No relationship between lipoprotein-associated phospholipase A2, proinflam-
matory cytokines, and neopterin in Alzheimers disease

S. Savas, C. Kabaroglu, A. Alpman, F. Sarac, M.A. Yalcin, Z. Parldar,

F. Ozkinay, E. Kumral, F. Akcicek

PII: S0531-5565(16)30014-6
DOI: doi: 10.1016/j.exger.2016.01.014
Reference: EXG 9774

To appear in: Experimental Gerontology

Received date: 26 November 2015

Revised date: 21 January 2016
Accepted date: 26 January 2016

Please cite this article as: Savas, S., Kabaroglu, C., Alpman, A., Sarac, F., Yalcin, M.A.,
Parldar, Z., Ozkinay, F., Kumral, E., Akcicek, F., No relationship between lipoprotein-
associated phospholipase A2, proinammatory cytokines, and neopterin in Alzheimers
disease, Experimental Gerontology (2016), doi: 10.1016/j.exger.2016.01.014

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 ACCEPTED MANUSCRIPT

No relationship between lipoprotein-associated phospholipase A2,

proinflammatory cytokines, and neopterin
in Alzheimers disease

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S. Savasa,*, C. Kabaroglub, A. Alpmanc, F. Saraca, M.A. Yalcina, Z. Parldarb, F. Ozkinayc, E. Kumrald,
F. Akciceka

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a
Division of Geriatric Medicine, Department of Internal Medicine, School of Medicine, Ege

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University, Izmir, Turkey
b
Department of Clinical Biochemistry, School of Medicine, Ege University, Izmir, Turkey

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c
Department of Medical Genetics, School of Medicine, Ege University, Izmir, Turkey
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Department of Neurology, School of Medicine, Ege University, Izmir, Turkey
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Corresponding author
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Sumru Savas MD, Division of Geriatric Medicine, Department of Internal Medicine, Faculty of
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Medicine, Ege University, Izmir, Turkey

Tel. +90-532.5135839
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Fax. +90-232.3437876
E-mail esumrusavas@yahoo.com
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 ACCEPTED MANUSCRIPT

No relationship between lipoprotein-associated phospholipase A2,

proinflammatory cytokines, and neopterin
in Alzheimers disease

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ABSTRACT

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Objective: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a reported risk factor for dementia.

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However, the relationship between Alzheimers disease (AD) and Lp-PLA2 is still debatable and, to
the best of our knowledge, no study has evaluated the associations between levels of Lp-PLA2,
proinflammatory cytokines, and neopterin in AD.

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Methods: In total, 59 patients with AD and 38 non-demented individuals were included in the case-
control study. Fasting serum concentrations of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-
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), neopterin, and Lp-PLA2 were determined using ELISA. The associations between AD and each of
the variables were analyzed by logistic regression.
Results: The median Lp-PLA2 levels in AD and controls were similar (P = 0.29, not significant).
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Median serum neopterin and IL-6 levels were significantly higher in patients with AD than in controls
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(P = 0.0001 and P = 0.03, respectively). In regression analyses, median neopterin levels, a lower level
of education, and female gender were significantly associated with AD when compared with controls
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(OR, 31.44, 95% CI 3.59275.28, P = 0.002; OR, 4.35, 95% CI 1.1316.61, P = 0.032; OR, 7.25, 95%
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CI 1.8828.00, P = 0.004, respectively).

Conclusion: In contrast to previous evidence suggesting its role in dementia and AD, Lp-PLA2 enzyme
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levels were higher in the controls, and no relationship between Lp-PLA2 and either proinflammatory
cytokines or neopterin was identified in AD. Elevated neopterin levels may be considered
inflammatory markers of AD.

