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Increased low-density lipoprotein oxidation, but not total plasma protein oxidation, in Alzheimer's disease
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Clinical Biochemistry 43 (2010) 267 – 271
Increased low-density lipoprotein oxidation, but not total plasma protein oxidation, in Alzheimer's disease Sarah Aldred a,⁎, Stuart Bennett a , Patrizia Mecocci b a
School of Sport and Exercise Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK b Institute of Geriatrics, University Hospital Perugia, Italy Received 15 July 2009; received in revised form 25 August 2009; accepted 27 August 2009 Available online 4 September 2009
Abstract Objectives: The two most common forms of dementia are Alzheimer's disease (AD), and vascular dementia (VaD). In the overlap of biochemical processes which have been identified in AD and VaD, oxidative stress is believed to contribute to the numerous pathologies of both dementias. Design and methods: This study assessed oxidative damage in total plasma proteins, and isolated LDL in AD patients and age matched controls, in addition total antioxidant capacity (TAC) was measured. Results: Significantly higher LDL protein carbonylation was observed in AD compared to age-matched controls (AD: 4.17 ± 0.73 vs. control: 3.85 ± 0.86 nmol/mg LDL; p = 0.05, 2-tailed Mann–Whitney), in addition to reduced TAC (AD: 924.708 ± 174.429 vs. control: 1078.536 ± 252.633 μM; p = 0.001, 2-tailed Mann–Whitney). No differences were seen in total plasma protein carbonyl content (AD: 3.88 ± 0.31 vs. control: 3.98 ± 0.48 nmol/mg protein). Conclusion: The results further support the view that oxidation events in AD may be specific in nature, and represent functional changes to proteins, rather than random global events. © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Dementia; Oxidative stress; Protein oxidation; Antioxidant
Introduction Alzheimer's disease (AD) is the most common form of dementia, characterized by loss of short term memory, spatial and temporal perception and disorientation. These symptoms are associated with the extra cellular deposition of β-amyloid protein as senile plaques. Recent data suggest that in 2006, 26.6 million individuals were suffering from AD, and using a multistate model postulated this to increase fourfold to 106.2 million by 2050 worldwide. Further, the model indicates that almost 1 in 2 persons of 85 years old or older will be inflicted with this disease by 2050 [1]. In recent years it has become clear that there are considerable overlaps between AD and vascular pathologies. The role that
⁎ Corresponding author. Fax: +44 0 1214144121. E-mail address: [email protected] (S. Aldred).
vascular diseases, and indeed that vascular dementia (VaD), may play in AD has received an increasing amount of attention. VaD and AD have been considered two separate diseases, but it is now becoming increasingly clear that vascular factors are an important part of AD [2]. A possible mechanism believed to contribute to both vascular pathologies and AD involves oxidative stress (for review see [3]). Oxidative stress is an environment where the balance of pro-oxidant species to antioxidant species is altered in favor of the former. Oxidative stress has been implicated in a number of diseases including Alzheimer's disease, vascular dementia, cardiovascular disease, rheumatoid arthritis and diabetes [4]. Free radicals or reactive oxygen species (ROS) are highly reactive molecules which are naturally occurring by-products of normal cellular oxidation processes. ROS are reported to act as transient signaling molecules in the Ras GDP/GTP cycle and MAP kinase cascades, through modification of protein bound redox sensitive thiol groups [5]. ROS can increase to
0009-9120/$ - see front matter © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2009.08.021
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S. Aldred et al. / Clinical Biochemistry 43 (2010) 267–271
high levels in some disease processes or where there is antioxidant deficiency, and may react with cellular constituents to cause damage, disruption of function, or degradation. An increase in oxidation products has been identified in Alzheimer's pathology including DNA damage seen as an increase in 8-oxo-dG in cortical tissue and lymphocytes [6], and markers of protein oxidation including protein carbonyl formation in Alzheimer's brains [7] and plasma proteins of Alzheimer's suffers [8]. Unfavorable lipoprotein profiles are associated with chronic vascular disease including heart disease and atherosclerosis, and are often associated with ageing. Decreased levels of high-density lipoprotein (HDL) cholesterol and increased levels of low-density lipoprotein (LDL) cholesterol are strong markers for risk of disease [9,10]. LDL carries cholesterol from the liver to the circulation, and is susceptible to oxidation by ROS. In addition, oxidative damage to LDL by ROS is a contributing factor in atherosclerosis [11]. In LDL oxidation, damage is seen both to the lipid and to the protein moiety. A number of studies have suggested that protein oxidation of the lipoprotein molecule can occur as a direct result of free radical action and as a secondary result of the free radical cascade brought about by initial lipid peroxidation [12]. Although the majority of lipoprotein oxidation research has been done on the lipid moiety, protein oxidation has a number of functional consequences as apolipoprotein B oxidation (the protein moiety associated with LDL) is pivotal to the modulation of LDL uptake and accumulation. Elevated levels of circulating oxidized LDL are reported in individuals suffering from cardiovascular disease [13,14], and as previously mentioned oxidative damage to LDL is a contributing factor in atherosclerosis [11]. Atherosclerosis has also been linked to an increased incidence of AD and VaD [15,16] and therefore risk factors for atherosclerosis may also represent risk factors for AD and VaD. Indeed, increased levels of LDL in AD serum [17], in addition to an increased susceptibility of lipoproteins to oxidation in AD CSF and plasma has been reported [18,19], which may suggest increased LDL oxidation is present in circulation of AD sufferers. The primary aim of this study, therefore, was to identify increased oxidative damage to key plasma proteins in AD when compared to age matched controls; specifically LDL protein.
