serum zinc, copper, insulin and lipids in alzheimer's disease epsilon 4 apolipoprotein e allele...

European Journal of Clinical Investigation (1999) 29, 637–642

Serum zinc, copper, insulin and lipids in Alzheimer’sdisease epsilon 4 apolipoprotein E allele carriers*

C. Gonzalez* , T. Martın†, J. Cacho†, M. T. Brenas†, T. Arroyo†, B. Garcıa-Berrocal†,J. A. Navajo† and J. M. Gonzalez-Buitrago*

†University Hospital and *University of Salamanca, Salamanca, Spain

Abstract Background Copper (Cu) and zinc (Zn) have been implicated in the development ofAlzheimer’s disease (AD) and, in this regard, Cu and Zn serum concentrations have beenanalysed but with inconclusive results. Serum insulin, glucose and cholesterol concentra-tions have been related to the apolipoprotein E genotype in non-AD populations.

Design In this study, we have analysed the relationship between serum Cu, Zn, insulin,glucose and lipid parameters (cholesterol, triglycerides, apoA and apoB apolipoproteins) inAD and AD epsilon 4 apolipoprotein E carriers by multivariate analysis using logisticregression, including the variables that showed a significance of P < 0·05 in the bivariateanalysis.

Results The results obtained show that epsilon 4 apoE allele is an independent AD riskfactor (OR ¼ 6·67, 95% CI ¼ 2·59–17·16). In AD epsilon 4 apoE allele carriers, we foundsignificantly higher Zn, Cu and insulin serum concentrations. Non-demented controlsubjects with at least one epsilon 4 apoE allele had the lowest serum insulin concentrations.There was no significant association between epsilon 4 apolipoprotein E allele and lipidparameters in the sample studied.

Conclusions In AD we have found a significant association between higher serum Zn, Cuand insulin concentrations and the presence of an epsilon 4 apoE allele, but only greaterserum Zn concentration appears to be an independent risk factor associated with thedevelopment of AD.

Keywords Alzheimer’s disease, apolipoprotein E, copper, insulin, lipids, zinc.Eur J Clin Invest 1999; 29 (7) 637–642

Introduction

Alzheimer’s disease (AD) is a heterogeneous neurodegen-erative disorder to date associated with three determinantor causal genes – amyloid precursor protein (APP) gene,presenilin 1 (PSEN1) gene and presenilin 2 (PSEN2) gene– and three susceptibility or risk genes – apolipoprotein E(ApoE) gene, LRP1, a gene encoding a neuronal receptor

for both apoE and APP, and A2M, the gene that codes fora2-macroglobulin [1,2]. There are probably various otherrisk factors that interact with the known susceptibilitygenes in the development of the disease [3]. In thisregard, preliminary findings suggest that the apoE epsilon4 allele may be associated with increased severity of chronictraumatic brain injury [4].

Copper (Cu) and zinc (Zn) have been implicated in thedevelopment of AD. Both appear to participate in the Ab

amyloid formation together with a number of metallothio-neins, metalloproteases and oxidative stress [5–8]. Copperand Zn are cofactors of Cu–Zn-dependent superoxidedismutase, an enzyme related to a possible vascular con-nection of AD [9]. Moreover, findings about Cu cerebro-spinal fluid and Zn in nails and plasma thymulin suggestmodified Zn and Cu metabolism in patients with AD[10–12]. However, the results of Cu and Zn in plasma areconfusing.

Positron emission tomography (PET) has revealedabnormalities in the cerebral metabolic rate of glucose in

Q 1999 Blackwell Science Ltd

Departamento de Bioquımica y Biologıa Molecular, Universidadde Salamanca, Salamanca, Spain (C. Gonzalez, J. M. Gonzalez-Buitrago); Servicios de Bioquımica (C. Gonzalez, T. Martın, M.T. Brenas, T. Arroyo, B. Garcıa-Berrocal, J. A. Navajo, J. M.Gonzalez-Buitraso) and Neurologıa (J. Cacho), HospitalUniversitario, Salamanca.*See Commentary on page 569.

