS most of time are defined erroneously as "free radicals", and even though some of them are in this
status, this is not the condition allowing them to be oxidants.
In fact, any element of the Mendeleev table is defined as a "free radical" if it contains a single e- instead of
2 e- in one or more orbitals (usually the external orbitals).
The atomic oxygen (O) in its most common conformation (168O) consists of 8 protons and 16 neutrons in the
nucleus, and 16 e- in its 9 orbitals, being the two external orbitals (2x and 2y) composed by a single e-
instead of 2 e-. Because of this, O can be defined a "bi-radical", and maintains the same characteristics even
in the biatomic molecular configuration as O2 .
This condition is the basis of its relative instability and avidity to capture electrons in an attempt to reach
the closer and simpler stable form of water (H2O).
O2 undergoes some modifications to reach the stable condition of H2O, following a 4 e- capture in 4
different steps. However, each step ends up with an instable compounds, both of radical (i.e. with an
unpaired electron in an orbital) and non-radical nature (i.e. with complete electronic orbitals), but still more
oxidizing then the original O2. All these compounds, formed before reaching the state of H2O, are called
reactive oxygen species (ROS).

Reactive oxygen species (ROS)

ROS in sequence are represented by: O2- (superoxide, following 1 e- capture), H2O2 (hydrogen peroxide,
following 2 e- capture), OH (hydroxyl radical, following 3 e- capture). The fourth e- capture will end up with
H2O which in essence consists of O2 that was picking up 4e- associated with 2 H+ (protons).
The radical nature of each of the ROS is indicated by the point at the top on the right (or left); does not
escape that H2O2 is not a radical, nevertheless is one of the most important oxidizing molecules of the
organism. An unstable intermediate represented as O2H is formed by the combination of O2- with a H+
and participates in the many reactions.
In nature exists also other ROS known as O and O (singlet oxygen, respectively of radical and non radical
nature) which are formed in the atmosphere only, generated by the impact between a photon and O2 [O
in case of orbital change following the impact (non radical) or O in case of a change of the electron spin


Other reactive species different from ROS

Reactive species non-related to O2 derive from other elements, and the most common are based on
nitrogen (N), carbon (C), sulfur (S), and chloride (Cl). These species are able to oxidize substrates with the
same efficiency as for the ROS. Molecules or macromolecules such as lipids or protein etc. can become
reactive species that are centered on the abstracting element, usually carbon or sulfur, that will be
indicated respectively as RC and RS.
Some of the most common RS reported in Table I.1.
Among these, nitrogen monoxide (NO) is the most important since it is actively synthesized by different
cellular systems (in particular from the endothelial cells) called NO synthetases or NOS, in order to induce
vasodilation, anti-platelet and signaling functions. It can be defined as a RNS (reactive nitrogen species)
together with is most dangerous adduct ONOO- (peroxinitrite) deriving from the reaction with O2- (NO +
O2- ). This last reaction has one of the highest rate constant known (7x 109 M-1s-1), thus NO toxicity is
predominantly linked to the ability to combine with superoxide anions since ONOO- is a powerful oxidising
agent that can cause DNA fragmentation and lipids oxidation. In particular environmental conditions (acidic
pH) ONOO- tends to be transformed into OH (which is by far the most reactive oxidizing in the organism).

Modalities of ROS formation

In humans the production of ROS takes place in every cell of then body, but more selectively in different
compartments of the cell, such as in cell membranes (outer and inner), in the cytoplasm (considering, also
corpuscles and the reticular-endothelial system), in the nucleus and in the mitochondria (Figure I.1).

Figure I.1. Some of the cellular localization of ROS/RNS production

Xanthine oxidase NAD(P)H oxidase

heme oxigenase lipoxigenase

uncoupled eNOS mitochomdria


In terms of quantity, mitochondria are the most important production sites of ROS (energetic formation),
followed by cell membranes (reactive formation) and finally by some metabolic process (metabolic

Energetic formation. In mitochondria the presence of O2 is fundamental for the production of ATP. As a
consequence there is a continuous generation of O2- which flow both in the mitochondrial matrix and in
the inter-membrane space, where it immediately undergo the transformation into H2O2 (dismutation, see
later) which has the capability to diffuse away from the place of production (the half life is about 15 s) . For
this reason, mitochondria are the real producers/exporters of H2O2, that in case of overproduction can
easily deplete both the mitochondrial and cytoplasmatic antioxidant reserves.

Reactive formation. This source of ROS belongs mainly by membranes.

In both inner and outer cell membranes, the O2- production occurs through the NAD (P)H oxidases which
are a variegated family of enzyme and cofactors, adapted to the membranes where they are located (ie
mitochondria, endoplasmic reticulum, cell particles, such as: cit P450, peroxisomes, etc..) and can be
activated by many different stimuli. Immediately after the O2- production is the dismutation to H2O2. A
part of the damaging oxidant activity H2O2 triggers a cascade of events causing the production of
constitutional factors (structural proteins), reactive mediators (pro-inflammatory cytokines or anti-
inflammatory), and finally protective compounds (antioxidant enzyme systems).

Metabolic formation. The metabolic source of ROS belongs to many different processes of transformation
or activation of different molecules (i.e. prostaglandin synthesis from arachidonic acid, noradrenalin
synthesis from dopamine, purine bases metabolism up to the formation of uric acid)
Among all these pathways, the production of ROS through the enzyme xanthine oxidase (XO) is considered
the most important. XO is a membrane enzyme belonging to the NAD(P)H oxidase family (competent for
the transformation of purine bases into uric acid), and is responsible for the overproduction of O2-
following the ischemia/reperfusion process.
This last can be considered a pathological process taking place when tissues in ischemic condition (because
of blood flow reduction) are immediately reperfused with blood bringing O2 into the ischemic territory.
Because of this incoming, flows an immediate activation of xanthine oxidase takes place and allows the
formation of an extremely high amount of O2-, such that a local aggressive OS is generated damaging
further the ischemic reperfused tissue. This process however does not belong to pathological processes
only, but is common during endurance sport training and smoking also.
After the production of O2-, no matter how it is generated, the dismutation process takes place under the
activity of the enzymes SODs (superoxidodismutases) that transform O2- into H2O2. In many instances this
new entity undergoes to a non-enzymatic reaction (Fenton reaction) that in presence of Fe2+ transforms
H2O2 into HO- and OH as follows: H2O2 + Fe2+ HO- and OH


The propagation of oxidation

Each biological molecule (proteins, lipids, nucleic acids, vitamins, antioxidants, etc.), once oxidized, has the
ability to oxidize another molecule acting in the same way of a reactive species (RS).
Among these RS of particular importance are the hydroperoxides (ROOH) formed from different sources
(lipid, protein, DNA) and can directly oxidize substrates containing SH groups (thiols) or trigger the
propagation of the oxidative process, operating as multipliers of the OS as classically represented by
unsaturated lipids oxidation.
The ROOH (see Table I.1) refers to all the compounds that have been combined with a molecule of O2 and
the most frequent ones are the fat derivatives, but also proteins, saccharides and nucleic acids. Therefore
the prefix R indicates different types of products.
These ROOH have the tendency to react with the transition metals (see below "Fenton reaction") assuming
the radical shape (RO or ROO), therefore they have cumulatively a considerable oxidant potential.

Table I.1 Some of the most common oxidant agents for human body.
Defined, in general, as reactive species (RS) and divided basing on the representative element.

Reactive Oxygen Species (ROS)

Radical nature Formula No-radical nature Formula
Oxygen Singlet Oxygen o O2

Superoxyde O2- Hydrogen peroxyde H2O2

Hydroxyl OH Ozone O3

Hydroperoxyl Hypocloric acid HOCl

Peroxyl RO2 Hypobromous acid HOBr

Alcoxyl RO Organic hydroperoxydes ROOH

Carbonate Peroxynitrite ion ONOO

Carbon di-oxide Peroxynitrous acid ONOOH

Chloride Reactive Species (RClS)

Chloride ion Hypochloric acid HOCl

Nitryl Chloride NO2Cl


Chlorammine RNHCl

Chloride Cl2

Nitrogen Reactive Species (RNS)

Nitric oxyde NO Nitrous acid HNO2

Nitric bi-oxide Peroxynitrous ONOOH

Alkylperoxynitrite ROONO

b -
Peroxynitrite ion ONOO

Nitryl chloride NO2Cl

a b
represented in the form of bi-radical; Some reactive species appears in more than one category because some are attributed to
the central role of different elements of the compound. The dot in the upper right (sometimes left) indicates the radical nature.

The oxidative paradigm

In a normal physiological condition, an equilibrium is taking place following a ROS (RS) production and the
protective network that modulate their activity for the allostasis. The driving principle of allostasis is to
control that ROS (RS) production will not exceed the necessary and beneficial amount, since an
overproduction would determine an irreversible cellular damage.
The presence of ROS follows the so-called oxidative paradigm, for which both the lack of ROS and the
excessive availability are equally harmful for cells. Therefore, a modulation (adjustment of their production)
is a necessary condition for the allostasis (Figure I.2).

Figure I.2 Relation between RS production and cellular function

The oxidative paradigm

cellular death
growth arrest
incomplete growth
no growth
ROS production [redox environment]

All the constitutive molecules of the living material (phospholipids, nucleic acids, proteins, etc.) are subject
to oxidation determined by RS with even though at a different degree of sensitivity.


The antioxidant potential

The oxidative and reductive processes alternation is the basis of birth, growth, differentiation and
functionality of living organisms. It is therefore misleading to deal only with oxidative process, but we must
refer to an oxidative equilibrium or "oxidative balance."
The potential danger of ROS implies the need for the cell to balance the potential harm with the
antioxidant reserve, in order to counteract the inevitable ROS diffusion and limiting the propagation
process. The antioxidant reserve is constituted primarily by enzymes with damping function (quenching) of
ROS, and by the non-enzymatic antioxidants.

