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CHAPTER – I

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General Introduction Chapter I

1.0 General Introduction

As the proportion of the average age of world population has been steadily

increasing in the last fifty years and with changing global demography, age

associated impairments in cognition and memory process are increasingly prevalent.

This increasing number of patients with cognitive impairments is one of the major

health problems of old age. One of the major causes of dementia in the elderly is

Alzheimer’s disease (AD). As there is no definite treatment or cure at present for

AD, it imposes a major financial burden on health care systems.

AD is clinically characterized by progressive memory loss and deficits in one

or more of the cognitive domains - aphasia (language disturbance), agnosia (failure

to recognize people or objects in the presence of intact sensory function), apraxia

(inability to perform motor acts in the presence of intact motor system), or executive

function (plan, organize, sequence actions or form abstraction). In AD patients these

deficits are severe enough to interfere with daily life or work and represent a

significant decline in earlier level of function (Brown et al., 2005)

1.1 History

The word “dementia” is derived from Latin the word which means without

mind. Dementia is a clinical syndrome, comprising an assembly of symptoms which

includes deterioration of intellectual function and other cognitive skills leading to

gradual decline in daily activities. (Lishman, 1987). The connection between

dementia and aging was stressed as early as 1896 by Kraepelin. The most commonly

used terms of dementia is given in the American Psychiatric Association Diagnostic

and Statistical Manual of Mental Disorders, 4th edition (DSM – IV, 1994) and World

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Health Organization’s (WHO) International Classification of Diseases (ICD – 10,

1992). The term dementia includes a number of different subtypes of which the most

common form is AD.

AD was named after the German psychiatrist, Alois Alzheimer (1864 - 1915)

by Emil Kraeplein in 1910. He was the first to describe this disease in 1907 with the

case study of a 51 year old woman, presented with paranoid delusions. At the first

visit he found memory impairment, as well as reading and writing difficulties. Later

in the course of the illness she developed hallucinations, dysphasia, dysgnosia,

dyspraxia and spatial disorientation. When she died in 1906 a post mortem

examination was performed on her brain. Alzheimer found gross cortical atrophy

and on a microscopic level, nerve cell loss, senile plaques and neurofibrillary tangles

(Alzheimer, 1907). Until 1950, arteriosclerosis was generally considered to be the

major cause of dementia in the elderly. In 1951, Fisher showed that senile dementia

was unrelated to cerebral arteriosclerosis (Fischer, 1951). This led to renewed

interest in the findings of Alzheimer (Blessed et al., 1968). The findings of two

abnormal pathological lesions, senile plaques (SP’s) or neuritic plaques (NP’s) and

neurofibrillary tangles (NFT) became the hallmarks of the disease (Maurer et al.,

1997). Today we realize that the case identified by Alzheimer was merely the tip of

the iceberg and that dementia is one of the major health problems of old age.

Moreover, even after 100 years we still do not understand the etiological, clinical

and pathological mechanism of the disease except the rare cases of genetic

mutations associated with the familial disease. This thesis reveals the detection of

the factors, biochemical markers of the disease in common clinical practice.

Majority of AD patients have no obvious family history of disease and is

therefore classified as so-called sporadic AD cases. There are however rare known

genetic forms of AD (Tanzi et al., 1996). These genetic forms of AD, collectively

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referred to as Familial Alzheimer’s Disease (FAD), are associated with specific

mutations that are inherited in an autosomal dominant manner. The discovery of

these mutations has been instrumental in supplying tools for studying the molecular

pathology of AD.

1.2 Epidemiology

Currently over 24 million people worldwide are estimated to be demented

and with the expected increase of elderly population will increase the number of

people demented in the coming years (Larson et al., 2008). Almost half of the

demented people, 46% live in Asia, 30% in Europe and 12% in North America

(Wimo et al., 2003). Most of the AD cases are sporadic cases with millions affected

world wide (Qiu et al., 2007). The familial or inherited form of AD represents only

1-10 % (Blennow et al., 2006). During the recent years incidence and prevalence

data have been reported in a large number of studies from several continents. The

prevalence and incidence of dementia in various studies are presented in Table 1.1.

