European Journal of Clinical Nutrition (2008) 62, 695–703 & 2008 Nature Publishing Group All rights reserved 0954-3007/08 $30.00 www.nature.com/ejcn ORIGINAL ARTICLE Food sources of plant sterols in the EPIC Norfolk population S Klingberg1, H Andersson1, A Mulligan2, A Bhaniani2, A Welch2, S Bingham3, K-T Khaw2,4, S Andersson1 and L Ellega˚rd1 1Department of Clinical Nutrition, Sahlgrenska Academy at Go¨teborg University, Go¨teborg, Sweden; 2Department of Public Health and Primary Care, University of Cambridge, Strangeways Site, Wort’s Causeway, Cambridge, UK; 3Medical Research Council Dunn Human Nutrition Unit, Cambridge, UK; 4Clinical Gerontology Unit, University of Cambridge School of Medicine, Addenbrooke’s Hospital, Cambridge, UK Objective: To investigate the intake of plant sterols and identify major dietary sources of plant sterols in the British diet. Subjects: A total of 24 798 men and women recruited during 1993–1997, participating in the European Prospective Investigation into Cancer (EPIC-Norfolk). Interventions: A database of the plant sterol (campesterol, b-sitosterol, stigmasterol, campestanol and b-sitostanol) content in foods, based on gas-liquid chromatography (GLC) analyses, was linked to nutritional intake data from food frequency questionnaires in the EPIC-Norfolk population. Results: The mean (s.d.) intake of total plant sterols was 300 (108) mg/d for men and 293 (100) mg/d for women. Bread and other cereals, vegetables and added fats were the three major food sources of plant sterols representing 18.6 (8.9), 18.4 (8.5) and 17.3 (10.4)% of the total plant sterol intake respectively. Women had a higher plant sterol density than men (36.4 vs 32.8 mg/1000 kJ, Po0.001) and in relation to energy intake higher intakes of plant sterols from vegetables, bread and other cereals, added fats, fruits and mixed dishes (all Po0.001), whilst men had higher intakes of plant sterols from cakes, scones and chocolate, potatoes (all Po0.001) and other foods (Po0.01). Conclusions: The intake of plant sterols in UK, mainly from bread, cereals, fats and vegetables, is much higher than previously reported but comparable to recent European studies. European Journal of Clinical Nutrition (2008) 62, 695–703; doi:10.1038/sj.ejcn.1602765; published online 18 April 2007 Keywords: plant sterols; food sources; population; diet Introduction The chemical structure of plant sterols resembles that of cholesterol but with an additional methyl- or ethyl group in Plant sterols are bioactive compounds found in all vegetable the side chain. More than 250 different plant sterols have foods at varying concentrations. There is, however, limited been identified (Piironen et al., 2000). Plant sterols are information on the plant sterol intake from common foods known to reduce the absorption of cholesterol and the serum in Britain. level of cholesterol, although the mechanisms are not fully understood. In addition to the well-known effect of decreased intestinal cholesterol absorption due to reduced Correspondence: S Klingberg, Department of Clinical Nutrition, Sahlgrenska incorporation of cholesterol into mixed micelles, plant Academy at Go¨teborg University, Box 459, Gothenburg 40530, Sweden. sterols are suggested to influence cellular cholesterol meta- E-mail: [email protected] bolism within the intestinal enterocytes (Plat and Mensink, Guarantor: S Klingberg. Contributors: K-TK and SB are principal investigators in the EPIC-Norfolk 2005). population study. AW and SB are responsible for the nutritional analyses. HA, It is well documented that commercial products enriched SA and LE developed the plant sterol nutrient database. AM and AB prepared with plant sterols reduce serum cholesterol levels. A the plant sterol data set in the EPIC cohort. SK was principal investigator for metaanalysis of 41 different studies has shown that a plant this study and wrote the paper with contributions from co-authors. Received 3 July 2006; revised 12 March 2007; accepted 14 March 2007; sterol dose of 2 g/day reduces low-density lipoprotein (LDL) published online 18 April 2007 cholesterol levels by 10% (Katan et al., 2003). In contrast to Food sources of plant sterols S Klingberg et al 696 these studies with products supplemented with plant sterols, 0.01 mg/100 g fresh material of the tested product. The much less is known about the effects of dietary plant sterol concentration of the five most frequently occurring plant intake (Ostlund, 2002). Traditionally plant sterols from the sterols (the unsaturated plant sterols campesterol (24a- habitual diet have not been considered to have any methyl-5-cholesten-3b-ol), stigmasterol (5,22-cholestadien- significant effect on the serum cholesterol level but this 24a-ethyl-3b-ol) b-sitosterol (24a-ethylcholest-5-en-3b-ol), view has been questioned (Fraser, 1994; Ostlund, 2002). and the saturated plant stanols campestanol (24a-methyl- Serum cholesterol lowering effects of the plant sterol intake 5a-cholestan-3b-ol), and b-sitostanol (24a-ethyl-5a-cholestan- from the natural