Keywords: Lipoprotein-associated phospholipase A2; Proinflammatory cytokine; Neopterin;

Alzheimers disease

Abbreviations: Lp-PLA2, Lipoprotein-associated phospholipase A2; TNF-, Tumor necrosis factor-

alpha; IL-6, Interleukin-6

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1. Introduction

The proximal mechanisms underlying dementia and Alzheimers disease (AD) are complex and
poorly understood. Although there are many hypotheses, chronic inflammation is known to contribute

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substantially to the etiopathology of AD (McGeer & McGeer, 2004). In previous studies, inconclusive

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results have been obtained regarding the role of lipoprotein-associated phospholipase A2 (Lp-PLA2),

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which may be an inflammatory marker in AD (Rader, 2000). The enzyme belongs to the
phospholipase A2 (PLA2) superfamily (Fitzpatrick et al., 2014), which forms inflammatory molecules

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by hydrolyzing oxidized low-density lipoprotein (LDL) (MacPhee et al., 1999). In addition, Lp-PLA2
may hydrolyze platelet-activating factor in platelets, monocytes, and macrophages (Tselepis et al.,

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2002). Its expression is regulated by inflammatory cytokines (Cao, Stafforini, Zimmerman, McIntyre,
& Prescott, 1998). High Lp-PLA2 expression is thought to be a predictor of coronary heart disease
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(Packard et al., 2000; Ballantyne et al., 2004; Caslake & Packard, 2003) and is associated with the risk
of stroke, independent of traditional cardiovascular risk factors (Oei et al., 2005; Fitzpatrick et al.,
2014). High Lp-PLA2 enzyme activity is a risk factor for dementia, independent of cardiovascular and
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inflammatory factors, based on the Rotterdam study (van Oijen et al., 2006). In agreement with this
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result, Fitzpatrick et al. (2014) concluded that Lp-PLA2 is a risk factor for dementia, independent of
cardiovascular disease and its risk factors. However, in the Framingham Heart Study, Lp-PLA2 mass
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was not associated with dementia or AD (van Himbergen et al., 2012).

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It is hypothesized that various risk factors such as dyslipidemia and oxidative stress promote
the accumulation of damage signals, which may be the earliest triggering event in AD. After the
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activation of microglias, tumor necrosis factor-alpha (TNF-), interleukin (IL)-6, and some trophic
factors are released (Maccioni, Rojo, Fernndez, & Kuljis, 2009). However, the cytokine network is
complex, and these molecules are biologically labile and rapidly disappear from circulation (Licastro
et al., 2000). Neopterin is an indicator of peripheral immune responses (Fuchs, Weiss, & Wachter,
1993) and enables effective monitoring of immune responses (Akgl et al., 2013). Increased neopterin
levels have been found in patients with AD (Leblhuber et al., 1999).
As chronic inflammation may play a substantial role in the etiopathology of AD, we
hypothesized that there is a relationship between Lp-PLA2, cytokines, and neopterin; accordingly, we
examined each of these factors in this study. These relationships may explain the inconclusive results
regarding the role of Lp-PLA2 in AD.

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2. Material and methods

2.1. Patients

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Patients with AD were recruited from the Neurology Department of the School of Medicine of

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Ege University, Izmir who fulfilled the criteria for probable and possible AD established by the

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National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's
disease and Related Disorders Association (Morris et al., 1989). Between December 2010 and March

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2011, 80 consecutive patients were recruited. Cognitive function was assessed using the adapted
Turkish Mini-Mental State Examination (MMSE) (Gngen, Ertan, Eker, Yaflar, & Engin, 1999) and

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AD Assessment Scale (ADAS-Cog) (Rosen, Mohs, & Davis, 1984). Patients with a history of
cerebrovascular disease, prior coronary revascularization, dialysis, liver disease, malignancy,
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congestive heart failure, or acute and chronic inflammatory diseases were excluded from the study.
Finally, 59 patients with MMSE scores between 10 and 24 were included in the study.
The control group consisted of non-demented individuals from the Internal Medicine
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Department of the same School of Medicine. Seventy consecutive individuals with non-specific
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complaints were evaluated. Individuals with an MMSE score of greater than 24 who did not meet the
exclusion criteria were considered normal controls. In total, 38 subjects were included in the study as
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the control group.

