Reliability of Fatty Acid Composition in Human Serum Phospholipids

Reliability of fatty acid composition in human serum phospholipids

A Zeleniuch-Jacquotte1,2*, V ChajeÁs3, AL Van Kappel3, E Riboli3 and P Toniolo1,2,4

1Nelson Institute of Environmental Medicine, New York University Medical Center, New York, USA; 2Kaplan Comprehensive Cancer Center, New York University Medical Center, New York, USA; 3International Agency for Research on Cancer, 150 cours Albert Thomas, 69372 Lyon Cedex 08, France; and 4Department of Obstetrics and Gynecology, New York University Medical Center, New York, USA

Objective: We examined the reliability of the fatty acid composition of serum phospholipids in the New York University Women's Health Study, a prospective study of sex hormones, diet and breast cancer. Design: Non-fasting serum samples collected at three yearly visits, in 46 healthy women, and stored at 80C for 7 ± 12 y, were included in the study. Serum phospholipid fatty acid composition was measured by capillary gas chromatography. Results: For the 20 individual fatty acids measured, the reliability coef®cients were less than 0.50 for four, between 0.50 and 0.70 for nine, and greater than 0.70 for seven. Among the major fatty acids, arachidonic and a-linolenic acids had high reliability coef®cients (0.71 and 0.72, respectively), palmitic, oleic, linoleic, eicosapentaenoic and docosahexaenoic acids had intermediate coef®cients (0.57, 0.69, 0.62, 0.64 and 0.66, respectively), whereas stearic acid had the lowest coef®cient (0.15). The reliability coef®cients for total monounsaturated fats, omega-6 and omega-3 fatty acids were moderately high (0.66, 0.53 and 0.66, respectively), whereas the coef®cients for total saturated fats and total polyunsaturated fats were low (0.31 and 0.43, respectively). Conclusions: These results indicate that the fatty acid composition of serum phospholipids can be a useful tool in epidemiologic studies, although for most fatty acids a single determination is associated with some error in measurement that should be taken into account at the design and analysis stages. Storage for up to 12 y at 80C preserved polyunsaturated fatty acids from oxidation very well. Descriptors: fatty acids; phospholipids; reliability; serum European Journal of Clinical Nutrition (2000) 54, 367±372

