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Non-cholesterol sterols in serum, lipoproteins, and red cells in statin-treated FH subjects off and on plant stanol and sterol ester spreads
Clinica Chimica Acta 353 (2005) 75 – 86 www.elsevier.com/locate/clinchim
Non-cholesterol sterols in serum, lipoproteins, and red cells in statin-treated FH subjects off and on plant stanol and sterol ester spreads Anna Ketom7kia,b, Helena Gyllingc,d, Tatu A. Miettinena,b,* a
Division of Internal Medicine, Department of Medicine, University of Helsinki, Finland b Biomedicum Helsinki, P.O. Box 700, FIN-00029 HUS, Finland c Department of Clinical Nutrition, University of Kuopio, Finland d Kuopio University Hospital, Kuopio, Finland
Received 9 September 2004; received in revised form 4 October 2004; accepted 8 October 2004
Abstract Background: Serum plant sterol levels are increased by consumption of statins and dietary plant sterols, and decreased by dietary plant stanols, but little is known about combination therapy of statin and plant sterols. Methods: We measured plant sterols in serum, lipoproteins, and red cells in subjects with familial hypercholesterolemia (FH) (n=18) treated with variable doses of statins off and on plant stanol (STA) and sterol ester (STE) spreads. Results: STA and STE spreads lowered LDL cholesterol ~15%. Plant sterols were decreased in serum, lipoproteins, and red cells by ~25% with STA and increased from 37% to 80% with STE, especially with high statin doses. The changes in serum were related to those in red cells. The baseline levels of serum plant sterols were negatively (r-range 0.639 to 0.935) and positively (r-range 0.526 to 0.598) correlated with the respective changes evoked by the STA and STE spreads. Conclusions: STE reduces LDL cholesterol, but increases serum, lipoprotein, and red cell plant sterol levels in statin-treated FH subjects, while all the respective values are decreased with STA. Recent predictions that elevated serum plant sterols pose an increased coronary risk suggest that increases of serum plant sterol levels should be avoided, especially in atherosclerosis-prone individuals, such as subjects with FH. D 2004 Elsevier B.V. All rights reserved. Keywords: Plant sterols; Plant stanols; Familial hypercholesterolemia; Non-cholesterol sterols; Statins; Red cells
1. Introduction * Corresponding author. Biomedicum Helsinki, P.O. Box 700, FIN-00029 HUS, Finland. Tel.: +358 9 4717 1852; fax: +358 9 4717 1851. E-mail address: [email protected] (T.A. Miettinen). 0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2004.10.008
Plant sterols (phytosterols), including sitosterol, campesterol, stigmasterol, and avenasterol, are constituents of plants and are not synthesized by human
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cells. They are present in the Western diet mainly in vegetables and vegetable oils in small amounts approx. 160–360 mg/day [1]. Small amounts (about 10%) of plant sterols are absorbed from the intestine, and thus serum contains low concentrations of plant sterols. The 5a-derivatives of phytosterols, i.e., plant stanols, comprise about 10% of daily phytosterol intake and are almost unabsorbable. Ratios of serum plant sterols and cholestanol to cholesterol reflect the efficiency of cholesterol absorption [2,3], i.e. the ratios are increased in subjects with high absorption of cholesterol. In addition, hypercholesterolemic subjects, especially those with familial hypercholesterolemia (FH), have higher serum plant sterol concentrations and ratios to cholesterol than those individuals with a normal serum lipid profile [4]. Inhibition of cholesterol synthesis by statins lowers the serum ratios of cholesterol precursors (squalene, D8-cholestenol, desmosterol, lathosterol) [5], which reflect the synthesis of cholesterol [3,6,7]. However, statins have a completely different effect on plant sterols in that they elevate their serum concentrations and the ratios of plant sterols to cholesterol [5,8–10]. Long duration [8] and high dose [9] of statin treatment (indicating effective inhibition of cholesterol synthesis), and high baseline absorption of sterols [8], are factors predicting the higher increase of serum plant sterols induced by statins. In addition, consumption of plant sterol ester-enriched food products can increase the serum plant sterol levels by ~35–65% in different hypercholesterolemic populations [11–16]. Both plant sterol and stanol ester spreads can decrease the absorption of cholesterol [16], but only plant stanol ester products inhibit the absorption of also plant sterols, leading to decreases in serum plant sterol concentrations and ratios to cholesterol [11,17]. High serum plant sterol levels with increased prevalence or incidence of coronary events [18,19] raise the question how safe are these elevated serum plant sterol levels. FH patients have high baseline serum plant sterol values and are usually treated with large statin doses for years to achieve long-term reduction of LDL cholesterol. Thus, this apparently increases correspondingly the serum contents of plant sterols more in FH than in non-FH subjects. However, since virtually all patients known to have FH are currently treated with statins, no controlled statin studies during consumption of
dietary phytosterols can be performed in subjects with FH. Thus, we studied first serum and red cells of DNA diagnosed adult FH subjects for squalene, precursor sterols, cholestanol, and plant sterols at baseline on variable dose statin treatment, i.e. when they had been using these drugs for years. Special attention was paid to the cholesterol absorption marker sterols, cholestanol and plant sterols. Second, it was interesting to examine the additional response of serum plant sterols in FH subjects to combination of the statin treatment with plant sterol and stanol ester spreads because stanol esters effectively reduce and sterol esters increase the serum plant sterol values in normal and hyperlipidemic subjects. Third, only a few studies have identified the distribution of noncholesterol sterols in different lipoproteins [10,20,21], but no data exist in FH subjects on statin treatment and during consumption of phytosterol esters. Accordingly, the limited information on the distribution of squalene and non-cholesterol sterols in different lipoproteins of FH patients prompted us to study more closely the distribution of these compounds in different lipoproteins during expected changes in cholesterol metabolism with statin alone or when combined with phytosterol esters.
2. Materials and methods 2.1. Subjects The study population comprised 18 adult subjects (6 males, 12 females) with FH with a mean age of 48F2 years (S.E.M.). They were recruited from the Outpatient Lipid Clinics of the Department of Medicine, University of Helsinki. The DNA diagnosis of FH was verified in 17 subjects, and 1 subject was clinically defined to have FH. Exclusion criteria included diabetes or kidney, liver, or thyroid diseases. The prior use of plant stanol or sterol ester spread was terminated at least 3 weeks before the first visit. Most of the subjects had used statin treatment for several years, and at least for the two last months the dose had remained unchanged. The mean daily dose of statin used in the study was equivalent to 40 mg of atorvastatin, such that the daily dose of nine subjects was b40 mg of atorvastatin (low-dose statin group), and of the other
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nine subjects z40 mg of atorvastatin (high-dose statin group) (Table 1). In addition, two subjects used bile acid binding sequestrants, resins, with 40 and 80 mg/day of atorvastatin; these are shown separately in Results. The clinical characteristics and medication of the subjects are shown in Table 1. Four of the subjects were smokers. Informed consent was obtained from all subjects. The study protocol was approved by the Ethics Committee of the Department of Medicine, University of Helsinki. 2.2. Study design This double-blinded, randomized, cross-over study consisted of two consecutive 4-week intervention periods. The patients were advised to replace 25 g of dietary fat with either the low fat (40%) plant stanol ester spreads (2 g of plant stanols daily) or a low fat (40%) plant sterol (2 g of plant sterols daily) ester
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spread in a random order. The plant stanol ester spread contained 66% of sitostanol and 29% of campestanol, and the sterol ester spread contained 45% of sitosterol, 26% of campesterol, 15% of stigmasterol, 5% of brassicasterol, and 3% of avenasterol. The fatty acid composition of the spreads was similar and included 22% of saturated fatty acids, 34% of monounsaturated fatty acids, and 44% of polyunsaturated fatty acids. All of the patients visited the Outpatient Clinics six times, twice at the beginning of the study 2 or 3 days apart, and twice at the end of the two periods. Fasting blood samples were taken on every visit. Physical examinations including weight, height, and blood pressure measurements were performed three times during the study. The empty spread containers were returned to the study nurse, and the daily intake of spread was approximated by weighing the unwashed spread containers.
Table 1 Clinical characteristics and medication of the subjects No.
