Untitled Document

Scientific Director, PROBIOX SA – Université de Liège, Tour de Pathologie 2ème étage Sart Tilman 4000 Liège, Belgium. Email : J.Pincemail@ulg.ac.be and J.Pincemail@probiox.com

I. Introduction

Under physiological conditions the element oxygen, indispensable to life, is constantly being converted in the mitochondria to toxic forms that alter cell integrity. These so-called 'activated oxygen species' (AOS), which notably include free radicals, have oxidising properties enabling them to react, in the environment where they are produced, with a whole range of biological substrates (lipids, proteins, DNA, glucose...). At molecular level, these OAS can also act as second messengers and activate factors or genes involved in the development of various pathologies.

To protect itself against this toxic effect of oxygen, our organism has developed defence systems that regulate AOS production. These systems consist of antioxidants (e.g.: vitamins A, C, and E), oligoelements, and proteins preventing iron from triggering AOS production. To this panoply are added proteolytic enzymes, whose role is to degrade oxidised substrates.

AOS are also generated by the action of environmental oxidants. Modern life confronts us with pollution, consumption of alcohol or medications, prolonged exposure to sunlight, and smoking. All of these situations cause overproduction of AOS in our organism. This leads to a weakening of our antioxidant defences (vitamins, oligoelements) and also causes cell damage. To complicate the situation, our current diet is not healthy or balanced enough and thus provides less and less of the natural antioxidants needed to control the harmful effects of oxygen. It should also be mentioned that intense physical exercise, when not correctly practiced and managed, can generate oxidative stress. Clinically, oxidative stress can also develop as a result of cardiovascular surgery, organ transplantation, or respiratory distress.

Generally speaking, oxidative stress is defined as the result of an imbalance between pro-oxidants and defence systems (antioxidants), resulting in (often irreversible) cell damage (Figure 1).

Figure 1 : Imbalance between antioxidants and pro-oxidants

Different individuals have different antioxidant potentials according to their lifestyle and genetic makeup or to the environment in which they live. Good eating habits also play a key role in maintaining an optimal antioxidant potential. For instance, the average plasma vitamin C concentration is :
· 31.12 ± 14.88 mmol/L in healthy subjects who eat no fruit,
· 36.45 ± 18.79 mmol/L in healthy subjects eating 3 to 4 fruits a week,
· 48.83 ± 23.68 mmol/L in healthy subjects eating between 1 and 4 fruits a day (8).

Determining an individual's oxidative stress (OS) is now becoming a priority in disease prevention because a great many studies (1) indicate that there is a close link between alteration of the organism's antioxidant defence systems and the development of over 200 different pathophysiologies, ranging from atherosclerosis to cancer and AIDS, inflammatory diseases, diabetes, and ageing (Figure 2).

Figure 2 : Pathologies involving oxidative stress

II. Definition of oxidative stress

1. The oxygen paradox

The mitochondrial respiratory chain plays a key role in the cell. It is responsible for the conversion of oxygen to two molecules of water. This direct reduction reaction involving four electrons is made possible by a complex system of proteins and enzymes (cytochromes) located in the inner membrane of the mitochondrion. The consequences of this mitochondrial activity are dual and paradoxical. On the one hand, the mitochondrion provides the cell with an important source of energy, since oxygen reduction generates 36 molecules of adenosine triphosphate, a high-energy compound. On the other hand, about 0.4 to 4% of the oxygen is not correctly converted to water, because of electron leakage resulting from imperfections in the respiratory chain. By reduction involving a single electron, oxygen gives rise to activated oxygen species (AOS), among which are free radicals such as the superoxide anion or the hydroxyl radical. In chemistry, a free radical is an atom or a molecule whose chemical structure is characterised by the presence of an unpaired electron that makes this chemical species much more reactive than the atom or molecule from which it derives. Other, non-radical oxygen-containing entities can also be produced, such as hydrogen peroxide (H2O2) or singlet oxygen (1O2). The formation of AOS requires the presence of transition metals such as iron or copper, acting as necessary catalysers throughout the chemistry of free radicals (Figure 3).

Figure 3 : Description of activated oxygen species

Our organism is thus constantly producing AOS (Figure 3). The advent of molecular biology has enabled scientists to show that AOS at low concentration play an important physiological role, acting as second messengers capable of:

- regulating apoptosis, or the programmed suicide of cells evolving towards a cancerous state (2)
- activating transcription factors (NFkB, p38-MAP kinase…) responsible for activating genes involved in the immune response (3),
- modulating the expression of structural genes coding for antioxidant enzymes (4).

The drawback is that when AOS are produced in excessive amount, they have harmful effects because they induce apoptosis in healthy cells or activate various genes coding for pro--inflammatory cytokines or adhesion proteins. In addition, their unstable nature makes them particularly reactive and capable of inflicting major cell damage :
· by causing breaks and mutations in deoxyribonucleic acid (DNA),
· by inactivating proteins and enzymes,
· by oxidising sugars (glucose),
· by inducing lipid peroxidation among the polyunsaturated fatty acids of lipoproteins or of the cell membrane (Figure 4).

