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Red cell and plasma plant sterols are related during consumption of plant stanol and sterol ester spreads in children with hypercholesterolemia
RED CELL AND PLASMA PLANT STEROLS ARE RELATED DURING CONSUMPTION OF PLANT STANOL AND STEROL ESTER SPREADS IN CHILDREN WITH HYPERCHOLESTEROLEMIA ANNA M. KETOMÄKI, MD, HELENA GYLLING, MD, PHD, MARJATTA ANTIKAINEN, MD, PHD, MARTTI A. SIIMES, MD, PHD, AND TATU A. MIETTINEN, MD, PHD
Objective To show whether the ratios of squalene and cholesterol precursor sterols to cholesterol and cholestanol and plant sterols to cholesterol change differently in plasma and especially in the red cells of hypercholesterolemic children during consumption of plant stanol and sterol ester spreads. Study design In a randomized, double-blind, crossover study, hypercholesterolemic children (n = 23) consumed low-fat plant stanol and sterol ester spreads for 5-week periods separated by a 5-week washout period. Plasma and red cell lipids, squalene, and noncholesterol sterols were measured before and at the end of each period. Results The plant stanol and sterol ester spreads lowered plasma total (–9% and –6%, respectively) and low-density lipoprotein (–12% and –9%) cholesterol but had no effect on red cell cholesterol, high-density lipoprotein cholesterol, or plasma triglycerides. The ratios of plasma and red cell sitosterol and campesterol to cholesterol decreased by 32% to 36% (P < .001) with the plant stanol ester and increased by 40% to 52% (P < .001) with the sterol ester spread. Conclusions Consumption of plant sterols increases and consumption of plant stanols decreases the ratios of plant sterols to cholesterol in red cells of hypercholesterolemic children proportionately to the respective changes in plasma. (J Pediatr 2003;142:524-31)
lant sterols, including sitosterol, campesterol, stigmasterol, and avenasterol, are present in the human diet, especially in vegetable oils. The amount of plant sterols ingested daily may be similar to the amount of dietary cholesterol, varying from 160 to 360 mg/d.1 Normally, only <10% of the plant sterols are absorbed from the intestine, whereas the 5α-saturated stanols are almost unabsorbable.2 Thus, human serum normally contains varying amounts of plant sterols. Subjects with high ratios of plant sterol to cholesterol in serum have high absorption of cholesterol and plant sterols. In general, the serum plant sterol to cholesterol ratios correlate positively with absorption and negatively with synthesis of cholesterol, two variables of cholesterol metabolism, with clear inheritable features.3,4 Plant sterols from normal food interfere with absorption of cholesterol and lower its levels in serum.5 Addition of plant sterols or stanols to the diet in free or in esterified form further inhibits cholesterol absorption and lowers concentrations of serum total and lowdensity lipoprotein (LDL) cholesterol.6–15 However, in children, a phytosterol-enriched diet increased the concentrations of serum plant sterols16 to the values observed in phytosterolemia.17 In adults also, intake of plant sterol esters increased the serum concentrations of plant sterols.18,19 Free serum cholesterol is rapidly exchanged with that in red cell membranes20 and, depending on its concentration, can modify red cell morphology and hemorrheology.21 In studies in vitro, plant sterols were also incorporated into the red cell membranes.22 Elevated contents of red cell sitosterol have been found in cases of phytosterolemia17 and in
P
FH GLC
524
Familial hypercholesterolemia Gas-liquid chromatography
HDL LDL
High-density lipoprotein Low-density lipoprotein
From the Division of Internal Medicine, Department of Medicine, and the Hospital for Children and Adolescents, University of Helsinki, and Biomedicum, Helsinki, and the Department of Clinical Nutrition, University of Kuopio, and Kuopio University Hospital, Finland. Supported by a grant from Raisio Benecol Ltd, Raisio, Finland and Finnish Heart Research Association. Submitted for publication Sept 20, 2002; revision received Dec 27, 2002; accepted Feb 14, 2003. Reprint requests:Tatu A. Miettinen, MD, Division of Internal Medicine, Department of Medicine, University of Helsinki, PO BOX 340, FIN-00029 HUS, Finland. E-mail: [email protected]. Copyright © 2003 Mosby, Inc. All rights reserved. 0022-3476/2003/$30.00 + 0
10.1067/mpd.2003.193
healthy adults eating a sitosterol-enriched diet.23 It may be assumed that the hemolysis observed in phytosterolemia24 results from accumulation of plant sterols in the red cell membranes. However, there are no systematic studies on the physiological occurrence of squalene and noncholesterol sterols (cholesterol precursors and plant sterols) in human red cells or reports of their reduction or accumulation in the red cell membranes during the consumption of varying phytosterol mixtures, especially in children, who have excessively high absorption of plant sterols, as suggested by an earlier study.16 We therefore investigated the plasma and red cell squalene and noncholesterol sterols and their changes in children with hypercholesterolemia before and during consumption of plant stanol and sterol ester spreads in a randomized, double-blind study, and compared the plasma and red cell values with each other.
METHODS Subjects The study population was 23 prepubertal children age 2 to 9 years (10 boys and 13 girls) with a mean age of 6.8 ± 0.5 (SEM) years, a mean height of 120.5 ± 3.5 cm, and a mean weight of 23.1 ± 1.5 kg who were patients at the Outpatient Clinic of the Hospital for Children and Adolescents, University of Helsinki. The only inclusion criteria were that the serum total cholesterol value should be >5.0 mmol/L (>194 mg/dL) and serum triglycerides <2.0 mmol/L (<176 mg/dL). Of the 23 subjects, none was on hypolipidemic medication. The use of the plant stanol or sterol ester spread was stopped 2 weeks before entry into the study. Patients with diabetes or with kidney, liver, or thyroid disease were excluded from the study. Informed consent was obtained from the parents. The study protocol was approved by the Ethics Committee of the Hospital for Children and Adolescents, University of Helsinki. Of the 23 children, 16 had familial hypercholesterolemia (FH) diagnosed by polymerase chain reaction25–27 for the four most common Finnish mutations (FH-Helsinki [n = 10], FH-North Karelia [n = 4], FH-Turku [n = 0], FH-Pori [n = 0]) or by the lymphocyte test (n = 2).28 One child without a Finnish ancestry had a family member with FH, and the child’s serum lipid levels and family history fit to FH. Accordingly, the child was included in the FH group. The six remaining children were negative for the four Finnish mutations and did not have a family history of FH.
Study Design The study had a double-blind, randomized, crossover design. There were two 5-week intervention periods with a 5week washout on an ad libitum home diet between the interventions. Half of the children started randomly with a scheduled low-fat (32%) plant stanol (2.0 g/d) ester spread and the other half with a similarly scheduled low-fat (35%) plant sterol (2.0 g/d) ester spread. The fatty acid compositions of the plant stanol (Light Benecol, Raisio Benecol Ltd, Raisio, Finland) and sterol ester spreads (Becel pro.activ, Unilever BestFoods, Purfleet, UK) were 16% and 25% of saturated fatty Red Cell and Plasma Plant Sterols are Related During Consumption of Plant Stanol and Sterol Ester Spreads in Children With Hypercholesterolemia
acids, 56% and 25% of monounsaturated fatty acids, and 28% and 50% of polyunsaturated fatty acids, respectively. Both spreads contained 6 mg/100g and 66 mg/100 g of vitamin E, respectively, but the amounts of supplemented vitamin A (900 µg/100g) and vitamin D (7.5 µg/100g) were similar. The patients were advised to replace 25 g of daily dietary fat with the respective spreads on pieces of bread, but when this was too satiating, the spreads were also added to such food as porridge. The subjects visited the Outpatient Clinic four times, at the beginning and end of the two study periods. At each visit, a blood sample was taken in the morning after an overnight fast. Height and weight were also measured. A routine physical examination was performed at the first and last visits. The spread containers were returned to the study nurse, and an approximation of the daily intake was calculated by weighing the returned unwashed containers.
