**2. Homocysteine and red blood cells**

#### **2.1. Red blood cells oxidative-reducing balance**

Red blood cells are responsible for oxygen transport from lung to tissues. Their function depends on reduced state of iron (Fe2+) in hemoglobin (Hb). Both element oxygen and iron are able to quickly shift their oxidative state in response to different emerging stimuli. Literature shows in [1] that hemoglobin may undergo oxidative reaction in the process of releasing oxygen. In this process iron is oxidized to Fe3+ and reactive oxygen species are generated. The bigger the reactive species release is the higher normal deformability and flexibility of erythrocytes is disturbed.

#### *2.1.1. Reactive species generation*

In erythrocyte are found together oxygen (O2), nitrogen oxide (NO) and iron (Fe2+). All these elements act in normal condition in well established mechanisms but they may generate alone or together reactive species, named radicals, that damage red blood cells as well as vascular endothelium.

A radical is a chemical species that possesses a single unpaired electron in outer orbitals, and is able to independently exist, known also as free radical. Radicals are highly reactive in extracting an electron from any neighbor molecule in order to complete theirs own orbitals. There are two main groups of free radicals: ROS or reactive species of oxygen, RNS or reactive nitrogen species. ROS and RNS can act together damaging cells and causing nitrosactive stress. Therefore, these two species are often collectively referred to as ROS/RNS.

Transitional metals (Fe belongs to this group) particularly behave. They have a single unpaired electrons in theirs outer orbitals, but they don't behave as free radicals because within cells they are attached to proteins in most cases. However they are able to catalyze electron transfer in many processes and sometimes generate radicals.

#### *2.1.2. Reactive oxygen species*

32 Blood Cell – An Overview of Studies in Hematology

protein targeting.

properties

not mutually exclusive; oxidative processes, decreasing NO bioavailability and specific

The already specified free radicals are not all "bad". NO● can be regarded as a "good radical" but it is inactivated by many Hcy-dependent Mechanisms that finally impair its vasodilatatory function. Thus damaging Hcy effects expands to the environment where erythrocytes move and act. As a consequence directly and indirectly Hcy has a big impact on erythrocytes whose deformability in shear stress is crucial for circulatory function. Taken into account all these factors pharmacological approaches envisage lowering homocysteine levels by different ways such as: vitamins B supplementation, antioxidant drugs, hypotensive agent, antithrombotic drugs etc. Some of these patterns such is vitamins supplementation proved to have limited clinical benefits while others as nitrite/nitrate are still in debates. Because most pathological processes mentioned above involve oxidative pathway mechanism, pharmacological presentation will focus on drugs with antioxidant

As a conclusion the effect of elevated homocysteine appears multifactorial affecting both the

Red blood cells are responsible for oxygen transport from lung to tissues. Their function depends on reduced state of iron (Fe2+) in hemoglobin (Hb). Both element oxygen and iron are able to quickly shift their oxidative state in response to different emerging stimuli. Literature shows in [1] that hemoglobin may undergo oxidative reaction in the process of releasing oxygen. In this process iron is oxidized to Fe3+ and reactive oxygen species are generated. The bigger the reactive species release is the higher normal deformability and

In erythrocyte are found together oxygen (O2), nitrogen oxide (NO) and iron (Fe2+). All these elements act in normal condition in well established mechanisms but they may generate alone or together reactive species, named radicals, that damage red blood cells as

A radical is a chemical species that possesses a single unpaired electron in outer orbitals, and is able to independently exist, known also as free radical. Radicals are highly reactive in extracting an electron from any neighbor molecule in order to complete theirs own orbitals. There are two main groups of free radicals: ROS or reactive species of oxygen, RNS or reactive nitrogen species. ROS and RNS can act together damaging cells and causing nitrosactive stress. Therefore, these two species are often collectively referred to as

vascular wall structure, function as well as erythrocytes metabolism.

**2. Homocysteine and red blood cells** 

flexibility of erythrocytes is disturbed.

*2.1.1. Reactive species generation* 

well as vascular endothelium.

ROS/RNS.

**2.1. Red blood cells oxidative-reducing balance** 

Reactive species of oxygen refers to a group of highly reactive O2 metabolites, including superoxide anion (O2•-), hydrogen peroxide (H2O2), singlet oxygen (1O2), and hydroxyl radical (OH•), that can be formed within cells. Reactive oxygen species are constantly formed as byproducts in normal enzymatic reaction in all human cells through normal aerobic processes as mitochondrial oxidative phosphorylation or as necessary products in neutrophils in order to kill invading pathogens. The above mentioned phenomena are consuming oxygen processes.

Erythrocytes must save oxygen for delivering it to the cells; as a consequence red blood cells lack mitochondrion the main oxygen consumer within cell. In this particular condition the source of ROS in erythrocyte may be the carried oxygen itself.

In order to understand oxygen behavior an inside in its structure is needed. The ground state of oxygen is triplet oxygen meaning that the molecule has two unpaired electrons occupying two different molecular orbitals.

**Figure 1.** The structure of oxygen molecule

These electrons can't travel both in the same orbital because they have parallel spin (they spin in the same direction). As a consequence molecular oxygen is paramagnetic and from this feature it was concluded that the structure in the right may be assigned to O2 (figure1). Although O2 is very reactive from thermodynamic standpoint its single electrons cannot react rapidly with already paired electrons in the covalent bond of organic molecules (abundant in living cells). As a consequence it is harmless to these molecules. Instead molecular oxygen can rapidly react with single unpaired electrons from transitory metals (e.g. Fe, Cu, Mn). One mole of properly chelated cooper could catalyze consumption of all of the oxygen in an average room within one second in [2].

In fact oxygen O2 is both kinetically stable thus not reactive and very reactive promoting fast reactions, depending the surrounding conditions.

Within cells where transitional metals are bounded to proteins (in metal containing proteins or enzymes) oxidative attack of O2 tend to be slow, meaning that a first single electron is relatively difficult to add. As a consequence superoxid radical (O2 ) will form very slow.

Homocysteine in Red Blood Cells Metabolism – Pharmacological Approaches 35

**)** and hydrogen

consequence it can diffuse through lipid membranes. No matter where it meets proteins (including hemoglobin) that contain transition metals Fe2+, Cu+, H2O2 generates OH• at the

Another reactive species (but not a radical) derived from molecular oxygen is singlet oxygen, designated as 1O2. Singlet oxygen (1O2), a highly excited state created when molecular oxygen absorbs sufficient energy to shift an unpaired electron to a higher orbital,

Singlet oxygen is even more reactive than the hydroxyl radical, although it is not a radical.

As a conclusion the most reactive radical is hydroxyl (OH •) which indiscriminately extracts

All the above reactions and processes take place in all human cells including erythrocytes. As for the other cells the main source for free radicals is mitochondrion, in the particular case of red blood cells the main source for radicals is the carried oxygen. Molecular oxygen is carried in order to be delivered to tissues and as a consequence it is found, for a short period of time, free, thus unbound. In this state it might be prone to generate the above described radicals.

Oxygen binds to hemoglobin at the ferrous iron. The ferrous state (Fe2+) of iron is a condition for hemoglobin normal function. However a small percent of Fe2+ is slowly converted by O2 to ferric form (Fe3+) in resulting methemoglobin. An enzymatic system, methemoglobin reductase quickly restores Fe3+ to Fe2+ and reduces methemoglobin back to hemoglobin. Binding of oxygen to the iron in the hem is considered not to change the oxidation state of the metal. However oxygenated hem has some of the electronic characteristics of a Fe3+OO peroxide anion [3]. Misra and Fridovich demonstrate that the Fe3+O2- complex is able to generate superoxide radical in [7] during the normal molecular oxygen transport to tissues through the hemoglobin auto-oxidation. Thus hemoglobin auto-oxidation causes superoxide

Other researchers show that hemoglobin may undergo oxidative reaction in the oxygen releasing process. Balagopalakrishna and coworkers demonstrate in [8] that at intermediate oxygen pressure, where hemoglobin partially releases molecular oxygen, the superoxide radical production increases. They show that superoxide radical is released in the hydrophobic hem pocket. The process in slow enough thus the formation of superoxide was followed for more than 15 min, and thus detected by low temperature electron

Being a radical superoxide reacts fast with other radicals or alternatively it is efficiently

reacts with itself generating molecular oxygen (O2) and hydrogen peroxide (H2O2)

gives birth to new reactive species as follows:

scavenged through the specific superoxide dismutase (SOD) activity.

+ 2H + H2O2 + 1O2

specific site where these metals are located thus damaging protein structure.

2 O2 

electrons from any other molecules around it whereas superoxide (O2

peroxide (H2O2) are more selective in their reactions with biological molecules [6].

can be formed from superoxide radical in [5]:

formation within erytrocyte.

paramagnetic resonance technique.

When collides with other radicals O2

a source for hydroxyl radical.


**Figure 2.** Superoxide radical generation

Once an electron acquired additional electrons are easier added, further reactions quickly occur in [3] and reactive species of oxygen are generated.

**Figure 3.** Reactive oxygen species generation

Superoxide (O2 ) is formed when molecular oxygen (O2) gains an additional electron, producing a molecule with only one unpaired electron (figure 2), generating a very reactive free radical. When accepts an electron, superoxide is reduced to hydrogen peroxide, which is not a radical. In the next one-electron reduction step hydrogen peroxide generates water and the hydroxyl radical (OH ) which is probably the most reactive free radical. A final electron acceptance (figure 3) reduces hydroxyl radical to water in [4].

Superoxide radical (O2 ) is a reactive radical, however it cannot diffuse to far having limited lipid solubility. Instead it might react in the presence of ferric iron with de hydrogen peroxide generating the most potent hydroxyl radical through a non-enzymatic reaction known as Haber-Weiss reaction.

The reaction takes place in two steps that involved ferric iron and superoxide as follows:

$$\begin{aligned} \mathrm{Oz}^{\bullet \ \bullet \ \bullet} + \mathrm{Fe^{3+}} &\rightarrow \mathrm{Oz} + \mathrm{Fe^{2+}} \\\\ \mathrm{Fe^{2+}} + \mathrm{HzOz} &\rightarrow \mathrm{Fe^{3+}} + \mathrm{OH^{-}} + \mathrm{OH^{-}} \end{aligned}$$

The general non-enzymatic reaction occurring in living cells is:

$$\bullet \bullet \bullet \bullet \bullet \bullet \bullet \bullet \bullet \bullet \bullet \bullet \bullet \bullet$$

It appears that superoxide radical is also a source of hydroxyl radical in ferric iron presence.