Correspondence to: Professor J. M. Gonzalez-Buitrago, Serviciode Bioquımica, Hospital Universitario, 37007 Salamanca, Spain.E-mail: [email protected]

Received 4 June 1998; accepted 5 December 1998

638 C. Gonzalez et al.

patients with AD. The inheritance of apoE epsilon 4 isassociated with reduced cerebral parietal metabolism andincreased asymmetry in non-demented relatives at riskfrom probable AD [13]. Likewise, in late middle age,cognitively normal subjects who are homozygous for theepsilon 4 allele for apoE have reduced glucose metabolismin the same regions of the brain as patients with probableAD [14]. More recently, it has been observed that the apoEepsilon 4 allele does not appear to be associated withspecific deficits in brain metabolism in patients with AD,despite evidence that the epsilon 4 allele is associated withpreclinical alterations [15]. Hyperinsulinaemia has beenobserved in patients with AD in a number of studies[16,17], and changes in glucose metabolism have beenobserved in patients with cardiovascular diseases in relationto apoE polymorphisms [18].

On the other hand, apoE polymorphisms explain part ofthe variability of plasma cholesterol and apolipoproteinconcentrations [19]. The epsilon 4 allele is associatedwith elevations in total cholesterol (TC) levels relative tothe epsilon 3 allele, whereas the epsilon 2 allele has theopposite effect. The epsilon 4 allele is also associated withatherosclerotic disease and myocardial infarction [20–22].More recently, the joint effects of TC levels and the apoEgenotype in AD have been studied [23]. The resultsobtained point to the existence of a complex interactionbetween cholesterol, epsilon 4, sex and risk of AD.

The objective of the present study was to evaluate therelationships between serum Zn and Cu concentrationsand apoE polymorphisms in AD. Also, we have analysedserum insulin and glucose concentrations in relation toapoE genotype and the association between epsilon 4 apoEallele and serum cholesterol, triglycerides, apoA1 and apoBconcentrations in patients with AD.

Methods

The patients were recruited from the Neurology Clinic ofSalamanca University Hospital, where they receive peri-odic revision of their disease. The diagnosis of probable ADwas carried out by standardized evaluations and accordingto the National Institute of Neurological Disorders andStroke – Alzheimer’s Diseases and Related DisordersAssociation Work Group criteria [24]. Patients with ‘prob-able’ AD have been selected, without including in theanalysed sample ‘possible’ AD. All of the patients studiedbelong to the late-onset group (> 60 years). Symptomatictreatment included piracetam, citicholine and nicardipinehydrochloride. The control group was formed with thecollaboration of the patients’ partners, in order to avoidpossible confusing factors associated with AD. All the sub-jects included in the control group were free ofneuropsychological and psychiatric clinical pathology, andtheir participation in the study was voluntary. All of thesubjects were informed about the objective of the study.

After an overnight fast, a blood sample was obtained.For metal analysis the blood was collected in specific

tubes (Becton Dickinson, Rutherford, NJ, USA). Aftercoagulation and centrifugation, serum was separatedfrom cells within 45 min from blood drawing and storedat ¹ 208C in previously washed tubes with 10% nitric aciduntil analysis. A second serum sample was stored at ¹ 208Cfor the measurement of the parameters related with lipidand carbohydrate metabolisms. In a blood sample obtainedin EDTA tubes, nucleated corpuscular elements wereremoved and stored in buffered saline solution, pH 7·4,at ¹ 208C until DNA extraction.

DNA extraction was carried out with the a DNA Extrac-tion Kit (Stratagene Cloning System, La Jolla, CA, USA).Exon 4 from apoE was amplified by PCR in a Gene AmpPCR System 9600 (Perkin-Elmer, Norwalk, CT, USA).The amplification process and further reverse hybridizationwith colorimetric detection was carried out according toinstructions provided by the manufacturer (Innolipa ApoE, Innogenetics, Zwijndrecht, Belgium). Serum glucose,insulin, cholesterol, apolipoprotein A-1, apolipoprotein Band triglycerides were measured by automated methods.Serum Cu and Zn measurements were carried out byatomic absorption spectrometry (Shimadzu AA-670, Shi-madzu, Kyoto, Japan) according to the Societe Francaisede Biologie Clinique recommendations [25].