Methods Plasma total and free cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol levels after precipitation of apolipoprotein-B–containing lipoproteins were analyzed by enzymatic color reactions with commercial kits (total and HDL cholesterol, CHOD-PAP; triglycerides, GPO-PAP, Roche, Basel, Switzerland; free cholesterol, COD-PAP, Wako Chemicals, Neuss, Germany) by using a semiautomatic Mira analyzer (Roche, Basel, Switzerland). LDL cholesterol was calculated according to Friedewald et al.29 The red cells were washed three times with 0.9% sodium chloride and centrifuged at 3000 rpm for 5 minutes. The plasma and red cell squalene and noncholesterol sterols, ie, lathosterol, ∆8-cholestenol, and desmosterol (the cholesterol precursor markers),30,31 and cholestanol and plant sterols (the cholesterol absorption markers)31–33 were analyzed by gas-liquid chromatography (GLC) on a 50-m capillary column (Ultra-2, 5890, Hewlett-Packard, Wilmington, Del).34,35 In brief, after addition of 5α-cholestane as internal standard, the samples were saponified, and the nonsaponifiable lipid extracts were silylated and injected into the gas chromatograph. The squalene and noncholesterol sterol values are expressed in terms of 102 mmol/mol cholesterol to eliminate the influence of varying cholesterol concentrations and are expressed as ratios unless otherwise mentioned. Serum cholesterol and triglycerides are given in mmol/L. For mg/dL, cholesterol was multiplied by 38.7 and triglycerides by 88.2. The plant stanols and sterols in the spreads were also analyzed by GLC. The plant stanol ester spread contained 1.8% sitosterol, 1.5% campesterol, 0% stigmasterol, 0% avenasterol, 0.05% cholesterol, 0.06% squalene, 66.8% sitostanol, and 29.8% campestanol. The respective values for the plant sterol ester spread were 51.5%, 24.3%, 20.5%, 1.3%, 0.4%, 0.1%, 1.2%, and 0.6%. No cholestanol was detected in either margarine.
Statistical Analysis All the statistical analyses were performed by using the NCSS statistical software package (NCSS, 2000, Kaysville, Utah). After studying the carry-over effect by analysis of vari525
Table I. Plasma lipids and their changes* Stanol† (n = 22)
Sterol‡ (n = 23)
Variable
Baseline
Change, %
Baseline
Change, %
Cholesterol Ester, % LDL cholesterol HDL cholesterol TG
6.73 ± 0.21 73.9 ± 0.9 5.15 ± 0.24 1.10 ± 0.07 1.05 ± 0.09
–9 ± 3§ –4 ± 2 –12 ± 3 6±6 5±7
6.72 ± 0.22 73.2 ± 0.5 5.15 ± 0.22 1.12 ± 0.06 1.0 ± 0.07
–6 ± 2§ –0.7 ± 0.6 –9 ± 3 6±5 1±8
*Mean ± SEM, mmol/L. For mg/dL, multiply cholesterol 38.7 and triglycerides 88.2. †During stanol ester spread. ‡During sterol ester spread. Significantly different from baseline, §P < .01, P < .001, paired t test.TG, Triglycerides.
Table II. Plasma baseline concentrations and % changes in cholesterol* and ratios† of squalene and noncholesterol sterols to cholesterol Stanol‡ (n = 22) Variable Cholesterol Squalene ∆8-cholestenol Desmosterol Lathosterol Campesterol Sitosterol Avenasterol Cholestanol Campestanol Sitostanol
Sterol§ (n = 23)
Baseline
Change, %
Baseline
Change, %
6.3 ± 0.2 25 ± 3 12 ± 1 88 ± 2 78 ± 5 437 ± 32 190 ± 12 49 ± 2 184 ± 8 1.7 ± 0.4 3.5 ± 0.7
–8 ± 2b 16 ± 7 25 ± 9 10 ± 4a 34 ± 9b –36 ± 4c –32 ± 4c –30 ± 3c –4 ± 3 350c 367c
6.3 ± 0.2 23 ± 2 11 ± 1 87 ± 2 75 ± 4 450 ± 26 201 ± 10 52 ± 2 183 ± 7 1.5 ± 0.3 2.2 ± 0.4
–7 ± 2b 29 ± 12 36 ± 16 16 ± 4c 30 ± 7c 52 ± 6c¶ 43 ± 5c¶ –10 ± 2c¶ –6 ± 3a 23¶ ±0
*mmol/L, mean ± SEM. For mg/dL, multiply 38.7. †102 mmol/mol cholesterol, mean ± SEM. ‡During stanol ester spread. §During sterol ester spread. Analyzed by GLC. ¶Significant difference between the stanol and sterol ester groups, P < .001, paired t test. Significantly different from before: a, P < .05; b, P < .01; c, P < .001, paired t test.
ance for repeated measures, we calculated the means (SEM) for the plant stanol and sterol groups. In the calculations, according to the intention-to-treat principle, we included the plant sterol ester spread data of one child who left the study before the last visit during the stanol ester period. After logarithmic transformations for the skewed variables, the paired t test was used to evaluate the differences during and between the treatments. The correlation coefficients were calculated by using Pearson product moment and Spearman rank correlation in appropriate cases. A P value <.05 was considered significant.
for the sterol ester spread. The children consumed daily an average of 19.9 g (range, 6.9–33.9 g) of the stanol ester spread and of 21.0 g (range, 10.1–28.6 g) of the sterol ester spread. Accordingly, the plant stanols and sterols consumed averaged 1.6 g and 1.7 g per day, respectively, corresponding to 80% and 85% of the scheduled dose. The children reported no adverse effects. There was a slight but insignificant increase in body weight and height during the 15-week study period. There was virtually no carry-over effect.
RESULTS
The baseline values and the changes in the plasma lipids are shown in Table I. The concentrations of total and LDL cholesterol tended to decrease more with the plant stanol ester
Final data were obtained from 22 children (nine boys, 13 girls) for the stanol ester and 23 children (10 boys, 13 girls) 526 Ketomäki et al
Plasma Lipids
The Journal of Pediatrics • May 2003
spread than with the sterol ester spread, by 0.64 ± 0.17 mmol/L (25 ± 7 mg/dL) and 0.43 ± 0.15 mmol/L (17 ± 6 mg/dL) for total cholesterol and by 0.69 ± 0.16 mmol/L (27 ± 6 mg/dL) and 0.51 ± 0.13 mmol/L (20 ± 5 mg/dL) for LDL cholesterol, respectively. No significant difference in the esterification percentages of total cholesterol was found between the groups during the study. The total and LDL cholesterol values decreased by >10% and >15%, respectively, in 11 children with the plant stanol ester spread and in eight children with the plant sterol ester spread. We observed correlations between the individual amounts of plant stanol and sterol ester spreads ingested and the decreases in LDL cholesterol (r = 0.448, r = 0.428, P < .05 for both), and also between the ingested amount of plant stanol ester spread ingested and the decrease in total cholesterol (r = 0.515, P < .05). No significant changes were detected in the HDL cholesterol or serum triglyceride concentrations. At the baseline, the total and LDL cholesterol values were insignificantly higher in the FH group than in the non-FH group, and the reductions in the total and LDL cholesterol concentrations during the spread periods were similar in the FH and non-FH groups and also in the sexes (data not shown).
Table III. Correlations between changes in plasma and red cell cholesterol and ratios of squalene and noncholesterol sterol to cholesterol with respective baseline values Change Baseline Cholesterol Squalene ∆8-cholestenol Desmosterol Lathosterol Campesterol Sitosterol
Plasma Squalene and Noncholesterol Sterols Of the ratios of noncholesterol sterols to cholesterol, only that of ∆8-cholestenol was higher and that of campestanol lower in the girls than in the boys, but the responses during the consumption of the spreads were similar in the sexes. The baseline ratios of the cholesterol precursors were negatively related to those of the absorption markers, eg, r = –0.471 (P < .05) for ∆8-cholestenol versus campesterol. The ratios of the cholesterol synthesis markers increased by as much as 36% with both spreads (Table II). The ratios of avenasterol, present only in the sterol ester spread, decreased more with the stanol ester than with the sterol ester spread. The ratios of cholestanol exhibited borderline reductions in both groups. The major difference between the two spread periods was that during the plant sterol ester period, the ratios of both campesterol and sitosterol increased by 52 ± 6% and 43 ± 5%, respectively (P < .001), but during the plant stanol ester period, they decreased by 36 ± 4% and 32 ± 4%, respectively (P < .001). The plasma ratios of campestanol and sitostanol increased several-fold only with the plant stanol ester spread (Table II), but the respective concentrations remained low, 19 µg/dL for campestanol and 31 µg/dL for sitostanol. Of the noncholesterol sterol ratios, only the baseline campesterol ratio predicted the subsequent decrease in the concentrations of plasma total and LDL cholesterol during the plant sterol ester spread period (r = 0.421 and r = 0.452, P < .05 for both), suggesting a better cholesterol-lowering response in the children with high than in children with low baseline cholesterol absorption. The lower the baseline ratios of the plasma precursors of cholesterol, the higher were their increments during both treatment periods (Table III). The baseline ratios of the cholesterol absorption markers were negatively related to the reRed Cell and Plasma Plant Sterols are Related During Consumption of Plant Stanol and Sterol Ester Spreads in Children With Hypercholesterolemia
Avenasterol Cholestanol Campestanol Sitostanol
Plasma Red cell Plasma Red cell Plasma Red cell Plasma Red cell Plasma Red cell Plasma Red cell Plasma Red cell Plasma Red cell Plasma Red cell Plasma Red cell Plasma Red cell
Stanol* (n = 22)
Sterol† (n = 23)
–0.358 –0.470a –0.424a –0.430a –0.617b –0.771c –0.399 –0.703c –0.497a –0.797c –0.625b –0.652c –0.264 –0.604b –0.420a –0.593c –0.632c –0.475a –0.341 0.018 –0.569b –0.246
–0.360 –0.655c –0.477a –0.365 –0.310 –0.813c –0.477a –0.658c –0.229 –0.401 0.014 0.054 0.489a 0.360 –0.488a –0.586b –0.361 –0.508a –0.597b –0.772c –0.541b –0.650c
*During stanol ester spread. †During sterol ester spread. a, P < .05; b, P < .01; c, P < .001.
spective changes caused by the stanol esters, whereas the higher the baseline sitosterol ratio, the higher was its increment caused by the sterol esters. Reductions in the avenasterol and cholestanol ratios occurred mainly in the children with the highest baseline values, whereas the increases in the campesterol ratios caused by the sterol esters were independent of the baseline values.
