**5.2. Lipid peroxidation**

30 Lipid Metabolism

pathological processes in the body.

destructions also without O2 activation.

considered that SOD itself is a stress protein.

**5. The effects of reactive oxygen species** 

**5.1. Oxygen free radicals – Intracellular messengers** 

of lipid peroxides [2, 11].

The objective of various experimental models was to study erythrocyte response to oxidant substances action. Erythrocyte characteristics and test substance dosing allowed the evaluation of OS; these experiments can be extrapolated to explain various physiological or

Oxidative stress is an ongoing process in the body, and under physiologic conditions there are effective mechanisms that negate its effects, thus high concentration of erythrocyte GSH

Erythrocyte congenital enzyme deficiencies confer erythrocytes an increased sensitivity to OS.

A section of the body intensely studied to assess OS is the liver due to its role in the metabolism of a wide range of endogenous and exogenous products. Thus, by the metabolism of aromatic compounds, drugs or carcinogenic hydrocarbons in the live, a large

Liver antioxidant systems are represented by SOD, GSH and dependent enzymes (transferase and peroxidase). Using ESR and spin trapping, FR resulting from chemical pollutants metabolism were identified, and a strong correlation between the functional impairment of the hepatic parenchyma, free radicals formation and decrease in GSH was noted. Under these conditions, free radicals of that substance occur which can cause tissue

The experimental poisoning of rats with alcohol (1.5 mmol/kg) showed significant decrease at one hour of ingestion of GSH, vitamin E and C along with hepatic necrosis and formation

GSH is an important protective factor against OS. Its level is interrelated with other

*Stress proteins*. Structurally altered intracellular protein group, whose synthesis is induced by oxidative stress has been named stress proteins group. Of particular interest is the 32 kDa protein whose synthesis is induced by the action of ionizing radiation, hydrogen peroxide. This protein is a marker of generalized response to oxidative stress. Free radicals affect cytokines (endogenous pyrogens: IL-1, IL-2, TNF-a, IFN) that play a role in regulating signal transmission in response to stress which will cause the synthesis of these proteins. It is

The tissue repair process is enzymatically catalyzed (repair enzymes) that break down damaged cellular particles, take intact aminoacids to synthesize new defense proteins.

Before discussing the negative effect of oxygen activation on the body, we should also take into consideration their involvement in certain physiological processes, when these ROS are

antioxidants (vitamin C and E) that stimulate its preservation in reduced form.

amount of FR occurs, which will initiate in the next phase OS from this level.

and related enzyme equipment provide a defense against ROS.

Formation of peroxides, especially lipid ones, is a consequence of the activation of O2, the interconversion of reactive species and natural systems protection overcoming. In biological environments, the most favorable substrate for peroxidation is represented by polyunsaturated fatty acids (PUFA), components of cell and subcellular membranes.

Peroxidation is a complex process that includes three phases: initiation, propagation, end-decomposition, which interpose, so that only end products can be determined chemically: aldehydes (malondialdehyde), polymerized carbonyl compounds (lipofuscin) [9].

A radical character initiator (which may have different structures and origins, including peroxy ROO. radicals) removes a hydrogen atom from polyunsaturated fatty acid diallyl carbon, forming a favorable reactive center for oxygen action. The peroxy ROO. radicals which become hydroperoxides result. In fact, due to side reactions, other locations of the peroxide group per PUFA molecule occur [28].

### **5.3. Cell structural alterations**

Since the formation of peroxides and their decomposition products, the sequence of reactions passes from a molecular level to a cellular one due to structural changes that occur in membranes: structural disorganization of the membrane and deterioration of pores crossing the double phospholipid layers. Peroxidation leads to changes in fatty acid qualitative composition of phospholipids composition with changing the ratio between PUFA and other acids. The first two effects induce the third, which consists in a decrease in membrane fluidity and altered active ion transport; these effects finally lead to changes in ion and other intracellular compounds concentration [26].

Numerous experimental studies have shown that tissue injury caused by free radicals determined at one point an imbalance of Ca2+ (i.e., increases in intracellular Ca2+ concentration). Under physiological conditions, there are effective homeostatic mechanisms (enzyme systems, protein transporters) to keep an optimum ratio between intracellular (0.1- 0.4 microM) and extracellular of the mM order concentration. Overcoming these mechanisms (in this case by producing free radicals) determines the accumulation of

#### 32 Lipid Metabolism

calcium in the cell which will lead to structural membrane alterations, production of unsaturated lipids, efflux of GSH, its transition to an oxidized form and the creation of an intracellular oxidative potential [21, 23].