Introduction as an alternative or a complement to these methods (Kaaks et al, 1997) Assessing dietary intake of fatty acids is subject to the same It is well established that the fatty acid composition of dif®culties as assessing other dietary exposures. Traditional stored triglycerides in adipose tissue re¯ects the long-term epidemiologic tools include food records or recalls, food- qualitative dietary intake of fatty acids (Field & Clandinin, frequency questionnaires and biochemical indicators of 1984; van Staveren et al, 1986). However, adipose tissue dietary intake. Food records or recalls need to be repeated aspiration is not widely acceptable to healthy subjects, and over several days throughout the year in order to provide epidemiologic studies using this procedure are open to the estimates representative of usual food intake, which pre- biases associated with limited participation rates. Because cludes their use in large epidemiologic studies. Assessment blood drawing is less invasive, assessment of fatty acid of fatty acid dietary intake through food-frequency ques- composition in various serum components has also been tionnaires requires identi®cation of brand names of food suggested as potential marker of dietary intake. The fatty items such as margarine and baked goods and is limited acid composition of human serum triacylglycerols re¯ects by the fact that fatty acid distribution of these items has the composition of the last few meals and has been used to changed considerably over time (Kohlmeier et al, 1997). monitor dietary changes in subjects following special diets Assessment of fatty acid composition in biological speci- (Moore et al, 1977), but is not appropriate to assess long- mens, such as adipose tissue or plasma, has been suggested term levels. The fatty acid compositions of serum phospholipids and cholesterol esters, on the other hand, re¯ect medium-term (weeks to months) qualitative dietary intake *Correspondence: A Zeleniuch-Jacquotte, Department of Environmental Medicine, NYU School of Medicine, 650 First Avenue, room 512, New of fatty acids (Riboli et al, 1987; Zock et al, 1997), York, NY 10016-3240, USA. suggesting that they may be a valuable method of assessing E-mail: [email protected] habitual qualitative dietary intake of fatty acids. Guarantor: A Zeleniuch-Jacquotte. Fatty acid composition in one of the serum components Contributors: A Zeleniuch-Jacquotte designed the study, selected the samples to be assayed and conducted the statistical analysis. V Chajes will be a useful marker in epidemiologic studies if, in addition developed the assay methodology and supervised the conduct of the to having the ability to measure long-term dietary intake, it is assays. AL van Kappel was in charge of the preperation of the samples. also reliable, ie, if measurements in specimens collected at E Riboli and P Toniolo had the original idea of the study. P Toniolo different time points in the same individual lead to very provided the serum samples which were analyzed in E Rinoli's laboratory. similar results (Kaaks et al, 1997). Lack of reliability will All authors contributed to the writing of the paper. Received 26 April 1999; revised 5 November 1999; accepted reduce the ability to detect disease ± exposure associations in 22 November 1999 studies using only a single measurement. Serum phospholipid fatty acids reliability A Zeleniuch-Jacquotte et al 368 Reliability will be good if the within-subject variance is samples of each subject were always included in the same small relative to the between-subject variance, the other batch. All samples were sent in dry ice in one shipment main variance component, assuming that the assay repro- to the Nutrition laboratory at IARC, where the fatty acid ducibility is high. Although phospholipid fatty acid com- composition of serum phospholipids was analyzed. Batches position of human serum has been proposed as an indicator were assayed on separate days. One serum control from of dietary fatty acid intake in epidemiologic studies, limited IARC was added to each batch. data are available on its long-term variability and, thus, on its potential usefulness as a biomarker of exposure. We Analysis of fatty acid composition report here the within-and between-subject variability of Serum lipids were extracted from serum samples (300 ml) by the fatty acid composition of serum phospholipids in the the method of Folch et al (1957). The serum sample was New York University (NYU) Women's Health Study, a shaken vigorously with 9 ml of chloroform ± methanol 2:1 prospective cohort study of sex hormones, diet and cancer. (v:v) containing 100 ml of antioxidant butylated hydroxyto- luene (BHT, 100 mg=ml in methanol). The extract was washed with 2.2 ml NaCl and the mixture was allowed to Materials and methods separate into two phases by standing. The separated chloro- The NYU Women's Health Study form layer was transferred to a tube and evaporated to Between March 1985 and June 1991, the NYU Women's dryness under nitrogen. The lipid extract was dissolved in Health Study enrolled a cohort of 14,275 healthy women, 200 ml of chloroform ± methanol 2:1 and directly used for the aged 34 ± 65 y, at a breast cancer screening center in New column procedure. Boron tri¯uoride (BF3), butylated hydro- York City (Toniolo et al, 1991, 1995). At the time of xytoluene (BHT), 23:0, chloroform and hexane (HPLC enrollment and at annual screening visits thereafter, in- grade) were obtained from Sigma (St Louis, MO, USA). formation on medical, anthropometric and reproductive Phospholipids were puri®ed by adsorption chromatogra- factors was collected through a self-administered question- phy on Silica tubes (Supelco, Bellefonte, PA). The column naire, and 30 ml of nonfasting peripheral venous blood was treated with 2 ml chloroform ± methanol 2:1. The lipid collected. After blood drawing, tubes were kept covered at sample was applied to the column and the tube was washed room temperature (70F) for 15 min, then at 4C for 60 min to with 100 ml of this same system solvent added to the allow clot retraction, and then centrifuged at 600g. Super- column. As shown by thin-layer chromatography (with natant serum was partitioned into 1 ml aliquots and immedi- hexane:diethylether:acetic acid, 80:20:1, v:v:v, as a devel- ately stored at 80C for future biochemical analyses. oping solvent), neutral lipids were eluted with 5 ml of Approximately half of the participants gave blood at sub- chloroform and phospholipids with 8 ml of methanol. sequent visits (mean number of blood donations 3, range 2 ± Methanol fractions containing phospholipids were col- 8). An average of twelve 1 ml serum aliquots were generated lected in screw-cap tubes with a te¯on seal. An internal per blood donation. Women have since been followed up for standard (23:0, 25 mg) was added to the collected fractions. cancer and cardiovascular endpoints. Nested case ± control Fractions obtained from the column procedure were eva- studies of breast, colon and endometrial cancer, as well as of porated to dryness under a stream of nitrogen. Fatty acids coronary artery disease are ongoing. were converted into fatty acid methyl esters by transester- i®cation using 1 ml of 14% boron tri¯uoride in methanol. Sample selection The tubes were tightly capped and placed into a block The Nutrition Laboratory at the International Agency for heater (100C) for 30 min. After cooling, fatty acid methyl Research on Cancer (IARC) had agreed to conduct a esters were extracted twice into hexane. Hexane was maximum of 150 assays for this study, including samples evaporated to dryness under nitrogen and the residue for quality control. We decided to use three serum samples dissolved in 1 ml hexane. from each woman because this number of replicates has Fatty acid methyl esters composition was determined by been shown to minimize the variance of the statistic of capillary gas chromatography. A 0.5 ml aliquot was injected interest, ie the reliability coef®cient, for coef®cients ran- through an on-column injector at 65C into a gas chroma- ging from 0.3 to 0.8, the range in which we expected most tograph with the aid of an autosampler (Hewlett Packard) of the coef®cients to fall (Walter et al, 1998). As a result, operated at column and detector temperatures of 220 we decided to include 48 women in the study. and 250C. A 30 m60.32 mm i.d. fused silica capillary Eligible for the present study were study participants who column with a 0.25 mm ®lm thickness was used for the had given blood on three or more occasions (with a yield of separation of fatty acid methyl esters (Supelco, Bellefonte, 11 or more aliquots per visit), had not been diagnosed with PA). Helium was used as a carrier gas at a ¯ow rate of cancer or cardiovascular disease, and had not been selected as 1ml=min with N2 as make-up gas for the ¯ame ionization a control in any case ± control study nested within the cohort. detector. The temperature was programmed to rise from Sampling was strati®ed by menopausal status, age at enroll- 65 to 135C at a rate of 5C=min, from 135 to 200Cat ment (35 ± 45 y, > 45 y for premenopausal and <55 y, 55 ± a rate of 2C=min, and from 200 to 220C at a rate of 65 y for postmenopausal women) and year of ®rst blood 3C=min, and then kept constant for 10 min. Identi®cation donation (1985 ± 87, 1988 ± 89). Twenty-four pre- and 24 of FAME was obtained by comparison with the relative postmenopausal women were selected. Two women were retention times of pure standard mixtures (Sigma, St Louis, excluded because of missing data, resulting in inclusion of 46 MO, USA). The relative amount of each fatty acid (per- women in the present report. centage of total fatty acids) was quanti®ed by integrating Samples were organized in batches of seven to eight the area under the peak and dividing the results by the total serum samples that included six samples drawn from two area for all fatty acids. subjects, and one or two samples from a standard pool for The within-day coef®cients of variation were based on quality control. Samples were identi®ed solely by a code the analysis of an independent series of 10 serum samples, number and ordered randomly within a batch. The three all extracted and analysed during the same day. The