MMSE as measure of global cognitive function and tests evaluating the following cognitive domains: (a) memory: Babcock Story Recall test and Rey's Auditory Verbal Learning test immediate (Rey-IR) and delayed recall (Rey-DR) to assess episodic memory, and verbal fluency with semantic cues (Category Naming Test, CNT) to estimate semantic abilities; (b) attention and executive functions: Trail-Making test part A (TMT-A) and B (TMT-B) to evaluate selective and divided attention, respectively, and Controlled Oral Word Association test (COWA) to estimate executive functioning; (c) visuospatial and constructional abilities: Copy Drawing test (CD). Details on administration procedures and Italian normative data for score adjustment for age and education, and normality cut-off scores (95% of the lower tolerance limit of the normal population distribution) were used for each test. Patients diagnosed as having AD were compared to healthy controls. After obtaining informed consent from subjects or their relatives, patients and controls underwent blood drawing. Blood was immediately centrifuged and plasma stored frozen at -80 °C until analysis. Isolation of LDL LDL from all plasma samples was isolated by density gradient centrifugation in a Beckman TL-100 based on the method of Chung et al., [22]. Bicinchoninic assay (BCA) Determination of protein concentration was undertaken using the bicinchoninic acid (BCA) assay [23]. Briefly, a stock solution of 1 mg/ml Bovine serum Albumin (BSA) was prepared and diluted to form a six point standard curve ranging from 0 to 1 mg/ml. A 4% w/v copper sulphate solution (250 μl) was added to 12.5 ml bicinchoninic acid to give BCA working solution. Standards or samples (10 μl) were added to 96 well microtitre plates and 200 μl of BCA working solution was added to each well for 30 min at 37 °C. Absorbance values were read at 490 nm (Lab system Multiskan MS) and concentrations of protein were expressed as mg/ml.
Methods
Protein carbonyl enzyme-linked immunosorbent assay (ELISA)
The study conformed to the principles outlined in the Declaration of Helsinki. Patients with diagnosis of dementia made on the basis of scores obtained to a full battery of cognitive, functional and behavioral tests were divided into two groups according to NINDS-AIREN [20] and NINCDSADRDA [21] criteria as follows: neuropsychological and functional assessment tests were administered by a trained physician, who was blind to the operative procedure, in a quiet environment in the hospital. The battery of tests included the
Oxidative modification of total plasma proteins and LDL were assessed by detection of carbonyl groups by ELISA using the method of Carty et al., [24]. Briefly, standards and samples were diluted in coating buffer (Sodium carbonate 50 mM, pH 9.2) to a final concentration of 0.05 mg/ml and 50 μl, in duplicate, was added to 96 well high binding microtitre plates, for 1 h at 37 °C. Following this, 50 μl of DNPH solution was added to each well for 1 h at room temperature; 200 μl of blocking buffer was added for 1 h at 37 °C, and mouse anti-
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DNP antibody (diluted 1:1000 in blocking buffer) was added for 2 h at 37 °C. Plates were washed with Tris buffered saline (TBS) with Tween (0.05%) and incubated with anti-mouse IgE conjugated horseradish peroxidase (diluted 1:5000 in blocking buffer) was added for 1 h at 37 °C. Following this 50 μl of substrate was added to each well and the reaction was stopped after 15 min using 2 M sulphuric acid (H2SO4). Absorbance values were measured at 490 nm. Measurement of total antioxidant capacity (TAC) Total antioxidant capacity was measured using a slight modification to the ferric reducing ability of plasma assay (FRAP) described by McAnulty et al [25]. Briefly, freshly prepared FRAP reagent (300 μl; 300 mM acetate buffer, pH 3.6, 10 mM 2, 4, 6-Tris (2-pyridyl)-S-triazine in 40 mM hydrochloric acid and 20 mM Ferric chloride (FeCl3) in ddH2O in ratio 10:1:1) was added to test (10 μl) or standard samples (10 μl) in triplicate, at room temperature. The absorbance was then measured at 650 nm after an 8-min reaction and FRAP values were expressed as FRAP (μM) as determined by linear regression using a range of ascorbic acid standards (01000 μM). As the direct reaction of ascorbic acid gives a change in absorbance double that of Fe(II), an ascorbic acid standard of 1000 μM is equivalent to 2000 μM of antioxidant power as FRAP [26]. Results In total, 144 patients and control volunteers were recruited into the study (72 in each group). The age ranges for both groups can be seen in Table 1, along with other subject characteristics: MMSE scores; gender; depression status. At the time of the study, the mean time since diagnosis was 1.3 years ± 0.8 in the AD group. The percentage of smokers in the AD group was 20% compared to 24.3% in the control group. Total plasma protein carbonyl concentration was assessed and there were no observed differences between the control and AD groups (AD: 3.88 ± 0.31 vs. control: 3.98 ± 0.48 nmol/mg protein) (Fig. 1). However when LDL was isolated from all samples and assessed for carbonylation a significantly higher level was observed in AD compared to controls (AD: 4.17 ± 0.73 vs. control: 3.85 ± 0.86 nmol/mg LDL; p = 0.05, 2-tailed Mann– Whitney) (Fig. 2). A subset of samples (47 Alzheimer's disease and 56 age matched controls) was assessed for TAC by FRAP. A significantly lower antioxidant capacity was observed in the AD group when compared to the age-matched control group. (AD: 924.708 ± 174.429 vs. control: 1078.536 ± 252.633 μM FRAP; p = 0.001, 2-tailed Mann–Whitney) (Fig. 3). Table 1 Subject characteristics. Subject group
Age
MMSE % Female Geriatric depression scale
Alzheimer's disease 80 ± 4 19 ± 4 Control 75 ± 6 27 ± 2
63 56
7.4 ± 3.6 5.5 ± 3.4
Fig. 1. Total protein carbonyl content in control subjects and AD patients as assessed by ELISA. Data are presented as a box (25th–75th percentiles) and whisker (10th and 90th percentiles) plot with mean (bold line) and median lines displayed and individual outliers. Values are expressed as nmol protein carbonyl/mg of protein present.