Sex
Age (years)
FH-mutationa
Clinical findings
LDL-Cholb
Medication
1 2 3
f f f
51 51 62
FH-Hki FH-Hki FH-Hki
AL, XA AL, XL, XA AL, XA, CAD
6.62 3.87 4.57
4 5 6
f f m
43 46 56
FH-Turku FH-Hki FH-NK
XA AL AL, XA, CAD
4.78 5.24 4.17
7 8 9
m f m
38 47 54
FH-Hki FH-Hki FH
XA AL, XL, XA AL, CAD
4.54 4.99 3.86
10 11 12 13 14 15 16 17 18
f m f m f m f f f
43 56 43 32 45 44 56 33 59
FH-Hki FH-NK FH-Hki FH-Hki FH-NK FH-Hki FH-Hki FH-Turku FH-NK
XA AL, AL, XA XA AL, AL, XA XA
3.90 4.24 4.73 3.64 5.73 4.41 5.36 3.66 2.74
simvastatin 40 mg, ACE inhibitor, diuretic atorvastatin 80 mgc atorvastatin 80 mgc, ASA, beta-blocker, Ca-blocker, nitrate, protein pump inhibitor pravastatin 40 mg simvastatin 80 mgc atorvastatin 80 mgc, cholestipole 5 mgx2, beta-blocker, nitrate, ASA atorvastatin 80 mgc lovastatin 40 mg atorvastatin 80 mgc, beta-blocker, ASA, anti-depressive medication atorvastatin 40 mgc atorvastatin 20 mg simvastatin 40 mg, ASA simvastatin 80 mgc atorvastatin 20 mg, anti-depressive medication lovastatin 40 mg, beta-blocker, ASA lovastatin 60 mg atorvastatin 20 mg, oral contraceptives atorvastatin 40 mgc, cholestyramine 4gx2, beta-blocker, nitrate, Ca-blocker, ASA, hormone replacement therapy
XA XA
XA, CAD XA
ACE, angiotensin-converting enzyme; AL, arcus lipoides; ASA, acetyl salisylic acid; Ca-blocker, calcium channel blocker; XA, xanthomas; XL, xanthelasmas; CAD, coronary artery disease. a FH, no specific mutation has been diagnosed, but the clinical findings are consistent with FH; FH-Hki, FH-Helsinki; FH-NK, FH-North Karelia. b mmol/l. c Included in the high-dose statin group (atorvastatin z40 mg/day).
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2.3. Methods Serum total and high density lipoprotein (HDL) cholesterol and triglycerides (TG) were analyzed by the routine methods in use in our hospital laboratory. LDL was calculated according to Friedewald. From six volunteer subjects, chylomicrons (CM) were separated after a 30-min ultracentrifugation (20,000 rpm) in a fixed-angle type 50 Ti rotor (Beckman, Palo Alto, CA) with carefully overlayered 1.006 g/ml NaCl salt solution. From the infranatant, very low density lipoprotein (VLDL) (db1.006 g/ml) was separated by further ultracentrifugation with type 50.4 Ti rotor (Beckman), (18 h, 40000 rpm), followed by separation of HDL by precipitation of apolipoprotein B containing lipoproteins with phosphotungstic acid and magnesium ions (Roche, Basel, Switzerland) from the infranatant. The cholesterol and TG contents in all lipoproteins were analysed from the six subjects by commercial kits (cholesterol CHOD-PAP, triglycerides: GPO-PAP, ABX-Diagnostics, Parc Eurome´decine, France). From all patients, red cells were separated from plasma with centrifugation for 10 min (3000 rpm) and washed with 0.9% sodium chloride three times with centrifugation at 3000 rpm for 5 min. Cholesterol, squalene, and non-cholesterol sterols (cholestanol, D8 -cholestenol, desmosterol, lathosterol, campesterol, sitosterol, avenasterol, campestanol, and sitostanol) were analysed from all subjects from the serum and red cell samples, and from the six subjects also from lipoproteins (CM, VLDL, LDL, and HDL) by gas–liquid chromatography (GLC) on a 50-m-long ULTRA-2 SE-30 column (Hewlett Packard, Wilmington, DE) using 5a-cholestane as an internal standard [22]. The serum, lipoprotein, and red cell squalene and non-cholesterol sterols are given in terms of 102 mmol/mol of cholesterol and called ratios in the following sections in order to eliminate the influence of varying cholesterol concentrations. The values before spread consumption are termed as baseline values. 2.4. Statistical analysis Data analysis were performed with NCSS statistical software package (NCSS, 2000, Kaysville, UT, USA). In case of serum lipids and serum and red cell non-cholesterol sterols, the effect of the treatment
sequence was evaluated by two-way analysis of variance (ANOVA) with treatment period (baseline, sterol ester, or stanol ester) as the repeated factor, and the sequence, in which the patients consumed the spread (stanol–sterol or sterol–stanol) as the bgroupQ factor. Since no significant interaction was found, the statistical significance was tested by paired t-test. Correlations were calculated with Pearson’s product moment correlation. When the distribution of a variable was skewed, logarithmic transformations were performed. A p value b0.05 was considered statistically significant. The values are given as meanFS.E.M.
3. Results All subjects completed the study with good compliance, and no side effects were reported. The consumed amounts of the stanol and the sterol ester spreads were 25.0F0.5 and 25.4F0.3 g/day, respectively. The weight and blood pressure of the subjects remained stable during the study (weight: at the baseline 74F3 kg, at the end 73F4 kg, blood pressure: at the baseline 131/81 mm Hg, at the end 125/78 mm Hg). 3.1. Serum lipids Consumption of both spreads decreased the serum total and LDL cholesterol levels from the baseline values (Table 2), while HDL cholesterol level was increased and TG level was decreased, significantly so only during the sterol ester period. The lipid changes were similar in males and females and in subjects with different FH mutations. The proportions of subjects having LDL cholesterol below 4.0 mmol/l was Table 2 Serum lipids (mmol/l) at baseline and during stanol (STA) and sterol (STE) ester spreads Variables
Baseline
STA
STE
Cholesterol LDL-cholesterol HDL-cholesterol Triglycerides
6.30F0.24 4.50F0.21 1.26F0.05 1.19F0.10
5.65F0.22* 3.81F0.18* 1.32F0.04 1.16F0.12
5.71F0.21* 3.86F0.19* 1.37F0.04** 1.05F0.09**
MeanFS.E.M. Significantly different from baseline, *pb0.05, **pb0.01 by paired t-test.
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Fig. 1. Correlations between baseline LDL cholesterol (top), serum campesterol to cholesterol ratio (middle), serum cholestanol to cholesterol ratio (bottom) and the corresponding stanol (STA) (closed triangles) and sterol (STE) ester (open circles) induced changes in statin-treated FH subjects. The gray-colored symbols depict subjects receiving also bile acid sequestrants. *pb0.05.
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increased from 33% at baseline to 67% and 72% by plant stanol and sterol ester spreads, respectively. The baseline LDL cholesterol was negatively correlated with its respective change so that the higher the baseline LDL cholesterol level, the larger was its reduction evoked by plant sterols and stanols (Fig. 1). HDL cholesterol was increased most in subjects with the lowest baseline HDL cholesterol levels (r= 0.708 by stanol ester and r= 0.593 by sterol ester spread, pb0.01).
At baseline, LDL cholesterol was only insignificantly correlated with the statin dose (r= 0.333, ns). One of the two resin-treated subjects had the lowest value of LDL cholesterol (2.74 mmol/l), but in the other the respective value (4.17 mmol/l) was within the range of the study group (Table 1). LDL cholesterol concentrations were lower at baseline and during the intervention periods in the high-dose statin group than in the low-dose statin group (Fig. 2). This difference in LDL cholesterol between the statin groups became
Fig. 2. LDL cholesterol and serum campesterol, sitosterol, and cholestanol ratios to cholesterol in FH-subjects with high-dose statin treatment (equivalent dose of atorvastatin z40 mg/day) (closed circle) and with low-dose statin treatment (equivalent dose of atorvastatin b40 mg/day) (open square) at baseline (BL) and during consumption of stanol (STA) and sterol ester (STE) spreads. The spreads were consumed in a random order. *pb0.05, **pb0.01, ***pb0.001 from baseline, by paired t-test. #pb0.05 between the statin groups, by unpaired t-test.