Oxidative Stress and Lipid Peroxidation – A Lipid Metabolism Dysfunction 33

At an intracellular level (especially in muscles and neurons), deposits of lipofuscin

These deposits are mainly localized in the myocardium, brain, and, by the age of 80, they

Experimental studies demonstrated that in 50 years a person accumulates 13.4 mg/lipofuscin/gram of myocardium, pigment formation taking place once with exceeding

There is an inversely proportional relationship between the formation of these products and

To control the effects of aging, ones requires a moderate diet, which reduces metabolic rate and O2 consumption with an optimal concentration of lipids and a quantitatively and qualitatively balanced intake of antioxidants and other factors that enhance assimilation and their metabolism. It is also necessary to achieve a balanced interaction of endogenous

The antioxidants level varies greatly depending on the age of the body, that organ and subcellular components; thus an increase of GSH-Px activity was noted in mitochondria of cardiac cells and erythrocytes in the elderly, and a decrease of activity in liver and kidneys. The decrease of SOD activity in the liver of the elderly was highlighted and no significant changes in the concentration of intramitochondrial SOD in the heart were

Also, there is a correlation between the intensity of DNA destructions caused by FR and xanthine oxidase concentration. This enzyme, present in low concentrations, in tissue or

Free radicals are responsible for the inactivation of enzymes especially of serine proteases, the fragmentation of macromolecules (collagen, proteoglycans, hyaluronic acid), the formation of dimers, the protein aggregates in the cytoplasmic membranes. The most susceptible amino acids to their action are tryptophan, tyrosine, phenylalanine, methionine

Transition metal ions (Fe, Cu, Ni, Co, Cd) have a pro-oxidant action by intensifying reactions in which FR are formed and those in which the decomposition of lipid peroxides takes place. At the molecular level, Fe2+ ion contributes to the induction of oxidative stress by increasing non-enzymatic oxidation of catecholamines and GSH, promoting lipid peroxide decomposition and the formation of the most toxic free radicals, the hydroxyl radical. Fe2+, under complexed form as transferrin, is inactive against peroxides. Fe2+ release from transferrin takes place under pH decrease as it does

represent 70% of cytoplasmic volume of neurons and 6% of that of myocardiocytes.

pigments, lipid peroxides and their breakdown products are formed.

the absorption of 0.6 free radicals micromoles/gram of tissue.

the concentration of vitamin E in the body.

plasma, increases under tissue injury.

**5.5. Effects on molecules** 

antioxidants.

noted.

and cysteine.

Experimental studies on isolated hepatocytes have shown the correlation between the cellular toxicity of calcium and the decrease of tocopherols levels, substances with strong antioxidant character.

Maintaining the cell functional state ultimately depends on the level of proteins containing SH groups. Thus, the role of GSH in protection against oxidative stress is precisely regeneration of protein SH groups which in turn will ensure intracellular calcium homeostasis. Vitamin E stabilizes ATPase activity dependent of calcium in the endoplasmic reticulum by maintaining SH groups in the structure of the enzyme in reduced state. Also, vitamin E is protective against the compounds resulting from lipid peroxidation: a molecule of alpha-tocopherol protects against 500 molecules of polyunsaturated fatty acids.

#### **5.4. DNA destruction**

The results of chromatographic technique used to determine the urinary excretion products resulting from scission of DNA in humans showed a normal excretion in average of 100 nmol products. This total represents 103 thymine molecules oxidized per day for each of the 6X1013 cells in the body.

Between eliminating these products and the specific metabolic rate (SMR) there is a linear correlation.

The specific metabolic rate of an organism is dependent on the O2 use rate by its tissues and it is proportional to the free radicals production rate. In this case, the ratio between the total concentration of antioxidants (enzymatic and non-enzymatic systems) and the metabolic rate represents the protection degree of a tissue or body to free radicals. It seems that there is a genetic programming of the metabolic rate for each species and individual.

Looking at the hypothesis on free radicals involvement in aging, it has been shown that there is an inversely proportional relationship between the metabolic rate, free radicals production, respectively, and he maximum lifespan potential (MLP). Thus, on the evolutionary scale, metabolic rate decreased and lifespan increased, in mammals their product being constant.

One can calculate the lifespan energy potential (LEP), expressed in kcal-kg as follows: LEP 2.70 X MLP X SMR. This potential is directly proportional to the total concentration of antioxidants.