Hydrogen peroxide (H2O2) is not a free radical but still classified as a ROS because it generates the most powerful hydroxyl radical (OH •) in the presence of transitional metals (Fe2+, Cu+), the reaction above. Hydrogen peroxide (H2O2) is lipid soluble and as a consequence it can diffuse through lipid membranes. No matter where it meets proteins (including hemoglobin) that contain transition metals Fe2+, Cu+, H2O2 generates OH• at the specific site where these metals are located thus damaging protein structure.

34 Blood Cell – An Overview of Studies in Hematology

**Figure 2.** Superoxide radical generation

**Figure 3.** Reactive oxygen species generation

Superoxide (O2

Superoxide radical (O2

known as Haber-Weiss reaction.

occur in [3] and reactive species of oxygen are generated.

Once an electron acquired additional electrons are easier added, further reactions quickly

producing a molecule with only one unpaired electron (figure 2), generating a very reactive free radical. When accepts an electron, superoxide is reduced to hydrogen peroxide, which is not a radical. In the next one-electron reduction step hydrogen peroxide generates water and the hydroxyl radical (OH ) which is probably the most reactive free radical. A final

limited lipid solubility. Instead it might react in the presence of ferric iron with de hydrogen peroxide generating the most potent hydroxyl radical through a non-enzymatic reaction

The reaction takes place in two steps that involved ferric iron and superoxide as follows:

+ Fe3+ → O2 + Fe2+

Fe2+ + H2O2 → Fe3+ + OH <sup>−</sup> + OH•

+ H2O2 → OH • + OH − + O2

It appears that superoxide radical is also a source of hydroxyl radical in ferric iron presence.

Hydrogen peroxide (H2O2) is not a free radical but still classified as a ROS because it generates the most powerful hydroxyl radical (OH •) in the presence of transitional metals (Fe2+, Cu+), the reaction above. Hydrogen peroxide (H2O2) is lipid soluble and as a

electron acceptance (figure 3) reduces hydroxyl radical to water in [4].

O2

The general non-enzymatic reaction occurring in living cells is:

O2

) is formed when molecular oxygen (O2) gains an additional electron,

) is a reactive radical, however it cannot diffuse to far having

Another reactive species (but not a radical) derived from molecular oxygen is singlet oxygen, designated as 1O2. Singlet oxygen (1O2), a highly excited state created when molecular oxygen absorbs sufficient energy to shift an unpaired electron to a higher orbital, can be formed from superoxide radical in [5]:

$$\text{2O2} \bullet \text{''} \bullet \text{2H} \text{'} \rightarrow \text{H2O2} + \text{1O2}$$

Singlet oxygen is even more reactive than the hydroxyl radical, although it is not a radical.

As a conclusion the most reactive radical is hydroxyl (OH •) which indiscriminately extracts electrons from any other molecules around it whereas superoxide (O2 **)** and hydrogen peroxide (H2O2) are more selective in their reactions with biological molecules [6].

All the above reactions and processes take place in all human cells including erythrocytes. As for the other cells the main source for free radicals is mitochondrion, in the particular case of red blood cells the main source for radicals is the carried oxygen. Molecular oxygen is carried in order to be delivered to tissues and as a consequence it is found, for a short period of time, free, thus unbound. In this state it might be prone to generate the above described radicals.

Oxygen binds to hemoglobin at the ferrous iron. The ferrous state (Fe2+) of iron is a condition for hemoglobin normal function. However a small percent of Fe2+ is slowly converted by O2 to ferric form (Fe3+) in resulting methemoglobin. An enzymatic system, methemoglobin reductase quickly restores Fe3+ to Fe2+ and reduces methemoglobin back to hemoglobin. Binding of oxygen to the iron in the hem is considered not to change the oxidation state of the metal. However oxygenated hem has some of the electronic characteristics of a Fe3+OO peroxide anion [3]. Misra and Fridovich demonstrate that the Fe3+O2- complex is able to generate superoxide radical in [7] during the normal molecular oxygen transport to tissues through the hemoglobin auto-oxidation. Thus hemoglobin auto-oxidation causes superoxide formation within erytrocyte.

Other researchers show that hemoglobin may undergo oxidative reaction in the oxygen releasing process. Balagopalakrishna and coworkers demonstrate in [8] that at intermediate oxygen pressure, where hemoglobin partially releases molecular oxygen, the superoxide radical production increases. They show that superoxide radical is released in the hydrophobic hem pocket. The process in slow enough thus the formation of superoxide was followed for more than 15 min, and thus detected by low temperature electron paramagnetic resonance technique.

Being a radical superoxide reacts fast with other radicals or alternatively it is efficiently scavenged through the specific superoxide dismutase (SOD) activity.

When collides with other radicals O2 gives birth to new reactive species as follows:


	- O2 reacts fast with NO• radical generating toxic peroxinitrite (ONOO−), a reactive nitrogen specie (RNS). At physiological pH, ONOO− rapidly protonates to peroxynitrous acid, ONOOH. This powerful oxidizing and nitrating agent can directly damage proteins and lipids.

Homocysteine in Red Blood Cells Metabolism – Pharmacological Approaches 37

ROS are considered "bad" radicals because can they indiscriminately interact with any biological molecule they meet causing DNA, lipids and protein damage. As a missing nucleus cell erythrocyte can undergoes the last two lesional processes but endothelium can undergoes all three mentioned injuries. Erythrocytes are particularly affected by oxidation of polyunsaturated fatty acids in membrane phospholipids which causes their peroxidation, degradation and fragmentation [15]. During lipid peroxidation other reactive species as peroxyl radicals are generated in a succession of chain reactions (fig.5). This reactive

**Figure 5.** Lipid peroxidation and radical and non-radical intermediates formation [taken from 15]

Erythrocytes also undergo the amino acids and/or whole proteins oxidation. Protein oxidation leads to inactivation (if targeted proteins are enzymes), fragmentation, aggregation of fragments and/or increased susceptibility to proteolysis [**15**]. In addition if injured proteins belong to erythrocytes skeleton the deformability of erythrocytes is impaired [16]. ROS attack doesn't limit to erythrocytes it also affect endothelium cells, which in turn influence erythrocyte metabolism. In nucleus containing endothelium cell beside lipids and proteins oxidation ROS cause DNA injuries. Free radicals can interact with DNA leading to strand breaks or structural changes such as adduct formation (figure 6) [15].

**Figure 6.** Over simplifying scheme of ROS damaging activity in erythrocytes and in endothelium cells

intermediates amplify the injury at the place were they are formed.

*2.1.3. Damaging effects of ROS* 

Thus, any system producing O<sup>2</sup> and NO• can cause biological damage, and erythrocytes make no exception in [6].

Hydrogen peroxide is not a radical because it doesn't have any unpaired electron. The limited reactivity of H2O2 allows it to cross membranes and to become widely dispersed. Even hydrogen peroxide is not a radical it can generates the short-lived but very active hydroxyl radicals via Harber-Weiss non-enzyme reaction in [9]. The hydroxyl radical (OH•), which is highly reactive, diffuses only a short distance before it reacts with whatever biomolecules it collides with. Recent study consider that the high and indiscriminate reactivity of the hydroxyl radical minimizes its ability to diffuse and makes it more damaging within cell or in the environment where it is generated in [10]. This consideration becomes more important when the oxidative events prevail within a specific cellular compartment. Hydroxyl radical are especially dangerous because it can initiate an autocatalytic radical chain reaction. Being so harmful cells carefully control hydroxyl radical by limiting the availability of both Fe2+ and H2O2 in [6].

The non-enzymatic decomposition of hydrogen peroxide described by Haber-Wiess and especially the mechanism through which hydroxyl radical (OH•) acts was highly debated.

Some researchers consider that hydroxyl radical is responsible for damaging cellular component on behalf of a radical mechanism. Others consider that ferryl ion (Fe(IV)O2+), an oxidizing species where iron is in high oxidation state (Fe IV) in [11,12] is an active intermediate responsible for chain reaction propagation. Another group consider that conditions inside cell dictates whether metallo-oxo species or hydroxyl radical (OH•) is the main oxidant [in 13]. Some other like Prousek concluded in [14] that both oxidising species can be formed in living cells.

As a general conclusion in erythrocytes ROS are produced both accidentally and physiologically in different enzyme-catalyzed reactions (figure4).

**Figure 4.** ROS generation in erythrocyte.

#### *2.1.3. Damaging effects of ROS*

36 Blood Cell – An Overview of Studies in Hematology

damage proteins and lipids.

by limiting the availability of both Fe2+ and H2O2 in [6].

physiologically in different enzyme-catalyzed reactions (figure4).

Thus, any system producing O<sup>2</sup>

make no exception in [6].

can be formed in living cells.

**Figure 4.** ROS generation in erythrocyte.

 reacts fast with NO• radical generating toxic peroxinitrite (ONOO−), a reactive nitrogen specie (RNS). At physiological pH, ONOO− rapidly protonates to peroxynitrous acid, ONOOH. This powerful oxidizing and nitrating agent can directly

Hydrogen peroxide is not a radical because it doesn't have any unpaired electron. The limited reactivity of H2O2 allows it to cross membranes and to become widely dispersed. Even hydrogen peroxide is not a radical it can generates the short-lived but very active hydroxyl radicals via Harber-Weiss non-enzyme reaction in [9]. The hydroxyl radical (OH•), which is highly reactive, diffuses only a short distance before it reacts with whatever biomolecules it collides with. Recent study consider that the high and indiscriminate reactivity of the hydroxyl radical minimizes its ability to diffuse and makes it more damaging within cell or in the environment where it is generated in [10]. This consideration becomes more important when the oxidative events prevail within a specific cellular compartment. Hydroxyl radical are especially dangerous because it can initiate an autocatalytic radical chain reaction. Being so harmful cells carefully control hydroxyl radical

The non-enzymatic decomposition of hydrogen peroxide described by Haber-Wiess and especially the mechanism through which hydroxyl radical (OH•) acts was highly debated.

