447Clinical Science (2002) 102, 447–456 (Printed in Great Britain)

A European multicentre, placebo-controlledsupplementation study with α-tocopherol,carotene-rich palm oil, lutein or lycopene:

analysis of serum responses

Begon4 a OLMEDILLA*, Fernando GRANADO*, Susan SOUTHON†,Anthony J. A. WRIGHT†, Inmaculada BLANCO*, Enrique GIL-MARTINEZ*,Henk VAN DEN BERG‡, David THURNHAM§, Bernice CORRIDANs, Mridula CHOPRA§and Isabelle HININGER¶*Clı!nica Puerta de Hierro, 28035-Madrid, Spain, †Institute of Food Research, Colney, Norwich NR4 7UA, U.K., ‡TNO Nutritionand Food Research Institute, 3700-Zeist, The Netherlands, §University of Ulster at Coleraine, Coleraine BT52 1SA, NorthernIreland, U.K., sUniversity College Cork, Cork, Republic of Ireland, and ¶UFR des Sciences, Pharmaceutiques et Biologiques,38700-La Tronche, France

A B S T R A C T

Increased levels of oxidative stress have been implicated in tissue damage and the development

of chronic diseases, and dietary antioxidants may reduce the risk of oxidative tissue damage. As

part of a European multicentre project, several studies were undertaken with the aim of testing

whether the consumption of foods rich in carotenoids reduces oxidative damage to human tissue

components. We describe here the serum response of carotenoids and tocopherols upon

supplementation with carotenoids from natural extracts (α-carotene­β-carotene, lutein or

lycopene; 15 mg/day) and/or with α-tocopherol (100 mg/day) in a multicentre, placebo-

controlled intervention study in 400 healthy male and female volunteers, aged 25–45 years, from

five European regions (France, Northern Ireland, Republic of Ireland, The Netherlands and

Spain). Supplementation with α-tocopherol increased serum α-tocopherol levels, while

producing a marked decrease in serum γ-tocopherol. Supplementation with α- ­ β-carotene

(carotene-rich palm oil) resulted in 14-fold and 5-fold increases respectively in serum levels of

these carotenoids. Supplementation with lutein (from marigold extracts) elevated serum lutein

(approx. 5-fold), zeaxanthin (approx. doubled) and ketocarotenoids (although these were not

present in the supplement), whereas lycopene supplementation (from tomato paste) resulted in

a 2-fold increase in serum lycopene. The isomer distributions of β-carotene and lycopene

in serum remained constant regardless of the isomer composition in the capsules. In Spanish

volunteers, additional data showed that the serum response to carotenoid supplementation

reached a plateau after 4 weeks, and no significant side effects (except carotenodermia) or

changes in biochemical or haematological indices were observed throughout the study. This part

of the study describes dose–time responses, isomer distribution, subject variability and side

effects during supplementation with the major dietary carotenoids in healthy subjects.

Key words : β-carotene, humans, intervention study, lutein, lycopene, α-tocopherol.Abbreviations : HDL, high-density lipoprotein ; LDL, low-density lipoprotein.Correspondence: Dr B. Olmedilla, Unidad de Vitaminas, Seccio! n de Nutricio! n, Clı!nica Puerta de Hierro, C}San Martı!n dePorres 4, 28035-Madrid, Spain (e-mail bolmedilla!hpth.insalud.es).

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448 B. Olmedilla and others

INTRODUCTION

Nutritional epidemiology has shown that the dietaryphytochemicals, i.e. carotenoids and α-tocopherol, areconsistently associated with a reduced risk of the mostprevalent chronic diseases in developed countries (i.e.cardiovascular disease and cancer) [1,2]. Increased levelsof oxidative stress have been implicated in tissue damageand the development of chronic diseases such as cardio-vascular disease and cancers. It has been proposed thatdietary antioxidants (i.e. carotenoids) provide protectionagainst oxidative stress and thus reduce the risk ofoxidative tissue damage. However, intervention trialswith vitamin E have provided little evidence that thisantioxidant can reduce the incidence of or mortality dueto coronary heart disease by primary prevention, al-though it may decrease the recurrence of non-fatalmyocardial infarction, but not fatal cardiovascular events[1]. Also, in spite of the overwhelming epidemiologicalassociation found in observational studies implying aprotective effect of β-carotene against lung cancer, theresults from large intervention trials do not support apreventive role of β-carotene at pharmacological doses,and even indicated an adverse effect in smokers [2]. Theseresults have led to changes in the design of supplement-ation studies, such as the use of other dietary caro-tenoids, mixtures of phytochemicals and doses close tothose achievable by diet, to mimic more closely the intakedue to the consumption of fruits and vegetables. More-over, the diverse biological activities of α-tocopherol andcarotenoids [i.e. antioxidant, (pro)vitamin, gene regu-lation, inmunomodulation], along with the selectivedistribution of some carotenoids in tissues (i.e. lutein}zeaxanthin in retina), provide possible biological mechan-isms by which these compounds may decrease the risk ofchronic diseases. This provided a rationale to expand ourinterest beyond vitamin E and β-carotene and to focus onother, less well studied, dietary carotenoids (lutein andlycopene) from natural extracts, as well as doses that arenon-pharmacological but are close to those achievable bydiet.