Main endogenous antioxidant systems

The first enzymatic systems involved in ROS production are the superoxide dismutase (SODs). Usually SODs
are considered as antioxidant enzymes, however this function does not correspond to the real duty of
these enzymes that transform by dismutation (modification of a substrate into two different products) an
oxidant such as O2- into another product potentially more dangerous represented by H2O2.
The more appropriate definition should be regulators of oxidation, since they act as "rheostats" shifting
from the oxidative aggression determined by O2- to a more regulatory/metabolic function fulfilled by H2O2.
Three different SODs (SOD 1, SOD2, and SOD3) are known in humans with different location. SOD1 is
located in the cytoplasm, nucleus and inter-membrane space of mitochondria and contains Cu-ZN; SOD 2 is
located in the mitochondrial matrix and contains Mn-Zn; SOD3 (or extracellular SOD) is located into the
Extracellular Matrix and contains Cu-Zn.
The different types of SOD and the reason of the different content of metals is still a matter of speculation.
As common characteristics, all SODs accelerate thousands of times the transformation in O 2- and H2O2 (the
spontaneous reaction would occur to slowly). However, since H2O2 has also a high oxidizing power, cells
limit its diffusion through other enzymatic systems with antioxidant action (for example: thioredoxin,
glutathione, catalase, etc.) or by using low molecular weight antioxidants (lipoic acid, coenzyme Q10 , L-
cysteine, ascorbic acid, vitamin E, polyphenols, etc.).
Another important antioxidant system is the heme oxygenase (HOs). The most important is HO-1 but other
forms of HO emerge such as HO-2 and HO-3 less active than HO-1.
HO-1 catalyzes the transformation of heme (deriving from erythrocytes) into biliverdin [1] and leads to the
formation of biliverdin later transformed into bilirubin.
Most diseases, from pulmonary to endocrine disorders, are characterized by an overproduction of HO-1,
[1]. Several regulatory elements are capable of stimulating the production of HO-1. Among these two
important transcription factors, like the nuclear factor NF-kB and the activator protein AP-1, activate genes


involved in OS. Identical stimuli can trigger both the MAP kinase and HO-1 [2], resulting respectively in the
production of inflammatory mediators and protective factors.
Because the cellular response to OS is apoptosis, it is reasonable to consider the HO-1 as an anti-apoptotic
enzyme. Indeed, experimental studies have confirmed that cell lines deficient in HO-1 are more vulnerable
to toxic insults that generate apoptosis, the same was applied to animals (mice) lacking the gene HO-1. It
has been demonstrated that this anti-apoptotic action is related to the CO production [3].
HO-1 prevents the release of Fe3+(strongly oxidizing) and implements the heme diversion which leads to
Fe2+ (non-oxidizing) and CO (carbon monoxide) for the production of biliverdin/bilirubin. Both the final
compounds (biliverdin and bilirubin) have antioxidant capacity. This heme diversion process takes place not
only in erythrocytes because the HOs, though in different forms, are an ubiquitous enzyme which are over-
regulated in almost every reactive condition, playing a protective role by increasing, in particular, the
production of ferritin that binds Fe2+ [1,4] and reducing its availability in the possible transformation of
The thioredoxin system is considered a redox sensor: it is located both in the cytosol and mitochondria. It is
composed of thioredoxin reductase (TrxR) and proper thioredoxin (Trx) that act in concert with the
peroxiredoxin (Prx) to reduce H2O2 to H2O (Figure I.3).

Figure I.3 Schematic representation

of thioredoxin system
NADPH(H) TrxR Trx Prx H2 O
NADP TrxR Trx Prx H2 O
SH S SH H 2 O2

The TrxR (thioredoxin reductase) is reduced by NAPH, which in turn gives the H
to Trx (thioredoxin), which then transfers it to the Prx (peroxiredoxin), which
finally reduces H 2O2 to H2O. SH represents cysteine; while Se represents a
seleno-cysteine. SOH represents a sulfenic acid residues which is produced by
oxidation of the thiol group of cysteine [modified from 5].

Trx in the reduced form, stimulates the production of hypoxic pulmonary factor (HIP). The latter is capable
of over-regulating an extremely consistent series of genes [6]: from those related to angiogenesis till the
tumors develop.

Peroxyredoxin (Prdx).
This enzyme exists in 6 different typologies (form Prdx1 to Prdx6) and belongs to the peroxidase family,
which in addition to the H2O2 detoxification, may protect against ONOO- and ROOH also.


As shown in Figure 3, the Prdx acts in concert with the TrxR/Trx system as a terminal for the transformation
of H2O2 in H2O. There are, however peroxyredoxines acting independently or in concert with other
enzymatic systems different from TrxR/Trx, such as sulfiredoxine.

This enzyme has similarity with thioredoxin but instead of Prdx it uses GSH (glutathione) as prosthetic
exchange terminal for the transformation of H2O2 into H2O. The final enzyme of the system is glutathione
peroxidase (GPx) that contains selenocysteine instead of cysteine. Selenocysteine (SeH) by oxidation due to
H2O2 is transformed into selenenic acid (Se-OH) (Figure I.4).

Figure I.4 Mechanism of glutaredoxin


GR (glutaredoxi n) is reduced from NAPH; which in turn yiel ds H to GSH

(glutathi one) which then yields it to GPx (glutathione peroxi dase), which finally
reduces H2 O 2 to H2 O. SeH represents a selenocysteine ; Se-OH represents the
selenenic residue products during selenocysteine oxidation. [modified from 5].

The presence of SeH allows then the enzyme to operate in a broad pH range than it would be with the only
cysteine; this may explain the presence of different peroxidase systems that are activated depending on the
cellular conditions.

Glutathione or GSH
In the reaction overexposed (Figure I.3, I.4) is reported the intervention of the GSH (glutathione). GSH
consists of a tripeptide, the -glutamilcysteinilglycin. It is produced in a substantial amount in each cell (1-
11 mM) and represents the most important endogenous component to control oxidation. GSH is not only a
prosthetic part of glutaredoxin, but is produced as an essential water-soluble antioxidant also. The
difference between the non-enzymatic and the enzymatic product resides in cysteine which is replaced by
selenocysteine in the enzymatic system. Each cell produces GSH in an autonomous way (including cells that
lack the nucleus such as erythrocytes and platelets) through a series of enzymes, of which the most
important is the -glutamilcisteinil synthetase (GCS). GSH yields its hydrogen and it is transformed into
GSSG which is then regenerated in GSH by the NADPH/NADP+ system. This is possible thanks to the energy
supplied by the pentose phosphate cycle located in cytoplasm. The GSH/GSSG ratio is considered an index
of antioxidant capacity. The process of detoxification that needs GSH for it to be accomplished is called S-
glutathionylation and it is particularly important for thiol proteins protection. This process involves also an


inactivation and elimination of harmful substances to the body and it is one of the main mechanisms of
protection of exposed organs (lung) or ones interested in the detoxification (liver) [7].

This enzyme belongs also to the family of peroxidases, and is ubiquitous but most is concentrated on
peroxisomes that are corpuscles located in the cellular cytoplasm. The structure consists of a tetramer
which contains in each of the polypeptide chains a heme group with Fe2+. It allows the enzyme to convert
H2O2 into H2O in the following reaction: 2H2O2 + O2 2H2O [8], but it is also capable of binding NO [9].

Paraoxonase (PON)
The paraoxonase (PON) family comprises 3 members of calcium-dependent hydrolytic enzymes, PON1,
PON2, PON3, which takes its name from its ability to hydrolyze paraoxon (the active metabolite of the
organophosphorous insecticide parathion). Considering that this activity is related to human-made
chemicals, it seems to be an ancillary rather than a primary fuction of the enzymes [10], there also being a
difference of some orders of magnitude among the three members (PON1 is much more active than PON2
and PON 3). In contrast, lactonase activity that utilizes natural substrates (and also some drugs) is almost
similar for all the members, and all three PONs efficiently metabolize lactons and hydroxy acid derivatives
of arachidonic acid and docosahexaenoic acid preventing the damage generated by these hydroperoxides.
The hydrolytic activity consists of protecting high-density lipoproteins (HDL) and other lipoproteins
(LDL,VLDL respectively low density and very low density lipoproteins) from oxidative propagation by
degrading lipid peroxides that are formed in membrane phospholipids [11].
In HDL the estrogens have no antioxidant capacity because the free phenolic groups required are present in
the form of esters. PONs efficiently hydrolize the estrogen esters of HDL leading them to regain antioxidant
activity [12]. The combination of the two hydrolitic processes, respectively of phospholipids hydroperoxides
and estrogen esters, seems to be responsible for the PONs activity in maintaining the HDL efficiency.
One important aspect of the PONs activity is the hydrolysis of homocysteine thiolactone (HCyTL) which is a
toxic metabolite of homocysteine. HcyTL can bind fibrinogen and make it resistant to lysis (thrombogenic)
and bind proteins eliciting an inflammatory auto-immune response [13]. Hyperhomocysteinemic subjects
have disfunctional HDL particles with attenuated antiatherogenetic activity (reduced cholesterol efflux and
antinflammatory effect) due to a parallel decrease of PON1 activity [14].
In humans, PON1 and PON3 are bound to HDL and PON2 is an intracellular enzyme only.
The most characterized enzyme of the family is PON1, which is a protein of 43 kDA containing a cysteine
(position 283) and has two amino acid polymorphisms (methionine/leucine [M/L] in position 55, and
arginine/glutamine [R/Q] in position 192), such that individuals 192R/R and 55L/L have greater hydrolytic


capabilities and less risk of coronary heart diseases [15]. There is at least a 40-fold variation in serum PON1
activity among individuals [13] due both to genetic reasons and exogenous factors (diet, smoking,
environmental heavy metals).
PON2 was shown to exert its antioxidant functions at the cellular level joining the host of the intracellular
antioxidant enzymes [16]. PON1 is synthesized essentially in the liver with the aim of participating in
oxidation by product elimination [17]. During the acute phase of inflammation, some enzymes in the HDL
decrease (PON, PAF-AH, LCAT) leading to the increase of hydroperoxydes, and HDL becomes pro-oxidant.
This means that this complex HDL antioxidant apparatus may have a "chameleon-like" behavior being anti-
inflammatory in the basal state and pro-inflammatory during the acute-phase response [18] when losing
the coordination of the antioxidant apparatus.

Non-enzymatic antioxidants
The non-enzymatic antioxidants can be derived from endogenous factors and food and they represent a
wide range of compounds that are reported in Table I.2.