Red Cell Squalene and Noncholesterol Sterols The red cell cholesterol concentrations remained constant during the treatment periods (Table IV), but some reduction occurred in the subjects with high baseline values, as revealed by the negative correlations in Table III. However, the ratios of the plant sterols decreased >30% during the plant stanol ester spread period but increased >40% during the consumption of the plant sterol ester spread. The ratios of avenasterol decreased during the plant stanol period and those of cholestanol during the plant sterol period, whereas the cholesterol synthesis markers increased variably by both spreads. The ratios of plant stanols increased several-fold by consumption of the plant stanol spread, 527
Table IV. Red cell baseline and percent changes in cholesterol* and ratios† of squalene and noncholesterol sterols to cholesterol Stanol‡ (n = 22) Variable
Sterol§ (n = 23)
Baseline Change, % Baseline Change, %
Cholesterol Squalene ∆8-cholestenol Desmosterol Lathosterol Campesterol Sitosterol Avenasterol Cholestanol Campestanol Sitostanol
117 ± 3 –1 ± 2 34 ± 6 21 ± 11 13 ± 1 44 ± 28 64 ± 2 13 ± 5a 208 ± 13 23 ± 10 514 ± 34 –36 ± 4c 224 ± 14 –32 ± 4c 66 ± 3 –22 ± 4c 212 ± 13 –3 ± 5 0.6 ± 0.3 1400c 6±2 283c
115 ± 2 31 ± 3 10 ± 1 63 ± 2 204 ± 11 528 ± 30 236 ± 13 66 ± 3 228 ± 12 0.6 ± 0.3 8±2
4±2 20 ± 10 158 ± 59b 17 ± 7a 21 ± 8 47 ± 6c 40 ± 5c 5 ± 6 –17 ± 4b ± 0 –38
*mg/dL, mean ± SEM. †102 mmol/mol cholesterol, mean ± SEM. ‡During stanol ester spread. §During sterol ester spread. Significant difference between the stanol and sterol ester groups, P < .001, paired t test. Significantly different from before: a, P < .05; b, P < .01; c, P < .001, paired t test.
but the respective concentrations remained low (11 ± 2 µg/dL for campestanol and 25 ± 2 µg/dL for sitostanol). In red cells, the baseline ratios of the cholesterol precursors and the absorption markers were related to their changes similarly as in plasma (Table III). Neither the total nor the free plasma cholesterol was correlated with red cell cholesterol. However, many baseline plasma and red cell noncholesterol sterol concentrations and ratios to cholesterol were positively correlated (Table V). In addition, with both spreads, their changes in the plasma predicted the respective changes in the red cells (Table V ), as shown individually for sitosterol in the Figure.
DISCUSSION The plant sterol ester spread was investigated as a cholesterol-lowering agent for the first time in a comparable study with a plant stanol ester spread in hypercholesterolemic children on an ad libitum home diet. A previous observation that, during a plant sterol-enriched diet, serum plant sterols were elevated in children16 resulted in our rationale to evaluate the effects of a sterol ester spread on the plasma and red cell sterols. The red cells served as markers of tissue cells and, accordingly, as indicators of sterol accumulation in the tissues. The new observations in the current study were that in hypercholesterolemic children, the plant sterol ratios were elevated in the red cells during consumption of the plant sterol ester spread, whereas during consumption of the stanol ester spread, their respective values decreased, with the changes related to those observed in plasma. 528 Ketomäki et al
Table V. Correlations between plasma baseline and red cell baseline values, plasma changes with red cell changes in cholesterol, and ratios of squalene and noncholesterol sterols to cholesterol
Baseline Red cell vs plasma n = 23 Cholesterol Squalene ∆8-cholestenol Desmosterol Lathosterol Campesterol Sitosterol Avenasterol Cholestanol Campestanol Sitostanol
–0.212 0.131 –0.498a 0.497a 0.767c 0.963c 0.981c 0.235 0.508a –0.249 –0.301
Changes Red cell vs plasma Stanol* (n = 22)
Sterol† (n = 23)
0.216 0.032 0.364 0.537b 0.807c 0.896c 0.923c 0.455a 0.343 0.517b 0.248
0.197 –0.104 0.228 0.748c 0.838c 0.822c 0.952c 0.431a 0.144 0.124 –0.060
*During stanol ester spread. †During sterol ester spread. a, P < .05; b, P < .01; c, P < .001.
Squalene and Noncholesterol Sterols in Red Cell Membranes The red cell noncholesterol sterols have not been previously related to those in plasma. The current baseline data showed that the red cell membranes contained most of the noncholesterol sterols in ratios proportional to those observed in the plasma lipoproteins, despite the fact that the red cells contain only free sterols and the plasma lipoproteins also contain esterified sterols. Actually, rough calculations from the data of one previous study36 showed similar values, eg, for free campesterol and sitosterol to free cholesterol in plasma and in red cells. Thus, similar to cholesterol, noncholesterol sterols seemed to exchange freely with the plasma lipoproteins and red cells. However, squalene was exchanged less frequently between plasma lipoproteins and red cells, resulting in higher ratios of squalene to free cholesterol in the plasma than in the red cells (ratios 103 vs 34 102 mmol/mol of cholesterol, respectively, from Tables II and IV).
