Lipoic Acid Mineral Complex Modulates Free Radicals in Neurodegenerative Diseases

Frank Antonawich, Ph.D.^{a, b,*} and Merrill Garnett, DDS^a

^aGarnett McKeen Laboratory, Inc, Bohemia, NY 11716-1735, USA

^bDepartment of Biology, St. Joseph’s College, Patchogue NY 11772, USA

E-mail: frank@garnettmckeenlabs.net

Introduction

Oxidative stress is caused by the chemical imbalance between reactive oxygen species (ROS) production and their breakdown by reducing agents or antioxidants. Over-abundance of ROS has been found during neuronal development, as well as in numerous neuropathological conditions. A predominant feature of neuronal injury is the onset of oxidative stress [Higgins et. al., 2010].

Oxidative stress and mitochondrial dysfunction have been closely associated in many sub-cellular, cellular, animal, and human studies of both acute brain injury such as ischemia and stroke and neurodegenerative processes such as Parkinson’s, Alzheimer’s and Huntington’s. While the oxidative stress occurs chronically in Alzheimer’s disease, it is more acute in ischemic reperfusion injury. The consequences of mitochondrial dysfunction include DNA and protein damage, lipid peroxidation, disruption of the mitochondrial permeability transition, Ca²⁺ homeostasis, and triggering apoptosis. It is essential to have healthy mitochondria contributing substantially to the physical, mental, and emotional elements needed to support the well-being of patients suffering from brain injury or neurodegenerative diseases [Krishnan et.al., 2011, 2012].

Energy metabolism, calcium regulation, and apoptosis-signaling pathways are the major roles of mitochondria. Energy requirements dictate the number of mitochondria in a cell [Alberts et. al., 1989; Voet D. & Voet J. G., 1995; Beattie, 2002; Nagley et. al., 2010]. Cardiac and skeletal muscles, the brain, and the liver have the most mitochondria because of their high metabolic activities. These cells are also exposed to the most oxidative stress because the source of free radical production is also the mitochondria. Due to low levels of antioxidants in neurons, they are intrinsically ill-equipped to defend against an increase in oxidative stress. Glial cells including astrocytes play a supplementary role in antioxidant defense of neurons [Higgins et. al., 2010].

Our search for an extremely safe (up to 40 mL/day, 0.037 M aqueous solution) and nontoxic therapeutic agent resulted in the development of a novel redox molecule, “Lipoic acid mineral complex,” that is active in mitochondrial cellular metabolism and other functions. The selection of the naturally occurring coenzyme, α-lipoic acid, as our ligand was based on its safety as well as its redox, antioxidant, and fatty acid properties. After selecting the ligand that plays a critical role in biological energy metabolism and numerous other functions, we wanted to tweak the properties of the ligand by complexing it with a metal that is safe and has very high catalytic and electronic properties. After numerous investigations with a variety of metals, the final selection was made to use palladium, a transition mineral.

The properties of the resulting lipoic acid mineral complex were remarkable in many ways and have been reviewed recently [Krishnan et. al., 2011]. Briefly, this complex enhances the enzymatic activities of Krebs cycle enzymes, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase and mitochondrial respiratory enzymes, complex I, complex II, complex III, and complex IV. These enzymatic activity enhancements by the metal complex were, in general, much greater than that of the ligand, α-lipoic acid. Coupling this increase in the efficiency of the aerobic metabolic cascade with its powerful antioxidant properties, such as scavenging of free radicals, lowering lipid peroxidation, increasing the levels of glutathione, glutathione peroxidase, manganese superoxide dismutase, and catalase, gave us a powerful weapon to combat fatigue associated with numerous mitochondrial abnormalities [Sudheesh et al., 2009, 2010; Ajith , 2014]. The complex also modulates mitochondrial dysfunction, acts as a prophylactic for neuronal protection from transient ischemic attack, repairs DNA damage resulting from radiation, acts as a prophylactic for protection from radiation, and improves the quality of life. The therapeutic ability/potential of this complex may be exploited in its applications for combating brain injury resulting from transient ischemic attack, death of neurons, and other progressive loss of structure or function of neurons associated with diseases such as Parkinson’s, Alzheimer’s, and Huntington’s.