Figure 4 : Membrane destruction caused by AOS attack

2. Antioxidant defences

The balance between positive and negative effects of AOS is thus particularly fragile. AOS production is strictly regulated by our organism, which has developed antioxidant defences that protect us against the potentially destructive effects of AOS. These defence systems consist of (Figure 5) :
- enzymes (Cu-Zn and Mn superoxide dismutases, catalase, glutathione peroxidases, the thioredoxin - thioredoxin reductase pair, haem oxygenase, heat shock proteins),
- iron- and copper-transporting proteins (transferrin, ferritin, ceruleoplasmin),
- small antioxidant molecules (glutathione, uric acid, bilirubin, glucose, vitamins A, C, E, ubiquinone, carotenoids, flavonoids),
- oligoelements (copper, zinc, selenium) indispensable to the activity of antioxidant enzymes.
Alongside this panoply of defences against AOS is a secondary defence system consisting of enzymes whose role is to prevent intracellular accumulation of oxidised DNA or proteins and to degrade their toxic fragments (Figure 5).

Figure 5: Balance between antioxidants and pro-oxidants

3. Factors that contribute to increasing oxidative stress

Oxidative stress is defined as an imbalance between antioxidants and pro-oxidants in favour of the latter (5). In vivo, several biochemical systems can cause increased AOS production. Alteration of the electron transport chain in the mitochondrion is a first cause of increased oxidative stress. This happens during ageing and in situations of ischaemia-reperfusion (e.g. organ transplantation). White blood cell activation is also a very important source of AOS production. When subjected to the action of foreign agents, these cells shift from a quiescent to an activated state, and this results in a 400% increase in their oxygen consumption. Various enzyme systems convert nearly all the oxygen to AOS, which can then attack healthy tissues - this is the phenomenon called inflammation. Other mechanisms also contribute to massive AOS production: xanthine oxidase activation, haemoglobin oxidation, release of free iron, stepped-up prostaglandin metabolism, and endothelial cell activation. In addition, many epidemiological studies show that homocysteine is an independent risk factor for myocardial infarction, stroke, and death from coronary disease. One of the mechanisms by which homocysteine may favour the appearance of atherosclerosis is its direct cytotoxic effect on endothelial cells, partly linked to the formation of free radicals when reduced homocysteine is oxidised by iron (6, 7).

Both the environment in which we live and our lifestyle can cause our organism's level of oxidative stress to rise. Here are a few examples :
- prolonged exposure to sunlight
- exposure to radiation
- contact with carcinogenic agents (e.g. asbestos)
- smoking (cigarette smoke contains 1019 AOS)
- taking medications or the contraceptive pill
- excessive or ill-managed physical exercise
- excessive alcohol consumption
- intellectual stress
- heat shock
- ozone therapy
- pollution
- infectious agents
- …

4. An unbalanced diet : a major oxidative stress factor

Fruits and vegetables are the principal dietary contributors of antioxidants, being particularly rich in vitamins (A,C,E), oligoelements, and polyphenols. It is currently accepted that five daily portions of fruits and vegetables can cover an individual's recommended daily intake (RDI) of these compounds. Yet this ideal situation is far from being a general reality. Several epidemiological studies show that over 20% of the population of industrialised countries never eats fruit. In a study of 123 healthy subjects inhabiting the Liège region, we confirmed this figure and showed that plasma levels of vitamin C are on the average 26.3% lower in subjects who never eat fruit than in subjects eating 1 to 4 fruits a day (8). In addition, the advent of fast food (regular consumption of hamburgers, pizza...) contributes, particularly in the young, to an increasingly unbalanced and antioxidant-poor diet.

The following figure shows how eating fruits and vegetables can reduce cell levels of oxidised DNA (9), whose role in cancer development has been clearly demonstrated (Figure 6):

Figure 6: Effect of diet on DNA oxidation

5. It is useful to evaluate your oxidative stress status

In 1991, the WHO/Monica study of the World Health Organisation (WHO) showed that men living in Southern Europe (whose diet is rich in fruit) have a lower risk of coronary disease than men living in Northern Europe (who eat les fruit). The results clearly show that the lower the plasma level of vitamin C (low levels being frequent in the North), the higher the mortality due to coronary disease (10). These observations on vitamin C were later confirmed in a large-scale epidemiological study focusing on over 1600 subjects (Figure 7)(11).

Figure 7: Vitamin C and cardiovascular disease

The vast majority of epidemiological studies indicate that there is a close correlation between alteration of the organism's antioxidant defences, an increased level of oxidative stress markers, and the development of over 200 different pathophysiologies ranging from atherosclerosis to cancer, AIDS, inflammatory disease, diabetes, and ageing (Figure 2). It would thus appear that antioxidant supplements could be useful in prevention of these pathologies. There is not yet any well-established evidence-based medicine in this area, but converging results of antioxidant studies highlight the potential interest of antioxidants in disease prevention. For instance, recent studies show that vitamin E can delay atherosclerosis of the carotid (12, 13).

In terms of disease prevention, it is thus useful to know your oxidative stress status (OSS) (14). Thanks to the development of quick, specific, sensitive analytical methods adapted to routine analysis, PROBIOX SA offers healthcare professionals a wide range of tests for evaluating a person's antioxidant defences, oligoelement status, iron metabolism (iron catalyses AOS-forming reactions), and also for determining markers of lipid, DNA, and protein oxidation. This makes it possible to detect the presence of abnormal oxidative stress in an individual and to recommend corrective measures such as a healthier diet or the intake of appropriate supplements.
PROBIOX SA designs and develops increasingly high-performance tools for the evaluation of oxidative stress. This mission is part of a scientific revolution that should lead in the not-so-distant future to a much more personalised therapeutic approach, based notably on the analysis of how each patient's genome responds to a situation of oxidative stress and on a precise evaluation of that patient's capacity to cope with the stress.

