European Journal of Clinical Nutrition (2007) 61, 896–905 & 2007 Nature Publishing Group All rights reserved 0954-3007/07 $30.00 www.nature.com/ejcn

ORIGINAL ARTICLE Similar serum plant responses of human subjects heterozygous for a mutation causing sitosterolemia and controls to diets enriched in plant or stanols

M Kratz1, F Kannenberg1, E Gramenz1, B Berning1, E Trautwein2, G Assmann1 and S Rust1

1Leibnitz-Institute of Arteriosclerosis Research at the University of Muenster, Germany; 2Unilever Food and Health Research Institute, Unilever Research & Development, The Netherlands

Objective: We investigated the serum responses of heterozygous relatives of sitosterolemia patients to diets enriched in or stanols. Design: Randomized double-blind crossover design. Setting: Muenster, Germany. Subjects: Eight heterozygous and 13 control subjects were recruited. One heterozygote and three controls dropped out. Interventions: Seven heterozygotes and 10 controls received daily portions of containing 2 g of plant sterols, 2 g of stanols or a control margarine for 6 weeks each in a randomized order. These phases were intercepted by wash-out periods of 6 weeks each. Results: Compared to the control period, serum phytosterol concentrations increased overall by more than 20% when subjects consumed the plant sterol margarine (F(1,15) ¼ 8.719, P ¼ 0.01), with no significant difference between heterozygotes (mean þ 14.5 (s.d. 17.2) mmol/l, þ 23.0%) and controls ( þ 4.9 (9.9) mmol/l, þ 20.5%; F(1,15) ¼ 2.168, P ¼ 0.162), but decreased when subjects consumed the stanol-enriched margarine (F(1,15) ¼ 12.124, P ¼ 0.003), again to a similar extent in heterozygotes (À34.2 (41.2) mmol/l, À54.2%) and controls (À12.2 (9.2) mmol/l, À50.6%; F(1,15) ¼ 2.729, P ¼ 0.119). The lowest total serum concentrations of and phytosterols were seen after the diet enriched in stanols. Serum stanol concentrations increased on this diet, but on a very low level and never exceeded 0.05% of serum cholesterol levels in any subject. Conclusions: Serum phytosterol concentrations increased only moderately in heterozygotes consuming a diet enriched in phytosterols, indicating that they retained considerable capacity to excrete phytosterols even at higher intakes. Sponsorship: Supported by a grant of the Stifterverband fu¨r die Deutsche Wissenschaft (project number TS022/12372/2002) to MK. European Journal of Clinical Nutrition (2007) 61, 896–905; doi:10.1038/sj.ejcn.1602598; published online 17 January 2007

Keywords: sitosterolemia; phytosterols; plant sterols; campesterol; sitosterol

Introduction Correspondence: Dr M Kratz, Division of Metabolism, Endocrinology, and Nutrition, School of Medicine, University of Washington, Box 356426, 1959 NE Pacific Street, Seattle, WA, 98195, USA. Phytosterols such as b-sitosterol and campesterol are plant- E-mail: [email protected] derived sterols that are structurally similar to cholesterol Guarantor: M Kratz. (Law, 2000; Ostlund, 2002). Although phytosterols and Contributors: MK was responsible for study design and execution, statistical cholesterol are absorbed by the same principle mechanism, analyses and writing of the manuscript; FK measured serum phytosterol and stanol concentrations, and was involved in manuscript preparation; EG and BB it is remarkable that the absorption rates for plant sterols and were involved in planning, designing and executing the study; ET and GA their hydrogenated derivatives, stanols, are much lower than were involved in designing the study and in manuscript preparation and SR those for cholesterol (Salen et al., 1992b; Heinemann et al., was responsible for measuring ABCG5/G8 genotypes, analysis and interpreta- 1993; Ostlund et al., 2002). This is owing to the action of tion of the results, and was involved in manuscript preparation. Received 21 April 2006; revised 3 November 2006; accepted 3 November two half-transporters, ATP-binding cassette transporters (ABC) 2006; published online 17 January 2007 G5 and G8, which form a heterodimer located in the apical Diet and heterozygous sitosterolemia M Kratz et al 897 cell membranes of enterocytes that is capable of pumping obligate heterozygotes than in controls. We set up the plant sterols and stanols back into the intestinal lumen present study to investigate whether serum plant sterol once they have been taken up (Lee et al., 2001). ABCG5 and concentrations increase distinctly above normal levels in ABCG8 are also expressed in hepatocytes (Graf et al., 2003), subjects heterozygous for mutations in ABCG5 or ABCG8 where they are involved in the excretion of sterols and after the consumption of a daily dose of 2 g plant sterols. As stanols into the bile (Sudhop et al., 2002b). Owing to the the ABCG5/G8 genotypes of our subjects differed from those high effectiveness of these transporters, plant sterols and of the subjects enrolled in the study by Kwiterovich et al., stanols are both poorly absorbed and quickly removed from their response to a diet enriched in plant sterols might be the human body (Sudhop et al., 2002b). The serum different. Also, we were interested to see how these patients’ concentration of total plant sterols is usually in the range serum plant sterol and stanol concentrations change in of 12–20 mmol/l, and therewith well below 1% of the total response to a daily dose of 2 g stanols. We further analyzed serum cholesterol level (Miettinen et al., 1990; Jones et al., all known common variants of ABCG5/G8 in our study 1997). participants, as several reports have suggested an impact of Plant sterols and stanols have become a focus of biome- some variants on transporter capacity. Other end points were dical research after it was reported that uptake of plant the concentrations of total cholesterol, LDL-cholesterol, sterols or stanols decreases low-density lipoprotein (LDL)- high-density lipoprotein (HDL)-cholesterol and triglycerides, cholesterol concentrations in serum (Law, 2000; Katan et al., which were measured in serum gained before and after each 2003). Because of these LDL-cholesterol-lowering effects, diet period. and other food products like milk and yoghurt that are fortified with either plant sterols or stanols have been marketed in many European countries, the US and Methods Australia in recent years. Typical daily doses of these products contain about 2 g of either plant sterols or stanols, Subjects which is the dose found most effective in lowering LDL- Our specific alternative hypothesis H1 stated that the cholesterol concentrations. absolute increase in serum total plant sterols following the Data from a rare recessive disorder, sitosterolemia, imply consumption of a plant sterol-enriched margarine would that plant sterols are more atherogenic than cholesterol. The be at least 25 mmol/l higher in subjects heterozygous for molecular basis for this disease is a defect in both the alleles critical mutations in ABCG5 or ABCG8 compared to controls. of either ABCG5 or ABCG8 (reviewed in Lee et al., 2001) that Sample size calculation revealed that we needed eight leads to a distinctly increased absorption and reduced biliary controls and eight heterozygotes to be able to show this excretion of plant sterols (Salen et al., 2000; Lee et al., 2001). difference with a power of 90% at Po0.05. Therefore, it was Total serum plant sterol concentrations are much higher in decided to include between eight and 13 subjects per group, affected patients than in controls and range from about 500 depending on availability, to be able to cope with dropouts. to 2250 mmol/l (Salen et al., 1992a). Clinically, most patients The heterozygous subjects were identified by screening the are characterized by typically located xanthomas, and many family members of two patients suffering from sitosterole- also suffer from markedly augmented atherosclerosis that mia. These two subjects had serum campesterol concentra- frequently leads to fatal cardiovascular events very early in tions of 130 and 213 mmol/l, respectively and b-sitosterol life (Salen et al., 1992a). concentrations of 102 and 233 mmol/l, respectively. One of Subjects that are heterozygous for a critical mutation the sitosterolemic patients was compound heterozygous for in either ABCG5 or ABCG8 also have somewhat higher two defects in the ABCG8 gene. One was a Q442X mutation baseline plant sterol concentrations than control subjects in exon 9, the other a L572P mutation in exon 11. Although (Kwiterovich et al., 2003). This indicates that plant sterol L572P has been described as a cause of sitosterolemia before absorption is higher, and/or that the biliary excretion is (Lu et al., 2001), Q442X is new and should give rise to a lower. Serum plant sterol concentrations might therefore truncated protein. We found either of these two mutations increase even more distinctly after consumption of plant in seven out of 11 screened relatives of this patient. One sterol-enriched food items in these individuals than in the of these seven did not want to participate. Therefore, six general population. A stronger increase in addition to the members of this family, three with the Q442X mutation and already higher baseline levels might lead to serum plant three with the L572P mutation, were included into this sterol concentrations that are well above what is normally study. The other phytosterolemic patient was compound observed in the population, and might put these subjects at heterozygous for two defects in ABCG5: one R446X muta- an increased risk of atherosclerosis. Kwiterovich et al. (2003) tion in exon 10 and one H510D mutation in exon 11. We were able to show that obligate heterozygote relatives of found one of these two mutations in three out of five patients with sitosterolemia and controls without critical screened relatives of this patient. One of these three did not mutations in ABCG5 and ABCG8 responded similarly to a want to participate in the study. Therefore, two members of diet enriched in phytosterols. However, absolute serum this family, one with the R446X mutation and the other with phytosterol concentrations were several-fold higher in the H510D mutation, were included into the study. Of the