In the statistical analysis of the data, variables associatedwith AD were identified by the Mann–Withney–Wilcoxonrank sum test (non-Gaussian distribution), Student’s t-test(normal distribution) and x2 or Fisher’s exact test (dichot-omous variables). Next, multivariate analysis using logisticregression was carried out by including the variables thatshowed a significance of P < 0·05 in the preceding bivariateanalysis.

Results

There were no statistically significant differences(P ¼ 0·056) in age between AD patients [74·5 (2·3) years]and control subjects [70·3 (4·0) years]. There was asignificant difference (P ¼ 0·0136) in sex between ADpatients (36 women and 15 men) and control subjects(18 women and 22 men), because the control subjectswere the partners of the patients.

Genotype frequency distribution and alleles of apoE areshown in Table 1. The epsilon 4 apoE allele is morefrequent in the AD group than in the control group(P < 0·00001), and the apoE genotype distribution is sig-nificantly different in patients and control subjects. Inepsilon 4 apoE allele carriers, there was no difference insex (P ¼ 0·795) between AD patients (nine men and 25women) and control subjects (two men and seven women).Likewise, we found no difference (P ¼ 0·255) in agebetween AD patients [74·3 (2·9) years] and control sub-jects [70·8 (5·9) years]. Logistic regression analysis demon-strated that the presence of epsilon 4 apoE allele is anindependent risk factor adjusting for other parametersanalysed and increases the risk of AD by 6·67(OR ¼ 6·67; 95% CI ¼ 2·59–17·16).

Q 1999 Blackwell Science Ltd, European Journal of Clinical Investigation, 29, 637–642

Cu, Zn, insulin and apoE genotype in AD 639


The results of the biochemical parameters studied inpatients and control groups are shown in Table 2. Most ofthe parameters measured do not differ between AD andcontrol groups. However, serum Cu concentrations arehigher in AD patients, a group that has more women. Thebiochemical parameters analysed for patients and controlsubjects with at least one epsilon 4 apoE allele are shown inTable 3. Serum Cu, Zn and insulin concentrations and theserum insulin/glucose ratio are significantly higher in ADpatients than in control subjects with the epsilon 4 apoEallele. These four parameters were introduced in the multiplelogistic regression analysis. A 25mg dL¹1 increase in serumZn concentration is independently associated with astatistically significant increase in the relative risk of AD(OR ¼ 13·56; 95% CI ¼ 1·07–186·82; P ¼ 0·0443).

Finally, we analysed serum glucose and insulin concen-trations as a function of apoE alleles both in the patient andin the control groups. Serum insulin concentration is lowerin the carriers of at least one epsilon 4 apoE allele (Fig. 1),although it is only significant in the control group (Table 4).The same analysis was made for the variables related tolipid metabolism (cholesterol, A and B apolipoproteins andtriglycerides) without obtaining a significant result.

Discussion

Epsilon 4 apoE allele confers susceptibility to AD, but does

not provide sufficient sensitivity and specificity to be usedalone as a diagnostic test for AD. However, when used incombination with clinical criteria, it improves the specifi-city of the diagnosis [26,27]. In our country, as well as inother southern European countries, epsilon 4 apoE allelefrequency is lower than in northern Europe [28–30]. Thisfact, together with the design of our study, could explainthe relatively high odds ratio that we have obtained. How-ever, our value is about average for Caucasian populationsaccording to a recent meta-analysis [31].

There are a number of other factors that can interactwith the apoE gene. Among other possible factors, Zn hasbeen studied, because it is important in brain function andparticipates in Ab amyloid formation [5]. In our study,serum Zn concentrations that lie within the old people’sreference range are higher in patients with AD who carryone epsilon 4 apoE allele and confer to them a significantrisk, although we are not aware of data in which thepossible relationship between plasma Zn levels and apoEpolymorphisms in patients with AD are described. Bush etal. [5] have pointed out the unfavourable effect caused byZn supplementation in the diet of AD patients.