European Journal of Clinical Nutrition Serum phospholipid fatty acids reliability A Zeleniuch-Jacquotte et al 369 coef®cients of variation ranged from 0.85% for large peaks to 7.6% for the smallest peaks. Coef®cients of variation (n 10) were: 1.2% for 16:0, 1.1% for 18:0, 1.4% for 18:1 n-9, 0.85% for 18:2 n-6, 7.6% for 18:3 n-3, 2.1% for 20:4 n-6, 1.4% for 20:5 n-3 and 1.2% for 22:6 n-3. The between- day coef®cients of variation were based on the analysis of two different serum controls on separate days (up to 24 days). For one serum control (n 17), coef®cients of variation ranged from 3.9% for large peaks such as 16:0 to 13.9% for the smallest peaks such as 20:5 n-3. For the second serum control (n 22), coef®cients of variation ranged from 3.6% to 31.1%.

Statistical analysis The intraclass correlation coef®cient, which is the propor- tion of the total variance due to between-subject variance, was used to quantify the reliability of the fatty acid composition in serum phospholipids. Estimation of the intraclass correlation coef®cients was performed assuming Figure 1 Gas chromatogram from phospholipid fatty acid methyl esters a one-way random effects model ANOVA and was carried obtained from one individual (NYU Women's Health Study). out using restricted maximum-likelihood techniques in SAS PROC VARCOMP. Approximate 95% con®dence intervals were calculated as described by Donner (1986). 0.69, 0.62, 0.64 and 0.66, respectively), whereas stearic acid (18:0) had the lowest coef®cient (0.15). The reliability correlation coef®cients for total monounsaturated fats, Results omega-6 and omega-3 fatty acids were moderately high Premenopausal women had a mean ( Æ s.d.) age at ®rst (0.66, 0.53 and 0.66, respectively), whereas the co- blood donation of 47.0 y ( Æ 4.4 y) and a mean Quetelet's ef®cients for total saturated fats and total polyunsaturated index of 23.8 kg=m2 ( Æ 4.0 kg=m2). For postmenopausal fats were low (0.31 and 0.43, respectively). women the mean age was 59.1 y ( Æ 4.3 y) and the mean Quetelet's index was 24.4 kg=m2 ( Æ 1.9 kg=m2). Mean Discussion times in storage of the serum samples were 9.5 y ( Æ 1.5 y), 8.5 y ( Æ 1.5 y), and 7.5 y ( Æ 1.5 y) for visits 1, 2 and 3, The potential oxidation of polyunsaturated fatty acids in respectively. samples stored for a long time was of concern. Such Figure 1 shows a gas chromatogram from phospholipid oxidation might explain the low erythrocyte levels (<5%) fatty acid methyl esters obtained from one individual. Up to of arachidonic acid observed in healthy subjects in some 24 fatty acids were identi®ed in serum phospholipids, studies (Angelico et al, 1980). To fully ascertain degrada- ranging in chain length from 14 to 24 carbon atoms. The tion of fatty acids associated with storage would require the major fatty acids detectable were: palmitic acid (16:0), comparison of measurements in fresh samples and stored stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2 samples taken from the same individuals at the same time. n-6), arachidonic acid (20:4 n-6), eicosapentaenoic acid Because fatty acid composition was not assayed at the time (20:5 n-3) and docosahexaenoic acid (22:6 n-3). Fatty acids of blood donation, we cannot assert that no degradation accounting for less than 1% of total area, such as took place. However, after up to 12 y storage at 80C, a-linolenic acid (18:3 n-3), were detected and integrated. only three samples in our study showed a high level of Only three chromatograms were not acceptable because of oxidation detected by a level of 18:2 n-6 or 20:4 n-6 less a high level of polyunsaturated fatty acids oxidation, than 5%, and by extra-peaks greater than 20%. Salo et al indicated by a level of 18:2 n-6 or 20:4 n-6 less than 5% (1986) reported a decrease of polyunsaturated fatty acids in and a sum of extra-peaks area more than 20%. plasma after storage for 3 y at 20C, and Vatten et al The mean fatty acid composition of serum phospholipids (1993) reported a degradation of long-chain polyunsatu- at each of the three blood sampling visits is given in Table rated fatty acids in serum phospholipids after 10 y storage 1. Saturated fatty acids accounted for 46% of total fatty at 25C. However, little is known on possible effects on acids, monounsaturated fatty acids for 16%, n-6 polyunsa- blood fatty acid composition of long-term storage at lower turated fatty acids for 30% and n-3 polyunsaturated fatty temperatures. Stanford et al (1991) observed no substantial acids for 6%. Linoleic acid, essential fatty acid of the n-6 change in n-3 or n-6 polyunsaturated fatty acids in red family, accounted for 52% of total polyunsaturated fatty blood cells after storage at 70C, but storage was limited acids, while a-linolenic acid, essential fatty acid of the n-3 to 1 y. Our results suggest that fatty acids are well pre- family, accounted for 0.6%. served when stored for up to 12 y at 80C. For the 20 individual fatty acids presented in Table 1, For 16 of the 20 fatty acids the reliability coef®cient the reliability coef®cients were less than 0.50 for four, over a 2 y period was greater than 0.50, indicating that between 0.50 and 0.70 for nine, and greater than 0.70, for assessment of fatty acid composition in serum phospholi- seven. Among the major fatty acids, arachidonic (20:4 n-6) pids can be of value in epidemiologic studies. However, the and a-linolenic (18:3 n-3) had high coef®cients (0.71 and reliability coef®cient was greater than 0.80 for only two of 0.72, respectively), palmitic (16:0), oleic (18:1 n-9), lino- the fatty acids (22:0 and 16:1 n-7), and the reliability leic (18:2 n-6), eicosapentaenoic (20:5 n-3) and docosa- coef®cients of the groups of fatty acids (saturated, etc) hexaenoic (22:6 n-3) had intermediate coef®cients (0.57, tended to be lower than those of individual fatty acids.