European Journal of Clinical Nutrition Diet and heterozygous sitosterolemia M Kratz et al 898 Table 1 Baseline characteristics of study subjectsa

All subjects Heterozygotes Controls Heterozygotes vs controls

N 17 7 10 Age (years) 32 (8) 34 (5) 30 (10) P ¼ 0.351 Body weight (kg) 68 (13) 70 (16) 66 (11) P ¼ 0.514 Height (m) 1.75 (0.11) 1.77 (0.14) 1.73 (0.09) P ¼ 0.524 Body-mass-index (kg/m2) 22.0 (2.4) 22.2 (2.1) 21.9 (2.7) P ¼ 0.833 Total cholesterolb (mmol/l) 5.37 (0.75) 5.81 (0.72) 5.07 (0.63) P ¼ 0.040 LDL-cholesterolb (mmol/l) 2.77 (0.58) 3.09 (0.49) 2.54 (0.55) P ¼ 0.051 HDL-cholesterolb (mmol/l) 1.78 (0.41) 1.71 (0.44) 1.83 (0.41) P ¼ 0.575 Triglyceridesc (mmol/l) 1.35 (0.88) 1.75 (1.25) 1.07 (0.38) P ¼ 0.102 b-Sitosterold (mmol/l) 10.1 (5.7) 14.6 (5.3) 6.9 (3.5) P ¼ 0.003 b-Sitosterol (% of cholesterol) 0.20 (0.10) 0.27 (0.10) 0.15 (0.07) P ¼ 0.009 Campesterole (mmol/l) 23.3 (16.0) 37.0 (14.7) 13.7 (8.0) P ¼ 0.001 Campesterol (% of cholesterol) 0.43 (0.26) 0.65 (0.22) 0.28 (0.16) P ¼ 0.001

aData are means (s.d.) bTo convert to mg/dl, multiply by 38.67. cTo convert to mg/dl, multiply by 88.57. dTo convert to mg/l, divide by 2.41. eTo convert to mg/l, divide by 2.5.

