some of the ROS. MnSOD or Cu/ZnSOD converts the O2

peroxynitrite [ONOO-

processes.

[Nagley, 2010].

substantial mitochondrial damage.

**Balance of life and death**

Fig. 1. Deleterious stress factors and restorative factors in neuronal homeostasis and neuronal death [Adpated from Nagley et. al., 2010]. ∆Ψm, mitochondrial membrane potential; MP, misfolded proteins; RONS, reactive oxygen and nitrogen species; UPS, ubiquitin-proteasome system; UPR, unfolded protein response; CMA, chaperone mediated autophagy; MA, macroautophagy. Proteins failing to fold into native structure produces inactive and usually toxic proteins. Several neurodegenerative and other diseases are believed to result from misfolded proteins (MP). The unfolded protein response (UPR) is activated in response to a stress arising from an accumulation of unfolded or MP in the lumen of the endoplasmic reticulum. UPR attempts initially to restore the normal function of the cell by halting protein translation. They also activate the signaling pathways that lead to increasing the production of molecular chaperones involved in protein folding. If these efforts are not successful, then it induces apoptotic effector proteins. Proteasomes are very large protein complexes whose main function is to degrade unneeded or damaged proteins by proteolysis and thus helping cells to regulate proteins and MP. Ubiquitin, a small protein, is used to tag the proteins that need to be degraded. The overall system of ubiquitination and proteasomal degradation is known as the ubiquitin proteosome system (UPS). Autophagy, such as micro- and macro-autophagy, refers to the degradation of intracellular components via the lysosome. Chaperone mediated autophagy, CMA, can degrade only certain proteins and not organelles and thus very selective in what it degrades. Also the substrates are translocated across the membrane on a one on one basis instead of engulfing the substrate in bulk

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 103

other cellular pathways including increased demyelination and inflammation in neurons

While remyelination is extensive in some MS lesions, it is absent or incomplete in others [Zambonin et. al. 2011]. Remyelination helps to restore conduction and helps protect the axons from further inflammation. As an adaptive process or compensatory mechanism, demyelination in the central nervous system causes an increase in the mitochondrial content within axons [Mahad et. al., 2009; Kiryu-Seo et. al., 2010]. This has been attributed to the axons from further inflammation. As an adaptive process or compensatory mechanism, response to the changes in energy needs of axons caused by redistribution of sodium channels. Demyelination compromises the ionic balance and structural integrity of the axons. It results in diffusely expressed Na+ channels with persistent Na+ leakage and forces the need for additional energy to operate the Na+/K+ ATPase pumps. The mitochondria were found to increase in the order of increased energy demand, myelinated, remyelinated and demyelinated axons [Mahad et. al., 2009; Kiryu-Seo et. al., 2010; Zambonin et.al., 2011]. The increase in mitochondrial content (mostly stationary) within remyelinated compared to myelinated axons was attributed to the increase in density of the porin elements. (Porin is a voltage gated anion channel located in the outer membrane of all mitochondria). This increase in mitochondrial content resulted in a corresponding increase in mitochondrial respiratory chain complex IV activity. The change in demyelinated axons was attributed to the change in size. While the number of mobile mitochondria in both remyelinated and myelinated axons were nearly the same, they were much less in demyelinated neurons. An approximately 4 fold increase in mitochondrial content has been observed in chronically demyelinated and non-degenerative axons [Zambonin et. al., 2011]. However, increased mitochondrial content does not necessarily mean more activity. It has been demonstrated that while the mass number may increase in amyloid precursor protein positive segments of demyelinated axons, they appear to harbor mitochondria with complex IV defects [Mahad et. al., 2009]. Within injured axons (non-phosphorylated neurofilaments: SMI32) mitochondrial depletion and decreased complex IV activity was evident, in contrast to chronically demyelinated SMI31 positive axons located in the relatively inactive areas of chronic multiple sclerosis

and tissues that are affected by multiple sclerosis [Mao & Reddy, 2010].

lesions exhibiting a significant increase in complex IV activity and mass [Table 1].

*Structural Changes (fission and fusion imbalance)*

*Abnormal Gene Expresion*

**Mitochondrial Abnormalities in Multiple Sclerosis**

*DNA Defects*

*Abnormal Enzyme Activity*

 *Deficient DNA Repair*

*Increased Free Radicals and Oxidative Damage*

Fig. 3. Mitochondrial abnormalities in multiple sclerosis [adapted from Mao & Reddy, 2010]

#### **7. Multiple sclerosis**

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. It is associated with demyelination and a variable degree of axonal and neuronal degeneration. Demyelination decreases nerve impulse conduction velocity. Also the axons become vulnerable to inflammatory conditions. The mechanisms of tissue injury and neurodegeneration in MS are still under active investigation. Most MS patients initially experience relapsing-remitting episodes of neurologic deficits that last for six to eight weeks. The initial relapse rate is about 0.3/year. This rate declines progressively with time [Siffrin et. al., 2010]. This is followed by a gradual progression of irreversible neurological impairment or secondary progressive multiple sclerosis (SPMS) [Campbell et. al., 2011]. With advancing disease the observed increase in gray matter atrophy, which is indicative of the loss of neurons and axons, correlates well with the corresponding clinical disability.

Fig. 2. Probable causal factors of multiple sclerosis [adapted from Mao & Reddy, 2010]

Apart from the consistent features, neuroaxonal injury and dysfunction in MS, the vascular aspects of MS, an increased risk for ischemic disease, global cerebral hypoperfusion, and a chronic state of impaired venous drainage are receiving a great deal of attention [D'haeseleer et. al., 2011; Filippi & Rocca, 2011].

The precise causal factors of multiple sclerosis are unknown. However, it is possible that multiple factors [Fig. 2] are involved in causing multiple sclerosis, including DNA defects in nuclear and mitochondrial genomes, viral infection, hypoxia and oxidative stress, lack of sunlight or sufficient levels of vitamin D, and increased macrophages and lymphocytes in the brain [Mao & Reddy, 2010].

Current research has shown that mitochondrial abnormalities are involved in the development and progression of multiple sclerosis [Fig. 3], including: mitochondrial DNA defects, abnormal mitochondrial gene expression, defective mitochondrial enzyme activities, abnormal or deficient mitochondrial DNA repair mechanisms, and mitochondrial dysfunction. Studies suggest that abnormal mitochondrial dynamics (imbalance in mitochondrial fission and fusion) plays a key role in tissues affected by multiple sclerosis. Furthermore, mitochondrial abnormalities and mitochondrial energy failure may impact

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. It is associated with demyelination and a variable degree of axonal and neuronal degeneration. Demyelination decreases nerve impulse conduction velocity. Also the axons become vulnerable to inflammatory conditions. The mechanisms of tissue injury and neurodegeneration in MS are still under active investigation. Most MS patients initially experience relapsing-remitting episodes of neurologic deficits that last for six to eight weeks. The initial relapse rate is about 0.3/year. This rate declines progressively with time [Siffrin et. al., 2010]. This is followed by a gradual progression of irreversible neurological impairment or secondary progressive multiple sclerosis (SPMS) [Campbell et. al., 2011]. With advancing disease the observed increase in gray matter atrophy, which is indicative of the loss of neurons and axons, correlates well with the corresponding clinical disability.

> *Accumulation of Macrophages and Lymphocytes in the Brain*

Fig. 2. Probable causal factors of multiple sclerosis [adapted from Mao & Reddy, 2010]

Apart from the consistent features, neuroaxonal injury and dysfunction in MS, the vascular aspects of MS, an increased risk for ischemic disease, global cerebral hypoperfusion, and a chronic state of impaired venous drainage are receiving a great deal of attention

The precise causal factors of multiple sclerosis are unknown. However, it is possible that multiple factors [Fig. 2] are involved in causing multiple sclerosis, including DNA defects in nuclear and mitochondrial genomes, viral infection, hypoxia and oxidative stress, lack of sunlight or sufficient levels of vitamin D, and increased macrophages and lymphocytes in

Current research has shown that mitochondrial abnormalities are involved in the development and progression of multiple sclerosis [Fig. 3], including: mitochondrial DNA defects, abnormal mitochondrial gene expression, defective mitochondrial enzyme activities, abnormal or deficient mitochondrial DNA repair mechanisms, and mitochondrial dysfunction. Studies suggest that abnormal mitochondrial dynamics (imbalance in mitochondrial fission and fusion) plays a key role in tissues affected by multiple sclerosis. Furthermore, mitochondrial abnormalities and mitochondrial energy failure may impact

*Multiple*

*Sclerosis*

*Genetic Factors*

> *Reduced Sunlight (↓Vitamin D)*

**7. Multiple sclerosis**

*Hypoxia and Oxidative Stress*

 *Viral Infection*

[D'haeseleer et. al., 2011; Filippi & Rocca, 2011].

the brain [Mao & Reddy, 2010].

other cellular pathways including increased demyelination and inflammation in neurons and tissues that are affected by multiple sclerosis [Mao & Reddy, 2010].

While remyelination is extensive in some MS lesions, it is absent or incomplete in others [Zambonin et. al. 2011]. Remyelination helps to restore conduction and helps protect the axons from further inflammation. As an adaptive process or compensatory mechanism, demyelination in the central nervous system causes an increase in the mitochondrial content within axons [Mahad et. al., 2009; Kiryu-Seo et. al., 2010]. This has been attributed to the axons from further inflammation. As an adaptive process or compensatory mechanism, response to the changes in energy needs of axons caused by redistribution of sodium channels. Demyelination compromises the ionic balance and structural integrity of the axons. It results in diffusely expressed Na+ channels with persistent Na+ leakage and forces the need for additional energy to operate the Na+/K+ ATPase pumps. The mitochondria were found to increase in the order of increased energy demand, myelinated, remyelinated and demyelinated axons [Mahad et. al., 2009; Kiryu-Seo et. al., 2010; Zambonin et.al., 2011]. The increase in mitochondrial content (mostly stationary) within remyelinated compared to myelinated axons was attributed to the increase in density of the porin elements. (Porin is a voltage gated anion channel located in the outer membrane of all mitochondria). This increase in mitochondrial content resulted in a corresponding increase in mitochondrial respiratory chain complex IV activity. The change in demyelinated axons was attributed to the change in size. While the number of mobile mitochondria in both remyelinated and myelinated axons were nearly the same, they were much less in demyelinated neurons. An approximately 4 fold increase in mitochondrial content has been observed in chronically demyelinated and non-degenerative axons [Zambonin et. al., 2011]. However, increased mitochondrial content does not necessarily mean more activity. It has been demonstrated that while the mass number may increase in amyloid precursor protein positive segments of demyelinated axons, they appear to harbor mitochondria with complex IV defects [Mahad et. al., 2009]. Within injured axons (non-phosphorylated neurofilaments: SMI32) mitochondrial depletion and decreased complex IV activity was evident, in contrast to chronically demyelinated SMI31 positive axons located in the relatively inactive areas of chronic multiple sclerosis lesions exhibiting a significant increase in complex IV activity and mass [Table 1].

Fig. 3. Mitochondrial abnormalities in multiple sclerosis [adapted from Mao & Reddy, 2010]

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 105

Median & (range)

testing; all values represent counted cells/mm2. Active lesions = classical actively demyelinating lesions; slowly expanding lesions = lesions with inactive lesion centre, surrounded by a small rim of activated microglia with recent myelin-degradation products; inactive lesions = lesions without any recent demyelinating activity; NAWM multiple sclerosis = normal-appearing white matter from patients with multiple sclerosis; white matter controls = normal white matter of all controls; APP = amyloid precursor protein reactive axonal spheroids or end bulbs; CD3 = T cells; percentage of E06 positive area = densitometric analysis of area covered by E06 immunoreactivity; E06 axon spheroids = axonal spheroids or end bulbs stained by E06 antibody; HLA-D = class II MHC-positive macrophages/microglia; MDA-2 OG = oligodendrocyte-like cells, immunoreactive for MDA-2; 8-OHdG nuclei = number of cell nuclei containing 8 hydroxy-D-guanosine immunoreactivity. Data show a highly significant accumulation of oxidized DNA and oxidized lipids in active multiple sclerosis lesions in comparison with controls. Oxidized DNA and lipids are predominantly seen in lesions with high T-cell and macrophage infiltrates and with profound microglia activation. (p values not included for brevity).

A recent review details the current immunomodulatory treatments for MS. Other alternatives beyond immune-directed approaches are also speculated in this review [Aktas et. al., 2010]. Increased concentrations of reactive oxygen and nitrogen species found in MS, for example, lead to inhibition of ATP production within the axon. The ATP deficiency leads to loss of Na+/K+ ATPase and collapse of transmembrane ionic gradients. There is also an increase in intracellular Ca2+ levels and a decrease in mRNA levels of mitochondrial genes. At the same time immune –related demyelination also takes place. These results have prompted ion channel homeostasis as a potential therapeutic target to ameliorate the failed

Peroxy nitrite formation at the site of inflammation has been measured using nitrotyrosine as a biochemical marker. Levels of nitrite and nitrate, the stable oxidation products of nitric oxide and peroxynitrite measured in cerebral fluid samples also revealed significantly

higher levels of nitrate during clinical relapses of MS [Cross et. al., 1998].

