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Hyperhomocysteinemia Is a Risk Factor for Alzheimer's Disease in an Algerian Population
Archives of Medical Research 45 (2014) 247e250
ORIGINAL ARTICLE
Hyperhomocysteinemia Is a Risk Factor for Alzheimer’s Disease in an Algerian Population Khaled Nazef,a Malika Khelil,a Hiba Chelouti,a Ghouti Kacimi,b Mohamed Bendini,c Meriem Tazir,d Soraya Belarbi,d Mohamed El Hadi Cherifi,e and Bahia Djerdjouria a
Departement de Biologie Cellulaire et Moleculaire, Faculte des Sciences Biologiques, Universite des Sciences et de la Technologie Houari Boumediene Alger, Algerie b Laboratoire de Biochimie, H^opital Central de l’Armee Mohamed Nekkache Alger, Algerie c Service de Neurologie, H^opital Central de l’Armee Mohamed Nekkache Alger, Algerie d Service de Neurologie, Centre Hospitalo-Universitaire (CHU) Mustapha Bacha Alger, Algerie e Laboratoire Central de Biologie, Centre Hospitalo-Universitaire (CHU) Nafissa Hamoud Alger, Algerie Received for publication July 19, 2013; accepted March 3, 2014 (ARCMED-D-13-00393).
Background and Aims. There is growing evidence that increased blood concentration of total homocysteine (tHcy) may be a risk factor for Alzheimer’s disease (AD). The present study was conducted to evaluate the association of serum tHcy and other biochemical risk factors with AD. Methods. This is a case-control study including 41 individuals diagnosed with AD and 46 nondemented controls. Serum levels of all studied biochemical parameters were performed. Results. Univariate logistic regression showed a significant increase of tHcy ( p 5 0.008), urea ( p 5 0.036) and a significant decrease of vitamin B12 ( p 5 0.012) in AD group vs. controls. Using multivariate logistic regression, tHcy ( p 5 0.007, OR 5 1.376) appeared as an independent risk factor predictor of AD. There was a significant positive correlation between tHcy and creatinine ( p !0.0001). A negative correlation was found between tHcy and vitamin B12 ( p !0.0001). Conclusions. Our findings support that hyperhomocysteinemia is a risk factor for AD in an Algerian population and is also associated with vitamin B12 deficiency. Ó 2014 IMSS. Published by Elsevier Inc. Key Words: Hyperhomocysteinemia, Alzheimer’s disease, Vitamin B12, Folate.
Introduction Alzheimer’s disease (AD) is the most common type of dementia and is a multifactorial disease resulting from progressive neurodegenerative disorders. AD can affect different people in different ways, but the most common symptoms are memory and behavior disorders (1), gradually evolving towards dementia. Postmortem brains of AD patients display two pathological hallmarks: amyloid-b (Ab) peptide plaques and neurofibrillary tangles (NFTs) (2,3). The exact causes of this disease are not yet known. However, several risk factors Address reprint requests to: Malika Khelil, Departement de Biologie Cellulaire et Moleculaire, Faculte des Sciences Biologiques, Universite, des Sciences et de la Technologie Houari Boumediene BP 32 El-Alia. 16111 Alger, Algerie; Phone: (213) 771239487; FAX: (213) 21247217; E-mail: [email protected]
for AD have been suggested (4). The most important objective of research on AD is the discovery of biomarkers predictive of disease onset and reliable ways to diagnose AD as early as possible before the loss of autonomy and to also find preventive interventions. Several studies indicated that increased blood concentrations of homocysteine (Hcy) may be a risk factor for AD (5e7). The metabolism of Hcy depends on two cofactors like vitamin B12 and folate (8). An inverse correlation between serum folate and vitamin B12 concentrations and the risk of AD was found in several reports (9,10). There is growing evidence that hyperhomocysteinemia (HHcy) is in part due to vitamin deficiency, especially in older people (11). Presence of Hcy has been shown in the brain of rats deficient in folate, the hippocampus and cortex containing the highest proportions (12). In addition, folate deficiency
0188-4409/$ - see front matter. Copyright Ó 2014 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2014.03.001