Some researchers consider that hydroxyl radical is responsible for damaging cellular component on behalf of a radical mechanism. Others consider that ferryl ion (Fe(IV)O2+), an oxidizing species where iron is in high oxidation state (Fe IV) in [11,12] is an active intermediate responsible for chain reaction propagation. Another group consider that conditions inside cell dictates whether metallo-oxo species or hydroxyl radical (OH•) is the main oxidant [in 13]. Some other like Prousek concluded in [14] that both oxidising species

As a general conclusion in erythrocytes ROS are produced both accidentally and

and NO• can cause biological damage, and erythrocytes


ROS are considered "bad" radicals because can they indiscriminately interact with any biological molecule they meet causing DNA, lipids and protein damage. As a missing nucleus cell erythrocyte can undergoes the last two lesional processes but endothelium can undergoes all three mentioned injuries. Erythrocytes are particularly affected by oxidation of polyunsaturated fatty acids in membrane phospholipids which causes their peroxidation, degradation and fragmentation [15]. During lipid peroxidation other reactive species as peroxyl radicals are generated in a succession of chain reactions (fig.5). This reactive intermediates amplify the injury at the place were they are formed.

**Figure 5.** Lipid peroxidation and radical and non-radical intermediates formation [taken from 15]

Erythrocytes also undergo the amino acids and/or whole proteins oxidation. Protein oxidation leads to inactivation (if targeted proteins are enzymes), fragmentation, aggregation of fragments and/or increased susceptibility to proteolysis [**15**]. In addition if injured proteins belong to erythrocytes skeleton the deformability of erythrocytes is impaired [16]. ROS attack doesn't limit to erythrocytes it also affect endothelium cells, which in turn influence erythrocyte metabolism. In nucleus containing endothelium cell beside lipids and proteins oxidation ROS cause DNA injuries. Free radicals can interact with DNA leading to strand breaks or structural changes such as adduct formation (figure 6) [15].

**Figure 6.** Over simplifying scheme of ROS damaging activity in erythrocytes and in endothelium cells

## *2.1.4. ROS signaling*

Until recently reactive oxygen species were considered only oxidizing damaging factors. But it was demonstrated by in [**15, 17**] that ROS can be also "good" as they act as signaling molecules. In fact both authors show that ROS are neither "good" nor "bad", temporal length and intensity of free radicals generation make the difference between physiological, adaptive or pathological effect. Thus oxidizing molecules is not the end "of the road" for reactive species alternatively they trigger cellular responses which depending on the intensity of ROS attack, prepare the cell to survive or on contrary trigger cell death (figure 7).

Homocysteine in Red Blood Cells Metabolism – Pharmacological Approaches 39

in Ca2+concentration contributes to the oxidative stress-mediated activation of PKC and to

the transcriptional induction of the AP-1 proteins c-Fos and c-Jun [20] (figure 8).

**Figure 8.** Major pathways activated by ROS generation (modified from **21)**.MAPK=mitogen-activated protein, JNK=c-Jun N-terminal kinases, ERK=extracellular signal-regulated kinases, NFκB=nuclear factor κB, AP-1=activator protein-1, HIF-1= hypoxia-inducible factor-1, PKC=protein kinase C

More details about the cellular response in ROS and other radical and non-radicals species

Erythrocytes have an impressing antioxidant enzyme and non-enzyme system that deals with an important amount of free radicals. Superoxide dismutase and glutathione

approximation of catalytic efficiency) of any known enzyme (~7 x 109 M−1s−1) [22]. SOD catalyst activity is limited only by the frequency of collision with superoxide. That means the reaction rate is limited only by the diffusion of superoxid radical. Diffusion limitation

by catalyzing its dismutation, one O2

by SOD is very efficient having the largest kcat/KM (an

peroxidase are the most efficient antioxidant enzyme in red blood cells.

becomes canceled in radicals over production thus activating the process.

attack in oxidative events can be found in [15,17,19**]**

Erythrocytes superoxide dismutase remove O2

**Figure 9.** Superoxide dismutase activity

The dismutation of superoxide O2

being reduced to H2O2 and another oxidized to O2 (figure 9).

*2.1.5. Scavenging ROS* 

**Figure 7.** Cells response under ROS attack (taken from 17)

Being highly reactive ROS can intercept cell signaling pathways within successive steps in cascade events modulating the functions of many enzymes and transcription factors. Oxidative stress triggers cellular response by activating many signaling pathways. ROS can directly or indirectly modulate a) the function of different types of enzymes, b) the transcription factors activity and c) the activity of ion-channels.


in Ca2+concentration contributes to the oxidative stress-mediated activation of PKC and to the transcriptional induction of the AP-1 proteins c-Fos and c-Jun [20] (figure 8).

**Figure 8.** Major pathways activated by ROS generation (modified from **21)**.MAPK=mitogen-activated protein, JNK=c-Jun N-terminal kinases, ERK=extracellular signal-regulated kinases, NFκB=nuclear factor κB, AP-1=activator protein-1, HIF-1= hypoxia-inducible factor-1, PKC=protein kinase C

More details about the cellular response in ROS and other radical and non-radicals species attack in oxidative events can be found in [15,17,19**]**

#### *2.1.5. Scavenging ROS*

38 Blood Cell – An Overview of Studies in Hematology

Until recently reactive oxygen species were considered only oxidizing damaging factors. But it was demonstrated by in [**15, 17**] that ROS can be also "good" as they act as signaling molecules. In fact both authors show that ROS are neither "good" nor "bad", temporal length and intensity of free radicals generation make the difference between physiological, adaptive or pathological effect. Thus oxidizing molecules is not the end "of the road" for reactive species alternatively they trigger cellular responses which depending on the intensity of ROS

Being highly reactive ROS can intercept cell signaling pathways within successive steps in cascade events modulating the functions of many enzymes and transcription factors. Oxidative stress triggers cellular response by activating many signaling pathways. ROS can directly or indirectly modulate a) the function of different types of enzymes, b) the

a. Enzymes modulated by ROS include both kinases and phosphatases. The big class of kinase includes both tyrosine kinase as Src, Ras, JAK2, Pyk2, PI3K, and the mitogenactivated protein kinase (MAPK). The three best-characterized MAPK subfamilies are c-Jun N-terminal kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK) [18]. All these MAPK pathways are structurally similar, but functionally distinct. Importantly, ERK, JNK and p38 MAPK have all been shown to be activated by oxidative stress [19]. ERK and JNK are important in recruiting c-Fos and c-Jun to the nucleus where they activate the transcription factor AP—1 (activator protein -1), whereas activation of p38 and inhibitory kappa kinases (IKK) is important in the transcriptional activation of NF-κB. Both of these factors are important in regulating the diverse genes, which play key roles in the pathogenesis of inflammation, and in regulation of cell cycle, proliferation, and apoptosis. ROS may inhibit tyrosine

phosphatase activity further contributing to tyrosine kinase activation.

b. ROS also influence gene and protein expression by activating transcription factors, such as the already mention NFkB and activator protein-1 (AP-1) and hypoxia-inducible

c. ROS stimulate ion channels, such as plasma membrane Ca2+ and K+ channels, leading to changes in cation concentration. The cytosolic Ca2+ level can be increased by ROS in various cell types, including epithelium cell, through the mobilization of intracellular Ca2+stores and/or through the influx of extracellular Ca2+ [15]. The ROS-mediated increase

attack, prepare the cell to survive or on contrary trigger cell death (figure 7).

**Figure 7.** Cells response under ROS attack (taken from 17)

transcription factors activity and c) the activity of ion-channels.

*2.1.4. ROS signaling* 

factor-1 (HIF-1).

Erythrocytes have an impressing antioxidant enzyme and non-enzyme system that deals with an important amount of free radicals. Superoxide dismutase and glutathione peroxidase are the most efficient antioxidant enzyme in red blood cells.

Erythrocytes superoxide dismutase remove O2 by catalyzing its dismutation, one O2 being reduced to H2O2 and another oxidized to O2 (figure 9).

$$\text{H}\_2\text{O}\_2^- + 2\text{H}^+ \xrightarrow{\text{Superoxide dismutase}} \text{O}\_2 + \text{H}\_2\text{O}\_2$$

**Figure 9.** Superoxide dismutase activity

The dismutation of superoxide O2 by SOD is very efficient having the largest kcat/KM (an approximation of catalytic efficiency) of any known enzyme (~7 x 109 M−1s−1) [22]. SOD catalyst activity is limited only by the frequency of collision with superoxide. That means the reaction rate is limited only by the diffusion of superoxid radical. Diffusion limitation becomes canceled in radicals over production thus activating the process.

As seen in upper reaction (fig.9) superoxide dismutase must work with enzymes that remove H2O2.

Homocysteine in Red Blood Cells Metabolism – Pharmacological Approaches 41

Generated NO is a gaseous molecule with unpaired electrons and as a consequence a radical; it is lipophilic and diffuses rapidly through membranes. NO is a messenger in many physiological processes: endothelial relaxation of the smooth muscle, inhibition of platelet aggregation, neurotransmission and cytoxicity in [25]. NO pathology includes both low and high concentrations as follows: insufficient NO production is involved in hypertension, the activation of platelet aggregation and atherogenesis while high NO

NO released from endothelial cells diffuses through blood or to the underlining smooth muscle cells in the media where it triggers vasodilatation. In blood stream NO will affect

a. e-NOS can produce NO not only in normal oxygenation (figure 12) but also decreased oxygenation gradient across vascular branches. In addition eNOS can sometimes be a

b. endothelial cells are not the only ones able to generate NO, blood erythrocytes are

c. Hemoglobin itself also "produces" NO from nitrite in order to modulate

**a.** It is only recently found that endothelial NOS may be a source of ROS generating O2

Endothelial nitric oxide synthetase activity is regulated by a combination of mechanisms that allow eNOS to modulate its activity under physio-pathological condition in [22]. eNOS contains 2 enzymatic domains, a flavin-containing reductase and a heme-containing oxygenase domain (Fe3+) connected by a regulatory calmodulin-binding domain. Binding of the Ca2+/calmodulin complex orients the other domains in such a position that NADPHderived electrons generated on the reductase domain flow to the oxygenase domain in [26].

