Archives of Medical Research 45 (2014) 247e250

ORIGINAL ARTICLE

Hyperhomocysteinemia Is a Risk Factor for Alzheimer’s Diseasein 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

aD�epartement de Biologie Cellulaire et Mol�eculaire, Facult�e des Sciences Biologiques, Universit�e des Sciences et de la Technologie Houari

Boumediene Alger, Alg�eriebLaboratoire de Biochimie, Hopital Central de l’Arm�ee Mohamed Nekkache Alger, Alg�eriecService de Neurologie, Hopital Central de l’Arm�ee Mohamed Nekkache Alger, Alg�erie

dService de Neurologie, Centre Hospitalo-Universitaire (CHU) Mustapha Bacha Alger, Alg�erieeLaboratoire Central de Biologie, Centre Hospitalo-Universitaire (CHU) Nafissa Hamoud Alger, Alg�erie

Received for publication July 19, 2013; accepted March 3, 2014 (ARCMED-D-13-00393).

Address reprint re

Cellulaire et Mol�ecul

des Sciences et de la

16111 Alger, Alg�erieE-mail: malikakhelil@

0188-4409/$ - see frohttp://dx.doi.org/10

Background and Aims. There is growing evidence that increased blood concentration oftotal homocysteine (tHcy) may be a risk factor for Alzheimer’s disease (AD). The presentstudy was conducted to evaluate the association of serum tHcy and other biochemical riskfactors with AD.

Methods. This is a case-control study including 41 individuals diagnosed with AD and46 nondemented controls. Serum levels of all studied biochemical parameterswere performed.

Results. Univariate logistic regression showed a significant increase of tHcy ( p5 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) appearedas an independent risk factor predictor of AD. There was a significant positive correlationbetween tHcy and creatinine ( p !0.0001). A negative correlation was found betweentHcy and vitamin B12 ( p !0.0001).

Conclusions. Our findings support that hyperhomocysteinemia is a risk factor for AD inan Algerian population and is also associated with vitamin B12 deficiency. � 2014IMSS. Published by Elsevier Inc.

Key Words: Hyperhomocysteinemia, Alzheimer’s disease, Vitamin B12, Folate.

Introduction

Alzheimer’s disease (AD) is the most common type of de-mentia and is a multifactorial disease resulting from progres-sive neurodegenerative disorders. AD can affect differentpeople in different ways, but the most common symptomsare memory and behavior disorders (1), gradually evolvingtowards dementia. Postmortem brains of AD patients displaytwo pathological hallmarks: amyloid-b (Ab) peptide plaquesand neurofibrillary tangles (NFTs) (2,3). The exact causes ofthis disease are not yet known. However, several risk factors

quests to: Malika Khelil, D�epartement de Biologie

aire, Facult�e des Sciences Biologiques, Universit�e,

Technologie Houari Boumediene BP 32 El-Alia.

; Phone: (213) 771239487; FAX: (213) 21247217;

hotmail.fr

nt matter. Copyright � 2014 IMSS. Published by Elsevier.1016/j.arcmed.2014.03.001

for AD have been suggested (4). The most important objec-tive of research on AD is the discovery of biomarkers predic-tive of disease onset and reliable ways to diagnose AD asearly as possible before the loss of autonomy and to also findpreventive interventions.

Several studies indicated that increased blood concentra-tions of homocysteine (Hcy) may be a risk factor for AD(5e7). The metabolism of Hcy depends on two cofactorslike vitamin B12 and folate (8). An inverse correlation be-tween serum folate and vitamin B12 concentrations and therisk of AD was found in several reports (9,10). There isgrowing evidence that hyperhomocysteinemia (HHcy) isin part due to vitamin deficiency, especially in older people(11). Presence of Hcy has been shown in the brain of ratsdeficient in folate, the hippocampus and cortex containingthe highest proportions (12). In addition, folate deficiency

Inc.

248 Nazef et al./ Archives of Medical Research 45 (2014) 247e250

in mice leads to alteration of synaptic plasticity, hippocam-pal neurogenesis, memory deficits and amyloid deposits incerebral vessels (13).