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Table 1.1 Prevalence and incidence of dementia in various studies

Author Year Place Age Prevalence rate

(%) Annual Incidence

rate (%)

McDowell 1994 Canada > 65 4.2 -

Boothby 1994 London > 65 - 2

Park 1994 Korea > 65 10.8 -

Komahashi 1994 Japan > 65 6.1 -

Ogura 1995 Japan > 65 6.7 -

Yoshitake 1995 Japan > 65 - 2

Gussekloo 1995 Netherland > 85 6.9 -

Henderson 1995 Australila > 70 7.3 -

Liu 1996 Taiwan > 65 4.3 -

Shaji 1996 India > 60 3.1 -

Prencipe 1996 Italy > 65 8 -

Fichter 1996 Germany > 85 - 11.6

Ogunniyi 1997 Nigeria > 65 1.1 -

Andersen 1997 Denmark > 65 7.1 -

Chandra 1998 India > 55

> 65

0.8

1.3 -

Herrera 1998 Brazil > 65 7.1 -

Boersma 1998 Holand > 65 6.5 -

Shiba 1999 Japan > 65 8.5 -

Breitner 1999 EUA > 65 9.6 -

Hebert 2003 USA 65-74 7.0 -

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1.3 Risk factors

While the etiology of sporadic AD is still unknown, a number of risk factors

for the development of AD have been identified. The most important risk factor for

the development of AD is increasing age (Qiu et al., 2005). Other well established

risk factors include family history of dementia, female gender, head trauma,

hypertension, high cholesterol, low physical activity, obesity, low education and the

presence of the E4 allele of apolipoprotein E (Kivipelto et al., 2001; Huang et al.,

2004; Kivipelto et al., 2005). The more recently proposed risk factors are

cardiovascular disease (Qiu et al., 2006) and anemia (Atti et al., 2006). In addition to

the most common sporadic form, AD can appear in early onset familial form

suggesting that genetics play an important role in the etiology of the disease (Tanzi,

1999).

1.4 Development of AD Pathology

1.4.1 Pathologic progression

It has been demonstrated that the degenerative process begins in specific

limbic areas of the cortex and then spreads in a predictable pattern across the

hippocampus to the neocortex and a number of sub cortical nuclei (Braak et al.,

1999). The alteration in the pattern and severity of the pathology permit the

distinction of stages in amyloid deposits (Stage A- C) and NFT (Stage I – IV)

(Braak and Braak et al., 1991). Both NP’s and NFT are regionally specific occurring

predominantly in the hippocampus, entorinal cortex and associated areas of the

neocortex (Figure 1.1).

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Figure 1.1 Plaques and tangles in the cerebral cortex in AD. Plaques are extracellular deposits of Aβ surrounded by dystrophic neurites, reactive astrocytes, and microglia, whereas tangles are intracellular aggregates composed of a hyperphosphorylated form of the microtubule-associated protein tau. (Adapted from Kaj Blennow lancet Vol 368 July 29, 2006)

1.4.2 Plaques or tangles which come first

There is an ongoing debate about AD, concerning the sequence of events

underlying the AD pathology. Whether plaques or tangles arise first and how they

are related to one another? It was noted that hyper phosphorylated tau is directly

associated with dementia (Alafuzoff et al., 1987; Arriagada et al., 1992) thus

prompting many researchers to believe that formation of NFT is the key event

leading to the neuronal loss in AD. However, a recent study has demonstrated that a

decrease in endogenous tau levels in an AD mouse model can reduce the toxic

effects of Aβ peptide (Roberson et al., 2007). Interestingly previous studies have

shown that cdk5 activity is increased in AD (Patrick et al.,1999) as well as

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demonstrated that Aβ may induce prolonged activation of cdk5 (Lopes et al., 2007).

In support of this view, many studies proved that, transgenic mice mutant with tau

and APP/tau mutants induce the formation of neurofibrillary tangles on injection

with Aβ1-42 into the mutant mice (Gotz et al., 2001). By using the triple transgenic

model of AD expressing mutant versions of APP, tau and presenilin, it is shown that

accumulation of amyloid occurs prior to the appearance of NFT in the brain (Oddo

et al., 2003). This implies that hyperphoshorylation of tau and formation of NFT

occurs downstream of Aβ generation. On the other hand tau hyperphosphorylation

and NFT formation occurs in other degenerative disorders, which develop in the

absence of amyloid plaques. These include fronto temporal dementia, Picks disease,

progressive supranuclear palsy, corticobasal degeneration and argyrophilic grain

disease (Williams et al., 2006).