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smaller when the subjects receiving resin treatment were excluded. 3.2. Serum squalene and non-cholesterol sterols The ratios of squalene and non-cholesterol sterols to cholesterol in serum at baseline and during the intervention periods are shown in Table 3. At baseline, the ratios of cholesterol absorption markers correlated positively with each other (r ranged from 0.518 to 0.947, pb0.05–0.001). Cholesterol synthesis markers (except squalene) correlated also positively with each other (r ranged from 0.485 to 0.753, pb0.05–0.001), but not with cholesterol absorption markers or with total and LDL cholesterol. However, cholesterol absorption markers were negatively correlated with total and LDL cholesterol (e.g., r= 0.583 for campesterol vs. LDL cholesterol). During the intervention periods, the level of lathosterol was increased in serum (14–19%) by both spreads, while desmosterol was increased only by stanol ester spread. During consumption of the plant stanol ester spread, campesterol, sitosterol, and aveTable 3 The ratios of serum and red cell cholesterol (mg/dl) and squalene and non-cholesterol sterols to cholesterol (102mmol/mol of cholesterol) at baseline and during consumption of plant stanol (STA) and sterol (STE) ester spreads Variables Cholesterol Squalene D8-cholestenol Desmosterol Lathosterol Campesterol Sitosterol Cholestanol Avenasterol
serum red cell serum red cell serum red cell serum red cell serum red cell serum red cell serum red cell serum red cell serum red cell
Baseline
STA
STE
212F8 108F2 28F2 41F4 14F2 14F2 67F7 63F10 66F7 132F17 508F50 580F58 265F29 304F33 195F15 190F14 60F6 74F6
192F7*** 110F1 30F3 47F6 14F2 16F2 72F5* 68F11* 75F7* 151F20* 387F39** 429F43* 190F19*** 207F21*** 182F13 182F13 42F3*** 56F3***
193F7*** 110F1 28F2 43F5 13F1 17F2 69F5 60F8 73F7* 162F28* 846F78***,# 965F87***,# 382F40***,# 415F41***,# 176F13* 186F11 60F6# 70F5#
MeanFS.E.M. Significantly different from baseline, *pb0.05, **pb0.01, ***pb0.001 by paired t-test. # Significantly different STE vs. STA, pb0.001 by paired t-test.
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nasterol ratios were decreased in serum by ~25%, while during consumption of plant sterol ester spread, the serum campesterol and sitosterol ratios were increased (48–74%). The baseline values of all plant sterols were negatively correlated with the respective change in serum (r ranged from 0.639 to 0.935, pb0.01– 0.001) (e.g., Fig. 1 for campesterol) by the plant stanol ester spread. In contrast, the changes of serum campesterol and sitosterol by sterol ester spread were positively baseline correlated with their baseline values (r ranged from 0.526 to 0.598, pb0.05) (Fig. 1). Serum cholestanol decreased significantly only during consumption of the sterol ester spread, and the baseline values were negatively correlated with the respective changes by both spreads (r ranged from 0.545 to 0.573, pb0.05) (Fig. 1). Serum plant stanols were increased by the plant stanol ester spread, but the amounts remained rather small (e.g., sitostanol from 18 to 43 Ag/dl). The ratios of cholestanol and less so plant sterols were higher in the subjects in the high-dose statin group than in the low-dose statin group (Fig. 2). This difference disappeared when the subjects on resin treatment were excluded. The changes of plant sterols and cholestanol during the spread periods were similar in the high and low statin groups. 3.3. Red cell squalene and non-cholesterol sterols At baseline, red cell desmosterol, lathosterol, cholestanol, and plant sterol ratios to cholesterol were significantly correlated with the respective ratios in serum (r ranged from 0.890 to 0.995, pb0.001). Rather similar changes were seen in red cell and serum with respect to squalene and non-cholesterol sterols during the intervention periods (Table 3), and the changes of lathosterol, campesterol, and sitosterol in serum and red cells were significantly positively correlated (r ranged from 0.802 to 0.988, pb0.001) with both spreads (e.g., Fig. 3 for campesterol and lathosterol). 3.4. Squalene and non-cholesterol sterols in different lipoproteins Since release of newly synthesized precursors, newly absorbed plant sterols or sterols released from peripheral tissues could be changed by statins without or with phytosterol consumption, we measured
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Fig. 3. Correlations between changes of serum and red cell campesterol (a) and lathosterol (b) to cholesterol ratios (102 mmol/mol of cholesterol) during stanol (STA) (closed triangles) and sterol ester (STE) (open circles) spreads. The gray-colored symbols depict subjects receiving also bile acid sequestrants. For STA and STE, the correlation coefficients are r=0.986 ( y=1.2x 9.6) and r=0.988 ( y=1.1x+9.3), respectively, for campesterol, and r=0.802 ( y=2.4x 4.5) and r=0.948 ( y=4.5x 2.4), respectively, for lathosterol, pb0.001 for all.
squalene and non-cholesterol sterols also in different lipoproteins. 3.4.1. Concentrations At baseline, about 45% of squalene was carried by LDL and 44% by HDL. During consumption of the sterol ester spread the proportion of LDL squalene was 51% but less, 36%, with the stanol ester spread (Fig. 4). LDL transported 62–75% of the noncholesterol sterols (except D8-cholestenol) at baseline and 57–60% during consumption of both spreads. For
HDL, the respective proportions were 20–25% at baseline and 23–34% during consumption of both spreads ( pb0.05 from baseline for desmosterol, campesterol, and sitosterol by both spreads). The plant sterol ester spread increased the concentrations of plant sterols in lipoproteins, especially in HDL (data not shown). 3.4.2. Ratios to cholesterol At baseline and during consumption of both spreads, the ratios of the cholesterol synthesis markers
Fig. 4. The percentage distribution of absolute amounts of cholesterol, campesterol, lathosterol, and squalene in lipoproteins of statin-treated FH subjects at baseline (BL) and during stanol (STA) and sterol (STE) ester spreads. *pb0.05 vs. baseline, #pb0.05 STA vs. STE. The absolute concentrations (meanFS.E.M.) in serum are given on the top of the columns, for cholesterol in mg/dl, for squalene and other sterols in Ag/dl.
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were most abundant in CM and VLDL, except desmosterol, which was lowest in VLDL (data not shown). The ratios of absorption markers were lowest in VLDL at baseline and also with both spreads, and they were significantly lower than in serum. HDL tended to have the highest values of the absorption
marker sterols. Plant stanols were mostly carried by CM and HDL as shown for campestanol in Table 4. The decrements and increments of plant sterols were similar in all lipoproteins by plant stanol and sterol ester spreads, respectively (Table 4).
Table 4 Concentration of cholesterol (mg/dl) and ratios of squalene, lathosterol, campesterol, sitosterol, cholestanol, and campestanol to cholesterol (102mmol/mol of cholesterol) in serum, chylomicron (CM), VLDL, LDL, and HDL at baseline and during consumption of plant stanol (STA) and sterol (STE) ester spreads in FH patients receiving statin treatment
4. Discussion
Variables
Baseline
STA
STE
Cholesterol
225F16 3F0.4 10F2 164F14 44F4 25F3 168F57*** 100F31 26F6 98F25*** 69F10 87F14*** 82F13*** 66F8 73F7 418F37 442F40*** 383F34*** 436F39 433F42 221F28 236F27 156F15*** 226F28 261F31*** 171F19 166F18 128F16*** 166F27 196F31 3F1 26F3*** 7F1*** 4F2 19F6***
210F10* 3F0.5 11F3 145F9 49F3* 22F3 160F36*** 87F45*** 23F5 118F48*** 63F6 103F17*** 84F9*** 67F6 79F13 325F44* 330F45* 287F39*,*** 330F44* 343F46* 168F23* 168F21* 110F13*,*** 167F21* 193F27*,*** 168F26 168F21 142F26*** 162F27 196F33 17F2* 30F2*** 16F3 16F4 36F4*
210F16* 4F1 10F3 140F10* 46F4** 30F3 121F19*** 83F26 33F4 97F34*** 72F5** 92F9*** 97F12*** 78F8 82F16 729F85*,** 749F80*,** 670F70*,**,*** 739F83*,** 781F83*,**,*** 331F41*,** 332F40*,** 218F26*,**,*** 326F39*,** 373F44*,**,*** 172F22 191F41 126F25*** 174F34 191F30 6F2** 25F9 11F4 5F1 25F5***
serum CM VLDL LDL HDL Squalene serum CM VLDL LDL HDL Lathosterol serum CM VLDL LDL HDL Campesterol serum CM VLDL LDL HDL Sitosterol serum CM VLDL LDL HDL Cholestanol serum CM VLDL LDL HDL Campestanol serum CM VLDL LDL HDL
MeanFS.E.M., n=6. 4 Significantly different from baseline, pb0.05 by paired t-test. ** Significantly different STA vs. STE, pb0.05 by paired t-test. *** Significantly different from the serum values, pb0.05 by unpaired t-test.