Mitochondrial Dysfunction in Neurodegenerative Diseases

Since different organs can rely on mitochondrial energy to different extents, mitochondrial defects can cause organ-specific phenotypes. The organ system most reliant on mitochondrial energy is the central nervous system. The consequences of mitochondrial dysfunction are numerous and include oxidative stress, loss of cellular Ca²⁺ homeostasis, promotion of apoptosis, and metabolic failure. Hence, evidence continues to accrue implicating mitochondrial dysfunction in the etiology of a number of neurodegenerative conditions such as Parkinson’s, Alzheimer’s, and transient ischemia.

In transient ischemia, a lack of oxygen and glucose delivery compromise the integrity of aerobic metabolism, while reperfusion potentiates injury via the generation of free radicals. Superoxide, nitric oxide and peroxynitrite production in the brain is increased during reperfusion following 30 minutes of global ischemia. In patients with Parkinson’s disease, excess Fe²⁺ can reduce peroxide and produce hydroxyl radicals

.

These radicals and their reactions cause oxidative stress and consequent mitochondrial damage resulting in mutations. Evidence for mitochondrial dysfunction in Alzheimer’s disease pathogenesis comes from impaired activities of three key Krebs Cycle enzyme complexes and reduced respiratory chain complex I, III, and IV activity observed in postmortem Alzheimer’s disease brain, and oxidative damage to both mtDNA and nDNA.

Apart from ATP production, mitochondria also produce reactive oxygen species (ROS). Some electrons escaping or leaking from the electron transport complexes, mainly from complexes I and III, during respiration react with oxygen to form superoxide radicals. However, it may be possible for mitochondrial DNA (mtDNA) mutations to disrupt the normal electron flow and seriously affect energy production. Oxidative damage and the resulting serious consequences have been extensively reviewed recently [Singh, 2006]. Compared to nuclear DNA (nDNA), mtDNA is far more susceptible to mutations due to their being present in a highly oxidative environment, a lack of protective histones and limited repair capacity [Carew & Huang, 2002; Singh, 2006]. In aging and neurodegenerative disorders, apart from inherited defects, mDNA deletions and point mutations within neurons are well recognized.

Alzheimer’s Disease

The most common cause of loss of brain function or dementia is Alzheimer’s disease (AD). It is an age-related progressive and irreversible neurodegenerative disease resulting in impairments of cognition, memory and behavior. Available drugs do not offer significant benefit and seem to be ineffective against progression of the disease [Barnes & Yaffe, 2011]. The Alzheimer’s Disease International Conference, held in Paris, France, show cased certain advancements for early detection of AD, via the high rate of falls, an early blood test of nine hormones and proteins that predict amyloid levels in the brain, and imaging of the retina to look for the width of certain blood vessels. A mathematical model revealed seven factors that contributed to the risk worldwide: lack of education (19.1%), smoking (13.9%), lack of physical activity (12.7%), depression (10.6%), high blood pressure at midlife (5.1%), diabetes (2.4%), and midlife obesity (2.0%) [Barnes & Yaffe, 2011].

Alzheimer’s disease is a protein misfolding disease resulting from the accumulation of abnormally folded amyloid-β protein (Aβ) and hyperphosphorylated aggregates of the microtubule-associated (tau) proteins in the brain. Beta-amyloid plaque, made up of small peptides, is a fragment from the larger amyloid precursor protein (APP) that penetrates through the neuron’s membrane. This type I transmembrane protein, APP, is needed for neuron growth, survival and post-injury repair. Enzymes divide this APP into fragments by proteolysis. One of these fragments forms fibrils of beta-amyloid. The extracellular deposition of diffuse and neuritic plaques containing amyloid-β peptide, the intracellular accrual of neurofibrillary tangles containing tau protein, and selective nerve cell loss in certain brain regions are the histopathological features of AD [LaFerla, 2002]. These are formed selectively in the hippocampus and neocortex of the brain.

Dysregulation of Ca²⁺ homeostasis is implicated in the pathogenesis of AD [Khachaturian, 1989]. Every gene that is known to increase the susceptibility to AD also modulates Ca²⁺ signaling [ Laferla, 2002]. Increased intracellular Ca²⁺ increases accumulation of amyloid-β, hyperphosphorylation of tau protein, and neuronal death. Disruption of Ca²⁺ regulation in the endoplasmic reticulum mediates signal transduction cascades that are associated with AD [LaFerla, 2002].