eight heterozygous subjects included, one dropped out because she had to undergo surgery that was not related to the study. The control subjects were found by placing an advertise- ment in a local newspaper. Eighty-one subjects responded to this advertisement, and 21 of these were chosen for a screening visit by drawing lots. Of these, three subjects were not eligible because of participation in another study or the intake of lipid-lowering drugs, and three subjects retracted before the screening visit. The remaining 15 subjects were questioned about their medical and dietary history, and a Figure 1 Design of the study. Each subject received a control venipuncture was performed. One of these subjects was margarine, a margarine enriched in plant sterols and a margarine excluded because of a history of alcohol abuse and one enriched in stanols in a randomized order for 6 weeks. The individual because of impaired glucose tolerance. Other exclusion margarine phases were intercepted by wash-out periods of also 6 weeks. Fasting blood samples were drawn during clinical visits on criteria were the presence of severe illnesses such as day 1 and day 4371 of each margarine phase. cardiovascular disease, diabetes mellitus, or cancer, drug abuse and any illness associated with malabsorption such as celiac disease or colitis ulcerosa. The genes encoding ABCG5 and G8 were sequenced of the remaining 13 subjects to weeks each were intercepted by two wash-out periods of 6 exclude critical mutations. All were included into the study. weeks each. In the study periods, the subjects were asked to Of these, one was excluded because she did not appear for consume 25 g per day of a low-fat control margarine (phase her first clinical visit, and two others were excluded because A), a low-fat margarine enriched in plant sterol esters (phase of lack of compliance with the study regimen. Thus, 10 B) or stanol esters (phase C) in addition to their habitual diet. healthy controls finished the study. The baseline character- The margarines contained 35.5% fat and 8% plant sterols or istics of the 10 controls and the seven heterozygous subjects stanols. The composition of the plant sterol margarine was who completed the study are shown in Table 1. b-sitosterol 56.7%, campesterol 24.7%, stigmasterol 8.8%, The protocol and the objectives of the study were sitostanol 3.0%, brassicasterol 2.9%, campestanol 0.7% and explained to the participants in detail. All gave written others 2.9%. The composition of the stanol margarine was consent. The study protocol was approved by the Ethics sitostanol 64.8%, campestanol 32.1%, b-sitosterol 1.3%, Committee of the University of Muenster. campesterol 0.6%, stigmasterol 0.3% and others 1.0%. Neither the study team nor the participants were aware of the allocations of the three margarines to the three Design and diets diet periods. At the beginning of the study, the subjects The study was conducted in a randomized double-blind were randomized into six groups. Each of these groups crossover design (Figure 1). Three main study periods of 6 received the margarines in a different order (Figure 1). This

European Journal of Clinical Nutrition Diet and heterozygous sitosterolemia M Kratz et al 899 randomization was performed separately for heterozygotes sitostanol and campestanol were 0.12 mmol/l. For statistical and controls. analyses, those below the detection limit were entered as The subjects were asked to retain their usual diet and 0.06 mmol/l. lifestyle throughout the study. In each of the three main dietary periods, they were instructed to complete a 3-day dietary record (2 weekdays, 1 weekend day). This dietary Determination of ABCG5 and ABCG8 genotypes record was used to calculate the habitual dietary nutrient For mutation screening, we created polymerase chain and sterol uptake. In addition to these records, the subjects reaction (PCR)-products of promoter and exon sequences, were instructed to keep a study diary of their margarine including at least 50 bp upstream and 20 bp downstream of consumption. Intakes of the control, phytosterol and stanol the exons to cover relevant sites for lariat-formation and margarines were 25.5 (1.5), 25.3 (1.7) and 25.3 (2.5) g per day splice site recognition. Primer sequences and PCR conditions (means (s.d.), n ¼ 17). As the subjects also consumed a small are available upon request. PCR products were sequenced amount of plant sterols with their habitual diet, the daily using BigDye Terminator technology on a 96-lane capillary plant sterol intakes were 97 (60), 2033 (162) and 170 (54) mg sequencer (Applied Biosystems, Darmstadt, Germany). in periods A, B and C (means (s.d.)). The average intake of Heterozygous subjects were identified by sequencing of plant stanols was 0 (0), 75 (5) and 1963 (196) mg per day in exons that contained the critical mutation in their periods A, B and C. sitosterolemic relative. The control subjects were completely Each subject was asked to visit the clinic on six occasions sequenced for the coding regions of ABCG5 and ABCG8 to throughout the study, on day 1 and day 4371 of each exclude occurrence of any mutations. Also, we analyzed all dietary period. On these days, the subjects were asked to participants of the study for the common polymorphisms arrive early in the morning after an overnight fast of at ABCG8 D19H, Y54C, T400K, A632V and ABCG5 Q604E, least 10 h. A venipuncture was performed, the subjects were which, except for Y54C, have been described to have a weighted in light clothing and a nutritionist of the study significant impact on sterol levels (Berge et al., 2002; team met with the subject to hand out the margarine Weggemans et al., 2002). Furthermore, we identified a containers or to collect leftovers. polymorphism in ABCG5, R50C (in 8 of 34 alleles), that was in strong linkage disequilibrium with ABCG8 D19H. In our study, all ABCG5 R50C alleles seemed to be located on Laboratory methods ABCG8 D19H chromosomes. Only one excess ABCG8 D19H Serum and ethylenediamine-N,N,N0,N0-tetraacetic acid allele without ABCG5 R50C was found. A table listing all (EDTA)-plasma was isolated immediately after the venipunc- genotypes is available online as Supplementary material. ture by centrifugation for 15 min at 2000g and 41C. The serum and EDTA-plasma samples were frozen to À701C and stored until analysis. Statistics Statistical analyses were performed using the Statistical Package for the Social Sciences (version 11, SPSS Inc., Concentrations of lipids and lipoproteins in serum Chicago, IL, USA). Serum concentrations of all variables Serum concentrations of total cholesterol and triglycerides except triglycerides and lathosterol were found to be were measured using enzymatic assays (based on Ro¨schlau approximately normally distributed, as confirmed by check- et al., 1974; Siedel et al., 1993). HDL- and LDL-cholesterol ing normal plots and histograms of the data and by concentrations were measured using precipitation methods performing Kolmogorov–Smirnov tests. Serum triglyceride (based on Armstrong and Seidel, 1985; Sugiuchi et al., 1995). and lathosterol concentrations were logarithmically trans- These measurements were performed in series within formed to normality. By means of repeated measures analysis 2 days on a Hitachi 917 autoanalyzer (Roche Diagnostics, of variance (RM-ANOVA), it was tested whether the three Mannheim, Germany). The methods are validated by regular time points at the beginning of each margarine period (visits analyses of reference sera supplied by the national German 1, 3, and 5) showed any difference. This was not the case for INSTAND proficiency-testing program and/or the interna- any of the outcome measures. Baseline characteristics of tional quality assurance program of the US Centers for heterozygotes were compared to those of controls by means Disease Control and Prevention. of a two-sample t-test. Again, RM-ANOVA was employed to compare the three time points at the end of the control period, the plant sterol Concentrations of plant sterols and stanols in serum margarine period and the stanol margarine period (visits 2, 4 Concentrations of plant sterols and stanols in serum and 6). In these tests, the data of visits 2, 4 and 6 for each were measured by gas chromatography-mass spectrometry variable were used as the three levels of the within-subject (GC-MS) as described before (Assmann et al., 2006). For factor (time). Group (heterozygous vs control) was used as a quantitation of campestanol the response factors of sitostanol between-subjects factor. In case this test indicated a statisti- were used. The detection limits for the measurement of cally significant difference, we conducted post hoc either