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in mice leads to alteration of synaptic plasticity, hippocampal neurogenesis, memory deficits and amyloid deposits in cerebral vessels (13). In the present work we aimed to examine the possible association between total serum homocysteine (tHcy) and other proposed risk factors for AD such as vitamin B12, folate, cholesterol, thyroid stimulating hormone (TSH), uric acid and other biochemical factors in an Algerian population. We also investigated the correlation between tHcy, folate and vitamin B12. Subjects and Methods Subjects Harvesting, selection and classification of patients who were included in this study were performed with neurologists from the Neurology Departments at the University Hospital CentereMustapha Bacha and Central Hospital of Army, Algiers. This study excluded patients and healthy individuals under medications that alter Hcy concentrations including vitamin B12 and folate. Individuals who had a Hcy level O50 mM were also excluded. After these exclusions, there were 41 subjects with AD and 46 control subjects. Cognitive impairment was evaluated by several tests including the Mini-Mental State Examination (MMSE). Inclusion criteria for elderly controls were a MMSE score $24 and the absence of any known neurological disorder or dementia. Informed consent was obtained from patients, controls or responsible caregivers. This study was approved by the local ethics committee. Biochemical Assays Blood samples were collected and serum was used for the analysis of tHcy, folate, vitamin B12 and other biochemical parameters. Serum tHcy was measured using fluorescence polarization immunoassay (Abbott AxSYM system, Abbott Laboratories). Serum folate, vitamin B12, TSH, T3 and T4 concentrations were measured by competitive binding assays with electrochemiluminescent detection using an auto-analyser (Elecsys 2010, Hitachi, Roche or COBAS e401). Serum concentrations of uric acid, creatinine, total cholesterol, triglycerides, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were measured spectrophotometrically using an auto-analyzer (COBAS c501). Serum glucose and urea were measured spectrophotometrically by the hexokinase/glucose-6-phosphate dehydrogenase enzymatic assay and by the urease/glutamine dehydrogenase enzymatic assay, respectively, using the COBAS c501 auto-analyzer. Statistical Analysis Data are expressed as mean standard deviation (SD). After testing data for normality (ShapiroeWilk testeStatistica
v.8.0), correlation between variables was performed using Spearman Rank order correlations. The strength of association between each variable and AD was assessed by calculation of crude odds ratios (OR) and 95% confidence intervals (CI). OR was used to assess the intensity of the association between the different variables and AD describing a protective effect to !1 (OR !1) or predicting factor to a value O1 (OR O1). Multiple logistic regression analysis was also performed to determine variables that are the best predictors of AD. All statistical tests were performed using SPSS for windows v.15.0 (SPSS Science, Chicago, IL); p !0.05 was considered statistically significant.
Results Demographic, clinical, cognitive and functional characteristics of the population study are summarized in Table 1.
Table 1. Demographic, clinical and biochemical characteristics of AD patients and controls Characteristcs
Patients (n 5 41)
Controls (n 5 46)
Age (mean SD) (years) Gender (males [%]) MMSE (mean SD) Homocysteine (mmol) B12 vitamin (pg/mL) Folate (ng/mL) Glucose (mmol) Triglyceride (mmol/l) Cholesterol (mmol/l) HDL (mmol/l) LDL (mmol/l) Creatinine (mmol) Urea (mmol/l) Uric acid (mmol) TSH (mmol) Calcium (mmol) T3 (nmol/l) T4 (nmol/l) Diabetes (%) Hypertension (%) Hypercholesterolemia Thyroid diseases (%) Heart diseases (%) Chronic inflammatory diseases (%)
72.61 8.92 43.8 15.131 6.23 19.126 6.93 301.766 113.54 8.291 2.1 5.74 1.38 3.362 0.37 4.903 1.244 0.31 2.82 0.69 78.822 20.51 5.577 1.31 283.87 68.22 1.638 0.82 2.229 0.13 1.94 0.46 112.193 19.78 23.1 41 15.4 7.7 17.9 23.1
66.3 9.6* 52.2 26.668 2.87*** 15.613 3.63** 399.103 197.85* 7.993 3.43 6.335 3.56 1.527 0.67 4.881 1.05 1.233 0.03 3.046 0.91 72.919 14.65 5.002 1.09* 269.18 34.55 1.951 1.27 2.263 0.16 2.011 0.32 111.42 20.23 22.7 50 13.6 9.1 4.5 25
AD, Alzheimer’s disease; MMSE, Mini-Mental State Examination; T3, triiodothyrone; T4, thyroxine; TSH, thyroid stimulating hormone; HDL, high-density lipoprotein; LDL, low-density lipoprotein. Results are expressed as mean SD. Comparisons between AD and controls were performed by two-tailed Student t test (for normally distributed data) or by Mann-Whitney U test (for non-normally distributed data). c2 and Fisher’s Exact Test were performed for frequencies analysis. *p !0.05; **p !0.01; ***p !0.001; n 5 number of individuals.
Hyperhomocysteinemia in Alzheimer’s Disease
249
We used the missing values option when some values were missed.