Discussion The study demonstrated increased LDL protein carbonylation in patients with AD. This corroborates the results of a number of previous studies, which have also shown that LDL in the periphery of AD patients has increased susceptibility to oxidation [18,19]. LDL oxidation is a pathological hallmark of atherosclerosis and atherosclerosis is an associated risk factor for AD. The vascular nature of AD has received increasing attention in recent years and the role of VaD in AD warrants further characterization.
Fig. 2. LDL protein carbonyl content in control subjects and AD patients. LDL was isolated by ultracentrifugation and assessed by ELISA. Data are presented as a box (25th–75th percentiles) and whisker (10th and 90th percentiles) plot with mean (bold line) and median lines displayed and individual outliers. Values are expressed as nmol protein carbonyl/mg of protein present. ⁎ dictates significantly different values (p = 0.05), AD compared to control, by 2-tailed Mann–Whitney non-parametric test.
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Fig. 3. Total antioxidant capacity (TAC) in control subjects and AD patients. TAC was assessed by the Ferric reducing ability of plasma assay (FRAP). Data is presented as a box (25th–75th percentiles) and whisker (10th and 90th percentiles) plot with mean (bold line) and median lines displayed and individual outliers. Values are expressed as FRAP (μM). ⁎⁎ dictates significantly different values (p = 0.001), AD compared to control, by 2-tailed Mann–Whitney non-parametric test.
In contrast to LDL carbonylation, there was no difference in total plasma protein carbonylation between the AD and control groups. Existing studies assessing plasma levels of protein oxidation in Alzheimer's disease are equivocal, with several studies reporting no change but with others reporting increased levels in AD. For example, Greilberger et al., [27] and Bermejo et al., [28], in separate studies reported increased plasma protein oxidation in a group consisting of mild cognitive impairment (MCI) and AD patients. These studies are in contrast to no differences reported in mild to moderate, and advanced AD [29] or in mild AD patients [30]. All of the studies use the same spectrophotometric methodology to assess plasmatic protein oxidation so differences in methodology cannot account for the disparity between studies. It is possible that the severity of the disease would influence protein oxidation levels as it has been suggested oxidative stress is prevalent in the earliest stages of the disease [31], however the studies mentioned above measure plasma protein oxidation in both MCI and early AD sufferers and in advanced AD. The current study recruited AD sufferers with varied severity of disease (MMSE range 6 to 24). Further, the absence of an increased plasma protein oxidation in AD in the current study may add to the evidence that suggests that oxidation events within AD are specific and critical events which take place to alter the function of certain proteins, and this may contribute to the pathology of AD, rather than oxidative damage occurring in a non-specific and random manor. Previous studies assessing oxidative changes in AD brain have found similar specific protein oxidation events [32,33]. In this study we observed a significant reduction in total antioxidant capacity (TAC) in the AD group compared to the control group, as measured by FRAP. This is in agreement to a recent study by Sekler et al [34], who used the same methodology to measure TAC and reported a significant reduction of FRAP in an AD group compared to a control group, albeit only in the most severe cases (the authors
suggested that the stage of the disease may influence TAC). Indeed, further studies have demonstrated a significant decrease of total antioxidant status (TAS) in advanced AD, but not in moderate AD [29] as well as a trend for decreased TAS in MCI and AD [30]. In addition, a 24% decrease in TAC in plasma from probable AD compared to controls [35] has been reported. Studies which assess levels of individual antioxidants in plasma from AD subjects report a depletion of several vitamins compared to control subjects [36,37] and a reduction in an extensive range of vitamins and carotenoids has more recently been reported in AD and VaD patients [38]. It maybe that the vascular or degenerative severity of the disease accounts for the depletion of particular antioxidants, and thus contributes to the varied data reported by different groups regarding total antioxidant capacity in AD. It is becoming increasingly accepted that patients with AD, more often than not, will have a vascular