In the present work we aimed to examine the possibleassociation between total serum homocysteine (tHcy) andother proposed risk factors for AD such as vitamin B12,folate, cholesterol, thyroid stimulating hormone (TSH), uricacid and other biochemical factors in an Algerian popula-tion. We also investigated the correlation between tHcy,folate and vitamin B12.

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) 72.61 � 8.92 66.3 � 9.6*

Gender (males [%]) 43.8 52.2

MMSE (mean � SD) 15.131 � 6.23 26.668 � 2.87***

Homocysteine (mmol) 19.126 � 6.93 15.613 � 3.63**

B12 vitamin (pg/mL) 301.766 � 113.54 399.103 � 197.85*

Folate (ng/mL) 8.291 � 2.1 7.993 � 3.43

Glucose (mmol) 5.74 � 1.38 6.335 � 3.56

Triglyceride (mmol/l) 3.362 � 0.37 1.527 � 0.67

Cholesterol (mmol/l) 4.903 4.881 � 1.05

HDL (mmol/l) 1.244 � 0.31 1.233 � 0.03

LDL (mmol/l) 2.82 � 0.69 3.046 � 0.91

Creatinine (mmol) 78.822 � 20.51 72.919 � 14.65

Urea (mmol/l) 5.577 � 1.31 5.002 � 1.09*

Uric acid (mmol) 283.87 � 68.22 269.18 � 34.55

TSH (mmol) 1.638 � 0.82 1.951 � 1.27

Calcium (mmol) 2.229 � 0.13 2.263 � 0.16

T3 (nmol/l) 1.94 � 0.46 2.011 � 0.32

T4 (nmol/l) 112.193 � 19.78 111.42 � 20.23

Diabetes (%) 23.1 22.7

Hypertension (%) 41 50

Hypercholesterolemia 15.4 13.6

Thyroid diseases (%) 7.7 9.1

Heart diseases (%) 17.9 4.5

Chronic inflammatory

diseases (%)

23.1 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 distrib-

uted 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.

Subjects and Methods

Subjects

Harvesting, selection and classification of patients whowere included in this study were performed with neurolo-gists from the Neurology Departments at the UniversityHospital CentereMustapha Bacha and Central Hospital ofArmy, Algiers. This study excluded patients and healthy in-dividuals under medications that alter Hcy concentrationsincluding vitamin B12 and folate. Individuals who had aHcy level O50 mM were also excluded. After these exclu-sions, there were 41 subjects with AD and 46 control sub-jects. Cognitive impairment was evaluated by several testsincluding the Mini-Mental State Examination (MMSE).

Inclusion criteria for elderly controls were a MMSEscore $24 and the absence of any known neurological dis-order or dementia. Informed consent was obtained from pa-tients, controls or responsible caregivers. This study wasapproved by the local ethics committee.

Biochemical Assays

Blood samples were collected and serum was used for theanalysis of tHcy, folate, vitamin B12 and other biochemicalparameters. Serum tHcy was measured using fluorescencepolarization immunoassay (Abbott AxSYM system, AbbottLaboratories). Serum folate, vitamin B12, TSH, T3 and T4concentrations were measured by competitive binding as-says with electrochemiluminescent detection using anauto-analyser (Elecsys 2010, Hitachi, Roche or COBASe401). Serum concentrations of uric acid, creatinine, totalcholesterol, triglycerides, low-density lipoprotein (LDL)and high-density lipoprotein (HDL) were measured spec-trophotometrically using an auto-analyzer (COBAS c501).Serum glucose and urea were measured spectrophoto-metrically by the hexokinase/glucose-6-phosphate dehydro-genase enzymatic assay and by the urease/glutaminedehydrogenase enzymatic assay, respectively, using theCOBAS c501 auto-analyzer.

Statistical Analysis

Data are expressed as mean � standard deviation (SD). Af-ter testing data for normality (ShapiroeWilk testeStatistica

v.8.0), correlation between variables was performed usingSpearman Rank order correlations. The strength of associ-ation between each variable and AD was assessed by calcu-lation of crude odds ratios (OR) and 95% confidenceintervals (CI).