Table 3. Multivariate logistic regression model
Univariate Logistic Regression
Homocysteine MMSE
Association between analyzed variables with AD was investigated first by univariate logistic regression (Table 2). Significant difference is noted between controls and AD patients regarding age and MMSE, respectively ( p 5 0.004, p 5 0.000). tHcy and urea were positively associated, whereas vitamin B12 was negatively associated with AD. Indeed, tHcy and urea were significantly higher in patients than in controls ( p 5 0.008; p 5 0.036, respectively) and concentration of vitamin B12 was significantly lower in patients than in controls ( p 5 0.012). No statistically significant differences were observed for all other studied variables.
Variables
OR
p
1.376 0.527
0.007 0.000
Adjusted for age, sex, MMSE, homocysteine, vitamin B12, folate, triglycerides, LDL, creatinine, urea, uric acid, TSH.
Correlation between Hcy and Other Biochemical Variables Spearman’s correlation coefficient was used to grasp the efficient interaction between tHcy concentration and the other variables (Table 4). Significant negative correlation was observed between tHcy and vitamin B12 (r 5 e0.379, p !0.0001). However, a significant positive correlation was observed between tHcy and creatinine (r 5 0.386, p !0.0001), a biochemical indicator of renal function.
Multivariate Logistic Regression Multivariate logistic regression analysis was chosen to find the best independent predictors (Table 3). The candidate predictors were variables with a p value !0.25 in the univariate logistic regression analysis (14). Folate concentration was also included as a candidate because it could affect tHcy concentration (5,15). After correction for multiple testing, statistical differences were still noted for tHcy ( p 5 0.007) and MMSE ( p !0.001). Table 2. Crude ratios (OR) and 95% confidence intervals (CI) according to different factors Factors Age (years) Males (%) MMSE Education level Homocysteine Vitamin B12 Folate Glucose Triglycerides Cholesterol HDL LDL Creatinine Urea Uric acid TSH Calcium T3 T4 Diabetes (%) Hypertension (%) Hypercholesterolemia (%) Thyroid diseases (%) Cardiopathy (%) Chronic inflammatory diseases
OR
95% CI
p
1.075 0.72 0.594 0.75 1.164 0.996 1.037 0.916 0.566 1.023 1.125 0.702 1.02 1.527 1.005 0.744 0.222 0.644 1.002 1.02 0.696 1.152 0.833 4.594 0.9
1.024e1.129 0.38e1.671 0.472e0.748 0.234e2.408 1.041e1.302 0.993e0.999 0.895e1.203 0.764e1.097 0.249e1.283 0.669e1.564 0.281e4.508 0.406e1.23 0.994e1.048 1.028e0.268 0.997e1.014 0.476e1.62 0.013e3.939 0.217e1.911 0.981e1.023 0.366e2.845 0.292e1.66 0.339e3.916 0.1758e3.978 0.893e23.62 0.328e2.472
0.004 0.441 0.000 0.629 0.008 0.012 0.628 0.339 0.173 0.917 0.867 0.205 0.135 0.036 0.206 0.193 0.305 0.428 0.857 0.97 0.413 0.821 0.819 0.068 0.838
OR, odds ratio; CI, confidence intervals.
Discussion The present findings demonstrate that high serum tHcy level has an independent relation with AD. Indeed, an association of high serum concentration of tHcy to AD was evident through multivariate logistic regression analysis. High concentration of tHcy could be an independent risk factor for AD, dementia, and cognitive decline (7,16). An increased tHcy level has been shown to be a strong independent risk factor for the development of AD (7). A positive correlation between plasma concentration of Hcy and the cerebrospinal fluid (CSF) concentration of Hcy was reported in AD (17). The implication of HHcy in pathogenesis of AD is not fully understood. In fact, chronic high plasma levels of Hcy may have deleterious effects on brain function (18). Moreover, Hcy may have a neurotoxic effect in AD because it is readily oxidized as a consequence of auto-oxidation leading to superoxide anion and hydrogen peroxide generation that are toxic when accumulated (19). Additionally, intake of polyphenols contained in an antioxidant beverage may decrease tHcy concentrations in AD patients (20). Inhibition of protein phosphatase 2A (PP2A) by Hcy also promotes tau hyperphosphorylation (21,22). We found that tHcy and urea were increased, whereas vitamin B12 decreased in AD patients vs. control subjects, which is in accordance with some previous studies (4,19). In our study, however, there was no deficiency of folate Table 4. Correlation between homocysteine and serum variables among patients and controls Variable 2 Variable 1 Homocysteine
B12
Folate
Creatinine
r 5 e0.379 p O0.0001
r 5 e0.2 p 5 0.064
r 5 0.386 p !0.0001
Data were not shown for the nonsignificant results (folate was included for its important role in homocysteine metabolism).