European Journal of Clinical Nutrition Serum phospholipid fatty acids reliability A Zeleniuch-Jacquotte et al 370 Table 1 Serum phospholipid fatty acid composition at three yearly visits in 46 NYU Women's Health Study participants, and corresponding reliability coef®cients

Mean (s.d.) Mean (s.d.) Mean (s.d.) Reliability correlation Fatty acids at visit 1 at visit 2 at visit 3 coef®cient (95% CI)

Saturated 14:0 0.94 (0.41) 0.89 (0.46) 0.91 (0.47) 0.75 (0.64 ± 0.86) 15:0 0.24 (0.11) 0.24 (0.20) 0.20 (0.06) 0.44 (0.26 ± 0.62) 16:0 24.04 (1.63) 24.13 (1.60) 23.72 (1.97) 0.57 (0.41 ± 0.73) 17:0 0.69 (0.14) 0.69 (0.14) 0.72 (0.24) 0.32 (0.13 ± 0.51) 18:0 18.35 (2.53) 18.50 (2.35) 18.64 (2.91) 0.15 (0.00 ± 0.34) 20:0 0.23 (0.08) 0.23 (0.08) 0.24 (0.10) 0.70 (0.58 ± 0.82) 22:0 0.59 (0.24) 0.68 (0.29) 0.64 (0.27) 0.81 (0.73 ± 0.89) 24:0 0.56 (0.28) 0.66 (0.33) 0.62 (0.33) 0.71 (0.59 ± 0.83) Total saturated 46.67 (3.10) 46.02 (3.42) 45.70 (4.09) 0.31 (0.12 ± 0.50)

Monounsaturated 16:1 n-7 0.75 (0.42) 0.73 (0.42) 0.70 (0.39) 0.82 (0.74 ± 0.90) 18:1 n-9t 4.35 (1.96) 4.39 (2.25) 4.50 (2.16) 0.52 (0.35 ± 0.69) 18:1 n-9c 11.16 (1.60) 11.00 (1.76) 11.11 (1.89) 0.74 (0.63 ± 0.85) Total 18:1 n-9 15.51 (2.82) 15.38 (3.49) 15.61 (3.18) 0.69 (0.56 ± 0.82) Total monounsaturated 16.25 (2.71) 16.11 (3.28) 16.31 (3.08) 0.66 (0.53 ± 0.79)

n-6 polyunsaturated 18:2 n-6 17.65 (3.81) 17.42 (3.11) 17.24 (3.36) 0.62 (0.48 ± 0.76) 18:3 n-6 0.36 (0.12) 0.36 (0.12) 0.37 (0.10) 0.21 (0.02 ± 0.40) 20:2 n-6 0.26 (0.09) 0.26 (0.09) 0.27 (0.10) 0.62 (0.48 ± 0.76) 20:3 n-6 2.27 (0.58) 2.29 (0.61) 2.26 (0.52) 0.58 (0.43 ± 0.73) 20:4 n-6 9.79 (2.49) 9.44 (2.51) 9.56 (2.44) 0.71 (0.59 ± 0.83) 22:4 n-6 0.62 (0.37) 0.67 (0.42) 0.78 (0.53) 0.63 (0.49 ± 0.77) Total n-6 30.95 (4.86) 30.44 (4.56) 30.48 (4.56) 0.53 (0.37 ± 0.69)

n-3 polyunsaturated 18:3 n-3 0.19 (0.09) 0.20 (0.10) 0.20 (0.12) 0.72 (0.60 ± 0.84) 20:5 n-3 1.19 (0.84) 1.22 (0.81) 1.21 (1.08) 0.64 (0.50 ± 0.78) 22:5 n-3 0.85 (0.36) 0.91 (0.44) 0.87 (0.39) 0.70 (0.58 ± 0.82) 22:6 n-3 3.92 (1.28) 4.16 (1.53) 4.10 (1.47) 0.66 (0.53 ± 0.79) Total n-3 6.14 (1.98) 6.49 (2.38) 6.37 (2.61) 0.66 (0.53 ± 0.79)