The oxygenase domain of eNOS contains an iron ion (Fe3+) that binds oxygen on reduction Fe2+, and this complex finally causes the conversion of L-arginine to NO and L-citrulline. This sequence of events properly rules if the cofactor BH4 "provides the connection"

production generates septic shock, stroke, and carcinogenesis.

depending the availability of its substrates within cell (figure 13) [23].

**Figure 12.** Nitric oxide generation

platelets, leucocytes and erythrocytes.

source of ROS generating O2

expressing functional NOS

Recent studies show that:

vasodilatation.

Glutathione peroxidase (GPx) removes H2O2 by coupling its reduction to water (figure10) with oxidation of reduced glutathione (GSH), a thiol-containing tripeptide (glu-cys-gly). The product, oxidized glutathione (GS-SG), consists of two GSH linked by a disulphide bridge, and can be converted back to GSH by glutathione reductase enzymes in [6].

$$\text{CH}\_2\text{O}\_2 + 2\text{GSH} \xrightarrow{\text{Glutathion\\_peroxidase}} \text{g}\text{SSSG} + 2\text{H}\_2\text{O}\_2$$

**Figure 10.** Glutathion peroxidase activity.

Beside enzyme antioxidant systems erythrocytes uses antioxidants agents (fig.4). These agents are preferentially oxidized by reactive species to preserve more important biomolecules and can be reversibly reduced back. For example, GSH and ascorbate can scavenge O2 **,**OH•, and also ONOOH. Tocopherols are good scavengers of peroxyl radicals and help to protect membranes against lipid peroxidation by interrupting the propagationchain reaction (figure 5) in [6].

#### *2.1.6. Reactive nitrogen species*

Nitrogen compounds found in the body comes from exogenous sources as nitrites/nitrates or from endogen production of nitric oxide (NO). The group of nitrogen derivatives includes:


$$\mathop{\text{NO}}\_{\mathop{\text{Niitate}}}^{\text{NO}} \underbrace{\mathop{\text{NO}}\_{\mathop{\text{Niitate}}}^{-}}\_{\mathop{\text{Niitate}}} \mathop{\text{NO}}\_{\mathop{\text{Niitice}}} \cdot \mathop{\text{NO}}\_{\mathop{\text{Niitice}}} \cdot \mathop{\text{O}}\_{\mathop{\text{Niitice}}} $$

#### **Figure 11.** Nitrogen derivatives

Nitrogen derivatives convert into each other forward and backward continuously under shifting conditions within cells (figure11).

Endogenous NO is synthesized by nitric oxide synthases (NOS) in the endothelial and other cells, where is involved in vascular physiology.

Endothelial nitric oxide synthases (eNOS) synthesizes NO (figure 12) from L-arginine with 1,5 consumption of 1.5 NADPH equivalents and two oxygen molecules per NO formed in [23]. The reaction requires the presence of Ca2+-Calmodulin and tetrahydrobiopterin (BH4) as cofactors in[24].

Homocysteine in Red Blood Cells Metabolism – Pharmacological Approaches 41

**Figure 12.** Nitric oxide generation

40 Blood Cell – An Overview of Studies in Hematology

**Figure 10.** Glutathion peroxidase activity.

chain reaction (figure 5) in [6].

*2.1.6. Reactive nitrogen species* 

vasodilatation in mammals

from nitrous acid HNO2)

**Figure 11.** Nitrogen derivatives

cofactors in[24].

shifting conditions within cells (figure11).

other cells, where is involved in vascular physiology.

remove H2O2.

scavenge O2

includes:


As seen in upper reaction (fig.9) superoxide dismutase must work with enzymes that

Glutathione peroxidase (GPx) removes H2O2 by coupling its reduction to water (figure10) with oxidation of reduced glutathione (GSH), a thiol-containing tripeptide (glu-cys-gly). The product, oxidized glutathione (GS-SG), consists of two GSH linked by a disulphide bridge,

Beside enzyme antioxidant systems erythrocytes uses antioxidants agents (fig.4). These agents are preferentially oxidized by reactive species to preserve more important biomolecules and can be reversibly reduced back. For example, GSH and ascorbate can

and help to protect membranes against lipid peroxidation by interrupting the propagation-

Nitrogen compounds found in the body comes from exogenous sources as nitrites/nitrates or from endogen production of nitric oxide (NO). The group of nitrogen derivatives


Nitrogen derivatives convert into each other forward and backward continuously under

Endogenous NO is synthesized by nitric oxide synthases (NOS) in the endothelial and

Endothelial nitric oxide synthases (eNOS) synthesizes NO (figure 12) from L-arginine with 1,5 consumption of 1.5 NADPH equivalents and two oxygen molecules per NO formed in [23]. The reaction requires the presence of Ca2+-Calmodulin and tetrahydrobiopterin (BH4) as


nitrogen dioxide or nitrite. In organism is found in its corresponding salts nitrites(

**,**OH•, and also ONOOH. Tocopherols are good scavengers of peroxyl radicals

and can be converted back to GSH by glutathione reductase enzymes in [6].

Generated NO is a gaseous molecule with unpaired electrons and as a consequence a radical; it is lipophilic and diffuses rapidly through membranes. NO is a messenger in many physiological processes: endothelial relaxation of the smooth muscle, inhibition of platelet aggregation, neurotransmission and cytoxicity in [25]. NO pathology includes both low and high concentrations as follows: insufficient NO production is involved in hypertension, the activation of platelet aggregation and atherogenesis while high NO production generates septic shock, stroke, and carcinogenesis.

NO released from endothelial cells diffuses through blood or to the underlining smooth muscle cells in the media where it triggers vasodilatation. In blood stream NO will affect platelets, leucocytes and erythrocytes.

Recent studies show that:


**a.** It is only recently found that endothelial NOS may be a source of ROS generating O2 depending the availability of its substrates within cell (figure 13) [23].

Endothelial nitric oxide synthetase activity is regulated by a combination of mechanisms that allow eNOS to modulate its activity under physio-pathological condition in [22]. eNOS contains 2 enzymatic domains, a flavin-containing reductase and a heme-containing oxygenase domain (Fe3+) connected by a regulatory calmodulin-binding domain. Binding of the Ca2+/calmodulin complex orients the other domains in such a position that NADPHderived electrons generated on the reductase domain flow to the oxygenase domain in [26].

The oxygenase domain of eNOS contains an iron ion (Fe3+) that binds oxygen on reduction Fe2+, and this complex finally causes the conversion of L-arginine to NO and L-citrulline. This sequence of events properly rules if the cofactor BH4 "provides the connection"

between the two domains. Deficiency of arginine or BH4 causes the reductase uncoupling from oxygenase. At the oxygenase domain intermediate Fe2+-O2 complex dissociates to form superoxide and the original Fe3+ group of the eNOS)[27]. Thus eNOS releases O2 instead of NO (figure 9).

Homocysteine in Red Blood Cells Metabolism – Pharmacological Approaches 43

To summarize NO can be produced by eNOS from either L-arginine in good oxygenation physiological state or from nitrite in hypoxia. In vitamins deficiency (low BH4 levels) eNOS produces superoxide radical. Recent studies demonstrate that endothelium cells surprisingly produce ROS under hypoxia. The primary site of reactive oxygen species production was demonstrated to be complex III in electron transport in mitochondria. The paradoxically increase in ROS production under low oxygenation is still not fully understood but it is considered that reactive oxygen species released during hypoxia act as signalling agents that trigger induction of erythropoietin, endothelial growth factor and glycolytic enzymes. Systemically, these responses enhance the delivery of O2 to cells and facilitate the production of glycolytic ATP instead of mitochondrion. Induction of these genes is mediated by "specialized" hypoxia inducible factor 1 (HIF-1) [33, 34]. As a conclusion in normal oxygenation NO is produced by eNOS. In hypoxia adaptive responses are onset; the release of NO from nitrite to sustain normal vascular function is one path. Alternatively when mitochondrion "senses" hypoxia it releases ROS as signaling molecules that activate diverse

functional responses, including activation of gene expression that promote cell survival.

observed on platelet aggregation when decreased NO levels promote thrombosis.

erythrocyte contribution to the regulation of vascular tonus

**Figure 16.** Oxyhemoglobin activity of scavenging the nitric oxide

considered to be limited only by the diffusion.

depending on its oxygen saturation, as follows:

**b.** Kleinbongard demonstrates in [35] that red blood cells express functional eNOS which is located in both the internal side of the plasma membrane and the cytoplasm with a higher expression in the membrane. The enzyme has a similar activity and regulatory mechanism as the endothelial-derived NOS. Besides its vasodilatation activity NO also regulates red blood cells deformability and inhibits platelet activation. In physiological condition where there is a normal supply of L-Arginine and subsequently a normal NO production, nitric oxide sustains red blood cells deformability. On contrary decreased NO levels reduces erythrocytes deformability preventing them to easily pass through microcirculation. The same effect was

Ulker demonstrates in [36] that red blood cell-NOS is activated by mechanical factors and that export of NO from erythrocytes is enhanced by mechanical stress thus pointing

**c**. NO is a short life species as a consequence it quickly reacts with any encountered molecules or it is rapidly oxidized by hemoglobin in blood. In fact the general accepted theory is that Hb in the red blood cells is an extremely effective NO scavenger in [37]. Oxigenated-Hb reacts with NO which is rapidly converted into nitrate (figure 16). After reacting oxygenated hemoglobin is converted to methemoglobin (met-Hb). This reaction is

Lundberg shows in [38] that NO can be alternatively produced in hypoxic condition by deoxi-hemoglobin from nitrite. Inside erythrocyte Hb can interact with NO in many ways

**Figure 13.** Endothelial NOS differently behaves generating either NO or O2 depending on the substrate availability.

Oxygen deficiency is known to halt the L-arginine cycle if the oxygen levels fall below a threshold level of ca [O2] ~ 10 μM [28]. However, eNOS is not wholly inactivated in hypoxia, instead, in the presence of nitrite (figure 14), it shifts again and produces NO in [29].