The high baseline levels of serum plant sterols in FH subjects, especially their further increase which occurs during statin treatment, prompted us to study the effects of plant stanol and sterol esters on serum, lipoprotein, and red cell plant sterol levels in FH subjects receiving long-term statin treatment. This study was carried out in FH subjects who had high baseline serum plant sterol levels and were treated with different doses of statins. Long-term statin treatment increases the serum plant sterol levels probably by decreasing the biliary excretion of sterols, leading to a subsequent increase in the absorption of dietary plant sterols from the reduced intestinal pool [10,23]. The present study was not designed to evaluate the effects of different statin doses. However, the baseline measurements did reveal that the subjects being treated with the highest doses of statins exhibited the highest levels of serum plant sterols and cholestanol, suggesting that they had a more effective cholesterol absorption efficiency. The consumption of plant sterol esters further increased the serum plant sterol levels. Even though the percentual increase calculated from Fig. 1 (middle panel) was lower in those with the higher baseline levels, the absolute increase was highest. Thus, the high statin dose increased serum plant sterol levels more than the low dose, resulting in an additional increase of serum plant sterols with dietary plant sterol esters. This suggests that subjects, such as those with FH, who need large doses of statins to control their lipid levels, are prone to present with high serum levels of plant sterols. On the contrary, plant stanol esters have been reported to lower serum plant sterol levels, even in combination with statins [23–25]. In the present study, consumption of the plant stanol ester spread lowered the serum plant sterol levels, by the greatest extent in those subjects with the highest baseline levels, a correlation shown earlier in FH subjects receiving a low statin dose [24]. Thus, the increase of serum plant
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sterols caused by statin treatment could be eradicated by plant stanol esters, and the serum levels of plant sterols in these FH subjects approached the levels found in normal population (228F11 102 mmol/mol of cholesterol) [18]. The highest total serum plant sterol level in the present study (approx. 3 mg/dl) is far below the levels found in phytosterolemia (20–94 mg/dl), which is an inherited disease caused by a mutation in the genes coding for the ATP binding cassette (ABC) transporters G5 and G8, leading to premature atherosclerosis similarly as in homozygous FH [1]. However, some evidence has been presented between increased serum plant sterol levels and the risk of coronary heart disease in non-phytosterolemic subjects [26,27]. In fact, an increased ratio of serum plant sterols to cholesterol was shown to be an independent risk factor for coronary heart disease in a randomly selected population of postmenopausal women [18]. In addition, recently even a modestly increased serum sitosterol concentration (0.2 mg/dl) was associated with an increased risk of coronary events in a 10-year follow-up population study [19]. Thus, even though the present mean serum sitosterol concentrations was minimal (up to 0.7 mg/dl) compared to respective cholesterol values, according to recent evidence, it cannot be ignored since it may represent a potential additional risk for coronary events, especially in the FH subjects even though these subjects show markedly reduced LDL cholesterol levels. In addition, in this study, in agreement with earlier studies in hypercholesterolemic children [13] and in non-FH hypercholesterolemic adults [12], plant sterols were increased in the red cells in a parallel fashion to those in serum by the sterol ester spread. In addition, the correlation in Fig. 3b indicates that small lathosterol changes in serum do modulate more effectively the lathosterol content in red cells. This is in accordance with an in vitro study that demonstrated that lathosterol was more efficiently taken up than cholesterol by fibroblasts [28]. The non-cholesterol sterols are capable of replacing cholesterol from red cells in animal experiments [29], but in the present as well as in previous studies, red cell cholesterol levels were unchanged or even tended to be increased, suggesting that increased plant sterols in serum could accumulate in red cells with cholesterol. In one study, the increased plant sterol levels in red cell membranes
did not evoke any changes in membrane rigidity after 1 year of treatment with plant sterol esters [12]. However, according to a recent study, free cholesterol transported by the red cells may destabilize the atheromatotic plaque and make it vulnerable to rupture [30]. It is not known whether increased levels of plant sterols in red cell membranes may also have a harmful effect on plaque stability; this is a topic which needs to be clarified. The present study revealed that changes in percentual proportions of non-cholesterol sterols in different lipoproteins mimicked those of cholesterol. Thus, at baseline similarly to the situation in children with FH and [21] statin-treated patients with type 2 diabetes [10], most of the squalene and non-cholesterol sterols were carried by LDL and HDL. The proportion of most non-cholesterol sterols transported by HDL became increased and that transported by LDL was decreased by both spreads according to the changes observed in the respective cholesterol levels. In addition, squalene content in HDL was decreased by the sterol ester spread for unknown reasons. These results suggest that non-cholesterol sterols as concentrations are carried in lipoproteins along with cholesterol, such that LDL carries most of non-cholesterol sterols, and their proportions are decreased when LDL cholesterol level is reduced. However, when expressed as ratios to cholesterol, the baseline results confirmed the earlier findings [10,21] that cholesterol precursors (except desmosterol) become accumulated in triglyceride-rich lipoproteins, which reflect the release of precursors from liver to the circulation. On the contrary, cholesterol absorption markers were most abundant in HDL and low in VLDL, which is in agreement with the findings that HDL participates in the transport of plant sterols from tissues to bile [20]. This distribution of non-cholesterol sterols in lipoproteins was not changed by the spreads, even though large decreases and increases of plant sterols were seen in all lipoproteins during consumption of plant stanol and sterol esters, respectively. The impairment of cholesterol absorption (i.e., ratio of cholestanol to cholesterol) was only moderate, and in some subjects the serum cholestanol ratio may have even increased (Fig. 1). The individual analysis revealed that the non-responders were not the same as the subjects who did not respond to the interventions
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by lowering their LDL cholesterol levels. However, an overall ~15% decrease in LDL cholesterol was achieved by the spreads. Similar or slightly larger reductions from 14% to 20% in LDL cholesterol have been achieved by combination of statin and ezetimibe, a new potent cholesterol absorption inhibitor [31]. Even though the goal of LDL cholesterol level b3.0 mmol/l was attained only by one subject, two out of every three subjects during the spreads instead of one out of three at baseline achieved LDL cholesterol below 4.0 mmol/l. The decrease in LDL cholesterol was highest in those subjects with the highest baseline LDL cholesterol values, suggesting that the subjects with low dose of statin or those responding insufficiently to statin treatment may obtain the most additional benefit from the cholesterol absorption inhibitor. Thus, subjects with both low and high doses of statins seem to benefit from additional plant stanol esters by decreasing LDL cholesterol and/or serum plant sterol levels, respectively. To conclude, consumption of plant stanol and sterol ester spreads for 4 weeks resulted in an additional ~15% decrease in LDL cholesterol in statin-treated FH subjects. The sterol ester spread increased the serum, lipoprotein, and red cell plant sterol ratios to cholesterol, while the plant stanol ester spread decreased the respective values. Concerning the recent data of the association of elevated serum plant sterol levels with CHD, the increments of serum plant sterols of this magnitude in FH subjects could be related to an increased risk for CHD. This further suggests that use of agents, such as plant stanol esters, which lower both LDL cholesterol and serum plant sterols, may be more beneficial especially for atherosclerosis-prone individuals receiving statin treatment.
Acknowledgements This study was financially supported by the Finnish Heart Research Foundation, the Finnish Medical Society Duodecim, the Research Foundation of Orion, the Biomedicum Helsinki Foundation, the Finnish Cultural Foundation, the Ida Montin Foundation, and Helsinki University Central Hospital. The spreads were kindly provided by Raisio Benecol, Raisio, Finland. The excellent technical
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assistance of Ms. Leena Kaipiainen, Anne Honkonen, Pia Hoffstrfm, and Ritva Nissil7 is thankfully acknowledged.