Total polyunsaturated 37.09 (4.93) 36.93 (5.43) 36.85 (5.58) 0.43 (0.25 ± 0.61)

P=S 0.82 (0.16) 0.81 (0.17) 0.82 (0.19) 0.37 (0.19 ± 0.55)

CI con®dence interval.

These results indicate that, for most fatty acids, measure- assessed in our study, than for phospholipids. The relia- ment error was not negligible, and should be taken into bility coef®cients were greater than 0.50 for 73% of the account in epidemiologic studies. A low reliability correla- fatty acids measured in phospholipids, a result similar to tion, re¯ecting large measurement error, will result in a ours (80%). However, there were differences in individual biased estimate of exposure-disease association (Arm- fatty acids reliability coef®cients, in particular for saturated strong, 1990). The association will be attenuated in the and n-3 polyunsaturated fatty acids: the reliability coef®- absence of adjustment for other variables, but could be in cients for palmitic and stearic acids were higher in Ma's either direction when adjusting for potential confounders study (0.72 and 0.67, respectively) than in our study (0.57 also measured with error (Greenland, 1980). It should be and 0.15, respectively). On the other hand, the reliability noted that our study subjects were on their usual diet rather coef®cient was higher in our study for 14:0 (0.75 vs 0.30), than on a standardized diet. The reliability coef®cients a-linolenic acid (0.72 vs 0.35) and docosahexaenoic acid would be expected to be higher if the diet of individuals (0.66 vs 0.46). We are not aware of any other systematic was controlled and similar at each blood donation because assessment of the long-term reliability of fatty acid compo- the within-subject variance would be expected to be smal- sition of serum phospholipids. Nikkari et al (1983) reported ler. Our results are therefore more relevant to epidemiolo- coef®cients for phospholipid fatty acids in serum samples gic studies whose focus is to assess exposures in free-living from 18 fasting Finnish men collected 5 months apart. individuals than diet-controlled studies. Correlation coef®cients were similar to the reliability Ma et al (1995a) examined the long-term reliability of coef®cients observed in our study for 18:2 n-6 (r 0.70), the fatty acid composition of plasma phospholipids and 20:3 n-6 (r 0.59) and 22:6 n-3 (r 0.63), but lower than cholesterol esters using fasting samples collected at two the ones we observed for all other fatty acids measured. time points approximately 3 y apart, in 50 subjects, ages Bjerve et al (1993) reported correlation coef®cients of 0.64 45 ± 64, from the Minneapolis area. The long-term relia- for docosahexaenoic acid and 0.49 for eicosapentaenoic bility coef®cients for the measurements in phospholipids acid in 211 newly diagnosed diabetics in Norway, and ranged from 0.30 to 0.81 and tended to be higher for the Moilanen et al (1992) reported correlation coef®cients in major plasma fatty acids than for the fatty acids that the range 0.30 ± 0.54 for various fatty acids assessed compose <1% of total fatty acids. Reliability coef®cients 6 y apart in 759 Finnish boys, but the fatty acid tended to be better for cholesterol esters, which were not composition was analyzed in cholesteryl esters rather than