European Journal of Clinical Nutrition Diet and heterozygous sitosterolemia M Kratz et al 900 another RM-ANOVA (using a reduced number of levels of the than in controls (À17.0 (11.3) mmol/l or À59.0%;

within-subject factor) or a paired t-test. These tests were two- F(1,15) ¼ 6.548; P ¼ 0.022 for the time  group interaction tailed, and the level of significance was set to Po0.05. in RM-ANOVA). We observed the same pattern for the individual plant sterols, campesterol and b-sitosterol (Table 2). Compared to Results the control margarine, the mean serum campesterol con- centration was 7.2 (10.9) mmol/l or 25% higher after the Plant sterol and stanol concentrations in serum consumption of the plant sterol-enriched margarine

Serum plant sterol concentrations (sum of campesterol þ (F(1,15) ¼ 9.405, P ¼ 0.008) and 15.5 (20.2) mmol/l or 54% b-sitosterol) were higher when subjects had consumed the lower after the consumption of the plant stanol margarine

plant sterol-enriched margarine (F(1,15) ¼ 8.719, P ¼ 0.01 for (F(1,15) ¼ 12.872, P ¼ 0.003). Serum levels of b-sitosterol time effect in RM-ANOVA). Although this increase was increased by 1.6 mmol/l or 14% during the diet rich in plant absolutely greater in heterozygous subjects (mean: þ 14.5 sterols (95% confidence interval (CI) 0.04 to 3.17 mmol/l,

(s.d.: 17.2) mmol/l) than in controls ( þ 4.9 (9.9) mmol/l), it T(16) ¼ 2.177, P ¼ 0.045, Table 2), and decreased by 5.7 mmol/l was similar in relative terms ( þ 23.0 vs þ 20.5%) and not or 50% during the diet rich in plant stanols (95% CI À1.44

significantly different between the groups (F(1,15) ¼ 2.168, to À10.06 mmol/l, T(16) ¼ 2.830, P ¼ 0.012) compared to the P ¼ 0.162 for time  group interaction in RM-ANOVA). In control diet. Accordingly, the difference was largest between contrast, serum plant sterol concentrations were lower the plant sterol and the stanol margarine period (7.3 mmol/l,

when subjects had consumed the stanol-enriched diet 95% CI 3.18 to 11.53 mmol/l, T(16) ¼ 3.737, P ¼ 0.002).

(F(1,15) ¼ 12.124, P ¼ 0.003 for time effect in RM-ANOVA). These changes in serum campesterol and b-sitosterol Again, the absolute difference was greater in heterozygotes concentrations to the three diets were independent of the (À34.2 (41.2) mmol/l) than in controls (À12.2 (9.2) mmol/l), subjects’ group affiliation, that is, heterozygous and control but similar in relative terms (À54.2 vs À50.6%) and did not subjects did not differ in their plant sterol responses to these differ significantly between heterozygotes and controls diets. The one exception was a significantly greater differ-

(F(1,15) ¼ 2.729, P ¼ 0.119 for time  group interaction in ence in serum campesterol concentrations in heterozygotes RM-ANOVA). When comparing this stanol-enriched diet (À36.7 (26.6) mmol/l or À64.2%) than in controls (À12.9 with the plant sterol-enriched diet, however, we observed a (8.5) mmol/l or À62.3%) when comparing the sterol-enriched

significantly greater difference in serum phytosterol con- period with the stanol–enriched period (F(1,15) ¼ 7.139, centrations in heterozygotes (À48.7 (37.7) mmol/l or À62.8%) P ¼ 0.017 for the interaction time  group in RM-ANOVA).

Table 2 Serum concentrations of campesterol, b-sitosterol, campestanol and sitostanol (mmol/l) during the studya

N Control margarine Plant sterol margarine Stanol margarine RM-ANOVAb

Time Time  group

Campesterol a b c All subjects 17 28.5 (25.3) 35.8 (27.8) 13.0 (8.4) F(1.5,22.2) ¼ 19.812; Po0.001 F(1.5,22.2) ¼ 4.534; P ¼ 0.032 Heterozygotes 7 45.5 (31.7) 57.3 (30.9) 20.5 (6.8) Controls 10 16.7 (9.6) 20.7 (11.0) 7.8 (4.6)

b-Sitosterol a b c All subjects 17 11.5 (10.1) 13.1 (10.1) 5.8 (2.9) F(1.2,18.2) ¼ 13.900; P ¼ 0.001 F(1.2,18.2) ¼ 3.277; P ¼ 0.080 Heterozygotes 7 17.6 (13.6) 20.3 (12.4) 8.4 (1.9) Controls 10 7.2 (3.0) 8.1 (3.1) 3.9 (1.8)

Campestanolc a a b All subjects 17 ND ND 0.41 (0.41) F(2,30) ¼ 20.389; Po0.001 F(2,30) ¼ 2.135; P ¼ 0.136 Heterozygotes 7 ND ND 0.58 (0.50) Controls 10 ND ND 0.30 (0.30)

Sitostanolcc a a b All subjects 17 ND ND 0.25 (0.27) F(2,30) ¼ 23.663; Po0.001 F(2,30) ¼ 6.405; P ¼ 0.005 Heterozygotes 7 ND ND 0.42 (0.31) Controls 10 ND ND 0.13 (0.16)

Abbreviation; RM-ANOVA, repeated measures analysis of variance. aData are means (s.d). bRM-ANOVA with three levels of the inner-subject factor time (control- vs plant sterol- vs stanol period) and group (heterozygotes vs controls) as between-subjects variable. Data with different letters differ from each other at Po0.05 in post hoc analysis. c‘ND’ indicates that levels were below the detection limit of 0.12 mmol/l.