**Figure 14.** Endothelial NOS may generate NO in both normal and low oxygenation

Stroes et al demonstrate in [30, 31] an intriguing activity for eNOS only, the simultaneous generation of both NO and superoxide, even in the presence of BH4 and L-arginine, under physiological conditions. The consequence is the production of peroxynitrite, a highly reactive molecule, by eNOS (figure 15).

Thus eNOS is a source of free radical producing "good" or "bad" radicals upon inside cell condition.

**Figure 15.** Peroxinitrite generation as a result of particularly eNOS activity

Peroxynitrite anion (ONOO−) is a reactive species of increasingly recognized biological relevance that contributes to oxidative tissue damage. Recent research indicates in [32] that peroxynitrite is able to cross the erythrocyte membrane by two different mechanisms: in the anionic form through the anion exchange channel, and in the protonated form by passive diffusion. Entering the erythrocyte peroxynitrite causes nitration of intracellular hemoglobin, in a process that is enhanced in thiol-depleted erythrocytes.

To summarize NO can be produced by eNOS from either L-arginine in good oxygenation physiological state or from nitrite in hypoxia. In vitamins deficiency (low BH4 levels) eNOS produces superoxide radical. Recent studies demonstrate that endothelium cells surprisingly produce ROS under hypoxia. The primary site of reactive oxygen species production was demonstrated to be complex III in electron transport in mitochondria. The paradoxically increase in ROS production under low oxygenation is still not fully understood but it is considered that reactive oxygen species released during hypoxia act as signalling agents that trigger induction of erythropoietin, endothelial growth factor and glycolytic enzymes. Systemically, these responses enhance the delivery of O2 to cells and facilitate the production of glycolytic ATP instead of mitochondrion. Induction of these genes is mediated by "specialized" hypoxia inducible factor 1 (HIF-1) [33, 34]. As a conclusion in normal oxygenation NO is produced by eNOS. In hypoxia adaptive responses are onset; the release of NO from nitrite to sustain normal vascular function is one path. Alternatively when mitochondrion "senses" hypoxia it releases ROS as signaling molecules that activate diverse functional responses, including activation of gene expression that promote cell survival.

42 Blood Cell – An Overview of Studies in Hematology

reactive molecule, by eNOS (figure 15).

condition.

NO (figure 9).

substrate availability.

between the two domains. Deficiency of arginine or BH4 causes the reductase uncoupling from oxygenase. At the oxygenase domain intermediate Fe2+-O2 complex dissociates to form

Oxygen deficiency is known to halt the L-arginine cycle if the oxygen levels fall below a threshold level of ca [O2] ~ 10 μM [28]. However, eNOS is not wholly inactivated in hypoxia,

Stroes et al demonstrate in [30, 31] an intriguing activity for eNOS only, the simultaneous generation of both NO and superoxide, even in the presence of BH4 and L-arginine, under physiological conditions. The consequence is the production of peroxynitrite, a highly

Thus eNOS is a source of free radical producing "good" or "bad" radicals upon inside cell

Peroxynitrite anion (ONOO−) is a reactive species of increasingly recognized biological relevance that contributes to oxidative tissue damage. Recent research indicates in [32] that peroxynitrite is able to cross the erythrocyte membrane by two different mechanisms: in the anionic form through the anion exchange channel, and in the protonated form by passive diffusion. Entering the erythrocyte peroxynitrite causes nitration of intracellular

instead, in the presence of nitrite (figure 14), it shifts again and produces NO in [29].

**Figure 14.** Endothelial NOS may generate NO in both normal and low oxygenation

**Figure 15.** Peroxinitrite generation as a result of particularly eNOS activity

hemoglobin, in a process that is enhanced in thiol-depleted erythrocytes.

instead of

depending on the

superoxide and the original Fe3+ group of the eNOS)[27]. Thus eNOS releases O2

**Figure 13.** Endothelial NOS differently behaves generating either NO or O2

**b.** Kleinbongard demonstrates in [35] that red blood cells express functional eNOS which is located in both the internal side of the plasma membrane and the cytoplasm with a higher expression in the membrane. The enzyme has a similar activity and regulatory mechanism as the endothelial-derived NOS. Besides its vasodilatation activity NO also regulates red blood cells deformability and inhibits platelet activation. In physiological condition where there is a normal supply of L-Arginine and subsequently a normal NO production, nitric oxide sustains red blood cells deformability. On contrary decreased NO levels reduces erythrocytes deformability preventing them to easily pass through microcirculation. The same effect was observed on platelet aggregation when decreased NO levels promote thrombosis.

Ulker demonstrates in [36] that red blood cell-NOS is activated by mechanical factors and that export of NO from erythrocytes is enhanced by mechanical stress thus pointing erythrocyte contribution to the regulation of vascular tonus

**c**. NO is a short life species as a consequence it quickly reacts with any encountered molecules or it is rapidly oxidized by hemoglobin in blood. In fact the general accepted theory is that Hb in the red blood cells is an extremely effective NO scavenger in [37]. Oxigenated-Hb reacts with NO which is rapidly converted into nitrate (figure 16). After reacting oxygenated hemoglobin is converted to methemoglobin (met-Hb). This reaction is considered to be limited only by the diffusion.

**Figure 16.** Oxyhemoglobin activity of scavenging the nitric oxide

Lundberg shows in [38] that NO can be alternatively produced in hypoxic condition by deoxi-hemoglobin from nitrite. Inside erythrocyte Hb can interact with NO in many ways depending on its oxygen saturation, as follows:


Homocysteine in Red Blood Cells Metabolism – Pharmacological Approaches 45

─/ NO pool" in full oxygenation

**Figure 19.** Hemoglobin uses NO to generate nitrate a supposed "NO2

branches through the way it uses/produces NO radical. [47]

oxygenation/deoxigenation status in [48,49].

**2.2. Homocysteine metabolism** 

hyperhomocysteinemia can also install.

and releases it with maximal activity at P50 oxygen saturation in hypoxia [modified from [38].

To summarize: when there is plenty of oxygen, NO is mainly produced by endothelial NOS or by erythrocyte own NOS. Thus nitric oxide radical maintains good erythrocytes deformability and probably oxy-Hb regulates the amount of NO by trapping its extra amount to form *S*-nitrosohemoglobin. When oxygen is scarce NO is synthesized in low amount (as a consequence of low eNOS activity where oxygen is cofactor). In this condition NO is saved through binding on deoxyHb which becomes a source of NO in vascular circulation, or deoxy-Hb "catalyses" the NO generation from nitrite. Behaving this way deoxy-Hb can maintain also red blood cell deformability in hypoxic condition. As a consequence hemoglobin may "regulates" membrane deformability along circulating

In addition two other supplementary mechanisms, endothelial xantinoxidase activity and blood pH dictate superoxid radical versus NO generation depending on the blood

Homocysteine is a metabolic compound formed in methionine and betaine metabolism. The already mention metabolisms require vitamin B12, B6 and folic acid involvement and the proper activity of two main enzymes cystathionine β-synthase (CBS) and methylenetetrahydrofolate reductase (MTHFR). Every deficiency in vitamins supplies or enzymes activity triggers the onset of different disease. Megaloblastic anemia or Biermer disease affect primarily the red blood cells. Secondary to the above mentioned illnesses


**Figure 17.** Hemoglobin "regulates" the NO use/consumption, from [38]

Two recent theories try to explain the Hb involvement in nitric oxide use/consumption in vasorelaxation process in [42].


**Figure 18.** Hemoglobin behavior depending on oxygen saturation.

J.O.Lunderg concluded in [42] that: "in just one decade, Hb has gone from being merely a NO scavenger to NO carrier and now NO generator".

Homocysteine in Red Blood Cells Metabolism – Pharmacological Approaches 45

**Figure 19.** Hemoglobin uses NO to generate nitrate a supposed "NO2 ─/ NO pool" in full oxygenation and releases it with maximal activity at P50 oxygen saturation in hypoxia [modified from [38].

To summarize: when there is plenty of oxygen, NO is mainly produced by endothelial NOS or by erythrocyte own NOS. Thus nitric oxide radical maintains good erythrocytes deformability and probably oxy-Hb regulates the amount of NO by trapping its extra amount to form *S*-nitrosohemoglobin. When oxygen is scarce NO is synthesized in low amount (as a consequence of low eNOS activity where oxygen is cofactor). In this condition NO is saved through binding on deoxyHb which becomes a source of NO in vascular circulation, or deoxy-Hb "catalyses" the NO generation from nitrite. Behaving this way deoxy-Hb can maintain also red blood cell deformability in hypoxic condition. As a consequence hemoglobin may "regulates" membrane deformability along circulating branches through the way it uses/produces NO radical. [47]

In addition two other supplementary mechanisms, endothelial xantinoxidase activity and blood pH dictate superoxid radical versus NO generation depending on the blood oxygenation/deoxigenation status in [48,49].

#### **2.2. Homocysteine metabolism**

44 Blood Cell – An Overview of Studies in Hematology

vasorelaxation process in [42].


**Figure 17.** Hemoglobin "regulates" the NO use/consumption, from [38]

NO release occurs when Hb is deoxygenated.

**Figure 18.** Hemoglobin behavior depending on oxygen saturation.

NO scavenger to NO carrier and now NO generator".

pathologic conditions (figure 19).


Two recent theories try to explain the Hb involvement in nitric oxide use/consumption in



J.O.Lunderg concluded in [42] that: "in just one decade, Hb has gone from being merely a

Homocysteine is a metabolic compound formed in methionine and betaine metabolism. The already mention metabolisms require vitamin B12, B6 and folic acid involvement and the proper activity of two main enzymes cystathionine β-synthase (CBS) and methylenetetrahydrofolate reductase (MTHFR). Every deficiency in vitamins supplies or enzymes activity triggers the onset of different disease. Megaloblastic anemia or Biermer disease affect primarily the red blood cells. Secondary to the above mentioned illnesses hyperhomocysteinemia can also install.