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Subjects were classified based on marital status (married/living with spouse or living alone/not
married/widow) and level of education (5 years or <5 years). Baseline examinations, including a
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questionnaire and blood chemistry were performed for all patients to confirm the referral diagnosis.
Prospectively recorded variables included age, gender, risk factors, blood pressure, and cardiac and
neurological findings. Hypertension was defined as either systolic pressure 140 mm Hg, diastolic
pressure 85 mm Hg, or taking antihypertensive drugs. Diabetes mellitus was specified as fasting
serum glucose 125 mg/dL, nonfasting serum glucose 200 mg/dL, or use of oral blood sugar-
lowering drugs or insulin. Hyperlipidemia was defined as total cholesterol (TC) 200 mg/dL,
triglycerides (TG) 160 mg/dL, high-density lipoprotein cholesterol (HDL-C) 40 mg/dL, LDL
cholesterol (LDL-C) 160 mg/dL, or the use of lipid-lowering drugs. Levels below 0.3 mg/dL for
high-sensitivity C-reactive protein (hs-CRP) were accepted as normal. Written informed consent was
obtained from all participants or the guardians of the patients, and the study was approved by the Ege
University Ethical Committee.

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2.2. Biochemical analysis

Venous blood samples were drawn after a 12-h overnight fasting period. Blood samples were
centrifuged at 3,000 g for 10 min within 30 min of collection. All biochemical analyses were

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performed on the sample collection day, except for IL-6, TNF-, neopterin, and Lp-PLA2 levels. For

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estimates of these parameters, fasting serum samples were stored at 80 C until use.
The fasting serum concentrations of IL-6, TNF-, and neopterin, and Lp-PLA2 were

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determined using enzyme-linked immunosorbent assays (ELISA) (DIA Source, Louvain-la-Neuve,

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Belgium; Thermo Scientific, Waltham, MA, USA, DRG Instruments GmbH, Marburg, Germany;
diaDexus, Inc., San Francisco, CA, USA, respectively). The lower detection limits were 2 pg/mL for

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IL-6, 15.6 pg/mL for TNF-, 0.2 ng/mL for neopterin, and 0.34 ng/mL for Lp-PLA2. Neopterin values
were expressed as nmol/L. The fasting concentrations of TC, TG, HDL-C, and hs-CRP were
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determined using commercially available kits (Modular Analytics, Roche Diagnostics, Basel,
Switzerland). LDL-C was calculated by the Friedewald equation.
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2.3. Statistical analyses

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The ShapiroWilk test was used to assess normality. For continuous variables, the differences
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between the AD and control groups were assessed using the MannWhitney U test. Spearmans
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correlation analyses were used to evaluate the correlations between continuous variables. The results
are expressed as medians, interquartile ranges (IQR), and means ( SD), where appropriate. The
associations between AD and the variables were analyzed by logistic regression analyses. Results are
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presented as odds ratio (OR) estimates with 95% confidence intervals (CI). Values of P < 0.05 were
considered statistically significant. All statistical analyses were performed using the Statistical Package
for Social Sciences (SPSS/Windows version 18.0, SPSS Inc., Chicago, IL, USA).

3. Results

No statistically significant differences in age or sex were observed between patients in the AD
group [age; median 76 (IQR) (9) years; 39 females, 20 males] and the control group (age; median
70.50 (9) years; 22 females, 16 males) (P = 0.06 and 0.41, respectively). The patients with AD had
statistically significantly fewer years of education than the control group (P = 0.03). There were no
significant differences between AD patients and the control group with respect to marital status,

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gender, presence of DM, HT, dyslipidemia, or hs-CRP levels. Sociodemographic characteristics and
clinical data of AD patients and the control group are summarized in Table 1.

Table 1. Socio-demographic characteristics and clinical data for patients with Alzheimers disease and
the control group.

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Characteristics Patients with AD Control Group OR (95% CI)

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(n = 59) (n = 38)

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Age a, y [+SD; range] 75 6.4 (5885) 72 5.9 (6585)

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Female, n (%) 39 (66) 22 (58) 1.42 (0.613.28)

Living alone, n (%) 21 (36) 10 (26) 1.55 (0.633.79)

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Education <5 years, n (%) 41 (70) 18 (47) * 2.53 (1.095.89)

DM, n (%) 17 (29) 7 (18) 1.79 (0.664.85)

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HT, n (%) 28 (48) 14 (37) 1.55 (0.673.57)