European Journal of Clinical Nutrition Diet and heterozygous sitosterolemia M Kratz et al 901 Table 3 Serum total, LDL-, and HDL-cholesterol and triglyceride concentrations (mmol/l) during the studya

N Control margarine Plant sterol margarine Stanol margarine RM-ANOVAb

Time Time  group

Total cholesterol a a,b b All subjects 17 5.03 (0.76) 4.91 (0.89) 4.72 (0.55) F(2,30) ¼ 3.481; P ¼ 0.044 F(2,30) ¼ 0.068; P ¼ 0.934 Heterozygotes 7 5.11 (1.09) 5.03 (1.14) 4.80 (0.64) Controls 10 4.97 (0.48) 4.82 (0.72) 4.67 (0.51)

LDL-cholesterol All subjects 17 2.52 (0.51) 2.42 (0.71) 2.29 (0.43) F(2,30) ¼ 3.086; P ¼ 0.060 F(2,30) ¼ 0.193; P ¼ 0.825 Heterozygotes 7 2.62 (0.66) 2.56 (0.85) 2.37 (0.56) Controls 10 2.45 (0.39) 2.32 (0.62) 2.24 (0.33)

HDL-cholesterol a,b a b All subjects 17 1.77 (0.45) 1.78 (0.43) 1.68 (0.45) F(2,30) ¼ 3.358; P ¼ 0.048 F(2,30) ¼ 0.384; P ¼ 0.685 Heterozygotes 7 1.67 (0.43) 1.69 (0.43) 1.62 (0.42) Controls 10 1.83 (0.47) 1.85 (0.43) 1.72 (0.49)

Triglycerides All subjects 17 1.31 (0.80) 1.32 (0.72) 1.32 (0.72) F(2,30) ¼ 0.074; P ¼ 0.929 F(2,30) ¼ 0.426; P ¼ 0.657 Heterozygotes 7 1.65 (1.18) 1.50 (0.96) 1.59 (1.00) Controls 10 1.07 (0.22) 1.19 (0.51) 1.13 (0.38)

Abbreviations; HDL, high-density lipoprotein; LDL, low-density lipoprotein; RM-ANOVA, repeated measures analysis of variance. aData are means (s.d.). bRM-ANOVA with three levels of the inner-subject factor time (control- vs plant sterol- vs stanol-margarine period) and group (heterozygotes vs controls) as between- subjects variable. Data with different letters differ from each other at Po0.05 in post hoc paired t-tests.

Albeit overall on a very low level, serum stanol concentra- control diet (mean difference: À0.31 mmol/l; 95% CI À0.56 tions (sum of campestanol þ sitostanol) were distinctly to À0.06 mmol/l; T(16) ¼ 2.599; P ¼ 0.019). HDL-cholesterol higher when subjects had consumed the stanol-enriched levels in serum were lower after the diet rich in plant stanols margarine compared to when they had consumed the other compared to the diet rich in plant sterols (mean difference: two margarines (F(2,30) ¼ 20.351, Po0.001 for time effect in À0.10 mmol/l; 95% CI À0.18 to À0.03 mmol/l; T(16) ¼ 2.848; RM-ANOVA). The difference between the stanol margarine P ¼ 0.012). The serum concentrations of LDL-cholesterol period and the other two margarine periods also was greater tended to be lower after subjects had consumed the diet in heterozygotes than in controls (F(2,30) ¼ 3.722, P ¼ 0.036 enriched in plant sterols and particularly stanols. Serum for time  group interaction in RM-ANOVA). The same was triglycerides did not change throughout the study. true for serum sitostanol concentrations, which were elevated after the subjects had consumed the diet enriched in stanols (F(1,15) ¼ 23.663, Po0.001, Table 2). As was the case Serum concentrations of cholesterol precursor sterols for total stanols, the difference between the plant stanol diet During the study, no statistically significant changes were and both the control and the plant sterol diet was greater for observed in the serum concentrations of desmosterol and heterozygous as compared to control subjects (F(1,15) ¼ 6.405, lathosterol (Table 4). P ¼ 0.023 for the interaction time  group in RM-ANOVA). In contrast, heterozygotes and controls did not differ in their campestanol response to the stanol-enriched diet, Discussion which increased to a similar degree in these two groups

(T(16) ¼ 4.180, P ¼ 0.001 for control vs stanol and plant sterol A major finding of this study was that the diet enriched in vs stanol, respectively, in post hoc paired t-tests). plant sterols increased serum campesterol and b-sitosterol concentrations in both groups. This increase was similar in heterozygous and control subjects in relative terms, as total Concentrations of lipids and lipoproteins in serum plant sterol concentrations increased by 23 and 21%, Overall, no statistically significant differences were observed respectively. We had hypothesized that the difference in between heterozygous and control subjects with regard to total serum plant sterol concentrations between the control their serum total, LDL- and HDL-cholesterol and triglyceride and the plant sterol margarine period would be at least responses to the study diets (Table 3). Serum total cholesterol 25 mmol/l higher in the heterozygous as compared to the concentrations were significantly lower after subjects had control subjects. We had assumed that a lower transporter consumed the diet rich in plant stanols compared to the activity combined with distinctly increased phytosterol

European Journal of Clinical Nutrition Diet and heterozygous sitosterolemia M Kratz et al 902 Table 4 Serum desmosterol and lathosterol concentrations (mmol/l) during the studya