E06 axon spheroids 1.3 (18.2) 1.2 (121.2) 0.4 (2.3) 0.0 (1.2) 0.00 (0.0) MDA-2 OG 1.6 (25.8) 0.9 (5.4) 0.9 (1.6) 0.4 (9.2) 0.00 (1.6) 8-OHdG nuclei 5.6 (78.1) 9.0 (19.0) 1.6 (6.8) 1.4 (12.0) 0.40 (10.4) APP 44.3 (197.6) 16.1 (40.0) 0.3 (1.5) 0.0 (1.0) 0.00 (0.8) CD3 73.6 (298.8) 51.2 (105.6) 10.0 (69.1) 4.8 (38.4) 9.60 (49.6) HLA-D 324.8 (585.6) 148.3 (398.0) 29.0 (246.5) 99.3 (190.0) 43.10 (114.4) Table 2. Quantification of oxidized lipids and oxidized DNA in different types of multiple sclerosis lesions in comparison with controls [Haider et. al., 2011]. Values represent medians and range (95th percentile range values); P-values are corrected by Bonferroni for multiple

Inactive lesion NAWM MS White matter

Median & (range)

Median & (range)

3.1(26.5) 1.6 (16.7) 0.6 (9.2) 0.8 (17.1) 0.06 (2.9)

controls

Median & (range)

Active lesion Slowly expanding Lesion

Median & (range)

[For details, please see Haider et. al., 2011]

energy metabolism.

Percentage of E06 positive area

Table 1. Quantification of the intensity of complex IV active elements and mitochondrial mass within axons [Mahad et. al., 2009]. The intensity of complex IV active elements represents the difference in densitometric value between background and complex IV active elements in inverted grey scale 100x brightfield images of cytochrome c oxidase or COX histochemistry. The percentage area of porin reactive elements within axons was calculated based on the total area of axonal porin reactive elements in triple labeled (porin, syntaphilin and axonal marker) and area of axons in confocal images. †P = 0.001 (versus NAWM) and ‡P = 0.002 (versus CON). #P<0.001 (versus SMI31 in CON, NAWM and lesion). \*P<0.001 (versus SMI31 in NAWM and CON as well as SMI32 in lesion and APP in lesion). [For details, please see Mahad et. al., 2009]

Mitochondrial injury and subsequent energy failure have been implicated in the pathogenesis of MS [Lu et. al., 2000; Dutta et. al., 2006; Mahad et. al., 2008, 2009; Haider et. al., 2011; Campbell et. al., 2011]. Proteins and DNA in mitochondria are highly vulnerable to free radical damage and consequent mitochondrial injury in MS. The likely candidates involved in tissue injury in MS are the ROS and nitric oxide intermediates. These are produced by activated macrophages and microglia. In the brain tissue of patients with MS, oxidized DNA and oxidized lipids have been detected [Lu et. al., 2000]. Oxidized phospholipids and malondialdehyde (lipid peroxidation-derived structures) data from MS lesions of different activity of patients with acute, relapsing, remitting and progressive disease were found to be concentrated in active MS plaques, in areas known as initial demyelinating lesion or the "prephagocytic" stage of active MS lesions [Haider et. al., 2011]. There was good correlation between inflammation and the extent of DNA and lipid oxidation. Data in table 2 indicate up to a 5 fold increase in the extent of DNA damage (8-OHdG staining) and lipid oxidation (E06 and MDA-2 staining) in active lesions versus inactive lesions, normal white matter in MS patients, and white matter controls. The oxidation is predominantly seen in lesions with high T-cell and macrophage infiltrates and with profound microglial activation (HLA-D staining).

So far the efforts for complete restoration of axonal mitochondria following remyelination have not been successful. Thus the need for preservation of myelinated axons is exemplified by the fact that remyelinated axons have increased energy demand. This may also result in deficient neurons and reach detrimental levels in the long term. Mitochondrial DNA deletions have been found in the neurons in the progressive stage of MS [Campbell et. al., 2011]. The pathological features of MS lesions include demyelination and oligodendrocyte apoptosis, preferential destruction of small- caliber axons, differentiation arrest of oligodendrocyte progenitor cells and remyelination failure, and astrocyte dysfunction [Haider et. al., 2011].

Control(SMI31) NAWM(SMI31) Lesion(APP) Lesion(SMI32) Lesion(SM131)

Brian 17.93±5.89 16.14±6.54 9.85±6.94# 10.26±5.43# 20.10±7.38†, ‡ Spinal cord 16.99±15.49 16.87±12.44 9.01±11.16# 15.93±13.01 23.04±11.07\*

Brian 6.74±.2.82 7.35±4.30 7.93±4.50 4.19±3.95† 11.15±5.23\* Spinal cord 5.93±4.38 3.46±3.92 8.26±7.40 1.64 ±1.39# 13.97±7.23\* Table 1. Quantification of the intensity of complex IV active elements and mitochondrial mass within axons [Mahad et. al., 2009]. The intensity of complex IV active elements represents the difference in densitometric value between background and complex IV active elements in inverted grey scale 100x brightfield images of cytochrome c oxidase or COX histochemistry. The percentage area of porin reactive elements within axons was calculated based on the total area of axonal porin reactive elements in triple labeled (porin, syntaphilin and axonal marker) and area of axons in confocal images. †P = 0.001 (versus NAWM) and ‡P = 0.002 (versus CON). #P<0.001 (versus SMI31 in CON, NAWM and lesion). \*P<0.001 (versus SMI31 in NAWM and CON as well as SMI32 in lesion and APP in lesion). [For details, please see

Mitochondrial injury and subsequent energy failure have been implicated in the pathogenesis of MS [Lu et. al., 2000; Dutta et. al., 2006; Mahad et. al., 2008, 2009; Haider et. al., 2011; Campbell et. al., 2011]. Proteins and DNA in mitochondria are highly vulnerable to free radical damage and consequent mitochondrial injury in MS. The likely candidates involved in tissue injury in MS are the ROS and nitric oxide intermediates. These are produced by activated macrophages and microglia. In the brain tissue of patients with MS, oxidized DNA and oxidized lipids have been detected [Lu et. al., 2000]. Oxidized phospholipids and malondialdehyde (lipid peroxidation-derived structures) data from MS lesions of different activity of patients with acute, relapsing, remitting and progressive disease were found to be concentrated in active MS plaques, in areas known as initial demyelinating lesion or the "prephagocytic" stage of active MS lesions [Haider et. al., 2011]. There was good correlation between inflammation and the extent of DNA and lipid oxidation. Data in table 2 indicate up to a 5 fold increase in the extent of DNA damage (8-OHdG staining) and lipid oxidation (E06 and MDA-2 staining) in active lesions versus inactive lesions, normal white matter in MS patients, and white matter controls. The oxidation is predominantly seen in lesions with high T-cell and macrophage infiltrates and

So far the efforts for complete restoration of axonal mitochondria following remyelination have not been successful. Thus the need for preservation of myelinated axons is exemplified by the fact that remyelinated axons have increased energy demand. This may also result in deficient neurons and reach detrimental levels in the long term. Mitochondrial DNA deletions have been found in the neurons in the progressive stage of MS [Campbell et. al., 2011]. The pathological features of MS lesions include demyelination and oligodendrocyte apoptosis, preferential destruction of small- caliber axons, differentiation arrest of oligodendrocyte progenitor cells and remyelination failure, and astrocyte dysfunction

**Complex IV Intensity**

Mahad et. al., 2009]

[Haider et. al., 2011].

with profound microglial activation (HLA-D staining).

**Porin Percent Area**

Table 2. Quantification of oxidized lipids and oxidized DNA in different types of multiple sclerosis lesions in comparison with controls [Haider et. al., 2011]. Values represent medians and range (95th percentile range values); P-values are corrected by Bonferroni for multiple testing; all values represent counted cells/mm2. Active lesions = classical actively demyelinating lesions; slowly expanding lesions = lesions with inactive lesion centre, surrounded by a small rim of activated microglia with recent myelin-degradation products; inactive lesions = lesions without any recent demyelinating activity; NAWM multiple sclerosis = normal-appearing white matter from patients with multiple sclerosis; white matter controls = normal white matter of all controls; APP = amyloid precursor protein reactive axonal spheroids or end bulbs; CD3 = T cells; percentage of E06 positive area = densitometric analysis of area covered by E06 immunoreactivity; E06 axon spheroids = axonal spheroids or end bulbs stained by E06 antibody; HLA-D = class II MHC-positive macrophages/microglia; MDA-2 OG = oligodendrocyte-like cells, immunoreactive for MDA-2; 8-OHdG nuclei = number of cell nuclei containing 8 hydroxy-D-guanosine immunoreactivity. Data show a highly significant accumulation of oxidized DNA and oxidized lipids in active multiple sclerosis lesions in comparison with controls. Oxidized DNA and lipids are predominantly seen in lesions with high T-cell and macrophage infiltrates and with profound microglia activation. (p values not included for brevity). [For details, please see Haider et. al., 2011]

A recent review details the current immunomodulatory treatments for MS. Other alternatives beyond immune-directed approaches are also speculated in this review [Aktas et. al., 2010]. Increased concentrations of reactive oxygen and nitrogen species found in MS, for example, lead to inhibition of ATP production within the axon. The ATP deficiency leads to loss of Na+/K+ ATPase and collapse of transmembrane ionic gradients. There is also an increase in intracellular Ca2+ levels and a decrease in mRNA levels of mitochondrial genes. At the same time immune –related demyelination also takes place. These results have prompted ion channel homeostasis as a potential therapeutic target to ameliorate the failed energy metabolism.

Peroxy nitrite formation at the site of inflammation has been measured using nitrotyrosine as a biochemical marker. Levels of nitrite and nitrate, the stable oxidation products of nitric oxide and peroxynitrite measured in cerebral fluid samples also revealed significantly higher levels of nitrate during clinical relapses of MS [Cross et. al., 1998].

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 107

Therapeutic quick intervention may reduce the infarct volume in the penumbra because the irreversible damage occurs relatively slowly [Sims & Muyderman, 2010]. The only embolic or thrombolytic agent used to reverse arterial occlusion within the first 3 h is tissue plasminogen activator (tPA). As an enzyme, it catalyzes the conversion of plasminogen to the enzyme responsible for clot breakdown, plasmin. In ~17 % of ischemic stroke patients spontaneous reversal of occlusion takes place in 6 h and in 40-50% of patients in 4 days. Quick restoration of normal blood flow to this region can result in substantial recovery of energy- related metabolites. However, in the post ischemic tissue, energy requirements are low and the glucose oxidation is due to limitations on the mitochondrial oxidation of pyruvate. This results in a secondary impairment of mitochondrial function and consequent

cell death. Normalcy of function can be maintained in the outermost viable tissue.

vessel, e.g. middle cerebral artery, resulting in primarily apoptotic cell death.

leading to apoptosis and necrosis.

nitric oxide (NO**.**

While focal ischemia is confined to a specific region of the brain, global ischemia encompasses wide areas of the brain tissue. Focal ischemia occurs in a region when a blood clot has occluded in a downstream region of artery in the brain (ischemic stroke). This occlusion or blockage may be caused by thrombosis (a blood clot formed locally obstructing the blood flow) or arterial embolism (obstruction of blood flow due to an embolus from elsewhere in the body). While ischemic strokes are caused by interruption of the blood supply, hemorrhagic strokes are the result of rupture of a blood vessel. Hemorrhagic strokes result in areas of friable tissue, containing areas of both viable and dead tissue. Transient global ischemia involves a brief interruption in blood flow usually in a larger cerebral

More than 80% of all strokes are due to focal ischemia. Unless treated, the occlusion of an artery produces tissue infarction resulting in a loss of all cells including neurons, astrocytes, oligodendrocytes, microglia and endothelial cell [Sims & Muyderman, 2010]. The stroke results in mitochondrial impairment because the blood flow becomes < 20% of the normal and a consequent reduction in glucose and oxygen supplies ensue. This attenuates ATP and reactive oxygen species production as well as apoptosis. Lack of ATP production disrupts the ionic gradients across the plasma membrane. The net result is marked losses of intracellular K+ and a large influx of Ca2+ into cells [Doyle et. al., 2008]. Thus there is heavy involvement of the impaired mitochondria in the development of the tissue injury after ischemic attack, due to modifications in ATP production and other mitochondrial changes

Further, a transient zone in-between the core and penumbra is likely to merge to the core if the cerebral blood flow is not restored early. The damage in this transient zone is a result of the release of cellular contents from those necrotic "core" cells, such as the oxidative enzymes of various organelles i.e. lysozomes and peroxisomes. Eventually this transient zone would contribute to the total infarct volume. The penumbral region is surrounded by a region of viable tissue [Mehta et. al., 2007]. Penumbra varies in size and can be rescued Ischemic cell death is also attributed to abnormal activation of enzymes such as poly-ADP ribose polymerase (PARP) and the caspases. Oxidative stress, which produces free radical

apoptosis in focal ischemia. Peroxynitrite is formed by the reaction of NO**.** with superoxide. Mitochondria are targeted by peroxynitrite and the resulting mitochondrial dysfunction during severe hypoxia-ischemia increases generation of oxygen free radicals. This leads to dysfunction of cellular membrane causing necrosis [Mehta et. al. 2007]. An additional consequence of ischemia involves the dissociation of the electron transport chain within minutes of the insult. Ubiquinone and cytochrome C, which serve as electron shuttles,

) and reactive peroxynitrite (ONOO-), is implicated in both necrosis and

In view of our data with palladium α-lipoic acid formulation (section 10) on the enhanced enzymatic activities of Krebs cycle and electron transport chain enzymes in animals, antioxidant activity and the ability of this formulation to repair DNA, we had decided, as a preliminary step, to investigate its usefulness in ameliorating the fatigue conditions in 15 MS patients. The study is expected to be completed soon.