Figure 1. Endogenous and Exogenous Triggers of Nerve Cell Death in Neurodegeneration [adapted from Behl & Moosmann, 2002]

Neuronal cell survival and death are regulated by the pivotal role of mitochondria that also regulate energy metabolism and cell death pathways. Numerous studies have established that mitochondrial dysfunction has an early and preponderant role in AD [Moreira et. al., 2010a, b]. Oxidative damage to both mtDNA and nDNA has been examined in several studies [Mecocci et al., 1994; Gibson et al., 2000; Castellani et. al., 2002; Bubber et. al., 2005]. Significant, three-fold, increases in the amount of 8-hydroxy-2’-deoxyguanosine have been demonstrated in the mtDNA within the parietal cortex of Alzheimer’s patients. A smaller, but significant increase, in oxidative damage to nDNA has also been observed [Mecocci et al., 1994]. A deficiency in cytochrome c oxidase has been reported in Alzheimer’s disease [Castellani et al., 2002]. In addition, significant decreases were observed in the activities of pyruvate dehydrogenase complex (-41%), isocitrate dehydrogenase (-27%), and α-ketoglutarate dehydrogenase complex (-57%). A strong correlation exists between the diminished activity of these enzymes and the Clinical Dementia Rating [Gibson et al., 2000; Bubber et al., 2005]. On the other hand, the activities of succinate dehydrogenase (complex II) (+44%) and malate dehydrogenase (+54%) were increased.

Decreased glucose metabolism has been found to correlate well with the clinical deterioration [Minoshima et. al., 1997]. Diminished metabolism leads to hyperphosphorylation of tau proteins and increased production of amyloid-β peptide [Bubber et. al., 2005]. Oxidative nerve cell death in Alzheimer’s disease and the role of antioxidants as neuroprotective compounds have been examined [Behl and Moosmann, 2002].

Alpha lipoic acid is extensively studied as a potent mitochondrial nutrient for improving memory deficit, oxidative stress and mitochondrial dysfunction [Liu, 2008]. It is used to treat peripheral neuropathy, cardiac autonomic neuropathy, insulin resistance in type-II diabetes, retinopathy and cataract, glaucoma and Alzheimer’s type dementia. All the data suggest that lipoic acid mineral complex functions are far superior to that of α-lipoic acid. The complex is extremely safe with practically no side effects and has numerous properties including enhancement of activities of Krebs cycle and mitochondrial respiratory chain enzymes, free radical scavenging properties, and DNA repair, we strongly believe in its potential for ameliorating some of the deleterious effects of AD. However, there are currently no clinical data available on its effects on cognitive functions except some preliminary promising data from patients with Stage IV cancer and are under palliative care.

Parkinson’s Disease

The second most common neurodegenerative disease after Alzheimer’s disease is Parkinson’s disease (PD). This disease increases with aging exponentially in incidence above the age of 65 [Chan et. al., 2009]. This disease, associated with movement disorder, is characterized by the loss or degeneration of dopaminergic neurons in the substantia nigra pars compacta. This leads to a dopamine deficit in the striatum. The most common motor symptoms such as bradykinesia, hypokinesia, rigidity, resting tremor, and postural instability are all manifestations of this deficiency resulting in the dysregulation of basal ganglia circuits. At the time of clinical presentation, ~ 50-70% of dopaminergic neurons in the nigrostriatal system have been lost [Orth & Schapira, 2002]. The presence (in the surviving neurons) of Lewy bodies or proteinaceous deposits within neuronal perykarya and processes (Lewy neurites), mainly composed of α-synuclein, ubiquitin, neurofilaments, and molecular chaperones are viewed as pathological hallmarks of PD [Schapira, 2007, 2008; Winklhofer & Haass, 2010]. Autosomal forms of PD seem associated with a gain of function mechanism arise from mutations in the genes encoding α-synuclein and leucine-rich repeat kinase 2 (LRRK2). Similarly, autosomal recessive PD seems connected to a loss of function mutations in the genes encoding parkin, PINK1, DJ-1 and Omi/HtrA2 [Gandhi et. al., 2009]. The presence of activated microglial cells in the Parkinsonian substantia nigra is reported [Ruberg et. al., 1998]. These cells synthesize a known apoptogenic substance in the immune system, cytokine TNF-α.

