Role of Oxidative Stress Markers F2-Isoprostanes and Presenilin-1 in Clinical Diagnosed Alzheimer’s Diseases

ABSTRACT

Introduction:

Oxidative stress is an important component in the pathogenesis of Alzheimer’s disease (AD).

Materils and Methods:

A comparative cross-sectional study was conducted on 140 participants who made up 70 subjects diagnosed with AD and 70 controls. Serum levels of F2-isoprostanes and Presenilin 1 were estimated using ELISA. These markers were significantly elevated in the serum of AD patients, suggesting them to be potential biomarkers for early diagnosis and disease progression.

Aim and Objective:

In this study, the level of F2-isoprostanes and Presenilin 1 was determined in patients clinically diagnosed with AD, thus establishing a possible relationship between heightened markers for oxidative stress and progression of AD.

Results:

The results showed significantly elevated levels of F2-isoprostanes (179.38 ± 3.01 pg/mL vs 85.89 ± 0.97 pg/mL, P < 0.0001) and Presenilin 1 (93.26 ± 2.39 ng/mL vs 49.46 ± 0.78 ng/mL, P < 0.0001) in AD patients compared to controls. Additionally, markers of lipid peroxidation, such as malondialdehyde (MDA), were elevated in AD patients, while antioxidant defense mechanisms, including glutathione (GSH) and superoxide dismutase (SOD), were significantly impaired (P < 0.0001).

Conclusion:

This study provides compelling evidence that oxidative stress, as shown by elevated levels of F2-isoprostanes and Presenilin 1, plays a crucial role in the onset of AD.

INTRODUCTION

Dementia is the most common form of Alzheimer’s disease (AD) in the elderly population across the world. AD has yet to determine its mechanism of progression but only recently has discovered a stress-induced oxidizing state component in disease pathology, such as in neurofibrillary tangles and amyloid plaques.[1] There are two biomarkers of oxidative stress, F2-isoprostanes and Presenilin 1, and their potential as markers for early diagnosis and disease progression in AD patients. The two prototypical lesions of AD are: 1) senile plaques, consisting of a nucleus of β-amyloid protein accumulation (Aβ42), as extra-cellular lesions and 2) neurofibrillary tangles (NFTs) formed of phosphorylated tau protein (P-tau) and constituting an intraneuronal finding.[2] It can also deposit in the walls of capillaries, arteries, and arterioles causing amyloid cerebral angiopathy, leading to degeneration of vascular wall components and worsening of flow besides making intraparenchymal hemorrhages more probable.[3]

AD is most characterized by progressive loss of episodic and cognitive functions that gradually lead to loss of language and visuospatial skills. Most often, these changes come along with behavioral disorders such as apathy, aggressiveness, and depression.[4] It should be noted that there is an important subgroup of AD patients who do not present a typically amnestic picture, manifesting nonamnestic deficits from the onset of symptoms.[5]

F2-isoprostanes in Alzheimer

F₂-isoprostanes are a group of 64 compounds isomeric in structure to cyclooxygenase-derived PGF₂^α. Other products of the isoprostane pathway are also formed in vivo by rearrangement of labile PGH₂-like isoprostane intermediates. These include E₂- and D₂-isoprostanes,[6] cyclopentenone-A₂- and J₂-isoprostanes,[7] and the highly reactive acyclic ketoaldehydes (isoketals).[8]

The family of 64 F2-isoprostanes are structurally isomeric to the cyclooxygenase-derived PGF2α. Other products of the isoprostane pathway that are also in vivo produced by rearrangement of labile PGH2-like isoprostane intermediates include the E2- and D2-isoprostanes, the cyclopentenone-A2- and J2-isoprostanes, and the highly reactive acyclic ketoaldehydes (isoketals).

Presenilin 1

Presenilin 1 (PSEN1) is a protein that plays a critical role in the development and maintenance of the nervous system. It is a transmembrane protein located in the endoplasmic reticulum and plasma membrane of cells and is primarily known for its involvement in the pathogenesis of AD.[1] PSEN1 was first identified in 1995 as the gene responsible for early-onset familial AD, a rare form of the disease that affects individuals before the age of 65. Mutations in the PSEN1 gene account for about 20–50% of familial AD cases and lead to the production of abnormal amyloid-beta protein, a hallmark feature of AD.[9]

METHODOLOGY

This study was conducted in the biochemistry department, Department of Psychiatry and Medicine at Jawaharlal Nehru Medical College Sawangi (Meghe) Wardha and Datta Meghe Institute of Higher Education and Research (DU) Sawangi (Meghe), Wardha in Maharashtra, India,

Study period: The study period will more than 3 years.