As part of a European multicentre project (AAIRprogramme), several studies were undertaken with theaim of testing whether the consumption of foods rich incarotenoids reduces oxidative damage to human tissuecomponents. To our knowledge, this is the first multi-centre, placebo-controlled European supplementationstudy using dietary carotenoids other than β-carotene(lutein and lycopene), both alone and combined withα-tocopherol, in well characterized healthy subjects.The subjects came from five regions [3] with differentlifestyle and food habits [4]. Work from some ofthe studies within this project has been already published[3–11].

In this paper, we describe serum responses uponsupplementation with the major dietary carotenoids

alone and combined with α-tocopherol in a multicentre,placebo-controlled supplementation study in apparentlyhealthy male and female volunteers from five Europeanregions.

METHODS

Human subjectsA total of 200 men and 200 women aged 25–45 infive European regions (Grenoble, France ; Coleraine,Northern Ireland; Cork, Republic of Ireland; Zeist,The Netherlands ; and Madrid, Spain) were recruitedby non-probabilistic sampling (volunteers) accordingto inclusion criteria. Subjects were enrolled ineach centre by local advertisement in universitydepartments, and through general practitioners list,local newspapers and radio. All volunteers were free-living and were residents in the downtown areas orthe outskirts of the cities of the participating centres.Inclusion was based on biochemical and haematologicalcriteria and personal interview on dietary habits ; noadditional selection was done on the basis of thesocio-economical and cultural status of the subjects.Sample size was estimated by considering 80% statis-tical power to detect 20% differences at a 5%significance level. This required, in the whole study,a total of 100 subjects per treatment and 80 subjects percentre.

Inclusion criteria were as follows: male and femaleapparently healthy volunteers ; age 25–45 years ; non-smokers ; no consumption of vitamin and}or mineralsupplements (or these were stopped at least 1 monthbefore starting the study) ; not taking prescribed medi-cation; following habitual mixed diets characteristic ofeach region (no vegetarians ; no slimming diets) ; normalbiochemical and haematological profile : serum chol-esterol ! 6.8 mmol}l, serum triacylglycerol (triglyceride)! 2.3 mmol}l [or 2.3–3.0 mmol}l with a low-densitylipoprotein (LDL)}high-density lipoprotein (HDL) ratio! 5.0], serum retinol " 1.0 µmol}l and serum α-toco-pherol " 16 µmol}l. The volunteers, numbering 80 ateach centre (approx. 40 male and 40 female), were checkedin January–February for a normal fasting blood bio-chemical profile. After allowing for exclusions (those

Figure 1 Carotenoid supplementation protocolVit., vitamin; carot., carotene.

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449Carotenoid supplementation at five European centres

Table 1 Subject characteristics at baseline, by treatment groupValues are means³S.E.M., with ranges in parentheses. m, male ; f, female. Statistical analysis was carried out using the one-way ANOVA post hoc test.

SexCarotene-rich palm oil(m¯ 35, f¯ 45)

Lutein(m¯ 42, f¯ 48)

Lycopene(m¯ 52, f¯ 41)

Placebo(m¯ 46, f¯ 40) ANOVA

Age (years) m 34.0³1.2 34.2³1.0 32.5³0.8 33.1³1.0 nsf 32.6³1.0 34.3³1.0 36.4³1.1 24.2³1.1 ns

Body mass index (kg/m2) m 24.1³0.6 24.2³0.5 24.4³0.4 24.3³0.5 ns(18.8–39.6) (18.3–30.4) (19.3–34.9) (17.8–31.7)

f 22.9³0.4 23.4³0.5 22.2³0.3 22.9³0.5 ns(18.3–29.8) (18.5–33.0) (18.2–27.8) (18.8–30.5)