Oxidative stress, the pathogenic outcome of reactive oxygen and nitrogen species overproduction beyond the capacity of their clearance (by antioxidants) in cells [Niizuma et. al., 2010], is implicated in the pathogenesis of Parkinson’s disease (PD) [Olanow & Lieberman, 1992; Weiner et al., 2007]. Inhibition of complex I activity of the electron transport chain induces PD. An age-dependent increase in somatic mtDNA deletions associated with a respiratory chain defect has been identified in dopaminergic neurons from the substantia nigra. Also, several genes associated with sporadic and familial PD interface with pathways regulating the mitochondrial function, morphology, and fission-fusion dynamics have been closely scrutinized [Parker et al., 1989; Greenamyre et. al., 1999; Schapira, 2007; Winklhofer & Haass, 2010].

Excess Fe²⁺, also found in patients with Parkinson’s disease, can reduce peroxide and produce hydroxyl radical (HO

-)

. The hydroxyl radical can lead to lipid peroxidation and alter the structural integrity of neural membranes. Dopamine undergoes auto-oxidation, producing superoxide, H₂O₂, semiquinone radical and finally a quinone [Olanow and Lieberman, 1992].

The central nervous system contains high levels of polyunsaturated fatty acids. They are susceptible to attack by free radicals, especially HO

^.

, because the high consumption of oxygen in the brain facilitates more free radical production. The brain is relatively deficient in defending against oxidative stress. It has relatively low catalase activity and moderate SOD and GSHPx activities [Moreira et. al., 2010a]. Lipid peroxidation by the hydroxyl radical can alter the structural integrity of membranes. The excess Fe²⁺ can reduce hydrogen peroxide and produce HO

^.

. These radicals and their reactions (reactions 13-20) cause oxidative stress and consequent mitochondrial damage resulting in mutations and probably cancer.

Vitamin E can break the chain propagation reaction by reacting with lipid peroxyl radicals. Other small molecules such as ascorbate (vitamin C), reduced coenzyme Q10 (CoQH₂), GSH, and α-lipoic acid also “repair” oxidizing radicals directly. Under normal physiological conditions, SOD converts the superoxide to H₂O₂ and O₂ catalytically, and H₂O₂ is cleared by GSH catalyzed by glutathione peroxidase. The oxidized glutathione (GSSG) is reduced back to glutathione by NADPH, which is catalyzed by glutathione reductase.

The probable mitochondrial alterations associated with PD are schematically shown in Figure 2. Neuronal cells may die because the structural integrity of neuronal membranes may be compromised by any one of these stress factors.

Figure 2. Probable mitochondrial alterations associated with Parkinson’s disease [adapted from Winklhofer & Haass, 2010].

There are reports of oxidative stress to dopaminergic neurons [Ruberg et. al., 1998]. The oxygen consumption of these neurons is very high because of the presence of a large number of mitochondria. There are reports of a decrease in glucose metabolism as evidenced by an increased lactate/N-acetyl aspartate ratio in the occipital cortex of demented patients with PD as well as an increase in lactate/creatine ratio in the striatum. Compex I activity or mitochondrial NADH ubiquinone reductase activity in post mortem tissue samples of the substantia nigra compacta of PD brains is also found to be about 35% less [Ruberg et. al., 1998; Chan et. al., 2009]. It is also reported that the post-mortem brains from PD patients contain increased levels of malondialdehyde, and lipid hydroperoxides. There are also reports of oxidative damage to DNA, lipids, proteins, and somatic DNA mutations in dopaminergic neurons [Chan et. al., 2009].

Autopsy sample of substantia nigra from PD brains showed increased levels of oxidants, and decreased antioxidants [Table 1]. Increased levels of free iron were seen in autopsy samples as well as in brains of living PD patients by iron-mediated contrast magnetic resonance imaging.

Control, μg/g fresh tissue Parkinson’s Disease, μg/g fresh tissue

Ferritin 223 ± 22 288 ± 27

Fe3+ 16 ± 4 42 ± 5

Glutathione 57 ± 13 30 ± 12

Ascorbic acid 309 ± 47 271 ± 15

—————————————————————————————————————————

Table 1. Levels of Ferritin, Fe³⁺, Glutathione and Ascorbic acid in Substantia Nigra from control and Parkinson’s Disease [Prasad et. al, 1999].