European Journal of Clinical Nutrition Serum phospholipid fatty acids reliability A Zeleniuch-Jacquotte et al 371 phospholipids. The differences between studies argue composition in serum may be a useful tool in epidemiologic against generalizability of the results and suggest that, studies, especially for studies focusing on the role of very- prior to launching epidemiologic studies investigating long-chain n-3 fatty acids. Error in measurement should be serum phospholipid fatty acid composition, the reliability taken into account at the design and analysis stages. of the measurements should be assessed within the speci®c populations to be studied. Acknowledgements ÐSupported by grants CA-34588, CA-13343 and CA- Lack of reliability can be taken into account at the 16087 from the National Cancer Institute, and grant ES-00260 from the design and=or analysis stages of epidemiologic studies. National Institute of Environmental Health Sciences. The authors thank all At the design stage, investigators may choose to increase women who generously volunteered for the study and are grateful to Bernard the total sample size or the number of measurements per sub- Pasternack, Philip Strax and Frances Mastrota for their contributions. ject. Increasing the total sample size will allow adequate power to be maintained for hypothesis testing purposes, but References will not correct for the bias due to measurement error. Andersen LF, Solvoll S & Drevon CA (1996): Very-long-chain n-3 fatty Increasing the number of measurements on each subject acids as biomarkers for intake of ®sh and n-3 fatty acids concentrates. will both increase the power of the study and reduce the Am. J. Clin. Nutr. 64, 305 ± 311. measurement error bias. 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The fatty acid composi- Research on Cancer. tion of stored triglycerides in adipose tissue is considered to Kipnis V, Carroll RJ, Freedman LS & Li L (1999): Implications of a new adequately re¯ect the long-term qualitative dietary intake dietary measurement error model for estimation of relative risk: of fatty acids, but adipose tissue aspiration is not widely application to four calibration studies. Am. J. Epidemiol. 150, 642 ± 651. Kohlmeier L, Simonsen N, van't Veer P, Strain JJ, Martin-Moreno JM, acceptable to healthy subjects, and its use in large epide- Margolin B, Huttunen JK, FernaÂndez-Crehuet Navajas J, Martin BC, miologic studies is thus limited. Our food frequency ques- Thamm M, Kardinaal AFM & Kok FJ (1997): Adipose tissue trans fatty tionnaire was limited to 69 food items and was not acids and breast cancer in the European Community Multicenter Study appropriate to compute dietary intake of fatty acids. How- on antioxidants, myocardial infarction and breast Cancer. 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For very-long-chain n-3 fatty acids, Communities (ARIC) Study Investigators (1995b): Plasma fatty acid the correlation coef®cients between dietary fatty acid intake composition as an indicator of habitual dietary fat in middle-aged and fatty acid level in serum (0.51 for 20:5, n-3 and 0.52 for adults. Am. J. Clin. Nutr. 62, 564 ± 571. 22:6, n-3) were similar to the correlation coef®cients Moilanen T, RaÈsaÈnen L, Viikari J, Akerblom HK & Nikkari T (1992): between dietary intake and fatty acid level in adipose Tracking of serum fatty acid composition: a 6-year follow-up study in Finnish youths. Am. J. Epidemiol. 136, 1487 ± 1492. tissue (0.52 for 20:5, n-3 and 0.49 for 22:6, n-3). On the Moore RA, Oppert S, Eaton P & Mann JL (1977): Triglyceride fatty other hand, the correlations for dietary intake of polyun- acids con®rm a change in dietary fat. Clin. Endocrinol. 7, 143 ± saturated fatty acids and linoleic acid were highest with 149. proportions in adipose tissue. Van Houwelingen et al Nikkari T, Salo M, Maatela J & Aromaa A (1983): Serum fatty acids in Finnish men. Atherosclerosis 49, 139 ± 148. (1989) reported similar correlations between the intake of Prentice RL (1996): Measurement error and results from analytic epide- eicosapentaenoic acid and docosahexaenoic acid and the miology: dietary fat and breast cancer. J Natl Cancer Inst. 88, relative amount of these fatty acids in total serum lipids, 1138 ± 1147. and Ma et al (1995b) and Andersen et al (1996) Riboli E, RoÈnnholm H & Saracci R (1987): Biological markers of diet. in cholesterol esters and phospholipids. Together with the Cancer Sur. 6, 685 ± 718. Salo MK, Gey F & Nikkari T (1986): Stability of plasma fatty acids at 20 relatively high reliability coef®cients observed in our study degrees C and its relationship to antioxidants. Int. J. Vitamin Nutr. Res. for n-3 fatty acids, these results indicate that fatty acid 56, 231 ± 239.

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