Hyperhomocysteinemia is considered to be involved in many diseases from cardiovascular to neurological illnesses. It is generally agreed that two general mechanisms cause hyperhomocysteinemia: one is low vitamins (B12, B6, folic acid) supplies and second the main enzymes deficiencies (cystathionine ß-synthase deficiency and methylenetetrahydrofolate reductase deficiency). Hypehomocysteinemia is today considered a severe risk factor in vascular illnesses. Many approaches envisage lowering homocysteine levels by vitamin B or oral folic acid supplementation but many recent studies show that vitamins administration fail to give a real clinical benefit and suggest that B vitamins might instead increase some cardiovascular risks in [50,51]. However not all patients with cardiovascular events or neurodegenerative diseases are enzymes deficient or poor vitamins supplied. The majority of research works report hyperhomocysteinemia associated to many diseases but the question what triggers hyperhomocysteinemia is yet to answer. An interesting hypothesis suggests that in fact hyperhomocysteinemia is more a secondary effect that amplifies in its turn the initial injury [52]. Brattström and Wilcken in [52] consider that impaired renal function due to hypertension and atherosclerosis is an important cause of the elevated plasma homocysteine found in vascular disease patients. The reasons are as follows. Atherogenesis and elevation of blood pressure commonly develop silently over many years before the emergence of clinically evident vascular events. These processes also lead to nephrosclerosis and a degree of deterioration of renal function, and this is highly relevant to the plasma clearance of homocysteine. For these reasons, the presence of vascular disease itself may contribute to an elevation in circulating homocysteine by leading to a decline in renal function. This means that because of reduced renal function, patients with either occult or clinically evident cardiovascular disease may have elevated circulating homocysteine concentrations (figure 20). This could also explain the relation between plasma homocysteine and the severity of atherosclerosis.

Homocysteine in Red Blood Cells Metabolism – Pharmacological Approaches 47

**2.3. Hyperhomocysteinemia a disturbing factor of the endothelial function** 

around 70% is protein bounded.

hyperhomocysteinemia in [54].

target level for therapy in [55].

(figure 21).

and act.

Homocysteine refers to all species that contain and can release homocystein including homocystine (the dimer of homocysteine) and mixed cysteine-homocysteine disulfide or homocysteine bound on proteins. In fact the major form of homocysteine in circulation,

In early data normal levels of homocysteine were admitted to be around 15μM/L. It was found that homocysteine slightly increases with age in [53]. Levels of 15-30 μM/L corresponds to mild, 30-100 μM/L to moderate and more than 100 μM/L to severe

Nowadays it is considered that concentrations below 9 micro mol/L are an appropriate

Nowadays it is generally accepted that homocysteine promotes thrombosis with simultaneously vasodilatation inhibition. It is considered that homocysteine triggers its effects by three distinct mechanisms which can possibly integrate and are not mutually exclusive; oxidative processes, decreasing NO bioavailability and specific protein targeting

**Figure 21.** Homocysteine adverse effects. (+/─ represent activation/inhibition processes)

Activator (tPA), annexin II. Amongst "bad" factor can be included: ADMA, O2

Even the precise path was not yet established, it seems that Hcys inhibits some "good" factors and activates same "bad" factors that finally influence the processes of thrombosisfibrinolysis and constriction–vasorelaxation and which are summarized in (figure 21) [56].

Amongst "good" factor can be included: NO, GPx, eNOS, protein C, tissue Plasminogen

Homocysteine directly and indirectly influences erythrocytes metabolism. It directly affects the erythrocyte antioxidant enzyme systems promoting free radical generation. Indirectly Hcy decreases NO bioavailability and modifies the environment where erythrocytes move

, H2O2

**Figure 20.** Proposed mechanisms for the causes of hyperhomocysteinemia (taken from [52] )

#### **2.3. Hyperhomocysteinemia a disturbing factor of the endothelial function**

46 Blood Cell – An Overview of Studies in Hematology

Hyperhomocysteinemia is considered to be involved in many diseases from cardiovascular to neurological illnesses. It is generally agreed that two general mechanisms cause hyperhomocysteinemia: one is low vitamins (B12, B6, folic acid) supplies and second the main enzymes deficiencies (cystathionine ß-synthase deficiency and methylenetetrahydrofolate reductase deficiency). Hypehomocysteinemia is today considered a severe risk factor in vascular illnesses. Many approaches envisage lowering homocysteine levels by vitamin B or oral folic acid supplementation but many recent studies show that vitamins administration fail to give a real clinical benefit and suggest that B vitamins might instead increase some cardiovascular risks in [50,51]. However not all patients with cardiovascular events or neurodegenerative diseases are enzymes deficient or poor vitamins supplied. The majority of research works report hyperhomocysteinemia associated to many diseases but the question what triggers hyperhomocysteinemia is yet to answer. An interesting hypothesis suggests that in fact hyperhomocysteinemia is more a secondary effect that amplifies in its turn the initial injury [52]. Brattström and Wilcken in [52] consider that impaired renal function due to hypertension and atherosclerosis is an important cause of the elevated plasma homocysteine found in vascular disease patients. The reasons are as follows. Atherogenesis and elevation of blood pressure commonly develop silently over many years before the emergence of clinically evident vascular events. These processes also lead to nephrosclerosis and a degree of deterioration of renal function, and this is highly relevant to the plasma clearance of homocysteine. For these reasons, the presence of vascular disease itself may contribute to an elevation in circulating homocysteine by leading to a decline in renal function. This means that because of reduced renal function, patients with either occult or clinically evident cardiovascular disease may have elevated circulating homocysteine concentrations (figure 20). This could also explain

the relation between plasma homocysteine and the severity of atherosclerosis.

**Figure 20.** Proposed mechanisms for the causes of hyperhomocysteinemia (taken from [52] )

Homocysteine refers to all species that contain and can release homocystein including homocystine (the dimer of homocysteine) and mixed cysteine-homocysteine disulfide or homocysteine bound on proteins. In fact the major form of homocysteine in circulation, around 70% is protein bounded.

In early data normal levels of homocysteine were admitted to be around 15μM/L. It was found that homocysteine slightly increases with age in [53]. Levels of 15-30 μM/L corresponds to mild, 30-100 μM/L to moderate and more than 100 μM/L to severe hyperhomocysteinemia in [54].

Nowadays it is considered that concentrations below 9 micro mol/L are an appropriate target level for therapy in [55].

Nowadays it is generally accepted that homocysteine promotes thrombosis with simultaneously vasodilatation inhibition. It is considered that homocysteine triggers its effects by three distinct mechanisms which can possibly integrate and are not mutually exclusive; oxidative processes, decreasing NO bioavailability and specific protein targeting (figure 21).

**Figure 21.** Homocysteine adverse effects. (+/─ represent activation/inhibition processes)

Even the precise path was not yet established, it seems that Hcys inhibits some "good" factors and activates same "bad" factors that finally influence the processes of thrombosisfibrinolysis and constriction–vasorelaxation and which are summarized in (figure 21) [56].

Amongst "good" factor can be included: NO, GPx, eNOS, protein C, tissue Plasminogen Activator (tPA), annexin II. Amongst "bad" factor can be included: ADMA, O2 , H2O2

Homocysteine directly and indirectly influences erythrocytes metabolism. It directly affects the erythrocyte antioxidant enzyme systems promoting free radical generation. Indirectly Hcy decreases NO bioavailability and modifies the environment where erythrocytes move and act.

## *2.3.1. Homocysteine pro-oxidative activity*

Homocysteine is involved in reducing–oxidative processes by reacting either with itself or with different compounds. In other words homocysteine can submit auto-oxidative as well as oxidative processes.

Homocysteine in Red Blood Cells Metabolism – Pharmacological Approaches 49

Homocysteine may affect glutathione peroxidase activity, thus altering the microenvironment in the propagation of ROS in [63]. Our study on GPx activity, in installed hyperhomocysteinemia, was consistent with these reported data. GPx activity in red blood cells lysate significantly decreases as a consequence of experimental induced hypehocysteinemia in rats. We considered that increased amount of free radicals consume the GSH enzyme cofactor which subsequently trigger the enzyme activity decay in [62]. As a consequence GPx activity is lowered in hyperhomocysteinemia thus disturbing the

Upchurch in[63] demonstrates that homocysteine reduces mRNA levels of glutathione peroxidase, indicating that the expression of this enzyme is inhibited and/or down-

Even it was attributed to different causes such as: a decrease in enzyme activity, a down regulation from high homocysteine levels or an inappropriate gene expression of GPx, the

Homocysteine-induced oxidative stress was proved to be generated within vascular cells in [64]. Our data show that in installed hyperhomocysteinemia the intracellular space is more affected than the extracellular, circulatory one. We found significant changes in antioxidant enzyme systems within erythrocyte (we worked on erythrocyte lysate) as compared with

As a conclusion hyperhomocysteinemia by promoting free radical generation affects both

The second hypothesis considers that Hcy acts to prevent NO bioavailability. This process is considered to have, at least partially, the same oxidative basis. In living organisms, including in human, endothelial-derived nitric oxide performs the following function: regulates vessel tone by promoting vasodilatation, inhibits platelet activation, adhesion and aggregation, limits smooth muscle proliferation and modulates endothelial–leukocyte interactions in [56]. Homoysteine was proved to limits NO bioavailability thus promoting

There are proposed many patterns for homocysteine impairing NO bioavailability (figure

A first process that limits NO bioavailability seems to be more a protecting mechanism than a harmful one. Homocysteine reacts with nitric oxide to form S-nitroso-homocysteine, which has some of the properties of nitric oxide. It markedly inhibits platelet aggregation, is a potent vasodilator and does not support hydrogen peroxide generation. This represents much more a protective mechanism against the adverse effects of homocysteine than a

the contrary processes: vasoconstriction, thrombosis and fibrinolysis inhibition.

detoxification process of H2O2 within cell.

decrease in GPx activity in Hcys presence is generally reported.

total antioxidant capacity (TAC) in plasma in [62].

erythrocytes and endothelial cells as well in [65,66].

*2.3.2. Homocysteine decreases NO bioavailability* 

limiting process in NO bioavailability in [56].

regulated.

22).

