Carotenoids in young and elderly healthy humans: dietary
Transcription
Carotenoids in young and elderly healthy humans: dietary
European Journal of Clinical Nutrition (1999) 53, 644±653 ß 1999 Stockton Press. All rights reserved 0954±3007/99 $12.00 http://www.stockton-press.co.uk/ejcn Carotenoids in young and elderly healthy humans: dietary intakes, biochemical status and diet-plasma relationships YL Carroll1, BM Corridan1 and PA Morrissey1* 1 Nutritional Sciences, Department of Food Science and Technology, University College Cork, Cork, Ireland Objective: To determine dietary carotenoid concentrations using an established and newly developed food frequency questionnaire (FFQ) method, to determine plasma carotenoid concentrations and to determine the relationship between these dietary and plasma variables in 24 ± 45 y and 65 y groups. Design: Descriptive assessment of (FFQ), 7 ± d estimated records, and plasma carotenoids and their relationships in 24 ± 45 y and 65 y groups. Setting: Free living urban adults in Ireland. Subjects: Sixty-four volunteers aged 24 ± 45 y and 54 volunteers aged 65 y. Results: b-carotene was the predominant plasma carotenoid, but older groups had lower plasma concentrations of several carotenoids compared to younger groups (P < 0.005). b-carotene and lycopene were the major dietary carotenoids reported by estimated records and FFQ. Several estimated record and plasma carotenoid concentrations were positively associated in younger groups but not in older groups. FFQ overestimated dietary carotenoids relative to estimated records (P 0.05), generally did not re¯ect estimated record carotenoid concentrations and showed positive associations with plasma carotenoids only in older men. Neither of the dietary methods revealed a positive association between plasma and dietary b-carotene concentrations, whereas b-cryptoxanthin was strongly associated. Conclusions: Dietary and plasma concentrations of individual carotenoids are documented in young and elderly groups of a European country. Estimated record data reveals positive associations between diet and plasma carotenoids in younger, but not elderly groups. Further work examining diet-plasma relationship in older groups and developing a common FFQ suitable for use in several European countries is required. Sponsorship: Commission of the European Communities: AAIR Project (AIR2-CT93-0888). Descriptors: carotenoids; dietary assessment; elderly; biomarkers Introduction High fruit and vegetable consumption is associated with reduced incidence of several chronic diseases. Evidence for a protective effect of greater vegetable consumption is consistent for cancers of the stomach, oesophagus, lung, oral cavity and pharynx, endometrium, pancreas and colon (Steinmetz & Potter, 1996). Mortality from coronary heart disease, has been reported by several prospective studies, to be inversely associated with consumption of fruit and vegetables (Knekt et al, 1994; Key et al, 1996). Biological concentrations of antioxidants derived from fruits and vegetables are also inversely associated with risk of coronary heart disease. An inverse association between serum carotenoids and risk of coronary heart disease, was reported in a prospective study (Morris et al, 1994), while a nested case-control study, concluded that low serum levels of carotenoids, were associated with an increased risk of subsequent myocardial infarction among smokers (Street et al, 1994). A cross-sectional, multicentre study, also reported an inverse association between high adipose *Correspondence: Prof PA Morrissey, Nutritional Sciences, Department of Food Science and Technology, University College Cork, Cork, Ireland. Received 7 October 1997; revised 18 February 1999; accepted 26 February 1999 tissue b-carotene concentrations and risk of myocardial infarction (Kardinaal et al, 1993). Data from several studies support a role for carotenoids in protection against chronic disease in elderly as well as middle-aged groups. A prospective study of residents of a retirement community, reported reduced risks of colon and all sites combined cancers, with increasing intake of vegetables and fruits in elderly women (Shibata et al, 1992). Another prospective study on cardiovascular disease mortality, reported that the bene®cial effects of increased fruit and vegetable consumption are apparent in the elderly (Gaziano et al, 1995). Age related macular degeneration is another condition which has been shown to be inversely associated with consumption of foods rich in carotenoids (Seddon et al, 1994). In view of the inverse relationships between carotenoids and disease, both dietary and biochemical status of carotenoids are individually worthy of further study. Little published data is available on tissue carotenoid concentrations in Irish population groups. Research studies generally tend to exclude those over the age of 65y and studies addressing this age group are warranted. Dietary data on consumption of carotenoids were, in the past, usually expressed as b-carotene, b-carotene equivalents or retinol equivalents. More recently, an extensive carotenoid food composition database listing values for the ®ve major carotenoids occurring in fruits, vegetables and multi-component foods, has been compiled in the USA (Mangels et Carotenoids in diet and plasma YL Carroll et al al, 1993; Chug-Ahuja et al, 1993). In Europe, analysis of dietary carotenoids, other than b-carotene, is dif®cult because many national food composition databases only provide limited values for individual carotenoids (Souci et al, 1987; Holland et al, 1991b). However, analysis of foods in Finland, Spain, the Netherlands and UK has generated an extensive body of published data on the carotenoid composition of foods consumed in Europe (Heinonen et al, 1988, 1989; Ollilainen et al, 1988, 1989; Granado et al, 1992; Olmedilla et al, 1993; Vollebregt & Feskens, 1993; Hart & Scott, 1995). As part in a multi-centre European study, a common carotenoid food composition database, based mainly on data from the above sources was agreed between