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in AD patients vs. controls. However, deficiencies of either or both of these 2 B vitamins are relatively common in AD (4,5,19). Our study also indicated that there was a negative interaction of tHcy with vitamin B12. However, several studies have reported different controversial results concerning biochemical factors and AD. This is may be due to the difficulty of diagnosis of AD and changes in mean ages or repartition of gender in different cohorts. The present results showed no association of serum uric acid and creatinine to AD. However, it has been reported that uric acid, urea and creatinine, indicators of renal function, interacted synergistically with Hcy in the association with AD (5). Indeed, alteration of serum uric acid concentration in AD is controversial. Our results showed a positive correlation between tHcy and creatinine. An elevation of an indicator of renal function in male AD patients was also related (5). Clinical hypo- and hyperthyroidism have been recognized as potentially reversible causes of cognitive impairment (23,24). However, we found no association of TSH, T3 or T4 with AD. Our results showed no effect of serum calcium (Ca2þ) in AD patients. No difference in serum Ca2þ concentration was found in accordance with the study of Shore et al. (25). It has been reported that cholesterol was a significant predictor of AD prevalence (26). However, elevated total cholesterol promotes increasing synthesis of Ab (27). HDL may have a protective effect on hippocampal atrophy and AD (28,29). In our study, however, no correlation was found between lipid profile and homocysteine in association with AD. Indeed, total cholesterol levels in the blood may have little relevance to cholesterol levels in the brain because cholesterol cannot cross the blood brain barrier (30). In conclusion, our data support the notion that serum tHcy plays a significant role in the development of AD. Hcy can be considered as a risk factor for AD. To the best of our knowledge, this is the first study of the relation between tHcy, folate and vitamin B12 concentrations and AD in an Algerian population. Monitoring tHcy concentration in the elderly may be relevant for prevention of AD.
References 1. Forstl H, Kurz A. Clinical features of Alzheimer’s disease. Eur Arch Psychiatry Clin Neurosci 1999;249:288e290. 2. Jellinger K. Morphology of Alzheimer’s disease and related disorders. In: Maurer K, Riederer P, Beckmann H, eds. Alzheimer’s Disease: Epidemiology, Neuropathology, Neurochemistry, and Clinics. Berlin: Springer-Verlag; 1990. pp. 61e77. 3. Selkoe DJ. Alzheimer’s disease: genotypes, phenotypes, and treatments. Science 1997;275:630e631. 4. Clarke R, Smith AD, Jobst KA, et al. Folate, vitamin B 12, and serum total homocysteine levels in confirmed Alzheimer disease. Arch Neurol 1998;55:1449e1455. 5. Cascalheira JF, Jo~ao SS, Pinhanc¸os SS, et al. Serum homocysteine: interplay with other circulating and genetic factors in association to Alzheimer’s type dementia. Clin Biochem 2009;42:783e790.
6. Hooshmand B, Solomon A, K areholt I, et al. Homocysteine and holotranscobalamin and the risk of Alzheimer disease: a longitudinal study. Neurology 2010;75:1408e1414. 7. Seshadri S, Beiser A, Selhub J, et al. Plasma homocysteine as a risk factor for dementia and Alzheimer’s disease. N Engl J Med 2002;346:476e483. 8. Mattson MP, Shea TB. Folate and homocysteine metabolism in neural plasticity and neurodegenerative disorders. Trends Neurosci 2003;26:137e146. 9. K€oseoglu E, Karaman Y. Relations between homocysteine, folate and vitamin B12 in vascular dementia and in Alzheimer disease. Clin Biochem 2007;40:859e863. 10. Quadri P, Fragicomo C, Pezzati R, et al. Homocysteine, folate and vitaminB-12 in mild cognitive impairment, Alzheimer disease and vascular dementia. Am J Clin Nutr 2004;80:114e122. 11. Selhub J, Jacques PF, Wilson PW, et al. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA 1993;270:2693e2698. 12. Ho PI, Ashline D, Dhitavat S, et al. Folate deprivation induces neurodegeneration: roles of oxidative stress and increased homocysteine. Neurobiol Dis 2003;14:32e42. 13. Zhuo JM, Pratico D. Acceleration of brain amyloidosis in an Alzheimer’s disease mouse model by a folate, vitamin B6 and B12-deficient diet. Exp Gerontol 2010;45:195e201. 14. Preux PM, Odermatt P, Perna A, et al. Qu’est-ce qu’une regression logistique? Rev Mal Respir 2005;22:159e162. 