1.5 Cognitive changes in AD

AD refers to the pathological process in the brain. However these

pathological changes in AD are followed by the evolution of cognitive changes or

changes in brain function. These changes in brain exist in continuum with normal

ageing process in which the person is free of symptoms of cognition and functional

decline (Asymptomatic phase). However as the pathological process continue, the

individual will experience cognitive and or functional difficulties. These often begin

with subjective memory complaints, which can also be benign and part of normal

ageing (Flicker et al., 1993) but in individuals with AD pathology these deficits will

be gradually manifested. At this stage (preclinical phase / onset of Mild cognitive

impairment (MCI)) the cognitive and or functional difficulties are noted by persons

who are closely associated with these individuals. In the early clinical phase of AD

the symptoms of impairment become more evident and the patient typically exhibits

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deficits in an array of cognitive domains including memory, verbal ability,

visuospatial skills, problem solving, attention, and abstract reasoning. It is often at

this stage that clinicians make a clinical diagnosis of AD. By this time the deficits

reach such magnitude that often the individual can no longer manage on their own.

1.5.1 Preclinical phase of AD (MCI)

An important challenge for the clinicians is to identify subjects with

developing AD in the pre clinical phase of the disease. There are several reasons

why this is of great importance; firstly it would facilitate the intervention of

therapeutic agents to slow down or to prevent the disease progression. The affected

individuals and care givers may also benefit from counseling on how to handle the

cognitive impairment. Secondly, subjects experiencing mild cognitive impairment

can receive a prognosis with regard to their outcome. This may bring relief and may

end uncertainty about their cognitive impairment and may give opportunities to

anticipate their future.

However the major clinical problem is that although the preclinical phase is

characterized my mild cognitive impairment (MCI), the individuals with MCI

represent a heterogeneous group with respect to both clinical presentation and

outcome after follow up. The majority of MCI patients will however progress to AD

at a 10 to 15 % annually (Levey et al., 2006) in these individuals the obvious decline

will manifest over intervals of approximately 2 to 4 years (Flicker et al., 1991). In

contrast some patients remain relatively stable for a long preclinical phase and

perhaps never progress to fulfill the dementia criteria.

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1.6 Diagnosis

1.6.1 Clinical diagnosis

There are several guidelines for the clinical diagnosis of AD: the National

Institute of Neurological and Communicative Disorders and Stroke - Alzheimer’s

Disease and Related Disorders Association Work Group (NINCDS-ADRDA)

criteria (McKhann et al., 1984), the Diagnostic and Statistical Manual of Mental

Disorders, 4th edition (DSM-IV) American Psychiatric Association 1994 and the

ICD-10. The NINCDS-ADRDA criteria have been most widely used in research

because they are well validated, provide high diagnostic accuracy and allow

comparison between studies (Blacker et al. 1994, Gearing et al. 1995). In these

studies, the sensitivity has been better than the specificity and the follow-up of

patients has improved the diagnostic accuracy. Disorders that may mimic or overlap

with AD include depression, normal pressure hydrocephalus, dementia with Lewy

bodies, vascular dementia and frontotemporal dementias.

1.6.2 Neuropsychological tests

1.6.2.1 DSM-IV

DSM-IV is based exclusively on clinical findings. It defines dementia as a

syndrome with multiple cognitive deficits including memory impairment and at least

one of the following cognitive disturbances: aphasia, apraxia, agnosia or

disturbances of executive functioning. These criteria are intended to serve only as

guidelines for diagnosis and their application is dependent on clinical judgment

(Erkinjuntti et al., 1997). Subtypes are early onset AD (EAD onset at < 65 years of

age) and late onset AD (LAD onset at >65 years of age), see Table 1.2.

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Table 1.2 DSM-IV criteria. (Reproduced from APA 1987)

I Development of multiple cognitive deficits manifested by both Memory impairment

(impaired ability to learn new information or to recall previously learned information).