participants. This database, providing data on lycopene, a-carotene, b-carotene, b-cryptoxanthin and lutein zeaxanthin concentrations in 106 food descriptors, was used in the present study. Biomarkers of dietary intakes of nutrients are becoming increasingly popular in nutrition research. The ability of plasma to act as a biomarker of dietary intakes of individual carotenoids is of interest. Several intervention studies have shown that plasma carotenoids are responsive to increased and reduced intakes of fruits and vegetables (Brown et al, 1989; Micozzi et al, 1992; Rock et al, 1992; Fuller et al, 1993; Yeum et al, 1996). However, data from the USA indicate that under usual dietary conditions, the associations between dietary and plasma carotenoid concentrations are moderate and generally do not exceed correlation coef®cients more than r 0.5 (Ascherio et al, 1992; Forman et al, 1993). As far as we are aware, in Europe, only one published study has examined the association between individual carotenoids in diet and serum and was limited to a group of women aged 50 ± 65 y (Scott et al, 1996). As participants in a multi-centre study, leading on to an intervention phase, volunteers were required to complete an especially developed, but not previously evaluated FFQ. The FFQ which was devised by reference to existing FFQ in use at participating centres, necessitated inclusion of a suf®ciently wide range of foods to facilitate its use in each of the ®ve participating European countries. The primary aim of this present study was to assess dietary concentrations of individual carotenoids using an established dietary method, to assess plasma concentrations of individual carotenoids and to examine the associations between these dietary and plasma carotenoid concentrations in younger and older groups of Irish adults. A secondary aim of this present study was to evaluate the performance of the FFQ, by reference to an established dietary method and by reference to plasma carotenoid concentrations, in younger and older groups of Irish volunteers. Methods As part of a multi-centre European study, 69 healthy volunteers aged between 25 ± 45y and 57 healthy volunteers 65 y were recruited for this study and two follow-on supplementation studies. The studies on the younger and older groups were carried out ten months apart. All volunteers were screened by a medical history, physical examination, and biochemical and haematological pro®le. Subjects had normal lipid metabolism as indicated by fasting serum cholesterol and triacylglycerol concentrations. Body Mass Index (BMI) ranged from 19 ± 31 kg=m2. Subjects were not adhering to any special diets and were non-smokers. Informed consent was given by all volunteers. All procedures were approved by the Clinical Research Ethics Committee at University College, Cork. Following screening, eligible subjects completed a 7d estimated record. The FFQ was administered on the day following completion of the estimated records. Fasting blood samples were obtained at screening and on the day after completing the 7d estimated record. Biochemical analysis of plasma carotenoids Samples were protected from natural light. Plasma was immediately separated and stored at 7 70 C until analysed. Plasma carotenoids, tocopherols and retinol were extracted from 0.2mL plasma with 0.2 mL 10 mmol SDS solution, 0.4mL ethanol and triplicate extractions with 0.4 mL n-hexane (0.05% w=v butylated hydroxy toluene (BHT)) (Burton et al, 1985). The hexane extracts were dried under nitrogen and reconstituted in 20mL dichloromethane followed by 180 mL acetonitrile-methanol (75:20 by volume). A 50mL sample was injected onto a temperature controlled (25 C) reverse phase HPLC system (Scott & Hart, 1993). The column system included Spherisorb ODS2 Guard Cartridges (Alltech, Lancashire, UK) in line with Spherisorb ODS-2, 5 mm, 150 mm64.5 mm pre-column (Alltech, Lancashire, UK) and Vydac 201TP54 250 mm64.5mm analytical column (Separations Group, California, USA). The mobile phase was acetonitrilemethanol-dichloromethane (75:20:5 by volume) containing 0.05% v=v triethylamine and using 0.05 mol ammonium acetate in the methanol component of the mobile phase. Detection involved two on-line UV=VIS detectors (Shimatzu, Japan), with all carotenoids detected at 450 nm, while the UV detector was programmed to change from wavelength 325 ± 295 nm during the analysis, in order to detect retinol, g-tocopherol and a-tocopherol. The data were processed using Millennium 2.1 software data processing package (Waters Corporation, Milford, USA). Six carotenoids including lutein, zeaxanthin, b-cryptoxanthin, all-trans lycopene, a-carotene and all-trans b-carotene were quanti®ed by reference to ®ve point calibration curves. Total plasma carotenoids were computed by summing the ®ve individual carotenoids. For each subject, triplicate plasma samples from screening and from the day following completion of the estimated records were run on the same day. Plasma b-carotene concentrations in samples from the Fat soluble Vitamin Quality Assurance Program deviated in the range 3 ± 8% from the mean of the analyte concentration, in the programme conducted by the National Institute of Standards and Technology (NIST, Gaithesburg, USA). By the criteria of this programme, our laboratories performance is evaluated as acceptable relative to the current state of the practice for measurement of b-carotene. Dietary analysis Volunteers completed seven consecutive days of estimated records. Detailed written and verbal instructions on how to record the amount of food and drink consumed during the 7 d were given to volunteers. Volunteers were visited four times during the recording period and any discrepancies corrected. The weights of all foods were calculated (g=d), with the aid of a photographic atlas and standard portion sizes (Ministry of Agriculture, Fisheries and Food, 1993). FFQ relating to eating habits over the previous three months was also completed by all volunteers. The FFQ was comprised of a list of 110 food items and was divided into 645 Carotenoids in diet and plasma