References [1] Bjfrkhem I, Boberg KM, Leitersdorf E. Inborn errors in bile acid biosynthesis and storage of sterols other than cholesterol. In: Scriver CS, Beaudet AL, Sly WS, Valle D, editors. The metabolic and molecular bases of inherited disease. New York7 McGraw-Hill, 2001. p. 2961 – 88. [2] Miettinen TA, Tilvis RS, Kes7niemi YA. Serum cholestanol and plant sterol levels in relation to cholesterol metabolism in middle-aged men. Metabolism 1989;38:136 – 40. [3] Miettinen TA, Tilvis RS, Kes7niemi 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. [4] Gylling H, Miettinen TA. Serum noncholesterol sterols related to cholesterol metabolism in familial hypercholesterolemia. Clin Chim Acta 1988;178:41 – 9. [5] Uusitupa MI, Miettinen TA, Happonen P, Ebeling T, Turtola H, Voutilainen E, et al. Lathosterol and other noncholesterol sterols during treatment of hypercholesterolemia with lovastatin alone and with cholestyramine or guar gum. Arterioscler Thromb 1992;12:807 – 13. [6] Miettinen TA. Serum squalene and methyl sterols as indicators of cholesterol synthesis in vivo. Life Sci 1969;8:713 – 21. [7] Kempen HJ, Glatz JF, Gevers Leuven JA, van der Voort HA, Katan MB. Serum lathosterol concentration is an indicator of whole-body cholesterol synthesis in humans. J Lipid Res 1988; 29:1149 – 55. [8] Miettinen TA, Strandberg TE, Gylling H. Noncholesterol sterols and cholesterol lowering by long-term simvastatin treatment in coronary patients: relation to basal serum cholestanol. Arterioscler Thromb Vasc Biol 2000;20:1340 – 6. [9] Miettinen TA, Gylling H, Lindbohm N, Miettinen TE, Rajaratnam RA, Relas H. Serum noncholesterol sterols during inhibition of cholesterol synthesis by statins. J Lab Clin Med 2003;141:131 – 7. [10] Miettinen TA, Gylling H. Synthesis and absorption markers of cholesterol in serum and lipoproteins during a large dose of statin treatment. Eur J Clin Invest 2003;33:976 – 82. [11] Hallikainen MA, Sarkkinen ES, Gylling H, Erkkil7 AT, Uusitupa MI. Comparison of the effects of plant sterol ester and plant stanol ester-enriched margarines in lowering serum cholesterol concentrations in hypercholesterolaemic subjects on a low-fat diet. Eur J Clin Nutr 2000;54:715 – 25. [12] Hendriks HF, Brink EJ, Meijer GW, Princen HM, Ntanios FY. Safety of long-term consumption of plant sterol estersenriched spread. Eur J Clin Nutr 2003;57:681 – 92. [13] Ketom7ki AM, Gylling H, Antikainen M, Siimes MA, Miettinen TA. Red cell and plasma plant sterols are related during consumption of plant stanol and sterol ester spreads in children with hypercholesterolemia. J Pediatr 2003;142:524 – 31.
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[14] Neil HA, Meijer GW, Roe LS. Randomised controlled trial of use by hypercholesterolaemic patients of a vegetable oil sterolenriched fat spread. Atherosclerosis 2001;156:329 – 37. [15] O’Neill FH, Brynes AE, Mandeno R, Seed M, Thompson GR. Head to head comparison of the cholesterol-lowering efficacy of plant sterol and plant stanol esters. Circulation 2002; 106:676 [Suppl., abstr]. [16] Jones PJ, Raeini-Sarjaz M, Ntanios FY, Vanstone CA, Feng WE, Parsons WE. Modulation of plasma lipid levels and cholesterol kinetics by phytosterol versus phytostanol esters. J Lipid Res 2000;41:697 – 705. [17] Gylling H, Puska P, Vartiainen E, Miettinen TA. Serum sterols during stanol ester feeding in a mildly hypercholesterolemic population. J Lipid Res 1999;40:593 – 600. [18] Rajaratnam RA, Gylling H, Miettinen TA. Independent association of serum squalene and noncholesterol sterols with coronary artery disease in postmenopausal women. J Am Coll Cardiol 2000;35:1185 – 91. [19] Assmann G, Cullen P, Erbey J, Ramey DR, Kannenberg F, Schulte H. Elevation in plasma sitosterol concentration is associated with an increased risk for coronary events in the PROCAM study. Circulation 2003;108:3300. [20] Robins SJ, Fasulo JM. High density lipoproteins, but not other lipoproteins, provide a vehicle for sterol transport to bile. J Clin Invest 1997;99:380 – 4. [21] Ketom7ki A, Gylling H, Siimes MA, Vuorio A, Miettinen TA. Squalene and noncholesterol sterols in serum and lipoproteins of children with and without familial hypercholesterolemia. Pediatr Res 2003;53:648 – 53. [22] Miettinen TA, Koivisto P. Non-cholesterol sterols and bile acid production in hypercholesterolaemic patients with ileal bypass. In: Paumgartner G, Stiehl A, Gerok W, editors. Bile acids and cholesterol in health disease. Lancaster7 MTP Press, 1983. p. 183 – 7. [23] Gylling H, Miettinen TA. Effects of inhibiting cholesterol absorption and synthesis on cholesterol and lipoprotein
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metabolism in hypercholesterolemic non-insulin-dependent diabetic men. J Lipid Res 1996;37:1776 – 85. 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. Arterioscler Thromb Vasc Biol 2000;20:500 – 6. Gylling H, Miettinen TA. Baseline intestinal absorption and synthesis of cholesterol regulate its response to hypolipidaemic treatments in coronary patients. Atherosclerosis 2002; 160:477 – 81. Glueck CJ, Speirs J, Tracy T, Streicher P, Illig E, Vandegrift 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. Sudhop T, Gottwald BM, von Bergmann K. Serum plant sterols as a potential risk factor for coronary heart disease. Metabolism 2002;51:1519 – 21. Leppim7ki P, Mattinen J, Slotte JP. Sterol-induced upregulation of phosphatidylcholine synthesis in cultured fibroblasts is affected by the double-bond position in the sterol tetracyclic ring structure. Eur J Biochem 2000;267:6385 – 94. Bruckdorfer KR, Demel RA, De Gier J, van Deenen LL. 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 – 345. Kolodgie FD, Gold HK, Burke AP, Fowler DR, Kruth HS, Weber DK, et al. Intraplaque hemorrhage and progression of coronary atheroma. N Engl J Med 2003;349:2316 – 25. Davidson MH, McGarry T, Bettis R, Melani L, Lipka LJ, Le Beaut AP, et al. Ezetimibe coadministered with simvastatin in patients with primary hypercholesterolemia. J Am Coll Cardiol 2002;40:2125 – 34.
Non-cholesterol sterols in serum, lipoproteins, and red cells in statin-treated FH subjects off and on plant stanol and sterol ester spreads Anna Ketom7kia,b, Helena Gyllingc,d, Tatu A. Miettinena,b,* a
Division of Internal Medicine, Department of Medicine, University of Helsinki, Finland b Biomedicum Helsinki, P.O. Box 700, FIN-00029 HUS, Finland c Department of Clinical Nutrition, University of Kuopio, Finland d Kuopio University Hospital, Kuopio, Finland
Received 9 September 2004; received in revised form 4 October 2004; accepted 8 October 2004
Abstract Background: Serum plant sterol levels are increased by consumption of statins and dietary plant sterols, and decreased by dietary plant stanols, but little is known about combination therapy of statin and plant sterols. Methods: We measured plant sterols in serum, lipoproteins, and red cells in subjects with familial hypercholesterolemia (FH) (n=18) treated with variable doses of statins off and on plant stanol (STA) and sterol ester (STE) spreads. Results: STA and STE spreads lowered LDL cholesterol ~15%. Plant sterols were decreased in serum, lipoproteins, and red cells by ~25% with STA and increased from 37% to 80% with STE, especially with high statin doses. The changes in serum were related to those in red cells. The baseline levels of serum plant sterols were negatively (r-range 0.639 to 0.935) and positively (r-range 0.526 to 0.598) correlated with the respective changes evoked by the STA and STE spreads. Conclusions: STE reduces LDL cholesterol, but increases serum, lipoprotein, and red cell plant sterol levels in statin-treated FH subjects, while all the respective values are decreased with STA. Recent predictions that elevated serum plant sterols pose an increased coronary risk suggest that increases of serum plant sterol levels should be avoided, especially in atherosclerosis-prone individuals, such as subjects with FH. D 2004 Elsevier B.V. All rights reserved. Keywords: Plant sterols; Plant stanols; Familial hypercholesterolemia; Non-cholesterol sterols; Statins; Red cells
1. Introduction * Corresponding author. Biomedicum Helsinki, P.O. Box 700, FIN-00029 HUS, Finland. Tel.: +358 9 4717 1852; fax: +358 9 4717 1851. E-mail address: [email protected] (T.A. Miettinen). 0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2004.10.008
Plant sterols (phytosterols), including sitosterol, campesterol, stigmasterol, and avenasterol, are constituents of plants and are not synthesized by human
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cells. They are present in the Western diet mainly in vegetables and vegetable oils in small amounts approx. 160–360 mg/day [1]. Small amounts (about 10%) of plant sterols are absorbed from the intestine, and thus serum contains low concentrations of plant sterols. The 5a-derivatives of phytosterols, i.e., plant stanols, comprise about 10% of daily phytosterol intake and are almost unabsorbable. Ratios of serum plant sterols and cholestanol to cholesterol reflect the efficiency of cholesterol absorption [2,3], i.e. the ratios are increased in subjects with high absorption of cholesterol. In addition, hypercholesterolemic subjects, especially those with familial hypercholesterolemia (FH), have higher serum plant sterol concentrations and ratios to cholesterol than those individuals with a normal serum lipid profile [4]. Inhibition of cholesterol synthesis by statins lowers the serum ratios of cholesterol precursors (squalene, D8-cholestenol, desmosterol, lathosterol) [5], which reflect the synthesis of cholesterol [3,6,7]. However, statins have a completely different effect on plant sterols in that they elevate their serum concentrations and the ratios of plant sterols to cholesterol [5,8–10]. Long duration [8] and high dose [9] of statin treatment (indicating effective inhibition of cholesterol synthesis), and high baseline absorption of sterols [8], are factors predicting the higher increase of serum plant sterols induced by statins. In addition, consumption of plant sterol ester-enriched food products can increase the serum plant sterol levels by ~35–65% in different hypercholesterolemic populations [11–16]. Both plant sterol and stanol ester spreads can decrease the absorption of cholesterol [16], but only plant stanol ester products inhibit the absorption of also plant sterols, leading to decreases in serum plant sterol concentrations and ratios to cholesterol [11,17]. High serum plant sterol levels with increased prevalence or incidence of coronary events [18,19] raise the question how safe are these elevated serum plant sterol levels. FH patients have high baseline serum plant sterol values and are usually treated with large statin doses for years to achieve long-term reduction of LDL cholesterol. Thus, this apparently increases correspondingly the serum contents of plant sterols more in FH than in non-FH subjects. However, since virtually all patients known to have FH are currently treated with statins, no controlled statin studies during consumption of
dietary phytosterols can be performed in subjects with FH. Thus, we studied first serum and red cells of DNA diagnosed adult FH subjects for squalene, precursor sterols, cholestanol, and plant sterols at baseline on variable dose statin treatment, i.e. when they had been using these drugs for years. Special attention was paid to the cholesterol absorption marker sterols, cholestanol and plant sterols. Second, it was interesting to examine the additional response of serum plant sterols in FH subjects to combination of the statin treatment with plant sterol and stanol ester spreads because stanol esters effectively reduce and sterol esters increase the serum plant sterol values in normal and hyperlipidemic subjects. Third, only a few studies have identified the distribution of noncholesterol sterols in different lipoproteins [10,20,21], but no data exist in FH subjects on statin treatment and during consumption of phytosterol esters. Accordingly, the limited information on the distribution of squalene and non-cholesterol sterols in different lipoproteins of FH patients prompted us to study more closely the distribution of these compounds in different lipoproteins during expected changes in cholesterol metabolism with statin alone or when combined with phytosterol esters.
