A metabolic link between S-adenosylhomocysteine and polyunsaturated fatty acid metabolism in Alzheimer's disease

Neurobiology of Aging 28 (2007) 1834–1839

A metabolic link between S-adenosylhomocysteine and polyunsaturated fatty acid metabolism in Alzheimer’s disease Michael L. Selley ∗ Angiogen Pharmaceuticals Pty. Ltd., Level 31, ABN AMRO Tower, 88 Phillip Street, Sydney, NSW 2000, Australia Received 3 June 2005; received in revised form 7 July 2006; accepted 15 August 2006 Available online 25 September 2006

Abstract There is evidence that vascular risk factors contribute to the pathology of Alzheimer’s disease. Increased concentrations of circulating homocysteine are associated with vascular risk factors and Alzheimer’s disease but the mechanisms involved are unclear. Homocysteine inhibits the hydrolysis of S-adenosylhomocysteine (SAH) which is a product inhibitor of S-adenosylmethionine (SAM) dependent methyltransferase reactions. It has been shown previously that SAH inhibits phosphatidylethanolamine N-methyltransferase (PEMT) in the liver. The activity of PEMT in the liver plays an important role in the methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) and the delivery of essential polyunsaturated fatty acids (PUFAs) to peripheral tissues. In the present study, the plasma concentrations of SAH, SAM and homocysteine and the erythrocyte composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and their respective polyunsaturated fatty acid concentrations were determined in 26 patients with Alzheimer’s disease and compared to those in 29 healthy control subjects. There was a significant increase in the plasma concentrations of SAH (p < 0.001) and homocysteine (p < 0.001) and a significant increase in the plasma concentrations of SAM (p < 0.001) in the Alzheimer’s patients. A significant positive correlation was found between the plasma concentrations of SAH and homocysteine (r = 0.738, p < 0.001). There was a negative correlation between the plasma concentrations of homocysteine and the ratio of SAM/SAH (r = −0.637, p < 0.01). There was a significant decrease in the erythrocyte content of PC (p < 0.001) and an increase in the erythrocyte content of PE (p < 0.001) in the Alzheimer’s patients. Plasma SAH concentrations were negatively related to erythrocyte PC concentrations (r = −0.286, p < 0.01) and positively related to erythrocyte PE concentrations (r = 0.429, p < 0.001). The erythrocyte PC from Alzheimer’s patients had a significant depletion of docosahexaenoic acid (DHA) (p < 0.001) while there was no significant difference in the DHA content of erythrocyte PE. There was a significant negative correlation between plasma SAH and the DHA composition of erythrocyte PC (r = −0.271, p < 0.001). This data may reflect the inhibition of hepatic PEMT activity by SAH in Alzheimer’s disease. The decreased mobilization of DHA from the liver into plasma and peripheral tissues may increases the risk of atherosclerosis and stroke leading to chronic cerebral hypoperfusion. The evidence suggests that a metabolic link between the increased production of SAH and phospholipid metabolism may contribute to cerebrovascular and neurodegenerative changes in Alzheimer’s disease. © 2006 Elsevier Inc. All rights reserved. Keywords: Alzheimer’s disease; S-Adenosylhomocysteine; Phospholipid; Polyunsaturated fatty acid

1. Introduction It has been demonstrated that increased blood concentrations of homocysteine are a strong risk factor for Alzheimer’s disease [10,41,56]. Hyperhomocysteinemia is associated with a number of risk factors for vascular disease including atherosclerosis [54], hypercholesterolemia [47], hyperten∗

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sion [60], type 1 diabetes [28], insulin resistance syndrome [43], type 2 diabetes [15], stroke [4] and atrial fibrillation [2]. These disorders are all risk factors for Alzheimer’s disease [24,35,16,37,49,26,48]. Vascular risk factors are associated with cerebral hypoperfusion which may contribute to the metabolic, cognitive and neurodegenerative pathology of Alzheimer’s disease [30,12,31]. S-Adenosylmethionine (SAM) is the methyl donor for most cellular methyltransferase reactions [9]. The transfer of the methyl group leads to the formation of S-adenosylhomo-