15. Houcher B, Candito M, Gibelin P, et al. Assessment of folate status: measurement of homocysteine versus vitamin B12 and folate. Pteridines 2003;14:27e33. 16. Ravaglia G, Forti P, Maioli F, et al. Homocysteine and folate as risk factors for dementia and Alzheimer disease. Am J Clin Nutr 2005;82:636e643. 17. Selley ML, Close DR, Stern SE. The effect of increased concentrations of homocysteine on the concentration of (E)-4-hydroxy-2-nonenal in the plasma and cerebrospinal fluid of patients with Alzheimer’s disease. Neurobiol Aging 2002;23:383e388. 18. Agnati LF, Genedani S, Rasio G, et al. Studies on homocysteine plasma levels in Alzheimer’s patients. Relevance for neurodegeneration. J Neural Transm 2005;112:163e169. 19. Loscalzo J. The oxidant stress of hyperhomocysteinemia. J Clin Invest 1996;98:5e7. 20. Morillas-Ruiz JM, Rubio-Perez JM, Albaladejo MD, et al. Effect of an antioxidant drink on homocysteine levels in Alzheimer’s patients. J Neurol Sci 2010;299:175e178. 21. Vafai SB, Stock JB. Protein phosphatase 2A methylation: a link between elevated plasma homocysteine and Alzheimer’s Disease. FEBS Lett 2002;518:1e4. 22. Zhang CE, Tian Q, Wei W, et al. Homocysteine induces tau phosphorylation by inactivating protein. Neurobiol Aging 2008;29:1654e1665. 23. Smith JS, Kiloh LG. The investigation of dementia: results in 200 consecutive admissions. Lancet 1981;1:824e827. 24. Cummings J, Benson DF, LoVerme S Jr. Reversible dementia: illustrative cases, definition, and review. JAMA 1980;243:2434e2439. 25. Shore D, Henkin RI, Nelson NR, et al. Hair and serum copper, zinc, calcium, and magnesium concentrations in Alzheimer-type dementia. J Am Geriatr Soc 1984;32:892e895. 26. Notkola IL, Sulkava R, Pekkanen J, et al. Serum total cholesterol, apolipoprotein E epsilon 4 allele, and Alzheimer’s disease. Neuroepidemiology 1998;17:14e20. 27. Kuo YM, Emmerling MR, Bisgaier CL, et al. Elevated low-density lipoprotein in Alzheimer’s disease correlates with brain abeta 1e42 levels. Biochem Biophys Res Commun 1998;252:711e715. 28. Sj€ogren M, Mielke M, Gustafson D, et al. Cholesterol and Alzheimer’s disease—is there a relation? Mech Ageing Dev 2006;127:138e147. 29. Wolf H, Hensel A, Arendt T, et al. Serum lipids and hippocampal volume: the link to Alzheimer’s disease? Ann Neurol 2004;56:745e748. 30. Reiss A. Cholesterol and apolipoprotein E in Alzheimer’s disease. Am J Alzheimers Dis Other Demen 2005;20:91e96.
ORIGINAL ARTICLE
Hyperhomocysteinemia Is a Risk Factor for Alzheimer’s Disease in an Algerian Population Khaled Nazef,a Malika Khelil,a Hiba Chelouti,a Ghouti Kacimi,b Mohamed Bendini,c Meriem Tazir,d Soraya Belarbi,d Mohamed El Hadi Cherifi,e and Bahia Djerdjouria a
Departement de Biologie Cellulaire et Moleculaire, Faculte des Sciences Biologiques, Universite des Sciences et de la Technologie Houari Boumediene Alger, Algerie b Laboratoire de Biochimie, H^opital Central de l’Armee Mohamed Nekkache Alger, Algerie c Service de Neurologie, H^opital Central de l’Armee Mohamed Nekkache Alger, Algerie d Service de Neurologie, Centre Hospitalo-Universitaire (CHU) Mustapha Bacha Alger, Algerie e Laboratoire Central de Biologie, Centre Hospitalo-Universitaire (CHU) Nafissa Hamoud Alger, Algerie Received for publication July 19, 2013; accepted March 3, 2014 (ARCMED-D-13-00393).
Background and Aims. There is growing evidence that increased blood concentration of total homocysteine (tHcy) may be a risk factor for Alzheimer’s disease (AD). The present study was conducted to evaluate the association of serum tHcy and other biochemical risk factors with AD. Methods. This is a case-control study including 41 individuals diagnosed with AD and 46 nondemented controls. Serum levels of all studied biochemical parameters were performed. Results. Univariate logistic regression showed a significant increase of tHcy ( p 5 0.008), urea ( p 5 0.036) and a significant decrease of vitamin B12 ( p 5 0.012) in AD group vs. controls. Using multivariate logistic regression, tHcy ( p 5 0.007, OR 5 1.376) appeared as an independent risk factor predictor of AD. There was a significant positive correlation between tHcy and creatinine ( p !0.0001). A negative correlation was found between tHcy and vitamin B12 ( p !0.0001). Conclusions. Our findings support that hyperhomocysteinemia is a risk factor for AD in an Algerian population and is also associated with vitamin B12 deficiency. Ó 2014 IMSS. Published by Elsevier Inc. Key Words: Hyperhomocysteinemia, Alzheimer’s disease, Vitamin B12, Folate.