One (or more) of the following cognitive disturbances:

a. Aphasia (Impaired ability to speech)

b. Apraxia (impaired ability to carry out motor activities despite intact motor function)

c. Agnosia (failure to recognize or identify objects despite intact sensory function)

d. Disturbance in executive functioning (i.e., planning, organizing, sequencing, abstracting)

II The cognitive deficits in criteria Ia and Ib each cause significant impairment in social or

occupational functioning and represent a significant decline from a previous level of

functioning.

III The course is characterized by gradual onset and continuing cognitive decline.

IV The cognitive deficits in criteria Ia and Ib are not due to any of the following:

(i) Other CNS conditions that cause progressive deficits in memory and cognition

(ii) Systemic conditions that are known to cause dementia (e.g., hypothyroidism,

vitamin B12 or folic acid deficiency, niacin deficiency, hypercalcemia,

neurosyphilis, HIV infection).

(iii) Substance-induced conditions.

V The deficits do not occur exclusively during the course of a delirium

VI The disturbance is not better accounted for by another axis I Disorder (e.g. Major

depressive Disorder, Schizophrenia).

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1.6.2.2 NINCDS-ADRDA

The NINCDS-ADRDA criterions define possible, probable and definite AD.

The differences between those three, reflects on the available clinical information.

Criteria for Possible AD is diagnosed when the patient has dementia with a clinical

course that resembles AD, but also has a second brain disorder or systemic illness

that is sufficient to produce dementia but is not considered to be the cause of the

dementia and the patient has a progressive deficit in the absence of any other cause.

Criteria for Probable AD requires deficits in two or more areas of cognition;

progressive worsening of memory and other cognitive functions, no disturbances of

consciousness, onset between the ages 40 to 90 and absence of systemic brain

disorders that could explain the memory or cognitive decline. The diagnosis is

supported by progressive deterioration of specific functions such as aphasia, apraxia,

agnosia and impaired ADL. Criteria for definite AD require histopathological

evidence of AD plus clinical criteria for probable AD. See Table 1.3. For an accurate

diagnosis of AD it is vital to rule out other disease that can mimic dementia. Hence

the clinical work-up must include a detailed medical history of concurrent somatic

and psychiatric diseases, heredity, social and professional status and a description of

the course of the actual symptoms. A thorough neurological examination as well as

an evaluation of the cognitive status is required. The latter should be done with the

help of at least the Mini-Mental State examination (MMSE) (Folstein et al., 1975)

but more sophisticated rating scales such as: the ADAS-cog scale (Rosen et al.,

1984), the Blessed-Roth Dementia scale (Blessed et al., 1968), the Clinical Rating

Test (CDR) (Morris, 1993) or the Gottfries-Bråne-Steen (GBS) (Gottfries et al.,

1982) can be used.

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Table 1.3 NINCDS-ADRDA criteria (Reproduced from McKhann 1984) I. The criteria for the clinical diagnosis of “probable AD” include:

• Dementia established by clinical examination and documented by the mental status testing and confirmed by neuropsychological tests.

• Deficits in two or more areas of cognition. • Progressive worsening of memory and other cognitive functions. • No disturbance of consciousness. • Onset between ages 40 and 90, most often after age 65. • Absence of systemic disorder or other brain diseases that in and of themselves could account

for the progressive deficits in memory and cognition. a. The diagnosis of “probable AD” is supported by:

• Progressive deterioration of specific cognitive functions such as aphasia, apraxia and agnosia.

• Impaired activities of daily living and altered patterns of behavior. • Family history of similar disorder, particularly if confirmed neuropathology. • Laboratory results of normal LP as evaluated by standard techniques. • Normal pattern or nonspecific changes in EEG, such as increase slow wave activity. • Evidence of cerebral atrophy on CT with progression documented by serial observation.

b. Other clinical features consistent with the diagnosis of “probable AD”:

• After exclusion of causes of dementia other than AD, include • Plateaus in the course of progression of the illness. • Associated symptoms of depression, insomnia, incontinence, delusions, illusions,

hallucinations, catastrophic verbal, emotional, or physical outbursts, sexual disorders, and weight loss.

• Other neurologic abnormalities in some patients, especially with more advanced disease and including motor signs such as increased muscle tone, myoclonus, or gait disorder.