III. How to evaluate oxidative stress

No single method can alone provide an adequate measure of OSS. Precise evaluation of the situation requires a battery of tests (1) aimed at determining levels of six major types of compounds:

· 1. Antioxidants - enzymes and low-molecular-weight compounds

A. Superoxide dismutases (SODs)

The enzyme superoxide dismutase ensures elimination of the superoxide anion, the first toxic species to be formed from oxygen. SOD thus carries out first-line defence against oxidative stress. To function correctly, the enzyme requires oligoelements such as copper and zinc (Cu-Zn SOD present in the cytosol) or manganese (Mn SOD present in the mitochondrion. There also exists an extracellular SOD.

Low SOD levels may reflect low levels of oligoelements, but there is no absolute correlation between the former and the latter. In the presence of oxidative stress, SOD shows two different behaviours (Figure 8). First, in response to a moderate level of oxidative stress (due, e.g., to physical exercise), the organism overexpresses SOD (15, 16). Then, if the stress persists and involves massive production of toxic AOS, SOD is destroyed and its concentration drops. Paradoxically, a too-high SOD concentration can be dangerous, because it leads to overproduction of hydrogen peroxide (paradoxical effect of antioxidants).

Normal blood concentration: 785 – 1570 UI/g haemoglobin

Figure 8 : Evolution of antioxidant enzymes as a function of the oxidative stress level

B. Glutathione peroxidase (GPx)

To function correctly, this enzyme requires glutathione and selenium. Its main role is to eliminate lipid peroxides resulting from the action of oxidative stress molecules on polyunsaturated fatty acids.

Like SOD, seleno-dependent glutathione peroxidase behaves in two different ways in the presence of oxidative stress: first the enzyme is overexpressed and then, if the oxidative stress persists, it is destroyed. A reduced GPx activity may reflect too little selenium in the diet.

The correlation between the plasma level of selenium and the GPx content of red blood cells is not significant unless the selenium level is below 60 µg/L (Figure 9). Above this threshold, the curve flattens out, indicating that the enzyme's requirement for selenium is met (17) .

Figure 9 : Relation between GPx activity and the plasma selenium level

Normal blood concentration: : 30 – 55 UI/g haemoglobin

C. Thioredoxins (TRx) and thioredoxin reductase (TRxR)

Thioredoxins are enzymes with intrinsic antioxidant activity, like all proteins possessing a thiol group (-SH). They also play a major role in the regulation of the immune system (18, 19). Once oxidised, thioredoxin is reduced by thioredoxin reductase (TRxR), an enzyme possessing a selenocysteine group in its active site. TRxR also intervenes in the degradation of lipid peroxides and hydrogen peroxide and in the regeneration of ascorbic acid from the ascorbyl radical.

D. Haem oxygenase

The haem oxygenase system (HO) consists of three isozymes : the inducible HO-1 form, the constitutive HO-2 form, and the HO-3 form, whose gene has been recently cloned. In biological systems, HO catalyses conversion of haem to carbon monoxide, biliverdin, and iron. The protective effect of HO against oxidative stress is indirect - once formed, biliverdin converts to bilirubin, endowed with strong antioxidant activity. In addition, the iron produced via the activity of HO stimulates the synthesis of ferritin, also involved in the antioxidant response (long-term action). Yet the activity of HO can have harmful short-term effects, since iron also acts as a pro-oxidant via catalysis of AOS production (20).

E. Heat shock proteins

Heat shock proteins (e.g. HSP70) form a family of chaperone proteins playing an essential role in protein translocation, stabilisation, and assembly. They also intervene in repairing oxidative-stress-induced damage to proteins. They enable cells to withstand a hostile environment by prolonging their viability until more favourable conditions prevail. An increased level of these proteins should thus be seen as an adaptive response to oxidative stress induced by various conditions such as hypo- or hyperthermia, acidosis, energy depletion, ischaemia-reperfusion, a viral infection, or physical exercise (21).

F. Carotenoids

Some carotenoids such as ß- carotene are degraded to, and thus serve as precursors of vitamin A, which notably plays a key role in visual perception. Most carotenoids and vitamin A interact with singlet oxygen and can thus prevent oxidation of several biological substrates, including polyunsaturated fatty acids.

Severe vitamin A deficiency is rare and observed only at levels below 10 mg/100 ml. Low levels (< 28.6 mg/100 ml in children and < 40.1 mg/100 ml in adults) correspond with a moderate risk of vitamin A deficiency. The famous WHO/MONICA study conducted in 8 European countries has shown that vitamin A concentrations above 63 - 80.2 mg/100 ml are associated with a decreased risk of cardiovascular disease (22).

Other carotenoids of interest because of their antioxidant properties include lycopene, present in tomato skin (23), lutein, ß- cryptoxanthine, zeaxanthine, …..

Normal plasma vitamin A concentration: 1200 – 3700 UI/L or 36.03 – 111 µg/dL
Normal plasma ß carotene concentration: 10 - 85 µg/dl
Normal plasma lycopene concentration: 10 - 33 µg/dl

G. Vitamin C

Vitamin C or ascorbic acid is not synthesised by the organism. Its plasma level depends strongly on the diet and on changes in hepatic flow (e.g. after physical exercise). It is an excellent AOS scavenger that can protect biological substrates (proteins, fatty acids, DNA) from oxidation. At physiological concentrations, vitamin C can prevent LDL oxidation caused by various AOS-generating systems (activated neutrophils, activated endothelial cells, myeloperoxidase). An intermediate in its oxidation to dehydroascorbic acid is the ascorbyl radical, which plays an essential role in the cascade that regenerates vitamin E from oxidised vitamin E (Figure 10).