The role of multiple antioxidants in the prevention and treatment of PD has been reviewed [Prasad et. al., 1999]. Since α-lipoic acid is heavily involved in the attenuation of free radical related disorders including Alzheimer’s disease, it is possible for it to have a positive influence in the amelioration of PD as well. But there are no well documented data. We have demonstrated that the properties of lipoic aciid mineral complex formulation are far superior to that of the ligand in every respect. Thus, we are confident that the oxidative stress associated with Parkinson’s disease may be attenuated by the use of this complex. In addition, based on our data with ischemia in gerbils, the efficacy of the remaining dopaminergic neurons may be potentiated.

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 multiple sclerosis (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.

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].

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

Based on current research, it has been proposed that mitochondrial abnormalities are involved in the development and progression of multiple sclerosis (Figure 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 other cellular pathways including increased demyelination and inflammation in neurons and tissues that are affected by multiple sclerosis [Mao & Reddy, 2010].

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 reactive oxygen species 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.

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].

In view of our data with palladium α-lipoic acid formulation on the enhanced enzymatic activities of Krebs cycle and electron transport chain enzymes in animals, anti-oxidant 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. At the completion of this IND study there was a statistically significant decrease in MS-associated fatigue in all three clinical scales evaluated.

Cerebral Ischemia

An insufficient or reduced blood flow to the brain to meet the metabolic demand will result in cerebral or brain ischemia. The normal cerebral blood flow is ~ 50 to 60 mL/100g/min. Death of brain tissue is a consequence of poor oxygen supply or cerebral hypoxia resulting from the insufficient blood flow. A prototype of brain damage during cerebral ischemia is shown in Figure 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.

Figure 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].

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.

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, 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.

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 2 [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. 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 (however 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.

—————————————————————————————————————————

Focal ischemia Reperfusion

————————— ————————–

Core Penumbra Core Penumbra

————————————————————————————————————————

Metabolites

ATP ↓↓↓ ↓↓ ↓↓ ↓

Adenylate energy charge ↓↓ ↓ ↓/N.C. N.C.

Total adenine nucleotides ↓↓ ↓↓ ↓↓ ↓

Phosphocreatine ↓↓↓ ↓ ↓ N.C.

Lactate ↑↑ ↑↑ ↑↑ ↑/N.C.

Glucose ↓↓↓ N.C. N.C. N.C.

Metabolic activity

Glucose use ↓↓↓* N.C. ↓↓ ↓↓

Oxidative metabolism ↓↓↓* ↓↓↓ ↓↓ ↓↓

———————————————————————————————————————-

Table 2. 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].

One neuroprotective approach involves 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, demonstrated that acute, post ischemic and prophylactic administration of lipoic aciid mineral complex formulation limits ischemic damage [Antonawich et. al., 2004]. Following bilateral carotid artery occlusion in the gerbil, the lipoic acid mineral complex formulation was administered intraperitoneally (IP) immediately after surgery, then once daily for 3 days. The control group received saline. Palladium lipoic acid complex formulation treatment significantly protected CA1 hippocampal pyramidal cells from transient global ischemia at 30, 50, and 70 mg/kg per 24 h.

While a delayed application of the lipoic acid mineral 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 approximately 3 days. Their nesting behavior was observed to improve significantly after treatment with palladium lipoic acid complex formulation (50 mg/kg every 24 h 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 lipoic acid mineral 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. Furthermore, immediate administration of lipoic aciid mineral 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]. This mechanism of action appears to be related to stabilizing the electron transport chain, in addition to quenching radical formation.

Conclusions

At the heart of a number of neurodegenerative disorders is mitochondrial dysfunction. Lipoic acid mineral complex 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.

Lipoic acid mineral complex facilitates aerobic metabolism much more than that of α-lipoic acid, by significantly enhancing the enzymatic activity of isocitrate dehydrogenase, α-ketoglutarate 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 lipoic acid mineral complex formulation enhances the activities of catalase and glutathione peroxidase more than that of α-lipoic acid. The level of glutathione also was significantly improved, and the level of lipid peroxidation was decreased in the heart mitochondria of aged rats.

Lipoic acid mineral complex formulation 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.

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