#### **8. Cerebral ischemia**

An insufficient or reduced blood flow to the brain to meet the metabolic demand will result in cerebral or brain ischemia.  A prototype of brain damage during cerebral ischemia is shown in Fig. 4. [adapted from Mehta et. al., 2007]. Maximum damage occurs as a result of ischemic necrosis (infarction) at the "core" or "focal" tissue region, where the blood flow is < 7 mL/100g/min. Since the cellular integrity is compromised during necrosis, cellular damage repair at the core is extremely hard. The blood flow in the surrounding less-severely ischemic boundary ("penumbral" or "perifocal" tissue) is ~ 7 to 17 mL/100g/min. The penumbra is metabolically active but electrically silent [Mehta et. al., 2007]. More moderate alterations develop in this region because of the near normal glucose use, but the oxidative metabolism is still impaired. Different mechanisms contribute to cell death in the core and penumbra due to differences in the severity of ischemia.

Fig. 4. A prototype brain damage during cerebral ischemia: core, a region where cells undergo necrosis. The region surrounding the core is called ischemic penumbra, a site of delayed mode of cell death (apoptosis) due to availability of ATP. Further, a transient zone in-between the core and penumbra is likely to merge to the core if the cerebral blood flow is not restored early. The penumbral region is surrounded by a region of viable tissue [adapted from Mehta et. al., 2007]

In view of our data with palladium α-lipoic acid formulation (section 10) on the enhanced enzymatic activities of Krebs cycle and electron transport chain enzymes in animals, antioxidant activity and the ability of this formulation to repair DNA, we had decided, as a preliminary step, to investigate its usefulness in ameliorating the fatigue conditions in 15 MS

An insufficient or reduced blood flow to the brain to meet the metabolic demand will result in cerebral or brain ischemia.  A prototype of brain damage during cerebral ischemia is shown in Fig. 4. [adapted from Mehta et. al., 2007]. Maximum damage occurs as a result of ischemic necrosis (infarction) at the "core" or "focal" tissue region, where the blood flow is < 7 mL/100g/min. Since the cellular integrity is compromised during necrosis, cellular damage repair at the core is extremely hard. The blood flow in the surrounding less-severely ischemic boundary ("penumbral" or "perifocal" tissue) is ~ 7 to 17 mL/100g/min. The penumbra is metabolically active but electrically silent [Mehta et. al., 2007]. More moderate alterations develop in this region because of the near normal glucose use, but the oxidative metabolism is still impaired. Different mechanisms contribute to cell death in the core and

Fig. 4. A prototype brain damage during cerebral ischemia: core, a region where cells undergo necrosis. The region surrounding the core is called ischemic penumbra, a site of delayed mode of cell death (apoptosis) due to availability of ATP. Further, a transient zone in-between the core and penumbra is likely to merge to the core if the cerebral blood flow is

not restored early. The penumbral region is surrounded by a region of viable tissue

[adapted from Mehta et. al., 2007]

patients. The study is expected to be completed soon.

penumbra due to differences in the severity of ischemia.

**8. Cerebral ischemia**

Therapeutic quick intervention may reduce the infarct volume in the penumbra because the irreversible damage occurs relatively slowly [Sims & Muyderman, 2010]. The only embolic or thrombolytic agent used to reverse arterial occlusion within the first 3 h is tissue plasminogen activator (tPA). As an enzyme, it catalyzes the conversion of plasminogen to the enzyme responsible for clot breakdown, plasmin. In ~17 % of ischemic stroke patients spontaneous reversal of occlusion takes place in 6 h and in 40-50% of patients in 4 days. Quick restoration of normal blood flow to this region can result in substantial recovery of energy- related metabolites. However, in the post ischemic tissue, energy requirements are low and the glucose oxidation is due to limitations on the mitochondrial oxidation of pyruvate. This results in a secondary impairment of mitochondrial function and consequent cell death. Normalcy of function can be maintained in the outermost viable tissue.

While focal ischemia is confined to a specific region of the brain, global ischemia encompasses wide areas of the brain tissue. Focal ischemia occurs in a region when a blood clot has occluded in a downstream region of artery in the brain (ischemic stroke). This occlusion or blockage may be caused by thrombosis (a blood clot formed locally obstructing the blood flow) or arterial embolism (obstruction of blood flow due to an embolus from elsewhere in the body). While ischemic strokes are caused by interruption of the blood supply, hemorrhagic strokes are the result of rupture of a blood vessel. Hemorrhagic strokes result in areas of friable tissue, containing areas of both viable and dead tissue. Transient global ischemia involves a brief interruption in blood flow usually in a larger cerebral vessel, e.g. middle cerebral artery, resulting in primarily apoptotic cell death.

More than 80% of all strokes are due to focal ischemia. Unless treated, the occlusion of an artery produces tissue infarction resulting in a loss of all cells including neurons, astrocytes, oligodendrocytes, microglia and endothelial cell [Sims & Muyderman, 2010]. The stroke results in mitochondrial impairment because the blood flow becomes < 20% of the normal and a consequent reduction in glucose and oxygen supplies ensue. This attenuates ATP and reactive oxygen species production as well as apoptosis. Lack of ATP production disrupts the ionic gradients across the plasma membrane. The net result is marked losses of intracellular K+ and a large influx of Ca2+ into cells [Doyle et. al., 2008]. Thus there is heavy involvement of the impaired mitochondria in the development of the tissue injury after ischemic attack, due to modifications in ATP production and other mitochondrial changes leading to apoptosis and necrosis.

Further, a transient zone in-between the core and penumbra is likely to merge to the core if the cerebral blood flow is not restored early. The damage in this transient zone is a result of the release of cellular contents from those necrotic "core" cells, such as the oxidative enzymes of various organelles i.e. lysozomes and peroxisomes. Eventually this transient zone would contribute to the total infarct volume. The penumbral region is surrounded by a region of viable tissue [Mehta et. al., 2007]. Penumbra varies in size and can be rescued

Ischemic cell death is also attributed to abnormal activation of enzymes such as poly-ADP ribose polymerase (PARP) and the caspases. Oxidative stress, which produces free radical nitric oxide (NO**.** ) and reactive peroxynitrite (ONOO-), is implicated in both necrosis and apoptosis in focal ischemia. Peroxynitrite is formed by the reaction of NO**.** with superoxide. Mitochondria are targeted by peroxynitrite and the resulting mitochondrial dysfunction during severe hypoxia-ischemia increases generation of oxygen free radicals. This leads to dysfunction of cellular membrane causing necrosis [Mehta et. al. 2007]. An additional consequence of ischemia involves the dissociation of the electron transport chain within minutes of the insult. Ubiquinone and cytochrome C, which serve as electron shuttles,

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 109

The adenylate energy charge decreases rapidly to ~ 0.4-0.5 during the initial hours and remains above 0.8 after 2 h compared to the normal value in the brain of ~ 0.93. While adenylate kinase catalyses the conversion of some ADP to AMP to meet short term energy needs of the brain, in ischemic tissue, the adenine nucleotide pool is depleted by the conversion of AMP to inosine and hypoxanthine. Phosphocreatine, the short term energy reserve of the brain falls quickly to <30% of normal during ischemia. Phosphocreatine stabilizes to about 70% of normal after ~ 2 h of ischemia. ATP regeneration from ADP is catalyzed by the enzyme creatine kinase. Lack of oxygen forces glucose to go the glycolytic pathway creating a 10-fold increase in lactate and consequent lowering of pH. Of course lack of removal of lactate due to limited blood flow may also be contributing to this accumulation. In addition, restricted blood flow appears to have a greater effect on the delivery of oxygen to the tissues versus glucose, since penumbral glucose levels are either the same or slightly higher, while lactate levels are much higher (but less than in the core). During the first 2 h following reperfusion, phosphocreatine and adenylate energy charge are recovered to >90% of normal compared to ATP values of 50-70%. This resistance of ATP for restoration is attributed to the depletion of the adenine nucleotide pool. In the penumbral tissue, phosphocreatine and adenine nucleotide balance, but not ATP are recovered almost completely within 1 h of reperfusion for ischemic periods of 3 h or longer [Sims & Muyderman, 2010]. The glucose utilization is less in the penumbral region during the first

hour of reperfusion. The lactate, on the other hand, is decreased during this period.

kinase enzyme [Sims & Muyderman, 2010].

irreversible cell dysfunction.

The reduction in ATP production in the ischemic brain may be associated with decreased neuronal activity of the post-ischemic brain as a result of the enzyme AMP-activated protein

It must be mentioned that there is a complete or near complete recovery of mitochondrial respiratory function in core and penumbral tissues within the first hour following reperfusion. This is followed by a secondary deterioration, indicative of the development of

Under normal physiological conditions, the channel within the N-methyl-D-aspartic acid (NMDA) glutamate receptor is blocked in a voltage dependent manner by Mg2+. The triggering of energy deficits and neuronal depolarization are the results of decreased cerebral blood flow. The mild depolarization results in the dislodging of Mg2+ and glutamate, which are consequently released in large amounts to the extracellular space. This leads to an over-activation of the NMDA and AMPA glutamate receptors. Since these receptors regulate Ca2+ ion channels, a calcium ionic imbalance occurs in neurons. This influx of Ca2+, is due to this increase in glutamate release from neurons and astrocytes induced by the ischemia. Apart from the traditional ionotropic glutamate receptors, the influx of Ca2+ is also attributed to some emerging metabotropic and channel mechanisms that include: sodium-calcium exchangers, hemi channels (unopposed half-gap junctions), acid sensing channels, volume-regulated anion channels, nonselective cation channels and signaling cascades that mediate crosstalk between redundant pathways of cell death [Besancon et al., 2008]. This abnormal intracellular accumulation of Ca2+ is involved in the triggering of cell death by up regulation of a wide range of cell death executioners that include ATPases that serve to further deplete energy stores, lipases that damage lipid membranes of organelles and the cell surface itself, proteases that dismantle the cytoarchitecture of the neuron, and DNAses that damage the nucleus [Besancon et. al. 2008]. Bioenergetics of cerebral ischemia (both focal and global) as well as gray and white matter ischemia were recently reviewed from a cellular perspective. A brief summary is given in

translocate from the inner mitochondrial membrane. This is of particular consequence upon restoration of blood flow. While reperfusion limits some damage, oxidative stress is increased under these conditions. It has been found that over expression of Mn2+-superoxide dismutase, which converts superoxide to hydrogen peroxide results in moderate reductions in the size of infarction in temporary ischemia [Sims & Muyderman, 2010]. Addition of the mitochondrial uncoupling agent, dinitrophenol, was found to modulate the Ca2+ content and production of free radicals in the mitochondria of penumbra [Korde et. al., 2005].

The reduced delivery of oxygen and glucose to the tissue in focal ischemia affects the function of the mitochondria. Mitochondrial properties undergo further changes depending on the severity and duration of ischemia and also following reperfusion. Development of cell death pathways depends on the impaired mitochondria's ability to generate ATP.

Table 3. The effects of focal ischemia for up to 2 h and of subsequent reperfusion for 1 h on the content of energy-related metabolites and pathways of energy metabolism [Sims & Muyderman, 2010]. Differences are shown compared to non-ischemic tissue. ↓: decreased to >65%; ↓↓: decreased to between 35% and 65% ; ↓↓↓: decreased to less than 35%; ↑: increased less than four-fold; ↑↑: increased greater than four-fold; N.C.: no significant change. Two symbols indicate findings that differ between published reports. \*, direct evaluation of these properties in severely ischemic tissue may not give reliable information. The magnitude of these reductions is assumed from the large decrease in substrate delivery and large changes in ATP and phosphocreatine content [Sims & Muyderman, 2010]

The changes in energy-related metabolites and in the contributing metabolic pathways in brain tissue in the first 2 h of ischemia and reperfusion for 1 h are summarized in Table 3 [Sims & Muyderman, 2010]. In the core, the ATP and glucose content falls significantly in the first 5 min of occlusion and then ATP stabilizes to ~ 15-30% of normal for at least the first 2 h and then reaches about 50%. The initial rapid decrease is attributed to the major redistribution of ions across the plasma membrane of cells. In view of our admittance data on the H2O2-Ca2+, H2O2-Na+ interactions, we strongly believe that ion-peroxide interactions must also be playing a major part in this process. The adenylate energy charge, a measure of intracellular balance between ATP, ADP, and AMP, is given by:

Adenylate energy charge = {[ATP] + 0.5 [ADP]/ [ATP] + [ADP] + [AMP]} (31)

translocate from the inner mitochondrial membrane. This is of particular consequence upon restoration of blood flow. While reperfusion limits some damage, oxidative stress is increased under these conditions. It has been found that over expression of Mn2+-superoxide dismutase, which converts superoxide to hydrogen peroxide results in moderate reductions in the size of infarction in temporary ischemia [Sims & Muyderman, 2010]. Addition of the mitochondrial uncoupling agent, dinitrophenol, was found to modulate the Ca2+ content and production of free radicals in the mitochondria of penumbra [Korde et. al., 2005]. The reduced delivery of oxygen and glucose to the tissue in focal ischemia affects the function of the mitochondria. Mitochondrial properties undergo further changes depending on the severity and duration of ischemia and also following reperfusion. Development of cell death pathways depends on the impaired mitochondria's ability to generate ATP.