Figure 10 : When lipid radicals react with vitamin E, the latter is converted to the tocopheryl radical.
Vitamin C reacts with the tocopheryl radical (oxidised vitamin E),
thus regenerating active vitamin E.

Vitamin C is consumed in the presence of oxidative stress. In addition, several studies have shown that vitamin C levels below 4 mg/mL are associated with an increased risk of developing cardiovascular disease (10) (see Figure 8).

Although very labile, vitamin C can be determined by routine analysis, provided the blood sample is properly treated.

Normal plasma concentrations : 6.21– 15.18 µg/mL (men) ; 8.6– 18.8 µg/mL (women)

H. Vitamin E

The term vitamin E refers to a family of compounds: the tocopherols (alpha, beta, gamma, delta). The hydrophobic character of vitamin E enables it to insert itself into lipiproteins and the fatty acids of the cell membraned, where it plays a protective role by preventing oxidative-stress--induced propagation of lipid peroxidation (Figure 11). Of all the tocopherols, alpha and gamma tocopherol (24) display the most interesting antioxidant properties.

Figure 11 : Insertion of vitamin E
into the lipid membrane.

Vitamin E is consumed in response to oxidative stress. A vitamin E level below 7 – 8 mg/ml corresponds with a moderate risk of dietary vitamin E deficiency. The European study WHO/MONICA (10) has shown that blood levels of vitamin E are significantly higher among healthy men (aged 40-49) living in European regions where mortality from coronary disease is low (Switzerland, southern Italy) than in regions where this mortality is average (Northern Ireland) to high (south-western Finland, Scotland).

Vitamin E being transported by lipids, its concentration must always be normalised with respect to cholesterol (the VitE/cholesterol ratio) or to the total lipid content.

Normal plasma level of vitamin E : 8 – 15 µg/mL
Normal value of the VitE/cholesterol ratio: 4.40 – 7.00 µg/g

I. Glutathione

This is a tripeptide that intervenes at various levels to combat oxidative stress. Glutathione (GSH) can interact directly with activated oxygen species, but it mainly serves as a substrate of glutathione peroxidase, the enzyme that eliminates lipid peroxides. GSH also plays a role in the expression of genes coding for pro- and anti-inflammatory proteins (Figure 12). Too-low GHS concentrations lead to a weakened immune defence.

Figure 12 : Central role of GSH in regulating the expression of pro- and anti-inflammatory genes

During oxidative stress, GSH is usually consumed. It is thus important to measure the level of oxidised GSH (GSSG) and to calculate the GSH/GSSG ratio in order to get a more precise idea of the level of oxidative stress. Jones et al (25) recently showed that during ageing (above 50) this ratio decreases gradually. It also decreases considerably after intense physical exercise (Figure 13) as a result of a rise in the level of oxidised glutathione. This reflects the presence of oxidative stress, as described previously (26).

Figure 13 : Evolution of the GSH/GSSG during intense
physical exercise (personal data)

Normal blood level of GSH : 753 – 958 µM
Normal blood level of GSSG : 1.17 – 5.32 µM
Normal value of the GSH/GSSG ratio: 156 - 705

J. Thiol proteins

Most proteins possess thiol groups (-SH) that react readily with activated oxygen species. Because of its abundance; albumin (which possesses thiol groups) can be seen as one of the major antioxidants present in plasma. This test is complementary to the GSH assay.

Normal plasma concentration : 216 – 556 µM

K. Uric acid

In primates, uric acid is the major end product of purine metabolism. Possessing antioxidant properties, it can interact with activated oxygen species and notably with the hydroxyl radical. It appears as the most efficient antioxidant in plasma in terms of reactivity towards AOS. Yet its oxidation products (e.g. allantoin) can in turn readily become oxidised, generating toxic oxygen species.

Uric acid increases during oxidative stress, principally during ischaemia-reperfusion.

Normal plasma concentrations : men: 34 -84 mg/L; women : 22 - 60 mg/L

L. Coenzyme Q10

Ubiquinone or coenzyme Q10 (CoQ10) is well known for its vital role in mitochondrial energy production. CoQ10, principally in its reduced form ubiquinol-10 or CoQ10H2, also possesses interesting antioxidant properties. Like vitamin E, it can inhibit lipid peroxidation (27, 28). It is necessary to study the CoQ10H2/CoQ10 ratio in order to evaluate correctly the importance of CoQ10 in protection against AOS attack (29). On the other hand, the CoQ10 level can be a good discriminating parameter for detecting patients at risk of developing coronary disease (Figure 14).

	control	patients with heart ischaemia
CoQ`10` (µM)	0.86 +/- 0.28	0.70 +/- 0.19
LDL choles (mM)	3.21 +/- 0,84	3.81 +/- 0.92
Tot Choles/HDL choles	3.81 +/- 1.16	4.59 +/- 1.21
LDLcholes/CoQ`10` x 10Ò	3.73 +/- 1.12	5.76 +/- 1.72

Figure 14 : LDL/CoQ10 in patients with cardiovascular disease
of ischaemic origin
Hanaki et al. Clin Invest 71 :S112-115, 1993

As shown by Ghirlanda et al (30), the level of CoQ10 in the blood is markedly diminished (+/- 40%) in patients taking statins to reduce their cholesterol level. Statins inhibit the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which acts on production of mevalonate, a common intermediate in the synthesis of cholesterol and CoQ10.