2. Materials and methods 2.1. Subjects The study population comprised 18 adult subjects (6 males, 12 females) with FH with a mean age of 48F2 years (S.E.M.). They were recruited from the Outpatient Lipid Clinics of the Department of Medicine, University of Helsinki. The DNA diagnosis of FH was verified in 17 subjects, and 1 subject was clinically defined to have FH. Exclusion criteria included diabetes or kidney, liver, or thyroid diseases. The prior use of plant stanol or sterol ester spread was terminated at least 3 weeks before the first visit. Most of the subjects had used statin treatment for several years, and at least for the two last months the dose had remained unchanged. The mean daily dose of statin used in the study was equivalent to 40 mg of atorvastatin, such that the daily dose of nine subjects was b40 mg of atorvastatin (low-dose statin group), and of the other
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nine subjects z40 mg of atorvastatin (high-dose statin group) (Table 1). In addition, two subjects used bile acid binding sequestrants, resins, with 40 and 80 mg/day of atorvastatin; these are shown separately in Results. The clinical characteristics and medication of the subjects are shown in Table 1. Four of the subjects were smokers. Informed consent was obtained from all subjects. The study protocol was approved by the Ethics Committee of the Department of Medicine, University of Helsinki. 2.2. Study design This double-blinded, randomized, cross-over study consisted of two consecutive 4-week intervention periods. The patients were advised to replace 25 g of dietary fat with either the low fat (40%) plant stanol ester spreads (2 g of plant stanols daily) or a low fat (40%) plant sterol (2 g of plant sterols daily) ester
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spread in a random order. The plant stanol ester spread contained 66% of sitostanol and 29% of campestanol, and the sterol ester spread contained 45% of sitosterol, 26% of campesterol, 15% of stigmasterol, 5% of brassicasterol, and 3% of avenasterol. The fatty acid composition of the spreads was similar and included 22% of saturated fatty acids, 34% of monounsaturated fatty acids, and 44% of polyunsaturated fatty acids. All of the patients visited the Outpatient Clinics six times, twice at the beginning of the study 2 or 3 days apart, and twice at the end of the two periods. Fasting blood samples were taken on every visit. Physical examinations including weight, height, and blood pressure measurements were performed three times during the study. The empty spread containers were returned to the study nurse, and the daily intake of spread was approximated by weighing the unwashed spread containers.
Table 1 Clinical characteristics and medication of the subjects No.
Sex
Age (years)
FH-mutationa
Clinical findings
LDL-Cholb
Medication
1 2 3
f f f
51 51 62
FH-Hki FH-Hki FH-Hki
AL, XA AL, XL, XA AL, XA, CAD
6.62 3.87 4.57
4 5 6
f f m
43 46 56
FH-Turku FH-Hki FH-NK
XA AL AL, XA, CAD
4.78 5.24 4.17
7 8 9
m f m
38 47 54
FH-Hki FH-Hki FH
XA AL, XL, XA AL, CAD
4.54 4.99 3.86
10 11 12 13 14 15 16 17 18
f m f m f m f f f
43 56 43 32 45 44 56 33 59
FH-Hki FH-NK FH-Hki FH-Hki FH-NK FH-Hki FH-Hki FH-Turku FH-NK
XA AL, AL, XA XA AL, AL, XA XA
3.90 4.24 4.73 3.64 5.73 4.41 5.36 3.66 2.74
simvastatin 40 mg, ACE inhibitor, diuretic atorvastatin 80 mgc atorvastatin 80 mgc, ASA, beta-blocker, Ca-blocker, nitrate, protein pump inhibitor pravastatin 40 mg simvastatin 80 mgc atorvastatin 80 mgc, cholestipole 5 mgx2, beta-blocker, nitrate, ASA atorvastatin 80 mgc lovastatin 40 mg atorvastatin 80 mgc, beta-blocker, ASA, anti-depressive medication atorvastatin 40 mgc atorvastatin 20 mg simvastatin 40 mg, ASA simvastatin 80 mgc atorvastatin 20 mg, anti-depressive medication lovastatin 40 mg, beta-blocker, ASA lovastatin 60 mg atorvastatin 20 mg, oral contraceptives atorvastatin 40 mgc, cholestyramine 4gx2, beta-blocker, nitrate, Ca-blocker, ASA, hormone replacement therapy
XA XA
XA, CAD XA
ACE, angiotensin-converting enzyme; AL, arcus lipoides; ASA, acetyl salisylic acid; Ca-blocker, calcium channel blocker; XA, xanthomas; XL, xanthelasmas; CAD, coronary artery disease. a FH, no specific mutation has been diagnosed, but the clinical findings are consistent with FH; FH-Hki, FH-Helsinki; FH-NK, FH-North Karelia. b mmol/l. c Included in the high-dose statin group (atorvastatin z40 mg/day).
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2.3. Methods Serum total and high density lipoprotein (HDL) cholesterol and triglycerides (TG) were analyzed by the routine methods in use in our hospital laboratory. LDL was calculated according to Friedewald. From six volunteer subjects, chylomicrons (CM) were separated after a 30-min ultracentrifugation (20,000 rpm) in a fixed-angle type 50 Ti rotor (Beckman, Palo Alto, CA) with carefully overlayered 1.006 g/ml NaCl salt solution. From the infranatant, very low density lipoprotein (VLDL) (db1.006 g/ml) was separated by further ultracentrifugation with type 50.4 Ti rotor (Beckman), (18 h, 40000 rpm), followed by separation of HDL by precipitation of apolipoprotein B containing lipoproteins with phosphotungstic acid and magnesium ions (Roche, Basel, Switzerland) from the infranatant. The cholesterol and TG contents in all lipoproteins were analysed from the six subjects by commercial kits (cholesterol CHOD-PAP, triglycerides: GPO-PAP, ABX-Diagnostics, Parc Eurome´decine, France). From all patients, red cells were separated from plasma with centrifugation for 10 min (3000 rpm) and washed with 0.9% sodium chloride three times with centrifugation at 3000 rpm for 5 min. Cholesterol, squalene, and non-cholesterol sterols (cholestanol, D8 -cholestenol, desmosterol, lathosterol, campesterol, sitosterol, avenasterol, campestanol, and sitostanol) were analysed from all subjects from the serum and red cell samples, and from the six subjects also from lipoproteins (CM, VLDL, LDL, and HDL) by gas–liquid chromatography (GLC) on a 50-m-long ULTRA-2 SE-30 column (Hewlett Packard, Wilmington, DE) using 5a-cholestane as an internal standard [22]. The serum, lipoprotein, and red cell squalene and non-cholesterol sterols are given in terms of 102 mmol/mol of cholesterol and called ratios in the following sections in order to eliminate the influence of varying cholesterol concentrations. The values before spread consumption are termed as baseline values. 2.4. Statistical analysis Data analysis were performed with NCSS statistical software package (NCSS, 2000, Kaysville, UT, USA). In case of serum lipids and serum and red cell non-cholesterol sterols, the effect of the treatment
sequence was evaluated by two-way analysis of variance (ANOVA) with treatment period (baseline, sterol ester, or stanol ester) as the repeated factor, and the sequence, in which the patients consumed the spread (stanol–sterol or sterol–stanol) as the bgroupQ factor. Since no significant interaction was found, the statistical significance was tested by paired t-test. Correlations were calculated with Pearson’s product moment correlation. When the distribution of a variable was skewed, logarithmic transformations were performed. A p value b0.05 was considered statistically significant. The values are given as meanFS.E.M.