OR was used to assess the intensity of the associationbetween the different variables and AD describing a pro-tective effect to !1 (OR !1) or predicting factor to avalue O1 (OR O1). Multiple logistic regression anal-ysis was also performed to determine variables that arethe best predictors of AD. All statistical tests were per-formed using SPSS for windows v.15.0 (SPSS Science,Chicago, IL); p !0.05 was considered statisticallysignificant.

Results

Demographic, clinical, cognitive and functional character-istics of the population study are summarized in Table 1.

Table 3. Multivariate logistic regression model

Variables OR p

Homocysteine 1.376 0.007

MMSE 0.527 0.000

Adjusted for age, sex, MMSE, homocysteine, vitamin B12, folate, triglyc-

erides, LDL, creatinine, urea, uric acid, TSH.

249Hyperhomocysteinemia in Alzheimer’s Disease

We used the missing values option when some values weremissed.

Univariate Logistic Regression

Association between analyzed variables with AD was inves-tigated first by univariate logistic regression (Table 2). Sig-nificant difference is noted between controls and ADpatients regarding age and MMSE, respectively( p 5 0.004, p 5 0.000).

tHcy and urea were positively associated, whereasvitamin B12 was negatively associated with AD. Indeed,tHcy and urea were significantly higher in patients than incontrols ( p5 0.008; p5 0.036, respectively) and concentra-tion of vitamin B12was significantly lower in patients than incontrols ( p5 0.012). No statistically significant differenceswere observed for all other studied variables.

Multivariate Logistic Regression

Multivariate logistic regression analysis was chosen tofind the best independent predictors (Table 3). The candi-date predictors were variables with a p value !0.25 inthe univariate logistic regression analysis (14). Folate con-centration was also included as a candidate because it couldaffect tHcy concentration (5,15). After correction for mul-tiple 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 OR 95% CI p

Age (years) 1.075 1.024e1.129 0.004

Males (%) 0.72 0.38e1.671 0.441

MMSE 0.594 0.472e0.748 0.000

Education level 0.75 0.234e2.408 0.629

Homocysteine 1.164 1.041e1.302 0.008

Vitamin B12 0.996 0.993e0.999 0.012

Folate 1.037 0.895e1.203 0.628

Glucose 0.916 0.764e1.097 0.339

Triglycerides 0.566 0.249e1.283 0.173

Cholesterol 1.023 0.669e1.564 0.917

HDL 1.125 0.281e4.508 0.867

LDL 0.702 0.406e1.23 0.205

Creatinine 1.02 0.994e1.048 0.135

Urea 1.527 1.028e0.268 0.036

Uric acid 1.005 0.997e1.014 0.206

TSH 0.744 0.476e1.62 0.193

Calcium 0.222 0.013e3.939 0.305

T3 0.644 0.217e1.911 0.428

T4 1.002 0.981e1.023 0.857

Diabetes (%) 1.02 0.366e2.845 0.97

Hypertension (%) 0.696 0.292e1.66 0.413

Hypercholesterolemia (%) 1.152 0.339e3.916 0.821

Thyroid diseases (%) 0.833 0.1758e3.978 0.819

Cardiopathy (%) 4.594 0.893e23.62 0.068

Chronic inflammatory diseases 0.9 0.328e2.472 0.838

OR, odds ratio; CI, confidence intervals.

Correlation between Hcy and Other BiochemicalVariables

Spearman’s correlation coefficient was used to grasp theefficient interaction between tHcy concentration and theother variables (Table 4). Significant negative correlationwas observed between tHcy and vitamin B12 (r 5 e0.379,p !0.0001). However, a significant positive correlationwas observed between tHcy and creatinine (r 5 0.386,p !0.0001), a biochemical indicator of renal function.

Discussion

The present findings demonstrate that high serum tHcy levelhas an independent relation with AD. Indeed, an associationof high serum concentration of tHcy to AD was evidentthrough multivariate logistic regression analysis. High con-centration of tHcy could be an independent risk factor forAD, dementia, and cognitive decline (7,16). An increasedtHcy level has been shown to be a strong independent riskfactor for the development of AD (7). A positive correlationbetween plasma concentration of Hcy and the cerebrospinalfluid (CSF) concentration of Hcy was reported in AD (17).