Normal plasma concentration: 0.40 – 1.2 µg/mL

M. Total antioxidant capacity

This test consists in evaluating the capacity of whole blood or plasma to inhibit AOS production in an in vitro AOS-generating system. It is thus a screening method that sums the various individual activities of all the antioxidants present in a biological medium (31). There exist several tests (LPIC, TEAC, FRAP, TRAP, ORAC, DCFH-DA*…) that differ by the AOS-generating system used, the biological target to be oxidised, and the chosen detection system. The literature mentions that the TEA test is the least reliable. Recently have appeared very quick chemiluminescence-based methods (an assay takes a few minutes) making it possible to distinguish the respective contributions of lipophilic and hydrophilic antioxidants to the total antioxidant capacity. Yet these tests should be interpreted with extreme caution, because they often reflect the blood level of uric acid (or albumin), which is the antioxidant reacting most readily with AOS. Each method has its limitations, and according to the method chosen the total antioxidant capacity may be over- or underestimated, or it might not even correlate with the level of oxidative stress.

·* LPIC : Lipid peroxidation inhibition capacity
· TEAC : Trolox equivalent antioxidant capacity
· FRAP : Ferric reducing antioxidant power
· TRAP : Total radical-trapping antioxidant potential
· ORAC : Oxygen radical absorbance capacity
· DCFH-DA : Dichlorofluorescein – diacetate assay

2. Oligoelements

A. Selenium

This oligoelement is not itself an antioxidant, but it participates in defence against AOS as a co-factor of glutathione peroxidase.

Several large-scale studies have shown that serum selenium levels below 45 – 50 µg/L are associated with the appearance of coronary disease (32, 33, 34).

Normal plasma concentration : 94 – 130 µg/L
(Please note: the serum level of selenium is 1.18 times higher than the plasma level)

B. Copper

This oligoelement is one of the essential co-factors of SOD. Yet like iron and as a transition metal, copper plays a major role in triggering reactions leading to AOS production. An excessive copper concentration may thus reflect the presence of oxidative stress. Several studies have shown an increase in the serum level of copper during ageing (35).

Normal plasma concentration: 0.70 to 1.40 mg/L

C. Zinc

This oligoelement is one of the essential co-factors of SOD. Zinc intake leads in the long term to induction of antioxidant proteins such as metallothioneins. Zinc also protects the thiol groups of proteins, and can partially inhibit AOS-generating reactions induced by iron or copper. This is why measurement of the copper/zinc level can provide useflul information regarding an individual's oxidative stress status.

Zinc deficiency generally results in increased sensitivity to oxidative stress. A recent study has shown that elderly people with degenerative diseases have a higher copper/zinc ratio than elderly people in good health (36).

Normal plasma zinc level: 0.70 – 1.20 mg/L
Normal plasma Cu/Zn ratio: 1.14 – 1.29

· 3. Biological makers of oxidative stress

AOS react with a whole range of biological substrates such as proteins, lipids, and deoxyribonucleic acid (DNA). Oxidised derivatives of these substrates thus constitute markers of oxidative stress.

A. Lipid peroxidation

The polyunsaturated fatty acids of cell membranes are the principal target of AOS. Attack results in formation of lipid peroxides (LPO) that can be measured in whole blood or plasma within certain sensitivity and specificity limits (37). Yet LPO undergo decomposition to sub-products such as malondialdehyde (MDA), 4-hydroxynonenal, ethane, and pentane. For many years determination of MDA with thiobarbituric acid (the TBARS method) was used to evaluate in vivo the presence of lipid peroxidation. The scientific community, however, agrees that this test is not specific and that it is subject to numerous artefacts. It is now possible to measure MDA by more accurate HPLC methods. Yet MDA represents only a small percentage (1%) of all lipid peroxide decomposition products, and is thus far from a reliable marker of oxidative stress.

AOS can also interact directly with fatty acids to form 8-epi-prostaglandin PGF 2a, a member of the isoprostane family. For example, the level of this compound in plasma or urine is higher in chronic smokers (38) than in nonsmokers (Figure 15).

Figure 15 :Isoprostanes as markers of lipid peroxidation

Polyunsaturated fatty acids are also essential constituents of low-density lipoproteins (LDL). LDL oxidation is particularly important in the development of atherosclerosis. As compared to a control population, patients having suffered a myocardial infarction have a particularly high level of oxidised LDL (39). Generally speaking, patients at high risk of stroke or heart attack (owing to hypertension, hypercholesterolaemia, obesity, kidney dialysis) have abnormally high oxidised LDL levels (Figure 16). Spectrophotometric measurement of oxidised LDL, based on determination of conjugated dienes, is less sensitive and less specific than the recently developed immunological methods.

Normal plasma level of oxidised LDL:: 26 – 117 U/L

Figure 16 : Oxidised LDL as a risk factor for cardiovascular disease in kidney dialysis patients

LDL oxidation leads to production of antibodies against oxidised LDL (Ab-ox-LDL). Several studies have shown that an increased Ab-ox-LDL level is closely associated with progression of atherosclerosis of the carotid (40, 41, 42). Particularly high antibody titres are regularly found in high-level athletes (43), who constitute the in vivo model par excellence of oxidative stress (Figure 17).