YL Carroll et al 646 seven sections: green, red-orange, white-yellow coloured vegetables, fruits, processed foods, dairy products, other foods. Eleven options for frequency of consumption including; 1, 2, 3, 4, 5, 6, 7 (times per week), fortnightly, monthly, seldom and never, were given in all sections except the dairy and other food sections. Foods were quanti®ed in the most appropriate units, for example, slices of cucumber, tablespoons of baked beans, with reference to standard portion sizes (Ministry of Agriculture, Fisheries and Food, 1993). Intake of foods from the FFQ was calculated in g=d. The estimated records and FFQ from both studies were coded by one person. Dietary carotenoids were quanti®ed by reference to a comprehensive database which was incorporated into a computerised dietary analysis programme, Comp-Eat (Nutrition Systems, London, UK). Compiled as part of the larger multi-centre study, this carotenoid food composition database was based on published values, many of which were established in laboratories of participants in this multi-centre study (Granado et al, 1992; Olmedilla et al, 1993; Hart & Scott, 1995). Most of the data was derived from Finnish, Dutch, Spanish and UK sources (Heinonen et al, 1988, 1989; Ollilainen et al, 1988, 1989; Granado et al, 1992; Olmedilla et al, 1993; Vollebregt & Feskens 1993; Hart & Scott, 1995), with reference to data from other sources limited to a small number of food items that had not been analysed in the above publications (Holland et al, 1991; Tonucci et al, 1995; Burlingame, 1993; Mangels et al, 1993). Values for ®ve categories of carotenoids including lutein zeaxanthin, b-carotene, a-carotene, lycopene and b-cryptoxanthin were included in the database. Statistical analysis Sixty four subjects in the younger and 54 subjects in the older groups, completed the studies. Five subjects in the younger groups were excluded from analysis because FFQ were not completed. Three subjects in the older group were excluded because they did not complete either the diet record or the FFQ. Statistical analysis was performed with Datadesk 4, 2 statistical software package (Data Description Inc., New York, USA) and SPSS (SPSS Inc, Chicago, USA) statistical software package (Norusis SPSS Inc.). The effects of age group and dietary methods on dietary carotenoid intakes were examined by ANOVA with twoway interaction. Differences between estimated record and FFQ carotenoid intakes in younger and older males and in younger and older females were examined by post-hoc Bonferroni tests. The Mann ± Whitney U test was used to examine differences between older and younger groups Table 1 carotenoid intakes expressed as nutrient densities. The Mann ± Whitney U test was also used to examine differences in plasma carotenoid concentrations in younger and older groups. Differences between male and female estimated record carotenoid intakes (expressed as nutrient densities) and differences in plasma carotenoids were examined by the Mann ± Whitney U test. Spearman's rank correlation coef®cients were calculated to investigate the associations between dietary and plasma data and between the two dietary methods. Plasma carotenoids were also adjusted for BMI, plasma triglycerides and plasma cholesterol by General Linear Models. Adjusting for these variables did not improve correlations and the crude correlations are presented. The ability of the FFQ to correctly classify volunteers into the highest and lowest tertiles of the estimated record carotenoid distribution was examined. The ability of each of the dietary methods to correctly classify volunteers into the highest and lowest tertiles of the plasma carotenoid distribution was also examined. The Cochran Q test was used to determine whether the percentage of volunteers correctly classi®ed by estimated records into the same tertile of the plasma carotenoid distributions were signi®cantly different from the percentage of volunteers correctly classi®ed by FFQ. The extent of misclassi®cation into opposite tertiles, was also recorded and examined by the Cochran Q test. The within- and between-person coef®cient of variation of individual carotenoids in plasma and estimated records were calculated by reference to values of within- and between person variances, obtained from repeated measures ANOVA. Results Characteristics of the study populations are shown in Table 1. The average age of volunteers was 31 y in the younger groups and 70 y in the older groups. All volunteers were in good health and had biochemical and haematological values within the reference range. Mean dietary carotenoid intakes of males and females, assessed by estimated records and FFQ, are presented in Table 2. In both age groups, the predominant dietary carotenoids reported by both estimated records and FFQ were b-carotene and lycopene. There was no signi®cant difference between males and females in absolute carotenoid intakes, assessed by either dietary method, in any of the age groups. The effects of age group and dietary method on dietary carotenoid intake in male and females were evaluated by ANOVA with two-way interactions. In males, dietary method had a signi®cant effect on the dietary Characteristics of groups aged 24 ± 45 y and 65 y 24 ± 45 y Males (n 32) Age (y) BMI (kg=m2) Plasma lipids (mmol=L) Total cholesterol HDL cholesterol Triacylglycerols Haemoglobin (g=dL) Albumin (g=L) 65 y Females (n 32) Males (n 25) Females (n 29) Mean s.d. Mean s.d. Mean s.d. Mean s.d. 31 24 6 2 32 24 7 3 70 26 4 2 71 26 4 3 4.7 1.3 1.1 15.4 46.9 0.9 0.3 0.4 0.7 2.3 4.8 1.7 0.9 13.3 44.9 0.7 0.4 0.3 0.6 2.2 5.3 1.1 1.2 14.7 42.8 0.9 0.3 0.6 0.8 2.2 5.9 1.3 1.2 13.3 42.6 0.7 0.3 0.4 0.8 1.9 Carotenoids in diet and plasma YL Carroll et al Table 2 647 Dietary carotenoid intakes (mg=d) assessed by estimated records and FFQ in groups of men and women aged 24 ± 45 y and 65y Younger Males (n 32) Estimate records a-carotene b-carotene b-cryptoxanthin Lycopene Lutein zeaxanathin Total carotenoids Food frequency questionnaire a-carotene b-carotene b-cryptoxanthin Lycopene Lutein zeaxanathin