Effects of Plant Stanol Ester The current study confirms the previous findings11,12 that during consumption of plant stanol ester spread by hypercholesterolemic children, plant sterols in the plasma decrease and cholesterol precursor sterols increase. A new finding was that proportional changes also occurred in red cells and showed a high correlation with the plasma values. Respective increments were higher in children with low baseline ratios of cholesterol synthesis markers in plasma and red cells than in children with The Journal of Pediatrics • May 2003
Figure. Correlations between the changes in the ratios of plasma and red cell sitosterol to cholesterol (102 mmol/mol) during the periods of plant stanol (closed triangles) and sterol ester (open circles) spreads. y = 1.3x + 6.6 for stanol ester and y = 1.03x + 1.8 for sterol ester
high baseline ratios, indicating that their cholesterol synthesis was more enhanced. The negative baseline correlation of the precursors with the absorption markers indicates that the children with the low baseline ratios of precursor sterols in their plasma and red cells have high respective ratios of the absorption markers as a sign of effective cholesterol absorption. During the plant stanol ester period, the respective reductions in the ratios of absorption markers were higher in those with high than in those with low respective baseline values, indicating more effective inhibition of cholesterol absorption. These results suggest that the high baseline absorption of cholesterol, and probably also of other sterols, was markedly inhibited by plant stanol esters and resulted in inhibition of sterol accumulation in the red cells. The mean plasma total and LDL cholesterol reductions, 9% and 12%, were of the same magnitude as those in the previous studies with plant stanol esters (1.5–2.9 g/d) in normocholesterolemic,13 in mildly hypercholesterolemic,14 and in FH children.12 In contrast with an 8-week37 and a 1-year38 study with plant sterol ester spread with no cholesterol-lowering effect, the current short-term study in children showed reductions in plasma lipid values, as in short-term studies in adults18,19,39 and in children with FH.40 Despite some major differences in the distribution of fatty acids, possibly also affecting the cho-
lesterol-lowering effect, the plasma lipid values tended to decrease more during consumption of plant stanol spread than during consumption of plant sterol ester spread. However, even though the plant sterol ratios in plasma were increased by as much as 50% during the plant sterol ester period, the individual plasma values failed to reach the levels described in children on the phytosterol-enriched diet.16 Specifically, after consumption of 1.7 g of plant sterols in the current study, the highest concentration of plasma phytosterols, calculated as the sum of sitosterol and campesterol, was 3.5 mg/dL (0.09 mmol/L), compared with values as high as 17 mg/dL (0.44 mmol/L) reported by Mellies et al16 with even smaller doses of dietary plant sterols (300–900 mg). The reason for those remarkable elevations remains unknown, but at least, the levels observed in the current study did not differ from those reported in adults,18,19 suggesting that the absorption of phytosterols in young hypercholesterolemic children is not different from that in adults. The increments of plant sterols in the red cells are in accord with earlier results for an adult patient on a diet enriched with free β-sitosterol23 and for phytosterolemic patients.17 Moreover, the increase in both plasma and red cell sitosterol was greater in the children with high baseline plasma sitosterol levels, suggesting that more sitosterol accumulates in the plasma and red cells of children with effective absorption of cholesterol and sterols than in those with low baseline absorp-
Red Cell and Plasma Plant Sterols are Related During Consumption of Plant Stanol and Sterol Ester Spreads in Children With Hypercholesterolemia
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Effects of Plant Sterol Esters
tion. It has been shown recently that cholesterol absorption efficiency is genetically regulated,3,4 suggesting that subjects with higher sterol absorption have higher serum plant sterol levels. Whether the high serum plant sterol levels in children with high sterol absorption is harmful is not known. There is only indirect evidence showing that nonphytosterolemic subjects and their relatives with higher serum plant sterol levels had a higher incidence of atherosclerosis.41 Absorption of avenasterol and cholestanol was mainly inhibited in children with high baseline absorption of the respective sterols in both groups, resulting in lowering of the respective sterol ratios in subjects with high baseline values. Stanol esters apparently inhibited avenasterol absorption from a normal diet only (no avenasterol in the spread), lowering the ratios of plasma and red cells, whereas the presence of avenasterol in the sterol ester spread resulted in inconsistent changes in the ratios of avenasterol in the plasma and red cells. Systematic studies are lacking on the effects of large amounts of dietary plant sterols on human red cells. High content of plant sterols in red cells results in hemolysis in at least phytosterolemic patients24 and may lead to increased osmotic fragility.22 In stroke-prone spontaneously hypertensive rats, a phytosterol-rich diet made red cells less deformable and shortened the life span.42 On the other hand, in apolipoproteinE–deficient mice, lowering of serum cholesterol by phytosterols appeared to protect red cells.43 Even though the safety of plant sterol ester spread has been evaluated in long-term38 and shortterm14,18 human studies, the long-term effects on red cells and vascular endothelial cells need further investigation. In contrast with the results for noncholesterol sterols, the cholesterol concentrations in the red cells remained unchanged during the two intervention periods. This observation may relate to the fact that the levels of free cholesterol remained unchanged during both intervention periods. Surprisingly, the red cell and plasma total and free cholesterol values were not related to each other, in contrast with the high respective correlations of plant sterols. The exchange of plasma and red cell plant sterols was higher than that of cholesterol, whereas in vitro from among different sterols, plasma cholesterol exhibited the highest exchange with red cell cholesterol,44–46 a finding possibly caused by lack of alkylation of the sterol side chain in cholesterol. However, some noncholesterol sterols appeared to replace cholesterol in the red cells,22,44 suggesting that large amounts of dietary plant sterols could probably replace cholesterol in red cells and would lead to higher ratios of plant sterols to cholesterol in the red cells, as shown in the Figure. The technical assistance of Ms Leena Kaipiainen, Pia Hoffström, Anne Honkonen, and Ritva Nissilä is gratefully acknowledged.
REFERENCES 1. Miettinen TA, Tilvis RS, Kesäniemi YA. Serum plant sterols and cholesterol precursors reflect cholesterol synthesis and absorption in volunteers of a randomly selected male population. Am J Epidemiol 1990;131:20–31. 2. Heinemann T, Leiss O, von Bergmann K. Mechanism of action of plant sterols in inhibition of cholesterol absorption: comparison of sitosterol and sitostanol. Eur J Clin Pharmacol 1991;40(suppl 1):S59–69.
530 Ketomäki et al
3. Berge KE, von Bergmann K, Lutjohann D, Guerra R, Grundy SM, Hobbs HH, et al. Heritability of plasma noncholesterol sterols and relationship to DNA sequence polymorphism in ABCG5 and ABCG8. J Lipid Res 2002; 43:486–94. 4. Gylling H, Miettinen TA. Inheritance of cholesterol metabolism of probands with high or low cholesterol absorption. J Lipid Res 2002;43:1472–6. 5. Gylling H, Miettinen TA. New biologically active lipids in food, health food and pharmaceuticals. Lipidforum; Scandinavian forum for lipid research and technology, 19th Nordic Lipid Symposium, Ronneby, Sweden, June 1997, 81–6. 6. Lees AM, Mok HY, Lees RS, McCluskey MA, Grundy SM. Plant sterols as cholesterol-lowering agents: clinical trials in patients with hypercholesterolemia and studies of sterol balance. Atherosclerosis 1977;28:325–38. 7. Grundy SC, Ahrens EH, Davignon J. The interaction of cholesterol absorption and cholesterol synthesis in man. J Lipid Res 1969;10:304–15. 8. Heinemann T, Leiss O, von Bergmann K. Effect of low-dose sitostanol on serum cholesterol in patients with hypercholesterolemia. Atherosclerosis 1986;61:219–23. 9. Mattson FH, Volpenhein RA, Erickson BA. Effect of plant sterol esters on the absorption of dietary cholesterol. J Nutr 1977;107:1139–46. 10. Vanhanen H, Blomqvist S, Ehnholm C, Hyvönen M, Jauhianen M, Torstila I, et al. Serum cholesterol, cholesterol precursors, and plant sterols in hypercholesterolemic subjects with different apoE phenotypes during dietary sitostanol ester treatment. J Lipid Res 1993;34:1535–44. 11. Miettinen TA, Puska P, Gylling H, Vanhanen H, Vartiainen E. Reduction of serum cholesterol with sitostanol-ester margarine in a mildly hypercholesterolemic population. N Engl J Med 1995;333:1308–12. 12. Gylling H, Siimes MA, Miettinen TA. Sitostanol ester margarine in dietary treatment of children with familial hypercholesterolemia. J Lipid Res 1995;36:1807–12. 13. Vuorio AF, Gylling H, Turtola H, Kontula K, Ketonen P, Miettinen TA. Stanol ester margarine alone and with simvastatin lowers serum cholesterol in families with familial hypercholesterolemia caused by the FH-North Karelia mutation. Arterioscl Thromb Vasc Biol 2000;20:500–6. 14. Tammi A, Rönnemaa T, Gylling H, Rask-Nissilä L, Viikari J, Tuominen J, et al. Plant stanol ester margarine lowers serum total and low-density lipoprotein cholesterol concentrations of healthy children: the STRIP project. J Pediatr 2000;136:503–10. 15. Williams CL, Bollella MC, Strobino BA, Boccia L, Campanaro L. Plant stanol ester and bran fiber in childhood: effects on lipids, stool weight and stool frequency in preschool children. J Am Coll Nutr 1999;18:572–81. 16. Mellies M, Glueck CJ, Sweeney C, Fallat RW, Tsang RC, Ishikawa TT. Plasma and dietary phytosterols in children. Pediatrics 1976;57:60–7. 17. Bhattacharaya AK, Conner WE. Beta-sitosterolemia and xanthomatosis: a newly described lipid storage disease in two sisters. J Clin Invest 1974;53:1033–43. 18. Weststrate JA, Meijer GW. Plant sterol-enriched margarines and reduction of plasma total- and LDL-cholesterol concentrations in normocholesterolemic and mildly hypercholesterolemic subjects. Eur J Clin Nutr 1998;52:334–43. 19. Hallikainen MA, Sarkkinen ES, Gylling H, Erkkilä AT, Uusitupa MIJ. Comparison of the effects of plant sterol ester and plant stanol ester-enriched margarines in lowering serum cholesterol concentrations in hypercholesterolemic subjects on a low fat diet. Eur J Clin Nutr 2000;54:715–25. 20. Ashworth LAE, Green C. The transfer of lipids between human αlipoprotein and erythrocytes. Biochim Biophys Acta 1964;84:182–8. 21. Kanakaraj P, Singh M. Influence of hypercholesterolemia on morphological and rheological characteristics of erythrocytes. Atherosclerosis 1989;76:209–18. 22. Bruckdorfer KR, Demel RA, de Gier J, van Deenen LLM. The effect of partial replacements of membrane cholesterol by other steroids on the osmotic fragility and glycerol permeability of erythrocytes. Biochim Biophys Acta 1969;183:334–45. 23. Salen G, Ahrens EH, Grundy SM. Metabolism of β-sitosterol in man. J Clin Invest 1970;49:952–67. 24. Björkhem I, Boberg KM. Inborn errors in bile acid biosynthesis and storage of sterols other than cholesterol. In: Scriver CS, Beaudet AL, Sly WS,