Study area: The study will be carried out at rural area in Vidarbha region.

Study design: Comparative cross-sectional study

Inclusion criteria

1. Participants aged 60 years and above.

2. Individuals with documented measures of oxidative stress markers.

3. Individuals with clinically diagnosed AD.

Exclusion criteria

1. Participants below the age of 60 years.

2. Individuals without a diagnosis of AD.

3. Individuals with other causes of dementia will be excluded.

4. Studies lacking quantifiable measures of oxidative stress markers.

5. Studies lacking essential clinical data or outcome measures relevant to oxidative stress markers.

Sample size

This comparative cross-sectional study or experimental study on clinically diagnosed AD patients is proposed to be conducted at JNMC and DMMC. As per available statistically data from national and state estimates from a nationwide study, January 1, 2023, the incidence rate of the AD in the age group of 60 years and above is 7.4%.

The sample size was computed using Epi Info software (version 7.0) with a precision of 7% and a confidence level of 95%, with a design effect of 1%. Exclusion criteria included 10% AD linked with concurrent pathology such as diabetes, hypertension, renal failure, and seriously unwell patients, as well as 10% nonresponsive patients. As a result, the ultimate sample size is 50 for the study group and 50 for the control group, using the following formula:

$$n=\frac{z^{2}p(1-p)}{e^2}$$

n is the sample size

z is the selected critical value of desired confidence level (CL95%=1.96)

p is the estimated proportion of an attribute that is present in the population (7.4% =0.074)

e is the desired level of precision (7% =0.07)

So, the final calculated sample size is 50 for the study group.

And 50 sample size for the control group.

Blood Sample Collection

The serum sample will be collected in a plain tube and allow samples to clot for 2 hours at room temperature or overnight at 2–8°C. Centrifuge at approximately 1000 × g (or 3000 rpm) for 15 minutes. Collect serum and assay immediately or aliquot and store samples at -20°C or -80°C for estimation F2-isoprostanes and Presenilin 1.

Biochemical Parameters

The study of F2-isoprostanes and presenilin-1 was done by ELISA method at Jawaharlal Nehru Medical College Sawangi (Meghe) Wardha and Datta Meghe Institute of Higher Education and Research (DU) Sawangi (Meghe), Wardha in Maharashtra, India.

Statistical analysis: All estimated results will be expressed as mean ± SD. Mean values will be assessed for significance by unpaired Student t-test. A statistical analysis will be performed using the Statistical Package for the Social Science program (SPSS, 24.0). Frequencies and percentages will be used for the categorical measures. Probability values P < 0.05 will be considered statistically significant.

RESULT

Table 1 shows agewise distribution of study and control groups. The age of the subjects of this study was in the range 65–74 years. It is shown that the maximum number of subjects was in the age group of 70–74 years, that is, 40 (57.14%). The least number of subjects was found in the age group of 65–69 years, that is, 42.85%.

Table 1.

Agewise group distribution of the study and control groups

Age	Study group	Control group
65-69	30 (42.85%)	37 (52.85)
70-74	40 (57.14%)	33 (47.14)

All data are expressed as mean and standard deviation. Student t-test was applied to data to find statistical significance. *P<0.0001 is statistically significant

Table 2 shows genderwise distribution of oxidative stress markers in clinical diagnosed ADs enrolled in the study group. The maximum number of oxidative stress markers, that is 37 (52.85%) subjects, were having male.

Table 2.

Sexwise group distribution of the study and control groups

Gender	Study Group	Control Group
Male	37 (52.85%)	36 (51.42%)
Female	33 (47.14%)	34 (48.57%)

All data are expressed as mean±SD. Student t test was applied to data to find statistical significance. *P<0.0001 is statistically significant T

Table 3 shows biochemical parameters in the control and study groups, oxidative stress marker Presenilin-1 (ng/mL), which correlated with F2-isoprostanes (pg/mL), significantly increased in oxidative stress markers patients. The elevated levels of Presenilin-1, F2-isoprostanes, MDA, GHS, and SOD were significantly increased (P < 0.0001), which is statistically significant.

Table 3.

