Tamotsu study: changes in the phospholipid composition of erythrocyte membranes in Alzheimer's disease

Changes in the phospholipid composition of erythrocyte membranes in Alzheimer's disease

Original scientific article
Dement Geriatr Cogn Disord Extra 2012;2:298-303
DOI: 10.1159/000341603

Published online: August 15, 2012
\u00a9 2012 S. Karger AG, Basel. www.karger.com/dee

The article is publicly available under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License (www.karger.com/OA-license ), which applies only to the online version of the article. Distribution is allowed only for non-commercial purposes

Shinji Omaa Shiro Mawatarib Kazuyuki Saitos Chikako Wakanas Yoshio Tsuboya Tatsuo Yamada a Takehiko Fujino

Department of Neurology, Fukuoka University Faculty of Medicine, Institute of Rheological Nutrition Function, and Boocs Fukuoka Clinic, Fukuoka, Japan

Keywords: Alzheimer's disease вАҐ Erythrocyte phospholipids Ethanolamine-plasmalogen вАҐ Sphingomyelin вАҐ Oxidative stress

Information about the article: Department of Neurology, Faculty of Medicine of Fukuoka University, Institute of Rheological Function of Nutrition, and Boocs Fukuoka Clinic, Fukuoka, Japan

There are several studies indicating a decrease in the content of ethanolamine plasmalogen (pl-PE) in brain tissues and serum in patients suffering from Alzheimer's disease (AD).

The present study is aimed at studying the composition of erythrocyte phospholipids, including pl-PE, in patients suffering from AD: the method of high-performance liquid chromatography (HPLC) which allows separating intact plasmalogens and all other classes of phospholipids in one chromatographic run.

Results: the content of pl-PE, phosphatidylethanolamine (PE) and phosphatidylserine (PS) was found to be low about sphingomyelin compared with the indicators of the control group of the corresponding age.

Conclusion: these changes in erythrocyte phospholipids may be a consequence of oxidative stress, which indicates a high degree of oxidative stress in the peripheral blood of patients suffering from AD.

Introduction

Alzheimer's disease (AD) is the most common cause of dementia in the elderly. As the average life expectancy increases, AD becomes a serious problem for world medicine.

Several studies indicate a decrease in the content of ethanolamine plasmalogen (pl-PE) in the brain tissues of patients suffering from AD [1-3]. It was also found that the serum of patients suffering from AD has a reduced pl-PE content and that a decrease in pl-PE content in serum correlates with the severity of dementia [4, 5].

In animals, plasmalogens are present mainly in the cell membrane of tissues, therefore, peripheral blood cells contain much more pl-PE than serum. We previously reported on the possibility of separating intact plasmalogens and other phospholipids in one run using high-performance liquid chromatography (HPLC) [6]. The HPLC method detects only traces of pl-PE of human serum but determines the pl-PE of human erythrocytes [6].

Anomalies of erythrocytes have been reported in patients suffering from AD [7-11] but anomalies of pl-PE erythrocytes have not yet been investigated.

In this study, we measured the phospholipid composition, including pl-PE, of erythrocytes in patients suffering from AD, and compared it with data from control subjects of the corresponding age group.

Table 1. Age, gender and score on the Multiple Mental Status Assessment Scale (MMSE)

	Control group (n=15)	Group with AD (n=16)
Age	73.1 ¬± 4.6	76.5 ¬± 8.1
Man / Woman	5/10	7/9
MMSE	30.0 ¬± 0	4.1¬±3.4

Patients and methods

Patients

The study, conducted by the Department of Neurology at Fukuoka University Hospital, Japan, involved sixteen patients suffering from AD and a control group of 15 healthy subjects of the appropriate age.

The study was conducted by the Helsinki Declaration and was approved by the Expert Council of the Fukuoka University Clinic. BA was diagnosed clinically, including by the Short Mental Status Assessment Scale (MMSE).

Patients with a high ratio (>2) of low-density lipoproteins to high-density lipoproteins, obesity, hypertension and high blood sugar were excluded from the control group, and patients with a high C-reactive protein index (>1 mg/100 ml) were excluded from both groups. Age, gender and MMSE are shown in Table 1.

The indicators of total serum cholesterol, low-density lipoproteins, high-density lipoproteins, triglycerides, blood sugar and C-reactive protein in patients with AD did not differ statistically from those of the control group.