Cholesterol (mmol/l) m 4.78³0.11 5.20³0.14 4.95³0.13 4.99³0.12 ns(3.7–6.4) (3.5–6.6) (2.7–6.6) (3.2–6.8)

f 4.89³0.13 4.92³0.11 5.02³0.11 4.90³0.13 ns(3.3–6.5) (3.4–6.5) (3.3–6.3) (3.3–6.6)

Triacylglycerol (mmol/l) m 1.07³0.08 1.19³0.08 1.03³0.05 1.06³0.07 ns(0.44–2.91) (0.43–2.33) (0.35–2.22) (0.40–2.28)

f 0.94³0.05 0.94³0.05 0.86³0.05 0.76³0.05 P! 0.05*(0.43–1.94) (0.46–1.91) (0.44–1.75) (0.39–2.12)

* Although the variance ratio is significant, the post hoc Scheffe! test indicates no difference between individual means.

volunteers either not meeting the inclusion criteria orfailing to complete the study), the final numbers of validvolunteers were : France, 75 (38 male and 37 female) ; N.Ireland, 65 (32 male and 33 female) ; Republic of Ireland,73 (40 male and 33 female) ; The Netherlands, 72 (33 maleand 39 female) ; Spain, 64 (32 male and 32 female).Baseline concentrations of serum carotenoids andvitamins A, E and C were published previously [3].Volunteers were sorted randomly into four groups (ofeach sex) at each centre, and received supplements over aperiod of 20 weeks, as shown in Figure 1. Subjectcharacteristics according to treatment allocation areshown in Table 1.

The procedures used were in accordance with theDeclaration of Helsinki (1989) of the World MedicalAssociation. Approval from each local Ethical Com-mittee, and from the Ministry of Health in Spain, wasobtained, and all participants gave their written informedconsent.

Supplements and materialsLycopene-rich tomato extract was supplied byMakhteshim (Beer Sheva, Israel), α-tocopherol was a giftfrom R. P. Scherer North America (St. Petersburg, FL,U.S.A.), and ‘carotene-rich palm oil ’ and lutein-richmarigold extract were obtained from Quest (Unilever,Vlardingeen, The Netherlands). Capsules (500 mg fillweight) contained approx. 100 mg of α-tocopherol incorn oil, 15 mg of carotenoids (carotene-rich palm oil,lutein or lycopene) in corn oil, or corn oil only (placebo).Supplements were encapsulated by R. P. Scherer NorthAmerica. Capsule carotenoid content was analysed by

Table 2 HPLC analysis of capsulesOther carotenoids detected in lycopene capsules (not quantified) were phytoene,phytofluene and ξ-carotene. The carotene-rich palm oil capsule contained 30%α-carotene and 66.5% total β-carotene (40.4% trans-β-carotene and 19.6%9-cis-β-carotene). n.d., not determined.

Metabolite

Concentration (mg/capsule)

LycopeneCarotene-richpalm oil Lutein Vitamin E Placebo

α-Tocopherol 3.5* 1.75* 3.3* 98.1 0.06γ-Tocopherol 2.5 – – 0.6 0.23Lycopene (total) 13.3 – – – –trans-Lycopene 11.4 – – – –trans-Lutein – – 12.1* – –13/15-cis-Lutein – – 3.2* – –α-Carotene – 3.7 – – –β-Carotene (total) 1.4 8.2 – – –trans-β-Carotene n.d. 5.0 – – –9-cis-β-Carotene n.d. 2.4 – – –

Total carotenoids† 14.2 12.4 16.3* – –

* From saponified extracts.

† Assessed spectophotometrically.

HPLC and spectrophotometrically at our laboratory,giving the following composition (Table 2) : carotene-rich palm oil, 30% α-carotene and 66.5% total β-carotene(40.4% trans-β-carotene and 19.6% 9-cis-β-carotene) ;lutein, a mixture of trans- (79%) and cis- (21%) isomers ;lycopene, a mixture of trans- (85%) and cis- (15%)isomers, plus approx. 10% β-carotene.

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450 B. Olmedilla and others

The capsule of α- and β-carotene supplied an amountequivalent to that contained in 100 g of cooked carrots ;the amount of lutein was similar to that present in200 g of cooked spinach; and the amount of lyco-pene was equivalent to that provided by 600 g of rawtomato or 100 g of tomato paste (estimated usingdata from [12]). Based on the median intakes of eachcarotenoid assessed in the participating volunteers ineach of the five countries [4], capsules provided between3 and 13 times the usual intake of α-carotene (rangeof medians 0.29–1.23 mg}day), 1.5–3.0 times that ofβ-carotene (2.96–5.84 mg}day), 5–10 times that oflutein (1.56–3.25 mg}day) and 2–8 times that of lycopene(1.64–5.01 mg}day).

