ORIGINAL COMMUNICATION Rapeseed Oil, Olive Oil, Plant Sterols, and Cholesterol Metabolism: an Ileostomy Study

ORIGINAL COMMUNICATION Rapeseed oil, olive oil, plant sterols, and cholesterol metabolism: an ileostomy study

L Ellega˚rd1*, H Andersson1 and I Bosaeus1

1Department of Clinical Nutrition, University of Go¨teborg, Sahlgrenska University Hospital, Go¨teborg, Sweden

Objective: To study whether olive oil and rapeseed oil have different effects on cholesterol metabolism. Design: Short-term experimental study, with controlled diets. Setting: Outpatients at a metabolic-ward kitchen. Subjects: A total of nine volunteers with conventional ileostomies. Interventions: Two 3-day diet periods; controlled diet including 75 g of rapeseed oil or olive oil. Main outcome measures: Cholesterol absorption, ileal excretion of cholesterol, and bile acids. Serum levels of cholesterol and bile acid metabolites. Differences between diets evaluated with Wilcoxon’s signed rank sum test. Results: Rapeseed oil diet contained 326 mg more plant sterols than the olive oil diet. Rapeseed oil tended to decrease cholesterol absorption by 11% (P ¼ 0.050), and increased excretion of cholesterol, bile acids, and their sum as sterols by 9% (P ¼ 0.021), 32% (P ¼ 0.038), and 51% (P ¼ 0.011) compared to olive oil. A serum marker for bile acid synthesis (7a-hydroxy-4- cholesten-3-one) increased by 28% (P ¼ 0.038) within 10 h of consumption, and serum cholesterol levels decreased by 7% (P ¼ 0.024), whereas a serum marker for cholesterol synthesis (lathosterol) as well as serum levels of plant sterols remained unchanged. Conclusions: Rapeseed oil and olive oil have different effects on cholesterol metabolism. Rapeseed oil, tends to decrease cholesterol absorption, increases excretion of cholesterol and bile acids, increases serum marker of bile acid synthesis, and decreases serum levels of cholesterol compared to olive oil. This could in part be explained by different concentrations of natural plant sterols. Sponsorship: Supported by the Go¨teborg Medical Society, the Swedish Medical Society, the Swedish Board for Agricultural Research (SJFR) grant 50.0444/98 and by University of Go¨teborg. European Journal of Clinical Nutrition (2005) 59, 1374–1378. doi:10.1038/sj.ejcn.1602249; published online 10 August 2005

Keywords: cholesterol absorption; sterol excretion; dietary fibre; dietary fat; plant sterols; ileostomy

Introduction but due to bacterial degradation and varying transit time, Cholesterol can only be eliminated from the body by faecal precision was insufficient to settle the question of dietary excretion, except for minor amounts of metabolites of effects on sterol excretion (Andersson & Bosaeus, 1993). steroid hormones in urine. Sterol balance studies have been We have readressed the question of dietary effects on sterol performed to evaluate the dietary effects on sterol excretion excretion by studying ileostomy subjects without resection (Connor et al, 1969; Grundy & Ahrens, 1969; Nestel et al, of the small intestine (Sandberg et al, 1981). In ileostomy 1975). These metabolic ward investigations were elaborate, subjects, transit time is both shorter and less variable, and with far less bacterial degradation than in normal subjects (Sandberg et al, 1981). Thus, even short time studies in ˚ *Correspondence: L Ellegard, Department of Clinical Nutrition, University ileostomy subjects are feasible, to examine dietary effects on of Go¨teborg, Sahlgrenska University Hospital, S-413 45 Go¨teborg, Sweden. cholesterol metabolism. (Andersson & Bosaeus, 1993). With E-mail: [email protected] this approach, diets with the potential to reduce serum Guarantor: L Ellega˚rd. cholesterol levels have been shown to increase sterol Contributors: LE was the principal investigator, IB and HA contributed excretion from the small bowel, either as bile acids or as to the study design, and all contributed to writing this paper. Received 30 December 2004; revised 1 May 2005; accepted 14 June 2005; cholesterol, within 2–4 days, in carefully controlled dietary published online 10 August 2005 studies. This has been shown with diets low in fat, or high Monounsaturated oils and cholesterol metabolism L Ellega˚rd et al 1375 in polyunsaturated fat (Bosaeus & Andersson, 1987), high in serum thyroxine levels. One subject was overweight, and monounsaturated fat (Bosaeus et al, 1992), pectin (Bosaeus had elevated serum glucose twice, and thus was diagnosed as et al, 1986), and with oat-bran (Lia et al, 1995). Reducing diabetic during the study. His results did not differ from cholesterol intake increases net cholesterol excretion as those of the other subjects. No subjects had any signs of shown previously (Ellega˚rd & Bosaeus, 1994). We have also anaemia, inflammation, hepatic, or renal disease as judged reported increased cholesterol excretion from a ‘prudent’ by history, hospital records, and standard laboratory tests, diet, that is, a diet in accordance with contemporary dietary except the oldest subject who had slightly elevated CRP- guidelines of less saturated fat and more fibre-rich foods levels because of arthritis. All subjects had stable bowel (Ellega˚rd & Bosaeus, 1991). The combination of both function at the time of the study. modifications reduces cholesterol absorption, and increases Informed consent was obtained from each participant. The cholesterol and sterol excretion from the small intestine study protocol was approved by the Ethics Committee of (Ellegard et al, 2000b). The decrease in cholesterol absorption Sahlgrenska University Hospital. was correlated to plant sterol excretion, implying dietary plant sterol content to be an important determinant of cholesterol metabolism, together with cholesterol itself (Ellegard et al, 2000b). Thus, we speculated whether plant sterol content may Materials be as important as fatty acid composition and dietary fat Diets content in the regulation of sterol balance in humans. The diets were composed of common food items with In order to investigate this relationship, we compared the identical dishes in the two diets. The compositions of the effects of two monounsaturated oils, rapeseed oil (high in diets, given in Table 2, were calculated from Swedish food plant sterols) and olive oil (low in plant sterols), of similar composition tables (Swedish National Food Administration, although not identical fatty acid composition, on cholesterol 1996) and from analyses of energy, plant sterols, and metabolism in subjects with conventional ileostomies.