N Control margarine Plant sterol margarine Stanol margarine RM-ANOVAb

Time Time  group

Desmosterol All subjects 17 4.1 (1.6) 3.8 (1.9) 4.1 (2.2) F(2,30) ¼ 0.332; P ¼ 0.720 F(2,30) ¼ 1.012; P ¼ 0.376 Heterozygotes 7 4.5 (2.0) 4.3 (2.3) 5.2 (3.0) Controls 10 3.8 (1.2) 3.5 (1.6) 3.3 (1.1)

Lathosterol All subjects 17 4.7 (2.6) 4.5 (2.8) 5.2 (3.9) F(2,30) ¼ 1.033; P ¼ 0.368 F(2,30) ¼ 0.732; P ¼ 0.489 Heterozygotes 7 4.8 (3.9) 4.8 (3.7) 6.5 (5.8) Controls 10 4.6 (1.3) 4.3 (2.3) 4.3 (1.3)

Abbreviation; RM-ANOVA, repeated measures analysis of variance. aData are means (s.d.). bRM-ANOVA with three levels of the inner-subject factor time (control- vs plant sterol- vs stanol period) and group (heterozygotes vs controls) as between-subjects variable.

intake would trigger a disproportional serum plant sterol whereas the studies carried out by Berge, Plat and colleagues elevation in heterozygotes compared to controls. Sample size compared the 604QQ homozygous normal individuals calculation revealed that finishing eight subjects per group to a group consisting of Q604E heterozygotes and 604EE would give us a power of 90% to find this difference. As we homozygotes, supposedly because of insufficient numbers of were unable to recruit more than eight heterozygotes, and 604EE subjects. Weggemans suggested that 604E is associated one of these dropped out of the study, only seven hetero- with elevated cholesterol absorption and hypothesized zygotes finished the study. However, the actually observed that the same would be true for phytosterol absorption. difference between control subjects and heterozygotes Unfortunately, data on phytosterols were not available in was only 9.6 mmol/l, and therewith distinctly lower than their study. hypothesized. If we assume, as mentioned above, that heterozygosity of a Our data clearly show that the ABCG5/G8 genotype was mutation or polymorphism is associated with a relatively more important in determining serum phytosterol concen- small effect on serum phytosterol concentrations, then this trations than the amount of phytosterols in the diet. Even might explain why those studies comparing homozygotes under the standardized dietary conditions of this study, for the more efficient transporter allele 604Q with a mixture serum plant sterol concentrations showed considerable of heterozygotes and 604EE homozygotes were not able to inter-individual variability, both in heterozygotes and in detect a significant impact on phytosterols. Application of controls. A possible explanation for this might be the large this paradigm also to the other common variant where the number of polymorphisms in the genes encoding ABCG5 less frequent allele (ABCG8 632V), has been shown to be a and ABCG8 that have been described. It has been weak transporter would predict that bigger effects may be reported previously that ABCG8 D19H, T400K and A632V seen in 632VV homozygotes than in the heterozygotes. affect serum phytosterol concentrations (Berge et al., 2002; We therefore analysed our samples in subgroups (Figure 2) to Plat et al., 2005). It is somewhat surprising that in these compare controls carrying at least one normal allele (control studies effects were evident even in subjects who were group 1) with controls that were homozygous carriers of heterozygous for the weaker transporter variant ABCG8 small effect variants (control group 2). Members of group 2 632 V, given that one fully functional allele seems to be were either ABCG5 604EE or ABCG8 632VV, and had sufficient to provide 90–95% of the normal transport distinctly higher serum phytosterol concentrations than capacity. This is suggested by the fact that serum phytosterol those carrying at least one fully functional allele at all time concentrations in subjects heterozygous for mutations points. Although it will be important to confirm this finding causing sitosterolemia resemble those seen in the normal in a bigger study specifically designed to test this hypothesis, population much more closely than those seen in we believe that this comparison might be of considerable homozygous patients. A larger study by Weggemans et al. importance as control group 2 represents about 8% of the focused on the effect of the ABCG5 Q604E variant on population (calculated for the respective two homozygous serum cholesterol concentrations. Their comparison of the variants ABCG5 604 EE and ABCG8 632 VV, using data different genotypes indicated that serum cholesterol levels published by Berge et al. (2002)). It is important to note that were similar in 604QQ and Q604E subjects, whereas the we had insufficient data to evaluate the effects of ABCG8 homozygous 604EE had significantly elevated baseline D19H (Berge et al., 2002) or ABCG8 T400K (Berge et al., cholesterol values. A larger number of 604EE homozygotes 2002), which have also been reported to affect serum in the study by Weggemans made this analysis possible, phytosterol levels.

European Journal of Clinical Nutrition Diet and heterozygous sitosterolemia M Kratz et al 903 50 * * * event (Jones, 2004). Thus, it is assumed that lowering of serum LDL-cholesterol concentrations by an increased intake of plant sterols or stanols should also lower the risk of 40 coronary heart disease (CHD). On the other hand, there is a lack of data on the atherogenic potential of plant sterols and particularly stanols. Therefore, it cannot be completely 30 excluded that the reduction in CHD risk attributed to lower serum LDL-cholesterol concentrations is compensated by the 20 2 increase in serum plant sterols or stanols. Particularly, this 2 might be the case in subjects with increased absorption and retention of sterols and stanols such as the relatives of 10 sitosterolemic patients studied here. This is implied by 2 1 1 observations in patients suffering from sitosterolemia, which 1 suggest that plant sterols are more atherogenic than