3. Results All subjects completed the study with good compliance, and no side effects were reported. The consumed amounts of the stanol and the sterol ester spreads were 25.0F0.5 and 25.4F0.3 g/day, respectively. The weight and blood pressure of the subjects remained stable during the study (weight: at the baseline 74F3 kg, at the end 73F4 kg, blood pressure: at the baseline 131/81 mm Hg, at the end 125/78 mm Hg). 3.1. Serum lipids Consumption of both spreads decreased the serum total and LDL cholesterol levels from the baseline values (Table 2), while HDL cholesterol level was increased and TG level was decreased, significantly so only during the sterol ester period. The lipid changes were similar in males and females and in subjects with different FH mutations. The proportions of subjects having LDL cholesterol below 4.0 mmol/l was Table 2 Serum lipids (mmol/l) at baseline and during stanol (STA) and sterol (STE) ester spreads Variables
Baseline
STA
STE
Cholesterol LDL-cholesterol HDL-cholesterol Triglycerides
6.30F0.24 4.50F0.21 1.26F0.05 1.19F0.10
5.65F0.22* 3.81F0.18* 1.32F0.04 1.16F0.12
5.71F0.21* 3.86F0.19* 1.37F0.04** 1.05F0.09**
MeanFS.E.M. Significantly different from baseline, *pb0.05, **pb0.01 by paired t-test.
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Fig. 1. Correlations between baseline LDL cholesterol (top), serum campesterol to cholesterol ratio (middle), serum cholestanol to cholesterol ratio (bottom) and the corresponding stanol (STA) (closed triangles) and sterol (STE) ester (open circles) induced changes in statin-treated FH subjects. The gray-colored symbols depict subjects receiving also bile acid sequestrants. *pb0.05.
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increased from 33% at baseline to 67% and 72% by plant stanol and sterol ester spreads, respectively. The baseline LDL cholesterol was negatively correlated with its respective change so that the higher the baseline LDL cholesterol level, the larger was its reduction evoked by plant sterols and stanols (Fig. 1). HDL cholesterol was increased most in subjects with the lowest baseline HDL cholesterol levels (r= 0.708 by stanol ester and r= 0.593 by sterol ester spread, pb0.01).
At baseline, LDL cholesterol was only insignificantly correlated with the statin dose (r= 0.333, ns). One of the two resin-treated subjects had the lowest value of LDL cholesterol (2.74 mmol/l), but in the other the respective value (4.17 mmol/l) was within the range of the study group (Table 1). LDL cholesterol concentrations were lower at baseline and during the intervention periods in the high-dose statin group than in the low-dose statin group (Fig. 2). This difference in LDL cholesterol between the statin groups became
Fig. 2. LDL cholesterol and serum campesterol, sitosterol, and cholestanol ratios to cholesterol in FH-subjects with high-dose statin treatment (equivalent dose of atorvastatin z40 mg/day) (closed circle) and with low-dose statin treatment (equivalent dose of atorvastatin b40 mg/day) (open square) at baseline (BL) and during consumption of stanol (STA) and sterol ester (STE) spreads. The spreads were consumed in a random order. *pb0.05, **pb0.01, ***pb0.001 from baseline, by paired t-test. #pb0.05 between the statin groups, by unpaired t-test.
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smaller when the subjects receiving resin treatment were excluded. 3.2. Serum squalene and non-cholesterol sterols The ratios of squalene and non-cholesterol sterols to cholesterol in serum at baseline and during the intervention periods are shown in Table 3. At baseline, the ratios of cholesterol absorption markers correlated positively with each other (r ranged from 0.518 to 0.947, pb0.05–0.001). Cholesterol synthesis markers (except squalene) correlated also positively with each other (r ranged from 0.485 to 0.753, pb0.05–0.001), but not with cholesterol absorption markers or with total and LDL cholesterol. However, cholesterol absorption markers were negatively correlated with total and LDL cholesterol (e.g., r= 0.583 for campesterol vs. LDL cholesterol). During the intervention periods, the level of lathosterol was increased in serum (14–19%) by both spreads, while desmosterol was increased only by stanol ester spread. During consumption of the plant stanol ester spread, campesterol, sitosterol, and aveTable 3 The ratios of serum and red cell cholesterol (mg/dl) and squalene and non-cholesterol sterols to cholesterol (102mmol/mol of cholesterol) at baseline and during consumption of plant stanol (STA) and sterol (STE) ester spreads Variables Cholesterol Squalene D8-cholestenol Desmosterol Lathosterol Campesterol Sitosterol Cholestanol Avenasterol
serum red cell serum red cell serum red cell serum red cell serum red cell serum red cell serum red cell serum red cell serum red cell
Baseline
STA
STE
212F8 108F2 28F2 41F4 14F2 14F2 67F7 63F10 66F7 132F17 508F50 580F58 265F29 304F33 195F15 190F14 60F6 74F6
192F7*** 110F1 30F3 47F6 14F2 16F2 72F5* 68F11* 75F7* 151F20* 387F39** 429F43* 190F19*** 207F21*** 182F13 182F13 42F3*** 56F3***
193F7*** 110F1 28F2 43F5 13F1 17F2 69F5 60F8 73F7* 162F28* 846F78***,# 965F87***,# 382F40***,# 415F41***,# 176F13* 186F11 60F6# 70F5#
MeanFS.E.M. Significantly different from baseline, *pb0.05, **pb0.01, ***pb0.001 by paired t-test. # Significantly different STE vs. STA, pb0.001 by paired t-test.
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nasterol ratios were decreased in serum by ~25%, while during consumption of plant sterol ester spread, the serum campesterol and sitosterol ratios were increased (48–74%). The baseline values of all plant sterols were negatively correlated with the respective change in serum (r ranged from 0.639 to 0.935, pb0.01– 0.001) (e.g., Fig. 1 for campesterol) by the plant stanol ester spread. In contrast, the changes of serum campesterol and sitosterol by sterol ester spread were positively baseline correlated with their baseline values (r ranged from 0.526 to 0.598, pb0.05) (Fig. 1). Serum cholestanol decreased significantly only during consumption of the sterol ester spread, and the baseline values were negatively correlated with the respective changes by both spreads (r ranged from 0.545 to 0.573, pb0.05) (Fig. 1). Serum plant stanols were increased by the plant stanol ester spread, but the amounts remained rather small (e.g., sitostanol from 18 to 43 Ag/dl). The ratios of cholestanol and less so plant sterols were higher in the subjects in the high-dose statin group than in the low-dose statin group (Fig. 2). This difference disappeared when the subjects on resin treatment were excluded. The changes of plant sterols and cholestanol during the spread periods were similar in the high and low statin groups. 3.3. Red cell squalene and non-cholesterol sterols At baseline, red cell desmosterol, lathosterol, cholestanol, and plant sterol ratios to cholesterol were significantly correlated with the respective ratios in serum (r ranged from 0.890 to 0.995, pb0.001). Rather similar changes were seen in red cell and serum with respect to squalene and non-cholesterol sterols during the intervention periods (Table 3), and the changes of lathosterol, campesterol, and sitosterol in serum and red cells were significantly positively correlated (r ranged from 0.802 to 0.988, pb0.001) with both spreads (e.g., Fig. 3 for campesterol and lathosterol). 3.4. Squalene and non-cholesterol sterols in different lipoproteins Since release of newly synthesized precursors, newly absorbed plant sterols or sterols released from peripheral tissues could be changed by statins without or with phytosterol consumption, we measured
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Fig. 3. Correlations between changes of serum and red cell campesterol (a) and lathosterol (b) to cholesterol ratios (102 mmol/mol of cholesterol) during stanol (STA) (closed triangles) and sterol ester (STE) (open circles) spreads. The gray-colored symbols depict subjects receiving also bile acid sequestrants. For STA and STE, the correlation coefficients are r=0.986 ( y=1.2x 9.6) and r=0.988 ( y=1.1x+9.3), respectively, for campesterol, and r=0.802 ( y=2.4x 4.5) and r=0.948 ( y=4.5x 2.4), respectively, for lathosterol, pb0.001 for all.