We have measured serum Cu levels for two reasons: bothZn and Cu are co-factors for the enzyme superoxidedismutase, and low cerebral amounts and high cerebrosp-inal fluid concentrations have been observed in AD[10,32]. We have observed a significant associationbetween serum Cu concentrations and AD. However, itcannot be demonstrated that Cu contributes independently

Table 1 Comparison of apolipoprotein E genotype and allele for case patients with AD vs. controlsubjects

Without AD (n ¼ 40) With AD (n ¼ 51) P-value

«2/«3 genotype (no/yes) 38/2 50/1 0·5802«2/«4 genotype (no/yes) 39/1 51/0 0·4396«3/«3 genotype (no/yes) 11/29 35/16 0·0001«3/«4 genotype (no/yes) 32/8 2/29 0·0004«4/«4 genotype (no/yes) 40/0 56/5 0·0647«4 allele (no/yes) 31/9 17/34 0·0000«3 allele (no/yes) 1/39 46/5 0·2241«2 allele (no/yes) 37/3 50/1 0·3162

Table 2 Serum levels for case patients with AD vs. control subjects


Apolipoprotein A (mg dL¹1)* 135·75 (129·46–142·03) 137·12 (130·98–143·26) 0·756Apolipoprotein B (mg dL¹1)† 119·50 (101·25–135·75) 111·00 (94·10–135·50) 0·151ApoA1/ApoB† 1·15 (0·89–1·43) 1·28 (0·92–1·44) 0·157Total cholesterol (mg dL¹1)* 216·35 (205·19–227·51) 216·94 (206·26–227·63) 0·939Triglycerides (mg dL¹1)† 99·50 (77·25–126·50) 110·00 (78·00–127·00) 0·576Glucose (mg dL¹1)† 89·00 (79·00–98·50) 93·00 (85·00–103·00) 0·065Insulin (mU dL¹1)† 5·50 (3·30–9·10) 7·20 (4·28–11·65) 0·121Insulin/glucose† 0·06 (0·04–0·11) 0·07 (0·04–0·14) 0·204Cu (mg dL¹1)* 97·69 (92·61–102·78) 105·67 (99·48–111·87) 0·048Zn (mg dL¹1)* 66·65 (62·12–71·19) 70·26 (67·30–73·23) 0·162

* Data are means (95% confidence interval).† Data are medians (25th and 75th percentiles).


to AD, perhaps because of the significantly higher numberof women in the AD patient group (women have higherreference values) and to the small size of the sample. Twomajor reasons may explain the last point.

1.The use of multiple tests of hypotheses on individualvariables increases the overall probability of finding at leastone statistically significant difference between two groupswhen, in fact, there are no differences (type I error). Thedifference observed in serum Cu concentration in theunivariate analysis may be due to this type I error.

2.The sample used to carry out the logistic regressionanalysis is not sufficiently large to detect the statisticallysignificant difference in serum Cu concentrations when, infact, it exists [33]. At the present time we are continuingour studies in order to increase the size of the sample.

Hyperinsulinaemia, greater resistance to insulin andlower sensitivity index to insulin in patients with AD havebeen described by some authors [16,17], whereas otherauthors have not found such changes [34]. In our study,patients with AD and an epsilon 4 apoE allele have greaterserum insulin concentration than control subjects with thesame allele, but we cannot discard its independence fromthe other variables analysed. In any case, in both groups(AD patients and control subjects) there was a lower insulinconcentration in epsilon 4 apoE carriers than in non-carriers, which was significant only in the control group.On the other hand, Berger et al. [35] have found greaterserum insulin concentrations in post-menopausal healthywomen epsilon 4 apoE allele carriers than in non-carriers.