All-trans-β-carotene, lycopene, all-trans-α-carotene,all-trans-retinol, retinyl acetate, retinyl palmitate, D,L-α-tocopherol and γ-tocopherol standards were obtainedfrom Sigma Chemical Co. (St. Louis, MO, U.S.A.).Lutein, 15-cis-13-cis- and 9-cis-β-carotene, zeaxanthinand β-cryptoxanthin were gifts from Hoffmann-LaRoche (Basel, Switzerland). Dichloromethane and tetra-hydrofuran were purchased from Carlo Erba; methanol,ethanol, hexane and acetonitrile were obtained fromMerck.

Supplementation protocolVolunteers supplemented their habitual diet as shown inFigure 1. The capsules were uniform in size and colour,and were supplied every month, with some extra capsuleseach time. Compliance was checked by counting pillsreturned at each visit, and was above 80%.

Blood collection, sample preparation andanalysisFasting venous blood samples were taken from allvolunteers after 0 (baseline), 4, 16 and 20 weeks ofsupplementation, into plain blood tubes having noanticoagulant, and serum was separated by centrifugation(630 g ; 10 min). Additional samples were obtained fromthe Spanish cohort at 8 and 12 weeks, and also approx.3 months after stopping supplementation. Serum sampleswere stored (®70 °C or liquid nitrogen) at each centreuntil the end of the study, and were then air-courieredover solid CO

#to one of the participating centres

(Seccio! n de Nutricio! n, Clı!nica Puerta de Hierro, Madrid,Spain) for centralized analysis of retinol, α- and γ-toco-pherol and individual carotenoids. Samples werestored at ®70 °C and analysed within 18 months ofcollection to ensure stability of the compounds.

Serum samples were analysed randomly according totheir code number (supplementation unknown), andsamples with the same volunteer code number from eachcentre were analysed simultaneously. Code numberswere analysed in consecutive order, and one out of everysix samples was analysed in duplicate.

Sample extraction and HPLC analysis were carried outas described elsewhere [13]. Briefly, the chromatographicsystem consisted of a Spheri-5-ODS column (AppliedBiosystems, San Jose! , CA, U.S.A.) used with a gradientelution of acetonitrile}methanol (85:15, v}v) for 5 min toacetonitrile}dichloromethane}methanol (7 :2 :1, by vol.)for 20 min. Ammonium acetate (0.025 M) was added tomethanol. Detection of compounds was carried out usinga photodiode array detector (Model 996; Waters Associ-ates, Milford, MA, U.S.A.) set at 450 nm for carotenoids,325 nm for retinyl acetate and 294 nm for α- andγ-tocopherols. Under these conditions, the followingcompounds are resolved: retinol, retinyl acetate (internalstandard) and retinyl palmitate, lutein, zeaxanthin, α- andβ-cryptoxanthin, lycopene (all-trans-, 5}9-cis-, 13-cis-and 15-cis-), α-carotene and β-carotene (all-trans-,9-cis- and 13}15-cis-), and δ-, γ- and α-tocopherol. In ad-dition, ketocarotenoids, 13}15-cis-lutein, zeaxanthin,anhydrolutein I and II, γ-carotene, ξ-carotene, neuro-sporene, phytofluene, phytoene and lutein esters canbe also resolved.

The accuracy and precision of the analytical methodemployed was assessed periodically through our par-ticipation in the ‘Quality Assurance Programme’ con-ducted by the National Institute of Standards andTechnology (NIST; Gaithersburg, MD, U.S.A.). For theperiod during which analysis of samples from the studywas carried out, our performance was rated as 1 or 2 byNIST (exceptional or acceptable values). Within-day andbetween-day coefficients of variation (blind duplicatesfrom NIST) for the compounds of interest within thephysiological range were : 2% and 3% respectively forα-tocopherol ; 2% and 9% respectively for γ-tocopherol ;1% and 4% respectively for β-carotene; 4% and 14%respectively for lycopene; and 0% and 5% respectivelyfor lutein.

Statistical analysisTocopherol and carotenoid data were log

"!-transformed

to normalize distributions and}or to produce homo-geneous variances. The effects of α-tocopherol andcarotenoid supplementation on serum concentrationswere assessed by one-way ANOVA (‘within-subject ’).A comparison, at weeks 16 and 20, between the effects ofβ-carotene, lycopene, lutein and placebo supplemen-tation on serum carotenoid concentrations was con-ducted using standard two-way ANOVA, with variablesof treatment and gender (‘between-subject ’). Whenassessing whether serum α-tocopherol (week 4) orcarotenoid (week 16) concentrations following supple-mentation were related to pre-treatment baseline values(week 0), the statistical problem of ‘regression to themean’ was avoided by correlating change in concen-tration against the average of the baseline and post-treatment measurements [14].