M.L. Selley / Neurobiology of Aging 28 (2007) 1834–1839

cysteine (SAH). SAH binds to the catalytic region of methyltransferases with higher affinity than SAM and is a potent inhibitor of cellular methylation. SAH is hydrolyzed to homocysteine and adenosine by the enzyme S-adenosylhomocysteine hydrolase (SAH hydrolase) [27]. Homocysteine is an inhibitor of SAH hydrolase and increased concentrations of homocysteine result in parallel increases in intracellular SAH and the inhibition of methyltransferases [65,8,29]. It has been shown that increased plasma concentrations of SAH are a more sensitive indicator of cardiovascular disease than homocysteine [34]. There is a positive association between a low ratio of SAM to SAH in erythrocytes and peripheral arterial disease [39]. The plasma concentrations of SAH in patients with atherosclerosis are positively correlated with DNA lymphocyte hypomethylation [7]. SAH concentrations also influence the activity of phosphatidylethanolamine N-methyltransferase (PEMT) in the liver [61]. The methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) plays an important role in the delivery of essential fatty acids from the liver to the plasma and peripheral tissues and may have implications in the development of atherosclerosis [63]. There is evidence that cerebrovascular and neurodegenerative changes occur in parallel in Alzheimer’s disease [12]. The loss of DHA in the brains of patients with Alzheimer’s disease is accompanied by a decrease in memory and learning [58]. There is an inverse relationship between the ratio of n-3 and n-6 fatty acids in erythrocyte membranes and cognitive decline in older persons [22]. Impairment of memory in normal aging in the rat is associated with a decrease in DHA concentrations in the brain [13,14,17]. The chronic administration of DHA enhances long-term memory in young and aged rats deficient in n-3 PUFAs [19,20]. DHA administration protects against the oxidative stress and loss of avoidance learning ability caused by the infusion of amyloid-␤ into the cerebral ventricle of rats on a low n-3 PUFA diet [21]. The depletion of DHA in the Tg2576 transgenic mouse model of Alzheimer’s disease is accompanied by postsynaptic caspasemediated actin cleavage and loss of the actin-regulating dendritic spine protein debrin [5]. It has been shown recently that an adequate dietary intake of DHA significantly decreases total insoluble amyloid-␤, plaque burden and APP processing pathways in aged Tg2576 mice [38]. It has been proposed that the increased expression of amyloid-␤ combined with a low dietary intake of n-3 PUFAs leads to further increases in oxidative stress and the accelerated depletion of DHA in postsynaptic dendrites [5]. It was found previously that increased concentrations of homocysteine are associated with decreased concentrations of adenosine in the plasma of patients with Alzheimer’s disease [55]. This suggests that increased homocysteine production in Alzheimer’s disease may inhibit SAH hydrolase and increase the production of SAH. It has been reported increased SAH concentrations in the brains of Alzheimer’s patients inhibit methyltransferases and that this was related to cognitive impairment [33]. In the present study, the plasma

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concentrations of SAH, SAM and homocysteine and the erythrocyte membrane content of PC and PE and their respective polyunsaturated fatty acid (PUFA) concentrations were determined in patients with Alzheimer’s disease and compared to those in healthy control subjects.

2. Methods 2.1. Study population The study population were matched for age and sex. Individuals with a history of diabetes, hypercholesterolemia, hypertension (including current antihypertensive medication), ischemic heart disease, stroke, cancer, rheumatoid arthritis, alcohol abuse, major depression, schizophrenia and hepatic or renal dysfunction were excluded from the study. None of the participants were current smokers or vegetarians. None were known to be taking Vitamin B12 or folate (including multivitamin preparations) or prescribed drugs known to affect circulating SAM or SAH concentrations. Twentynine patients were diagnosed with Alzheimer’s disease in accordance with the NINCDS-ADRDA and DSM-IV criteria [1,42]. The patients had mild to moderate dementia as defined by a score of 11–24 on the mini-mental state examination [46] and a score of >12 on the cognitive subscale of the Alzheimer’s disease assessment score [53]. The mean age of the Alzheimer’s disease group was 71.9 years (range 62.1–81.2 years). There were 15 males and 14 females. The control group consisted of 26 subjects with no symptoms of cognitive dysfunction assessed using the mini-mental state examination [18]. The mean age of the control group was 71.3 years (range 61.7–86.2 years). There were 14 males and 12 females. The protocol was approved by the Ethics Committee at each site in accordance with the Declaration of Helsinki. 2.2. Sample collection Blood samples (10 ml) were obtained by venipuncture into EDTA tubes following an overnight fast and the blood placed on ice and centrifuged within 30 min to stabilize homocysteine concentrations [36,46]. Plasma and erythrocytes were separated by centrifugation at 2000 × g for 15 min at 4 ◦ C. The erythocytes were suspended in phosphate buffered saline and centrifuged at 2000 × g for 15 min at 4 ◦ C to remove excess plasma. The plasma and erythrocytes were stored under argon at −80 ◦ C until analysis. 2.3. Determination of S-adenosylmethionine and S-adenosyhomocysteine in plasma The plasma concentrations of SAM and SAH were determined simultaneously by liquid chromatography electrospray tandem mass spectrometry (LC–MS/MS) using 13 C5 SAH as the internal standard [59].