Table I.2 The antioxidant network

Function /structure Type of product

Vitamins vitamin A, vitamin E, vitamin C, nicotinamide, riboflavin, niacin

Lipids omega 3, omega 6, squalene, cholesterol
Aminoacids/thiol taurin, L-arginine, histidine, glycine, cysteine, glutamine, methionin, N-acetyl
Metabolites cysteine, S-adenosil-L-methionine, lypoic acid, uric acid, bilirubin, squalene
Peptides carnosine, gamma-glutamil-cysteinil glycine (GSH)
Proteins albumine, thioredoxin, peroxiredoxin, lactoferrin, transferrin, ceruloplasmin
Plant derivatives Polyphenols (hydroxycinnamic acid-derivated, hydroxybenzoic acid-
derivated, flavonoids, stilbenes, lignans, tannins, ellagic acid), glucosinolates,
carotenoids (, , , -carotene, lycopene, lutein, xeaxantin, canthaxantin,
astaxanthin), phytic acid, allicin, polycosanols.
Minerals zinc, iron, cupper, selenium, chromo

Most of the plant derivatives, such as those extracted from Goji berries, Garcinia fruits, curcumin, ginkgo
biloba, berberin, etc., that are proposed as antioxidants, contain compounds that are included in the
overexposed table.



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Part II.
Human whole blood and plasma

Blood composition/characteristics
Blood is the life-maintaining fluid that circulates through the body's heart, arteries, veins and capillaries and
is responsible for transporting/exchanging of nutrients, oxygen, carbon dioxide and waste products to and
away from the cells [1,2,3].
It represents approximately the 8 % of an adult's body weight (females 4-5 L; males 5-6 L). It has a pH of
7.3-7.5, making it slightly basic and with a mean temperature of 38C.
Whole blood is viscous fluids and this feature make it more resistant to flow than water.
Blood in the arteries is a brighter red than blood in the veins because of the higher levels of oxygen; plasma
constitutes 55 % of total blood volume [1,2,3] is instead the yellow liquid component of blood.
Human blood consists of about 22 % solids and 78 % water. The components of human blood are reported
in Figure II.1.

Figure II.1. Main components of blood

To summarize:
-blood plasma is a mixture of proteins, enzymes, nutrients, wastes, hormones, antioxidants and gases. The
main plasma proteins are: albumins which are the smallest and most abundant plasma proteins; globulins
subdivided into three classes: alpha, beta and gamma globulins and comprising lipoproteins also such as


chilomicrons, VLDL, LDH, HDL, remnants that are characterized by a membrane made of phospholipids and
proteins; fibrinogen which a soluble precursor of fibrin (blood clot and coagulation)[1];
Blood contains also formed elements that have a definite structure and shape. Except for the platelets and
all formed elements are cells and include: red blood cells (RBCs), white blood cells (WBCs) and platelets

Blood main functions

- Transport: gases (O2, N2, CO2); hormones, waste products (uric acid, creatinine and metabolites),
enzymes, nutrients (glucose, amino acids, fatty acids) and minerals.
- Maintain body temperature
- Control of pH and water balance: the pH of blood must remain in the range 6.8 to 7.4 to allow
appropriate cell physiology and enzymatic activities.
- Transport of toxins for elimination: toxins are removed from the blood by liver and intestine, or by
kidneys to be excreted into the urine, or even by saliva sweat and tears.
- Regulation of body fluid electrolytes: salt excess is removed from the body via urine (around 10 g
per day).
- Protection: blood has several roles in coagulation, inflammation, infection, and cancer cells control.
Leukocytes destroy exogenous microorganisms and cancer cells, antibodies and other proteins
destroy pathogenic substances, platelet and coagulation factors initiate blood clotting and help
minimize blood loss. Among the protection factors the antioxidant network is fundamental to
defend the organism against the dangerous and harmful effect of ROS produced during biological
process and cellular reactivity [4].

The importance of blood plasma antioxidants

Mammals have evolved complex AOs system to use oxygen and minimize the dangerous effects of reactive
species (RS). AOs present in each compartment of the body (cells, extracellular fluid,) and can be up-
regulated and mobilized to neutralize an excessive RS formation mainly represented by ROS. Blood have a
central role in the maintaining of the redox balance during oxidative conditions (chronic inflammation,
drugs, smoking, diets poor in antioxidant, pro-oxidant substances etc). It transports and shares the
antioxidants in each compartment of the body [4,5,6]. Plasma AOs can scavenge ROS to prevent reaction
with metal ions [7,8,9] or to reconstitute the oxidized vitamin C [10,11,12]. The synergic interaction
between different AOs ensures protection against ROS as in the case of the triangular mechanism of
regeneration of ascorbate by GSH [13] and -tocopherol by ascorbate [14]. Plasma AOs capacity is
modulated both by ROS excess and by dietary intake.


The recommended Dietary Reference Intakes of vitamin C is 75 mg per day for women and 90 mg per day
for men 1950 years of age. For vitamin E the intake suggested is 15 mg per day for both men and women,
whereas vitamin A daily intake is limited to 700 g per day for women and 900 g for men.
Plasma maintains the homeostasis of the oxidative status but in case the intake of these vitamins (or
Physiological Modulators with antioxidant activity) lower than recommended dose, even for few days, end
up with a lower antioxidant capacity and a higher lipid peroxidation status. Smokers with limited intake of
AOs with the diet compared to nonsmokers showed low blood levels of antioxidants such as vitamin C, -
tocopherol and antioxidant enzymes such as superoxide dismutase (SOD) [15-20]. This condition is
characterized by elevated levels of oxidative stress biomarkers [21-23].
The link between high fruit and vegetable intake and reduced chronic disease has been attributed to the
antioxidant protection.

Soluble antioxidant reserve

AOs are present in three different locations: tissues, formed elements (cells, platelets, RBCs, WBCs) and
plasma. All formed elements are capable to produce different AOs according to their needs and antioxidant
enzymes such as SOD and free GSH are ubiquitous. Among tissues, liver, kidney and endothelium are
making actively the synthesis of AOs and the former are involved in metabolic processes where the binding
with GSH (glutathionylation) is a common step to eliminate toxic metabolites. Endothelial cells are
producing AOs mainly by stimulation of circulating formed elements and lipoproteins, or by reactive
components present in plasma (hormones, coagulation and complement factors, eicosanoids, autacoids).
Liposoluble AOs such as tocopherols and carotenoids are located mainly on membranes, whereas soluble
AOs are not membrane-bound and circulate both in cytosol and plasma. Most of them are used also to
rigenerate liposoluble AOs, that once oxidized can behave as triggers of the propagation of the oxidative
Among the plasma antioxidant, transferring, ceruloplasmin can be considered among the liposolule AOs,
whereas albumin and uric despite belonging to the class of hydrosoluble AOs are not capable to regenerate
tocopherols or carotenoids. All these molecules can be considered in the class of shock adsorbers since
they behave as antioxidant substituting and supporting the AOs. The only liposoluble antioxidant that can
regenerate tocopherols is coenzyme Q10 that is produced by the cells during the cholesterol synthesis.
Because of these characteristics the soluble antioxidant reserve is constituted mainly by uric acid, vitamin
C, free GSH, albumins and those products such as polyphenols deriving by an active intake (food or
supplements). For this reason the soluble antioxidants are important and can be a reliable mirror of the
oxidative balance and their reduction indicates the threat of OS explosion.


However, a particular condition has to be considered in relation to AOs. In case of tissue damage and/or
RBCs hemolysis the cytosol antioxidants are released into the plasma and may increase the AOs reserve
that in this case is not indicating an healthy condition but at the opposite is a reactive condition. For this
reason the level of soluble antioxidant reserve per se has to be considered with prudence and measured
in concomitance with a marker of OS. In other terms an increase of OS concomitant to an increase of
plasma antioxidant levels is a marker of a redox inflammatory (RI) condition [24].

Kinetics of antioxidants
Following a normal constant daily intake of physiological modulators with antioxidant activity the plasma
level of antioxidant capacity can be relatively constant with fluctuations compatible with a steady state.
The AOs of the body are supposed to follow the kinetic model reported in Figure II.1.

Figure II.1. Kinetics model of antioxidant ADME (adsorption, distribution, metabolism, excretion).
Metabolism take place in every compartments.

Kinetics of AOs

Other secretions


Saliva Plasma Tissues


Very simply, after the absorption the AOs are distributed into the tissues where they can be used to
counteract the oxidative damage caused by RS. The amount which is not used is metabolized and/or
excreted through urine and saliva. Plasma levels of AOs tend to be in a steady state condition, even when
the AOs intake is reduced for a short period of time (days). Saliva and urine are the two most important
compartments to discharge the excess of AOs and have similar volume of daily excretion (about 1.5 L/day).
Sweat (and tears also) is an excretion compartment that is particularly active according to the seasonal
temperature (in summer is supposed to be more active).
In case of an abundant intake the surplus is excreted (via urine, saliva, sweat and other secretions)[export],
whereas in case of shortage due to both an increase of OS or diet poor of PMs with antioxidant activity,

the tissue reserve is implemented with components deriving from plasma [import] as reported in Figure
During the kinetics studies using SAT, PAT, UAT (respectively saliva and plasma and urine antioxidant
capacity) it was shown that following an acute administration of an pool of PMs, the plasma levels
modification can be limited, particularly when the levels of plasmatic antioxidant were normal, whereas
the increase of concentration in urine and saliva was much more consistent (internal data). As a
consequence the saliva and urinary levels should be also determined to obtain a more precise indication of
the bioavailability of the PMs.
These findings indicates that a modulation of the plasma antioxidant levels is in place to control the
oxidative processes that are normally underway in the blood.

Figure II.2. Model of AOs bioavailability: when plasma levels are low there is a need to import antioxidants (antioxidant
capacity) from the tissues; on the contrary, in case of surplus due to a large intake the antioxidants are excreted.