Getting red blood cells

The patients' venous blood, taken on an empty stomach, was placed in test tubes containing EDTA-2Na. The blood was cooled in an ice bath, kept in the refrigerator and processed for 48 hours. Plasma and leukocyte film was removed after centrifugation at 1000 g for 5 min at 4°C, erythrocytes were washed three times in cold isotonic saline solution at 1000 g for 5 min at 4 ° C. At each washing, a small part of the top layer was removed.

Determination of the lipid composition of erythrocytes

The extraction of lipids from erythrocytes was carried out by the method described in the works of Mawatari et al. [6] and Dise et al. [12]. 500 ml of compacted erythrocytes were hemolysed in an equal volume of phosphate buffer solution of 10 mmol (pH 7.4). Four milliliters of methanol were injected into the lysate, and then after 40 minutes 4 ml of chloroform. After another 30 minutes, the extract was centrifuged, and the remainder was re-extracted with 4 ml of a mixture of methanol and chloroform (1:1, by volume). Methanol and chloroform were used in the extraction of lipids containing butylhydroxytoluene (50 mg/l). The combined extracts were washed with 10 ml of KCl 10 mmol to obtain a two-phase mixture. One milliliter of the lower phase of the lipid extract was dried with N2 gas.

The separation of phospholipid classes, including plasmalogens, by HPLC was carried out according to the method described by us [6,13]. The Agilent HPLC HPLC system (Agilent Technologies, Tokyo, Japan) with an evaporative nephelometric detector was used. The system was connected to the Chem Station (Agilent Technology) workstation, which was used for monitoring and analyzing chromatograms. The dried total lipids were dissolved in 200 µl of a mixture of hexane and 2-propanol (1:1, vol/vol), and after filtration through a 0.2 µm filter, 20 µl of the solution was placed into the HPLC system.

Fig. 1. Representative HPLC chromatogram of human erythrocyte lipids. Lipids are detected by an evaporative nephelometric detector. This HPLC method revealed several peaks of sphingomyelin; the two largest peaks are designated SM-1 and SM-2. Chol = cholesterol.

Statistical analysis

We analyzed the data using a paired (two-sided) t-test with a significance level of p<0.05.

Results

Classes of phospholipids in erythrocyte membranes, including pl-PE and cholesterol, were determined simultaneously in one HPLC run (Fig. 1).

The relative composition of phospholipid classes was calculated from the chromatographic region of each of them, after which the ratios of glycerophospholipids to sphingomyelin (CM) were determined (Table 2). The ratio of pl-PE to CM in patients with AD was lower than in the control group (Table 2).

The ratio of phosphatidylethanolamine (PE) and phosphatidylserine (PS) to CM was also lowered. The ratio of phosphatidylcholine (PC) to CM was the same.

Table 2. Ratio of glycerophospholipids to sphingomyelin

	Control group (n=15)	Group with AD (n=16)
pl-PE/CM (x100)	40,0 ¬± 3.6	35.2 ¬± 3.6
PE/CM (x100)	50.4  ¬±  7.6	45.1  ¬±  6.5
PS/CM(x100)	14.6  ¬±  6.9	8.5 ¬± 7.1
PC/CM(x100)	110.0  ¬±  17.6	102.4 ¬±  10.4

Analysis of results

Circulating human erythrocytes do not contain intracellular organelles, therefore, lipids are present only in the cell membrane. Therefore, lipids extracted from washed erythrocytes are lipids of erythrocyte membranes.

CM in the membrane of human erythrocytes contains a small number of polyunsaturated fatty acids (PUFA); at the same time, PS contains a large amount of docosahexaenoic and arachidonic acid [13, 14]. In the membrane of human erythrocytes, PS contains more PUFA than PE, and PC contains much less PUFA than PS and PE [13,14]. Phospholipids with a high PUFA content are known to be susceptible to oxidative stress [15,16]. Our past studies have shown that the relative content of CM and PC increased, in contrast to PS, PE and pl-PE, when human erythrocytes were incubated in tert-butylhydroperoxide (tBHP) [6].

Thus, the ratio of each glycerophospholipid to CM is a reliable indicator of the degree of peroxide change of each glycerophospholipid [6,14].

In our previous study [13], pl-PE in human erythrocytes had a higher PUFA content than PS and PE, and the decrease in pl-PE content in human erythrocytes due to tBHP oxidation was approximately twice as high as the decrease in PE and PS [6]. In the present study, the degree of pl-PE reduction was the highest among glycerophospholipids, and the degree of PS reduction was greater than the degree of PE reduction (Table 2). There was no decrease in PC.

These results are very close to the results of the oxidation of human erythrocytes by tBHP [6]. Consequently, it appears that changes in phospholipid erythrocytes in AD correspond to changes from oxidative stress.