Introduction Alzheimer’s disease (AD) is the most common type of dementia and is a multifactorial disease resulting from progressive neurodegenerative disorders. AD can affect different people in different ways, but the most common symptoms are memory and behavior disorders (1), gradually evolving towards dementia. Postmortem brains of AD patients display two pathological hallmarks: amyloid-b (Ab) peptide plaques and neurofibrillary tangles (NFTs) (2,3). The exact causes of this disease are not yet known. However, several risk factors Address reprint requests to: Malika Khelil, Departement de Biologie Cellulaire et Moleculaire, Faculte des Sciences Biologiques, Universite, des Sciences et de la Technologie Houari Boumediene BP 32 El-Alia. 16111 Alger, Algerie; Phone: (213) 771239487; FAX: (213) 21247217; E-mail: [email protected]
for AD have been suggested (4). The most important objective of research on AD is the discovery of biomarkers predictive of disease onset and reliable ways to diagnose AD as early as possible before the loss of autonomy and to also find preventive interventions. Several studies indicated that increased blood concentrations of homocysteine (Hcy) may be a risk factor for AD (5e7). The metabolism of Hcy depends on two cofactors like vitamin B12 and folate (8). An inverse correlation between serum folate and vitamin B12 concentrations and the risk of AD was found in several reports (9,10). There is growing evidence that hyperhomocysteinemia (HHcy) is in part due to vitamin deficiency, especially in older people (11). Presence of Hcy has been shown in the brain of rats deficient in folate, the hippocampus and cortex containing the highest proportions (12). In addition, folate deficiency
0188-4409/$ - see front matter. Copyright Ó 2014 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2014.03.001
248
Nazef et al./ Archives of Medical Research 45 (2014) 247e250
in mice leads to alteration of synaptic plasticity, hippocampal neurogenesis, memory deficits and amyloid deposits in cerebral vessels (13). In the present work we aimed to examine the possible association between total serum homocysteine (tHcy) and other proposed risk factors for AD such as vitamin B12, folate, cholesterol, thyroid stimulating hormone (TSH), uric acid and other biochemical factors in an Algerian population. We also investigated the correlation between tHcy, folate and vitamin B12. Subjects and Methods Subjects Harvesting, selection and classification of patients who were included in this study were performed with neurologists from the Neurology Departments at the University Hospital CentereMustapha Bacha and Central Hospital of Army, Algiers. This study excluded patients and healthy individuals under medications that alter Hcy concentrations including vitamin B12 and folate. Individuals who had a Hcy level O50 mM were also excluded. After these exclusions, there were 41 subjects with AD and 46 control subjects. Cognitive impairment was evaluated by several tests including the Mini-Mental State Examination (MMSE). Inclusion criteria for elderly controls were a MMSE score $24 and the absence of any known neurological disorder or dementia. Informed consent was obtained from patients, controls or responsible caregivers. This study was approved by the local ethics committee. Biochemical Assays Blood samples were collected and serum was used for the analysis of tHcy, folate, vitamin B12 and other biochemical parameters. Serum tHcy was measured using fluorescence polarization immunoassay (Abbott AxSYM system, Abbott Laboratories). Serum folate, vitamin B12, TSH, T3 and T4 concentrations were measured by competitive binding assays with electrochemiluminescent detection using an auto-analyser (Elecsys 2010, Hitachi, Roche or COBAS e401). Serum concentrations of uric acid, creatinine, total cholesterol, triglycerides, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were measured spectrophotometrically using an auto-analyzer (COBAS c501). Serum glucose and urea were measured spectrophotometrically by the hexokinase/glucose-6-phosphate dehydrogenase enzymatic assay and by the urease/glutamine dehydrogenase enzymatic assay, respectively, using the COBAS c501 auto-analyzer. Statistical Analysis Data are expressed as mean standard deviation (SD). After testing data for normality (ShapiroeWilk testeStatistica
v.8.0), correlation between variables was performed using Spearman Rank order correlations. The strength of association between each variable and AD was assessed by calculation of crude odds ratios (OR) and 95% confidence intervals (CI). OR was used to assess the intensity of the association between the different variables and AD describing a protective effect to !1 (OR !1) or predicting factor to a value O1 (OR O1). Multiple logistic regression analysis was also performed to determine variables that are the best predictors of AD. All statistical tests were performed using SPSS for windows v.15.0 (SPSS Science, Chicago, IL); p !0.05 was considered statistically significant.