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et al, editors. The metabolic and molecular bases of inherited disease. 7th ed. New York: McGraw-Hill; 1995. p. 2073–99. 25. Aalto-Setälä K, Helve E, Kovanen PT, Kontula K. Finnish type of low density lipoprotein receptor mutation (FH-Helsinki) deletes exons encoding the carboxy-terminal part of the receptor and creates an internalization-defective phenotype. J Clin Invest 1989;84:499–505. 26. Koivisto U-M, Turtola H, Aalto-Setälä K, Top B, Frants RR, Kovanen PT, et al. The familial hypercholesterolemia (FH)-North Karelia mutation of the low density lipoprotein receptor gene deletes seven nucleotides of exon 6 and is a common cause of FH in Finland. J Clin Invest 1992;90:219–28. 27. Koivisto U-M, Viikari JS, Kontula K. Molecular characterization of minor gene rearrangements in Finnish patients with heterozygous familial hypercholesterolemia: identification of two common missense mutations (Gly823→Asp and Leu380→His) and eight rare mutations of the LDL receptor gene. Am J Hum Genet 1995;57:789–97. 28. Cuthbert JA, East CA, Bilheimer DW, Lipsky PE. Detection of familial hypercholesterolemia by assaying functional low-density-lipoprotein receptors on lymphocytes. N Engl J Med 1986;314:879–83. 29. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low density lipoprotein in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972;18:499–502. 30. Miettinen TA. Serum squalene and methyl sterols as indicators of cholesterol synthesis in vivo. Life Sci 1969;8:713–21. 31. Miettinen TA, Tilvis RS, Kesäniemi YA. Serum plant sterols and cholesterol precursors reflect cholesterol absorption and synthesis in volunteers of a randomly selected male population. Am J Epidemiol 1990;131:20–31. 32. Tilvis RS, Miettinen TA. Serum plant sterols and their relation to cholesterol absorption. Am J Clin Nutr 1986;43:92–7. 33. Miettinen TA, Tilvis RS, Kesäniemi YA. Serum cholestanol and plant sterol levels in relation to cholesterol metabolism in middle-aged men. Metabolism 1989;38:136–40. 34. Miettinen TA, Koivisto PVI. Non-cholesterol sterols and bile-acid production in hypercholesterolemic patients with ileal bypass. In: Paumgartner G, Stiehl A, Gerok W, editors. Bile acids and cholesterol in health and disease. Boston (MA): MTP Press; 1983. p. 183–7. 35. Miettinen TA. Cholesterol metabolism during ketoconazole treatment in man. J Lipid Res 1988;29:43–51.
36. Strandberg TE, Tilvis RS, Miettinen TA. Metabolic variables of cholesterol during squalene feeding in humans: comparison with cholestyramine treatment. J Lipid Res 1990;31:1637–43. 37. Davidson MH, Maki KC, Umporowicz DM, Ingram KA, Dicklin MR, Schaefer E, et al. Safety and tolerability of esterified phytosterols administered in reduced-fat spread and salad dressing to healthy adult men and women. J Am Coll Nutr 2001;20:307–19. 38. Brink EJ, Hendricks HFJ. Long-term follow-up study on the use of a spread enriched with plant sterols. TNO report, V99.869. Zeist (The Netherlands): TNO Nutrition and Food Research Institute Report; March 2000. 39. Jones PJ, Raeini-Sarjaz M, Ntanios FY, Vanstone CA, Feng JY, Parsons WE. Modulation of plasma lipid levels and cholesterol kinetics by phytosterol versus phytostanol esters. J Lipid Res 2000;41:697–705. 40. Amundsen ÅL, Ose L, Nenseter MS, Ntanios F. Plant sterol ester-enriched spread lowers plasma total- and LDL-cholesterol in children with familial hypercholesterolemia. Am J Clin Nutr 2002;76:338–44. 41. Glueck CJ, Speirs J, Tracy T, Streicher P, Illig E, Vandergrift J. Relationships of serum plant sterols (phytosterols) and cholesterol in 595 hypercholesterolemic subjects, and familial aggregation of phytosterols, cholesterol, and premature coronary heart disease in hyperphytosterolemic probands and their first-degree relatives. Metabolism 1991;40:842–8. 42. Ratnayake WM, L’Abbe MR, Mueller R, Hayward S, Plouffe L, Hollywood R, et al. Vegetable oils high in phytosterols make erythrocytes less deformable and shorten the life span of stroke-prone spontaneously hypertensive rats. J Nutr 2000;130:1166–78. 43. Moghadasian MH, Nguyen LB, Shefer S, McManus BM, Frohlich JJ. Histologic, hematologic, and biochemical characteristics of apo E-deficient mice: effects of dietary cholesterol and phytosterols. Lab Invest 1999;79:355–64. 44. Child P, Kuksis A. Differential uptake of cholesterol and plant sterols by rat erythrocytes in vitro. Lipids 1982;17:748–54. 45. Child P, Kuksis A. Uptake of 7-dehydro derivatives of cholesterol, campesterol, and β-sitosterol by rat erythrocytes, jejunal villus cells, and brush border membranes. J Lipid Res 1983;24:552–65. 46. Boberg KM, Skrede B, Skrede S. Metabolism of 24-ethyl-4cholesten-3one and 24-ethyl-5-cholesten-3β-ol (sitosterol) after intraperitoneal injection in the rat. Scand J Clin Lab Invest 1986;46(suppl 184):47–54.
Red Cell and Plasma Plant Sterols are Related During Consumption of Plant Stanol and Sterol Ester Spreads in Children With Hypercholesterolemia
531
Objective To show whether the ratios of squalene and cholesterol precursor sterols to cholesterol and cholestanol and plant sterols to cholesterol change differently in plasma and especially in the red cells of hypercholesterolemic children during consumption of plant stanol and sterol ester spreads. Study design In a randomized, double-blind, crossover study, hypercholesterolemic children (n = 23) consumed low-fat plant stanol and sterol ester spreads for 5-week periods separated by a 5-week washout period. Plasma and red cell lipids, squalene, and noncholesterol sterols were measured before and at the end of each period. Results The plant stanol and sterol ester spreads lowered plasma total (–9% and –6%, respectively) and low-density lipoprotein (–12% and –9%) cholesterol but had no effect on red cell cholesterol, high-density lipoprotein cholesterol, or plasma triglycerides. The ratios of plasma and red cell sitosterol and campesterol to cholesterol decreased by 32% to 36% (P < .001) with the plant stanol ester and increased by 40% to 52% (P < .001) with the sterol ester spread. Conclusions Consumption of plant sterols increases and consumption of plant stanols decreases the ratios of plant sterols to cholesterol in red cells of hypercholesterolemic children proportionately to the respective changes in plasma. (J Pediatr 2003;142:524-31)
lant sterols, including sitosterol, campesterol, stigmasterol, and avenasterol, are present in the human diet, especially in vegetable oils. The amount of plant sterols ingested daily may be similar to the amount of dietary cholesterol, varying from 160 to 360 mg/d.1 Normally, only <10% of the plant sterols are absorbed from the intestine, whereas the 5α-saturated stanols are almost unabsorbable.2 Thus, human serum normally contains varying amounts of plant sterols. Subjects with high ratios of plant sterol to cholesterol in serum have high absorption of cholesterol and plant sterols. In general, the serum plant sterol to cholesterol ratios correlate positively with absorption and negatively with synthesis of cholesterol, two variables of cholesterol metabolism, with clear inheritable features.3,4 Plant sterols from normal food interfere with absorption of cholesterol and lower its levels in serum.5 Addition of plant sterols or stanols to the diet in free or in esterified form further inhibits cholesterol absorption and lowers concentrations of serum total and lowdensity lipoprotein (LDL) cholesterol.6–15 However, in children, a phytosterol-enriched diet increased the concentrations of serum plant sterols16 to the values observed in phytosterolemia.17 In adults also, intake of plant sterol esters increased the serum concentrations of plant sterols.18,19 Free serum cholesterol is rapidly exchanged with that in red cell membranes20 and, depending on its concentration, can modify red cell morphology and hemorrheology.21 In studies in vitro, plant sterols were also incorporated into the red cell membranes.22 Elevated contents of red cell sitosterol have been found in cases of phytosterolemia17 and in
P
FH GLC
524
Familial hypercholesterolemia Gas-liquid chromatography
HDL LDL
High-density lipoprotein Low-density lipoprotein
From the Division of Internal Medicine, Department of Medicine, and the Hospital for Children and Adolescents, University of Helsinki, and Biomedicum, Helsinki, and the Department of Clinical Nutrition, University of Kuopio, and Kuopio University Hospital, Finland. Supported by a grant from Raisio Benecol Ltd, Raisio, Finland and Finnish Heart Research Association. Submitted for publication Sept 20, 2002; revision received Dec 27, 2002; accepted Feb 14, 2003. Reprint requests:Tatu A. Miettinen, MD, Division of Internal Medicine, Department of Medicine, University of Helsinki, PO BOX 340, FIN-00029 HUS, Finland. E-mail: [email protected]. Copyright © 2003 Mosby, Inc. All rights reserved. 0022-3476/2003/$30.00 + 0
10.1067/mpd.2003.193
healthy adults eating a sitosterol-enriched diet.23 It may be assumed that the hemolysis observed in phytosterolemia24 results from accumulation of plant sterols in the red cell membranes. However, there are no systematic studies on the physiological occurrence of squalene and noncholesterol sterols (cholesterol precursors and plant sterols) in human red cells or reports of their reduction or accumulation in the red cell membranes during the consumption of varying phytosterol mixtures, especially in children, who have excessively high absorption of plant sterols, as suggested by an earlier study.16 We therefore investigated the plasma and red cell squalene and noncholesterol sterols and their changes in children with hypercholesterolemia before and during consumption of plant stanol and sterol ester spreads in a randomized, double-blind study, and compared the plasma and red cell values with each other.