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Hypercholesterolemia b, n (%) 23 (47) 23 (61) 0.58 (0.241.36)

Hypertriglyceridemia b, n (%)
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11 (22) 11 (29) 0.71 (0.271.88)

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High LDL-C b, n (%) 33 (73) 31 (84) 0.53 (0.181.59)

Low HDL-C b, n (%) 10 (23) 4 (11) 2.28 (0.658.02)

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High hs-CRP b, n (%) 24 (62) 25 (70) 0.70 (0.271.84)

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*Significant difference between groups

**Values in parentheses are given as percentages

a
Values are expressed as means SD.

b
Missing data

Y, years; DM, diabetes mellitus; HT, hypertension; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density
lipoprotein cholesterol; hs-CRP, high-sensitivity C-reactive protein; AD, Alzheimers disease.

Lp-PLA2, IL-6, TNF-, and neopterin levels in patients with AD and the control group are
summarized in Table 2. The median Lp-PLA2 level was higher in the controls than in AD patients (P =

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0.29). Serum neopterin and IL-6 levels were significantly higher in the AD group than the control
group (P = 0.0001 and P = 0.03, respectively) (Figure 1). There was no relationship between Lp-PLA2
and either inflammatory cytokines or neopterin, as well as hs-CRP and age in both groups and the
whole population (data not shown).

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Table 2. Lipoprotein-associated phospholipase A2, proinflammatory cytokine, and neopterin levels in
patients with Alzheimers disease and the control group.

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Factor a Patients with AD Control group Significance

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Lp-PLA2 (ng/mL) 217.89 (60.8) 221.42 (62) P = 0.29

IL-6 (pg/mL) 2.82 (4.3) 1.55 (2.2) P = 0.03

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TNF- (pg/mL) 9.42 (6.4) 8.01 (9.5) P = 0.48

Neopterin (nmol/L) 17.37 (30.1) 8.89 (6.1) P = 0.0001

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________________________________________________________________________________________________
Lp-PLA2, Lipoprotein-associated phospholipase A2; IL-6, Interleukin-6; TNF-, Tumor necrosis factor-alpha; AD,
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Alzheimers disease.
a
Values are expressed as medians (interquartile range).
Bold values indicate statistically significant relationships (P < 0.05).
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Fig. 1. IL-6, TNF-, and neopterin levels in patients with Alzheimers disease and the control group.
AD, Alzheimers disease; IL-6, Interleukin-6; TNF-, Tumor necrosis factor-alpha.

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In the AD group, age was not correlated with any of the estimated parameters (data not shown).
However, in the whole population, age was positively correlated with neopterin (r = 0.235, P = 0.035)
(Figure 2) and negatively correlated with both TC and LDL-C, (r = -0.262, P = 0.014 and r = -0.292, P
= 0.008, respectively). When the age of the whole population was classified as 6074 years or 75

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years, neopterin was significantly higher in the 75 years old group (P = 0.012).

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Fig. 2. Neopterin levels are associated with age.

In multiple stepwise logistic regression analyses, median neopterin levels, low education level,
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and female gender were significantly associated with AD when compared with controls (OR, 31.44,
95% CI 3.59275.28, P = 0.002; OR 4.35, 95% CI 1.1316.61, P = 0.032; OR, 7.25, 95% CI 1.88
28.00, P = 0.004, respectively).

4. Discussion

AD is characterized by the presence of neurofibrillary tangles, senile plaques, and neuron and
synapse loss, which are observed in normal brain aging. However, these changes are more severe in
AD and the degeneration initially takes place in specific regions of the cerebral cortex. (D'Andrea,
2003). It is still unclear why and how AD develops. Accordingly, the relationship between chronic
inflammation and AD is of great interest and understanding the mechanisms that mediate this
relationship may provide a basis for preventative methods and novel therapies. In the present study, we

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investigated the associations of Lp-PLA2, proinflammatory cytokines, and neopterin with AD. We
determined that Lp-PLA2 is not associated with either AD or levels of proinflammatory cytokines and
neopterin.
Various PLA2 subtypes have been identified according to their biochemical features (Clark et