The Bogalusa study [36] has demonstrated that epsilon4 apoE allele carriers have higher serum total cholesteroland low-density lipoprotein (LDL)-cholesterol concentra-tions but not serum insulin concentrations. Similar changeshave been observed in a biethnic population that includesHispanic subjects [37]. In old Hispanic subjects, especiallypost-menopausal women and octogenarians, the relation-ship between apoE polymorphisms and the lipid para-meters, total cholesterol, LDL-cholesterol and apoE,disappears [38,39]. Jarvik et al. [23] demonstrated a com-plex interaction between total cholesterol, epsilon 4, sexand risk of AD. They demonstrated that the three-wayapoE genotype, for total cholesterol and sex, and the apoE

genotype, for total cholesterol and age interaction, have thepotential to improve the prediction of AD. We haveexamined these interactions and their lower order terms(apoE genotype for total cholesterol, apoE genotype forsex, apoE genotype for age, and total cholesterol for age)using the strategy called the hierarchical backward elim-ination procedure. To contrast nested models we used alikelihood ratio test. In our data set, the three-way inter-action terms and the two-way products were not signifi-cant, perhaps because of the small size of the sample. Theseresults are different to those found by Jarvik et al. [23] inAD patients but are in accordance with those obtained in ageneral old population [37,38].


Table 3 Serum levels in «4 allele carrier patients: comparison of case patients with AD and control subjects


Apolipoprotein A (mg dL¹1)* 139·67 (120·71–158·62) 136·28 (130·05–142·51) 0·643Apolipoprotein B (mg dL¹1)† 116·00 (97·20–127·00) 116·00 (95·73–132·25) 0·777Apo A1/Apo B† 1·23 (0·92–1·50) 1·21 (0·90–1·44) 0·788Total cholesterol (mg dL¹1)* 219·33 (190·20–248·46) 222·68 (209·91–235·44) 0·810Triglycerides (mg dL¹1)† 96·00 (65·00–101·00) 102·50 (77·00–128·25) 0·165Glucose (mg dL¹1)† 84·00 (81·50–98·00) 93·00 (86·50–108·75) 0·204Insulin (mU dL¹1)† 4·10 (2·65–4·90) 6·40 (4·25–9·95) 0·018Insulin/glucose† 0·04 (0·02–0·06) 0·07 (0·04–0·10) 0·036Cu (mg dL¹1)* 91·71 (82·83–100·59) 107·48 (99·84–115·13) 0·045Zn (mg dL¹1)* 61·46 (53·19–69·74) 70·43 (66·76–74·09) 0·029

* Data are means (95% confidence interval).† Data are medians (25th and 75th percentiles).

Figure 1 Comparative boxplots of espisilon 4 association withinsulin and insulin/glucose ratio for case patients and controlsubjects. The median value is represented by the line drawn inthe interior of the box, and outliers are represented by asterisks.The proportion of observed data contained in the box representsthe middle 50% of the observation.

Cu, Zn, insulin and apoE genotype in AD 641


In summary, in our population, the epsilon 4 apoE alleleincreased the risk of Alzheimer disease by 6·4-fold. In ADpatients who carried one epsilon 4 apoE allele, Zn serumconcentrations were higher than in control subjects whocarried the same allele, and could act as an independentrisk factor. In AD, we have found a significant associationbetween higher serum Cu and insulin concentrations andthe presence of an epsilon 4 apoE allele. Serum insulinconcentration wass lower in the control subjects whocarried an epsilon 4 apoE allele than in the subjects whodid not carry it. In our sample of old people analysed, inboth patient and control groups, the association betweenepsilon 4 apoE allele and serum total cholesterol andapolipoprotein A and B concentrations was lost.

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Table 4 Association of «4 allele (one or two alleles) with glucose, insulin and insulin/glucose

P-value

Control group Non-carriers (n ¼ 31)* Carriers (n ¼ 9)*

Glucose (mg dL¹1) 89·00 (78·00–102·00) 84·00 (81·50–98·00) 0·7458Insulin (mU dL¹1) 6·15 (3·62–9·63) 4·10 (2·65–4·90) 0·0454Insulin/glucose 0·07 (0·04–0·12) 0·04 (0·02–0·06) 0·0532

Group with AD Non-carriers (n ¼ 17)* Carriers (n ¼ 34)*

Glucose (mg dL¹1) 94·00 (82·50–100·50) 93·00 (86·50–108·75) 0·4779Insulin (mU dL¹1) 8·00 (4·25–19·90) 6·40 (4·25–9·95) 0·3620Insulin/glucose 0·11 (0·05–0·21) 0·07 (0·04–0·10) 0·2006


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