Bioavailability of AOs


range plasma


low normal high




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Part III
The methods for OS balance evaluation

OS evaluation in plasma (or serum)

For long time researchers have tried to develop methods for the assessment of the OS. The only known
system that captures the reactive species is the ESR (electric spin resonance) that is considered as a golden
standard because it may detect O2- and HO (and ONOO- also) at the moment of their generation into the
tissues. These methods use resins that capture these RS and are called spin traps. However, they are
hardly applicable in vivo because they have to be administered parenterally to reach blood and tissues. This
is possible in experimental animals only and not in humans because of their toxicity. Therefore, research
has turned to derivatization systems that allow the determination of those endogenous substances that
have been modified by reaction with (RS) and defined as adducts (Table III.1).

Table III.1. Some main methods used for OS evaluation.

Method Derivate
oxDNA Oxidized deoxyribonucleic acid
SPC Carbonylated Proteins
ROOH Hydroperoxydes
TBARS Thiobarbituric acid reactive substances
LNO2 nitrolinoleate
MDA malonyldialdheide
4-HNE 4-hydroxynonenal
IsoPs F2/D2/E2/isoprostanes
NeuroPs F3/F4 isoprostanes
H2O2 Hydrogen peroxides
BH Respiratory Hydrocarbons
ONOO- a Peroxynitrite
PTN a Alfa-fenyl-N-tert-butyltirone
AHS a Aromatic hydroxylation of salicylate
"spin trap" methods which involve resins or potentially toxic substances administration.

It is generally recognized that an ideal method should be a reliable early indicator of OS, easy to carry
out, with limited costs.

Whereas the tests based on spin traps are not permitted for human use, all the most common
derivatization methods reported in table III.1 are safe and non invasive. They are however time consuming
and quite expensive, and for these reasons most of them are used exclusively for research and rarely for
monitoring diseases in humans.
In medical literature the most common methods are related to TBARS, MDA, 4-HNE, urinary/blood
isoprostanes (F2), carbonylated proteins, DNA oxidized and hydroperoxides.
Each method has some limitations. The analysis of TBARs, MDA and 4-HNE are indicators that emerge when
high levels of blood glucose (diabetes) undergo oxidation and escape the circulating AO reserve. They
increase following inflammatory processes but cannot be considered early indicators because the AO
reserve creates a barrier to their formation. In other words, when these indicators are altered, a
pathological state is present but they do not allow for early diagnosis and also are characterized by high
coefficient of variation (CV).
Isoprostanes, carbonylated proteins and DNA can be considered reliable markers, but they suffer two
important limitations: the first is relatively high coefficient of variation (about 50 -60 %) that makes difficult
the comparison of groups of data generated unless they have large average differences; the second is that
methods are quite complex and data coming from different labs may carry a consistent amount of errors.
A completely different value has to be given to the hydroperoxides measurement (ROOH) that are direct
markers of most of the derivatives that can modify the redox signaling (lipids, proteins and DNA).
The ROOH measurement can be carried out using the d-ROMs test. The validity of d-ROMs test has been
analyzed in comparison to the other most common tests used for the measurement of RS such as
isoprostanes, carbonylated proteins, oxidized DNA and hydroperoxides. Furthermore the values of all the
tests were compared with C-reactive protein (hsCRP) as inflammation index.
The experience [1] was conducted on healthy volunteers in condition "mimicking" an oxidative stress. In
such conditions the marker that was changing in a more homogeneous way (with smaller inter-individual
variation) was considered the most reliable. The hydroperoxides determination through d-ROMs test was
showing the lowest coefficient of variation (CV < 15 %) compared to isoprostanes, carbonyl proteins and
oxidized DNA (CV between 50 to 60 %). The results were not unexpected, but confirmed that the ROOH are
a derivatization of the oxidative modification of both PL (and lipids), proteins and DNA, while the other
tests are unidirectional and they only pick up their specific derivatization. In other clinical experiences
ROOH levels were shown to be correlated positively and significantly with isoprostanes levels [2] and with
hsCRP, and also with the MDA levels in critically ill patients in intensive care [3] indicating that d-ROMs test
is ideal to determine the Redox inflammatory condition (RI).


Up to now at least 600 trials have used the d-ROMs test to determine the oxidative stress. The test is the
most used by the clinical communities. The inter laboratory variation can be minimized since it can be
carried out using a dedicated portable device with an extremely simple and low cost set of reagents.
Since the ROOH can be detectable by the d-ROMs test only [4], it is believed that this test will be extremely
valuable for the monitoring of various diseases and for following the patient's clinical outcomes to
modulate the necessary therapies. The d-ROMs test can be carried out, beside in the labs, in the medical
office of the doctor using a drop of capillary blood and the dedicated instrument FRAS 4 Evolvo.

AOs evaluation (in plasma or serum)

In order to define the complete oxidative profile, the increase of markers related to the OS has to be
completed with the measurement of the antioxidant capacity.
Many methods have been studied, and the most known are reported in Table III.2

Table III.2 The most common test for the evaluation of the antioxidant capacity

Method Derivate
FRAP assay Ferric-ion Reducing Antioxidant Power
TRAP assay Total Radical trapping Antioxidant Parameter
OXY-absorbent Oxygen radical absorbance capacity
ORAC Oxygen Radical Absorbance Capacity
TEAC Trolox Equivalent Antioxidant Capacity
FCR Total phenols assay by Folin-Ciocalteu Reagent
BAP Biological Antioxidant potential
PAT Plasma Antioxidant capacity
SAT Saliva Antioxidant capacity
CUPRAC Cupric Reducing Antioxidant capacity

Depending upon the reactions involved, these assays can be classified into two types: assays based on
hydrogen atom transfer (HAT) reactions, and assays based on electron transfer (ET). The HAT-based assays
apply a competitive reaction scheme, in which antioxidant and substrate compete; this group includes:
ORAC, TRAP and crocin bleaching assays [5,6,7,8]. ORAC (Oxygen Radical Absorbance Capacity) is a
standardized and most common method for determining the antioxidants capacity of any substances
(lipophilic, hydrophilic and total antioxidant capacity). The ORAC assay is based upon the inhibition of the
peroxylradical-induced oxidation initiated by thermal decomposition of azocompounds [6] and use
fluorescein as the probe for the reaction [7].


This method was used to determine the antioxidant capacity of foods and was used quite extensively as a
standards measure for the antioxidant capacity.
Recently (May 2012) the USDAs Nutrient Data Laboratory (NDL) removed the USDA ORAC Database for
Selected Foods from the NDL website due to mounting evidence that the values indicating antioxidant
capacity have no relevance to the effects of specific bioactive compounds, including polyphenols on human
health since it was defined that the antioxidant activity measured with ORAC has absolutely no predicting
value of the antioxidant capacity in humans [9].
On the other hand ET-based assays are usually colorimetric assay based on the ability of a coloured solution
to changes color in the presence of an antioxidant. The degree of color change is correlated with the
sample's antioxidant concentrations. This group includes: FCR, TEAC, FRAP, CUPRAC and BAP test
[10,11,12,13,14]. The most used are FRAP and BAP test, two colorimetric assay measuring the ferric
reducing ability of plasma. In addition, other new innovative assays has been discovered basing on the
same principle but with a reduced time of execution, higher precision and low cost: PAT test and SAT test
[13,14,15] respectively for plasma and saliva. These last two test makes the measurement of the
antioxidant power of, respectively, plasma and saliva, a simple, precise and fast test. The two tests based
on the ability of an red-brown iron-thiocyanate solution to decolour in the presence of antioxidants in the
biological fluid. With its specific composition, PAT test avoids the interferences of phosphates present in
blood plasma and detect the real AOs capacity. As reported in part IV and V PAT test is sensible to AOs
variations with clinical relevance.

Some critical variables

The measurement of the antioxidant capacity can be influenced by different factors and many
discrepancies among the data published in the medical literature are still matter of debate. Some critical
variables can interfere in plasma AO capacity evaluation:
-Diet rich in fruit, vegetables, tea, coffee, juice,
-Physical activity. Sports and physical activities, in particular aerobic exercises increase oxygen
consumption, which is directly related to ROS production. The increased production of AOs can be viewed
as a defense mechanisms.
-Diseases/genetics Plasma AOs capacity can be influenced by diseases, for example, kidney dysfunction
may increase the uric acid concentration and thus the AOs capacity or at the opposite the loss of AOs
reserve for decrease of reabsorption.
-Phosphate content. It has been demonstrated that phosphates may overestimate the antioxidant capacity
of plasma. Phosphates content in plasma is extremely variable depending on diet, diseases and exogenous
exposures or extreme physical effort (endurance).


An adult male has approximately 600 g of phosphates expressed as phosphorous. In plasma, the
phosphorus content in normal subjects is between 2.6 and 4.5 mg/dL (0.84-1.45 mmol/L) corresponding to
values of phosphate (PO43-, H2PO4-, HPO42-), respectively, equal to 7.8 and 13.5 mg/dL [16]. Plasma
phosphate exists in the form of monovalent and bivalent phosphate anions. Approximately 10% is attached
to proteins, 35% is a sodium, calcium and magnesium complex and the remaining 55% is present in free-
form. Although both organic and inorganic phosphates are present in the serum, only the inorganic
phosphate is measured as the organic part is primarily located in the hematic cells.
However, among mineral complexes, phosphates are the only capable to bind iron and interfere with TAA
measurement [17]. As a result, the biochemical dosage for assessing the total plasma antioxidant capacity
based on iron reduction (e.g. BAP [18]) can be influenced by the plasma concentration of the phosphates
leading to an overestimation of the plasma antioxidant capacity (Table III.3.)

Table III.3.TAA evaluation and AOs contents in normal and in diabetes subject [17].

Variable Measure Normal Diabetes Diabetes

Subject [12] Controlled [9] Uncontrolled [11]

K mmol/L 7 25 21

Ca mg/dL 1.3 1.0 1.1

P mg/dL 8.1 20.0 14.7

Mg mg/dL 1.1 0.7 0.48

Prot T mg/dL 27 85 66

Uric acid mg/dL 0.9 1.4 1.8

TAA mmol/L 0.39 0.69 0.61

This variable makes extremely erratic any comparison between clinical trials aimed to measure the AO
capacity of plasma. Particularly in case of P interference with the methods to determine the AO capacity.
The consequence of this bias makes complex to define if different diseases/pathologies show a real
modification of AO capacity.
Antioxidants can be consider a defense mechanism against OS [19-22]. The antioxidant nutrients have
been shown through animal and laboratory studies of brain tissue to decrease lipid peroxidation [23,24],
and the oxidation of proteins [25,26], inhibit the production of ROS [27], prevent mitochondrial

dysfunction [28] and DNA damage [29], reduce neurotoxicity [30,31], apoptosis [32], and neuronal death
[33, 34], atherosclerotic processes [35] and different other mechanism related to diseases development.