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451Carotenoid supplementation at five European centres

RESULTS

Within gender, there were no differences in the baselinecharacteristics of the final volunteers in the four sup-plemental treatment groups, except for small (althoughstatistically significant) differences in female haemo-globin and erythrocyte concentrations (results notshown).

Supplementation with α-tocopherol (alone, or com-bined with carotenoids) resulted in an increase in serumα-tocopherol levels (approx. 35%; Figure 2), whileproducing a marked decrease in γ-tocopherol (Figure 3) :

Figure 2 Fasting serum α-tocopherol concentrations(µmol/l) at 0, 4, 16 and 20 weeksGeometric means and 95% confidence intervals are shown for male (n ¯ 175)and female (n ¯ 174) volunteers from the five countries (France, Spain,N. Ireland, Netherlands and Republic of Ireland). Inset : Spanish subset (includingdata at 8 and 12 weeks).

Figure 3 Fasting serum γ-tocopherol concentrations(µmol/l) at 0, 4, 16 and 20 weeksGeometric means and 95% confidence intervals are shown for male (n ¯ 175)and female (n ¯ 174) volunteers from the five countries (France, Spain,N. Ireland, Netherlands and Republic of Ireland). Inset : Spanish subset (includingdata at 8 and 12 weeks).

Figure 4 Fasting serum β-carotene concentrations (µmol/l)at 0, 4, 16 and 20 weeksGeometric means and 95% confidence intervals are shown for male (n ¯ 35) andfemale (n ¯ 45) volunteers from the five countries (France, Spain, N. Ireland,Netherlands and Republic of Ireland). Inset : Spanish subset (including data at 8and 12 weeks).

Figure 5 Fasting serum α-carotene concentrations (µmol/l)at 0, 4, 16 and 20 weeksGeometric means and 95% confidence intervals are shown for male (n ¯ 35) andfemale (n ¯ 45) volunteers from the five countries (France, Spain, N. Ireland,Netherlands and Republic of Ireland). Inset : Spanish subset (including data at 8and 12 weeks).

approx. 63% on average when supplied alone (weeks0–4), and approx. 55% when supplied combined withcarotenoids (weeks 17–20). Serum concentrations ofα- and γ-tocopherol had almost returned to baselinevalues 1 month after discontinuing α-tocopherol sup-plementation, as shown in the Spanish cohort (Figures 2and 3, insets) [15]. Supplementation with carotene-richpalm oil, containing β-carotene and α-carotene, resultedin 5-fold (Figure 4) and 14-fold (Figure 5) increasesrespectively in the serum levels of these carotenoids.Supplementation with lutein elevated serum lutein signifi-cantly (approx. 5-fold; Figure 6) and serum zeaxanthin(approx. double). Lycopene supplementation resultedin a 2-fold increase in serum lycopene (Figure 7), but

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452 B. Olmedilla and others

Figure 6 Fasting serum lutein concentrations (µmol/l) at 0,4, 16 and 20 weeksGeometric means and 95% confidence intervals are shown for male (n ¯ 42) andfemale (n ¯ 48) volunteers from the five countries (France, Spain, N. Ireland,Netherlands and Republic of Ireland). Inset : Spanish subset (including data at 8and 12 weeks).

Figure 7 Fasting serum total lycopene concentrations(µmol/l) at 0, 4, 16 and 20 weeksGeometric means and 95% confidence intervals are shown for male (n ¯ 52) andfemale (n ¯ 41) volunteers from the five countries (France, Spain, N. Ireland,Netherlands and Republic of Ireland). Inset : Spanish subset (including data at 8and 12 weeks).

Table 3 Markers of oxidative stress measured before and after supplementation with lutein, lycopene and β-carotene (aloneand with α-tocopherol) and the effects of supplementationSOD, superoxide dismutase ; Se-GSH-Px, erythrocyte Se-dependent glutathione peroxidase.

End point Markers of oxidative stress Response (effect of supplementation) Ref.