element to their disease pathology. The observed higher LDL oxidation level in this study concurs with this perception. Almost all healthy aged individuals will have atherosclerotic plaque formation, and an associated increase circulating oxidized LDL, if compared to younger controls. However this study presented healthy aged individuals as controls, and thus the finding that LDL oxidation was significantly higher in AD, when compared to age-matched controls goes some way to demonstrating increased LDL oxidation is part of AD pathology. Acknowledgments This research was funded by the Alzheimer's Society. References [1] Brookmeyer R, Johnson E, Ziegler-Graham K, Arrighi HM. Forecasting the global burden of Alzheimer's disease. Alzheimers Dement 2007;3(3): 186–91. [2] Launer LJ. Demonstrating the case that AD is a vascular disease: epidemiologic evidence. Ageing Res Rev 2002;1(1):61–77. [3] Bennett S, Grant MM, Aldred S. Oxidative stress in vascular dementia and Alzheimer's disease: a common pathology. J Alzheimers Dis 2009 Feb 16. [4] Telci A, Cakatay U, Kayali R, et al. Oxidative protein damage in plasma of type 2 diabetic patients. Horm Metab Res 2000;32(1):40–3. [5] Accorsi K, Giglione C, Vanoni M, Parmeggiani A. The Ras GDP/GTP cycle is regulated by oxidizing agents at the level of Ras regulators and effectors. FEBS Lett 2001;492(1-2):139–45. [6] Mecocci P, Polidori MC, Cherubini A, et al. Lymphocyte oxidative DNA damage and plasma antioxidants in Alzheimer disease. Arch Neurol 2002;59(5):794–8. [7] Korolainen MA, Goldsteins G, Alafuzoff I, Koistinaho J, Pirttila T. Proteomic analysis of protein oxidation in Alzheimer's disease brain. Electrophoresis 2002;23:3428–33. [8] Choi J, Malakowsky CA, Talent JM, Conrad CC, Gracy RW. Identification of oxidised plasma proteins in Alzheimer's disease. Biochem Biophys Res Commun 2002;293:1566–70. [9] Fuster V, Badimon JJ, Badimon L. Clinical-pathological correlations of coronary disease progression and regression. Circulation 1992;86(6 Suppl): III1–III11. [10] Poulter N. Global risk of cardiovascular disease. Heart 2003;89:ii2–5. [11] Raitakari OT, Pitkanen OP, Lehtimaki T, et al. In vivo low density lipoprotein oxidation relates to coronary reactivity in young men. J Am Coll Cardiol 1997;30(1):97–102.
S. Aldred et al. / Clinical Biochemistry 43 (2010) 267–271 [12] Berlett BS, Stadtman ER. Protein oxidation in aging, disease and oxidative stress. J Biol Chem 1997;272:20313–6. [13] Holvoet P, Mertens A, Verhamme P, et al. Circulating oxidized LDL is a useful marker for identifying patients with coronary artery disease. Arterioscler Thromb Vasc Biol 2001;21(5):844–8. [14] Kita T, Kume N, Minami M, et al. Role of oxidized LDL in atherosclerosis. Ann NY Acad Sci 2001;947:199–205. [15] Hofman A, Ott A, Breteler MM, et al. Atherosclerosis, apolipoprotein E, and prevalence of dementia and Alzheimer's disease in the Rotterdam Study. Lancet 1997;349(9046):151–4. [16] van OM, de Jong FJ, Witteman JC, Hofman A, Koudstaal PJ, Breteler MM. Atherosclerosis and risk for dementia. Ann Neurol 2007;61(5): 403–10. [17] Kuo YM, Emmerling MR, Bisgaier CL, et al. Elevated low-density lipoprotein in Alzheimer's disease correlates with brain abeta 1-42 levels. Biochem Biophys Res Commun 1998;252(3):711–5. [18] Bassett CN, Neely MD, Sidell KR, Markesbery WR, Swift LL, Montine TJ. Cerebrospinal fluid lipoproteins are more vulnerable to oxidation in Alzheimer's disease and are neurotoxic when oxidized ex vivo. Lipids 1999;34(12):1273–80. [19] Schippling S, Kontush A, Arlt S, et al. Increased lipoproetin oxidation in Alzheimers disease. Free Radic Biol Med 2000;28:351–60. [20] Wetterling T, Kanitz RD, Borgis KJ. Comparison of different diagnostic criteria for vascular dementia (ADDTC, DSM-IV, ICD-10, NINDS-AIREN). Stroke 1996 Jan;27(1):30–6. [21] Criteria for the clinical diagnosis of Alzheimer's disease. Excerpts from the NINCDS-ADRDA Work Group report. J Am Geriatr Soc 1985 Jan;33(1): 2–3. [22] Chung BH, Segrest JP, Ray MJ, et al. Single vertical spin density gradient centrifugation. Methods Enzymol 1986;128:181–209. [23] Smith PK, Krohn RI, Hermanson GT, et al. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150(1):76–85. [24] Carty JL, Bevan R, Waller H, et al. The effects of vitamin C supplementation on protein oxidation in healthy volunteers. Biochem Biophys Res Commun 2000;273(2):729–35. [25] McAnulty SR, McAnulty LS, Nieman DC, Morrow JD, Utter AC, Dumke CL. Effect of resistance exercise and carbohydrate ingestion on oxidative stress. Free Radic Res 2005 Nov;39(11):1219–24. [26] Benzie IF, Strain JJ. Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol 1999;299:15–27.