• Seizures in advanced disease. • CT normal for age.

c. Features that make the diagnosis “probable AD” uncertain or unlikely include:

• Sudden apoplectic onset. • Focal neurological findings such as hemi paresis, sensory loss, visual field deficits, and in

coordination early in the course of the illness. • Seizures and gait disturbances at the onset or very early in the course of the illness.

II. Clinical diagnosis of “possible AD”:

• May be made on the basis of the dementia syndrome, in the absence of other neurologic, psychiatric, or systemic disorder sufficient to cause dementia, and in the presence of variations in the onset, in the presentation, or in the clinical course.

• May be made in the presence of a second systemic or brain disorder sufficient to produce dementia, which is not considered to be the cause of the dementia should be used in research studies when a single, gradual progressive severe cognitive deficit is identified in the absence of other identifiable cause.

III. Criteria for diagnosis of “definite AD”.

• The clinical criteria for probable AD and histopathologic evidence obtained from a biopsy or autopsy.

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1.6.3 Laboratory tests

1.6.3.1 Blood and Urine tests

The laboratory evaluation that is routinely performed includes: Erythrocyte

sedimentation rate, C-reactive protein (CRP), complete blood count, serum

electrolytes (including calcium), glucose, creatinine, liver function tests, thyroid

stimulating hormone (TSH), serum vitamin B12 and urine analysis. Optional tests

are serum folate, methylmalonic acid and blood serology for Borrelia, HIV or other

viruses, urine collection for heavy metals or toxicology (Olafsdottir and Marcusson,

1996).

1.6.3.2 Brain imaging

Brain imaging is an important part of the standard investigation. The use of

computed tomography (CT) does not differentiate early AD from normal ageing

with high diagnostic accuracy (DeCarli et al., 1990; 1992) as there is a substantial

overlap of the atrophy seen in AD with normal ageing (George et al., 1983; Creasey

et al., 1986) but the rate of progression of the atrophy is estimated to be ten times

greater than that seen in normal ageing (Smith, 1996). Magnetic resonance imaging

(MRI) offers a superior anatomic discrimination power and permits accurate

imaging of the affected regions. Many studies have shown that volumetric MRI of

hippocampus and entorhinal cortex may be helpful in the diagnosis of AD

(Juottonen et al., 1999). The decrease in hippocampal volume has a diagnostic

accuracy from 85 % to 100 % in the differentiation of AD patients from non

demented controls (Jack et al., 1997; Laakso et al., 1998) Figure 1.2.

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Normal Brain AD Brain

Figure 1.2 Normal and AD Brian pathology

The differentiation of AD patients from other dementias using hippocampal

volumetry has not yet achieved a high diagnostic accuracy (Laakso et al., 1996). The

use of MRI volumetry as a routine method is quite time consuming and requires

normalization and calibrations in departments specialized in the field (Jack et al.,

1995). MRI is an expensive method and is seen more as a complement to CT in

clinical routine diagnosis.

1.7 Diagnostic issues in recognizing the preclinical phase (MCI)

The available evidence suggests that, diagnostic accuracy is much lower at

the early clinical and especially presymptomatic stages of AD. In recent years, the

clinical phase of AD with MCI the so-called preclinical stage, has gained increased

attention in the medical community as it truly represents a high-risk pre-dementia

state (Solomon et al., 2008; Nestor et al., 2004). As a consequence, the preclinical

AD is an important factor to consider in studies assessing cognitive function.

Pathological and clinical data have accumulated in the last few years showing that

some biological indicators of AD might be used to distinguish those MCI patients

who will progress from those who will not. Thus, new diagnostic tools for the

diagnosis of early and preclinical AD would be of great importance.

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1.8 Biomarkers and early diagnosis of Alzheimer’s disease

Tremendous efforts have been made in recent years to identify the

neuropathological, biochemical and genetic biomarkers of diseases so that the

diagnosis could be established in earlier stages. The search for relevant biomarkers

of AD in living patients has been an active part of clinical research in the last

decade. The assumption for such enterprise is that a biomarker provides at least an

indirect link to the disease process or ideally directly relates to the primary

mechanism of the disease. According to a recent consensus report (Trojanowski et

al., 1998), an ideal biomarker for AD should fulfill the following criteria a) detect a

fundamental feature of the neuropathology; b) be validated in neuropathologically

confirmed cases; c) have a sensitivity higher than 85% for detecting AD and have a

specificity of more than 75% for distinguishing AD from other causes of dementia,

preferably established by two independent studies appropriately powered; d) be

precise, reliable and inexpensive; e) be convenient to use and not harmful to the

patient.