Thiols (RSH) can auto-oxidize in the presence of transition metal catalysts and molecular oxygen, leading to the formation of reactive oxygen species (ROS). Hcy like all containing thiol group undergoes oxidation to disulfide (RS-SR) in O2 presence at normal pH. It was found that cooper catalyses Hcy (noted with general formula RSH) auto-oxidation, even in low homocysteine concentration, yielding hydrogen peroxide and thus promoting ROS generation in both extra and intracellular compartments through reaction proposed by Starkebaum in [57]:

2 Homocysteine (RSH) + O2 → Homocystine (RS-SR)

Homocystine (RS-SR) + superoxid (O2 **)**→ H2O2

Hydrogen peroxide generated by the copper catalysed auto-oxidation of homocysteine was involved in the mechanism of toxicity by the demonstration of the reduction in endothelial damage with the addition of catalase in [58].

Homocysteine was proved to generate superoxide radicals which promote vasoconstriction. Lang et al. demonstrates in [59] that the inhibitory effect of homocysteine on endotheliumdependent relaxation is caused by an increase of the intracellular levels of O2 in the endothelial cell and provide a possible mechanism for the endothelial dysfunction associated with hyperhomocysteinemia.

Cysteine is also a thiol circulating aminoacid related to homocysteine and its concentration is 20 to 30 times higher than Hcys one. In fact Cys is the main circulatory thiol but there was found no correlation of Cys with free radicals generation. Instead a strong association of hyperhomocysteinemia with F2-isoprostane was found. F2-isoprostane is an indicator of in vivo lipid-peroxidation and its association with Hcy lead to the conclusion that this amino acid is involved in free radicals generation in [60] thus pointing Hcy as pro-oxidative agent.

Hcy involvement in ROS generation was also indirectly proved in connection with antioxidant enzyme system modulation SOD and GPx.

The activity of superoxide dismutase, an important antioxidant enzyme in vascular tissue, was measured along with homocysteine in homocystinuric patients and found to be positively associated with homocysteine levels. This strong relationship can be regarded as a protective antioxidant response to homocysteine-induced oxidative action and as indirect evidence that Hcy represents a source of free radicals in [61]. In our study we found an increased superoxide dismutase activity in red blood cells lysate in experimental induced hyperhocysteinemia in rats. We consider this increased response in enzyme activity as evidence for free radicals' production in [62].

Homocysteine may affect glutathione peroxidase activity, thus altering the microenvironment in the propagation of ROS in [63]. Our study on GPx activity, in installed hyperhomocysteinemia, was consistent with these reported data. GPx activity in red blood cells lysate significantly decreases as a consequence of experimental induced hypehocysteinemia in rats. We considered that increased amount of free radicals consume the GSH enzyme cofactor which subsequently trigger the enzyme activity decay in [62]. As a consequence GPx activity is lowered in hyperhomocysteinemia thus disturbing the detoxification process of H2O2 within cell.

Upchurch in[63] demonstrates that homocysteine reduces mRNA levels of glutathione peroxidase, indicating that the expression of this enzyme is inhibited and/or downregulated.

Even it was attributed to different causes such as: a decrease in enzyme activity, a down regulation from high homocysteine levels or an inappropriate gene expression of GPx, the decrease in GPx activity in Hcys presence is generally reported.

Homocysteine-induced oxidative stress was proved to be generated within vascular cells in [64]. Our data show that in installed hyperhomocysteinemia the intracellular space is more affected than the extracellular, circulatory one. We found significant changes in antioxidant enzyme systems within erythrocyte (we worked on erythrocyte lysate) as compared with total antioxidant capacity (TAC) in plasma in [62].

As a conclusion hyperhomocysteinemia by promoting free radical generation affects both erythrocytes and endothelial cells as well in [65,66].

## *2.3.2. Homocysteine decreases NO bioavailability*

48 Blood Cell – An Overview of Studies in Hematology

as oxidative processes.

Starkebaum in [57]:

*2.3.1. Homocysteine pro-oxidative activity* 

damage with the addition of catalase in [58].

associated with hyperhomocysteinemia.

antioxidant enzyme system modulation SOD and GPx.

evidence for free radicals' production in [62].

Homocysteine is involved in reducing–oxidative processes by reacting either with itself or with different compounds. In other words homocysteine can submit auto-oxidative as well

Thiols (RSH) can auto-oxidize in the presence of transition metal catalysts and molecular oxygen, leading to the formation of reactive oxygen species (ROS). Hcy like all containing thiol group undergoes oxidation to disulfide (RS-SR) in O2 presence at normal pH. It was found that cooper catalyses Hcy (noted with general formula RSH) auto-oxidation, even in low homocysteine concentration, yielding hydrogen peroxide and thus promoting ROS generation in both extra and intracellular compartments through reaction proposed by

2 Homocysteine (RSH) + O2 → Homocystine (RS-SR)

Hydrogen peroxide generated by the copper catalysed auto-oxidation of homocysteine was involved in the mechanism of toxicity by the demonstration of the reduction in endothelial

Homocysteine was proved to generate superoxide radicals which promote vasoconstriction. Lang et al. demonstrates in [59] that the inhibitory effect of homocysteine on endothelium-

endothelial cell and provide a possible mechanism for the endothelial dysfunction

Cysteine is also a thiol circulating aminoacid related to homocysteine and its concentration is 20 to 30 times higher than Hcys one. In fact Cys is the main circulatory thiol but there was found no correlation of Cys with free radicals generation. Instead a strong association of hyperhomocysteinemia with F2-isoprostane was found. F2-isoprostane is an indicator of in vivo lipid-peroxidation and its association with Hcy lead to the conclusion that this amino acid is involved in free radicals generation in [60] thus pointing Hcy as pro-oxidative agent.

Hcy involvement in ROS generation was also indirectly proved in connection with

The activity of superoxide dismutase, an important antioxidant enzyme in vascular tissue, was measured along with homocysteine in homocystinuric patients and found to be positively associated with homocysteine levels. This strong relationship can be regarded as a protective antioxidant response to homocysteine-induced oxidative action and as indirect evidence that Hcy represents a source of free radicals in [61]. In our study we found an increased superoxide dismutase activity in red blood cells lysate in experimental induced hyperhocysteinemia in rats. We consider this increased response in enzyme activity as

**)**→ H2O2

in the

Homocystine (RS-SR) + superoxid (O2

dependent relaxation is caused by an increase of the intracellular levels of O2

The second hypothesis considers that Hcy acts to prevent NO bioavailability. This process is considered to have, at least partially, the same oxidative basis. In living organisms, including in human, endothelial-derived nitric oxide performs the following function: regulates vessel tone by promoting vasodilatation, inhibits platelet activation, adhesion and aggregation, limits smooth muscle proliferation and modulates endothelial–leukocyte interactions in [56]. Homoysteine was proved to limits NO bioavailability thus promoting the contrary processes: vasoconstriction, thrombosis and fibrinolysis inhibition.

There are proposed many patterns for homocysteine impairing NO bioavailability (figure 22).

A first process that limits NO bioavailability seems to be more a protecting mechanism than a harmful one. Homocysteine reacts with nitric oxide to form S-nitroso-homocysteine, which has some of the properties of nitric oxide. It markedly inhibits platelet aggregation, is a potent vasodilator and does not support hydrogen peroxide generation. This represents much more a protective mechanism against the adverse effects of homocysteine than a limiting process in NO bioavailability in [56].

However prolonged exposure to high homocysteine concentrations impairs nitric oxide production. Thus in hyperhomocysteinemia the limited bioavailability of nitric oxide could be due to S-nitrosothiol formation in [67].

Homocysteine in Red Blood Cells Metabolism – Pharmacological Approaches 51

Methionine loading was proved to induce hyperhomocysteinemia. In methionine loading there is S-adenosylmethionine accumulation and as a consequence a high proteins methylation. Asymmetrical dimethylarginine (ADMA) the product of degradation from methylated proteins competitively inhibits NO-synthetase activity. Elevated ADMA levels found in hyperhomocysteinemia are supposed to inhibit NO synthesis thus decreasing the

A third way that homocysteine acts is targeting specific proteins which are located within

Two important intracellular proteins, already mentioned, targeted by Hcy are the antioxidant enzyme GPx which activity decreases and SOD which activity increases in homocysteine presence (fig22). Hcy alters other intracellular proteins disturbing the redox potential of endoplasmic reticulum and Golgi apparatus thus inhibiting the surface

Hcy targets proteins located on both membrane surface and within cell. Jacobsen considers that circulatory oxidized form of Hcy enters the cell were it is converted back to reduced Hcy, in reducing environment within cell. Under reduced form Hcy impairs the binding of tissue plasminogen activator (tPA), a protein involved in the breakdown of blood clots, to annexin II in [71] by forming a disulfide bridge with Cys9 on annexin II. Thus Hcy limits the plasminogen conversion to plasmin. This results in a decreased fibrinolysis. The circulating reduced Hcy acts in the same manner with annexin II on the membrane from the vascular endothelium. Homocysteine was found to be the only circulating thiol that impairs the

The atherogenic factor lipoprotein (a) [Lp (a)] competitively inhibits the binding of plasminogen to fibrin. Fibrin is a cofactor for plasminogen activation to plasmin, an important enzyme that degrades fibrin clot. Homocysteine was found to interfere in this process. Hcy and lipoprotein (a) seems to act in the same direction: homocysteine promotes lipoprotein(a) binding to fibrin and lipoprotein(a) competitively inhibits the binding of plasminogen to fibrin. The final effect is the decrease of fibrinolysis. The combination of Lp (a) plus homocysteine is a possible mechanisms for the occurrence of thrombosis in

Protein C is an example of circulating proteins whose activity is inhibited by Hcy. The protein C enzyme system appears to be one of the most important anticoagulant pathways in the blood. Its activation depends on the complex thrombomodulin-trombin. Thrombomodulin is an integral membrane protein expressed on the surface of endothelial cells where it serves as a receptor for thrombin. The complex thrombomodulin-trombin activates protein C thus raising its activity. Homocysteine inhibits the function of thrombomodulin. Both thrombomodulin and protein C contain disulfide-rich domains. Reduction of these disulfide bonds by homocysteine may disrupt important structures

NO availability in [70].

*2.3.3. Homocysteine own action* 

cell, on cell membrane or in the extracellular space.

expression and secretion of proteins in [71,72].

binding of tissue-plasminogen activator.

hyperhomocysteinemia in [73].

**Figure 22.** Proposed mechanism through which Hcy inhibits some "good" factors and activates same "bad" factors thus influencing thrombosis-fibrinolysis respectively constriction–vasorelaxation processes. tPA, ADMA represents tissue plasminogen activator/respectively asymmetrical dimethyl arginine.