**Focal ischemia Reperfusion**

ATP ↓↓↓ ↓↓ ↓↓ ↓ Adenylate energy charge ↓↓ ↓ ↓/N.C. N.C. Total adenine nucleotides ↓↓ ↓↓ ↓↓ ↓ Phosphocreatine ↓↓↓ ↓ ↓ N.C. Lactate ↑↑ ↑↑ ↑↑ ↑/N.C. Glucose ↓↓↓ N.C. N.C. N.C.

Glucose use ↓↓↓\* N.C. ↓↓ ↓↓ Oxidative metabolism ↓↓↓\* ↓↓↓ ↓↓ ↓↓

in ATP and phosphocreatine content [Sims & Muyderman, 2010]

intracellular balance between ATP, ADP, and AMP, is given by:

Table 3. The effects of focal ischemia for up to 2 h and of subsequent reperfusion for 1 h on the content of energy-related metabolites and pathways of energy metabolism [Sims & Muyderman, 2010]. Differences are shown compared to non-ischemic tissue. ↓: decreased to >65%; ↓↓: decreased to between 35% and 65% ; ↓↓↓: decreased to less than 35%; ↑: increased less than four-fold; ↑↑: increased greater than four-fold; N.C.: no significant change. Two symbols indicate findings that differ between published reports. \*, direct evaluation of these properties in severely ischemic tissue may not give reliable information. The magnitude of these reductions is assumed from the large decrease in substrate delivery and large changes

The changes in energy-related metabolites and in the contributing metabolic pathways in brain tissue in the first 2 h of ischemia and reperfusion for 1 h are summarized in Table 3 [Sims & Muyderman, 2010]. In the core, the ATP and glucose content falls significantly in the first 5 min of occlusion and then ATP stabilizes to ~ 15-30% of normal for at least the first 2 h and then reaches about 50%. The initial rapid decrease is attributed to the major redistribution of ions across the plasma membrane of cells. In view of our admittance data on the H2O2-Ca2+, H2O2-Na+ interactions, we strongly believe that ion-peroxide interactions must also be playing a major part in this process. The adenylate energy charge, a measure of

Adenylate energy charge = {[ATP] + 0.5 [ADP]/ [ATP] + [ADP] + [AMP]} (31)

**Metabolites**

**Metabolic activity**

Core Penumbra Core Penumbra

The adenylate energy charge decreases rapidly to ~ 0.4-0.5 during the initial hours and remains above 0.8 after 2 h compared to the normal value in the brain of ~ 0.93. While adenylate kinase catalyses the conversion of some ADP to AMP to meet short term energy needs of the brain, in ischemic tissue, the adenine nucleotide pool is depleted by the conversion of AMP to inosine and hypoxanthine. Phosphocreatine, the short term energy reserve of the brain falls quickly to <30% of normal during ischemia. Phosphocreatine stabilizes to about 70% of normal after ~ 2 h of ischemia. ATP regeneration from ADP is catalyzed by the enzyme creatine kinase. Lack of oxygen forces glucose to go the glycolytic pathway creating a 10-fold increase in lactate and consequent lowering of pH. Of course lack of removal of lactate due to limited blood flow may also be contributing to this accumulation. In addition, restricted blood flow appears to have a greater effect on the delivery of oxygen to the tissues versus glucose, since penumbral glucose levels are either the same or slightly higher, while lactate levels are much higher (but less than in the core).

During the first 2 h following reperfusion, phosphocreatine and adenylate energy charge are recovered to >90% of normal compared to ATP values of 50-70%. This resistance of ATP for restoration is attributed to the depletion of the adenine nucleotide pool. In the penumbral tissue, phosphocreatine and adenine nucleotide balance, but not ATP are recovered almost completely within 1 h of reperfusion for ischemic periods of 3 h or longer [Sims & Muyderman, 2010]. The glucose utilization is less in the penumbral region during the first hour of reperfusion. The lactate, on the other hand, is decreased during this period.

The reduction in ATP production in the ischemic brain may be associated with decreased neuronal activity of the post-ischemic brain as a result of the enzyme AMP-activated protein kinase enzyme [Sims & Muyderman, 2010].

It must be mentioned that there is a complete or near complete recovery of mitochondrial respiratory function in core and penumbral tissues within the first hour following reperfusion. This is followed by a secondary deterioration, indicative of the development of irreversible cell dysfunction.

Under normal physiological conditions, the channel within the N-methyl-D-aspartic acid (NMDA) glutamate receptor is blocked in a voltage dependent manner by Mg2+. The triggering of energy deficits and neuronal depolarization are the results of decreased cerebral blood flow. The mild depolarization results in the dislodging of Mg2+ and glutamate, which are consequently released in large amounts to the extracellular space. This leads to an over-activation of the NMDA and AMPA glutamate receptors. Since these receptors regulate Ca2+ ion channels, a calcium ionic imbalance occurs in neurons. This influx of Ca2+, is due to this increase in glutamate release from neurons and astrocytes induced by the ischemia. Apart from the traditional ionotropic glutamate receptors, the influx of Ca2+ is also attributed to some emerging metabotropic and channel mechanisms that include: sodium-calcium exchangers, hemi channels (unopposed half-gap junctions), acid sensing channels, volume-regulated anion channels, nonselective cation channels and signaling cascades that mediate crosstalk between redundant pathways of cell death [Besancon et al., 2008]. This abnormal intracellular accumulation of Ca2+ is involved in the triggering of cell death by up regulation of a wide range of cell death executioners that include ATPases that serve to further deplete energy stores, lipases that damage lipid membranes of organelles and the cell surface itself, proteases that dismantle the cytoarchitecture of the neuron, and DNAses that damage the nucleus [Besancon et. al. 2008]. Bioenergetics of cerebral ischemia (both focal and global) as well as gray and white matter ischemia were recently reviewed from a cellular perspective. A brief summary is given in

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 111

Five minutes of carotid artery occlusion was sufficient to hinder the characteristic nesting behavior of gerbils for ~ 3 days. Their nesting behavior was observed to improve significantly after treatment with palladium lipoic acid complex formulation (50 mg/kg every 24h and 30 mg/kg /24h at 24 and 72 hours after ischemia. The lack of nesting behavior at 70 mg/kg-treated animals was attributed to their excessive energy and

It was observed that preventive or prophylactic treatment with 10 mg/kg gerbil (or allometric scaling equivalent of 10 mL-human dosage) offered significant behavioral and

Our studies demonstrate a greater protective effect of palladium α-lipoic acid complex versus α-lipoic acid alone (Cao & Phillis, 1995). Four times more α-lipoic acid and for a longer period of pre-treatment were necessary to obtain morphologiocal protection. Further more immediate administration of palladium α-lipoic acid complex formulation protected over 70%of the CA1 neurons, and administration delayed up to 24 hours after the TIA still offered significant protection (30% of the CA1 pyramidal cells) [Antonawich et. al., 2004].

Alpha- lipoic acid is a very unique and simple biological molecule. It has a carboxylic acid group with a pKa of 4.7. It is ionized at biological pH, and it has a cyclic disulfide or dithiolane ring [Baumgartner et. al., 1996; Patel & Packer, 2008]. It exists intracellularly as the reduced form, (±)-dihydrolipoic acid. Lipoic acid occurs naturally as a coenzyme in both prokaryotic and eukaryotic cells, as well as in plants, and animals including humans. It is

α-Lipoic acid, absorbed intact from the diet, is readily converted into dihydrolipoic acid in many tissues. In the intracellular environment, two or more enzymes reduce the exogenous lipoic acid. The reversible reduction to dihydrolipoic acid is favored by the presence of the ring strain in the 1, 2-dithiolane ring of about 15-25 kJmol-1 [Baumgartner et. al., 1996]. α-Lipoic acid exits as R(+)-and S(-)- enantiomers due to the presence of an asymmetric carbon. The biologically active enantiomer is mostly the former one. Since its first isolation in 1951, numerous investigations have been carried out to decipher the uniqueness of this

Molecular mechanisms and therapeutic potential of α-lipoic acid, a dietary supplement, have been reviewed recently [Shay et. al., 2009]. Lipoic acid can cross the blood-brain barrier. The biological effects of lipoic acid are attributed to its redox property, the antioxidant capacity and the fatty acid properties. There is ample evidence indicating the usefulness of the lipoic acid/dihydrolipoic acid redox couple as a therapeutic agent for diabetes, ischemia-reperfusion injury, heavy metal poisoning, modulator of various inflammatory signaling pathways, age associated cardiovascular, cognitive, and neuromuscular deficits, protection from radiation damage, neurodegeneration, and HIV infection [Packer et. al., 1995; Patel & Packer, 2008; Shay et.al., 2009]. Dihydrolipoic acid can regenerate or recycle the antioxidants CoQ (ubiquinol), vitamins C and E (via glutathione), and glutathione without itself becoming one in the process. Dihydrolipoic acid also prevents lipid peroxidation by regenerating glutathione. [Packer et. al., 1995; Patel & Packer, 2008]. Lipoic acid and dihydrolipoic acid are efficiently transported in and out of both mitochondria and cells. Compared to this, the transport of disulfides such as cystine that is needed in modulating glutathione (GSH) levels in cells is very inefficient. The mitochondrial

consequent ignoring of the nesting material.

morphological improvement from transient global ischemia.

**9. The powerful super-antioxidant,** α**-lipoic acid**

enzymatically synthesized from octanoic acid in the mitochondrion.

simple but elegant molecule [Reed 2001; Patel & Packer, 2008].

Table 4 [Hertz, 2008]. During the early stages of ischemia, fatal injury is observed for neurons and oligodendrocytes. They are very sensitive to the excitotoxicity of glutamate due to their cell process expression of NMDA receptors and cell body expression of AMPA/kinase receptors. Astrocytes and endothelial cells seem to survive longer. Neurons are damaged from lack of astrocyte support. Axonal injury is due to channel mediated Na+ uptake followed by Na+/Ca2+ exchange.

Table 4. Bioenergetic mechanisms involved in ischemic death of different cell types and constituents [Hertz, 2008]

Studies have demonstrated that ischemic damage may be reduced by blockade of ionotropic glutamate receptors using glutamate receptor antagonists [Mehta et. al., 2007; Besancon et. al., 2008; Doyle et. al., 2008; Sims & Muyderman, 2010]. The most extensively evaluated neuroprotectors that include calcium channel blockers, glutamate antagonists, GABA agonists, antioxidants and radical scavengers, and NO**.** signal down regulator, have been critically reviewed recently [Ginsberg, 2008].

Other neuroprotective approaches involve the use of anti-oxidants. As an example, α-lipoic acid reduced the mortality rate of male Sprague-Dawley rats from 78% to 26% during 24 hours of reperfusion. It was found effective in improving survival and protecting the rat brain against reperfusion injury following cerebral ischemia [Panigrahi et. al., 1996]. In another study rats that received subcutaneous treatment of R-or S-lipoic acid for 2 hours before ischemia significantly reduced the infarct volume [Wolz & Krieglstein, 1996]. Similar results with mice were obtained with 100 mg/kg of lipoic acid given subcutaneously 1.5 hours before ischemia [Clark et. al., 2001]. Transient global ischemia also benefits from pretreatment with α-lipoic acid. Administration of 40 mg/kg for 7 days protected from ischemic damage when gerbils were tested for locomotor behavior and morphological damage to the CA1 region of the hippocampus [Cao & Phillis, 1995].

Animal studies, using adult male Mongolian gerbils, used as controls or treatment group with palladium α-lipoic acid complex formulation (PdLA), demonstrated that acute, post ischemic and prophylactic administration of PdLA limits ischemic damage [Antonawich et. al., 2004]. Following bilateral carotid artery occlusion in the gerbil, the PdLA was administered intraperitoneally (IP) immediately after surgery, then once daily for 3 days. The control group received saline. PdLA treatment significantly protected hippocampal pyramidal cells (CA1) from transient global ischemia at 30, 50, and 70 mg/kg per 24 h.

While a delayed application of the palladium α-lipoic acid complex formulation after 48 hours of ischemic attack had no significant effect in protecting CA 1 cells, a delayed administration after 6 hours of ischemic attack was as good as giving it immediately after ischemic attack in minimizing cell death.

Five minutes of carotid artery occlusion was sufficient to hinder the characteristic nesting behavior of gerbils for ~ 3 days. Their nesting behavior was observed to improve significantly after treatment with palladium lipoic acid complex formulation (50 mg/kg every 24h and 30 mg/kg /24h at 24 and 72 hours after ischemia. The lack of nesting behavior at 70 mg/kg-treated animals was attributed to their excessive energy and consequent ignoring of the nesting material.

It was observed that preventive or prophylactic treatment with 10 mg/kg gerbil (or allometric scaling equivalent of 10 mL-human dosage) offered significant behavioral and morphological improvement from transient global ischemia.