Figure 17: Levels of antibodies against oxidised LDL in high-level athletes
(Pincemail et al. Free Rad Biol Med 28 :559-565, 2000)

Normal serum level of antibodies against oxidised LDL: 200 – 600 UI/L

B. Oxidised proteins

In the presence of AOS, proteins can undergo denaturation or fragmentation or they can lose their primary and secondary structures. Oxidative damage to proteins (and amino acids) can manifest itself in various ways (44, 45) :
- appearance of hydroperoxide groups (-OOH),
- oxidation of the carbon skeleton of the polypeptide chain, leading to protein fragmentation and the appearance of carbonyl groups,
- oxidation of amino-acid side chains with formation of disulfide bonds, methionine sulfoxide, and carbonyl groups. Some amino acids (Phe, Tyr, His) undergo hydroxylation. In the case of Phe, this generates ortho - and meta-tyrosine, whilst oxidation of Tyr leads to dityrosine formation through dimerisation of two tyrosyl radicals.
- formation of chlorinated derivatives (e.g. 3 –chlorotyrosine, 3,5-dichlorotyrosine) and nitro derivatives (nitrotyrosine) upon contact of tyrosine with, respectively, the MPO/H202 system and the nitrogen monoxide radical (NO°).

Detection of carbonyl groups in proteins is the method most often used (46), but the appearance of these groups reflects merely an overall modification of the protein. It is probably not as specifically representative of oxidative stress as the detection of hydroxylated tyrosine.

Normal plasma concentrations

C. 8-hydroxy-2’deoxyguanosine

AOS show high-affinity reactivity towards certain constitutive bases of DNA. Guanine is readily transformed to 8-hydroxy-2’-deoxyguanosine (8-OH-dG), which is normally eliminated by DNA repair enzymes. When the body's DNA repair systems are deficient, 8-OH-dG accumulates in the DNA (Figure 18), causing mutations involved in cancer development (47). The 8-OH-dG concentration must be normalised with respect to creatinin when it is measured in urine.

Figure 18 : Effect of the environment on DNA oxidation

Normal level in urine: 0 – 20 µg 8OH-dG/gr creatinin ( ELISA)

· 4. The iron status

Iron acts as a catalyser in AOS formation. Under normal physiological conditions, it is not present in the free form responsible for this effect. Analysis of iron transport proteins is thus an important element in establishing the OSS. A great many papers suggest an involvement of transition metals like iron and copper in the development of atherosclerosis (48).

A. Free iron

The serum iron measured in standard blood tests respresents the pool of iron bound to proteins and to chelating agents such as polyphosphates. It is not the notoriously toxic free iron (49, 50). The latter can be determined by routine analysis, with a few technical constraints. Under normal physiological conditions, free iron is not detectable.

Normal plasma concentration of free iron : 0 µmol/L

B. Ferritin

This protein is the major storage site of non-metabolised iron. It thus plays a paramount role in regulating the availability of free iron, catalyser of reactions leading to AOS formation. Several studies have shown that an increased ferritin level (notably in the skin) is a response to oxidative stress (51).

Normal plasma level : 30 – 300 ng/mL (men) ;15 – 150 ng/mL (women)

C. Transferrin and its iron saturation capacity

Under normal physiological conditions, transferrin is between 25% and 30% saturated in iron. A higher level of saturation indicates that iron has been released in a free form. For example, the cardiopulmonary bypass (CPB) procedures used in coronary bypass surgery cause a large amount of iron to be released from red blood cells. Several studies have shown that the iron saturation level of transferrin increases gradually during CPB up to 80 or 90%, in association with an increase in oxidative stress markers (52, 53).

Normal plasma transferrin concentration: 1.60 – 3.50 g/L
Normal iron saturation level of transferrin : 20 – 40%

· 5. The nitrogen monoxide radical

This peculiar radical derived from nitrogen (NO°) is produced by endothelial cells and plays a prime physiological role in the regulation of blood pressure. Oxidative stress can lead to dysfunctioning of endothelial cells, causing them to produce the nitrogen monoxide radical in excess. The radical may then react with oxygenated free radicals to form substances that are highly toxic to the organism: peroxynitric derivatives (HOONOH).

Once formed, NO can also be converted to nitrites or nitrates. An increase in the nitrite/nitrate level reflects nitrogen monoxide production. NO can also interact with the tyrosine residue of proteins (nitration) and with gamma tocopherol. These properties are exploited to detect the presence of NO in biological samples (54, 55).

· 6. Electron paramagnetic resonance (EPR)

This is the ideal technique for visualising free radicals as directly as possible. Free radicals possess an unpaired electron which spins like a top and thereby induces a magnetic field (Figure 19). If the free radical is placed in a magnetic field generated by two powerful magnets, energy is absorbed and this can be visualised as a spectrum. The signal obtained will be typical of each type of radical.

Figure 19 : EPR apparatus for visualising oxygenated free radicals
as directly as possible

In addition, the absorption intensity will be proportional to the quantity of free radicals in the biological sample analysed, thus providing a quantitative measurement of free radical production. With a few (not unmanageable) technical constraints (the use of spin traps), EPR can be used in routine clinical analysis to detect production of the superoxide radical in a plasma sample.