Total carotenoids Older Males (n 25) Younger Females (n 32) Mean s.d. Mean s.d. Mean 775 2921 189 3198 1005 8089 656 1797 288 4129 675 5002 812 3099 116 2092 778 6899 651 2060 153 1809 320 4624 771 2850 165 2877 943 7605 2285 8077 471 7642 2323 20800 1470* 4463* 466* 6262* 1436* 9563* 1217 5277 295 2026 1877 10693 1375 4607 346 2211 1462* 8475 2426 8795 727 8045 2615 22608 s.d. Older Females (n 29) Mean s.d. 598 1539 157 2066 385 3007 973 3358 108 2285 1015 7739 530 1447 160 2367 463 3245 1872* 6022* 803* 9393* 2120* 16368* 1223 5496 317 4615 2120 13773 797 3035* 338 5368 1277* 8823* *P 0.05; signi®cantly different from estimated records by post hoc Bonferroni tests. intake of all carotenoids examined. Age group had a signi®cant effect on the dietary intake of the following: a-carotene (P 0.016), b-carotene (P 0.05), lycopene (P 0.007) and total carotenoids (P 0.001). There was also a signi®cant interaction between age group and dietary method for these carotenoids. In females, dietary method had a signi®cant effect on the dietary intake of all carotenoids examined. Age group had a signi®cant effect on the dietary intake of the following: a-carotene (P 0.014), bcarotene (P 0.034), b-cryptoxanthin (P 0.006) and total carotenoids (P 0.015). There was also a signi®cant interaction between age group and dietary method for these carotenoids. In the younger group, FFQ gave approximately 3-fold higher estimates of carotenoid intakes than estimated records which the post-hoc Bonferroni test found to be signi®cant (Table 2). In the older group, FFQ carotenoid intakes were signi®cantly higher than estimated records for all carotenoids except a-carotene, b-cryptoxanthin and lycopene. The most consistent association between FFQ and estimated records was observed for b-cryptoxanthin (Table 3). Spearman correlation coef®cients ranged from r 0.55 ± 0.62 (P 0.001) and 59 ± 75% of volunteers were classi®ed into the same tertiles of the estimated record b-cryptoxanthin distribution. With the exception of younger males, there was little association between intakes of most carotenoids, assessed by estimated records and FFQ, in the Table 3 Effect of age and dietary method on dietary carotenoid intake in groups of men and women aged 24 ± 45 y and 65 y Males a-carotene b-carotene b-cryptoxanthin Lycopene Lutein zeaxanthin Total carotenoids Females a-carotene b-carotene b-cryptoxanthin Lycopene Lutein zeaxanthin Total carotenoids Effect of age group P Effect of dietary method P Age group6 dietary method P 0.016 0.048 0.055 0.001 0.106 0.001 0.001 0.001 0.001 0.007 0.001 0.001 0.010 0.025 0.418 0.005 0.597 0.002 0.014 0.034 0.006 0.054 0.368 0.015 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.004 0.036 0.172 0.227 0.012 other groups. The percentage of volunteers classi®ed into the same tertiles of the estimated record carotenoid distributions also tended to be higher in younger men than in other groups. A high proportion of volunteers (6 ± 36%) were misclassi®ed by FFQ into opposite tertiles of the estimated records carotenoid distributions. In younger women, there was more misclassi®cation by FFQ into opposite (36%), than correct classi®cation (32%) into the same tertiles of the estimated record b-carotene distribution. In younger volunteers, there was no signi®cant difference between men and women in estimated record carotenoid intakes expressed in terms of nutrient density (Table 4). In the older groups, the intakes of a-carotene (P 0.05), lutein zeaxanthin (P 0.01) and total carotenoids (P 0.05), expressed as nutrient densities, were higher in women than in men (Table 5). The intakes of carotenoids, assessed by estimated records and expressed as nutrient densities, did not differ signi®cantly between the older and younger groups. The plasma concentrations at screening and baseline were averaged and the mean plasma concentrations are shown in Table 6. Spearman correlation coef®cients in the range r 0.63 ± 0.91 were observed between repeat screening and baseline plasma carotenoid concentrations in both age groups. In the younger groups, plasma carotenoids were ranked in the order: b-carotene, lycopene and lutein in males, whereas in women the order was: b-carotene, bcryptoxanthin and lycopene. In the older groups, b-carotene, lutein and a-carotene were the predominant plasma carotenoids in men and women. There were no signi®cant differences observed between plasma carotenoid concentrations in men and women in any of the age groups. With the exception of a-carotene and b-carotene, the younger men and women had higher plasma carotenoid concentrations than the older men and women (P < 0.005). Plasma acarotene concentrations were signi®cantly higher in older women and in the total older group than in younger women and the total younger group (P < 0.005). In both younger men and women, positive correlations between estimated record and plasma values were observed for several carotenoids, with the exception of b-carotene (Tables 7 and 8). In the older groups, there was little association between estimated record and plasma carotenoid concentrations, except for b-cryptoxanthin in men (r 0.46, P 0.04) and total carotenoids in women Carotenoids in diet and plasma YL Carroll et al 648 Table 4 Spearman's rank order correlation coef®cients and percentage of subjects correctly classi®ed in same tertile and misclassi®ed in opposite tertile for dietary carotenoid concentrations assessed by estimated records and FFQ in groups of men and women aged 24 ± 45y and 65y 24 ± 45y Spearman correlation coef®cients r Same tertiles 0.29 0.27* 0.55*** 0.30*** 0.37** 0.39** 0.06 0.03 0.61*** 0.32 0.32 0.42 Males a-carotene b-carotene b-cryptoxanthin Lycopene Lutein zeaxanathin Total carotenoids Females a-carotene b-carotene b-cryptoxanthin Lycopene Lutein zeaxanathin Total carotenoids 65y % of classi®cation Opposite tertile Spearman correlation coef®cients r % of classi®cation Same tertiles Opposite tertile 55 41 64 45 50 64 27 27 9 23 23 23 0.25 0.23 0.62*** 0.12 0.40 0.27 38 38 75 31 44 58 25 19 6 31 13 19 32 32 59 45 41 55 27 36 14 23 18 18 0.14 0.24 