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[27] Greilberger J, Koidl C, Greilberger M, et al. Malondialdehyde, carbonyl proteins and albumin-disulphide as useful oxidative markers in mild cognitive impairment and Alzheimer's disease. Free Radic Res 2008;42 (7):633–8. [28] Bermejo P, Martin-Aragon S, Benedi J, et al. Peripheral levels of glutathione and protein oxidation as markers in the development of Alzheimer's disease from mild cognitive impairment. Free Radic Res 2008 Feb;42(2):162–70. [29] Zafrilla P, Mulero J, Xandri JM, Santo E, Caravaca G, Morillas JM. Oxidative stress in Alzheimer patients in different stages of the disease. Curr Med Chem 2006;13(9):1075–83. [30] Baldeiras I, Santana I, Proenca MT, et al. Peripheral oxidative damage in mild cognitive impairment and mild Alzheimer's disease. J Alzheimers Dis 2008 Sep;15(1):117–28. [31] Nunomura A, Perry G, Aliev G, et al. Oxidative damage is the earliest event in Alzheimer disease. J Neuropathol Exp Neurol 2001 Aug;60(8): 759–67. [32] Castegna A, Akensov M, Thongboonkerd V, et al. Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part II, dihydropyrimidinase-related protein 2, alpha-enolase and heat shock cognate 71. J Neurochem 2002;82:1524–32. [33] Castegna A, Akensov M, Thongboonkerd V, et al. Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part I: Creatinine kinase BB, Glutamine synthase, and Ubiquitin carboxyl terminal hydrolase L-1. Free Radic Biol Med 2002;33(4):562–71. [34] Sekler A, Jimenez JM, Rojo L, et al. Cognitive impairment and Alzheimer's disease: Links with oxidative stress and cholesterol metabolism. Neuropsychiatr Dis Treat 2008;4(4):715–22. [35] Repetto MG, Reides CG, Evelson P, Kohan S, de Lustig ES, Llesuy SF. Peripheral markers of oxidative stress in probable Alzheimer patients. Eur J Clin Invest 1999 Jul;29(7):643–9. [36] Rinaldi P, Polidori MC, Metastasio A, et al. Plasma antioxidants are similarly depleted in mild cognitive impairment and in Alzheimer's disease. Neurobiol Aging 2003;24(7):915–9. [37] Bourdel-Marchasson I, Delmas-Beauvieux MC, Peuchant E, et al. Antioxidant defences and oxidative stress markers in erythrocytes and plasma from normally nourished elderly Alzheimer patients. Age Ageing 2001 May;30(3):235–41. [38] Polidori MC, Mattioli P, Aldred S, et al. Plasma antioxidant status, immunoglobulin g oxidation and lipid peroxidation in demented patients: relevance to Alzheimer disease and vascular dementia. Dement Geriatr Cogn Disord 2004;18(3-4):265–70.
Clinical Biochemistry 43 (2010) 267 – 271
Increased low-density lipoprotein oxidation, but not total plasma protein oxidation, in Alzheimer's disease Sarah Aldred a,⁎, Stuart Bennett a , Patrizia Mecocci b a
School of Sport and Exercise Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK b Institute of Geriatrics, University Hospital Perugia, Italy Received 15 July 2009; received in revised form 25 August 2009; accepted 27 August 2009 Available online 4 September 2009
Abstract Objectives: The two most common forms of dementia are Alzheimer's disease (AD), and vascular dementia (VaD). In the overlap of biochemical processes which have been identified in AD and VaD, oxidative stress is believed to contribute to the numerous pathologies of both dementias. Design and methods: This study assessed oxidative damage in total plasma proteins, and isolated LDL in AD patients and age matched controls, in addition total antioxidant capacity (TAC) was measured. Results: Significantly higher LDL protein carbonylation was observed in AD compared to age-matched controls (AD: 4.17 ± 0.73 vs. control: 3.85 ± 0.86 nmol/mg LDL; p = 0.05, 2-tailed Mann–Whitney), in addition to reduced TAC (AD: 924.708 ± 174.429 vs. control: 1078.536 ± 252.633 μM; p = 0.001, 2-tailed Mann–Whitney). No differences were seen in total plasma protein carbonyl content (AD: 3.88 ± 0.31 vs. control: 3.98 ± 0.48 nmol/mg protein). Conclusion: The results further support the view that oxidation events in AD may be specific in nature, and represent functional changes to proteins, rather than random global events. © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Dementia; Oxidative stress; Protein oxidation; Antioxidant
Introduction Alzheimer's disease (AD) is the most common form of dementia, characterized by loss of short term memory, spatial and temporal perception and disorientation. These symptoms are associated with the extra cellular deposition of β-amyloid protein as senile plaques. Recent data suggest that in 2006, 26.6 million individuals were suffering from AD, and using a multistate model postulated this to increase fourfold to 106.2 million by 2050 worldwide. Further, the model indicates that almost 1 in 2 persons of 85 years old or older will be inflicted with this disease by 2050 [1]. In recent years it has become clear that there are considerable overlaps between AD and vascular pathologies. The role that
⁎ Corresponding author. Fax: +44 0 1214144121. E-mail address: [email protected] (S. Aldred).