Our studies demonstrate a greater protective effect of palladium α-lipoic acid complex versus α-lipoic acid alone (Cao & Phillis, 1995). Four times more α-lipoic acid and for a longer period of pre-treatment were necessary to obtain morphologiocal protection. Further more immediate administration of palladium α-lipoic acid complex formulation protected over 70%of the CA1 neurons, and administration delayed up to 24 hours after the TIA still offered significant protection (30% of the CA1 pyramidal cells) [Antonawich et. al., 2004].

#### **9. The powerful super-antioxidant,** α**-lipoic acid**

110 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

Table 4 [Hertz, 2008]. During the early stages of ischemia, fatal injury is observed for neurons and oligodendrocytes. They are very sensitive to the excitotoxicity of glutamate due to their cell process expression of NMDA receptors and cell body expression of AMPA/kinase receptors. Astrocytes and endothelial cells seem to survive longer. Neurons are damaged from lack of astrocyte support. Axonal injury is due to channel mediated Na+

Neurons Axons Oligodendrocytes Astrocytes Endothelial

Table 4. Bioenergetic mechanisms involved in ischemic death of different cell types and

Studies have demonstrated that ischemic damage may be reduced by blockade of ionotropic glutamate receptors using glutamate receptor antagonists [Mehta et. al., 2007; Besancon et. al., 2008; Doyle et. al., 2008; Sims & Muyderman, 2010]. The most extensively evaluated neuroprotectors that include calcium channel blockers, glutamate antagonists, GABA

Other neuroprotective approaches involve the use of anti-oxidants. As an example, α-lipoic acid reduced the mortality rate of male Sprague-Dawley rats from 78% to 26% during 24 hours of reperfusion. It was found effective in improving survival and protecting the rat brain against reperfusion injury following cerebral ischemia [Panigrahi et. al., 1996]. In another study rats that received subcutaneous treatment of R-or S-lipoic acid for 2 hours before ischemia significantly reduced the infarct volume [Wolz & Krieglstein, 1996]. Similar results with mice were obtained with 100 mg/kg of lipoic acid given subcutaneously 1.5 hours before ischemia [Clark et. al., 2001]. Transient global ischemia also benefits from pretreatment with α-lipoic acid. Administration of 40 mg/kg for 7 days protected from ischemic damage when gerbils were tested for locomotor behavior and morphological

Animal studies, using adult male Mongolian gerbils, used as controls or treatment group with palladium α-lipoic acid complex formulation (PdLA), demonstrated that acute, post ischemic and prophylactic administration of PdLA limits ischemic damage [Antonawich et. al., 2004]. Following bilateral carotid artery occlusion in the gerbil, the PdLA was administered intraperitoneally (IP) immediately after surgery, then once daily for 3 days. The control group received saline. PdLA treatment significantly protected hippocampal pyramidal cells (CA1) from transient global ischemia at 30, 50, and 70 mg/kg per 24 h. While a delayed application of the palladium α-lipoic acid complex formulation after 48 hours of ischemic attack had no significant effect in protecting CA 1 cells, a delayed administration after 6 hours of ischemic attack was as good as giving it immediately after

Intracellular Na+ increase X X X X X Increased metabolism X ? ? X ? Intracellular Ca2+ increase X X X X ? Mitochondrial damage X X X ? Formation of ROS X X (X) X

cells

signal down regulator, have been

uptake followed by Na+/Ca2+ exchange.

agonists, antioxidants and radical scavengers, and NO**.**

damage to the CA1 region of the hippocampus [Cao & Phillis, 1995].

critically reviewed recently [Ginsberg, 2008].

ischemic attack in minimizing cell death.

constituents [Hertz, 2008]

Alpha- lipoic acid is a very unique and simple biological molecule. It has a carboxylic acid group with a pKa of 4.7. It is ionized at biological pH, and it has a cyclic disulfide or dithiolane ring [Baumgartner et. al., 1996; Patel & Packer, 2008]. It exists intracellularly as the reduced form, (±)-dihydrolipoic acid. Lipoic acid occurs naturally as a coenzyme in both prokaryotic and eukaryotic cells, as well as in plants, and animals including humans. It is enzymatically synthesized from octanoic acid in the mitochondrion.

α-Lipoic acid, absorbed intact from the diet, is readily converted into dihydrolipoic acid in many tissues. In the intracellular environment, two or more enzymes reduce the exogenous lipoic acid. The reversible reduction to dihydrolipoic acid is favored by the presence of the ring strain in the 1, 2-dithiolane ring of about 15-25 kJmol-1 [Baumgartner et. al., 1996]. α-Lipoic acid exits as R(+)-and S(-)- enantiomers due to the presence of an asymmetric carbon. The biologically active enantiomer is mostly the former one. Since its first isolation in 1951, numerous investigations have been carried out to decipher the uniqueness of this simple but elegant molecule [Reed 2001; Patel & Packer, 2008].

Molecular mechanisms and therapeutic potential of α-lipoic acid, a dietary supplement, have been reviewed recently [Shay et. al., 2009]. Lipoic acid can cross the blood-brain barrier. The biological effects of lipoic acid are attributed to its redox property, the antioxidant capacity and the fatty acid properties. There is ample evidence indicating the usefulness of the lipoic acid/dihydrolipoic acid redox couple as a therapeutic agent for diabetes, ischemia-reperfusion injury, heavy metal poisoning, modulator of various inflammatory signaling pathways, age associated cardiovascular, cognitive, and neuromuscular deficits, protection from radiation damage, neurodegeneration, and HIV infection [Packer et. al., 1995; Patel & Packer, 2008; Shay et.al., 2009]. Dihydrolipoic acid can regenerate or recycle the antioxidants CoQ (ubiquinol), vitamins C and E (via glutathione), and glutathione without itself becoming one in the process. Dihydrolipoic acid also prevents lipid peroxidation by regenerating glutathione. [Packer et. al., 1995; Patel & Packer, 2008].

Lipoic acid and dihydrolipoic acid are efficiently transported in and out of both mitochondria and cells. Compared to this, the transport of disulfides such as cystine that is needed in modulating glutathione (GSH) levels in cells is very inefficient. The mitochondrial

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 113

and superoxide radical. The reduction potential for the α-lipoic acid/dihydrolipoic acid couple of -320 mV or -290 mV [Krishnan et. al., 2011] and the GSSG/GSH) couple of -240 mV indicate that dihydrolipoic acid can react with GSSG and regenerate GSH [Packer et. al., 1995]. Thus lipoic acid helps to maintain GSH/GSSG ratio (about 100 to 10,000 times greater than other redox couples such as NAD+/NADH, and NADP+/NADPH), an estimate of

Treatment with lipoic acid increases the GSH levels in human cell lines and primary cells including T cells, erythrocytes, lymphocytes, and glial and neuroblastoma cells.. This is explained by 1) facile transport of lipoic acid into cells, where it is reduced by NADH or NADPH dependent pathways to dihydrolipoic acid. 2) Dihydrolipoic acid is transported back into the extracellular media where it is oxidized by cysteine regenerating lipoic acid and producing cysteine, the limiting substrate on GSH synthesis. 3) Compared to cystine, cysteine is more easily transported into the cell and aids the synthesis of GSH [Patel &

Thus elevated levels of GSH and ascorbic acid, which in turn regenerates vitamin E, are all indicative of lipoic acid acting as an inducer of endogenous antioxidants. It has been reported that lipoic acid is also an effective regulator of signaling pathways and induces synthesis of GSH transcriptionally. Lipoic acid reverses the decline in transcriptional activity

The pharmacokinetics of R-lipoic acid, reviewed recently [Patel & Packer, 2008; Shay et.al., 2009], revealed a plasma level concentration, Cmax, of 1.154 μg/mL from 1 g R-lipoic acid compared to the proposed therapeutic range of 10-20 μg/mL or 50-100 μM (Carlson et al., 2008). A dose of 600-800 mg sodium R-lipoate gave plasma levels of 8-18 μg/mL, which is within the therapeutic range. The upper limit suggested for therapeutic action of 45 μg/mL or 225 µM is reached by a dose of about 1.2 g of racemic-α-lipoic acid. The no adverse observed effect level (NOAEL) of racemic lipoic acid is considered to be 60 mg/kg body mass/day. Therapeutic and energy production applications of this powerful antioxidant

Located within the mitochondrial matrix are lipoic acid requiring enzymes: three α-keto acid dehydrogenase complexes that catalyze the oxidative decarboxylation of α-keto acids such as pyruvate, α-ketoglutarate, and branched chain α-ketoacids [Voet D. and Voet J. G., 1995]. In organisms, hydrogen atom transfer and acyl group transfer take place in the oxidative decarboxylation of α-ketoacids with the aid of α-lipoic acid. The reversible redox reaction between α-lipoic acid and dihydrolipoic acid is thus a very important biochemical reaction. The reversible reduction to dihydrolipoic acid is favored by the presence of the

The multienzyme complex, pyruvate dehydrogenase, consists of three enzymes, pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3) [Voet D. and Voet J. G., 1995]. This enzyme complex participates in five sequential reactions during the conversion of pyruvate to acetyl-CoA. The R- lipoic acid is covalently linked to a ε-amino group of lysine residue via an amide linkage. These lipoic acid

The multienzyme complex, α-ketoglutarate dehydrogenase, also consists of three enzymes, α-ketoglutarate dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase (E3) [Voet D. and Voet J. G., 1995]. The branched chain α-ketoacid dehydrogenase is also a multienzyme complex resembling the other two enzymes

ring strain in the 1,2-dithiolane ring of about 15-25 kJmol-1 [Patel & Packer, 2008].

containing enzymes participate in four out of the five reactions.

redox state, in cells [Patel & Packer, 2008].

of Nrf2 caused by age-related loss of GSH [Suh et. al, 2004].

have been explored extensively [Patel & Packer, 2008].

Packer, 2008].

β-oxidation of lipoic acid has been attributed to its fatty acid properties, similar to that of octanoic acid [Patel & Packer, 2008]. Redox as well as biological antioxidant effects have been attributed to the β-oxidation products of lipoic acid, the oxidized and reduced forms of bisnorlipoic acid and tetranorlipoic acid [Patel & Packer, 2008].

Table 5. Antioxidant activities of lipoic acid (LA) and dihydrolipoic acid (DHLA) [detailed references in Shay et. al., 2009]

The α-lipoic acid/dihydrolipoic acid couple is called a "universal antioxidant" because it fulfills several criteria used to evaluate the antioxidant potential as well as preventive or therapeutic applications of a compound such as specificity of free radical quenching, metal chelating ability, interaction with other antioxidants, effects on gene expression, absorption and bioavailability, concentration in tissues, cells, and extracellular fluid, and location (in aqueous or membrane domains or in both) [Packer et.al., 1995].

The free radical scavenging activities of lipoic acid and dihydrolipoic acid are given in Table 5 [Shay et.al., 2009]. Both are capable of scavenging peroxynitrite, peroxyl radical and hypochlorous acid, but not hydrogen peroxide. There are conflicting data for singlet oxygen and radicals such as hydroxyl, superoxide, and peroxyl.

Questions have been raised regarding the direct-acting antioxidant status of LA/DHLA in vivo based on the invitro data [Shay et.al., 2009]. This is due to the large dosage, 200-600 mg, used for invivo studies and the amounts found in the plasma (both area under the curve and Cmax are in the range of microgram to nanogram levels per mL). The administration of lipoic acid in acid form or salt form, oral or intravenous, with a meal or without meal also contributed to the fluctuations in the data. Also the invitro data cannot imitate the invivo clearance of 98% of LA excretion in the urine within 24 hours.

α-Lipoic acid was found to protect hematopoietic tissues in mice from radiation damage [Ramakrishnan et. al., 1992]. It was also found that α-lipoic acid offered protection from radiation for children affected by the Chernobyl nuclear accident [Korkina et al., 1993]. α-Lipoic acid scavenges hydroxyl radicals but is not effective against hydrogen peroxide

β-oxidation of lipoic acid has been attributed to its fatty acid properties, similar to that of octanoic acid [Patel & Packer, 2008]. Redox as well as biological antioxidant effects have been attributed to the β-oxidation products of lipoic acid, the oxidized and reduced forms of

Scavenged or not by DHLA

and rate constant

and rate constant

Peroxynitrite Yes, 1.4 x 103 M-1 S-1 Yes, 2.5 x 102 M-1 S-1 Nitric oxide No Yes, 3.19 M-1 S-1

Yes, 3.3 x 105 M-1 S-1

Peroxyl radical Yes, 1.8 x 108 M-1 S-1 Yes, 2.3 x 107 M-1 S-1

Table 5. Antioxidant activities of lipoic acid (LA) and dihydrolipoic acid (DHLA) [detailed

The α-lipoic acid/dihydrolipoic acid couple is called a "universal antioxidant" because it fulfills several criteria used to evaluate the antioxidant potential as well as preventive or therapeutic applications of a compound such as specificity of free radical quenching, metal chelating ability, interaction with other antioxidants, effects on gene expression, absorption and bioavailability, concentration in tissues, cells, and extracellular fluid, and location (in

The free radical scavenging activities of lipoic acid and dihydrolipoic acid are given in Table 5 [Shay et.al., 2009]. Both are capable of scavenging peroxynitrite, peroxyl radical and hypochlorous acid, but not hydrogen peroxide. There are conflicting data for singlet oxygen

Questions have been raised regarding the direct-acting antioxidant status of LA/DHLA in vivo based on the invitro data [Shay et.al., 2009]. This is due to the large dosage, 200-600 mg, used for invivo studies and the amounts found in the plasma (both area under the curve and Cmax are in the range of microgram to nanogram levels per mL). The administration of lipoic acid in acid form or salt form, oral or intravenous, with a meal or without meal also contributed to the fluctuations in the data. Also the invitro data cannot imitate the invivo

α-Lipoic acid was found to protect hematopoietic tissues in mice from radiation damage [Ramakrishnan et. al., 1992]. It was also found that α-lipoic acid offered protection from radiation for children affected by the Chernobyl nuclear accident [Korkina et al., 1993]. α-Lipoic acid scavenges hydroxyl radicals but is not effective against hydrogen peroxide

bisnorlipoic acid and tetranorlipoic acid [Patel & Packer, 2008].