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M.L. Selley / Neurobiology of Aging 28 (2007) 1834–1839

2.4. Determination of homocysteine in plasma The plasma concentrations of homocysteine were determined using LC–MS/MS with homocystine-d8 as the internal standard [40].

separately for Alzheimer’s patients and control subjects, and standard p-values for significance were calculated. In addition, scatterplots of each pair of concentrations were examined for departures from linearity. The relationships were re-assessed using a Spearman rank correlation to establish consistency with the Pearson correlations.

2.5. Determination of phosphatidylethanolamine, phosphatidylcholine and n-3 polyunsaturated fatty acids 3. Results The total lipids were extracted from erythrocytes and the PC, PE and total phospholipid concentrations determined by high performance liquid chromatography with evaporative light scattering detection [62,64]. The PUFA content of the PE and PC fractions was analyzed by capillary column gas liquid chromatography of the methyl esters [11]. 2.6. Statistics 2.6.1. Mean differences The mean differences between plasma concentrations of SAH, homocysteine and SAM and the erythrocyte concentrations of PE and PC as well as the PUFA content for the Alzheimer’s patients and the control subjects were assessed using a standard two-sample t-test assuming equal variances in the two groups. In addition, the data were graphically examined for possible departures from normality which might invalidate the results of the t-test. Also, Wilcoxon ranksum tests were performed to establish consistency of the given t-test p-values. 2.6.2. Correlations Standard Pearson correlation coefficients were calculated for each of the concentration variables in the given dataset,

The mean differences in the concentrations of SAH, homocysteine and SAM are presented in Table 1. The results indicate a significant increase in the plasma concentrations of SAH, homocysteine and SAM among the Alzheimer’s patients compared to the control subjects. The SAM/SAH ratio was decreased in the Alzheimer’s group compared to the control group. Table 2 shows the mean differences in the erythrocyte concentrations of PC, PE and PUFAs. The erythrocyte concentration of PC decreased while the erythrocyte concentration of PE increased in the Alzheimer’s patients compared to the control subjects. There was a significant depletion of DHA from erythrocyte PC in the Alzheimer’s patients compared to the control subjects. There was a decrease in arachidonic acid concentrations in erythrocyte PC from the Alzheimer’s patients. The DHA composition of erythrocyte PE from the Alzheimer’s patients was similar to that in the control subjects. There was an increase in the arachidonic acid and ␣-linolenic acid concentrations in the erythrocyte PE from Alzheimer’s patients compared to the control group. There were no differences in the concentrations of linoleic and eicosapentaenoic acid in PC and PE from the Alzheimer’s patients and the controls.

Table 1 Plasma concentrations of SAH, homocysteine and SAM in Alzheimer’s patients and control subjects Concentration

Alzheimer’s patients (mean ± S.E.)

SAH (nmol/L) Homocysteine (␮mol/L) SAM (nmol/L) SAM/SAH

51.3 15.62 97.10 1.93

± ± ± ±

1.9 0.68 3.20 0.21

Control subjects (mean ± S.E.) 22.8 9.57 53.37 2.41

± ± ± ±

1.7 0.84 1.05 0.18

p-Value <0.001 <0.001 <0.001 <0.05

Table 2 Concentrations of PE, PC and PE and PC PUFAs in red blood cells in Alzheimer’s patients and control subjects Concentration (g/100 g PUFA)

Alzheimer’s patients (mean ± S.E.)

PE PC PE DHA PC DHA PE linoleic acid PC linoleic acid PE arachidonic acid PC arachidonic acid PE ␣-linolenic acid PC ␣-linolenic acid PE eicosapentaenoic acid PC eicosapentaenoic acid

12.06 58.29 5.68 1.32 7.67 18.71 23.96 2.36 0.42 0.19 1.21 0.60

± ± ± ± ± ± ± ± ± ± ± ±

0.21 3.02 0.94 0.05 0.33 1.63 0.71 0.64 0.03 0.07 0.15 0.05

Control subjects (mean ± S.E.) 9.93 79.36 6.94 3.21 7.05 16.08 15.38 5.85 0.34 0.31 1.13 0.55

± ± ± ± ± ± ± ± ± ± ± ±

0.24 2.81 0.85 0.07 0.412 2.53 0.72 0.52 0.09 0.07 0.51 0.04

p-Value <0.001 <0.001 ns <0.001 ns ns <0.001 <0.001 <0.01 ns ns ns

M.L. Selley / Neurobiology of Aging 28 (2007) 1834–1839 Table 3 Correlations between the plasma concentrations of SAH and homocysteine and the PC and PE concentrations and the DHA content of PC in erythrocytes in Alzheimer’s patients

SAH Homocysteine * **

PC

PE

DHA

−0.286*

0.429**

0.271** 0.349**

−0.374*

0.315**

Significant at the p < 0.01 level. Significant at the p < 0.001 level.