vascular diseases, and indeed that vascular dementia (VaD), may play in AD has received an increasing amount of attention. VaD and AD have been considered two separate diseases, but it is now becoming increasingly clear that vascular factors are an important part of AD [2]. A possible mechanism believed to contribute to both vascular pathologies and AD involves oxidative stress (for review see [3]). Oxidative stress is an environment where the balance of pro-oxidant species to antioxidant species is altered in favor of the former. Oxidative stress has been implicated in a number of diseases including Alzheimer's disease, vascular dementia, cardiovascular disease, rheumatoid arthritis and diabetes [4]. Free radicals or reactive oxygen species (ROS) are highly reactive molecules which are naturally occurring by-products of normal cellular oxidation processes. ROS are reported to act as transient signaling molecules in the Ras GDP/GTP cycle and MAP kinase cascades, through modification of protein bound redox sensitive thiol groups [5]. ROS can increase to
0009-9120/$ - see front matter © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2009.08.021
268
S. Aldred et al. / Clinical Biochemistry 43 (2010) 267–271
high levels in some disease processes or where there is antioxidant deficiency, and may react with cellular constituents to cause damage, disruption of function, or degradation. An increase in oxidation products has been identified in Alzheimer's pathology including DNA damage seen as an increase in 8-oxo-dG in cortical tissue and lymphocytes [6], and markers of protein oxidation including protein carbonyl formation in Alzheimer's brains [7] and plasma proteins of Alzheimer's suffers [8]. Unfavorable lipoprotein profiles are associated with chronic vascular disease including heart disease and atherosclerosis, and are often associated with ageing. Decreased levels of high-density lipoprotein (HDL) cholesterol and increased levels of low-density lipoprotein (LDL) cholesterol are strong markers for risk of disease [9,10]. LDL carries cholesterol from the liver to the circulation, and is susceptible to oxidation by ROS. In addition, oxidative damage to LDL by ROS is a contributing factor in atherosclerosis [11]. In LDL oxidation, damage is seen both to the lipid and to the protein moiety. A number of studies have suggested that protein oxidation of the lipoprotein molecule can occur as a direct result of free radical action and as a secondary result of the free radical cascade brought about by initial lipid peroxidation [12]. Although the majority of lipoprotein oxidation research has been done on the lipid moiety, protein oxidation has a number of functional consequences as apolipoprotein B oxidation (the protein moiety associated with LDL) is pivotal to the modulation of LDL uptake and accumulation. Elevated levels of circulating oxidized LDL are reported in individuals suffering from cardiovascular disease [13,14], and as previously mentioned oxidative damage to LDL is a contributing factor in atherosclerosis [11]. Atherosclerosis has also been linked to an increased incidence of AD and VaD [15,16] and therefore risk factors for atherosclerosis may also represent risk factors for AD and VaD. Indeed, increased levels of LDL in AD serum [17], in addition to an increased susceptibility of lipoproteins to oxidation in AD CSF and plasma has been reported [18,19], which may suggest increased LDL oxidation is present in circulation of AD sufferers. The primary aim of this study, therefore, was to identify increased oxidative damage to key plasma proteins in AD when compared to age matched controls; specifically LDL protein.
MMSE as measure of global cognitive function and tests evaluating the following cognitive domains: (a) memory: Babcock Story Recall test and Rey's Auditory Verbal Learning test immediate (Rey-IR) and delayed recall (Rey-DR) to assess episodic memory, and verbal fluency with semantic cues (Category Naming Test, CNT) to estimate semantic abilities; (b) attention and executive functions: Trail-Making test part A (TMT-A) and B (TMT-B) to evaluate selective and divided attention, respectively, and Controlled Oral Word Association test (COWA) to estimate executive functioning; (c) visuospatial and constructional abilities: Copy Drawing test (CD). Details on administration procedures and Italian normative data for score adjustment for age and education, and normality cut-off scores (95% of the lower tolerance limit of the normal population distribution) were used for each test. Patients diagnosed as having AD were compared to healthy controls. After obtaining informed consent from subjects or their relatives, patients and controls underwent blood drawing. Blood was immediately centrifuged and plasma stored frozen at -80 °C until analysis. Isolation of LDL LDL from all plasma samples was isolated by density gradient centrifugation in a Beckman TL-100 based on the method of Chung et al., [22]. Bicinchoninic assay (BCA) Determination of protein concentration was undertaken using the bicinchoninic acid (BCA) assay [23]. Briefly, a stock solution of 1 mg/ml Bovine serum Albumin (BSA) was prepared and diluted to form a six point standard curve ranging from 0 to 1 mg/ml. A 4% w/v copper sulphate solution (250 μl) was added to 12.5 ml bicinchoninic acid to give BCA working solution. Standards or samples (10 μl) were added to 96 well microtitre plates and 200 μl of BCA working solution was added to each well for 30 min at 37 °C. Absorbance values were read at 490 nm (Lab system Multiskan MS) and concentrations of protein were expressed as mg/ml.