METHODS Subjects The study population was 23 prepubertal children age 2 to 9 years (10 boys and 13 girls) with a mean age of 6.8 ± 0.5 (SEM) years, a mean height of 120.5 ± 3.5 cm, and a mean weight of 23.1 ± 1.5 kg who were patients at the Outpatient Clinic of the Hospital for Children and Adolescents, University of Helsinki. The only inclusion criteria were that the serum total cholesterol value should be >5.0 mmol/L (>194 mg/dL) and serum triglycerides <2.0 mmol/L (<176 mg/dL). Of the 23 subjects, none was on hypolipidemic medication. The use of the plant stanol or sterol ester spread was stopped 2 weeks before entry into the study. Patients with diabetes or with kidney, liver, or thyroid disease were excluded from the study. Informed consent was obtained from the parents. The study protocol was approved by the Ethics Committee of the Hospital for Children and Adolescents, University of Helsinki. Of the 23 children, 16 had familial hypercholesterolemia (FH) diagnosed by polymerase chain reaction25–27 for the four most common Finnish mutations (FH-Helsinki [n = 10], FH-North Karelia [n = 4], FH-Turku [n = 0], FH-Pori [n = 0]) or by the lymphocyte test (n = 2).28 One child without a Finnish ancestry had a family member with FH, and the child’s serum lipid levels and family history fit to FH. Accordingly, the child was included in the FH group. The six remaining children were negative for the four Finnish mutations and did not have a family history of FH.
Study Design The study had a double-blind, randomized, crossover design. There were two 5-week intervention periods with a 5week washout on an ad libitum home diet between the interventions. Half of the children started randomly with a scheduled low-fat (32%) plant stanol (2.0 g/d) ester spread and the other half with a similarly scheduled low-fat (35%) plant sterol (2.0 g/d) ester spread. The fatty acid compositions of the plant stanol (Light Benecol, Raisio Benecol Ltd, Raisio, Finland) and sterol ester spreads (Becel pro.activ, Unilever BestFoods, Purfleet, UK) were 16% and 25% of saturated fatty Red Cell and Plasma Plant Sterols are Related During Consumption of Plant Stanol and Sterol Ester Spreads in Children With Hypercholesterolemia
acids, 56% and 25% of monounsaturated fatty acids, and 28% and 50% of polyunsaturated fatty acids, respectively. Both spreads contained 6 mg/100g and 66 mg/100 g of vitamin E, respectively, but the amounts of supplemented vitamin A (900 µg/100g) and vitamin D (7.5 µg/100g) were similar. The patients were advised to replace 25 g of daily dietary fat with the respective spreads on pieces of bread, but when this was too satiating, the spreads were also added to such food as porridge. The subjects visited the Outpatient Clinic four times, at the beginning and end of the two study periods. At each visit, a blood sample was taken in the morning after an overnight fast. Height and weight were also measured. A routine physical examination was performed at the first and last visits. The spread containers were returned to the study nurse, and an approximation of the daily intake was calculated by weighing the returned unwashed containers.
Methods Plasma total and free cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol levels after precipitation of apolipoprotein-B–containing lipoproteins were analyzed by enzymatic color reactions with commercial kits (total and HDL cholesterol, CHOD-PAP; triglycerides, GPO-PAP, Roche, Basel, Switzerland; free cholesterol, COD-PAP, Wako Chemicals, Neuss, Germany) by using a semiautomatic Mira analyzer (Roche, Basel, Switzerland). LDL cholesterol was calculated according to Friedewald et al.29 The red cells were washed three times with 0.9% sodium chloride and centrifuged at 3000 rpm for 5 minutes. The plasma and red cell squalene and noncholesterol sterols, ie, lathosterol, ∆8-cholestenol, and desmosterol (the cholesterol precursor markers),30,31 and cholestanol and plant sterols (the cholesterol absorption markers)31–33 were analyzed by gas-liquid chromatography (GLC) on a 50-m capillary column (Ultra-2, 5890, Hewlett-Packard, Wilmington, Del).34,35 In brief, after addition of 5α-cholestane as internal standard, the samples were saponified, and the nonsaponifiable lipid extracts were silylated and injected into the gas chromatograph. The squalene and noncholesterol sterol values are expressed in terms of 102 mmol/mol cholesterol to eliminate the influence of varying cholesterol concentrations and are expressed as ratios unless otherwise mentioned. Serum cholesterol and triglycerides are given in mmol/L. For mg/dL, cholesterol was multiplied by 38.7 and triglycerides by 88.2. The plant stanols and sterols in the spreads were also analyzed by GLC. The plant stanol ester spread contained 1.8% sitosterol, 1.5% campesterol, 0% stigmasterol, 0% avenasterol, 0.05% cholesterol, 0.06% squalene, 66.8% sitostanol, and 29.8% campestanol. The respective values for the plant sterol ester spread were 51.5%, 24.3%, 20.5%, 1.3%, 0.4%, 0.1%, 1.2%, and 0.6%. No cholestanol was detected in either margarine.
Statistical Analysis All the statistical analyses were performed by using the NCSS statistical software package (NCSS, 2000, Kaysville, Utah). After studying the carry-over effect by analysis of vari525
Table I. Plasma lipids and their changes* Stanol† (n = 22)
Sterol‡ (n = 23)
Variable
Baseline
Change, %
Baseline
Change, %
Cholesterol Ester, % LDL cholesterol HDL cholesterol TG
6.73 ± 0.21 73.9 ± 0.9 5.15 ± 0.24 1.10 ± 0.07 1.05 ± 0.09
–9 ± 3§ –4 ± 2 –12 ± 3 6±6 5±7
6.72 ± 0.22 73.2 ± 0.5 5.15 ± 0.22 1.12 ± 0.06 1.0 ± 0.07
–6 ± 2§ –0.7 ± 0.6 –9 ± 3 6±5 1±8
*Mean ± SEM, mmol/L. For mg/dL, multiply cholesterol 38.7 and triglycerides 88.2. †During stanol ester spread. ‡During sterol ester spread. Significantly different from baseline, §P < .01, P < .001, paired t test.TG, Triglycerides.