0.48 0.28 0.44* 0.33 30 50 60 45 55 50 30 25 10 20 10 25 *P 0.05; **P 0.01; ***P 0.01 signi®cant correlation between estimated records and FFQ. Table 5 Energy intakes (MJ=d) (excluding alcohol) and nutrient densities (mg=MJ) measured by estimated records in groups of men and women aged 24 ± 45 y and 65y Younger males (n 32) Energy a-carotene b-carotene b-cryptoxanthin Lycopene Lutein zeaxanathin Total carotenoids Younger females (n 32) Older males (n 25) Older females (n 29) Mean s.d. Mean s.d. Mean s.d. Mean s.d. 10.4 85 320 19 314 111 2.4 73 205 24 346 85 8.6 101 370 21 379 119 1.5 82 222 19 302 49 9.4 87 335 13 227 86 1.9 63 203 17 201 34 7.8 129 441 13 307 135 1.4 80* 216 18 357 68** 852 486 989 480 749 475 1025 517* *P 0.05; **P 0.01 signi®cant difference between males and females in median carotenoid intakes expressed in mg=MJ energy (Mann Whitney U test). Table 6 Average plasma carotenoid concentrations (nmol=L) of screening and baseline plasma samples in groups of men and women aged 24 ± 45 y and 65y Younger males (n 32) Mean a-carotene 92 b-carotene 393 b-cryptoxanthin 191 Lycopene 297 Lutein 207 Zeaxanathin 97 Lutein zeaxanathin 304 Total carotenoids 1693 s.d. Younger females (n 32) Older males (n 25) Older females (n 29) Mean s.d. Mean s.d. Mean s.d. 43 107 194 462 113* 296 125** 253 70** 237 40** 83 99** 320 473** 1813 53* 186 226* 110** 73** 25** 94** 567** 122 81 166 94 472 222 553 254 117 96 123 62 91 69 111 61 140 62 170 56 49 25 57 19 187 73 223 73 992 395 1177 436 *P 0.01; **P 0.001 signi®cant difference between older and younger groups (Mann Whitney U test). (r 0.27, P 0.05). In younger men and women, the only signi®cant association between FFQ and plasma carotenoids was observed for b-cryptoxanthin and this correlation was evident in males (r 0.68, P < 0.0001) and females (r 0.53, P 0.0025). In the group of older men, positive correlations were observed for several carotenoids comparing FFQ and plasma carotenoid concentrations. In the group of older women, the only positive correlation between FFQ and plasma carotenoids occurred for bcryptoxanthin (r 0.65, P 0.0001). More younger (36 ± 73%) than older volunteers (20 ± 60%) were correctly classi®ed by estimated records into plasma tertiles. Within the younger groups and within the older groups, there was no signi®cant difference between estimated records and FFQ in their ability to correctly classify volunteers into the same tertiles of the plasma carotenoid distribution. Similarly, within the younger and within the older groups, the percentage of volunteers misclassi®ed by estimated records into opposite tertiles of the plasma carotenoid distributions, did not differ signi®cantly from the percentage misclassi®ed by FFQ. In the younger groups, a mean of 46 d elapsed between collection of screening and baseline plasma samples, with a minimum interval of 13 d and a maximum interval of 57 d. 85% of screening and baseline samples were collected within 40 ± 57d of one another in the younger age groups, with a median interval of 51 d. In the older groups, a mean of 38 d elapsed between collection of screening and baseline plasma samples, with a minimum interval of 19 d and a maximum interval of 56d. The median interval was 39 d in the older groups. The within-person coef®cient of variation was consistently lower than the between-person coef®cient of variation in individual carotenoids, in both plasma and estimated records, as shown in Table 9. Within- and between-person variability in dietary carotenoids assessed by estimated records, exceeded the variability in plasma carotenoid concentrations. In all groups, the ratio of withinperson:between-person variability in estimated records, was lowest for b-cryptoxanthin. Discussion Data on the consumption and plasma concentrations of individual carotenoids and the associations between these Carotenoids in diet and plasma YL Carroll et al Table 7 Spearman's rank order correlation coef®cients and percentage of men correctly classi®ed in same tertile and misclassi®ed in opposite tertile between average plasma and estimated records or FFQ carotenoid concentrations in groups of men aged 24 ± 45y and 65 y Younger males Spearman correlation coef®cients r Estimated records a-carotene b-carotene b-cryptoxanthin Lycopene Lutein zeaxanathin Total carotenoids Food frequency questionnaire a-carotene b-carotene b-cryptoxanthin Lycopene Lutein zeaxanathin Total carotenoids Older males % of classi®cation Same tertiles Opposite tertile 0.60* 0.05 0.66*** 0.52* 0.44* 0.07 68 36 59 55 55 36 5 27 14 14 9 23 0.31 7 0.09 0.68*** 0.26 0.27 7 0.16 45 32 68 50 45 23 18 36 5 18 23 36 Spearman correlation coef®cients r % of classi®cation Same tertiles Opposite tertile 0.32 0.04 0.46* 0.14 0.07 0.11 44 31 50 32 31 50 19 25 13 38 25 19 0.70*** 0.34 0.54** 0.47* 0.39 0.55* 63 50 50 50 50 44 6 19 13 13 13 6 *P 0.05; **P 0.01; *** P 0.001 signi®cant correlation between carotenoid concentrations in plasma and diet assessment method. Table 8 Spearman's rank order correlation coef®cients and percentage of women correctly classi®ed in same tertile and misclassi®ed in opposite tertile between average plasma and estimated records or FFQ carotenoid concentrations in groups of men aged 24 ± 45y and 65 y Younger females Spearman correlation coef®cients r Estimated records a-carotene b-carotene b-cryptoxanthin Lycopene Lutein zeaxanathin Total carotenoids Food frequency questionnaire a-carotene b-carotene b-cryptoxanthin Lycopene Lutein zeaxanathin Total carotenoids 0.42** 0.29 0.74*** 0.43** 0.32 0.41* 0.24 0.11 0.53** 0.50 7 0.02 0.23 Older females % of classi®cation Same tertiles Opposite tertile 55 50 73 59 50 45 18 27 5 14 27 23 45 32 59 59 36 45 23 41 14 9 32 27 Spearman correlation coef®cients r % of classi®cation Same tertiles Opposite tertile 0.11 7 0.09 0.52 0.32 0.14 0.27* 45 20 60 40 40 45 35 45 10 30 25 25 7 0.14 0.12 0.65*** 0.44 0.10 0.34 30 40 75 60 35 55 35 25 0 10 25 20 *P 0.05; **P 0.01; ***P 0.001 signi®cant correlation between carotenoid concentrations in plasma and diet assessment method. variables, are not well documented