Currently, the success of therapeutic intervention is determined through

psychosocial testing, which is, unfortunately, unable to detect changes over short

periods of time. Furthermore, the various psychosocial tests used to assess the trial

are highly susceptible to variations among investigators, test administrators and day-

to-day fluctuations in the patient’s mood or behavior. Scores are often affected by

educational level, culture, subject age and the ability of the patient to learn the test

when repeated over a period of time. It is also difficult to determine whether small

changes in scores over short periods of time are meaningful or significant and testing

is generally required over a period of 6 months to a year or even longer.

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Another major limitation to the present methods of testing is their inability to

diagnose AD early in its development. Primary care physicians, who generally see

the patients during the early stages of the disease, seem to widely under-diagnose the

disease, because it appears there is considerable brain damage prior to observable

symptoms of AD. It is extremely important to identify AD in its early stages and

ultimately presymptomatically. Early therapeutic intervention may be able to halt or

even reverse AD before extensive brain damage has occurred. Although some tests

are recently introduced to aid diagnosis, such as tau protein, Aβ1-42 or neural thread

protein found in the cerebrospinal fluid (CSF) (Kahle et al., 2000; Blennow 2004;

Ibach et al., 2006). Unfortunately, all these tests are based on CSF samples (obtained

by lumbar puncture), which is not a routine or simple testing procedure.

By using patient history data, brain imaging (Mosconi et al., 2007) and

psychosocial testing (Anton et al 2007) 80 % of patients can be correctly diagnosed

at the present time. However, none of the assessments and testing required to

achieve this accuracy are simple, quick or inexpensive and cannot be used for

routine screening of the general population for AD. Furthermore, the currently

available tests are generally unable to diagnose AD early in its development and are

also inadequate for measuring the impact of different AD therapies.

1.8.1 Biochemical markers

The search for a specific biochemical marker is therefore still essential for

the assessment and management of AD. A quick and simple test based on a

biochemical marker could also reduce the cost and time of current diagnosis. Ante-

mortem biochemical markers have been sought for years in different peripheral

tissues and cells such as erythrocytes (Bosman et al., 1991), lymphocytes (Pirttila et

al., 1992), urine (Ghanbari et al., 1998), hair (De Berker et al., 1997) and skin

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(Soininen et al., 1992; Heinonen et al., 1994). However, until now there has been no

consensus for biochemical markers for AD that would allow pre-symptomatic

detection or definitive pre-morbid diagnosis. In view of the wide spectrum of search

on diagnostic markers, a considerable amount of work has been devoted in the thesis

to unravel the biochemical markers in the extraneuronal tissues.

1.8.1.1 Oxidative stress markers

Growing data from experimental models and human brain studies suggest

that oxidative stress plays an important role in neurodegeneration in AD

(Markesbery et al., 1999). There is increasing evidence that Aβ not only can induce

oxidative stress but its generation is also increased as a result of oxidative stress

(Chauhan and Chauhan 2006). In this thesis, markers of oxidative stress such as

reactive oxygen species (ROS) levels and the DNA oxidation product 8- hydroxy

guanosine (8-OHdG) were measured in lymphocytes, superoxide dismutase (SOD),

glutathione peroxidase (GPx), were measured in red blood cells and glutathione

redox system (reduced glutathione (GSH), oxidized glutathione (GSSG)) were

measured in the plasma from probable/possible AD patients along with age matched

non demented healthy subjects.

1.8.1.2 Platelet APP isoform ratio

Amyloid precursor protein (APP) processing abnormalities are believed to be

a very early change in AD. Since platelets show concentrations of APP isoforms

equivalent to those found in brain, they represent an important peripheral source of

APP to study the APP metabolic changes in AD (Di Luca et al., 2000; Liu et al.,

2007). To unravel the pathogenic mechanism of AD, current thesis focuses on the

detection of platelet APP isoform ratio in AD compared to age matched non

demented healthy subjects.