Table II. Plasma baseline concentrations and % changes in cholesterol* and ratios† of squalene and noncholesterol sterols to cholesterol Stanol‡ (n = 22) Variable Cholesterol Squalene ∆8-cholestenol Desmosterol Lathosterol Campesterol Sitosterol Avenasterol Cholestanol Campestanol Sitostanol
Sterol§ (n = 23)
Baseline
Change, %
Baseline
Change, %
6.3 ± 0.2 25 ± 3 12 ± 1 88 ± 2 78 ± 5 437 ± 32 190 ± 12 49 ± 2 184 ± 8 1.7 ± 0.4 3.5 ± 0.7
–8 ± 2b 16 ± 7 25 ± 9 10 ± 4a 34 ± 9b –36 ± 4c –32 ± 4c –30 ± 3c –4 ± 3 350c 367c
6.3 ± 0.2 23 ± 2 11 ± 1 87 ± 2 75 ± 4 450 ± 26 201 ± 10 52 ± 2 183 ± 7 1.5 ± 0.3 2.2 ± 0.4
–7 ± 2b 29 ± 12 36 ± 16 16 ± 4c 30 ± 7c 52 ± 6c¶ 43 ± 5c¶ –10 ± 2c¶ –6 ± 3a 23¶ ±0
*mmol/L, mean ± SEM. For mg/dL, multiply 38.7. †102 mmol/mol cholesterol, mean ± SEM. ‡During stanol ester spread. §During sterol ester spread. Analyzed by GLC. ¶Significant difference between the stanol and sterol ester groups, P < .001, paired t test. Significantly different from before: a, P < .05; b, P < .01; c, P < .001, paired t test.
ance for repeated measures, we calculated the means (SEM) for the plant stanol and sterol groups. In the calculations, according to the intention-to-treat principle, we included the plant sterol ester spread data of one child who left the study before the last visit during the stanol ester period. After logarithmic transformations for the skewed variables, the paired t test was used to evaluate the differences during and between the treatments. The correlation coefficients were calculated by using Pearson product moment and Spearman rank correlation in appropriate cases. A P value <.05 was considered significant.
for the sterol ester spread. The children consumed daily an average of 19.9 g (range, 6.9–33.9 g) of the stanol ester spread and of 21.0 g (range, 10.1–28.6 g) of the sterol ester spread. Accordingly, the plant stanols and sterols consumed averaged 1.6 g and 1.7 g per day, respectively, corresponding to 80% and 85% of the scheduled dose. The children reported no adverse effects. There was a slight but insignificant increase in body weight and height during the 15-week study period. There was virtually no carry-over effect.
RESULTS
The baseline values and the changes in the plasma lipids are shown in Table I. The concentrations of total and LDL cholesterol tended to decrease more with the plant stanol ester
Final data were obtained from 22 children (nine boys, 13 girls) for the stanol ester and 23 children (10 boys, 13 girls) 526 Ketomäki et al
Plasma Lipids
The Journal of Pediatrics • May 2003
spread than with the sterol ester spread, by 0.64 ± 0.17 mmol/L (25 ± 7 mg/dL) and 0.43 ± 0.15 mmol/L (17 ± 6 mg/dL) for total cholesterol and by 0.69 ± 0.16 mmol/L (27 ± 6 mg/dL) and 0.51 ± 0.13 mmol/L (20 ± 5 mg/dL) for LDL cholesterol, respectively. No significant difference in the esterification percentages of total cholesterol was found between the groups during the study. The total and LDL cholesterol values decreased by >10% and >15%, respectively, in 11 children with the plant stanol ester spread and in eight children with the plant sterol ester spread. We observed correlations between the individual amounts of plant stanol and sterol ester spreads ingested and the decreases in LDL cholesterol (r = 0.448, r = 0.428, P < .05 for both), and also between the ingested amount of plant stanol ester spread ingested and the decrease in total cholesterol (r = 0.515, P < .05). No significant changes were detected in the HDL cholesterol or serum triglyceride concentrations. At the baseline, the total and LDL cholesterol values were insignificantly higher in the FH group than in the non-FH group, and the reductions in the total and LDL cholesterol concentrations during the spread periods were similar in the FH and non-FH groups and also in the sexes (data not shown).
Table III. Correlations between changes in plasma and red cell cholesterol and ratios of squalene and noncholesterol sterol to cholesterol with respective baseline values Change Baseline Cholesterol Squalene ∆8-cholestenol Desmosterol Lathosterol Campesterol Sitosterol
Plasma Squalene and Noncholesterol Sterols Of the ratios of noncholesterol sterols to cholesterol, only that of ∆8-cholestenol was higher and that of campestanol lower in the girls than in the boys, but the responses during the consumption of the spreads were similar in the sexes. The baseline ratios of the cholesterol precursors were negatively related to those of the absorption markers, eg, r = –0.471 (P < .05) for ∆8-cholestenol versus campesterol. The ratios of the cholesterol synthesis markers increased by as much as 36% with both spreads (Table II). The ratios of avenasterol, present only in the sterol ester spread, decreased more with the stanol ester than with the sterol ester spread. The ratios of cholestanol exhibited borderline reductions in both groups. The major difference between the two spread periods was that during the plant sterol ester period, the ratios of both campesterol and sitosterol increased by 52 ± 6% and 43 ± 5%, respectively (P < .001), but during the plant stanol ester period, they decreased by 36 ± 4% and 32 ± 4%, respectively (P < .001). The plasma ratios of campestanol and sitostanol increased several-fold only with the plant stanol ester spread (Table II), but the respective concentrations remained low, 19 µg/dL for campestanol and 31 µg/dL for sitostanol. Of the noncholesterol sterol ratios, only the baseline campesterol ratio predicted the subsequent decrease in the concentrations of plasma total and LDL cholesterol during the plant sterol ester spread period (r = 0.421 and r = 0.452, P < .05 for both), suggesting a better cholesterol-lowering response in the children with high than in children with low baseline cholesterol absorption. The lower the baseline ratios of the plasma precursors of cholesterol, the higher were their increments during both treatment periods (Table III). The baseline ratios of the cholesterol absorption markers were negatively related to the reRed Cell and Plasma Plant Sterols are Related During Consumption of Plant Stanol and Sterol Ester Spreads in Children With Hypercholesterolemia
Avenasterol Cholestanol Campestanol Sitostanol
Plasma Red cell Plasma Red cell Plasma Red cell Plasma Red cell Plasma Red cell Plasma Red cell Plasma Red cell Plasma Red cell Plasma Red cell Plasma Red cell Plasma Red cell
Stanol* (n = 22)
Sterol† (n = 23)
–0.358 –0.470a –0.424a –0.430a –0.617b –0.771c –0.399 –0.703c –0.497a –0.797c –0.625b –0.652c –0.264 –0.604b –0.420a –0.593c –0.632c –0.475a –0.341 0.018 –0.569b –0.246
–0.360 –0.655c –0.477a –0.365 –0.310 –0.813c –0.477a –0.658c –0.229 –0.401 0.014 0.054 0.489a 0.360 –0.488a –0.586b –0.361 –0.508a –0.597b –0.772c –0.541b –0.650c
*During stanol ester spread. †During sterol ester spread. a, P < .05; b, P < .01; c, P < .001.
spective changes caused by the stanol esters, whereas the higher the baseline sitosterol ratio, the higher was its increment caused by the sterol esters. Reductions in the avenasterol and cholestanol ratios occurred mainly in the children with the highest baseline values, whereas the increases in the campesterol ratios caused by the sterol esters were independent of the baseline values.
Red Cell Squalene and Noncholesterol Sterols The red cell cholesterol concentrations remained constant during the treatment periods (Table IV), but some reduction occurred in the subjects with high baseline values, as revealed by the negative correlations in Table III. However, the ratios of the plant sterols decreased >30% during the plant stanol ester spread period but increased >40% during the consumption of the plant sterol ester spread. The ratios of avenasterol decreased during the plant stanol period and those of cholestanol during the plant sterol period, whereas the cholesterol synthesis markers increased variably by both spreads. The ratios of plant stanols increased several-fold by consumption of the plant stanol spread, 527
Table IV. Red cell baseline and percent changes in cholesterol* and ratios† of squalene and noncholesterol sterols to cholesterol Stanol‡ (n = 22) Variable
Sterol§ (n = 23)
Baseline Change, % Baseline Change, %
Cholesterol Squalene ∆8-cholestenol Desmosterol Lathosterol Campesterol Sitosterol Avenasterol Cholestanol Campestanol Sitostanol
117 ± 3 –1 ± 2 34 ± 6 21 ± 11 13 ± 1 44 ± 28 64 ± 2 13 ± 5a 208 ± 13 23 ± 10 514 ± 34 –36 ± 4c 224 ± 14 –32 ± 4c 66 ± 3 –22 ± 4c 212 ± 13 –3 ± 5 0.6 ± 0.3 1400c 6±2 283c
115 ± 2 31 ± 3 10 ± 1 63 ± 2 204 ± 11 528 ± 30 236 ± 13 66 ± 3 228 ± 12 0.6 ± 0.3 8±2
4±2 20 ± 10 158 ± 59b 17 ± 7a 21 ± 8 47 ± 6c 40 ± 5c 5 ± 6 –17 ± 4b ± 0 –38
*mg/dL, mean ± SEM. †102 mmol/mol cholesterol, mean ± SEM. ‡During stanol ester spread. §During sterol ester spread. Significant difference between the stanol and sterol ester groups, P < .001, paired t test. Significantly different from before: a, P < .05; b, P < .01; c, P < .001, paired t test.
but the respective concentrations remained low (11 ± 2 µg/dL for campestanol and 25 ± 2 µg/dL for sitostanol). In red cells, the baseline ratios of the cholesterol precursors and the absorption markers were related to their changes similarly as in plasma (Table III). Neither the total nor the free plasma cholesterol was correlated with red cell cholesterol. However, many baseline plasma and red cell noncholesterol sterol concentrations and ratios to cholesterol were positively correlated (Table V). In addition, with both spreads, their changes in the plasma predicted the respective changes in the red cells (Table V ), as shown individually for sitosterol in the Figure.