in European populations. The primary purpose of the present study was to assess plasma carotenoid concentrations, to assess dietary intakes of individual carotenoids using an established dietary method, and to examine the relationship between these dietary and plasma variables in groups of younger and older volunteers. In agreement with other studies, the predominant dietary carotenoids reported by estimated records in both the younger and older groups are b-carotene and lycopene (Forman et al, 1993; Yong et al, 1994; Scott et al, 1996). The concentrations of total carotenoids reported by estimated records in both age groups are similar to those reported in the USA (Forman et al, 1993; Yong et al, 1994). However, the intake of b-carotene assessed by estimated records was 1.5 ± 4-times higher than the levels reported in older women in the UK, but it should be noted that the discrepancy between Irish and UK data is smaller than the discrepancies within UK data (Maisey et al, 1995; Scott et al, 1996). These differences between the various studies may be attributable to the use of different food composition databases, different dietary assessment methods and population differences. In this present study, a modi®ed version of the carotenoid food composition database developed by Scott et al (1996) was used. Weighed records, which are known to be associated with underreporting of food intakes (Black et al, 1993) and analysis of only fruit and vegetable sources of carotenoids (Scott et al, 1996), may also account for these differences in carotenoid intakes between Ireland and the UK. The estimated record data in the present study shows that composite dishes, for example, soups and pizza are signi®cant sources of carotenoids. In this present study, some carotenoids were also partly derived from animal foods with butter, dairy 649 Carotenoids in diet and plasma YL Carroll et al 650 Table 9 Within-(CWw) and between-subject (CVb) coef®cient of variation in plasma and estimated records of men and women aged 24 ± 45 y and 65y Younger males (n 32) Plasma a-carotene b-carotene b-cryptoxanthin Lycopene Lutein zeaxanthin Total carotenoids Estimated Records a-carotene b-carotene b-cryptoxanthin Lycopene Lutein zeaxanathin Total carotenoids Younger females (n 32) Older males (n 25) Older females (n 29) CVw CVb CVw CVb CVw CVb CVw CVb 27.03 29.44 31.02 28.02 16.52 17.63 66.66 69.61 83.69 59.35 45.88 39.49 33.39 25.21 46.66 25.99 14.50 15.74 70.42 56.92 107.88 61.51 41.58 44.20 25.06 30.02 74.16 33.24 19.26 26.18 94.09 66.52 115.85 106.32 55.55 56.44 24.14 25.82 29.24 32.82 11.24 18.03 80.30 65.06 72.18 77.97 46.18 52.40 162.47 114.26 137.44 175.88 84.63 90.87 224.21 162.77 404.17 341.64 177.47 163.62 151.57 104.78 116.00 182.39 107.19 95.87 204.98 142.89 251.99 189.99 108.15 104.60 118.33 84.44 110.50 99.01 54.94 68.11 212.05 175.90 348.07 228.81 108.79 177.34 121.87 90.27 139.60 224.12 93.31 98.64 144.44 114.02 393.11 274.10 120.60 110.95 products and eggs and egg dishes identi®ed as signi®cant sources of lutein and b-carotene. This agrees with a report from the USA, where dairy products contribute approximately 7% of the total pro-vitamin A carotenoid intake (Block, 1994). Similarly, data from Finland indicate that margarines, oils, butter, milk products and eggs provide 24 ± 25% of b-carotene and lutein intakes in men (Jarvinen, 1995). In this present study, the inter- and intra-individual coef®cients of variation in estimated record carotenoid intakes are high in all age groups, but variation in carotenoid intakes was always greater between individuals than within individuals. In estimated records, b-cryptoxanthin tended to show that lowest ratio of within-person:betweenperson variability in carotenoid intakes and this may account for its stronger associations with plasma b-cryptoxanthin concentrations. b-Carotene was the predominant plasma carotenoid in all groups in this present study. Plasma carotenoid concentrations were ranked in the order: b-carotene > lutein > a-carotene > b-cryptoxanthin > lycopene > zeaxanthin in older groups. This contrasts with the pro®le of the younger groups in which plasma lycopene concentrations were considerably higher and ranked second or third to b-carotene concentrations. Others have shown that lycopene and b-carotene were the major plasma carotenoids in younger and older subjects respectively (Yeum et al, 1996). In this present study, all individual plasma carotenoid concentrations, with the exception of a- and bcarotene, were higher in younger groups than older groups. Plasma a-carotene concentrations were higher in older than younger volunteers. It has been shown elsewhere that age is inversely related to plasma lycopene concentrations (Brady et al, 1996). Others have shown that age is directly associated with serum b-carotene concentrations, although an age effect on plasma b-carotene has not always been reported (Hallfrisch et al, 1994; Brady et al, 1996; Santos et al, 1996). Whether these effects of age on plasma carotenoid concentrations are due to physiological mechanisms, or are merely attributable to different dietary patterns in younger and older groups, has not yet been established. The relative order of plasma carotenoid concentrations appears to be population speci®c. b-Cryptoxanthin is the predominant plasma carotenoid in Spanish women, whereas data from the US indicate that lycopene is the predominant plasma carotenoid (Micozzi et al, 1992; Olmedilla et al, 1994). Plasma concentrations of individuals carotenoids are moderately stable, as shown by correlations in the range r 0.63 ± 0.91, between repeat plasma samples and withinperson coef®cients of variation generally less than 30% in this present study. In the younger group, there were positive associations between most plasma and estimated record concentrations of carotenoids, except b-carotene. Although moderate, the magnitude of the correlations between estimated records and individual plasma carotenoid concentrations observed in the younger groups compare well with those reported elsewhere, by estimated records, weighed