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• Investigators evaluated peripheral levels of a broad spectrum of non-enzymatic and enzymatic antioxidants, of nitrogen oxidative species and lipid and protein oxidation markers in a homogenous and clinically well characterized group of Mild Cognitive Impairment (MCI) and early stage Alzheimer's disease (AD) patients (mild AD) compared with age-matched healthy subjects. 
• The presence of the apolipoprotein E (ApoE) ε4 allele, a major risk factor for sporadic AD, was also analyzed in order to verify the relationship between oxidative parameters and ApoE genotype 
• Investigators also searched for possible correlations between oxidative and clinical variables including age, gender and cognitive evaluation.

• All patients underwent a thorough biochemical, neurological and neuropsychological evaluation and were in a stable condition without acute comorbidities
• The control group consisted of healthy volunteers, matched for age, with no neurological or psychiatric history, A CDR of zero and without cognitive impairment on MMSE using Portuguese normative data.

No specific exclusion criteria was provided.

Recruitment

The study subjects were recruited at the Dementia Clinic, Neurology Department of Coimbra University Hospital. 

Design

Case Control Study 

Blinding used

Implied with measurements 

Intervention

Not applicable 

Statistical Analysis

• For comparisons of variables between diagnostic groups, one-way ANOVA followed by the Bonferroni (for variables showing a normal distribution) or the Games-Howell post-test (for variables that do not have normal distribution) was used
• Comparison within groups were made using either the T test for variables with homogenous variances or the Mann-Whitney U test for non-homogenous variables
• Pearson test was used for correlation between parameters and x² analysis for frequency evaluation.

Timing of Measurements

Laboratory determinations were made after fasting blood samples were obtained.

Dependent Variables

• Uric acid plasma levels
• Plasma levels of vitamins A and E
• Vitamin E present in red blood cells
• Plasma and erythrocyte levels of reduced glutathione (GSH) and oxidized glutathione (GSSG)
• Total antioxidant status (TAS)
• Erythrocyte glutathione peroxidase (GIPx)
• Erythrocyte glutathione reductase (GIRed)
• Levels of lipid peroxidation measured by the formation of a thiobarbituric acid (TBA) adduct of malondialdehyde (MDA)
• Plasma protein carbonyl content
• Plasma levels of nitric oxide (NO).

Independent Variables

• MCI group
• Mild AD group
• Control group. 

Control Variables

• Gender
• Age
• Age at onset
• Education
• MMSE
• ADAS-Cog
• AoE ε₄ carrier.

 

• Initial N: 164
• Attrition (final N): 164
• Age:
   

  	Controls	MCI	Mild AD
  Age	68.4±1.8	71.1±0.8	73±1.2

• Ethnicity: Not specified
• Other relevant demographics:
   

  	Controls	MCI	Mild AD
  Age at onset	N/A	67.9±0.9	69.9±1.3
  Education	6.5±0.6	6.2±0.5	5.6±0.7
  MMSE	28.4±0.5	27±0.6	20.9±0.6
  ADAS-Cog	N/A	10±0.5	21.6±1.4

• Anthropometrics:
  

  	Controls	MCI	Mild AD
  ApoE ε4 carrier [n (%)]	9 (24)	31 (36)	20 (48)

• Location: Coimbra, Portugal.

 

Key Findings

ApoE-ε₄ allele:

The AD group showed an increased distribution of the ApoE-ε₄ allele relative to controls (P<0.05), whereas the MCI group, although presenting a higher distribution of ApoE-ε₄ allele did not prove to be statistically different from the controls.

Plasma Antioxidants:

• Mild AD patients showed decreased levels of plasmatic antioxidants relative to controls and MCI patients.  In particular, mild AD patients showed statistically significant decreased plasma levels of vitamin E (P<0.05) and a slight decrease although not statistically significant of vitamin A.
• Comparative to controls, but not to the MCI group, AD patients also present higher levels of the oxidized form of glutathione (GSSG) which were not accompanied by a decrease of the tripeptide reduced form (GSH) (P<0.005).
• Statistically significant higher levels of plasma GSSG were found in MCI patients when compared to controls, similar to those found in mild AD patients (P<0.05)
• There was a trend for a decrease in uric acid levels and total antioxidant status in MCI and mild AD patients that failed to reach statistical significance. Also, intracellular levels of vitamin E, GSH and GSSG showed no differences between patients and controls as well as the activity of the antioxidant enzymes GIPx and GIRed. 
• There were no changes in the plasma levels of protein carbonyls or of the lipid oxidation marker between patients and controls. In erythrocytes, there was a significant increase in lipid oxidation both in mild AD and MCI patients compared to controls (P<0.05). Also, MCI and mild AD patients presented increased plasma levels of NO₂+NO₃ as compared to controls, indicative of an increase in nitrogen reactive species (P<0.05). 

Gender:

• Females had significantly higher plasma levels of the antioxidant vitamin E (P<0.05). In MCI patients, the level of another plasma antioxidant defense, GSH, was also increased in females (P<0.05). In AD patients, there was an increased activity of the erythrocytes antioxidant enzyme GIPx in females (P<0.05).
• Nevertheless, these differences were not accomplished by a higher cognitive performance in females as better MMSE scores were found in males which were not related to asymmetries in education (P<0.05). In the control population, there were no differences in the levels of any of the peripheral antioxidants and oxidative markers studied between genders.