DISCUSSION The plant sterol ester spread was investigated as a cholesterol-lowering agent for the first time in a comparable study with a plant stanol ester spread in hypercholesterolemic children on an ad libitum home diet. A previous observation that, during a plant sterol-enriched diet, serum plant sterols were elevated in children16 resulted in our rationale to evaluate the effects of a sterol ester spread on the plasma and red cell sterols. The red cells served as markers of tissue cells and, accordingly, as indicators of sterol accumulation in the tissues. The new observations in the current study were that in hypercholesterolemic children, the plant sterol ratios were elevated in the red cells during consumption of the plant sterol ester spread, whereas during consumption of the stanol ester spread, their respective values decreased, with the changes related to those observed in plasma. 528 Ketomäki et al
Table V. Correlations between plasma baseline and red cell baseline values, plasma changes with red cell changes in cholesterol, and ratios of squalene and noncholesterol sterols to cholesterol
Baseline Red cell vs plasma n = 23 Cholesterol Squalene ∆8-cholestenol Desmosterol Lathosterol Campesterol Sitosterol Avenasterol Cholestanol Campestanol Sitostanol
–0.212 0.131 –0.498a 0.497a 0.767c 0.963c 0.981c 0.235 0.508a –0.249 –0.301
Changes Red cell vs plasma Stanol* (n = 22)
Sterol† (n = 23)
0.216 0.032 0.364 0.537b 0.807c 0.896c 0.923c 0.455a 0.343 0.517b 0.248
0.197 –0.104 0.228 0.748c 0.838c 0.822c 0.952c 0.431a 0.144 0.124 –0.060
*During stanol ester spread. †During sterol ester spread. a, P < .05; b, P < .01; c, P < .001.
Squalene and Noncholesterol Sterols in Red Cell Membranes The red cell noncholesterol sterols have not been previously related to those in plasma. The current baseline data showed that the red cell membranes contained most of the noncholesterol sterols in ratios proportional to those observed in the plasma lipoproteins, despite the fact that the red cells contain only free sterols and the plasma lipoproteins also contain esterified sterols. Actually, rough calculations from the data of one previous study36 showed similar values, eg, for free campesterol and sitosterol to free cholesterol in plasma and in red cells. Thus, similar to cholesterol, noncholesterol sterols seemed to exchange freely with the plasma lipoproteins and red cells. However, squalene was exchanged less frequently between plasma lipoproteins and red cells, resulting in higher ratios of squalene to free cholesterol in the plasma than in the red cells (ratios 103 vs 34 102 mmol/mol of cholesterol, respectively, from Tables II and IV).
Effects of Plant Stanol Ester The current study confirms the previous findings11,12 that during consumption of plant stanol ester spread by hypercholesterolemic children, plant sterols in the plasma decrease and cholesterol precursor sterols increase. A new finding was that proportional changes also occurred in red cells and showed a high correlation with the plasma values. Respective increments were higher in children with low baseline ratios of cholesterol synthesis markers in plasma and red cells than in children with The Journal of Pediatrics • May 2003
Figure. Correlations between the changes in the ratios of plasma and red cell sitosterol to cholesterol (102 mmol/mol) during the periods of plant stanol (closed triangles) and sterol ester (open circles) spreads. y = 1.3x + 6.6 for stanol ester and y = 1.03x + 1.8 for sterol ester
high baseline ratios, indicating that their cholesterol synthesis was more enhanced. The negative baseline correlation of the precursors with the absorption markers indicates that the children with the low baseline ratios of precursor sterols in their plasma and red cells have high respective ratios of the absorption markers as a sign of effective cholesterol absorption. During the plant stanol ester period, the respective reductions in the ratios of absorption markers were higher in those with high than in those with low respective baseline values, indicating more effective inhibition of cholesterol absorption. These results suggest that the high baseline absorption of cholesterol, and probably also of other sterols, was markedly inhibited by plant stanol esters and resulted in inhibition of sterol accumulation in the red cells. The mean plasma total and LDL cholesterol reductions, 9% and 12%, were of the same magnitude as those in the previous studies with plant stanol esters (1.5–2.9 g/d) in normocholesterolemic,13 in mildly hypercholesterolemic,14 and in FH children.12 In contrast with an 8-week37 and a 1-year38 study with plant sterol ester spread with no cholesterol-lowering effect, the current short-term study in children showed reductions in plasma lipid values, as in short-term studies in adults18,19,39 and in children with FH.40 Despite some major differences in the distribution of fatty acids, possibly also affecting the cho-
lesterol-lowering effect, the plasma lipid values tended to decrease more during consumption of plant stanol spread than during consumption of plant sterol ester spread. However, even though the plant sterol ratios in plasma were increased by as much as 50% during the plant sterol ester period, the individual plasma values failed to reach the levels described in children on the phytosterol-enriched diet.16 Specifically, after consumption of 1.7 g of plant sterols in the current study, the highest concentration of plasma phytosterols, calculated as the sum of sitosterol and campesterol, was 3.5 mg/dL (0.09 mmol/L), compared with values as high as 17 mg/dL (0.44 mmol/L) reported by Mellies et al16 with even smaller doses of dietary plant sterols (300–900 mg). The reason for those remarkable elevations remains unknown, but at least, the levels observed in the current study did not differ from those reported in adults,18,19 suggesting that the absorption of phytosterols in young hypercholesterolemic children is not different from that in adults. The increments of plant sterols in the red cells are in accord with earlier results for an adult patient on a diet enriched with free β-sitosterol23 and for phytosterolemic patients.17 Moreover, the increase in both plasma and red cell sitosterol was greater in the children with high baseline plasma sitosterol levels, suggesting that more sitosterol accumulates in the plasma and red cells of children with effective absorption of cholesterol and sterols than in those with low baseline absorp-
Red Cell and Plasma Plant Sterols are Related During Consumption of Plant Stanol and Sterol Ester Spreads in Children With Hypercholesterolemia
529
Effects of Plant Sterol Esters
tion. It has been shown recently that cholesterol absorption efficiency is genetically regulated,3,4 suggesting that subjects with higher sterol absorption have higher serum plant sterol levels. Whether the high serum plant sterol levels in children with high sterol absorption is harmful is not known. There is only indirect evidence showing that nonphytosterolemic subjects and their relatives with higher serum plant sterol levels had a higher incidence of atherosclerosis.41 Absorption of avenasterol and cholestanol was mainly inhibited in children with high baseline absorption of the respective sterols in both groups, resulting in lowering of the respective sterol ratios in subjects with high baseline values. Stanol esters apparently inhibited avenasterol absorption from a normal diet only (no avenasterol in the spread), lowering the ratios of plasma and red cells, whereas the presence of avenasterol in the sterol ester spread resulted in inconsistent changes in the ratios of avenasterol in the plasma and red cells. Systematic studies are lacking on the effects of large amounts of dietary plant sterols on human red cells. High content of plant sterols in red cells results in hemolysis in at least phytosterolemic patients24 and may lead to increased osmotic fragility.22 In stroke-prone spontaneously hypertensive rats, a phytosterol-rich diet made red cells less deformable and shortened the life span.42 On the other hand, in apolipoproteinE–deficient mice, lowering of serum cholesterol by phytosterols appeared to protect red cells.43 Even though the safety of plant sterol ester spread has been evaluated in long-term38 and shortterm14,18 human studies, the long-term effects on red cells and vascular endothelial cells need further investigation. In contrast with the results for noncholesterol sterols, the cholesterol concentrations in the red cells remained unchanged during the two intervention periods. This observation may relate to the fact that the levels of free cholesterol remained unchanged during both intervention periods. Surprisingly, the red cell and plasma total and free cholesterol values were not related to each other, in contrast with the high respective correlations of plant sterols. The exchange of plasma and red cell plant sterols was higher than that of cholesterol, whereas in vitro from among different sterols, plasma cholesterol exhibited the highest exchange with red cell cholesterol,44–46 a finding possibly caused by lack of alkylation of the sterol side chain in cholesterol. However, some noncholesterol sterols appeared to replace cholesterol in the red cells,22,44 suggesting that large amounts of dietary plant sterols could probably replace cholesterol in red cells and would lead to higher ratios of plant sterols to cholesterol in the red cells, as shown in the Figure. The technical assistance of Ms Leena Kaipiainen, Pia Hoffström, Anne Honkonen, and Ritva Nissilä is gratefully acknowledged.
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