intakes and diet histories (Forman et al, 1993; van Staveren et al, 1994; Yong et al, 1994; Scott et al, 1996). In the present study, good reproducibility of plasma carotenoids was observed as shown by correlations in the range r 0.65 ± 0.85, and coef®cients of variation generally ranging from 20 ± 30%. This level of reproducibility is similar to that observed by others for plasma carotenoids (Campbell et al 1994, Yong et al, 1994, Apgar et al, 1996, Scott et al, 1996). Therefore, the interval between screening and baseline plasma samples is unlikely to account for the observation of only moderate associations between estimated record and plasma carotenoids. In the younger groups, the association between estimated record and plasma carotenoid concentrations was strongest for b-cryptoxanthin, and was stronger than that reported elsewhere (Forman et al, 1993; van Staveren et al, 1994; Yong et al, 1994; Scott et al, 1996). No association between estimated record and plasma b-carotene concentrations was evident in any of the age groups in the present study. Others report signi®cant, but moderate, correlations for b-carotene in the range r 0.27 ± 0.52 (van Staveren et al, 1994; Yong et al, 1994; Scott et al, 1996), but not all studies have observed signi®cant correlations between plasma and estimated record b-carotene concentrations (Forman et al, 1993). Within- and between-person variation in estimated record and plasma b-carotene concentrations was similar to that observed for other carotenoids and may not explain the lack of association between estimated record and plasma b-carotene concentrations. A possible explanation for the poor association between estimated record and plasma b-carotene concentrations, may be the wide range of foods in which it is distributed. Data from the present study indicate that approximately 10 foods account for 90% of b-carotene consumption compared to approximately 4 foods accounting for 90% of lycopene and b-cryptoxanthin intakes. Other studies also Carotenoids in diet and plasma YL Carroll et al show that the number of foods contributing to b-carotene intake is considerably greater than that of lycopene and bcryptoxanthin (Granado et al, 1996). This extended number of sources of b-carotene has implications for absorption, matrix composition and quanti®cation by the dietary assessment tool, and may thereby affect the relationship between dietary and plasma b-carotene. The food matrix in which the carotenoids are provided may affect the absorption and physical inaccessibility of carotenoids in plant tissues, for example in pigment protein, complexes may reduce their bioavailability (Brown et al, 1989; de Pee et al, 1995). By contrast with the many positive correlations observed in the younger groups, only b-cryptoxanthin in men and total carotenoids in women showed signi®cant associations between plasma and estimated record carotenoid concentrations, in the older groups. The lack of association between estimated record and plasma carotenoid distributions in the older groups was also re¯ected in the poor ability of estimated records to correctly classify individuals into extremes of the plasma carotenoid distributions, with as many as 45% being misclassi®ed into opposite tertiles. This low level of association between plasma and estimated records in the older groups, does not agree with the trends of signi®cant associations observed for several carotenoids in the younger groups, in this present study and in other studies (Ascherio et al, 1992; Forman et al, 1993; Yong et al, 1994). The poor agreement between estimated record and plasma carotenoid concentrations in the older group was unanticipated and possible explanations may include physiological differences, differences in validity of estimated records in younger and older volunteers, and seasonal differences in consumption of carotenoids. An alteration in gastric acid secretion with age could be complicating the relationship between dietary and plasma carotenoid concentrations (Hartz et al, 1992; Tang et al, 1996). However, a study of patients positive for Helicobacter pylori, (a condition associated with atrophic gastritis and hypochlorhydria) has shown that these patients do not have low concentrations of a-carotene, b-carotene or lycopene (Sanderson et al, 1997). Furthermore, in this present study, screening excluded volunteers with gastrointestinal disorders, so it is unlikely that this is an adequate explanation. Female gender and age over 45y have been shown to be predictors of underreporting of food consumption (Hirvonen et al, 1997) and may be another reason why the associations between estimated record and plasma carotenoids were poor in the older groups. Underreporting, as de®ned by energy intakes less than 1.1 times the basal metabolic rate (Goldberg et al, 1991), was evident in the present study. However, similar percentages of volunteers in both age groups (16% of men and 9 ± 10% of women in both age groups) underreported energy intakes in the present study and this degree of underreporting is not unusual in dietary studies (Black et al, 1991). If underreporting was distorting the diet-plasma relationship, this bias should also have in¯uenced the associations between plasma and estimated record carotenoid concentrations in the younger groups. Spanish data revealed that marked seasonality in some food items, caused differences in the dietary supply of b-cryptoxanthin and lycopene (Granado et al, 1996). This group also reported signi®cant seasonal variation in some, but not all, serum carotenoid concentrations of Spanish men and women (Olinedilla et al, 1994). It remains to be determined whether seasonal effects are profound in the Irish population. In the UK, no seasonal difference was found in the intake of b-carotene (Scott et al, 1996). However, this UK study did note that the strength of the association between dietary and plasma b-carotene concentrations varied according to season, with no signi®cant association observed in winter, compared to a signi®cant association in spring (Scott et al, 1996). Dietary