There was a significant positive correlation between the plasma concentrations of SAH and homocysteine in the Alzheimer’s patients (r = 0.738, p < 0.001). The main PUFAs affected by disruption of PEMT are DHA and arachidonic acid [63] therefore correlations were performed only for these PUFAs. Table 3 shows the correlations between the plasma concentrations of SAH and homocysteine and the concentrations of PC and PE in erythrocytes and the DHA concentration in PC in the Alzheimer’s patients. There was a significant negative correlation between SAH and PC and a significant positive correlation between SAH and PE. The correlation between SAH and DHA was significant and negative. No other significant correlations with the plasma concentrations of SAH were found. Spearman rank correlations were in close agreement with all the correlations, thereby confirming the results.

4. Discussion Hyperhomocysteinemia is a strong risk factor for Alzheimer’s disease [10,41,56] and is linked to a number of vascular disease risk factors [54,47,60,28,43,15,4,2]. These vascular disease risk factors are associated with the development of Alzheimer’s disease [24,35,16,37,49,26,48]. This suggests that homocysteine may be a common factor in the pathogenesis of both Alzheimer’s disease and vascular disease. One mechanism by which homocysteine may contribute to adverse vascular events is by reversal of the SAH hydrolase reaction leading to the accumulation of SAH and the inhibition of cellular methylation reactions [65,8,29,34,39,7]. The plasma concentrations of SAH in the Alzheimer’s patients were inversely related to the concentrations of erythrocyte PC suggesting that SAH inhibits the activity of hepatic PEMT. The decrease in the ratio of SAM/SAH is a further indication of hypomethylation in the Alzheimer’s patients [39]. There is epidemiological evidence of an association between a reduced risk of Alzheimer’s disease and a diet high in DHA [32,3,45]. It has been shown that DHA and arachidonic acid are depleted in hepatic and plasma PC in PEMT-deficient mice [63]. The activity of PEMT plays a critical role in the mobilization of DHA from the liver into the plasma and its distribution into other tissues [63]. There was a significant decrease in the DHA concentrations in the erythrocyte PC of patients with Alzheimer’s disease. It is widely accepted that n-3 PUFAs protect against atherosclerosis and thrombo-

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sis [23]. Atherosclerosis of the circle of Willis and the large leptomeningeal arteries are positively associated with the neuropathological lesions of Alzheimer’s disease [57,51,52]. The inhibition of DHA delivery from the liver to the blood and arterial tissues may be an important mechanism by which SAH contributes to cerebral atherosclerosis and infarction leading to the development of Alzheimer’s disease. There is a correlation between the fatty acid composition of the brain and that of circulating erythrocytes [6,50]. The depletion of DHA in erythrocyte PC in the present study suggests the inhibition of hepatic PEMT by SAH in Alzheimer’s disease may decrease the flux of DHA from the liver into the circulation and the brain. The decreased concentrations of DHA in n-3 PUFA deficiency can be reversed by the administration of a DHA diet [44]. However, this may not correct the deficiency of DHA because the inhibition of PEMT by SAH could still inhibit the uptake of DHA into lipoproteins and impair its transport to peripheral tissues [63]. The increase in circulating homocysteine in Alzheimer’s disease is associated with a decrease in folic acid and Vitamin B12 [10]. Dietary supplementation with folic acid and Vitamin B12 is a simple and effective means of lowering circulating homocysteine concentrations [25]. The use of a combination of n-3 PUFAs, folic acid and Vitamin B12 may be a more effective means of increasing the uptake of DHA into the brain than n-3 PUFAs alone. There was also a decrease in the concentration of erythrocyte PC arachidonic acid in the Alzheimer’s patients. The PEMT pathway plays an important role in the mobilization of arachidonic acid into plasma [63]. The inhibition of hepatic PEMT by SAH may influence the production of arachidonic acid-derived regulatory lipids such as prostaglndins. In conclusion, this study offers preliminary evidence that increased circulating concentrations of SAH may be metabolically linked to altered phospholipid metabolism which contributes to parallel cerebrovascular and neurodegenerative pathology in Alzheimer’s disease. More research needs to be carried out the confirm these results which have potential implications in the prevention and treatment of Alzheimer’s disease. Acknowledgements The author thanks Dr. Diana Close for patient recruitment and assessment, Dr. Lars H¨arstr¨om for assistance in mass spectrometry, Dr. Steven Stern for the statistical analysis and Ms. Cindy Smith, Ms. Anne-Marie Ekstr¨om and Mr. Gregg Thompson for excellent technical assistance. References [1] American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 3rd ed. Washington, DC: American Psychiatric Press; 1994. [2] Ay H, Arsava EM, Tokgozoglu SL, Ozer N, Saribas O. Hyperhomocysteinemia is associated with the presence of left atrial thrombus in

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