DNA oxidative damage 8-Oxo-deoxyguanosine in lymphocyte DNA ; Comet assay Lack of protective effect [7,8]Antioxidant status Glutathione (GSH and GSSG) ; erythrocyte antioxidant enzyme

activities (Cu-Zn SOD, Se-GSH-Px)No beneficial or adverse effects [11]

Plasma ascorbic acid Unchanged [11]Lipid oxidative damage Susceptibility of LDL to copper-induced oxidation Failed to enhance the resistance of LDL to oxidation or to

modify the LDL polyunsaturated/saturated fatty acid ratio[11]

Protein oxidative damage -SH groups Unchanged [11]

did not affect serum levels of β-carotene significantly,although a small increase (30–50%) in β-caroteneconcentration was observed. Upon supplementation,the proportions of cis}trans isomers of β-caroteneand lycopene in serum were not altered.

When α-tocopherol was administered together withcarotenes, lutein or lycopene (weeks 17–20), there was noeffect on serum concentrations of the supplementedcarotenoids. Conversely, there was no effect of caro-tenoid supplementation (carotene-rich palm oil, luteinand lycopene) on the rise in serum α-tocopherol con-centration which was seen after 4 weeks of supplemen-tation with α-tocopherol alone. Supplementation withplacebo did not modify baseline serum carotenoid ortocopherol concentrations.

Additional data from sample analysis at 8, 12 and 32weeks in Spanish volunteers indicated that the serumresponse to carotenoid supplementation (carotene-richpalm oil, lutein or lycopene) reached a plateau after4 weeks of supplementation (Figures 4–7, insets). At3 months after the end of the supplementation period(approx. week 32), serum α- and β-carotene levelsremained higher (3- and 2-fold respectively) than baselinelevels in the ‘palm oil carotene’-supplemented group,while serum lutein and lycopene levels had returned tobaseline in the lutein- and lycopene-supplementedgroups. In these subjects (Spanish cohort), no significantchanges were observed during the supplementationperiod in total cholesterol, HDL-cholesterol or LDL-cholesterol, or in the general biochemical and haemato-logical profile, at any time point assessed in any treatmentgroup (results not shown). In the Spanish cohort, minorside effects were reported by volunteers, the mostfrequent of which was the presence of carotenodermia ;this was reported by 95% of subjects supplemented withα-}β-carotene, 40% of those in the lutein group and 25%of those taking lycopene capsules.

The changes in serum α-tocopherol and carotenoidconcentrations after compared with before supplemen-tation [week 4 minus baseline (week 0) in the case ofα-tocopherol ; week 16 minus baseline (week 0) in thecase of the carotenoids], when correlated against the aver-

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453Carotenoid supplementation at five European centres

age of the two measurements, indicated that increasesin serum α-tocopherol (n ¯ 348; 175 males and 173females), β-carotene (n ¯ 80; 35 males and 45 females)and lutein (n ¯ 90; 42 males and 48 females) levels werepositively correlated with their pre-treatment serumconcentrations (r#¯ 0.247, P ! 0.001; r#¯ 0.834, P !0.001; r#¯ 0.852, P ! 0.001 respectively). However, theincrease in serum lycopene concentration (n ¯ 93; 52males and 41 females) was not associated with its pre-treatment value (r#¯ 0.028, P ¯ 0.107). Since there wereno significant gender differences for any of the asso-ciations, overall associations were assessed using pooleddata.

The changes in serum α-tocopherol and carotenoidconcentrations after compared with before supplemen-tation [week 4 minus baseline (week 0) in the case ofα-tocopherol ; week 16 minus baseline (week 0) in thecase of the carotenoids] showed either no association(α-tocopherol, lycopene) or little association (β-carotene,r#¯ 0.06; lutein, r#¯ 0.07) with baseline cholesterolvalues.

The effects of the lutein, lycopene and β-carotene(alone and plus α-tocopherol) supplementation on severalmarkers of oxidative stress selected to test the hypothesisof this project are summarized in Table 3 [7,8,11].

DISCUSSION

In the present study, we used as supplements naturalextracts of three major serum carotenoids, includinglutein and lycopene (which have not been widely testedin previous studies), in amounts that would be achievableby diet. The study was conducted using identical timeprotocols and capsule preparations and very similardoses, allowing relative comparisons between each caro-tenoid treatment. The study was performed in wellcharacterized subjects from five European countries whodisplayed different dietary habits [4]. Treatment allo-cation at each centre and amounts used reduced con-siderably potential confounding effects due to dietarymodifications induced by participation and seasonaldietary changes during enrolment in the study. Inaddition, the centralization, randomization and qualitycontrol of analysis will have eliminated inter-laboratoryanalytical bias and improved the reliability of the results.