Methods
Protein carbonyl enzyme-linked immunosorbent assay (ELISA)
The study conformed to the principles outlined in the Declaration of Helsinki. Patients with diagnosis of dementia made on the basis of scores obtained to a full battery of cognitive, functional and behavioral tests were divided into two groups according to NINDS-AIREN [20] and NINCDSADRDA [21] criteria as follows: neuropsychological and functional assessment tests were administered by a trained physician, who was blind to the operative procedure, in a quiet environment in the hospital. The battery of tests included the
Oxidative modification of total plasma proteins and LDL were assessed by detection of carbonyl groups by ELISA using the method of Carty et al., [24]. Briefly, standards and samples were diluted in coating buffer (Sodium carbonate 50 mM, pH 9.2) to a final concentration of 0.05 mg/ml and 50 μl, in duplicate, was added to 96 well high binding microtitre plates, for 1 h at 37 °C. Following this, 50 μl of DNPH solution was added to each well for 1 h at room temperature; 200 μl of blocking buffer was added for 1 h at 37 °C, and mouse anti-
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DNP antibody (diluted 1:1000 in blocking buffer) was added for 2 h at 37 °C. Plates were washed with Tris buffered saline (TBS) with Tween (0.05%) and incubated with anti-mouse IgE conjugated horseradish peroxidase (diluted 1:5000 in blocking buffer) was added for 1 h at 37 °C. Following this 50 μl of substrate was added to each well and the reaction was stopped after 15 min using 2 M sulphuric acid (H2SO4). Absorbance values were measured at 490 nm. Measurement of total antioxidant capacity (TAC) Total antioxidant capacity was measured using a slight modification to the ferric reducing ability of plasma assay (FRAP) described by McAnulty et al [25]. Briefly, freshly prepared FRAP reagent (300 μl; 300 mM acetate buffer, pH 3.6, 10 mM 2, 4, 6-Tris (2-pyridyl)-S-triazine in 40 mM hydrochloric acid and 20 mM Ferric chloride (FeCl3) in ddH2O in ratio 10:1:1) was added to test (10 μl) or standard samples (10 μl) in triplicate, at room temperature. The absorbance was then measured at 650 nm after an 8-min reaction and FRAP values were expressed as FRAP (μM) as determined by linear regression using a range of ascorbic acid standards (01000 μM). As the direct reaction of ascorbic acid gives a change in absorbance double that of Fe(II), an ascorbic acid standard of 1000 μM is equivalent to 2000 μM of antioxidant power as FRAP [26]. Results In total, 144 patients and control volunteers were recruited into the study (72 in each group). The age ranges for both groups can be seen in Table 1, along with other subject characteristics: MMSE scores; gender; depression status. At the time of the study, the mean time since diagnosis was 1.3 years ± 0.8 in the AD group. The percentage of smokers in the AD group was 20% compared to 24.3% in the control group. Total plasma protein carbonyl concentration was assessed and there were no observed differences between the control and AD groups (AD: 3.88 ± 0.31 vs. control: 3.98 ± 0.48 nmol/mg protein) (Fig. 1). However when LDL was isolated from all samples and assessed for carbonylation a significantly higher level was observed in AD compared to controls (AD: 4.17 ± 0.73 vs. control: 3.85 ± 0.86 nmol/mg LDL; p = 0.05, 2-tailed Mann– Whitney) (Fig. 2). A subset of samples (47 Alzheimer's disease and 56 age matched controls) was assessed for TAC by FRAP. A significantly lower antioxidant capacity was observed in the AD group when compared to the age-matched control group. (AD: 924.708 ± 174.429 vs. control: 1078.536 ± 252.633 μM FRAP; p = 0.001, 2-tailed Mann–Whitney) (Fig. 3). Table 1 Subject characteristics. Subject group
Age
MMSE % Female Geriatric depression scale
Alzheimer's disease 80 ± 4 19 ± 4 Control 75 ± 6 27 ± 2
63 56
7.4 ± 3.6 5.5 ± 3.4
Fig. 1. Total protein carbonyl content in control subjects and AD patients as assessed by ELISA. Data are presented as a box (25th–75th percentiles) and whisker (10th and 90th percentiles) plot with mean (bold line) and median lines displayed and individual outliers. Values are expressed as nmol protein carbonyl/mg of protein present.
Discussion The study demonstrated increased LDL protein carbonylation in patients with AD. This corroborates the results of a number of previous studies, which have also shown that LDL in the periphery of AD patients has increased susceptibility to oxidation [18,19]. LDL oxidation is a pathological hallmark of atherosclerosis and atherosclerosis is an associated risk factor for AD. The vascular nature of AD has received increasing attention in recent years and the role of VaD in AD warrants further characterization.
Fig. 2. LDL protein carbonyl content in control subjects and AD patients. LDL was isolated by ultracentrifugation and assessed by ELISA. Data are presented as a box (25th–75th percentiles) and whisker (10th and 90th percentiles) plot with mean (bold line) and median lines displayed and individual outliers. Values are expressed as nmol protein carbonyl/mg of protein present. ⁎ dictates significantly different values (p = 0.05), AD compared to control, by 2-tailed Mann–Whitney non-parametric test.
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Fig. 3. Total antioxidant capacity (TAC) in control subjects and AD patients. TAC was assessed by the Ferric reducing ability of plasma assay (FRAP). Data is presented as a box (25th–75th percentiles) and whisker (10th and 90th percentiles) plot with mean (bold line) and median lines displayed and individual outliers. Values are expressed as FRAP (μM). ⁎⁎ dictates significantly different values (p = 0.001), AD compared to control, by 2-tailed Mann–Whitney non-parametric test.