Table 2 Composition of study diets Subjects, materials and methods Rapeseed Olive Subjects Nine persons (three female, six male) volunteered for the Energy* (MJ) 9.070.1 8.770.05 study. Clinical data on the subjects are presented in Table 1. Total fat (E%) 38 38 Saturated fat (E%) 5 7 All subjects had previously undergone proctocolectomy for Monounsaturated fat (E%) 20 24 ulcerative colitis (n ¼ 7) or Crohn’s disease (n ¼ 2) and had Polyunsaturated fat (E%) 11 5 well-functioning ileostomies for at least 2 y. No additional Dietary fibre (g) 35 35 small-bowel resections had been performed in any of the Cholesterol* (mg) 19876 20275 Plant sterols* (mg) 58477 258724 subjects. Seven of the nine subjects used some medication (listed in Table 1), but these medications were kept constant Calculated from food tables (Swedish National Food Administration, 1996). during the study. Subjects using thyroxine had normal *Analysed values, mean7s.e.

Table 1 Clinical data on ileostomy subjects

Serum Serum Subject BMI cholesterol Serum HDL Serum LDL triglycerides ApoE Serum CRP number Sex Age (y) (mmol/l) (mmol/l) (mmol/l) (mmol/l) (mmol/l) phenotype (mg/l) Medication

1 M 80 24.6 6 1.2 4.4 1.0 3/3 8 Ibuprofen, ACEI 2 F 59 22.5 4.9 1.4 2.9 1.4 3/3 o5 Thyroxine 3 M 44 23.4 4.2 1 2.7 1.1 4/3 o5 Metoprolol 4 M 55 25.1 5.1 1.5 3.1 1.1 3/3 o5 Thyroxine, spironolactone, PPI 5 F 63 21.7 5.7 2.6 2.8 0.7 3/3 o5 6 M 63 27.9 6.2 1.9 3.7 1.3 3/3 o5 7 M 47 29.3 5.7 1.1 3.8 1.7 4/3 o5 Atropiumbromide, allopurinol 8 F 37 21.3 4 1.7 2.0 0.6 3/3 o5 Antacids, oestrogen, alimemazin 9 M 34 27.5 5.5 1 3.6 2.0 4/3 o5 thyroxine, DHEA

Median 55 24.6 5.5 1.4 3.1 1.1 Range 34–80 21.3–29.3 4.4–7.8 1–2.6 2–4.4 0.6–2.0

European Journal of Clinical Nutrition Monounsaturated oils and cholesterol metabolism L Ellega˚rd et al 1376 cholesterol. To meet energy demands, one subject (#7) had 7a-hydroxy-4-cholesten-3-one in serum was determined 50% larger than normal portions of all items on the menu. by HPLC (Axelson et al, 1988). The variation coefficient for duplicate samples was 7.4%.