EPR can also detect in plasma the ascorbyl radical (Figure 20), an intermediate in the pathway of ascorbic acid oxidation to the end product dehydroascorbic acid. Measurement of the ascorbyl radical is interesting, because it shows whether a low vitamin C level is due to a low dietary intake or to oxidative stress. A low plasma vitamin C concentration associated with a normal vitC/ascorbyl radical ratio is indicative of low intake, whilst a low vitC/ascorbyl radical ratio, even when the vitamin C level is normal, is a sign of oxidative stress (56).

Figure 20 : EPR spectrum of the ascorbyl radical

Normal concentration of the ascorbyl radical in plasma : 0.28 – 0.44 arbitrary units*
Normal value of theVitC/ascorbyl radical ratio in plasma: 20.73 – 3.,29 arbitrary units
(* mesurement with a Jeol FR300)

7. Other markers

A. Homocysteine

The sulfur-containing amino acid homocysteine is an intermediate in methionine and cysteine metabolism (Figure 21). Although many laboratories mention that the normal level of homocysteine ranges from 5 to 15 µmol/L, recent prospective studies have shown that a homocysteine level above 6.3 µmol/L is associated with a 35% increase in the risk of myocardial infarction (Figure 22) (57). Each 5-µmol/l increment in homocysteine, equivalent to a 20-mg/dL increment in plasma cholesterol, increases the risk of coronary infarction by 60% in men and 80 % in women. Homocysteine is also readily oxidisable, generating AOS that can oxidise LDL, particularly in the presence of metals like iron or copper (6).

Normal plasma concentration: 5 – 15 µmol/L

Figure 21 : Homocysteine metabolism

Figure 22 : Relation between homocysteine and cardiovascular disease

B. Glucose

Glucose produces large quantities of AOS and glyoxal through auto-oxidation (Figure 23). Glyoxal binds to amino groups of proteins, and this leads to the appearance of carboxymethyl-lysine residues (« old proteins »). The latter have the capacity to bind copper and thus induce lipid peroxidation, this leading to an increase in glyoxal production. Glucose itself can combine with haemoglobin to yield glycosylated haemoglobin. An increase in these markers is observed in diabetics, diabetes being clearly associated with a high level of oxidative stress (58). Yet these markers can also accumulate in the organism during ageing.

Figure 23 : Interaction of glucose with activated oxygen species

Normal plasma concentration : 0.60 to 1.10 g/L

C. Myeloperoxidase

When activated, neutrophils increase their oxygen consumption by 400%. A consequence is massive AOS production and the release of proteolytic enzymes (elastase) and myeloperoxidase (MPO). The latter enzyme is involved in the development of oxidative stress, since its enzymatic activity is responsible for formation of hypochlorous acid, a powerful oxidant. An increased plasma MPO level is thus a specific indicator of neutrophil activation and thus indirectly of AOS production, events that always accompany the inflammatory process (59).

Normal plasma concentration : 170 – 498 µg/L

D. Fatty acid profile

Our diet contains large quantities of w6 polyunsaturated fatty acids (linoleic acid and arachidonic acid). In the presence of AOS, these molecules are readily oxidised to lipid peroxides. In addition, the biosynthesis of these fatty acids leads to formation of pro-inflammatory prostaglandins (Figure 24). On the other hand, our diet is rather poor in fish, which unlike meat has many w3 polyunsaturated fatty acids (alpha-linolenic acid and docosahexaenoic acid). The latter are less readily peroxidised, and their biosynthesis gives rise to anti-inflammatory prostaglandins. Scientists agree that the overall w6/w3 ratio should not exceed 4/1 to 8/1, values far below those actually observed in occidental populations. De Lorgeril et al have shown that a Cretan diet rich in alpha-linolenic acid can reduce by over 70% the recurrence of cardiovascular incidents in patients having suffered a myocardial infarction (60).

Figure 24 : Biosynthesis of polyunsaturated fatty acids

IV. What is the best marker of oxidative stress?

The answer to this question is simple: there is none.

From the beginning of research on oxidative stress, scientists have been obsessed with discovering a biomarker that would be a sure sign of oxidative stress in various experimental or clinical situations. The measurement of malondialdehyde was long viewed as the reference test. Unfortunately, MDA detection with thiobarbituric acid (the TBARS test) lacks specificity and above all is subject to numerous artefacts. On the basis of this simple test, many errors of interpretation were committed, sometimes discrediting the importance of oxidative stress in human medicine. Not until the 1980's did new, more reliable techniques emerge for detecting oxidative stress in vivo. Currently over 8 assays are proposed, only about thirty of which are suitable for routine clinical use. Each method has its specificities and limitations. This is why it is utopic to want to detect oxidative stress with a single analysis, even if it is presented to doctors as attractive because it is quick and easy. It is important not to repeat the past mistakes committed with MDA determination.

In-depth knowledge of the scientific literature is thus necessary to maintain a critical view of all available means of investigation (61). Evaluating an individual's oxidative stress requires a battery of complementary tests. The main avenues of investigation include :
1° analysis of antioxidants
2° determination of oligoelements
3° detection of oxidative damage to lipids, proteins, DNA, and glucose
4° investigation of iron metabolism

Doctors, however, are often at a loss when it comes to deciding which tests are needed. One of the missions of PROBIOX SA is to guide them in this choice. Our scientific and clinical expertise has taught us that some markers are more informative than others in a given physiological or pathological situation. PROBIOX SA thus proposes a variety of oxidative stress indicator profiles, each based a set of blood tests appropriate for a specific condition involving oxidative stress (patent application pending).