Total phytosterol serum concentration (µmol/L) phytosterol Total 0 cholesterol. Also, new data from our group suggest that Stanol margarine Control margarine Plant sterol margarine elevated serum sitosterol concentrations may be a risk factor Figure 2 Serum concentrations of total phytosterols for CHD (Assmann et al., 2006). This finding indicates that (campesterol þ b-sitosterol) in control subjects with at least one fully not only pathologically high serum phytosterol concentra- functional allele (subgroup 1, n ¼ 6), and control subjects homo- zygous for a common variant known to affect transporter function tions as seen in sitosterolemia but also slightly elevated (ABCG5 604EE or ABCG8 632VV, subgroup 2, n ¼ 4). Data shown concentrations might be associated with an increased are means and standard errors of the means. *Po0.05 in long-term risk of cardiovascular events. In support of this independent samples t-tests. hypothesis, Glueck et al. (1991) found an association between serum phytosterol concentrations and family history of CHD. A similar finding was published by Sudhop The major findings of our study are in good agreement et al. (2002a) who reported that CHD patients with a family with the study published recently by Kwiterovich et al. history of CHD had higher serum plant sterol concentrations (2003). In addition, we found that the diet enriched in than patients without a family history of CHD (22.1 vs stanols led to reduced plant sterol levels in all participants. 17.0 mmol/l). It should be noted that the phytosterol- Particularly, our findings suggest that stanols are an effective enriched margarine elevated serum plant sterols (sum of means of reducing elevated serum plant sterol concentra- campesterol and b-sitosterol) to 28.8 mmol/l in the control tions as seen in our heterozygous subjects. Both controls and group and even to 43.3 mmol/l in the control subgroup 2, heterozygotes seemed to have sufficient capacity to excrete which is clearly above the concentration that has previously stanols as suggested by the very low stanol serum levels, even been linked to an increased risk of CHD. after the subjects had consumed the diet enriched in stanols. On the other hand, no relationship between plasma levels This is in contrast to sitosterolemia patients, who show of phytosterols and atherosclerosis or CHD risk was found distinctly higher serum levels of sitostanol and campestanol by Wilund et al. (2004), who investigated this question in on diets enriched in stanols (Connor et al., 2005). both mice and humans. Also, the findings discussed above A considerable number of studies have consistently shown do not prove a causal relationship between CHD and that a daily consumption of 2 g or more of plant sterols or serum plant sterol levels, as these associations might be stanols is capable of reducing serum concentrations of LDL- explained by underlying genetic defects that affect both cholesterol by about 10% (Law, 2000; Katan et al., 2003). serum plant sterol concentrations and the risk of CHD. Even During the stanol diet period, we observed a similar more so, it remains unclear whether a diet-induced increase reduction in LDL-cholesterol, whereas the effect seen during in serum plant sterol concentrations would increase the risk the plant sterol diet was somewhat less. However, these of CHD. Also, it is still unclear whether stanols have any effects were not statistically significant in this study. atherogenic potential. As these are the least absorbed sterols, Apparently, this was owing to the fact that our power the total serum concentrations of cholesterol and plant calculation was based on a different outcome measure. Thus, sterols were lowest when subjects consumed a diet fortified the sample size likely was too small to detect effects of the in stanols, whereas serum stanol concentrations did not diets on LDL-cholesterol concentrations. exceed 0.05% of serum cholesterol levels in any of the At present, it is difficult to assess the relevance of our subjects studied. findings. It is known from large epidemiological studies that Generally, most heterozygous subjects in our study as well the serum concentration of LDL-cholesterol shows a strong as in the study carried out by Kwiterovich et al. (2003) seem positive correlation with cardiovascular disease (Cullen and to have a substantial capacity to excrete sterols and stanols. Assmann, 1999). Also, lowering of serum LDL-cholesterol However, one heterozygous subject in our study, the only concentrations, for example by means of statin treatment, one with the H510D mutation, developed serum total has been associated with a reduced risk of a cardiovascular phytosterol concentrations during the control and the plant