During aging, the formation of free radicals amplifies by exposure to prooxidant factors from the environment, and by the decreased antioxidant defense capacity.

At an intracellular level (especially in muscles and neurons), deposits of lipofuscin pigments, lipid peroxides and their breakdown products are formed.

These deposits are mainly localized in the myocardium, brain, and, by the age of 80, they represent 70% of cytoplasmic volume of neurons and 6% of that of myocardiocytes.

Experimental studies demonstrated that in 50 years a person accumulates 13.4 mg/lipofuscin/gram of myocardium, pigment formation taking place once with exceeding the absorption of 0.6 free radicals micromoles/gram of tissue.

There is an inversely proportional relationship between the formation of these products and the concentration of vitamin E in the body.

To control the effects of aging, ones requires a moderate diet, which reduces metabolic rate and O2 consumption with an optimal concentration of lipids and a quantitatively and qualitatively balanced intake of antioxidants and other factors that enhance assimilation and their metabolism. It is also necessary to achieve a balanced interaction of endogenous antioxidants.

The antioxidants level varies greatly depending on the age of the body, that organ and subcellular components; thus an increase of GSH-Px activity was noted in mitochondria of cardiac cells and erythrocytes in the elderly, and a decrease of activity in liver and kidneys. The decrease of SOD activity in the liver of the elderly was highlighted and no significant changes in the concentration of intramitochondrial SOD in the heart were noted.

Also, there is a correlation between the intensity of DNA destructions caused by FR and xanthine oxidase concentration. This enzyme, present in low concentrations, in tissue or plasma, increases under tissue injury.

#### **5.5. Effects on molecules**

32 Lipid Metabolism

antioxidant character.

polyunsaturated fatty acids.

**5.4. DNA destruction** 

6X1013 cells in the body.

product being constant.

antioxidants.

correlation.

intracellular oxidative potential [21, 23].

calcium in the cell which will lead to structural membrane alterations, production of unsaturated lipids, efflux of GSH, its transition to an oxidized form and the creation of an

Experimental studies on isolated hepatocytes have shown the correlation between the cellular toxicity of calcium and the decrease of tocopherols levels, substances with strong

Maintaining the cell functional state ultimately depends on the level of proteins containing SH groups. Thus, the role of GSH in protection against oxidative stress is precisely regeneration of protein SH groups which in turn will ensure intracellular calcium homeostasis. Vitamin E stabilizes ATPase activity dependent of calcium in the endoplasmic reticulum by maintaining SH groups in the structure of the enzyme in reduced state. Also, vitamin E is protective against the compounds resulting from lipid peroxidation: a molecule of alpha-tocopherol protects against 500 molecules of

The results of chromatographic technique used to determine the urinary excretion products resulting from scission of DNA in humans showed a normal excretion in average of 100 nmol products. This total represents 103 thymine molecules oxidized per day for each of the

Between eliminating these products and the specific metabolic rate (SMR) there is a linear

The specific metabolic rate of an organism is dependent on the O2 use rate by its tissues and it is proportional to the free radicals production rate. In this case, the ratio between the total concentration of antioxidants (enzymatic and non-enzymatic systems) and the metabolic rate represents the protection degree of a tissue or body to free radicals. It seems that there is

Looking at the hypothesis on free radicals involvement in aging, it has been shown that there is an inversely proportional relationship between the metabolic rate, free radicals production, respectively, and he maximum lifespan potential (MLP). Thus, on the evolutionary scale, metabolic rate decreased and lifespan increased, in mammals their

One can calculate the lifespan energy potential (LEP), expressed in kcal-kg as follows: LEP 2.70 X MLP X SMR. This potential is directly proportional to the total concentration of

During aging, the formation of free radicals amplifies by exposure to prooxidant factors

a genetic programming of the metabolic rate for each species and individual.

from the environment, and by the decreased antioxidant defense capacity.

Free radicals are responsible for the inactivation of enzymes especially of serine proteases, the fragmentation of macromolecules (collagen, proteoglycans, hyaluronic acid), the formation of dimers, the protein aggregates in the cytoplasmic membranes. The most susceptible amino acids to their action are tryptophan, tyrosine, phenylalanine, methionine and cysteine.