diet could probably have been under- 3b-ol)) was analysed. The sum of the five plant sterols estimated (Ostlund, 2002). As recently shown in a large-scale constitutes ‘total plant sterols’. All foods were analysed in epidemiological study using the current database, a high duplicate. All fruits and vegetables were bought in 1996, intake of natural dietary plant sterols is significantly cereals in 1997 and fatty foods in 1997 and 2001 in two inversely related to total and LDL cholesterol levels shops in the Gothenburg area. Specific British food items (Andersson et al., 2004). were bought in Cambridge in 2004 and 2005. Fruits and Knowledge of major food sources of plant sterols could be vegetables were analysed as a mix of two different samples an important tool when trying to further improve the and analysis of cereal and fatty foods were made from a mix natural intake of plant sterols as a component in the of two to seven samples. British food items were analysed in prevention of cardiovascular disease. The aim of the current duplicate from a single sample. Seasonal variation in fruits study was to investigate the natural intake of plant sterols in and vegetables was not taken into account. the Norfolk population of The European Prospective In- vestigation of Cancer (EPIC) and to identify major dietary sources of plant sterols in the British diet. Plant sterol database Analyses of more than 330 food items were collected in a plant sterol database and this database was used to estimate Methods plant sterol intake. This database includes vegetables, fruits, cereals, bread, fats, nuts, confectionery, beverages and The study population specific British food items. Food items from this database The EPIC-Norfolk cohort recruited approximately 25 000 were matched with the food items present in the FFQ. The men and women aged 40–79 years of age between 1993 and plant sterol data on vegetables, fruits, cereals and fatty foods 1997 (Day et al., 1999). EPIC-Norfolk is part of the Europe- have been published (Norme´n et al., 1999, 2002, 2007). Plant wide EPIC study designed to investigate dietary and other sterol content of specific British food items used in this study determinants of cancer. The EPIC-Norfolk cohort also is shown in Appendix 1. included end points other than cancer, for example cardio- vascular disease. Informed consent was given by all partici- pants and the study was approved by the Norfolk and Food frequency questionnaire Norwich Hospital Ethics Committee. Food intake and At recruitment participants completed a FFQ consisting of nutrient data from food frequency questionnaires (FFQ) a food list of 130 questionnaire lines corresponding to were available for 24 838 participants. 275 food items (Bingham et al., 2001; Welch et al., 2005). The FFQ was validated against a 16-day weighed records and the biomarkers 24-h urine N and K, plasma carotenoids and Plant sterol analysis plasma vitamin C (Bingham et al., 1994, 1995, 1997). The Analyses of the food items were performed at the Depart- questions about intake of different fruits and vegetables were ment of Clinical Nutrition, Go¨teborg University, Sweden, detailed and types of bread, pasta, breakfast cereals and types using a gas-liquid chromatography (GLC) procedure of fat used for frying, baking and spreading had been modified after Jonker et al. (1985) and validated with gas specified which assisted in the correct assignment of plant chromatography mass spectrometry (Dutta and Norme´n, sterol values. Based either on pure animal origin or on 1998). In short, the method comprised acid hydrolysis (6 M ingredients not containing plant sterols, 63 food items were HCl), alkaline saponification (96% ethanolic KOH), lipid set to zero. For plant sterol containing food items the plant extraction with toluene, and a final step of washing in sterol value was based on direct analysis in 119 food items. de-ionized water until neutral pH was reached. Internal Fifty-six food items were assigned a value from similar standard, containing 5a-cholestane, was added to all samples analysed products or proportions of analysed products. For before saponification to quantify the sterols. Samples were 23 food items the plant sterol content was based on dehydrated with Na2SO4, filtered and evaporated under calculations of British standard receipts with analysed vacuum at 501C. The residue was dissolved in chloroform ingredients. Fourteen food items were assigned plant sterol and stored at À201C. Silylation of sterols to trimethylsilyl values from the UK food composition database ‘McCance ether derivatives was performed before analysis by gas liquid and Widdowson’s The composition of Foods’ (Food Stan- chromatography (GLC). The limit of detection was set to dards Agency, 2002). As plant sterol enriched products were European Journal of Clinical Nutrition Food sources of plant sterols S Klingberg et al 697 not launched until 1999 in the UK, no such products were higher intakes of plant sterols from cakes, scones and present in the FFQ:s.