Hydroxyl radical Yes, 4.7 x 1010 M-1 S-1 No Yes Yes Superoxide No No

 Yes Singlet oxygen Yes, 1.3 x 108 M-1 S-1 No

 No Yes Hypochlorous acid Yes Yes Hydrogen peroxide No No

aqueous or membrane domains or in both) [Packer et.al., 1995].

and radicals such as hydroxyl, superoxide, and peroxyl.

clearance of 98% of LA excretion in the urine within 24 hours.

Yes

references in Shay et. al., 2009]

Oxidant Scavenged or not by LA

and superoxide radical. The reduction potential for the α-lipoic acid/dihydrolipoic acid couple of -320 mV or -290 mV [Krishnan et. al., 2011] and the GSSG/GSH) couple of -240 mV indicate that dihydrolipoic acid can react with GSSG and regenerate GSH [Packer et. al., 1995]. Thus lipoic acid helps to maintain GSH/GSSG ratio (about 100 to 10,000 times greater than other redox couples such as NAD+/NADH, and NADP+/NADPH), an estimate of redox state, in cells [Patel & Packer, 2008].

Treatment with lipoic acid increases the GSH levels in human cell lines and primary cells including T cells, erythrocytes, lymphocytes, and glial and neuroblastoma cells.. This is explained by 1) facile transport of lipoic acid into cells, where it is reduced by NADH or NADPH dependent pathways to dihydrolipoic acid. 2) Dihydrolipoic acid is transported back into the extracellular media where it is oxidized by cysteine regenerating lipoic acid and producing cysteine, the limiting substrate on GSH synthesis. 3) Compared to cystine, cysteine is more easily transported into the cell and aids the synthesis of GSH [Patel & Packer, 2008].

Thus elevated levels of GSH and ascorbic acid, which in turn regenerates vitamin E, are all indicative of lipoic acid acting as an inducer of endogenous antioxidants. It has been reported that lipoic acid is also an effective regulator of signaling pathways and induces synthesis of GSH transcriptionally. Lipoic acid reverses the decline in transcriptional activity of Nrf2 caused by age-related loss of GSH [Suh et. al, 2004].

The pharmacokinetics of R-lipoic acid, reviewed recently [Patel & Packer, 2008; Shay et.al., 2009], revealed a plasma level concentration, Cmax, of 1.154 μg/mL from 1 g R-lipoic acid compared to the proposed therapeutic range of 10-20 μg/mL or 50-100 μM (Carlson et al., 2008). A dose of 600-800 mg sodium R-lipoate gave plasma levels of 8-18 μg/mL, which is within the therapeutic range. The upper limit suggested for therapeutic action of 45 μg/mL or 225 µM is reached by a dose of about 1.2 g of racemic-α-lipoic acid. The no adverse observed effect level (NOAEL) of racemic lipoic acid is considered to be 60 mg/kg body mass/day. Therapeutic and energy production applications of this powerful antioxidant have been explored extensively [Patel & Packer, 2008].

Located within the mitochondrial matrix are lipoic acid requiring enzymes: three α-keto acid dehydrogenase complexes that catalyze the oxidative decarboxylation of α-keto acids such as pyruvate, α-ketoglutarate, and branched chain α-ketoacids [Voet D. and Voet J. G., 1995]. In organisms, hydrogen atom transfer and acyl group transfer take place in the oxidative decarboxylation of α-ketoacids with the aid of α-lipoic acid. The reversible redox reaction between α-lipoic acid and dihydrolipoic acid is thus a very important biochemical reaction. The reversible reduction to dihydrolipoic acid is favored by the presence of the ring strain in the 1,2-dithiolane ring of about 15-25 kJmol-1 [Patel & Packer, 2008].

The multienzyme complex, pyruvate dehydrogenase, consists of three enzymes, pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3) [Voet D. and Voet J. G., 1995]. This enzyme complex participates in five sequential reactions during the conversion of pyruvate to acetyl-CoA. The R- lipoic acid is covalently linked to a ε-amino group of lysine residue via an amide linkage. These lipoic acid containing enzymes participate in four out of the five reactions.

The multienzyme complex, α-ketoglutarate dehydrogenase, also consists of three enzymes, α-ketoglutarate dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase (E3) [Voet D. and Voet J. G., 1995]. The branched chain α-ketoacid dehydrogenase is also a multienzyme complex resembling the other two enzymes

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 115

the concentration of lipoic acid and the scanning potential, radicals and polymers are formed [Krishnan & Garnett 2011]. Also our data suggest the need for a revalidation of the

Fig. 5. Cyclic voltammogram of 1 mM α-lipoic acid (sodium salt), pH 7.2, scan 2.0 to -2.0 V and back, scan 3, scan rate a and c, 100mV/sec; b. scan rates 1) 400 2) 200 3) 100 4) 50

in a. [Krishnan & Garnett 2011]

**complex**

mV/sec; **c.** 1) scan 2.0 to -2.0 V and back, 2) scan 1.0 to -0.5V and back; peak 7 same as peak 7

Our electrochemical data also suggested a caution in deciding on the dosage of oral supplements of lipoic acid because of its tendency to form dimers and higher polymers under reducing biological conditions. The pharmacokinetic data mentioned earlier is also

**10. Catalytic, electronic, and therapeutic properties of palladium α-lipoic acid**

The coordination chemistry of palladium complexes have recently been reviewed with an emphasis on cancer therapy[Gao et. al., 2009]. Even though there are many structural and thermodynamic similarities between the complexes of palladium and platinum, the palladium(II) complexes seem to exhibit biological action very different from those of the

subject to these complexities depending on the dosage chosen for the studies.

reported redox potential of the lipoic acid/dihydrolipoic acid couple.

mentioned above. These three enzymes have the same dihydrolipoyl dehydrogenase and employ the coenzymes thiamine pyrophosphate, lipoamide, FAD and the terminal oxidizing agent NAD+ [Voet D. and Voet J. G., 1995]. The importance of lipoic acid in the energy metabolism is illustrated by these three enzymes.

Disulfide bonds in proteins are formed between the thiol groups of cysteine residues. The other sulfur-containing amino acid, methionine, cannot form disulfide bonds. More aggressive oxidants convert cysteine to the corresponding sulfinic acid and sulfonic acid. A variety of oxidants promote this reaction including air and hydrogen peroxide. Such reactions are thought to proceed via sulfenic acid intermediates.

The oxidized form α-lipoic acid can undergo further oxidation at sulfur or get reduced. This property, which is somewhat similar to an intermediate oxidation state in a transition metal or a nonmetal, makes this molecule very unique compared to other biological molecules. A biological oxidation product of α-lipoic acid (lipoic acid S-oxide or a thiolsulfinate) is known as β-lipoic acid or Protogen-B [Baumgartner et. al., 1996]. It has not been possible to conclusively prove which sulfur is oxidized [Stary et. al. 1975].

The oxidation of α-lipoic acid was found to be a one-electron charge transfer process, pH independent, and an irreversible process [Corduneanu et. al., 2009]. Most voltammetric studies were centered on the oxidation of α-lipoic acid and studies related to its reduction to dihydrolipoic acid were limited [Rogers & Mallett, 1983]. To understand the electrochemical behavior of α-lipoic acid, we had explored potential regions beyond the normal range of the mercury electrode, both on the cathodic side as well as on the anodic side. The advantage in using the mercury electrode is the ease with which we can obtain a fresh consistent surface and drop. We have not observed (visually) any passivation of mercury on the anodic side when lipoic acid was present without any background electrolyte. We had investigated the cathodic side, the normal potential region for mercury working electrode, in much greater detail.

The complexity of the redox process of α-lipoic acid is shown in the cyclic voltammograms in Fig. 5 [Krishnan & Garnett, 2011]. We had assigned the three cathodic peaks to 1) reduction of lipoic acid S-oxide, 2) reduction of lipoic acid to most probably dihydrolipoic acid, and 3) probable reduction of lipoic acid dimers or higher polymers. We have assigned the five anodic peaks to 4) the oxidation product of lipoic acid dimers or higher polymers, 5) oxidation product of dihydrolipoic acid, 6) formation of S-oxide of lipoic acid dimers or higher polymers, 7) formation of lipoic acid S-oxide, and 8) further oxidation of lipoic acid S-oxide/the formation of thiolsulfonates. The peak due to the formation of lipoic acid S-oxide (peak 7) is missing in Figure 3a because of the formation of S-oxide of lipoic acid dimers or higher polymers (peak 6). However, when the formation of lipoic acid polymers is minimized by restricting the scan to less cathodic potentials (to -0.5 V instead of -2.0 V) the formation of S-oxide of lipoic acid dimers or higher polymers is minimized and thus allowing the formation of lipoic acid S-oxide (peak 7) [Krishnan & Garnett, 2011]. Other complexities were expressed at differing scan rates, at higher concentrations and by the presence of electrolytes. The data shown in Fig. 5 are for 1mM α-lipoic acid where solutesolute interactions are minimized and in the absence of electrolytes so that the influence of ion-dipole interactions can be more readily investigated at the double layer.

The molecule, α-lipoic acid, is very unique because of its ability to form a variety of radicals, dimers and higher polymers as well as a variety of lipoic acid S-oxides. This complexity is reflected in its electrochemical redox behavior. Our data demonstrated that depending on

mentioned above. These three enzymes have the same dihydrolipoyl dehydrogenase and employ the coenzymes thiamine pyrophosphate, lipoamide, FAD and the terminal oxidizing agent NAD+ [Voet D. and Voet J. G., 1995]. The importance of lipoic acid in the energy

Disulfide bonds in proteins are formed between the thiol groups of cysteine residues. The other sulfur-containing amino acid, methionine, cannot form disulfide bonds. More aggressive oxidants convert cysteine to the corresponding sulfinic acid and sulfonic acid. A variety of oxidants promote this reaction including air and hydrogen peroxide. Such

The oxidized form α-lipoic acid can undergo further oxidation at sulfur or get reduced. This property, which is somewhat similar to an intermediate oxidation state in a transition metal or a nonmetal, makes this molecule very unique compared to other biological molecules. A biological oxidation product of α-lipoic acid (lipoic acid S-oxide or a thiolsulfinate) is known as β-lipoic acid or Protogen-B [Baumgartner et. al., 1996]. It has not been possible to

The oxidation of α-lipoic acid was found to be a one-electron charge transfer process, pH independent, and an irreversible process [Corduneanu et. al., 2009]. Most voltammetric studies were centered on the oxidation of α-lipoic acid and studies related to its reduction to dihydrolipoic acid were limited [Rogers & Mallett, 1983]. To understand the electrochemical behavior of α-lipoic acid, we had explored potential regions beyond the normal range of the mercury electrode, both on the cathodic side as well as on the anodic side. The advantage in using the mercury electrode is the ease with which we can obtain a fresh consistent surface and drop. We have not observed (visually) any passivation of mercury on the anodic side when lipoic acid was present without any background electrolyte. We had investigated the cathodic side, the normal potential region for mercury working electrode, in much greater

The complexity of the redox process of α-lipoic acid is shown in the cyclic voltammograms in Fig. 5 [Krishnan & Garnett, 2011]. We had assigned the three cathodic peaks to 1) reduction of lipoic acid S-oxide, 2) reduction of lipoic acid to most probably dihydrolipoic acid, and 3) probable reduction of lipoic acid dimers or higher polymers. We have assigned the five anodic peaks to 4) the oxidation product of lipoic acid dimers or higher polymers, 5) oxidation product of dihydrolipoic acid, 6) formation of S-oxide of lipoic acid dimers or higher polymers, 7) formation of lipoic acid S-oxide, and 8) further oxidation of lipoic acid S-oxide/the formation of thiolsulfonates. The peak due to the formation of lipoic acid S-oxide (peak 7) is missing in Figure 3a because of the formation of S-oxide of lipoic acid dimers or higher polymers (peak 6). However, when the formation of lipoic acid polymers is minimized by restricting the scan to less cathodic potentials (to -0.5 V instead of -2.0 V) the formation of S-oxide of lipoic acid dimers or higher polymers is minimized and thus allowing the formation of lipoic acid S-oxide (peak 7) [Krishnan & Garnett, 2011]. Other complexities were expressed at differing scan rates, at higher concentrations and by the presence of electrolytes. The data shown in Fig. 5 are for 1mM α-lipoic acid where solutesolute interactions are minimized and in the absence of electrolytes so that the influence of

ion-dipole interactions can be more readily investigated at the double layer.