Transition metal ions (Fe, Cu, Ni, Co, Cd) have a pro-oxidant action by intensifying reactions in which FR are formed and those in which the decomposition of lipid peroxides takes place. At the molecular level, Fe2+ ion contributes to the induction of oxidative stress by increasing non-enzymatic oxidation of catecholamines and GSH, promoting lipid peroxide decomposition and the formation of the most toxic free radicals, the hydroxyl radical. Fe2+, under complexed form as transferrin, is inactive against peroxides. Fe2+ release from transferrin takes place under pH decrease as it does

#### 34 Lipid Metabolism

in hypoxia, leukocyte activation or in muscle tissue during strenuous physical exercise. Another source of free Fe2+ is represented by hemoglobin, which at low concentrations acts as a pro-oxidant favoring PUFA peroxidation. Proteins that bind Fe2+ have a different action: thus, ferritin has a pro-oxidant capacity, while hemosiderin and lactoferrin are antioxidants.

Oxidative Stress and Lipid Peroxidation – A Lipid Metabolism Dysfunction 35

Highly reactive aldehydes are one of the major causative factors in oxidative related cardiovascular pathology and ageing. Specific aldehydes (e.g., 4-hydroxynonenal acetaldehyde, acrolein) were reported to be transiently increased in the settings of heart failure and ischemia-reperfusion injury [13] and to interfere with transcriptional regulation of endogenous anti-oxidant networks in mitochondria [1]. Recently, accumulation of reactive aldehydes was studied from the point of view of the subsequent protein

On the other hand, decreased antioxidant defense further contributes to the oxidative damage. Low concentration of GSH-peroxidase in the vascular wall creates conditions favorable to the actions of hydrogen peroxide and other FR on lipids and lipoproteins [28]. In physiological conditions, nitric oxide acts as an antioxidant, inhibiting LDL peroxidation and their destructive effect on interstitial proteoglycans. With the increased production of FR, NO may become a prooxidant factor, stimulating LDL peroxidation by a mechanism involving myoglobin. Deficiency of other protective factors will favor oxidative injury. Lipid-soluble antioxidants such as tocopherols and ubiquinol are present in the hydrophobic environment of the lipoproteins in order to protect PUFA from FR attack. *In vitro* experimental data showed that: i) exposure of LDL to oxidative stress will trigger lipid peroxidation only after the loss of its above mentioned antioxidants and ii) enrichment of LDL with vitamin E will make LDL oxidation more

Accordingly, the beneficial role of antioxidant supplementation has been extensively investigated in the past decades in a variety of animal models. Most investigators reported beneficial effects, i.e., prevention of atherosclerotic lesions with vitamin E supplementation, yet an early study by Keaney et al. mentioned a deleterious effect of high doses of tocopherol on endothelial-dependent relaxation in cholesterol fed rabbits [11]. Unfortunately, despite the promising observational experimental data, several prospective, double-blind, placebo-controlled trials did not support a causal relationship between vitamin C and E supplementation and a lower risk of coronary heart disease [21]. Similarly, lack of beneficial effect with long term vitamin E supplementation was recently reported in large clinical trial (the Women's Health Study) that addressed the role of antioxidant

These negative results may be related to the fact that antioxidant supplements could abolish the physiological role of ROS as signaling molecules [18], especially when considering that most cardiovascular patients are treated with "pleiotropic" drugs such as statins, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, that besides their major effects are reported to reduce ROS formation [23]. Indeed, a large body of evidence demonstrated unequivocally that reduced amounts of reactive oxgen species, most probably of mitochondrial origin [17] but not exclusively, are essential in regulating cardiovascular homeostasis [19] as well as the powerful mechanisms of endogenous cardioprotection at postischemic reperfusion, namely pre- and

carbonylation and its implication in cardiovascular pathophysiology [4].

therapy in the primary prevention of heart failure [2].

difficult [6].

postconditioning [20].

Bilirubin, resulting from the metabolism of hemoglobin, as transition metal ions, causes alterations in the membrane structure by initiating PUFA peroxidation. Bilirubin crosses the blood-brain barrier, inhibits oxidative phosphorylation and decreases AMPc and GSH concentration. Thus, the encephalopathy caused by intense hemolytic jaundice in neonates is correlated with elevated levels of bilirubin, blood lipid peroxides and GSH decrease.

The same changes were observed in hepatitis of various etiologies (viral, ethanolic) and were correlated with graded morphological changes of the steatosis type, up to the irreversible ones, cirrhosis, caused by exceeding the protective antioxidant systems.

The bilirubin has an antioxidant effect, enhanced by binding to albumin, its plasma transport form. This different behavior of bilirubin depends on the concentration and the environment, like ascorbic acid, which features a pro- and antioxidant character, widely accepted today.