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[34]Turconi G, Simonetti P, Brusamolino A et al. Nutritional and Plasma Antioxidant Status Assessment in a
Group of Old Alzheimers Inpatients J Nutr Food Sci 2011, 1:1
[35] Halliwell B. Lipid peroxidation, antioxidants and cardiovascular disease: how should we move forward?
Cardiovascular Research 2000;47: 410418.


Part IV.
PAT test

Principle and validation

PAT (Plasma Antioxidant) test is a new photometric test used as a specific method for the determination of
antioxidants (AOs) in plasma samples. In aerobic living organism the production of reactive species (RS), in
particular reactive oxygen species (ROS) is under the control of the antioxidant network (RAO). Antioxidants
can derive from either endogenous or exogenous agents and they counterbalance the ROS production (in
particular hydroperoxides) [1].
An higher level of ROS and a consequent lower level of antioxidants lead to a condition of oxidative stress
(OS) an emerging risk factor for the development of many diseases (inflammatory, infectious and
degenerative disease) [1-6].
PAT test allows to evaluate the plasma antioxidant capacity based on the ability of a the plasma sample to
reduce ferric ions (Fe3+) to ferrous ions (Fe2+). The quantified antioxidant potential is attributable to the
major component of plasma barrier to oxidation (vitamin C, vitamin E, uric acid, bilirubin) [2,7-9].
A chemical and unspecific standard reaction between iron (III) and thiocyanate has been used extensively
[2] as a measure of the antioxidant capacity of fluids and plasma. However, phosphate content in the
samples can interfere with the reaction generating false positive results.
As reported in Part III, plasma phosphates have the capacity to bind iron and form a complex. As a
consequence, the biochemical dosage based on iron reduction leads to an overestimation of the plasma
antioxidant capacity.
This overestimation can be negated introducing in the reaction the correct quantity of zirconium salts,
which have the capacity to bind the phosphates without causing precipitation.
PAT test contains in its solution a particular zirconium salt that binds phosphates the antioxidant content
will be proportional to the real capacity of plasma to reduce Fe3+ and decolour the iron-thiocyanate
complex, from red-brown solution to colorless.
Although the reagents solution is made stable by adding a stabilizer aimed to avoid an uncontrolled
reaction and making compounds free from polarity modifications. In this way the prevalent and stable
species of Fe(SCN)3 will be formed. Characteristics of this species is the red-brown color that is detectable
at 505 nm. Furthermore, the stabilizer allows to make the reaction pH-independent also such to avoids the
plasma proteins precipitation..
The PAT test is performed by the addiction of 40 l of R2 (ferric solution) to the cuvette containing the R1
(tiocyanate) reagent and then 10 l of sample. The photometrical reading is performed at 505 nm after 1
minute incubation at 25-30 C (and values are minimally modified from 20 to 37 C ).


Analytical performance
PAT test Is commercially available as pre-dosed diagnostic kit and it should be performed both with
common laboratory instruments (photometer or multi-analyzer) or with an easy dedicated instrument, the
FRAS system (H&D srl, Parma).
The analytical parameters considered were the following:
- phosphates interference (in vitro) on the iron reduction;
- comparison between BAP test (the common test used for the evaluation of the antioxidant capacity and
blood) and PAT test in presence of different phosphate concentration;
-linearity, repeatability, reproducibility and the accuracy.

In order to interchange the value obtained with BAP (where the results is influenced by phosphates) and
the values obtained with PAT test, an experiment was carried out as follow: in the same subjects and on the
same blood sample, both the BAP test (expressed in mol/L of vitamin C) and the PAT test (expressed in
UCor or Cornelli units where 1 UCor correspond to mol/L of vitamin C obtained with the formula:
PAT value x 1.4) were performed. The values obtained both with BAP test and PAT test result comparable.

a) The presence of phosphates in the sample makes an interference during the evaluation of the
antioxidant capacity based on iron (Fe3+) reduction.
b) The use of zirconium salts (PAT test) avoids this interference and allows the phosphate clearance in
plasma physiological concentration.
c) The observed difference between BAP and PAT test comes definitely from the phosphate
interferences, and the plasma antioxidant capacity dosage with BAP test results overestimated in
direct relation of the plasma phosphates levels. An increased BAP values due to phosphates
correspond to an increased differences between BAP and PAT value (correlation coefficient
R=0.812, p=0.001). To allow the comparison with the data so far reported in literature about the
BAP test, an appropriate algorithm has been applied that allows the interchange of PAT and BAP
values. A new unit has been introduced , U Cor. The U Cor (Cornelli units) has been determined
considering the mean phosphates plasma levels (mean between 2.6 e 4.5 mg/dL) leads to an
overestimation equal to ~700 mol/L of Vit C. One U Cor. corresponds to 1.4 mol/L of Vit C. The
multiplication factor of 1.4 (corresponding to a 40% increase of the vitamin C mol/L) has been
determined following the contemporaneous evaluation of the phosphates and the BAP test. It has
been demonstrated that BAP values were overestimated on average of 40%, with oscillation
between 25 and 55 %. Because of this variability, it is possible that in subjects with higher


phosphates levels, the percentage might be > 40%. In other terms, it may happen that BAP and PAT
are not identical depending upon the phosphate content in the plasma.
Although PAT test delivers the result in 1 minute (instead of 5 minutes with BAP)
d) Different concentrations of vitamin C, starting from three different stock solutions of 35.22 mg/mL
vitamin C, are used in order to develop the calibration curve. All measurement were performed on
the same day at a controlled room temperature (25C). PAT test resulted linear in the range from
500 to 6000 mol/L of Vitamin C (R2 = 0.99).
e) Both the intra-assay and inter-assay tests confirmed the HIGH PRECISION, ACCURACY and
REPRODUCIBILITY of the method, with a comparable coefficient of variation (CV 4.17%).

General consideration on PAT test

1) PAT test is an innovative substitute of the common BAP test, usually used to quantify the
antioxidant contents in biological fluids.
2) The test needs a relatively simple equipment (a photometer and a centrifuge) commonly present in
each analytical laboratory; furthermore, the test can be performed with a dedicated
instrumentation (FRAS-Evolvo systems) also.
3) It can be carried out with the dedicated instrumentation, which requires an extremely limited
manuality, with a considerable reduction errors and time of execution.

PAT values in normal subjects

As explain before a new unit has been introduced , UCor where 1UCor corresponds to mol/L of Vit C
obtained with the algorithm: PAT value x 1.40. For these reason the reference values (Table IV.1) can
be the same as the BAP test, as shown making the evaluation in the same samples.

Table IV.1. PAT test reference values

> 2800 Very high value

2200 2800 U Cor Normal value
2200 2000 U Cor Border line low range
2000 1800 U Cor Slight deficiency status
<1400 U Cor Deficiency status
Unit of measurement (Vitamin C equivalents) in terms of U.Cor.
1 U.Cor = 1.4 mol/L of ascorbic acid



1. Halliwell B. Free radicals, antioxidant and human disease: curiosity, cause or consequence. Lancet
1. Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the
FRAP assay. Analytical Biochemistry 1996;239:70-76.
2. Bildik A, Kargin F, Syrek K et al. Oxidative stress and non-enzymatic antioxidative status in dogs with
visceral Leishmaniosis. Research in Veterinary Science 2004; 77(1):63-6.
3. Colombo C, Muti P, Pala V et al. Plant-based diet, serum fatty acid profile, and free radicals in
postmenopausal women: the diet and androgens (DIANA) randomized trial. The International Journal of
Biological Markers 2004;20(3):169-76.
4. Kumaraguruparan R, Balachandran C, Murali Manohar, B et al. Altered oxidant-antioxidant profile in
canine mammary tumors, Veterinary Research Communications 2005;29:287-296.
5. Kumaraguruparan R, Balachandran C, Murali Manohar, B et al. Altered oxidant-antioxidant profile in
canine mammary tumors, Veterinary Research Communications 2005;29:287-296.
6. Vajdovich P, Kriska T, Mezes M et al. Redox status of dogs with non-hodgkin lymphomas. An ESR study.
Cancer Letters 2005: 28:339-46.
7. Pasquini A, Lucchetti E, Merchetti V et al. Analytical performances of d-ROMs test and BAP test in canine
plasma. Definition of the normal range in healthy Labrador dog. Veterinary Research Communication 2008;
8. Dohi K, Satoh K, Ohtaki H et al. Elevated plasma levels of bilirubin in patients with neurotrauma reflect its
pathophysiological role in free radical scavenging. In Vivo 2005;19(5):855-60.
9.Hetyey CS, Manczur F, Dudas-Gyorky Z et al. Plasma antioxidant capacity in dogs with naturally occurring
heart disease. Journal of Veterinary Medicine series A 2007; 54:36-39.
10. Ganong WF Review of Medical Physiology Lange Medical Book/Mc Grow-Hill, 21st Edition- 2001.


Part V. Clinical applications

PAT test is an innovative new test so few experiences are documented. In any case the basic principle of
the test is the same as for other common colorimetric test. PAT test is improved in terms of precision,
accuracy, robustness, simplicity and time of execution. All these characteristics make the PAT test on the
same levels as the other test commonly used even with some new improvements.
OS can be consider an asymptomatic pathology and the possibility to monitor its development and to
prevent it is becoming a necessity for different chronic diseases. The imbalance between ROS/AOs can be
observed in different disease and in different fields. Here we report just few notion about the imbalance of
The Part V will be divided into two chapters: Chapter I, or application of PAT test already documented;
Chapter II, or possible application of PAT test.