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1.8.1.3 Plasma Aβ1-42 levels

Beta amyloid (Aβ) can normally be detected in plasma but the levels are

100-fold lower than in CSF. The source of Aβ in plasma is unknown. Previous

studies have shown that Aβ injected into the ventricular CSF of rat is rapidly cleared

into blood, most likely by sub ependymal capillaries and the choroid plexuses

(Ghersi-Egea et al., 1996). Other studies have suggested peripheral sources, such as

platelets, for Aβ in plasma (Chen et al., 1995). The significance of Aβ levels in

plasma in relation to the Aβ accumulation in brain is unclear. Current thesis focuses

on the detection of Aβ1-42 levels in plasma as a biochemical marker of AD.

1.8.1.4 Red blood cells Aβ1-42

Aβ is transported both ways across the blood brain barrier (DeMattos et al.,

2002), resulting in a dynamic equilibrium between brain Aβ and circulatory Aβ. Red

Blood cells particularly in AD subjects are therefore exposed to appreciable levels of

Aβ despite low levels of Aβ reported to be associated with RBC’s (Kuo et al.,

2000). Furthermore recent literature reveals that RBC’s bind Aβ fibrils in vitro

(Jayakumar et al., 2003). The interaction of Aβ fibrils with RBC and the role of

RBC in the removal of Aβ fibrils from the circulation has been studied in a murine

model (Ravi et al., 2005). Based on these studies, the present study focuses on the

red cell interaction with Aβ and the usefulness of RBC bound Aβ for the diagnosis

of AD, the progression of disease and for monitoring drug responses in clinical

trials.

1.9 Aim of the Thesis

The aim of this thesis is to determine the levels of oxidant and antioxidants

in red blood cells, lymphocytes and plasma. Platelet APP ratio, plasma and red

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blood cell bound Aβ1-42 levels as biochemical markers for AD in peripheral venous

blood during common clinical practice.

1.9.1 Specific objectives of the present study are

• To study the oxidant and antioxidant levels (ROS, 8-OHdG, SOD, GPx,

GSH, and GSSG) in peripheral tissues such as lymphocytes, RBC and

plasma, during the disease process

• To investigate the alterations in APP metabolism/concentration in peripheral

tissues such as platelets in diagnosis of AD

• To study the potential of plasma Aβ 1−42 as a biochemical marker of AD

• To study the Aβ interaction with RBC and the usefulness of RBC bound Aβ

1-42 levels as a biochemical marker for the diagnosis of AD in clinical

practice

• To evaluate the utility by combining these parameters as biochemical

markers for the diagnosis of AD in clinical practice

1.10 Subjects and samples

1.10.1 Selection of patients and controls

The study was performed on 42 patients with probable/possible AD and 60

age matched healthy controls. Participants were from the neurological center at Sri

Ramachandra Hospital, a tertiary care hospital attached to Sri Ramachandra Medical

College and Research Institute, Porur, Chennai, India. The patient evaluations were

performed in a standardized way, including the following items. The study was

conducted in accordance with local clinical research regulations and informed

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consent was obtained from all subjects and caregivers. All participants received a

medical, epidemiological, and neuropsychological assessment.

1.10.1.1 Clinical examination

The clinical assessment included a medical history obtained from the patient

and from relatives or caregivers. Medical examinations, which include a somatic,

neuropsychiatric and neurological status was performed on all patients.

1.10.1.2 Neuropsychological assessment

The following assessments were measured: expressive and impressive

language ability, spatial ability, ability of abstract and logic thinking, executive

ability, and short-term memory.

1.10.1.3 Blood and urine samples

Blood was drawn from an antecubital vein for determination of complete

blood cell count, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP),

serum folate, serum B12, Liver function tests, blood glucose, serum electrolytes

including calcium, creatinine, thyroid stimulating hormone (TSH). Urine samples

were collected for urine routine examination.

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1.10.1.4 Brain Imaging

To rule out other causes of cognitive impairment (e.g. brain tumor or

hydrocephalus); CT or MRI of the brain was performed on all patients. Cortical

atrophy, ventricular enlargement, white matter lesions (WML) and focal lesions

(infarcts and lacunes) were recorded.

1.10.1.5 Diagnostic criteria

In the final diagnostic assessment, Diagnosis was coded according to the

International Classification of Diseases, 10th edition. In patients with dementia or

psychiatric diseases the conditions were defined using DSM-IV criteria.