The molecule, α-lipoic acid, is very unique because of its ability to form a variety of radicals, dimers and higher polymers as well as a variety of lipoic acid S-oxides. This complexity is reflected in its electrochemical redox behavior. Our data demonstrated that depending on

metabolism is illustrated by these three enzymes.

detail.

reactions are thought to proceed via sulfenic acid intermediates.

conclusively prove which sulfur is oxidized [Stary et. al. 1975].

the concentration of lipoic acid and the scanning potential, radicals and polymers are formed [Krishnan & Garnett 2011]. Also our data suggest the need for a revalidation of the reported redox potential of the lipoic acid/dihydrolipoic acid couple.

Fig. 5. Cyclic voltammogram of 1 mM α-lipoic acid (sodium salt), pH 7.2, scan 2.0 to -2.0 V and back, scan 3, scan rate a and c, 100mV/sec; b. scan rates 1) 400 2) 200 3) 100 4) 50 mV/sec; **c.** 1) scan 2.0 to -2.0 V and back, 2) scan 1.0 to -0.5V and back; peak 7 same as peak 7 in a. [Krishnan & Garnett 2011]

Our electrochemical data also suggested a caution in deciding on the dosage of oral supplements of lipoic acid because of its tendency to form dimers and higher polymers under reducing biological conditions. The pharmacokinetic data mentioned earlier is also subject to these complexities depending on the dosage chosen for the studies.

#### **10. Catalytic, electronic, and therapeutic properties of palladium α-lipoic acid complex**

The coordination chemistry of palladium complexes have recently been reviewed with an emphasis on cancer therapy[Gao et. al., 2009]. Even though there are many structural and thermodynamic similarities between the complexes of palladium and platinum, the palladium(II) complexes seem to exhibit biological action very different from those of the

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 117

The antioxidant status in the liver, kidney and brain of mice after exposure to 2-6 Gy radiation were also measured recently [Menon et. al., 2009]. The results were similar to the ones observed in the heart of rats without radiation. Also an analysis of their blood leukocytes and bone marrow for DNA damage using alkaline single cell gel electrophoresis (alkaline comet assay) revealed DNA repair from the lowering of comet assay parameters, DNA in tail, tail length, tail moment, and olive tail moment. Administration of the complex also reduced the mortality of the mice and also aided recovery from the radiation induced weight loss [Ramachandran et. al., 2010]. DNA repair was also observed in human blood leukocytes with palladium α-lipoic acid complex treatment immediately after exposure to

The mechanism of the superiority of the metal complex compared to that of the ligand is still an unsolved puzzle. Compared to the high oral dose, the available plasma concentration of α-lipoic acid is very small. One possibility for the higher activity of the metal complex is probably due to its increased concentration in the plasma. This needs experimental verification. It is also known that 98% of α-lipoic acid is excreted in urine within 24 hours and since it takes 4-6 weeks for serum and urine clearance of palladium, the previous suggestion seems justified. Another possibility is the chemistry of the transition metal playing a dominant role in the enzymatic activity. The starting material in the synthesis of the palladium α-lipoic acid complex is palladium(II). The final complex is also palladium(II), based on preliminary ESR data. Palladium(II) complexes are diamagnetic. The complex concentration dependent electrochemical characteristics of α-lipoic acid suggest the possibility of free radical formation by one electron reduction under physiological conditions. In such a case the electron spin may be involved in the enzymatic process. The impedance characteristics of the palladium α-lipoic acid as well as that of the α-lipoic acid,

Another possibility for the superiority of the palladium α-lipoic acid complex compared to α-lipoic acid is its ability to form self-assembled structures, such as the one shown in Fig. 6. No self-assembly was observed for sodium lipoate. We want to point out that binding of a lysine residue in the protein to the lipoyl group of E2 in 2-oxoacid dehydrogenases results in a long flexible arm that can oscillate a distance of ~200 Å. This arm is utilized during the catalytic cycle [Patel & Packer, 2008]. It is obvious that the self assembled palladium α-lipoic

The physics and chemistry of non-equilibrium systems have been utilized to understand some of the spatial patterns and temporal patterning observed in biological processes such as bacterial colonies shaped by diffusive instabilities and calcium waves governed by

In homogeneous systems, spiral waves and spatiotemporal phenomena are formed from autocatalytic reactions and diffusion resulting from chemical instabilities. Our data suggest that the propagation of electrical signaling among the packing units and extending to long

We have utilized the technique of electrochemical impedance spectroscopy extensively to probe spatiotemporal phenomena in biological systems. This technique is routinely used to study corrosion and fuel cells. We have used admittance measurements for understanding solute-solvent interactions, "π –way" conduction, ion pair formation, water-structure enforced ion pair formation, potential induced and solvent mediated ion pair formation at the double layer, and semi conduction characteristics of simple biological molecules

nonlinear amplification during intracellular signaling [Levine & Jacob, 2004].

distances by global coupling is viable by such self-assembled systems.

radiation [Menon & Nair, 2011].

described in this section, strongly suggest this possibility.

acid complex can make this process more facile.

toxic platinum complexes. Copper, zinc, and arsenic complexes of α-lipoic acid and palladium α-lipoic acid complexes with 1:1 and 1:2 stoichiometry have been reported [Garnett, 1995a, 1995b; Strasdeit et. al., 1995; Baumgartner et. al., 1996].

Palladium α-lipoic acid complex has demonstrated numerous antitumor activities against various cell lines. Also it was found to halt the growth of glioblastoma in nude mice. Clinical veterinary studies indicated its effectiveness as a complimentary support to chemotherapy. A recent Phase I, dose escalation study, has revealed safety in humans up to 40 mL 0.037 M of this complex with no severe adverse events, and minor adverse events i.e. mild gastrointestinal irritation, and aversion to taste. Washout periods for palladium, monitored in blood serum and urine, ranged from three to seventeen weeks after cessation of the formulation [Krishnan et. al., 2011].

The electrochemical characteristics of α-lipoic acid and palladium α-lipoic acid complex have been explored using glassy carbon and highly oriented pyrolytic graphite electrodes [Corduneanu et al., 2007, 2009]. Palladium complex was found to dissociate at negative potentials with deposition of Pd(0) nanoparticles. The application of a positive potential induced the oxidation of the palladium complex and the formation of a mixed layer of lipoic acid and palladium oxides.

Superior free radical scavenging capacity, as measured by oxygen radical absorbance capacity or ORAC analysis, was observed for palladium α-lipoic acid complex formulation (5.65) compared to vitamin E (1.0, normalized value), vitamin A (1.6), vitamin C (1.12), and α-lipoic acid (1.4) [Krishnan et.al., 2011]. This may be compared to the superior antitumor activities observed for metal complexes compared to that of their ligands [Matesanz et. al., 1999; Maloň et. al., 2001].

The enhanced energy effects observed in gerbils during transient ischemia studies [Antonawich et. al., 2004] prompted us to investigate the influence of palladium α-lipoic acid complex on the activities of enzymes involved in mitochondrial energy production. The activities of four Krebs cycle enzymes, isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (α-KGDH), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH) and mitochondrial complexes I, II, III, and IV in aged male albino rats were investigated [Sudheesh et. al., 2009]. The enhanced activity of the metal complex was superior to the activity of lipoic acid, the ligand.

The antioxidant status in the heart of aged male Albino Wistar rats, measured by MnSOD, CAT, and GSHPx, were higher with the palladium lipoic acid treated group than with the α-lipoic acid group [Sudheesh et al., 2010]. This was also true in alloxan induced diabetic rats [Sudheesh et. al. 2011]. Similarly the lipid peroxidation levels were lowered and the GSH levels were increased in the palladium α-lipoic acid treated group. It is not clear at this time whether scavenging some free radicals by either α-lipoic acid or palladium α-lipoic acid complex formulation is connected in any way to the enhanced activities of Krebs cycle and mitochondrial enzymes.

A specific example is illustrated here. The α-ketoglutarate dehydrogenase complex (KGDH) is a critical component of Krebs cycle and of glutamate metabolism. Glutamate is an excitotoxic neurotransmitter. Reactive oxygen species modify the activity of KGDH. It is also known that the activity of KGDH is lower than that of any other enzyme in the brain. Deficiencies in KGDH lead to brain neurological syndromes. Palladium α-lipoic acid complex increases the activity of KGDH and thus helps in the removal of the glutamate.

toxic platinum complexes. Copper, zinc, and arsenic complexes of α-lipoic acid and palladium α-lipoic acid complexes with 1:1 and 1:2 stoichiometry have been reported

Palladium α-lipoic acid complex has demonstrated numerous antitumor activities against various cell lines. Also it was found to halt the growth of glioblastoma in nude mice. Clinical veterinary studies indicated its effectiveness as a complimentary support to chemotherapy. A recent Phase I, dose escalation study, has revealed safety in humans up to 40 mL 0.037 M of this complex with no severe adverse events, and minor adverse events i.e. mild gastrointestinal irritation, and aversion to taste. Washout periods for palladium, monitored in blood serum and urine, ranged from three to seventeen weeks after cessation of the

The electrochemical characteristics of α-lipoic acid and palladium α-lipoic acid complex have been explored using glassy carbon and highly oriented pyrolytic graphite electrodes [Corduneanu et al., 2007, 2009]. Palladium complex was found to dissociate at negative potentials with deposition of Pd(0) nanoparticles. The application of a positive potential induced the oxidation of the palladium complex and the formation of a mixed layer of lipoic

Superior free radical scavenging capacity, as measured by oxygen radical absorbance capacity or ORAC analysis, was observed for palladium α-lipoic acid complex formulation (5.65) compared to vitamin E (1.0, normalized value), vitamin A (1.6), vitamin C (1.12), and α-lipoic acid (1.4) [Krishnan et.al., 2011]. This may be compared to the superior antitumor activities observed for metal complexes compared to that of their ligands [Matesanz et. al.,

The enhanced energy effects observed in gerbils during transient ischemia studies [Antonawich et. al., 2004] prompted us to investigate the influence of palladium α-lipoic acid complex on the activities of enzymes involved in mitochondrial energy production. The activities of four Krebs cycle enzymes, isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (α-KGDH), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH) and mitochondrial complexes I, II, III, and IV in aged male albino rats were investigated [Sudheesh et. al., 2009]. The enhanced activity of the metal complex was

The antioxidant status in the heart of aged male Albino Wistar rats, measured by MnSOD, CAT, and GSHPx, were higher with the palladium lipoic acid treated group than with the α-lipoic acid group [Sudheesh et al., 2010]. This was also true in alloxan induced diabetic rats [Sudheesh et. al. 2011]. Similarly the lipid peroxidation levels were lowered and the GSH levels were increased in the palladium α-lipoic acid treated group. It is not clear at this time whether scavenging some free radicals by either α-lipoic acid or palladium α-lipoic acid complex formulation is connected in any way to the enhanced activities of Krebs cycle

A specific example is illustrated here. The α-ketoglutarate dehydrogenase complex (KGDH) is a critical component of Krebs cycle and of glutamate metabolism. Glutamate is an excitotoxic neurotransmitter. Reactive oxygen species modify the activity of KGDH. It is also known that the activity of KGDH is lower than that of any other enzyme in the brain. Deficiencies in KGDH lead to brain neurological syndromes. Palladium α-lipoic acid complex increases the activity of KGDH and thus helps in the removal of the

[Garnett, 1995a, 1995b; Strasdeit et. al., 1995; Baumgartner et. al., 1996].

formulation [Krishnan et. al., 2011].

acid and palladium oxides.

1999; Maloň et. al., 2001].

and mitochondrial enzymes.

glutamate.

superior to the activity of lipoic acid, the ligand.

The antioxidant status in the liver, kidney and brain of mice after exposure to 2-6 Gy radiation were also measured recently [Menon et. al., 2009]. The results were similar to the ones observed in the heart of rats without radiation. Also an analysis of their blood leukocytes and bone marrow for DNA damage using alkaline single cell gel electrophoresis (alkaline comet assay) revealed DNA repair from the lowering of comet assay parameters, DNA in tail, tail length, tail moment, and olive tail moment. Administration of the complex also reduced the mortality of the mice and also aided recovery from the radiation induced weight loss [Ramachandran et. al., 2010]. DNA repair was also observed in human blood leukocytes with palladium α-lipoic acid complex treatment immediately after exposure to radiation [Menon & Nair, 2011].

The mechanism of the superiority of the metal complex compared to that of the ligand is still an unsolved puzzle. Compared to the high oral dose, the available plasma concentration of α-lipoic acid is very small. One possibility for the higher activity of the metal complex is probably due to its increased concentration in the plasma. This needs experimental verification. It is also known that 98% of α-lipoic acid is excreted in urine within 24 hours and since it takes 4-6 weeks for serum and urine clearance of palladium, the previous suggestion seems justified. Another possibility is the chemistry of the transition metal playing a dominant role in the enzymatic activity. The starting material in the synthesis of the palladium α-lipoic acid complex is palladium(II). The final complex is also palladium(II), based on preliminary ESR data. Palladium(II) complexes are diamagnetic. The complex concentration dependent electrochemical characteristics of α-lipoic acid suggest the possibility of free radical formation by one electron reduction under physiological conditions. In such a case the electron spin may be involved in the enzymatic process. The impedance characteristics of the palladium α-lipoic acid as well as that of the α-lipoic acid, described in this section, strongly suggest this possibility.