Chapter I. Clinical applications

Comparison between different test for measure the oxidative balance conditions
[Cornelli et al. The Oxidative Stress Balance Measured in Humans with Different Markers, Following a Single
Oral Antioxidants Supplementation or a Diet Poor of Antioxidants. JCDSA 2011;1:64-70]

Epidemiological studies indicates that a constant fruits and vegetables intake reduces the incidence of
cardiovascular diseases [1] and maybe of some cancer [2], whereas the use of food supplements containing
vitamins usually end up with negative or controversial results [3].
The problem of those debates have as possible cause the evaluation of oxidative stress (OS) and the use of
antioxidant as remedies for many diseases. Reviews are available reporting different methods to study the
OS [4] and antioxidant capacity [5], however, a direct comparison among the different method in relation
to the intake of antioxidant are very scarce, as it is the kinetics of the antioxidant capacity of blood. A study
was designed to integrate the antioxidant power (measured by PAT, SAT and UAT) with the most common
methods for OS evaluation, and to determine the behavior (kinetics) of antioxidants capacity in three
different conditions: a) during a normal diet; b) after a single administration of a food supplement; c)
after a diet poor in antioxidants. In the same conditions 4 different markers of oxidative stress [OS] (8-
OHdG in urine, 8-Iso-PGF, hydroperoxides and carbonylated proteins in plasma), a new marker of


antioxidant capacity (AC) in plasma/urine/saliva, and hs-CRP were determined concomitantly in twelve
apparently healthy volunteers.
All the markers were determined at 8 am, 10 am, 12 am in three different moments: after a week of normal
diet (baseline), after an acute supplementation with an antioxidant pool, and finally following a week of a
diet poor in antioxidant.
The supplementation of antioxidants determined a significant (t test p < 0.05) decrease of 8-OHdG in urine
and of carbonylated proteins in plasma, whereas hydroperoxides and 8-Iso-PGF were unmodified; the
antioxidant capacity increased significantly (t test p < 0.05) up to 19%, 78%, and 67%, respectively in
plasma, urine and saliva. Hs-CRP was unchanged.
The diet poor in antioxidant caused a significant increases (t test p< 0.01) of hydroperoxides, 8-Iso-PGF,
carbonylated proteins, 8-OHdG and hs-CRP increase.
The antioxidant surplus determined by a single antioxidants pool administration seems to protect DNA and
proteins from oxidation. The AO capacity in plasma tends to be constant, and the limitation of antioxidants
intake is followed by reduction of AOs in urine and saliva. On the contrary a surplus of AOs tend to be
eliminated rapidly though saliva and urine (Table V.3). This is the confirmation of as explain before about
the AOs kinetic mechanism.

Table V.3. Behavior of antioxidants capacity in human plasma in three different dietary conditions

PAT values (mol/L Vit C)

Periods Run in Normal diet Antioxidant Normal diet Low antioxidant diet

8 am 1350 167.8 1290 99.1 1270 120.8

10 am 1290 102.3 1535 149.0* 1335 84.7

12 am 1320 119.6 1480 184.6* 1327 103.5

*t test for interdependent data; the value are significantly higher (p < 0.01) vs same period of normal diet

In this regards, it has been demonstrated that after the antioxidants pool administration, was revealed an
increase in both saliva and urine AOs pool. Instead during the low antioxidant intake the decrease was
significantly higher in urine than in saliva (respectively 31 % and 47 %; t test p< 0.01) and may give support
to the aspect of saliva recycling.
This may mean that in a condition of AO abundance, plasma export AOs in saliva and urine; on the contrary
when an AOs deficiency occur, the AOs in saliva and urine are imported in plasma. In fact, a statistically
significant correlation between plasma and saliva was found in every determination, whereas the


correlation between plasma and urine was not. Sweat and tears should be added to the list of possible
excretion compartment, and the former is more important since it may the increase in relation to the
temperature of the body and environment.
In conclusion this clinical trial has shown that a normal diet, rich in fruit and vegetables in combination with
other antioxidants in foods and beverages (e.i. coffee, tea, fruit juice, chocolate) can be helpful to avoid the
OS progression.
However, all these findings need confirmation in long terms studies, and evaluation during different
diseases in which the OS, antioxidant capacity and inflammatory markers should be measured

1. Buckland G, Gonzlez CA, Agudo A et al. Adherence to the Mediterranian diet and risk of coronary heart
disease in the Spanish EPIC cohort study. Am J Epidemiol 2009;170:1518-1529.
2. Koushik A, Hunter DJ, Spiegleman D ety al. Fruits, vegetables and colon cancer risk in a pooled analysis of
14 cohort studies. J Natl Cancer Inst 2007;99:1417-1483.
3. Huang HY, Caballero S, Chang S et al. The efficacy and safety of multivitamin and mineral supplements
use to prevent cancer and chronic disease in adults: a systematic review for the National Institute of Health
State-of-the-Science Conference. Ann Int Med 2006;145:372-387.
4. Hwang ES, Kim GH. Biomarkers for oxidative stress status of DNA, lipids, and proteins in vitro and in vivo
cancer research. Toxicology;2006;229:1-10.
5. Niki E. Assessment of antioxidant capacity in vitro and in vivo. Free Rad Biol Med 2010;49:503-515.


Application of PAT test in odontoiatry

[Cornelli U, Belcaro G, Nardi GM et al. Action of an antioxidant complex on the antioxidant power of saliva.
Panminerva Med. 2010;52(2 Suppl 1):69-73].

In order to maintain a correct balance between AOs/ROS in the body and in the oral cavity, in particular
conditions of lower levels of AOs or, on the contrary, in an inflammatory conditions, the use of specific
physiological modulators (PM) should be helpfulness. A registry study has been conducted to evaluate the
efficacy of a mix of antioxidants in plasma, saliva and urine. Several physiological modulators (PMs) with
antioxidants activity [2] are included in this product MF Odontovis (Vit E, beta-carotene, Vit A, Vit C,
polyphenols, cathechins, ellagic acid, antocyanins, coenzime Q10, piridoxin in association with Se, Zn, L-
cisteine) (Table V.1). Marine AOs is present in the formulation and consists of a combination of extremely
powerful and purified antioxidants from marine algae, mainly of carotenoid nature (95 %).
MF Odontovis is a mix of four different kind of PMs: circulating antioxidants, membrane antioxidants,
intracellular antioxidants, and system antioxidants. All these products generate a protective shield against
reactive species. However, it is important to underline the importance of circulating antioxidants that may
reach saliva more easily.
Table V.1. PMs composition

The formulation has been studied specifically for the oral cavity, in fact, the red-fruit extract contains
antocyanins, catechols, polyphenols and ellagic acid which have an important anti-bacterial action against
Streptococcus mutans and other patogenic bacterial elements of the mouth [1]. Whether the action is
effective against bacterial adhesion (either bacteriostatic or bactericidal) will have to be determined in
specific studies involving caries or parodontosis or other oral infective processes.

MF Odontovis transports into saliva an antioxidant complex rather than a single product/compound, but
the AOs surplus is also export in the systemic cycle.
Dosages aimed at producing antioxidant action are kept low on purpose, and compounds including RDA
have been kept at 100% RDA to avoid an unwanted pro-oxidant action that might result from using doses
much higher than normal physiological intake, this is the concept of physiological modulation [2].
Twenty-six apparently healthy volunteers (13 males and 13 females; age 28-34) were included in the trials.
Inclusion criteria: no smokers, no therapy under way a part from oral contraceptives. The subjects were
treated for one week with MF Odontovis or placebo in a cross-over double-blind design.
All subjects completed the study. Compliance was very good (>98 %), and no tolerability problems were
reported. MF Odontovis increases the baseline levels of AOs in plasma and the antioxidant capacity
increased at 4 hours after treatment (P<0.05) with minimal effects at 2 hours.
Data concerning PAT is reported in Table V.2. The evening administration of PMs in fact increases in the
morning: PAT for 20 % (p < 0.05) and SAT for 40 % (p < 0.05), instead UAT increase (6 %) was not significant.
To evaluate the acute effects of MF Odontovis, the treatment was administered in the morning after the
last evening administration; this administration induced an increase of the antioxidant power both in
plasma and in saliva, urine did not show any change. PAT increases up to 34% (4 hours after administration;
P<0.05); the increase was clear one hour after administration, showing a fast absorption. Comparable
increases were observed in saliva (SAT): the values increased up to 44%; after 2 hours they increased 58%;
after 4 hours the increase was 28%. Therefore, hydrosoluble antioxidants are quickly transferred into saliva.

Table V.2. Values of PAT (mol/L Vit C) following the treatment with a physiological modulators
( MF Odontovis) pool and placebo: average values SD.


These tests (SAT, PAT and UAT) may indicate how antioxidant levels can be increased with the use of
specific nutritional supplements within physiological modulation and results useful to monitor the efficacy
of particular treatment.

1. Smullen J, Koutsu GA, Foster HA et al. The antibacterial activity of plant extract containing polyphenols
against Streptococcus mutans. Caries Res 2007;41:342-349.
2. Olson JA Benefits and liabilities of vitamin A and carotenoids. J Nutr 1996;126:1208S-1212S.


CHAPTER II. Some possible applications


Cardiovascular diseases provide the paradigmatic examples for understanding the pathogenic role of
ROS/AOs and represent one of the most fertile fields in which the PAT test should be successfully applied.
A good evidence that oxidative damage contributes to the pathology of atherosclerosis and vascular
dysfunction generally exists, and that ROS are involved in myocardial ischemia-reperfusion injury [1-4].
Increased vegetable and fruits consumptions can decrease levels of oxidative DNA damage in humans [1].
Untreated hypertensive patients (EAH) show serum levels of ROOH significantly higher than those detected
in normotensive subjects [5,6]. These EAH subjects in spite of their normal antioxidant defenses are more
prone than normotensive subjects to oxidative stress because of an increased ROS production. OS
evaluation in EAH patients seems therefore useful in order to evaluate the progression of the disease and
to monitor the efficacy of pharmacological therapy [5,6].
In patients affected by peripheral arterial diseases the use of Physiological Modulators (PMs) with
antioxidant activity improves both the blood pressure reduction and the OS condition [7]. This indicates
that OS can be reduced with low-dosage combination of antioxidants with helps the organism to re-
equilibrate the ROS/AO balance [7].
Antioxidants might be also useful for treatment and prevention of cardiovascular disease for example
associated with metabolic syndrome (MS) because to retard the progression of cardiovascular and renal
disease in patients with the MS all the used drugs have antioxidant effects [8].
The antioxidant drug dipyridamole shows a concentration-dependent antioxidant effect, it protects
membranes from oxidation [9]. Dipyridamole was used in a placebo-controlled study where it has been
shown that in patients affected by tight carotid stenosis undergoing endarterectomy the treatment was
able to reduce serum ROS levels. The transient cerebral hypoxia causing the ischemia reperfusion that
inevitably accompanies this type of intervention was limited by the treatment with dipyridamole [10].
An extremely significant data concerns the finding that in subjects with cardiovascular disease with a
significant imbalance between ROS/AO are prognostic for cardiovascular mortality [11] and therefore, the
use of predictable in vitro test can be useful to determine the cardiovascular risk [12].