In contrast to LDL carbonylation, there was no difference in total plasma protein carbonylation between the AD and control groups. Existing studies assessing plasma levels of protein oxidation in Alzheimer's disease are equivocal, with several studies reporting no change but with others reporting increased levels in AD. For example, Greilberger et al., [27] and Bermejo et al., [28], in separate studies reported increased plasma protein oxidation in a group consisting of mild cognitive impairment (MCI) and AD patients. These studies are in contrast to no differences reported in mild to moderate, and advanced AD [29] or in mild AD patients [30]. All of the studies use the same spectrophotometric methodology to assess plasmatic protein oxidation so differences in methodology cannot account for the disparity between studies. It is possible that the severity of the disease would influence protein oxidation levels as it has been suggested oxidative stress is prevalent in the earliest stages of the disease [31], however the studies mentioned above measure plasma protein oxidation in both MCI and early AD sufferers and in advanced AD. The current study recruited AD sufferers with varied severity of disease (MMSE range 6 to 24). Further, the absence of an increased plasma protein oxidation in AD in the current study may add to the evidence that suggests that oxidation events within AD are specific and critical events which take place to alter the function of certain proteins, and this may contribute to the pathology of AD, rather than oxidative damage occurring in a non-specific and random manor. Previous studies assessing oxidative changes in AD brain have found similar specific protein oxidation events [32,33]. In this study we observed a significant reduction in total antioxidant capacity (TAC) in the AD group compared to the control group, as measured by FRAP. This is in agreement to a recent study by Sekler et al [34], who used the same methodology to measure TAC and reported a significant reduction of FRAP in an AD group compared to a control group, albeit only in the most severe cases (the authors
suggested that the stage of the disease may influence TAC). Indeed, further studies have demonstrated a significant decrease of total antioxidant status (TAS) in advanced AD, but not in moderate AD [29] as well as a trend for decreased TAS in MCI and AD [30]. In addition, a 24% decrease in TAC in plasma from probable AD compared to controls [35] has been reported. Studies which assess levels of individual antioxidants in plasma from AD subjects report a depletion of several vitamins compared to control subjects [36,37] and a reduction in an extensive range of vitamins and carotenoids has more recently been reported in AD and VaD patients [38]. It maybe that the vascular or degenerative severity of the disease accounts for the depletion of particular antioxidants, and thus contributes to the varied data reported by different groups regarding total antioxidant capacity in AD. It is becoming increasingly accepted that patients with AD, more often than not, will have a vascular element to their disease pathology. The observed higher LDL oxidation level in this study concurs with this perception. Almost all healthy aged individuals will have atherosclerotic plaque formation, and an associated increase circulating oxidized LDL, if compared to younger controls. However this study presented healthy aged individuals as controls, and thus the finding that LDL oxidation was significantly higher in AD, when compared to age-matched controls goes some way to demonstrating increased LDL oxidation is part of AD pathology. Acknowledgments This research was funded by the Alzheimer's Society. References [1] Brookmeyer R, Johnson E, Ziegler-Graham K, Arrighi HM. Forecasting the global burden of Alzheimer's disease. Alzheimers Dement 2007;3(3): 186–91. [2] Launer LJ. Demonstrating the case that AD is a vascular disease: epidemiologic evidence. Ageing Res Rev 2002;1(1):61–77. [3] Bennett S, Grant MM, Aldred S. Oxidative stress in vascular dementia and Alzheimer's disease: a common pathology. J Alzheimers Dis 2009 Feb 16. [4] Telci A, Cakatay U, Kayali R, et al. Oxidative protein damage in plasma of type 2 diabetic patients. Horm Metab Res 2000;32(1):40–3. [5] Accorsi K, Giglione C, Vanoni M, Parmeggiani A. The Ras GDP/GTP cycle is regulated by oxidizing agents at the level of Ras regulators and effectors. FEBS Lett 2001;492(1-2):139–45. [6] Mecocci P, Polidori MC, Cherubini A, et al. Lymphocyte oxidative DNA damage and plasma antioxidants in Alzheimer disease. Arch Neurol 2002;59(5):794–8. [7] Korolainen MA, Goldsteins G, Alafuzoff I, Koistinaho J, Pirttila T. Proteomic analysis of protein oxidation in Alzheimer's disease brain. Electrophoresis 2002;23:3428–33. [8] Choi J, Malakowsky CA, Talent JM, Conrad CC, Gracy RW. Identification of oxidised plasma proteins in Alzheimer's disease. Biochem Biophys Res Commun 2002;293:1566–70. [9] Fuster V, Badimon JJ, Badimon L. Clinical-pathological correlations of coronary disease progression and regression. Circulation 1992;86(6 Suppl): III1–III11. [10] Poulter N. Global risk of cardiovascular disease. Heart 2003;89:ii2–5. [11] Raitakari OT, Pitkanen OP, Lehtimaki T, et al. In vivo low density lipoprotein oxidation relates to coronary reactivity in young men. J Am Coll Cardiol 1997;30(1):97–102.
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