A second process that limits NO bioavailability is nitric oxide trapping/degradation by other radical species. NO is trapped by superoxide to form peroxinitrite thus being inactivated. This mechanism was confirmed by many experimental data in [63, 68].

Nitric oxide can be alternatively degraded by hydrogen peroxide as a consequence of GPx activity inhibition through Hcy-dependent mechanism. Homocysteine seems to be the only amino acid amongst all circulating others capable to inhibit glutathione peroxidase activity in vitro. Cysteine is also capable of generating free radicals and is present in serum at concentrations four times higher than homocysteine but cysteine doesn't prove inhibiting properties on GPx activity. Experimental data show that Hcy inhibits GPx activity and also suppresses the cellular GPx expression thus promoting the increase of hydrogen peroxide concentration in [64]. Hydrogen peroxide promotes in its turn free radicals generation and peroxinitrite production thus decreasing NO availability.

A third mechanism that limits NO bioavailability is the decrease in NO synthesis through Hcy-dependent asymmetrical dimethylarginine (ADMA) generation. ADMA is produced by methylation of specific arginine residues of certain cellular proteins. Most of these proteins are found in the nucleus. When the proteins are degraded, ADMA and other isomers are released to the extracellular space where especially ADMA acts as potent endogenous inhibitors of NOS enzyme in [69].

Methionine loading was proved to induce hyperhomocysteinemia. In methionine loading there is S-adenosylmethionine accumulation and as a consequence a high proteins methylation. Asymmetrical dimethylarginine (ADMA) the product of degradation from methylated proteins competitively inhibits NO-synthetase activity. Elevated ADMA levels found in hyperhomocysteinemia are supposed to inhibit NO synthesis thus decreasing the NO availability in [70].

#### *2.3.3. Homocysteine own action*

50 Blood Cell – An Overview of Studies in Hematology

be due to S-nitrosothiol formation in [67].

arginine.

However prolonged exposure to high homocysteine concentrations impairs nitric oxide production. Thus in hyperhomocysteinemia the limited bioavailability of nitric oxide could

**Figure 22.** Proposed mechanism through which Hcy inhibits some "good" factors and activates same "bad" factors thus influencing thrombosis-fibrinolysis respectively constriction–vasorelaxation processes. tPA, ADMA represents tissue plasminogen activator/respectively asymmetrical dimethyl

A second process that limits NO bioavailability is nitric oxide trapping/degradation by other radical species. NO is trapped by superoxide to form peroxinitrite thus being

Nitric oxide can be alternatively degraded by hydrogen peroxide as a consequence of GPx activity inhibition through Hcy-dependent mechanism. Homocysteine seems to be the only amino acid amongst all circulating others capable to inhibit glutathione peroxidase activity in vitro. Cysteine is also capable of generating free radicals and is present in serum at concentrations four times higher than homocysteine but cysteine doesn't prove inhibiting properties on GPx activity. Experimental data show that Hcy inhibits GPx activity and also suppresses the cellular GPx expression thus promoting the increase of hydrogen peroxide concentration in [64]. Hydrogen peroxide promotes in its turn free radicals generation and

A third mechanism that limits NO bioavailability is the decrease in NO synthesis through Hcy-dependent asymmetrical dimethylarginine (ADMA) generation. ADMA is produced by methylation of specific arginine residues of certain cellular proteins. Most of these proteins are found in the nucleus. When the proteins are degraded, ADMA and other isomers are released to the extracellular space where especially ADMA acts as potent endogenous

inactivated. This mechanism was confirmed by many experimental data in [63, 68].

peroxinitrite production thus decreasing NO availability.

inhibitors of NOS enzyme in [69].

A third way that homocysteine acts is targeting specific proteins which are located within cell, on cell membrane or in the extracellular space.

Two important intracellular proteins, already mentioned, targeted by Hcy are the antioxidant enzyme GPx which activity decreases and SOD which activity increases in homocysteine presence (fig22). Hcy alters other intracellular proteins disturbing the redox potential of endoplasmic reticulum and Golgi apparatus thus inhibiting the surface expression and secretion of proteins in [71,72].

Hcy targets proteins located on both membrane surface and within cell. Jacobsen considers that circulatory oxidized form of Hcy enters the cell were it is converted back to reduced Hcy, in reducing environment within cell. Under reduced form Hcy impairs the binding of tissue plasminogen activator (tPA), a protein involved in the breakdown of blood clots, to annexin II in [71] by forming a disulfide bridge with Cys9 on annexin II. Thus Hcy limits the plasminogen conversion to plasmin. This results in a decreased fibrinolysis. The circulating reduced Hcy acts in the same manner with annexin II on the membrane from the vascular endothelium. Homocysteine was found to be the only circulating thiol that impairs the binding of tissue-plasminogen activator.

The atherogenic factor lipoprotein (a) [Lp (a)] competitively inhibits the binding of plasminogen to fibrin. Fibrin is a cofactor for plasminogen activation to plasmin, an important enzyme that degrades fibrin clot. Homocysteine was found to interfere in this process. Hcy and lipoprotein (a) seems to act in the same direction: homocysteine promotes lipoprotein(a) binding to fibrin and lipoprotein(a) competitively inhibits the binding of plasminogen to fibrin. The final effect is the decrease of fibrinolysis. The combination of Lp (a) plus homocysteine is a possible mechanisms for the occurrence of thrombosis in hyperhomocysteinemia in [73].

Protein C is an example of circulating proteins whose activity is inhibited by Hcy. The protein C enzyme system appears to be one of the most important anticoagulant pathways in the blood. Its activation depends on the complex thrombomodulin-trombin. Thrombomodulin is an integral membrane protein expressed on the surface of endothelial cells where it serves as a receptor for thrombin. The complex thrombomodulin-trombin activates protein C thus raising its activity. Homocysteine inhibits the function of thrombomodulin. Both thrombomodulin and protein C contain disulfide-rich domains. Reduction of these disulfide bonds by homocysteine may disrupt important structures

within these domains, resulting in impaired function in [74]. The result is the promotion of thrombotic process.

Homocysteine in Red Blood Cells Metabolism – Pharmacological Approaches 53

When erythrocytes are undergo shear stress in constricted vessels, they release ATP which

Also, when their hemoglobin molecules are deoxygenated, erythrocytes release **S**nitrosothiols which acts to dilate vessels, thus directing more blood to areas of the body

Using L-arginine as substrate, erythrocytes can also synthesize nitric oxide enzymatically, just like endothelial cells. The nitric oxide synthase is activated when the erythrocytes are exposure to physiological levels of shear stress, thus, nitric oxide is synthesized, exported

Another mechanism that involves the erythrocytes in relaxing vessel walls is the production of hydrogen sulfide. It works as a signaling gas. It is believed that the cardioprotective effects of garlic are due to erythrocytes converting its sulfur compounds into hydrogen

The free radicals released by erythrocytes when they are lysed by pathogens break down the pathogen's cell wall and cell membrane, and so, they are killing them. This represents the

On the other hand, as response of injury after several stressors, including oxidative stress, energy depletion, as well as a wide variety of endogenous mediators and xenobiotics, the erythrocytes can initiate the self suicidal death (eryptosis). Eryptosis is characterized by cell shrinkage, membrane blebbing, activation of proteases, and phosphatidylserine exposure at the outer membrane leaflet. This can make the macrophages to recognized and engulf erythrocyte to be degraded. Eryptosis can be considered a mechanism of defective erythrocytes to escape hemolysis. Conversely, excessive eryptosis favors the development of anemia. Conditions with excessive eryptosis include iron deficiency, lead or mercury intoxication, sickle cell anemia, thalassemia, glucose 6- phosphate dehydrogenase deficiency, malaria, and infection with hemolysis-forming pathogens. Inhibitors of eryptosis include erythropoietin, nitric oxide, catecholamine and high concentrations of urea in [82,

The red blood cell SOD activity has been found to be useful in evaluating the biochemical index of copper, zinc and manganese nutrition. The largest amount of SOD enzyme is found in liver and erythrocytes. There are two forms of SOD in human tissue. One form is present in cytosol and it is a protein containing two atoms each of copper and zinc. The other form is a much larger molecule containing four atoms of manganese and it is found in mitochondria and cytosol. Significant changes in cellular concentration of copper, manganese and zinc have the potential of altering the antioxidant activity of SOD. On the other hands, the correlation between of copper and zinc plasma level, the oxidase activity of ceruloplasmin in serum, and Cu,Zn-SOD activity in erythrocytes can be a way to investigate involvement of oxidative stress in pathological conditions, as atherosclerosis

causes the vessel walls to relax and dilate so as to promote normal blood flow [78].

and it may contribute to the regulation of vascular tonus [79].

involving of erythrocytes in the body's immune response in [81].

depleted of oxygen in [35].

sulfide. [80]

83]

obliterans [84]

Hcy acts on both endothelial cells and smooth muscle where it generates contrasting effect. On endothelium it promotes injury and impairs DNA repair, in smooth muscle Hcy stimulates proliferation in [69]. Md S. Jamaluddin considers that Hcys promotes vascular injury through hypomethylation. When Hcys accumulates it uses adenosine, a normal constituent of all cells, to form S-adenosyl-homocysteine (SAH) a potent inhibitor of cellular methylation. By impairing methylation Hcy arrests cell growth, increases cellular SAH concentration in endothelial cells (EC) and decreased DNA synthesis thus decreasing cellular repair. This chain of events was not found in vascular smooth muscle cells in [75].

Erythrocytes are also affected by homocysteine-induced hypometilation. High intracellular SAH impairs the posttranslational methylation of membrane proteins. Reduction in membrane protein methylation was particularly observed for erythrocyte cytoskeletal component ankyrin, which is known to be involved in membrane stability and integrity. Because of hypomethylation, structural damages accumulate in erythrocyte membrane proteins, and are not adequately repaired thus affecting membrane physical properties. Erythrocyte deformability is a crucial properties for circulatory function in [76].

As a conclusion the effect of elevated homocysteine appears multifactorial affecting both the vascular wall structure and the blood coagulation system as well as erythrocytes metabolism in [77].