[1] Halliwell B. Lipid peroxidation, antioxidants and cardiovascular disease: how should we move forward?
Cardiovascular research 2000;47:410-418.


[2] Halliwell B, Gutteridge JMC. Free radicals in biology and medicine, ed. 3, Oxford: Oxford University
Press, Oxford, 1999.
[3] Rosenfeld ME. Inflammation, lipids and free radicals: lessons learned from the atherogenic process.
Semin Reprod Endocrinol 1998;16:249261.
[4] Ferrari R, Agnoletti L, Comini L et al. Oxidative stress during myocardial ischaemia and heart failure. Eur
Heart J 1998;19(Suppl. B):B211.
[5] Digiesi V, Oliviero C, Gianno V et al. Reactive metabolites of oxygen, lipid peroxidation, total antioxidant
capacity and vitamin E in essential arterial hypertension. Clin Ter. 1997; 148 (11): 515-9.
[6] Digiesi V, Fiorillo C, Cosmi L et al. Reactive oxygen species and antioxidant status in essential arterial
hypertension during therapy with dihydropyridine calcium channel antagonists. Clin Ter 2000;151 (1):15-8.
[7] Cornelli U. Antioxidant use in nutraceuticals. Clin Dermat 2009;27:175-194.
[8] Hutcheson R, Rocic P. The metabolic syndrome, oxidative stress, environment, and cardiovascular
disease: the great exploration. Exp Diabetes Res. 2012;2012:271028. Epub 2012 Jul 9.
[9] Kusmic C, Picano E, Busceti CL, Petersen C, Barsacchi R. The antioxidant drug dipyridamole spares the
vitamin E and thiols in red blood cells after oxidative stress. Cardiovasc Res. 2000 Aug 18;47(3):510-4.
[10] Kusmic C, Petersen C, Picano E et al. Antioxidant effect of oral dipyramidole during cerebral
hypoperfusion with human carotid endarterectomy. Journal of Cardiovascular Pharmacology 2000;36 (2):
[11] Vassalle C, Boni C, Di Cecco P et al. Elevated hydroperoxide levels as a prognostic predictor of mortality
in a cohort of patients with cardiovascular disease. Int J Cardiol. 2006;110(3):415-6.
[12]Vassalle C, Landi P, Boni C, Zucchelli G. Oxidative stress evaluated using an automated method for
hydroperoxides estimation in patients with coronary artery disease. Clin Chem Lab. 2007;45:367-371.


Metabolic diseases, such as MS and diabetes might be considered sufficiently similar in regard to the OS
condition. Each of the above illnesses has obvious peculiar features that may share the common risk for
represented by increased cardiovascular mortality.

Metabolic syndrome (MS) is multi-factorial that underlines the risk of diabetes and obesity. There are
many hypotheses on the genesis of MS and OS [1] as well as the inflammatory ground which plays a
prominent role [2].
One of the constant variables of the MS is the oxidized lipoproteins (LDLox) [2-4] that are believed to be a
reliable predictor. However, it has been recently observed that the metabolic and oxidative damages of the


endothelium seem to be distinct despite they tend to add up [5]. The vascular dysfunction in MS is
highlighted by the ROOH increase and AOs decrease [6].
MS is commonly treated with drugs with antioxidant activity so an evaluation of the therapy can be
quantified with the PAT test as a valid tool for monitoring the MS and the therapy efficacy [7].

[1] Luo ZC, Xiao L, Nuyt AM. Mechanism of developmental programming of the metabolic syndrome and
related disorders. World J Diab 2010;15:89-98.
[2] Holvoet P. Relation between metabolic syndrome, oxidative stress, and inflammation and
cardiovascular disease. Verh K Acad Geneeskd Belg 2008;70:193-219.
[3] Rao VS, Nagaraj RK, Hebbagodi S et al. Association of inflammatory and oxidative stress markers with
metabolic syndrome in Asian Indians in India. Cardiol Res Pract 2010;28:952-976.
[4]Holvoet P, De Keyzer D, Jacobs DR. Oxidized LDL and the metabolic syndrome. Future Lipidol 2008;3:637-
[5] Vaidva D, Sxklo M, Cushman M et al. Association of endothelial and oxidative stress with metabolic
syndrome and subclinical atherosclerosis: multi-ethic study of atherosclerosis. Eur J Clin Nutr 2011;65:818-
[6] Simo ANC, Lozovoy MAB, Simo TNC et al. Immunological and biochemical parameters of patients with
metabolic syndrome and the participation of oxidative and nitroactive stress. Braz J Med Biol Res
[7] Hutcheson R, Rocic P. The metabolic syndrome, oxidative stress, environment, and cardiovascular
disease: the great exploration. Exp Diabetes Res. 2012;2012:271028. Epub 2012 Jul 9.

In diabetes, the massive production of ROS has been documented in sufficient detail [1]. Hyperglycemia
causes an increased production of mitochondrial O2- and deplete the reducing capacity made available by
GSH [2].
This may happen for the necessity of transforming glucose through the polyol pathway. The glucose
becomes, in a first step, sorbitol (through the aldose reductase), and then it is oxidized to fructose. The
aldose reductase consumes NAD(P)H as cofactor, which needs GSH to regenerate itself. The excess of
glucose causes also the formation of AGEs (advanced glycation end-products) which in turn stimulate the
production of ROS [3,4] and activate the NAD(P)H oxidase [5].
Overall, the condition of OS in diabetes represents a major risk factor for the development of endothelial
dysfunction and cardiovascular complications of diabetes [2]. OS increases in diabetes type I and II [6,7] can


predict the onset of complications both of renal [8] or more generically vascular [9]. It has been
demonstrated to be useful to follow the evolution of antioxidants therapies to prevent or reduce the
typical complications of diabetes type II [10,11].


[1] Baines JW. Role of oxidative stress in development and complication in diabetes. Diabetes 1991;40:405-
[2] Wolff SP, Jiang ZY, Hunt JV. Protein glycation and oxidative stress in diabetes mellitus and aging. Free
Rad Med Biol 1991;10:339-352.
[3] Mullarkey CJ, Edelstein D, Browlee M. Free radical generation by early glycation products: a mechanism
for accelerated atherogenesis in diabetes. Biochem Biophys Res Commun 1990;173:932-939.
[4] Inoguchi T, Sonta T, Tsubouchi H et al. Protein kinase C-dependent increase in reactive oxigen species
(ROS) production in vascular tissue of diabetes: role of NAD(P)H oxidase. J Am Soc Nephrol 2003;14:S227-
[5] Brownlee M. The pathobiology of diabetes complications: a unifying mechanism. Diabetes
[6] Tanganelli I, Ciccoli L, Tansi R et al. Markers of oxidative stress in diabetic patients. Diabetes Research
and Clinical Practice. 2000;50(Suppl 1): S1.
[7] Kotani K, Sakane N, Tsuzaki K et al. Lifestyles and oxidative stress in type 2 diabetic patients. Scand J Clin
Lab Invest. 2008;68(7):516-8.
[8] De Cosmo S, Lamacchia O, Rauseo A et al. Cigarette smoking is associated with low glomerular filtration
rate in male patients with type 2 diabetes. Diabetes Care.2006;29(11):2467-70.
[9] Dominguez LJ, Galioto A, Pineo A et al. Age, homocysteine, and oxidative stress: relation to
hypertension and type 2 diabetes mellitus. J Am Coll Nutr. 2010;29(1):1-6.
[10] Ivkovic S, Zabcic D. Antioxidative therapy: nanotechnology product TMA-ZEOLITE reduces oxidative
stress in cancer and diabetic patients. Free Radical Biology and Medicine 2002;33 (Suppl 2).
[11] Gianturco V, Bellomo A, D'Ottavio E et al. Impact of therapy with alpha-lipoic acid (ALA) on the
oxidative stress in the controlled NIDDM: a possible preventive way against the organ dysfunction? Arch
Gerontol Geriatr. 2009;49 Suppl 1:129-33.



Dyslipidemia is the most frequent dysmetabolic disease characterized by an increase in the levels of
cholesterol and/or triglycerides that are determinants for atherosclerosis. However, atherosclerosis is a
complex disease that is determined by many concomitant factors, and among them vessel reactivity and
inflammatory processes have the greatest responsibility.
The hypothesis of the relationship between OS and atherosclerosis [1] has been confirmed by the presence
of oxidized lipoproteins that modify the contractility and vascular reactivity [2] and create an inflammatory
The cause of the vascular contraction, damaging the arterial wall, is believed to be the distraction of NO
due to the reaction with O2- (absolutely the faster reaction in the organism 1x10-9 min) to form
peroxynitrite (ONOO-) [3]. Hypercholesterolemia is known to reduce the availability of NO and to impair
endothelial function [4].
Overall, there are no doubts that dyslipidemia is accompanied by an increase of hydroperoxides (ROOH)
since lipids and proteins represent the main molecular entities of lipoproteins and can be considered both
an index of OS and a mirror of the advancement of the atherosclerotic process [5].
In addition to endothelial function, which may be compromised by oxidative condition, in dyslipidemia
there is also the problem of the RBCs, which are circulating and in contact with lipoproteins.
Its well known that the erythrocytes are subjected to membranes lipid peroxidation which limits their
reducing ability [6]. The efficiency of the intrinsic reducing system determines the duration of life of the
RBCs [7]. The dyslipidemia is characterized also by a reduction of RBCs antioxidant capacity which takes
place in parallel with lipid peroxidation [8], and the increase of their resistance to OS is beneficial for the
hypertriglyceridemia treatment [9].
Finally it must be considered that the therapies with antioxidant products are believed to be valid in the
treatment of dyslipidemia [10] and that the use of statins [11] and other lipid-lowering [12] also reduce the
oxidative burden caused by the disease.
The PAT test, together with the normal lipemic index, can be considered an appropriate new test to
determine the general and oxidative balance in any kind of dyslipidemia.