Methods Protocol Isotope analyses Each subject was studied during two 3-day periods, on the Isotope activities were assayed by liquid scintillation count- same weekday on each diet. The first day of each period was ing in a Beckman Tricarb 1900TR as we have described used for adaptation and to minimise any carryover effects previously (Ellega˚rd & Bosaeus, 1994). The variation coeffi- from the habitual diets of the participants. There was a wash- cients for duplicate analyses were 4.5% for [3H]-cholesterol out period of at least 4 days between the dietary periods. The and 3.4% for [14C]-b -sitosterol. order of the diets was randomised for each subject, and the diets were blinded for the laboratory staff, the investigators and the subjects. 14 Calculations and statistical methods On the second day, 8.7 kBq of beta-4-[ C]-sitosterol and Values of wet weight, dry weight, cholesterol, and bile acids n 3 17.4 kBq of 1a, 2a- -[ H]-cholesterol were added at breakfast, are reported as medians with ranges for each dietary period. lunch, and dinner, in three divided doses, to measure Cholesterol excretion minus dietary cholesterol intake is cholesterol absorption. Isotopes were purchased from Amer- reported as net cholesterol excretion. Total bile acid plus net sham International, Buckinghamshire, UK. cholesterol excretion is termed net sterol excretion, which would correspond to cholesterol synthesis under steady-state conditions. Collection of ileostomy contents Fractional cholesterol absorption was calculated from Ileostomy contents were collected during the last 2 days of isotope ratios in ileostomy effluent, as described previously each period. Ileostomy bags were changed every second hour (Ellega˚rd & Bosaeus, 1994). during the daytime and directly after awakening. The bags All descriptive values are given as medians for 24 h with were immediately frozen on dry ice in Dewar vessels which ranges, unless otherwise stated. Differences between diets the subjects kept at home. Each morning, the bags were were assessed by Wilcoxon’s signed rank test, using the delivered to the metabolic ward and freeze dried before s s computer package SYSTAT version 7.0.1 for Windows chemical analysis. (SPSS Inc., 1997).

Chemical analyses Wet weight and dry weight of ileostomy effluent were Results determined as previously described (Sandberg et al, 1981). Both diets were well tolerated and consumed without Energy of the diets was determined by combustion in a leftovers. Excretion data are summarised in Table 3. Gallenkamp bomb calorimeter (Loughborough, Leicester- Rapeseed oil tended to decrease cholesterol absorption by shire, UK). Determinations of cholesterol and plant sterols in median 11% (P ¼ 0.050) compared to olive oil. Conse- the diets, as well as cholesterol, bile acids, and plant sterols quently, net excretion of cholesterol rose by median 9% in the ileostomy contents were made by GLC, as previously (P ¼ 0.021), bile acid excretion rose by 32% (P ¼ 0.038), and described (Bosaeus & Andersson, 1987). Variation coeffi- total net sterol excretion increased by median 51% cients in duplicate samples from food and ileostomy excreta (P ¼ 0.011) on rapeseed oil compared to olive oil. were 3.6% for cholesterol and 3.4% for bile acids. Before starting the study, standard laboratory tests including total serum cholesterol, triglycerides, and apo E pheno- Table 3 Sterol excretion and cholesterol absorption in nine ileostomy type, were determined by current methods at the Central subjects on rapeseed oil and olive oil diets Laboratory for Clinical Chemistry, Sahlgrenska University Rapeseed oil diet Olive oil diet Hospital. Serum samples for analyses specific to the study were collected on the second day of the diet period after Wet weight (g) 667 (575–962) 656 (589–1016) overnight fast, before breakfast at 0800 hours and before Dry weight (g) 83.0 (70.7–104.3) 81.3 (67.7–105.4) supper at 1800 hours. Serum concentrations of lathosterol, Plant sterols (mg) 631 (500–937) 243 (206–337) cholesterol, and plant sterols (beta-sitosterol, stigmasterol Net cholesterol (mg) 681 (540–1229) 623 (346–883) and campesterol) were determined by GLC (Miettinen, Bile acids (mg) 634 (174–970) 482 (111–692) 1982). Lathosterol was expressed in` ımol/100 mmol choles- Net sterols (mg) 1460 (896–1863) 964 (702–1575) terol as simultaneously derived during analysis. Variation Cholesterol absorption (%) 50 (31–59) 56 (27–69) coefficients in duplicate samples of sera were 4.4% for Values are medians and ranges for 24 h. Bold values indicate Po0.05 by lathosterol, 3.6% for cholesterol, and 4.9% for plant sterols. Wilcoxon’s signed rank sum test.