Another possibility for the superiority of the palladium α-lipoic acid complex compared to α-lipoic acid is its ability to form self-assembled structures, such as the one shown in Fig. 6. No self-assembly was observed for sodium lipoate. We want to point out that binding of a lysine residue in the protein to the lipoyl group of E2 in 2-oxoacid dehydrogenases results in a long flexible arm that can oscillate a distance of ~200 Å. This arm is utilized during the catalytic cycle [Patel & Packer, 2008]. It is obvious that the self assembled palladium α-lipoic acid complex can make this process more facile.

The physics and chemistry of non-equilibrium systems have been utilized to understand some of the spatial patterns and temporal patterning observed in biological processes such as bacterial colonies shaped by diffusive instabilities and calcium waves governed by nonlinear amplification during intracellular signaling [Levine & Jacob, 2004].

In homogeneous systems, spiral waves and spatiotemporal phenomena are formed from autocatalytic reactions and diffusion resulting from chemical instabilities. Our data suggest that the propagation of electrical signaling among the packing units and extending to long distances by global coupling is viable by such self-assembled systems.

We have utilized the technique of electrochemical impedance spectroscopy extensively to probe spatiotemporal phenomena in biological systems. This technique is routinely used to study corrosion and fuel cells. We have used admittance measurements for understanding solute-solvent interactions, "π –way" conduction, ion pair formation, water-structure enforced ion pair formation, potential induced and solvent mediated ion pair formation at the double layer, and semi conduction characteristics of simple biological molecules

Free Radicals in Neurodegenerative Diseases: Modulation by Palladium α-Lipoic Acid Complex 119

quadrant in the plot. However, if there is inductance, the data will require the first and fourth quadrants. In corrosion studies, the oxides formed at passivation potentials exhibit, quite often, semiconduction characteristics and their impedance data will be in both first and second quadrants. The impedance in the second quadrant may be compared to the negative differential resistance (NDR) observed in the I-V curves of tunnel diodes and some enzymes. Impedance spectra spanning more than two quadrants and especially four quadrants are unusual and are often explained by nonequilibrium phenomena and

The impedance spectra for α-lipoic acid and its modulation by complexing with palladium are shown in Fig. 7. While α-lipoic acid exhibits NDR and shows impedance in only 3 quadrants (chaotic in quadrant 3), the spectra of the metal complex is extended to 4 quadrants and much more smoothly by complexation with palladium. Of course the NDR behavior can be optimized by slightly tweaking the applied potential. This enhancement in NDR behavior may be compared to the enhanced Krebs cycle and mitochondrial enzymatic

We have reason to believe that the self-assembled structure of the complex, by providing a spatial extension of the membrane with much more surface area, may be catalyzing the electron transfer process by enhancing spin coupling. This may account, for example, the enhanced complex I and complex II activities activities of PdLA by 151% and 212% more

Another important aspect of this system is the fact α-lipoic acid is linked to lysine by an amide bond in the multienzyme complexes of pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and branched chain α-ketoglutarate dehydrogenase. Thus both α-lipoic acid and lysine have heavy involvement in the electronic aspects of the enzymatic process.

Oxidative stress is caused by the chemical imbalance between ROS production and their breakdown by antioxidants. Over-abundance of ROS has been found during neuronal

Fig. 7. a) Nyquist plot for 0.0373 M sodium lipoate, -1.15V, pH 7.79, NDR at 4.81Hz. b) Modulation of lipoate impedance by palladium in 0.0373M palladium α-lipoic acid (1:1

complex) in 0.1792 M NaCl, -1.18V, pH 7.78, NDR at 66Hz [Krishnan et. al., 2011]

compared to spatiotemporal oscillations in biological systems.

than that of α-lipoic acid.

**11. Conclusions**

activities of the palladium α-lipoic acid compared to that of the ligand.

[Krishnan & Garnett, 2006; 2011; Krishnan et al., 2007a,b; 2008a,b,c,d; 2009a,b; 2011]. Simple molecules such as arginine, histidine, lysine, flavin adenine dinucleotide, riboflavin, cysteine, lidocaine hydrochloride, α-lipoic acid, and hydrogen peroxide exhibit negative differential resistance, a characteristic of a tunnel diode.

Fig. 6. Phase microscopy of palladium α-lipoic acid complex, 300X [Krishnan & Garnett, 2006]

In this technique a perturbing sinusoidal voltage E = Eosin(ωt) is applied at angular frequency ω (2πf, where f is the conventional frequency in Hz) to the electrode system consisting of a working electrode, counter electrode and reference electrode. The measurements reported in this chapter were made using and EG & G PARC Model 303A SMDE trielectrode system (mercury working electrode, platinum counter electrode and Ag/AgCl saturated KCl reference electrode) along with Autolab ecochemie. The measurements were carried out in the range 1000Hz to 30 mHz. The amplitude of the sinusoidal perturbation was 10 mV. The response of the applied sinusoidal voltage is analyzed in terms of the resultant current I = Iosin(ωt + Ф), where Ф represents a characteristic phase angle shift. In the plane of Cartesian coordinates, an impedance is expressed by its real (Z′) and imaginary (Z′′) parts. The modulus ⎢Z ⎢and phase angle Ф of **Z**(ω) can be obtained from ⎢Z ⎢= [Z′ 2 + Z′′2]1/2 and Ф = tan-1 [Z′′/Z′], respectively [Macdonald & Johnson, 2005]. Admittance and impedance are interrelated:

$$
\mathbb{Z}'/\mathbb{Y}' = \mathbb{Z}''/\mathbb{Y}'' = (\mathbb{Z}')^2 + (\mathbb{Z}'')^2 = 1/[(\mathbb{Y}')^2 + (\mathbb{Y}'')^2] \tag{32}
$$

Over a frequency bandwidth of interest, there are various ways of representing the impedance spectrum. Most often, the well known Nyquist or Cole-Cole plot (Z′′ as the Y-axis and Z′ as the X-axis for the range of frequencies explored at a fixed potential) and Bode plot (⎢Z ⎢and Ф vs. logω) are employed to represent the data. In simple terms, impedance is like a frequency dependent generalized resistance and admittance is like a frequency dependent conductance. In electrochemistry, the imaginary impedance is almost always capacitive and therefore negative. Majority of impedance data require only the first quadrant in the plot. However, if there is inductance, the data will require the first and fourth quadrants. In corrosion studies, the oxides formed at passivation potentials exhibit, quite often, semiconduction characteristics and their impedance data will be in both first and second quadrants. The impedance in the second quadrant may be compared to the negative differential resistance (NDR) observed in the I-V curves of tunnel diodes and some enzymes. Impedance spectra spanning more than two quadrants and especially four quadrants are unusual and are often explained by nonequilibrium phenomena and compared to spatiotemporal oscillations in biological systems.

The impedance spectra for α-lipoic acid and its modulation by complexing with palladium are shown in Fig. 7. While α-lipoic acid exhibits NDR and shows impedance in only 3 quadrants (chaotic in quadrant 3), the spectra of the metal complex is extended to 4 quadrants and much more smoothly by complexation with palladium. Of course the NDR behavior can be optimized by slightly tweaking the applied potential. This enhancement in NDR behavior may be compared to the enhanced Krebs cycle and mitochondrial enzymatic activities of the palladium α-lipoic acid compared to that of the ligand.

We have reason to believe that the self-assembled structure of the complex, by providing a spatial extension of the membrane with much more surface area, may be catalyzing the electron transfer process by enhancing spin coupling. This may account, for example, the enhanced complex I and complex II activities activities of PdLA by 151% and 212% more than that of α-lipoic acid.

Fig. 7. a) Nyquist plot for 0.0373 M sodium lipoate, -1.15V, pH 7.79, NDR at 4.81Hz. b) Modulation of lipoate impedance by palladium in 0.0373M palladium α-lipoic acid (1:1 complex) in 0.1792 M NaCl, -1.18V, pH 7.78, NDR at 66Hz [Krishnan et. al., 2011]

Another important aspect of this system is the fact α-lipoic acid is linked to lysine by an amide bond in the multienzyme complexes of pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and branched chain α-ketoglutarate dehydrogenase. Thus both α-lipoic acid and lysine have heavy involvement in the electronic aspects of the enzymatic process.

#### **11. Conclusions**

118 Neurodegenerative Diseases – Processes, Prevention, Protection and Monitoring

[Krishnan & Garnett, 2006; 2011; Krishnan et al., 2007a,b; 2008a,b,c,d; 2009a,b; 2011]. Simple molecules such as arginine, histidine, lysine, flavin adenine dinucleotide, riboflavin, cysteine, lidocaine hydrochloride, α-lipoic acid, and hydrogen peroxide exhibit negative

Fig. 6. Phase microscopy of palladium α-lipoic acid complex, 300X [Krishnan & Garnett,

The modulus ⎢Z ⎢and phase angle Ф of **Z**(ω) can be obtained from ⎢Z ⎢= [Z′

In this technique a perturbing sinusoidal voltage E = Eosin(ωt) is applied at angular frequency ω (2πf, where f is the conventional frequency in Hz) to the electrode system consisting of a working electrode, counter electrode and reference electrode. The measurements reported in this chapter were made using and EG & G PARC Model 303A SMDE trielectrode system (mercury working electrode, platinum counter electrode and Ag/AgCl saturated KCl reference electrode) along with Autolab ecochemie. The measurements were carried out in the range 1000Hz to 30 mHz. The amplitude of the sinusoidal perturbation was 10 mV. The response of the applied sinusoidal voltage is analyzed in terms of the resultant current I = Iosin(ωt + Ф), where Ф represents a characteristic phase angle shift. In the plane of Cartesian coordinates, an impedance is expressed by its real (Z′) and imaginary (Z′′) parts.

Ф = tan-1 [Z′′/Z′], respectively [Macdonald & Johnson, 2005]. Admittance and impedance

 Z′/Y′ = Z′′/Y′′ = (Z′)2 + (Z′′)2 = 1/[(Y′)2+ (Y′′)2 (32) Over a frequency bandwidth of interest, there are various ways of representing the impedance spectrum. Most often, the well known Nyquist or Cole-Cole plot (Z′′ as the Y-axis and Z′ as the X-axis for the range of frequencies explored at a fixed potential) and Bode plot (⎢Z ⎢and Ф vs. logω) are employed to represent the data. In simple terms, impedance is like a frequency dependent generalized resistance and admittance is like a frequency dependent conductance. In electrochemistry, the imaginary impedance is almost always capacitive and therefore negative. Majority of impedance data require only the first

2 + Z′′2]1/2 and

differential resistance, a characteristic of a tunnel diode.

2006]

are interrelated:

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development, as well as in numerous neuropathological conditions. Oxidative stress and mitochondrial dysfunction have been closely associated in brain injury such as ischemia and stroke and neurodegenerative processes such as Multiple Sclerosis, Parkinson's, Alzheimer's, and Huntington's.

Lipoic acid was found to be effective in modulating many neurodegenerative disorders. The development of palladium α-lipoic acid complex was intended to augment the properties of this ligand by the catalytic properties of the transition metal. It is formulated to combat mitochondrial dysfunction. The unique electronic properties of palladium modulating the properties of α-lipoic acid appear to be a key to this physiological effectiveness. This is exemplified in our electrochemical impedance spectroscopic studies of α-lipoic acid and palladium α-lipoic acid complex.

Palladium α-lipoic acid complex facilitates aerobic metabolism much more than that of αlipoic acid, by significantly enhancing the enzymatic activity of isocitrate dehydrogenase, αketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase at the Krebs cycle and mitochondrial complexes I, II, III, and IV of the electron transport chain. The electronic properties of palladium also appear to modulate the antioxidant properties of α-lipoic acid in that PdLA enhances the activities of catalase and glutathione peroxidase more than that of α-lipoic acid. The level of GSH also was significantly improved and the level of lipid peroxidation was decreased in the heart mitochondria of aged rats.

PdLA is similar to a multi-spectrum drug. Since it targets the mitochondria it is able to carry out several functions such as combating age-related as well as disease-associated fatigue, and minimizes the effects of ischemic injury. Being a powerful free radical scavenger, it may also be effective in combating death of neurons and other progressive loss of structure or function of neurons caused by free radicals.

PdLA is able to protect from radiation exposure and repair DNA. It also seems to ward off radiation exposure-associated weight loss in mice, possibly protecting susceptible gastrointestinal tract.

#### **12. Acknowledgements**

The area covered in this chapter is vast and warrants citation of numerous publications. The authors regret being unable to cite many publications due to space limitations. The authors wish to express their sincere thanks and gratitude for many fruitful discussions and collaborations in various aspects of the work presented in this chapter: B. Chu, K. K. Janardhanan, T.A. Ajith, C.K.K. Nair, N.P. Sudheesh, A. Menon, and L. Ramachandran.

#### **13. References**

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Palladium α-lipoic acid complex facilitates aerobic metabolism much more than that of αlipoic acid, by significantly enhancing the enzymatic activity of isocitrate dehydrogenase, αketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase at the Krebs cycle and mitochondrial complexes I, II, III, and IV of the electron transport chain. The electronic properties of palladium also appear to modulate the antioxidant properties of α-lipoic acid in that PdLA enhances the activities of catalase and glutathione peroxidase more than that of α-lipoic acid. The level of GSH also was significantly improved and the

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