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al., 1991; Chen, Engle, Seilhamer, & Tischfield, 1994; Cupillard, Koumanov, Mattei, Lazdunski, &

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Lambeau, 1997). PLA2 participates in various physiological processes, including the remodeling of cell
membranes, intracellular signaling, and phospholipid metabolism. PLA2 functions in the inflammatory

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response by contributing to the release of arachidonic acid from membrane phospholipids in most cell

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types, which is a fundamental step in the synthesis of major mediators of inflammation (Gattaz,
Forlenza, Talib, Barbosa, & Bottino, 2004). Lp-PLA2 contributes to the generation of

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lysophosphatidylcholine and oxidized fatty acids by oxidizing phospholipids, which are believed to
have proinflammatory properties (MacPhee et al., 1999). It is an inflammatory biomarker (Rader,
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2000) that is expressed in macrophages and foam cells (Rubinstein & Izkhakov, 2011) and circulates
in the blood associated with LDL-cholesterol (Stafforini, McIntyre, Zimmerman, & Prescott, 1997).
Lp-PLA2 is also known as platelet-activating factor acetylhydrolase and is a potential predictor of
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coronary heart disease (Ballantyne et al., 2004). Likewise, Lp-PLA2 is associated with strokes (Oei et
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al., 2005). In male SpragueDawley rats, chronic mild vascular risk factors over months induce small
lesions of brain capillaries in the cortex, which may contribute to the development of vascular
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dementia and AD (Ehrlich & Humpel, 2012). Although present data suggest that the management of
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cardiovascular risk factors may reduce cognitive decline, their impact on the development of AD is
less certain (Giordano et al., 2007). Additionally, it has been reported that the Lp-PLA2 enzyme is a
risk factor for dementia, irrespective of cardiovascular and inflammatory factors and risk factors of
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cardiovascular diseases (van Oijen et al., 2006; Fitzpatrick et al., 2014). However, analyses of data
from the Framingham Study failed to replicate this association; in this prospective cohort study of
dementia-free Framingham Heart Study participants, Lp-PLA2 levels were not associated with all-
cause dementia or AD (van Himbergen et al., 2012). In a recent cross-sectional study, plasma Lp-PLA2
was not associated with a diagnosis of AD or amnestic mild cognitive impairment (Davidson et al.,
2012). Consistent with these previous results, we found no association between serum Lp-PLA2 levels
and AD in the current study. Furthermore, Lp-PLA2 levels were higher in the control group than in the
AD group; however, the difference was not statistically significant.
Inflammation and immune activation may play an important role in the development and
progression of dementia (McGeer & McGeer, 2004). Brain inflammation is mainly caused by the
activation of glia cells that produce proinflammatory and neurotoxic factors (Bernstein et al. 2005;
Sharma, Al-Omran, & Parvathy, 2007; Coppus, Fekkes, Verhoeven, Tuinier, & van Duijn, 2010).

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Many studies have investigated the levels of various cytokines and other inflammatory markers
(Swardfager et al., 2010). In the Framingham study, subjects with higher spontaneous production of
IL-1 or TNF- by peripheral blood mononuclear cells were at an increased risk of developing AD (Tan
et al., 2007). In a prospective cohort study of patients with mild to severe AD, high baseline levels of

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TNF- were associated with an increase in the rate of cognitive decline (Holmes et al., 2009). In

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addition, increased peripheral levels of IL-6 have been reported in patients with AD in various studies
(Singh & Guthikonda, 1997; Licastro et al., 2000). However, in a longitudinal study with a small

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sample, no significant differences were observed between AD subjects and controls with respect to the

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mean serum levels of IL-1, IL-1 receptor antagonist, IL-6, TNF-, the soluble TNF receptors type I
and II, and alpha 1-antichymotrypsin (Lanzrein et al., 1998). Additionally, although there is evidence