European Journal of Clinical Nutrition Monounsaturated oils and cholesterol metabolism L Ellega˚rd et al 1377 Table 4 Serum levels of phytosterols, cholesterol, lathosterol, lathoster- relation between dietary cholesterol and serum cholesterol ol/cholesterol ratio (L/C), and 7a-hydroxy-4-cholesten-3-one (7a-HC) in levels. nine ileostomy subjects after 10 h with rapeseed oil vs olive oil diet In this study, bile acid excretion rose by 32% on rapeseed Rapeseed oil Olive oil oil compared to olive oil, which was mirrored by a 8% increase in serum 7a-hydroxy-4-cholesten-3-one, a marker Plant sterols (mmol/l) 32.1 (10–58) 23.7 (8–58) Cholesterol (mmol/l) 5.5 (4.2–6.6) 5.9 (4.6–6.8) for bile acid synthesis. This increase could be detected within Lathosterol (mmol/l) 11.8 (6.5–22) 12.3 (6.8–22.9) 10 h after consumption. This rapid response was first described by us in a controlled experiment in healthy m L/C ratio ( mol/l/100 mmol) subjects (Andersson et al, 2002). Plant sterols in high doses cholesterol 225 (127–352) 229 (162–340) 7a-HC (ng/ml) 29 (17–71) 27 (18–58) have been shown to decrease cholesterol absorption (Nor- me´n et al, 2000), as well as to increase excretion of Values are medians and ranges. Bold values indicate Po0.05 by Wilcoxon’s cholesterol (Miettinen et al, 2000), but whether plant sterols signed rank sum test. naturally occurring in normal food would have these effects have not been reported until recently. The plant sterol content of ordinary foods seems to influence cholesterol Plant sterol excretion was 160% higher on rapeseed oil absorption, as found in single-meal experiments (Ostlund than on olive oil, reflecting the higher plant sterol content of et al, 2002, 2003). We have earlier found a negative the rapeseed oil. correlation between natural plant sterols in food and Data on serum analyses are presented in Table 4. Serum cholesterol absorption in ileostomy subjects (Ellegard et al, cholesterol concentration as determined by GLC during 2000b). Interestingly, the decline in cholesterol absorption— analysis for lathosterol, decreased by 7% (P ¼ 0.024) within although of borderline statistical significance—is close to 10 h of consumption of rapeseed oil compared to olive oil that predicted (Ellegard et al, 2000b). We also found a diet. 7a-hydroxy-4-cholesten-3-one, as a marker for bile acid positive correlation between changes in net sterol excretion synthesis, increased by median 8% (P ¼ 0.038) within 10 h of cholesterol and plant sterol excretion in a retrospective on rapeseed oil diet compared to olive oil. The lathosterol/ analysis of 14 different dietary interventions in ileostomy cholesterol ratio, as well as the absolute lathosterol concen- subjects (Ellega˚rd et al, 2000a). This supports the fact that tration in serum, as markers for cholesterol synthesis, part of the effect of cholesterol-lowering dietary interven- remained unchanged after 10 h of consumption of the tions on sterol metabolism could be attributed to the plant rapeseed oil compared to olive oil, as did the phytosterol sterol content. Dietary intake of natural plant sterols has concentration in serum. been shown to be inversely related to serum cholesterol levels in men and women in the EPIC-Norfolk population (Andersson et al, 2004). Thus, the present study, together with previous reported experiences, indicates that plant Discussion sterols intrinsic in vegetables, fatty foods, and cereals are Rapeseed oil and olive oil are both good sources of biologically active in cholesterol metabolism, to increase monounsaturated oleic acid and are often, from a dietetic sterol excretion, and to a lesser extent, decrease cholesterol perspective, considered equal with respect to cholesterol absorption. metabolism (Wood et al, 1998). However, as has been recently reviewed in this journal, oils high in monounsaturated fatty acids do not have the same effect on plasma Acknowledgements cholesterol (Truswell & Choudhury, 1998). The expertise and technical assistance provided by Vibeke This small but controlled study shows that rapeseed oil Malmros is greatly appreciated. Supported by the Gothen- might have more impact on cholesterol metabolism than burg Medical Society, Grant number: 91/00, the Swedish olive oil. Thus, rapeseed oil tended to decrease cholesterol Board for Agricultural Research (SJFR) Grant 50.0444/98, the absorption, increased cholesterol excretion, and actually Swedish Medical Society, Grant number 2002-405 and from decreased serum cholesterol levels within 10 h of consump- the Sahlgrenska University Hospital under the LUA-agree- tion of the rapeseed oil diet. This is supported by findings of ment. Pedersen et al (2000)who reported rapeseed oil to be more effective than olive oil in reducing LDL-cholesterol, and attributed the differences in part to the squalene and plant References sterol differences between the oils. The small but rapid 7% Andersson H & Bosaeus I (1993): Sterol balance studies in man. A decrease in serum cholesterol by rapeseed oil with 326 mg critical review. Eur. J. Clin. 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