data were recorded in November in the younger groups and in January in the older groups, and in the context of the ®ndings in the UK study, this may help to explain why no positive associations were observed for b-carotene with any of the dietary methods in the present study. As part of a multicentre study the carotenoid food composition database used in the present study was compiled from published analysis of foods sourced mainly in Europe. This could in¯uence the diet-plasma relationships observed in the present study which was con®ned to Irish population groups. A carotenoid food composition database, based on analysis of foods sourced in Ireland, is not available. Many carotenoid food composition databases are compiled from published data relating to foods sourced in several countries (Mangels et al, 1993; West & Poortvliet, 1993). Comparison of carotenoid intakes estimated using several food composition databases has shown that the use of a single database may give misleading carotenoid intake values (Granado et al, 1997). Analysis of dietary data by reference to two different carotenoid composition databases, indicated that although estimates of carotenoid intake differed signi®cantly, only minor differences in carotenoid rankings and diet-serum correlations were observed using either data source (Vandenlangenberg et al, 1996). A secondary purpose of this present study was to assess the validity of a FFQ developed for assessing dietary carotenoid intakes in several European countries. Validity was assessed by comparison of mean intakes with estimated records, by examining correlations with estimated records, by observing the extent of correct classi®cation and gross misclassi®cation into tertiles of estimated records carotenoid concentrations, by examining correlations with plasma and by observing the extent of correct classi®cation and gross misclassi®cation into tertiles of plasma carotenoid concentrations. The FFQ concentrations of dietary carotenoids were approximately 2 ± 3-fold higher than estimated records in the present study. Furthermore, the FFQ total carotenoid and b-carotene intakes in both age groups, in this present study, are at least 2-fold higher than those reported by FFQ in other studies (Forman et al, 1993; Mares-Perlman et al, 1993; Vandenlangenberg et al, 1996; Ocke et al, 1997). Overestimation of carotenoid intakes by FFQ by 10 ± 30% of estimated records and by 38 ± 50% of weighed record concentrations have been observed (Yong et al, 1994; Bingham et al, 1994). This is attributed to a greater reported frequency of consumption in the questionnaire methods than actually measured by the weighed records (Bingham et al, 1994). In this present study, correlations between dietary intakes of most carotenoids assessed by estimated records and FFQ, were poor in most groups except younger men. In contrast to this present study, modest, but signi®cant correlations between estimated record and FFQ have been observed for several carotenoids in young men and women (Rimm et al, 1992; Forman et al, 1993; Yong et al, 1994). Misclassi®cation by FFQ in opposite tertiles of the estimated record carotenoid distributions ranged from 19 ± 36%, and is much greater than that reported elsewhere (Bingham et al, 1994; Bonifacj et al, 1997). The only group in which several 651 Carotenoids in diet and plasma YL Carroll et al 652 signi®cant associations between FFQ and plasma carotenoids were observed was in older men. Other studies have reported signi®cant associations between several plasma and dietary carotenoids, estimated by FFQ (Coates et al, 1991; Ascheno et al, 1992; Vandenlangenberg et al, 1996). Conversely, several studies failed to note a positive relationship between plasma and lycopene or b-carotene concentrations assessed by FFQ (Coates et al, 1991; Ascherio et al, 1992; Forman et al, 1993; Kardinaal et al, 1995; Ocke et al, 1997). b-Cryptoxanthin was the only carotenoid for which signi®cant correlations were observed between plasma and FFQ in both age groups. Other studies have also shown that correlations between plasma and FFQ were stronger for b-cryptoxanthin than other carotenoids (Forman et al, 1993; Vandenlangenberg et al, 1996). There was no statistical evidence, in the present study, that FFQ were better than estimated records in correctly classifying individuals into extremes of the plasma carotenoid distribution. The 110 item FFQ administered in these studies included only foods high in carotenoids, and this emphasis combined with the lack of representation of other foods, may have accounted for its poor performance. It has been shown in another study that adding a list of carotenoid-rich foods to a standard FFQ, did not improve the validity of the questionnaire (Enger et al, 1995). The presence of irrelevant nutrient sources on a questionnaire may contribute to increased misclassi®cation rather than to increased precision (Block, 1994). It has also been shown that fruit and vegetables, which are perceived as healthy foods, are most often overreported by FFQ, while meats and dairy products, which are considered to be less healthy are most often underreported (Feskanich et al, 1993). Further development is required to establish a valid FFQ suitable for use in several European countries. Conclusions Estimated records indicate that b-carotene and lycopene are the major dietary carotenoids. The pro®le of plasma carotenoids show that b-carotene is the major carotenoid in both age groups. Younger groups have higher plasma concentrations of lycopene, b-cryptoxanthin, lutein zeaxanthin and total carotenoids than older groups. Moderate positive associations, similar to those observed in other countries, exist between several plasma and estimated record dietary carotenoid concentrations in younger, but not older groups. Plasma b-cryptoxanthin concentrations were strongly associated with both estimated record and FFQ concentrations in both age groups. b-Carotene in plasma is not associated with estimated record concentrations in any age group. 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