Responses to carotenoids may be linked to factorsaffecting their bioavailability and which are related bothto the carotenoid supplied (i.e. chemical structure, pro-vitamin A activity, dose, preferential uptake by tissues)and to the characteristics of the subject (i.e. nutritionalstatus, absorption modifiers, genetic factors). Compara-tively, based on the carotenoid content of the capsules,α-carotene was the carotenoid that showed the greatestrelative response in serum, followed by β-carotene, lutein

and lycopene. Changes in the serum carotenoid profilewere observed mostly in the lutein arm of the study.Upon lutein supplementation, an increase in the putativeoxidation products of lutein}zeaxanthin (referred to asketocarotenoids) [15–17] was also observed. Theirabsence from the supplement and during samplepreparation, and their parallel increment in the lutein-supplemented group only, suggests that these compoundsare formed in vivo. In accordance with previous obser-vations in Spanish participants, in subjects reachingrelatively high serum lutein levels (" 1.05 µmol}l) wealso detected the presence of a chromatographic peak thatwas tentatively identified as lutein monopalmitate. Thispeak may be indicative of a ‘ceiling effect ’ on, orsaturation of, the transport capacity of lutein, which maybe re-esterified in vivo when it is supplied in excess ofnormal dietary intake [18].

Supplementation with lycopene from tomato extractprovoked a 2-fold increase in serum lycopene, the lowestincrement compared with that with the other carotenoids.The lower response of serum lycopene may have beencaused by the high content of other carotenes in thelycopene supplement (i.e. phytofluene, phytoene) or bymore efficient transfer to tissues. Lycopene capsulesalso supplied β-carotene and, proportional to the amountsupplied, the change in serum β-carotene was com-paratively higher than that observed for lycopene, anobservation that could be related to an enhancement ofβ-carotene bioavailability by lycopene [19]. However,because this change in serum β-carotene was not signifi-cantly different from concentrations in the placebogroup, this effect could not be ascribed directly to thesupplementation.

Carotene-rich palm oil capsules contained approx.70% β-carotene and 30% α-carotene, but the serumresponse was much higher for α-carotene (14-fold) thanfor β-carotene (5-fold). The different increments may berelated to the relatively lower baseline concentration ofα-carotene, but also may be due partly to preferentialabsorption and}or conversion into retinol, interactionsduring absorption, and differential storage and}or uptakefrom tissues. Serum concentrations of 9-cis-β-carotenedid not change, even though this isomer accounted for30% of total β-carotene supplied by the capsules.According to several authors [20–22], 9-cis-β-carotene isabsorbed similarly to the trans-isomer, and thus the lackof an increase in serum β-carotene in the present studymay have been due to its isomerization during absorption[21].

Carotenoid absorption from single large (30 mg) oraldoses has been reported to be low (3.50%, 2.63% and2.56% for β-carotene, lycopene and lutein respectively),at least on the basis of appearance in the plasmatriacylglycerol-rich lipoprotein fraction [6], althoughhigher values (approx. 15%) have been also reported[23]. However, a lack of a change in the plasma carotenoid

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454 B. Olmedilla and others

concentration may not necessarily imply poor absorp-tion. In the case of ‘physiological ’ doses (10 mg) ofβ-carotene, results from mass balance studies using hu-man ileostomy volunteers [20] have suggested that meanabsorption can exceed 90% in the absence of signifi-cant alterations in the plasma carotenoid concentration.

In the present study, changes in both β-carotene andlutein concentrations were strongly and positively associ-ated with initial pre-supplement plasma concentrations.There was no association between the increase in theserum lycopene concentration after supplementation andthe initial baseline concentration. This may be becauseof the much smaller elevation in the fasting plasma con-centration following lycopene supplementation, a factorthat may have more to do with rapid plasma clearance toan unobservable pool than with poor absorption. This issupported by the observation that the appearance oflycopene in the plasma triacylglycerol-rich lipoproteinfraction, after a single oral load, is of a similar speed andmagnitude to that of an equivalent dose of β-carotene[24].

All participants in the present study showed serumincrements (although to a different extent) of the caro-tenoid supplied, suggesting that the protocol used waseffective in increasing serum concentrations in (target)control subjects, while being without significant sideeffects. Our results are also consistent with the conceptthat each carotenoid shows individual metabolism,and that several (ill-understood) factors influence the ab-sorption, transport, serum clearance and}or tissue de-position and utilization of each carotenoid within thebody. In agreement with previous observations in smallergroups of volunteers [25–27], the proportions of cis}transisomers of β-carotene and lycopene in serum were notaltered after supplementation, indicating that the serumisomer distribution is independent of that in the supple-ment}diet. Also, all carotenoids supplied (lutein, ly-copene, α-carotene and β-carotene) showed a plateauconcentration in serum at 4 weeks of supplementation(Spanish cohort), which was mantained throughout therest of the study. However, it is interesting to notethe different frequencies of carotenodermia observedin the Spanish participants (95% with α­β-carotene,40% with lutein and 25% with lycopene), suggestingthat all carotenoids are not equally deposited in theskin, and}or that other tissues are primary targets forlutein and lycopene deposition.