Comparison of biochemical parameters in the study and control groups

Biochemical Parameters	Group I (Study)	Group II (Control)	P
Presenilin-1 (ng/mL)	93.26±239	49.46±0.78	<0.0001
F2-Isoprostanes (pg/mL)	179.38±3.01	85.89±0.97	<0.0001
MDA (nmol/mL)	5.73±0.31	2.23±0.16	<0.0001
GSH (μmol/L)	1.36±0.18	3.45±0.14	<0.0001
SOD (U/mL)	86.42±1.93	147.17±1.46	<0.0001

All data are expressed as mean±SD. Student t-test was applied to data to find statistical significance. *P<0.0001 is statistically significant

DISCUSSION

This study explored the relationship between oxidative stress markers—Presenilin 1, F2-Isoprostanes, Malondialdehyde (MDA), Glutathione (GSH), and Superoxide Dismutase (SOD)—in clinically diagnosed AD patients compared to a control group. The findings revealed significant alterations in oxidative stress markers in AD patients, supporting the hypothesis that oxidative stress plays a critical role in AD pathology.

Presenilin 1 and AD pathology

Our study results show significantly higher levels of Presenilin 1 in AD patients, compared to controls (93.26 ± 2.39 ng/mL vs 49.46 ± 0.78 ng/mL, P < 0.0001). This was in line with previous research suggesting that Presenilin 1 (PSEN1) mutations resulted in the onset of FAD and the progression of these diseases. According to Hardy and Selkoe.,[10] the most well-studied mutations of PSEN1 have been shown to be associated with an increase in the production of amyloid-beta (Guarantano’s mean Aβ42) peptides that forms such toxic aggregates and amyloid plaques forming a hallmark of AD. Consistent with this observation, our detection of elevated Presenilin 1 levels [Table 3] is consistent with oxidative stress producing the observed effect through an inhibition of amyloid deposition which could be caused by a defect in γ-secretase complex function mediated through oxidation specifically of Presenilin 1.

F2-isoprostanes and lipid peroxidation

The levels of F2-isoprostanes in AD patients were significantly high in our study. This is evidenced by 179.38 ± 3.01 pg/mL versus 85.89 ± 0.97 pg/mL, P < 0.0001. This further supports the idea that lipid peroxidation is an important pathological feature of AD. Montine et al.[11] similarly described the findings of F2-Isoprostanes, which are derived from the nonenzymatic oxidation of arachidonic acid, to be some of the useful biomarkers for oxidative stress within neurodegenerative conditions. In AD, increased levels of F2-Isoprostanes reflected elevated lipid peroxidation, which might even escalate neuronal damage.

Malondialdehyde (MDA) as a marker of oxidative stress

MDA levels were significantly elevated in the study group, that is, AD group (5.73 ± 0.31 nmol/mL vs 2.23 ± 0.16 nmol/mL, P < 0.0001). MDA is a marker of lipid peroxidation and is one of the most used indicators of oxidative damage in neurodegenerative diseases. Our results are consistent with those of Butterfield et al.[12] (2007), who established that MDA was significantly higher in the brains of subjects affected by AD than in control subjects, because lipid oxidation would be increased, thereby causing neuronal dysfunction and cell death.

Glutathione (GSH) and antioxidant defense

We have found that the levels of glutathione (GSH), the major endogenous antioxidant, are significantly lower in AD patients compared to controls, indicating that in AD, the mechanisms of antioxidant defense are impaired. Consistent with such findings, Chen JJ et al.[13] (2022) have been accustomed to report that lowered GSH levels in AD patients correspond to increased oxidative damage. Depletion of GSH exacerbates oxidative stress as it limits the body’s capacity to effectively neutralize ROS, which in turn causes damage to the neurons and other cells.

Superoxide Dismutase (SOD) and oxidative stress

In our study, the activity of SOD was significantly lowered in AD patients (86.42 ± 1.93 U/mL vs 147.17 ± 1.46 U/mL, P < 0.0001), indicating defective enzymatic antioxidant defense. SOD is the primary enzyme responsible for the dismutation of superoxide radicals into oxygen and hydrogen peroxide, thus protecting cells from oxidative damage. Keller et al.[14] (2000), in which SOD activity was significantly decreased in AD patients, reduced antioxidant capacity continues to play a determinant role for increased oxidative stress and neuronal injury.

CONCLUSION

It has been indicated that oxidative stress plays a critical function in the formation of AD and suggested that the upregulated levels here are a vital factor for each biomarker MDA, GSH, SOD, and Presenilin 1. AD patients were considered to be controls with increased levels of F2-isoprostanes and Presenilin 1 and probably as biomarkers that could contribute to the development of several key events adjusting amyloid plaque or oxidative stress in pathogenesis if AD. Oxidative stress had an effect on lipid peroxidation resulting in a significant increase of MDA levels; the link between oxidative stress and neuronal damage in AD was well established by this study.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.