Results Demographic, clinical, cognitive and functional characteristics of the population study are summarized in Table 1.
Table 1. Demographic, clinical and biochemical characteristics of AD patients and controls Characteristcs
Patients (n 5 41)
Controls (n 5 46)
Age (mean SD) (years) Gender (males [%]) MMSE (mean SD) Homocysteine (mmol) B12 vitamin (pg/mL) Folate (ng/mL) Glucose (mmol) Triglyceride (mmol/l) Cholesterol (mmol/l) HDL (mmol/l) LDL (mmol/l) Creatinine (mmol) Urea (mmol/l) Uric acid (mmol) TSH (mmol) Calcium (mmol) T3 (nmol/l) T4 (nmol/l) Diabetes (%) Hypertension (%) Hypercholesterolemia Thyroid diseases (%) Heart diseases (%) Chronic inflammatory diseases (%)
72.61 8.92 43.8 15.131 6.23 19.126 6.93 301.766 113.54 8.291 2.1 5.74 1.38 3.362 0.37 4.903 1.244 0.31 2.82 0.69 78.822 20.51 5.577 1.31 283.87 68.22 1.638 0.82 2.229 0.13 1.94 0.46 112.193 19.78 23.1 41 15.4 7.7 17.9 23.1
66.3 9.6* 52.2 26.668 2.87*** 15.613 3.63** 399.103 197.85* 7.993 3.43 6.335 3.56 1.527 0.67 4.881 1.05 1.233 0.03 3.046 0.91 72.919 14.65 5.002 1.09* 269.18 34.55 1.951 1.27 2.263 0.16 2.011 0.32 111.42 20.23 22.7 50 13.6 9.1 4.5 25
AD, Alzheimer’s disease; MMSE, Mini-Mental State Examination; T3, triiodothyrone; T4, thyroxine; TSH, thyroid stimulating hormone; HDL, high-density lipoprotein; LDL, low-density lipoprotein. Results are expressed as mean SD. Comparisons between AD and controls were performed by two-tailed Student t test (for normally distributed data) or by Mann-Whitney U test (for non-normally distributed data). c2 and Fisher’s Exact Test were performed for frequencies analysis. *p !0.05; **p !0.01; ***p !0.001; n 5 number of individuals.
Hyperhomocysteinemia in Alzheimer’s Disease
249
We used the missing values option when some values were missed.
Table 3. Multivariate logistic regression model
Univariate Logistic Regression
Homocysteine MMSE
Association between analyzed variables with AD was investigated first by univariate logistic regression (Table 2). Significant difference is noted between controls and AD patients regarding age and MMSE, respectively ( p 5 0.004, p 5 0.000). tHcy and urea were positively associated, whereas vitamin B12 was negatively associated with AD. Indeed, tHcy and urea were significantly higher in patients than in controls ( p 5 0.008; p 5 0.036, respectively) and concentration of vitamin B12 was significantly lower in patients than in controls ( p 5 0.012). No statistically significant differences were observed for all other studied variables.
Variables
OR
p
1.376 0.527
0.007 0.000
Adjusted for age, sex, MMSE, homocysteine, vitamin B12, folate, triglycerides, LDL, creatinine, urea, uric acid, TSH.
Correlation between Hcy and Other Biochemical Variables Spearman’s correlation coefficient was used to grasp the efficient interaction between tHcy concentration and the other variables (Table 4). Significant negative correlation was observed between tHcy and vitamin B12 (r 5 e0.379, p !0.0001). However, a significant positive correlation was observed between tHcy and creatinine (r 5 0.386, p !0.0001), a biochemical indicator of renal function.