The implication of HHcy in pathogenesis of AD is notfully understood. In fact, chronic high plasma levels ofHcy may have deleterious effects on brain function (18).Moreover, Hcy may have a neurotoxic effect in AD becauseit is readily oxidized as a consequence of auto-oxidationleading to superoxide anion and hydrogen peroxide gener-ation that are toxic when accumulated (19). Additionally,intake of polyphenols contained in an antioxidant beveragemay decrease tHcy concentrations in AD patients (20). In-hibition of protein phosphatase 2A (PP2A) by Hcy also pro-motes tau hyperphosphorylation (21,22).

We found that tHcy and urea were increased, whereasvitamin 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 B12 Folate Creatinine

Variable 1 r 5 e0.379 r 5 e0.2 r 5 0.386

Homocysteine p O0.0001 p 5 0.064 p !0.0001

Data were not shown for the nonsignificant results (folate was included for

its important role in homocysteine metabolism).

250 Nazef et al./ Archives of Medical Research 45 (2014) 247e250

in AD patients vs. controls. However, deficiencies of eitheror both of these 2 B vitamins are relatively common in AD(4,5,19). Our study also indicated that there was a negativeinteraction of tHcy with vitamin B12. However, severalstudies have reported different controversial results con-cerning biochemical factors and AD. This is may be dueto the difficulty of diagnosis of AD and changes in meanages or repartition of gender in different cohorts.

The present results showed no association of serum uricacid and creatinine to AD. However, it has been reported thaturic acid, urea and creatinine, indicators of renal function, in-teracted synergistically with Hcy in the association with AD(5). Indeed, alteration of serum uric acid concentration in ADis controversial. Our results showed a positive correlation be-tween tHcy and creatinine. An elevation of an indicator ofrenal function in male AD patients was also related (5).

Clinical hypo- and hyperthyroidism have been recog-nized as potentially reversible causes of cognitive impair-ment (23,24). However, we found no association of TSH,T3 or T4 with AD.

Our results showed no effect of serum calcium (Ca2þ) inAD patients. No difference in serum Ca2þ concentrationwas found in accordance with the study of Shore et al. (25).

It has been reported that cholesterol was a significant pre-dictor ofADprevalence (26). However, elevated total choles-terol promotes increasing synthesis of Ab (27). HDL mayhave a protective effect on hippocampal atrophy and AD(28,29). In our study, however, no correlation was found be-tween lipid profile and homocysteine in association with AD.Indeed, total cholesterol levels in the blood may have littlerelevance to cholesterol levels in the brain because choles-terol cannot cross the blood brain barrier (30).

In conclusion, our data support the notion that serumtHcy plays a significant role in the development of AD.Hcy can be considered as a risk factor for AD. To the bestof our knowledge, this is the first study of the relation be-tween tHcy, folate and vitamin B12 concentrations andAD in an Algerian population. Monitoring tHcy concentra-tion in the elderly may be relevant for prevention of AD.

References1. 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 treat-

ments. 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 Neu-

rol 1998;55:1449e1455.

5. Cascalheira JF, Jo~ao SS, Pinhancos 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 hol-

otranscobalamin 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. MattsonMP, SheaTB. Folate and homocysteinemetabolism in neural plas-

ticity andneurodegenerativedisorders.TrendsNeurosci 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 neuro-

degeneration: roles of oxidative stress and increased homocysteine.

Neurobiol Dis 2003;14:32e42.

13. Zhuo JM, Pratic�o 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 r�egression

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. Pteri-

dines 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 concentra-

tions of homocysteine on the concentration of (E)-4-hydroxy-2-none-

nal 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 neurodegenera-

tion. 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 be-

tween elevated plasma homocysteine and Alzheimer’s Disease. FEBS

Lett 2002;518:1e4.

22. Zhang CE, Tian Q, Wei W, et al. Homocysteine induces tau phosphor-

ylation 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: illustra-

tive 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. Neuroepi-

demiology 1998;17:14e20.

27. Kuo YM, Emmerling MR, Bisgaier CL, et al. Elevated low-density

lipoprotein in Alzheimer’s disease correlates with brain abeta 1e42levels. 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 vol-

ume: 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.