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suggesting that hs-CRP is associated with cognitive decline (Yaffe et al., 2003; Noble et al., 2010) or
the risk of dementia (Engelhart et al., 2004), and other investigations do not support this association
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(Kravitz, Corrada, & Kawas, 2009; Tan et al., 2007; van Himbergen et al., 2012). Finally, in a meta-
analysis including 40 studies that measured peripheral blood cytokine concentrations and 14 studies
that measured cerebrospinal fluid cytokine concentrations, significantly higher concentrations of IL-6,
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TNF-, IL-1, transforming growth factor-, IL-12, and IL-18 were observed in patients with AD than
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in control subjects; however, this difference was not observed for IL-4, IL-8, IL-10, interferon-, or
CRP in peripheral blood (Swardfager et al., 2010). Similarly, our data showed that AD patients had
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significantly higher blood levels of IL-6, but did not have higher concentrations of hs-CRP.
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Additionally, studies investigating the levels of different cytokines are limited owing to the biological
instability and short half-lives of these molecules (Hagberg et al., 2010; Parker et al., 2013). However,
neopterin has a long half-life in biological fluids (Hagberg et al., 2010) and can be used to indirectly
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monitor peripheral immune responses (Fuchs et al., 1993). Previous studies have reported elevated
serum neopterin levels in several diseases, such as autoimmune and malignant tumor diseases,
neurological, and cardiovascular diseases, as well as in infections (Balzs, Trke, & Vmos, 2012;
Nyamweya et al., 2012; De Rosa et al., 2011). Increased serum neopterin levels have also been found
in patients with AD (Leblhuber et al., 1999). Consistent with this, in a recent study, subjects with AD
had significantly higher plasma neopterin values compared with those observed in patients with
amnestic mild cognitive impairment and normal subjects (Parker et al., 2013). However, Licastro et al.
(2000) found that the plasma levels of neopterin were slightly lower in AD patients than in controls;
however, the differences were not statistically significant. In the present study, we detected elevated
serum neopterin levels in patients with AD. In the whole population, age was positively correlated
with neopterin and negatively correlated with TC and LDL-C. The decreased levels of TC and LDL-C
may be explained by the anorexia of ageing or by the use of lipid-lowering drugs.

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In a recent study, elevated plasma Lp-PLA2 activity was associated with an increase in
inflammatory cytokines, particularly IL-6 and oxidized LDL, in women with metabolic syndrome
(Chae et al., 2011). Additionally, in another recent study, no strong correlations were detected between
Lp-PLA2 activity and cerebrospinal fluid markers of AD; the main clinical correlates of Lp-PLA2

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activity were variables associated with lipid metabolism (Davidson et al., 2012). Few studies have

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examined the relationship between neopterin and Lp-PLA2 levels. Kabarolu et al. (2013) did not
observe a statistically significant relationship between Lp-PLA2, neopterin, IL-6, fibrinogen, and hs-

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CRP. Similarly, we did not detect a relationship between Lp-PLA2 enzyme levels and proinflammatory

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cytokines, neopterin, or hs-CRP in patients with AD. Female gender and low education levels were
associated with AD, in agreement with the findings of previous studies (Launer et al., 1999). The

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limitations of the current study include the limited number of study subjects and the missing data
regarding lipid parameters and hs-CRP.
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5. Conclusions
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We did not detect a relationship between Lp-PLA2 enzyme levels and AD. However, serum
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neopterin and IL-6 levels were significantly higher in AD patients than in the control group. Elevated
neopterin levels may be used as an inflammatory marker in patients with AD. Further studies of
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neopterin as a biomarker for diagnosis or for monitoring of disease progression and larger studies
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investigating the relationship between AD and Lp-PLA2 and neopterin are needed. The genes that
encode PLA2 enzymes could play a role in disease susceptibility; accordingly, polymorphisms in genes
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that act as regulatory factors of strategic molecules like cytokines and Lp-PLA2 should be examined.

Funding

This study was supported by funding from Ege University Scientific Research Projects
(Number: 2010-TIP-023).

Conflict of interest

All authors declare that there are no personal or financial conflicts of interest.

Acknowledgements

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We would like to acknowledge Hayal Boyacioglu, PhD and Hatice Uluer for their contributions
to the statistical analysis.

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Highlights

We studied Lp-PLA2, TNF-, IL-6, and neopterin in Alzheimers disease (AD) cases.
Neopterin, low level of education, and female gender were associated with AD.

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Elevated neopterin levels are potential inflammatory markers of AD.

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