1.10.1.6 AD

Probable and possible AD was defined according to the National Institute of

Neurological Disorders and Communicative Disorders - Alzheimer’s disease and

Related Disorders Association (NINCDS-ADRDA) criteria (McKhann et al., 1984).

All AD patients with symptoms or signs of other dementia disorders, such as

vascular dementia and alcoholic dementia were excluded. An inclusion criterion was

also a MMSE score of below 25.

1.10.1.7 Controls

The control groups consisted of aged matched non demented healthy

individuals. These individuals didn’t have any family histories, symptoms or signs

of psychiatric or neurological disease, malignant disease or systemic disorders (e.g.

collagenosis, rheumatoid arthritis or infectious disease). None of the controls were

receiving pharmacological therapy for neurological or psychiatric disorders. Their

cognitive status was examined using the MMSE (Folstein et al., 1975). Individuals

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with scores below 24 were not included. All controls were examined at Sri

Ramachandra Hospital, a tertiary care hospital attached to Sri Ramachandra Medical

College and Research Institute, Porur, Chennai, India.

1.10.1.8 Exclusion criteria

The following exclusion criteria were designed to ensure that participants

had probable/possible AD as the cause of their dementia:

• Major depressive disorder, bipolar disorder, schizophrenia, substance use

disorder, or mental retardation according to criteria of the DSM-IV.

• Cerebro vascular disorders, hydrocephalus, and intra-cranial mass,

documented by CT or MRI within the past 12 months.

• Abnormalities in serum folate and vitamin B12, syphilis serology, or thyroid

hormone levels.

• History of traumatic brain injury or other neurological disease (e.g.,

Parkinson disease, Huntington disease, seizure disorders).

• Remarkable medical problems (e.g., cancer within the past 5 years, clinically

significant hepatic or pulmonary disorders).

• Alcohol abuse.

Normal entered the study only if neurological examination was negative

In order to avoid potential pharmacological confounding effects on platelet

physiology and coagulation, patients and controls on psychotropic agents,

cholinergic or anticholinergic agents, antiplatelet agents and anticoagulants entered

the study only when drug free for at least 14 days before blood collection and

platelet preparation.

22


The demographic and clinical characteristics of the study population are

presented in Table 1.4. The list of parameters and the type of specimen collected in

the present study and a brief description on sample processing is presented on

Table 1.5.

Table 1.4 Demographic and clinical characteristics of the study population. Data are given as mean ± SD except where noted otherwise. Control: Age matched non demented healthy controls; Probable/Possible AD: Alzheimer’s disease; MMSE: Mini Mental State Examination. Age of onset of the dementia was estimated at the age at which memory loss or changing behavior was first noted by the next of kin or any other relative or partner.

CONTROLS PATIENTS

VARIABLES Non demented age matched

healthy individuals

Probable/Possible AD P value

No. of participants 60 42 -

Age, Mean ± SD, y 71±9.8 73±9.6 NS

Sex (M / F ratio) 39/21 28/14 NS

Age of onset - 69±9.4 NS

Diabetes (%) 11 28 -

Hypertension (%) 23 25 -

Smoking (%) 10 22 -

Education years 11 ± 4 7 ± 3 NS

MMSE Score >25 <24 NS

23


Samples

Table 1.5 List of parameters and the type of specimen collected for processing the samples in the present study.

ANALYTE DRAW TUBE

(Vacutainer) PROCESSING NOTES

Superoxide dismutase (SOD) Heparin Processed on the same

day of blood collection.

Glutathione peroxidase (GPx) Heparin Processed on the same


Reduced glutathione (GSH) Heparin Processed on the same


Oxidised glutathione (GSSG) Heparin Processed on the same


Reactive oxygen species

(ROS) Heparin

Processed on the same


8-Hydroxy guanosine Heparin Samples were stored at –

80oC until assay.

Platelet APP ratio Citrate

Platelets were isolated on

the same day of blood

collection and stored at –

80oC until assay.

Red blood cells Aβ1-42 EDTA Samples were stored at 2-

8oC for a week.

Plasma Aβ1-42 EDTA

Plasma was separated

and stored at – 80oC until

assay.

24

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