V. Immediate processing of samples and cold chain: two indispensable elements for obtaining quality results

The interpretation of oxidative stress profiles must imperatively rely on very careful processing of blood samples. Most assays proposed for the evaluation of oxidative stress are highly sensitive to sample preservation conditions (type of anticoagulant, temperature...). An example is the determination of reduced and oxidised glutathione. Figure 25 shows clearly how these two markers evolve if the analysis is carried out after storage of the blood for 1 hour or more at room temperature. In this case, the only way to have a good-quality, interpretable analysis is to place the blood immediately in contact with stabilisers and freeze it.

Figure 25 : Evolution of the GSH/GSSG ratio in blood samples kept at room temperature for 1 to 24 hours.

T0 : sample processed immediately after it was taken
Blue rectangle: reference interval

Generally speaking, these observations are valid also for all markers reflecting the presence of oxidative stress. Figure 26 shows that the conservation kinetics of oxidised lipoproteins fluctuates considerably, even in whole blood samples stored at +4°C. A reliable result is obtained only if the sample is centrifuged immediately and the plasma frozen at – 20°C.

Figure 26 .

When scientists publish results on the evolution of plasma markers of oxidative stress (e.g. vitamin E), they are required to specify in the "Materials and Methods" section of the paper that the blood samples were immediately centrifuged and the plasma stored at –20°C until the marker of interest was analysed. Intent on respecting this directive scrupulously, PROBIOX SA has made available to users a highly detailed protocol for blood sample collection and processing and for sample transfer to the laboratory on dry ice and in suitable containers.

VI. Future developments: genomics and proteomics

PROBIOX SA also devotes considerable effort to developing these new tools so as to better understand the involvement of oxidative stress in the development of various diseases. As compared to standard methods, these methods may provide more information regarding the cause of the oxidative stress and the specific type of organ confronted with AOS overproduction.

One of the most recent and also most exciting developments in the field of oxidative stress is the discovery that the expression of genes and proteins is regulated differentially in the presence of oxidative stress. The real-time polymerase chain reaction technique (RT-PCR) involves producing the cDNA corresponding to the gene of interest by reverse transcription of cellular RNA (in the presence of reverse transcriptase) and then amplifying the cDNA with the help of two primers and a polymerase. Detection of amplification is done in real time by a specific fluorescent probe. More recently, DNA chips (microarrays) have made their appearance. They offer the advantage of enabling us to visualise, all at once, differences of expression between hundreds of genes coding for oxidative stress proteins (62), on the scale of the whole human genome. The chip is made by depositing on a glass slide cDNA fragments (corresponding to the genes to be studied) amplified beforehand by PCR. The cDNA is then denatured to single-stranded DNA, so that it will hybridise with a complementary strand in the probe or studied target (cells, pieces of tissue, etc.). From cells subjected or not (controls) to oxidative stress, RNA is extracted and converted to cDNA by reverse transcriptase. The cDNA of control cells is labelled with a green fluorescent dye and that of cells having undergone oxidative stress is labelled with a red fluorochrome. The hybridisation step consists in mixing the DNA of the two types of cells and placing them on the chip. The chip is incubated overnight at 42°C to enable a cDNA of the probe to hybridise with its complementary strand deposited on the chip and thus form double-stranded DNA. Then scanning of the slide provides fluorescence intensity values corresponding to the red and green signals. The red-to-green signal ratio will be 1 if the gene of interest is equally expressed under the test and control conditions. If the value is higher than 1, this means that the gene is overexpressed under the test conditions. If the value is lower than one, then the gene is underexpressed in the presence of oxidative stress (Figure 27).

Figure 27 : Principle of DNA chips

VII. Conclusions

It is currently accepted that AOS cause major cell damage that may impair organ function. In this perspective, oxidative stress is increasingly implicated in the ageing process, in the appearance of clinical complications, and in the development of age-associated diseases (atherosclerosis, cancer, neurodegenerative diseases). An important field of investigation is thus opening in the area of oxidative stress, its diagnosis, and therapies for limiting its harmful effects. If well applied, this new discipline should revolutionise tomorrow's medicine and have a major economic impact in terms of healthcare. For this it is necessary to have specific, high-performance tools. Whereas the first laboratory studies of oxidative stress were based solely on the determination of malondialdehyde as an in vivo marker of lipid peroxidation, the past five years have seen a real explosion of methods for the routine clinical evaluation of an individual's oxidative stress status (OSS). Each method without exception has its limitations, and hence cannot alone reflect an OSS. It will thus be necessary to use batteries of tests, but since oxidative stress has different manifestations according to the physiological or pathological condition considered, the tests must be chosen accordingly. In this perspective, PROBIOX SA has patented oxidative stress profiles adapted to specific situations. In addition, the researchers of PROBIOX SA pay special attention to the procedures used to take blood samples and and process them, because most of the molecules to be assayed are unstable. Centrifuging the blood samples immediately after they are taken and also maintaining a cold chain for plasmas and sera up to the time of analysis are two (not unmanageable) constraints that constitute the main guarantees of quality results (see the PROBIOX Charter of Quality). They make possible the best possible interpretation of results on the basis of over 20 years' experience in the field of oxidative stress.

The research tool described here will, for example, enable healthcare professionals to detect any abnormal antioxidant concentrations in their patients and to correct them by means of a better diet or appropriate supplements. What's more, the genomics and proteomics research in progress in the R&D department of PROBIOX SA will change considerably our understanding of oxidative stress and should lead to improved, individualised corrections making it possible to reduce even more effectively the harmful effects of oxidative stress.

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