Supplementation with α-tocopherol increased serumvitamin E status (α-tocopherol and α-tocopherol}cholesterol ratio), but negatively affected other com-pounds that potentially protect against cardiovasculardisease (i.e. γ-tocopherol), as described previously[28–31]. This effect was almost completely reversedwithin 1 month of discontinuing the treatment (asobserved in the Spanish cohort ; insets of Figures 2 and 3).High concentrations of α-tocopherol have been as-

sociated with a lower risk of cardiovascular diseases[1,32], and ‘optimum levels ’ have been proposed thatwill achieve a protective effect [33,34]. However, theα-tocopherol concentration achievable in serum is de-termined in part by serum lipid concentrations. In thepresent study, the values of the α-tocopherol}cholesterolratio achieved after 1 month of α-tocopherol supplemen-tation (results not shown) were very similar betweensubjects. Because of the design of the protocol, it is notpossible to assess if these reflect a ceiling effect orwhether they could be surpassed upon continous sup-plementation in normolipaemic subjects. Also, whencarotenoids were supplied together with α-tocopherol,there were no changes in serum levels of the supple-mented carotenoid or alterations in the serum increase inα-tocopherol. Thus there is no clear evidence of asynergistic effect between carotenoids and α-tocopherol.

If emphasis is placed on achieving tissue concentrationsof carotenoids present in those populations with a lowerrisk of chronic disease via an increase in fruit andvegetable intake, this will also have the effect of increasingthe intake of all other potentially beneficial compoundspresent in these foods, and will avoid antagonistic effectson other potentially beneficial agents (i.e. γ-tocopherol).Although the association between high levels of fruit andvegetable consumption and a lower risk of chronic diseaseis extremely strong, it is highly likely that there aremultiple reasons for this association, of which thecarotenoid content of the diet may only be one small part.Consistent with this assumption are other results fromthis European project, showing a decrease in LDLoxidability in a group of smokers consuming foods withcarotenoid levels similar to those in the capsules providedin the present study [5], and the unexpected lack of aneffect on oxidative stress biomarkers in the subjectsdescribed in the present study after supplementation withcarotenoid capsules (Table 3). However, it should benoted that the importance of individual carotenoids, orother phytochemicals present in carotenoid-rich foods,in diet–health relationships may be distinct in differentpopulations, depending on the mixture of fruits andvegetables consumed and other lifestyle factors con-tributing to health outcome. Although the amounts ofcarotenoids supplied in the present study may beachievable by dietary means, they are probably notpresent in the diet on a daily basis in European countries[4,10,35–37]. Most importantly, it should be noted thatthe bioavailabity of carotenoids from dietary sources islower than from supplements [38,39]. However, the‘ceiling’ effect in serum at 1 month observed inthe present study suggests that cumulative lower doses(i.e. dietary ranges) consumed on a long-term basis maybe used succesfully to reach, and maintain, high serumlevels of carotenoids. Thus increasing fruit and vegetableconsumption can be considered as a long-term, sustain-able and safe approach to achieving increased serum and

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455Carotenoid supplementation at five European centres

tissue levels of these compounds (or other biologicallyactive associated phytochemicals), which are inverselycorrelated with DNA damage [8] and associated epi-demiologically with potential health benefits in humans.

ACKNOWLEDGMENTS

The participation and enthusiasm of all volunteers iswarmly acknowledged. We acknowledge the con-tribution made by researchers at study centres in therecruitment of subjects and the collection and processingof blood samples ; in particular, Teresa Motilla and PilarMartı!nez (Spain). We thank Dr E. Rojas-Hidalgo for hisenthusiastic support, and Dr T. Gea Malpica. We alsothank Anne Ma Roussel (France), Maura O’Neill (N.Ireland), Yvonne Carroll, Siobha! n Higgins and Sine! adMcCarthy (Republic of Ireland) and Frank van Schaik(The Netherlands) for supervised collection of thesamples. We are grateful to Quest, Makhteshim and R. P.Scherer for supplying and encapsulating the supplements.

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Received 18 May 2001/19 September 2001; accepted 20 November 2001

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