European Journal of Clinical Nutrition Diet and heterozygous sitosterolemia M Kratz et al 904 sterol-enriched diets that came close to the levels we with sitosterolemia and xanthomatosis and in rat tissues. Lipids measured in her sitosterolemic son (161 and 166 vs 40, 919–923. 232 mmol/l). These levels are much higher than those Cullen P, Assmann G (1999). High risk strategies for atherosclerosis. Clin Chim Acta 286, 31–45. observed in heterozygotes for nonsense mutations. The Glueck CJ, Speirs J, Tracy T, Streicher P, Illig E, Vandegrift J (1991). effect might be owing to efficient incorporation of this Relationships of serum plant sterols (phytosterols) and cholesterol mutant protein into an inactive ABCG5/G8 complex (domi- in 595 hypercholesterolemic subjects, and familial aggregation of nant negative effect), leading to a larger impact of this phytosterols, cholesterol, and premature coronary heart disease in hyperphytosterolemic probands and their first-degree relatives. variant than that of other missense and especially nonsense Metabolism 40, 842–848. variants. It should be noted that the diet enriched in stanols Graf GA, Yu L, Li WP, Gerard R, Tuma PL, Cohen JC et al. (2003). led to a distinct reduction in this patient’s total serum plant ABCG5 and ABCG8 are obligate heterodimers for protein sterol concentration (36 mmol/l). trafficking and biliary cholesterol excretion. J Biol Chem 278, 48275–48282. Taken together, diets enriched in either plant sterols or Heinemann T, Axtmann G, von Bergmann K (1993). Comparison of stanols have previously been shown to lower the serum intestinal absorption of cholesterol with different plant sterols in concentration of LDL-cholesterol and the LDL-/HDL-choles- man. Eur J Clin Invest 23, 827–831. terol ratio. Data presented here now show that the Jones PH (2004). Low-density lipoprotein cholesterol reduction and cardiovascular disease prevention: the search for superior treat- concentration of plant sterols in serum of subjects hetero- ment. Am J Med 116 (Suppl 6A), 17S–25S. zygous for critical mutations in ABCG5 and/or ABCG8 and Jones PJ, MacDougall DE, Ntanios F, Vanstone CA (1997). Dietary controls is elevated moderately and to a similar extent phytosterols as cholesterol-lowering agents in humans. Can J by consuming 2 g of plant sterols daily, but lowered by Physiol Pharmacol 75, 217–227. Katan MB, Grundy SM, Jones P, Law M, Miettinen T, Paoletti R consuming 2 g of stanols daily. Very small amounts of stanols (2003). Efficacy and safety of plant stanols and sterols in can be detected in the serum of most subjects after the the management of blood cholesterol levels. Mayo Clin Proc 78, consumption of the stanol-enriched diet. As the data about 965–978. atherogenicity as well as other potential health effects of Kwiterovich Jr PO, Chen SC, Virgil DG, Schweitzer A, Arnold DR, plant sterols and stanols is incomplete, it is hard to weigh the Kratz LE (2003). Response of obligate heterozygotes for phytoster- olemia to a low-fat diet and to a plant sterol ester dietary potential benefits and risks associated with an intake of large challenge. J Lipid Res 44, 1143–1155. amounts of plant sterols and stanols against each other. Law M (2000). Plant sterol and stanol margarines and health. BMJ Whatever their net health effect might be, serum levels of 320, 861–864. plant sterols and stanols will be affected by genotype, dietary Lee MH, Lu K, Patel SB (2001). Genetic basis of sitosterolemia. Curr Opin Lipidol 12, 141–149. habits and enrichment of the diet with phytosterols or Lu K, Lee MH, Hazard S, Brooks-Wilson A, Hidaka H, Kojima H et al. stanols. (2001). Two genes that map to the STSL locus cause sitosterolemia: genomic structure and spectrum of mutations involving sterolin-1 and sterolin-2, encoded by ABCG5 and ABCG8, respectively. Am J Hum Genet 69, 278–290. Acknowledgements Miettinen TA, Tilvis RS, Kesaniemi YA (1990). Serum plant sterols and cholesterol precursors reflect cholesterol absorption and synthesis in volunteers of a randomly selected male population. Am J We thank R Hagel, E Jung, A Mischke, M Schreiner, M Epidemiol 131, 20–31. Toebbe, and S Wentker for excellent technical assistance, Ostlund Jr RE (2002). Phytosterols in human nutrition. Annu Rev Nutr and the study subjects for participation. Supported by a grant 22, 533–549. Ostlund Jr RE, McGill JB, Zeng CM, Covey DF, Stearns J, Stenson WF of the Stifterverband fu¨r die Deutsche Wissenschaft (project et al. (2002). Gastrointestinal absorption and plasma kinetics of number TS 022/12372/2002) to MK. Study margarines were soy delta(5)-phytosterols and phytostanols in humans. Am J provided by Unilever, Rotterdam, The Netherlands. Physiol Endocrinol Metab 282, E911–E916. Plat J, Bragt MC, Mensink RP (2005). Common sequence variations in ABCG8 are related to plant sterol metabolism in healthy volunteers. J Lipid Res 46, 68–75. References Ro¨schlau P, Bernt E, Gruber Z (1974). Enzymatische Bestimmung des Gesamtcholesterins im Serum. Z Klin Chem Klin Biochem 12, Armstrong V, Seidel D (1985). Evaluation of a commercial kit for the 403–408. determination of LDL-cholesterol in serum based on precipitation Salen G, Shefer S, Nguyen L, Ness GC, Tint GS, Shore V (1992a). of LDL with dextran sulfate. A¨rztl Lab 31, 325–330. Sitosterolemia. J Lipid Res 33, 945–955. Assmann G, Cullen P, Erbey J, Ramey DR, Kannenberg F, Schulte H Salen G, Tint GS, Shefer S, Shore V, Nguyen L (1992b). Increased (2006). Plasma sitosterol elevations are associated with an sitosterol absorption is offset by rapid elimination to prevent increased incidence of coronary events in men: results of a nested accumulation in heterozygotes with sitosterolemia. Arterioscler case–control analysis of the Prospective Cardiovascular Munster Thromb 12, 563–568. (PROCAM) study. Nutr Metab Cardiovasc Dis 16, 13–21. Salen G, Xu G, Tint GS, Batta AK, Shefer S (2000). Hyperabsorption Berge KE, von Bergmann K, Lutjohann D, Guerra R, Grundy SM, and retention of campestanol in a sitosterolemic homozygote: Hobbs HH et al. (2002). Heritability of plasma noncholesterol comparison with her mother and three control subjects. J Lipid Res sterols and relationship to DNA sequence polymorphism in 41, 1883–1889. ABCG5 and ABCG8. J Lipid Res 43, 486–494. Siedel J, Schmuck R, Staepels J, Town MH (1993). Long term stable, Connor WE, Lin DS, Pappu AS, Frohlich J, Gerhard G (2005). Dietary liquid ready-to-use monoreagent for the enzymatic assay of serum sitostanol and campestanol: accumulation in the blood of humans or plasma triglycerides (GPO-PAP-method). Clin Chem 39, 1127.

European Journal of Clinical Nutrition Diet and heterozygous sitosterolemia M Kratz et al 905 Sudhop T, Gottwald BM, von Bergmann K (2002a). Serum plant in serum with polyethylene glycol-modified enzymes and sulfated sterols as a potential risk factor for coronary heart disease. alpha-cyclodextrin. Clin Chem 41, 717–723. Metabolism 51, 1519–1521. Weggemans RM, Zock PL, Tai ES, Ordovas JM, Molhuizen HO, Katan Sudhop T, Sahin Y, Lindenthal B, Hahn C, Luers C, Berthold HK et al. MB (2002). ATP binding cassette G5 C1950G polymorphism may (2002b). Comparison of the hepatic clearances of campesterol, affect blood cholesterol concentrations in humans. Clin Genet 62, sitosterol, and cholesterol in healthy subjects suggests that efflux 226–229. transporters controlling intestinal sterol absorption also regulate Wilund KR, Yu L, Xu F, Vega GL, Grundy SM, Cohen JC et al. (2004). biliary secretion. Gut 51, 860–863. No association between plasma levels of plant sterols and Sugiuchi H, Uji Y, Okabe H, Irie T, Uekama K, Kayahara N et al. atherosclerosis in mice and men. Arterioscler Thromb Vasc Biol 24, (1995). Direct measurement of high-density lipoprotein cholesterol 2326–2332.

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