The Influence of ApoE-ε₄ Genotype on the Peripheral Oxidative Status:

• Patients and controls were divided into subgroups according to the absence or presence of the allele. It was observed for both MCI and mild AD patients that the presence of the allele was associated in red blood cells with an increase in lipid peroxidation as assessed by the levels of MDA (P<0.05) and a decrease in the levels of non-enzymatic antioxidant defense GSH (P<0.05).
• In both patients groups, a trend for decreased plasmatic levels of vitamin E in ε₄ carriers was found (P>0.05). ApoE-ε₄ genotype did not influence the peripheral oxidative status of the controls.

Correlations Between Antioxidant Levels, Oxidative Markers and Clinical Variables:

In all groups, significant positive correlations were found between:

• Plasma uric acid levels and total antioxidant status (P<0.001)
• The levels of vitamin E and A in plasma (P<0.05)
• The plasma and erythrocytes levels of MDA (for controls and AD, P<0.05 and for MCI, P<0.01)  
• Between the activity of GIPx and the levels of GSSG in erythrocytes (P<0.05) were found. 

Additionally, the following negative correlations were found between:

• The levels of lipid peroxidation (MDA) and the non-enzymatic antioxidants, GSH in controls (P<0.05) and MCI patients (P<0.01) 
• Vitamin E in mild AD patients (P<0.01).

Considering mild AD patients, significant negative correlations between plasma antioxidant levels of vitamin E or GSH and cognitive function, assessed by MMSE score were found (r= -0.369; P=0.029; r= -0.446, P=0.008), while these same variables exhibited positive correlation with ADAS-Cog scores (r=0.363, P=0.028; r=0.397, P=+0.023).

Also, a tendency for a positive correlation between MDA levels in erythrocytes and MMSE was also observed (r=0.298; P=0.074). On the contrary, in MCI patients, erythrocytes levels on GSH were positively correlated with MMSE scores (r=0.37; P=0.002) and negatively related with ADAS-Cog scores (r= -0.354; P=0.006). Moreover, in MCI patients, a negative correlation was found between plasma vitamin E levels and duration of disease (r= -0.403; P<0.001). 

 

The main finding of this study was that most of the oxidative changes found in mild AD patients are already present in the MCI group. Both groups presented in comparison to age matched controls with increased levels of plasmatic nitrogen reactive species and markers of lipid peroxidation in red blood cells.  Furthermore, there was an increase in the oxidized form of the plasma antioxidant defense glutathione in MCI and mild AD patients. However, the plasma levels of vitamin E were markedly reduced in mild AD patients while the MCI group showed levels similar to controls.

A statistically significant increase in the plasmatic oxidized form of glutathione similar to that occurring in mild AD patients was found in MCI patients relative to controls. Also, the NO₂ and NO₃ plasma levels observed in MCI and mild AD patients suggested an increase in the production of NO that can react with anion superoxide, generating peroxynitrite. The evolvement of NO in AD pathogenesis has previously been recognized.

An expected result was that the presence of the Apo ε4 allele was increased in AD patients while in MCI patients it was higher, although not statistically, than the control population. In MCI and mild AD patients, the presence of a ε4 allele was associated with an increase in lipid peroxidation and a decrease in the levels of GSH in red blood cells and a trend for decreased plasma levels of vitamin E. This tendency for more marked indexes of oxidative stress in ε4 carriers had already been suggested for AD patients. This study widens these findings to the MCI population.

In this study, the decrease in the plasmatic antioxidant defense vitamin E in the male population of mild AD patients was associated with improved cognitive function. In fact, there was a negative correlation between the plasma levels of vitamin E or GSH and cognitive status, as quantified by the MMSE in mild AD patients whereas a trend for a positive correlation between MDA levels in red blood cells and MMSE score was also observed. These findings suggest that susceptibility to oxidative stress, due to a decrease in antioxidant defenses and increase in both lipid and nucleic acids oxidative markers, might be an early event in AD.

This is the first time that a correlation between cognitive deterioration and antioxidant depletion was reported in MCI patients. These findings are in accordance with the hypothesis that oxidative stress is an early event in AD pathology. This hypothesis should be tested in a longitudinal analysis of MCI patients where the progression of oxidative damage and the progression to AD could be followed. 

One of the most important applications of this type of study is the development of strategies that prevent or delay cognitive deterioration and dementia. This study and other studies show that oxidative stress is a very early event in AD which occurs in the pre-symptomatic phase. Other epidemiological prospective cohort studies show that dietary intake of vitamin C and E may lower the risk of AD and a similar effect has also been reported with ß-carotene and flavonoids. New antioxidant strategies appear to be an encouraging focus of therapeutic intervention and should be proposed as primary prevention measures, maybe years before the dementia age risk.

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Portuguese Society of Neurology

• Study limitations were only discussed briefly when comparing this study to other studies. Only mild AD patients were included in this study compared to other studies with older patients with much more advanced disease stages. Also, there may have been differences between this study and other studies in the way in which TAS was used to determine antioxidants and uric acid levels.
• There were some differences between the groups, but they were inherent to the group assignment, such has members of the MCI and mild AD groups having lower cognition than the control group.
• There was no discussion of confounding factors.