Multivariate Logistic Regression Multivariate logistic regression analysis was chosen to find the best independent predictors (Table 3). The candidate predictors were variables with a p value !0.25 in the univariate logistic regression analysis (14). Folate concentration was also included as a candidate because it could affect tHcy concentration (5,15). After correction for multiple testing, statistical differences were still noted for tHcy ( p 5 0.007) and MMSE ( p !0.001). Table 2. Crude ratios (OR) and 95% confidence intervals (CI) according to different factors Factors Age (years) Males (%) MMSE Education level Homocysteine Vitamin B12 Folate Glucose Triglycerides Cholesterol HDL LDL Creatinine Urea Uric acid TSH Calcium T3 T4 Diabetes (%) Hypertension (%) Hypercholesterolemia (%) Thyroid diseases (%) Cardiopathy (%) Chronic inflammatory diseases
OR
95% CI
p
1.075 0.72 0.594 0.75 1.164 0.996 1.037 0.916 0.566 1.023 1.125 0.702 1.02 1.527 1.005 0.744 0.222 0.644 1.002 1.02 0.696 1.152 0.833 4.594 0.9
1.024e1.129 0.38e1.671 0.472e0.748 0.234e2.408 1.041e1.302 0.993e0.999 0.895e1.203 0.764e1.097 0.249e1.283 0.669e1.564 0.281e4.508 0.406e1.23 0.994e1.048 1.028e0.268 0.997e1.014 0.476e1.62 0.013e3.939 0.217e1.911 0.981e1.023 0.366e2.845 0.292e1.66 0.339e3.916 0.1758e3.978 0.893e23.62 0.328e2.472
0.004 0.441 0.000 0.629 0.008 0.012 0.628 0.339 0.173 0.917 0.867 0.205 0.135 0.036 0.206 0.193 0.305 0.428 0.857 0.97 0.413 0.821 0.819 0.068 0.838
OR, odds ratio; CI, confidence intervals.
Discussion The present findings demonstrate that high serum tHcy level has an independent relation with AD. Indeed, an association of high serum concentration of tHcy to AD was evident through multivariate logistic regression analysis. High concentration of tHcy could be an independent risk factor for AD, dementia, and cognitive decline (7,16). An increased tHcy level has been shown to be a strong independent risk factor for the development of AD (7). A positive correlation between plasma concentration of Hcy and the cerebrospinal fluid (CSF) concentration of Hcy was reported in AD (17). The implication of HHcy in pathogenesis of AD is not fully understood. In fact, chronic high plasma levels of Hcy may have deleterious effects on brain function (18). Moreover, Hcy may have a neurotoxic effect in AD because it is readily oxidized as a consequence of auto-oxidation leading to superoxide anion and hydrogen peroxide generation that are toxic when accumulated (19). Additionally, intake of polyphenols contained in an antioxidant beverage may decrease tHcy concentrations in AD patients (20). Inhibition of protein phosphatase 2A (PP2A) by Hcy also promotes tau hyperphosphorylation (21,22). We found that tHcy and urea were increased, whereas vitamin B12 decreased in AD patients vs. control subjects, which is in accordance with some previous studies (4,19). In our study, however, there was no deficiency of folate Table 4. Correlation between homocysteine and serum variables among patients and controls Variable 2 Variable 1 Homocysteine
B12
Folate
Creatinine
r 5 e0.379 p O0.0001
r 5 e0.2 p 5 0.064
r 5 0.386 p !0.0001
Data were not shown for the nonsignificant results (folate was included for its important role in homocysteine metabolism).
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Nazef et al./ Archives of Medical Research 45 (2014) 247e250
in AD patients vs. controls. However, deficiencies of either or both of these 2 B vitamins are relatively common in AD (4,5,19). Our study also indicated that there was a negative interaction of tHcy with vitamin B12. However, several studies have reported different controversial results concerning biochemical factors and AD. This is may be due to the difficulty of diagnosis of AD and changes in mean ages or repartition of gender in different cohorts. The present results showed no association of serum uric acid and creatinine to AD. However, it has been reported that uric acid, urea and creatinine, indicators of renal function, interacted synergistically with Hcy in the association with AD (5). Indeed, alteration of serum uric acid concentration in AD is controversial. Our results showed a positive correlation between tHcy and creatinine. An elevation of an indicator of renal function in male AD patients was also related (5). Clinical hypo- and hyperthyroidism have been recognized as potentially reversible causes of cognitive impairment (23,24). However, we found no association of TSH, T3 or T4 with AD. Our results showed no effect of serum calcium (Ca2þ) in AD patients. No difference in serum Ca2þ concentration was found in accordance with the study of Shore et al. (25). It has been reported that cholesterol was a significant predictor of AD prevalence (26). However, elevated total cholesterol promotes increasing synthesis of Ab (27). HDL may have a protective effect on hippocampal atrophy and AD (28,29). In our study, however, no correlation was found between lipid profile and homocysteine in association with AD. Indeed, total cholesterol levels in the blood may have little relevance to cholesterol levels in the brain because cholesterol cannot cross the blood brain barrier (30). In conclusion, our data support the notion that serum tHcy plays a significant role in the development of AD. Hcy can be considered as a risk factor for AD. To the best of our knowledge, this is the first study of the relation between tHcy, folate and vitamin B12 concentrations and AD in an Algerian population. Monitoring tHcy concentration in the elderly may be relevant for prevention of AD.
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