[1] Witzum JL. The oxidation hypothesis of atherosclerosis. Lancet 1994;334:793-795.

[2] Cox DA, Cohen ML. Effects of oxidized low-density lipoprotein on vascular contraction ad relaxation:
clinical and pharmacological implication in atherosclerosis. Pharmacological Rev 1996;48:3-19.


[3] Kojda G, Harrison D. Interaction between NO and reactive oxygen species: pathophysiological
importance in atherosclerosis, hypertension, diabetes and heart failure. Cardiovasc Res 1999;43:562-571.
[4] Maas R, Schwdhelm E, Kahl L et al. Simultaneous assessment of endothelial function, nitric oxide
synthase activity, nitric oxide-mediated signaling, and oxidative stress in individuals with and without
hypercholesterolemia. Clin Chem 2008;54:292-300.
[5] Kinoshita M, Oikawa S, Hayasaka K et al. Age-related increases in plasma phosphatidylcholine
hydroperoxide concentrations in control subjects and patients with hyperlipidemia. Clin Chem
[6] Korgun DK, Bilmen S, Yesilkaya A. Alteration in the erythrocyte antioxidant system of blood stored in
blood bugs. Res Commun Mol Pathol 2001;109:357-363.
[7] Kurata M, Suzuki M, Agar NS. Antioxidant system and erythrocyte life-span in mammals. Comp Biochem
Physiol B 1993;106:477-487.
[8] Efe H, Kirci D, Deger O et al. Erythrocyte antioxidant enzymes activities and lipid peroxidation in patients
with type IIb and IV hyperlipoproteinemias. Tohoku J Exp Med 2004;201:163-172.
[9] Mabile L, Piolot A, Boulet L et al. Moderate intake of n-3 fatty acids is associated with stable erythrocyte
resistance to oxidative stress in hypertriglyceridemic subjects. Am J Clin Nutr 2001;74:449-456.
[10] Accinni R, Rosina M, Bamonti F et al. Effects of combined dietary supplementation on oxidative and
antiinflammatory status in dyslipemic subjects. Nutr Metab Cardiovasc Dis 2006;16:121-127.
[11] Yoshino G, Hirano T, Kazumi T et al. Fluvastatin increase LDL particle size and reduces oxidative stress
in patients with hyperlipidemia. J Atheroscler Thromb 2003;10:343-347.
[12] Frits HAF, Onkers IJAM, Schwedhelm E et al. Normal oxidative stress and enhanced lipoprotein
resistance to in vitro oxidation in hypertriglyceridemia. Effect of benzofibrate therapy. Arteriocler Thromb
Vasc Bio 2000;2:2434-2440.

Use of PAT test in other fields

PAT test can be used as a predictable markers of AOs excess or deficiency not only in patient with chronic
diseases but as index of AO balance in commonly activities, such as in sport and physical exercises.

Physical activity increases the ATP requirement and the consequent ROS production and AOs imbalance
[1]. Although, the increased use of energetic substrates (carbohydrates, lipids, proteins) leads to an
overproduction of ROS via all the three common modalities (energetic, metabolic, and reactive). The
temporary increase of an energetic pathway is usually balanced by the AOs. However, when the muscle
effort is strenuous [2], the increase can be determined by the metabolic pathway (ischemia/reperfusion)


and by the reactive cells activation (lymphocytes, macrophages) retrieved from tissue and leads to vascular
In other terms, during the continued/exhausting effort takes place all the three known conditions for ROS
production (energetic, metabolic, and reactive) and it might be possible that the antioxidant reserve is
insufficient to balance the OS. In this case, if this condition continues even for a short period of time
(minutes), the organism falls into the RI condition. The RI condition is characterized by an increase of
reactive interleukins (IL-6) and inflammatory indexes, as: -TNF, IFN-, hsCRP [3] and by the activation of
TLR (tall like receptors) of the innate immunity on the macrophages [4].
In untrained subjects also, the incongruous physical activity is associated with an increase of ROS.
In case the sport training is performed in a congruous way, it can oppose the development of the RI [5],
limiting the stimulation of the TLR receptors [6] and inducing positive effects also in case of chronic disease
The exercise as a healthy support for chronic disease has to be implemented properly to avoid damages.
The oxidative status evaluation becomes essential to maximize the benefit/ risk ratio.
ROS production increases during ultra-marathon race and re-turn to basal levels after the race thanks to
the antioxidant network [10]. In particular it has been observed in different studies that marathon and
ultra-marathon athletes showed increased marker of cellular damage [11-16].
The increased use of O2, in particular during the ultra-marathon race, is considered the main cause of OS
Redox homeostasis of the ultra-endurance running athletes was evaluated in a short and long-term. It has
been observed that ultra-endurance exercise causes OS, characterized by an increase of OS markers and a
reduction of AOs (GSH) for at least one month after the race [18].
It has been also observed in another study that during a cycling race the OS levels increased till an critical
ROS levels. Cyclists treated for 10 days after the race with AO supplements reduces OS values [19].
The evaluation of the oxidative balance in athletes results fundamental [20] in order to detect RI and
prevent cellular and tissues damage. The use of innovative methods of measurement of OS balance allows
to evaluate the possible need to modify the training regimen, nutrition and lifestyles of the athletes
although further studies are needed to provide a single view of the problem.
In this regards the new innovative PAT test should add value for the research because of the simplicity of
the determination, that allows for the monitoring of the athlete condition very easily because of the
portable dedicated system (Fras Evolvo) and the minimal amount of blood needed (a tip finger test).


[1] Reid MB. Invited Review: redox modulation of skeletal muscle contraction: what we know and what we
don't. J Appl Physiol 2001;90:724-731.
[2] Cases N, Sureda A, Maestre I et al. Response of antioxidant defenses to oxidative stress induced by
prolonged exercise: antioxidant enzyme gene expression in lymphocytes. Eur J Apll Physiol 2006;98:263-
[3] Margeli A, Skenderi K, Tsironi M et al. Dramatic elevation of interleukin-6 and acute phase reactants in
athletes participating in ultradistance foot race spartathlon: severe systemic inflammation and lipid and
lipoprotein changes in protracted exercise. J Clin Endocrinol Metab 2005;90:3914-3918.
[4] Simpson RJ, McFarlin BK, McSporran C eyt al. Toll-like receptors expression on classic and
proinflammatory blood monocytes after acute exercise in humans. Brain Behav Immun 2009;23:232-239.
[5] Libardi CA, de Souza GV, Cavaglieri CR et al. Effect of resistance, endurance and concurrent training on
TNF-, IL-6 and CRP. Med Sci Sport Exerc. 2011; Jun 21 [Epub ahead of print].
[6] Gleeson M, McFarlin B, Flynn M. Exercise and Toll-like receptors. Exerc Immunol Rev 2006;12:34-53.
[7] Leung FP, Yung LM, Laher I et al. Exercise, vascular wall and cardiovascular diseases: an update (Part 1)
Spots Med 2008;38:1009-1024.
[8] Ng LW, Mackney J, Jenkins S, Hill K. Does exercise training change physical activity in people with COPD?
A systematic review and metanalysis. Chron Resp Dis 2011;Dec 22 [Epub ahead to print]
[9] Ribeiro F, Alves AJ, Teixeira M et al. Rev Port Cardiol 2012 Jan 4 [Epub ahead to print]
[10] Hattori N, Hayashi T, Nakachi K et al. Changes of ROS during a two-day ultra-marathon race. Int J Sports
Med 2009; 30(6):426-9.
[11] Marzatico F, Pansarasa O, Bertorelli L et al. Blood free radical antioxidant enzymes and lipid peroxides
following long-distance and lactacidemic performances in highly trained aerobic and sprint athletes. J
Sports Med Phys Fitness. 1997; 37(4):235-9.
[12] Mastaloudis A, Leonard SW, Traber MG. Oxidative stress in athletes during extreme endurance
exercise. Free Radic Biol Med. 2001; 31(7):911-22.
[13] Mastaloudis A, Morrow JD, Hopkins DW et al. Antioxidant supplementation prevents exercise-induced
lipid peroxidation, but not inflammation, in ultramarathon runners. Free Radic Biol Med. 2004a;
[14] Mastaloudis A, Traber MG, Carstensen K et al. Antioxidants did not prevent muscle damage in
response to an ultramarathon run. Med Sci Sports Exerc. 2006; 38(1):72-80.


[15] Mastaloudis A, Yu TW, O'Donnell RP et al. Endurance exercise results in DNA damage as detected by
the comet assay. Free Radic Biol Med. 2004b; 36(8):966-75.
[16] Fisher-Wellman K and Bloomer RJ. Acute exercise and oxidative stress: a 30 year history. Dynamic
Medicine 2009; 8:1.
[17] Sen CK, Atalay M, Hanninen O. Exercise-induced oxidative stress: glutathione supplementation and
deficiency. J Appl Physiol 2000; 77:2177-2187.
[18] Turner JE, Hodges NJ, Bosch JA, Aldred S.Prolonged depletion of antioxidant capacity after
ultraendurance exercise. Med Sci Sports Exerc. 2011 Sep;43(9):1770-6.
[19] Beltrami G, Fanton F, Schiavottiello G. Determination of free radicals in different sporting activities and
their application. Report of Italian Baseball And Softball Medical Federal Commission. Scientific Institute for
Sport Italian National Olympic Committee (CONI), Rome, Italy. CLINICAL REPORT, 1999.
[20] Corsetti R, Villa M, Pasturenzi M et al. Redox State in Professional Cyclists Following Competitive Sports
Activity. The Open Sports Medicine Journal. 2012;6:40-47.


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