squalene and non-cholesterol sterols also in different lipoproteins. 3.4.1. Concentrations At baseline, about 45% of squalene was carried by LDL and 44% by HDL. During consumption of the sterol ester spread the proportion of LDL squalene was 51% but less, 36%, with the stanol ester spread (Fig. 4). LDL transported 62–75% of the noncholesterol sterols (except D8-cholestenol) at baseline and 57–60% during consumption of both spreads. For
HDL, the respective proportions were 20–25% at baseline and 23–34% during consumption of both spreads ( pb0.05 from baseline for desmosterol, campesterol, and sitosterol by both spreads). The plant sterol ester spread increased the concentrations of plant sterols in lipoproteins, especially in HDL (data not shown). 3.4.2. Ratios to cholesterol At baseline and during consumption of both spreads, the ratios of the cholesterol synthesis markers
Fig. 4. The percentage distribution of absolute amounts of cholesterol, campesterol, lathosterol, and squalene in lipoproteins of statin-treated FH subjects at baseline (BL) and during stanol (STA) and sterol (STE) ester spreads. *pb0.05 vs. baseline, #pb0.05 STA vs. STE. The absolute concentrations (meanFS.E.M.) in serum are given on the top of the columns, for cholesterol in mg/dl, for squalene and other sterols in Ag/dl.
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were most abundant in CM and VLDL, except desmosterol, which was lowest in VLDL (data not shown). The ratios of absorption markers were lowest in VLDL at baseline and also with both spreads, and they were significantly lower than in serum. HDL tended to have the highest values of the absorption
marker sterols. Plant stanols were mostly carried by CM and HDL as shown for campestanol in Table 4. The decrements and increments of plant sterols were similar in all lipoproteins by plant stanol and sterol ester spreads, respectively (Table 4).
Table 4 Concentration of cholesterol (mg/dl) and ratios of squalene, lathosterol, campesterol, sitosterol, cholestanol, and campestanol to cholesterol (102mmol/mol of cholesterol) in serum, chylomicron (CM), VLDL, LDL, and HDL at baseline and during consumption of plant stanol (STA) and sterol (STE) ester spreads in FH patients receiving statin treatment
4. Discussion
Variables
Baseline
STA
STE
Cholesterol
225F16 3F0.4 10F2 164F14 44F4 25F3 168F57*** 100F31 26F6 98F25*** 69F10 87F14*** 82F13*** 66F8 73F7 418F37 442F40*** 383F34*** 436F39 433F42 221F28 236F27 156F15*** 226F28 261F31*** 171F19 166F18 128F16*** 166F27 196F31 3F1 26F3*** 7F1*** 4F2 19F6***
210F10* 3F0.5 11F3 145F9 49F3* 22F3 160F36*** 87F45*** 23F5 118F48*** 63F6 103F17*** 84F9*** 67F6 79F13 325F44* 330F45* 287F39*,*** 330F44* 343F46* 168F23* 168F21* 110F13*,*** 167F21* 193F27*,*** 168F26 168F21 142F26*** 162F27 196F33 17F2* 30F2*** 16F3 16F4 36F4*
210F16* 4F1 10F3 140F10* 46F4** 30F3 121F19*** 83F26 33F4 97F34*** 72F5** 92F9*** 97F12*** 78F8 82F16 729F85*,** 749F80*,** 670F70*,**,*** 739F83*,** 781F83*,**,*** 331F41*,** 332F40*,** 218F26*,**,*** 326F39*,** 373F44*,**,*** 172F22 191F41 126F25*** 174F34 191F30 6F2** 25F9 11F4 5F1 25F5***
serum CM VLDL LDL HDL Squalene serum CM VLDL LDL HDL Lathosterol serum CM VLDL LDL HDL Campesterol serum CM VLDL LDL HDL Sitosterol serum CM VLDL LDL HDL Cholestanol serum CM VLDL LDL HDL Campestanol serum CM VLDL LDL HDL
MeanFS.E.M., n=6. 4 Significantly different from baseline, pb0.05 by paired t-test. ** Significantly different STA vs. STE, pb0.05 by paired t-test. *** Significantly different from the serum values, pb0.05 by unpaired t-test.
The high baseline levels of serum plant sterols in FH subjects, especially their further increase which occurs during statin treatment, prompted us to study the effects of plant stanol and sterol esters on serum, lipoprotein, and red cell plant sterol levels in FH subjects receiving long-term statin treatment. This study was carried out in FH subjects who had high baseline serum plant sterol levels and were treated with different doses of statins. Long-term statin treatment increases the serum plant sterol levels probably by decreasing the biliary excretion of sterols, leading to a subsequent increase in the absorption of dietary plant sterols from the reduced intestinal pool [10,23]. The present study was not designed to evaluate the effects of different statin doses. However, the baseline measurements did reveal that the subjects being treated with the highest doses of statins exhibited the highest levels of serum plant sterols and cholestanol, suggesting that they had a more effective cholesterol absorption efficiency. The consumption of plant sterol esters further increased the serum plant sterol levels. Even though the percentual increase calculated from Fig. 1 (middle panel) was lower in those with the higher baseline levels, the absolute increase was highest. Thus, the high statin dose increased serum plant sterol levels more than the low dose, resulting in an additional increase of serum plant sterols with dietary plant sterol esters. This suggests that subjects, such as those with FH, who need large doses of statins to control their lipid levels, are prone to present with high serum levels of plant sterols. On the contrary, plant stanol esters have been reported to lower serum plant sterol levels, even in combination with statins [23–25]. In the present study, consumption of the plant stanol ester spread lowered the serum plant sterol levels, by the greatest extent in those subjects with the highest baseline levels, a correlation shown earlier in FH subjects receiving a low statin dose [24]. Thus, the increase of serum plant
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sterols caused by statin treatment could be eradicated by plant stanol esters, and the serum levels of plant sterols in these FH subjects approached the levels found in normal population (228F11 102 mmol/mol of cholesterol) [18]. The highest total serum plant sterol level in the present study (approx. 3 mg/dl) is far below the levels found in phytosterolemia (20–94 mg/dl), which is an inherited disease caused by a mutation in the genes coding for the ATP binding cassette (ABC) transporters G5 and G8, leading to premature atherosclerosis similarly as in homozygous FH [1]. However, some evidence has been presented between increased serum plant sterol levels and the risk of coronary heart disease in non-phytosterolemic subjects [26,27]. In fact, an increased ratio of serum plant sterols to cholesterol was shown to be an independent risk factor for coronary heart disease in a randomly selected population of postmenopausal women [18]. In addition, recently even a modestly increased serum sitosterol concentration (0.2 mg/dl) was associated with an increased risk of coronary events in a 10-year follow-up population study [19]. Thus, even though the present mean serum sitosterol concentrations was minimal (up to 0.7 mg/dl) compared to respective cholesterol values, according to recent evidence, it cannot be ignored since it may represent a potential additional risk for coronary events, especially in the FH subjects even though these subjects show markedly reduced LDL cholesterol levels. In addition, in this study, in agreement with earlier studies in hypercholesterolemic children [13] and in non-FH hypercholesterolemic adults [12], plant sterols were increased in the red cells in a parallel fashion to those in serum by the sterol ester spread. In addition, the correlation in Fig. 3b indicates that small lathosterol changes in serum do modulate more effectively the lathosterol content in red cells. This is in accordance with an in vitro study that demonstrated that lathosterol was more efficiently taken up than cholesterol by fibroblasts [28]. The non-cholesterol sterols are capable of replacing cholesterol from red cells in animal experiments [29], but in the present as well as in previous studies, red cell cholesterol levels were unchanged or even tended to be increased, suggesting that increased plant sterols in serum could accumulate in red cells with cholesterol. In one study, the increased plant sterol levels in red cell membranes
did not evoke any changes in membrane rigidity after 1 year of treatment with plant sterol esters [12]. However, according to a recent study, free cholesterol transported by the red cells may destabilize the atheromatotic plaque and make it vulnerable to rupture [30]. It is not known whether increased levels of plant sterols in red cell membranes may also have a harmful effect on plaque stability; this is a topic which needs to be clarified. The present study revealed that changes in percentual proportions of non-cholesterol sterols in different lipoproteins mimicked those of cholesterol. Thus, at baseline similarly to the situation in children with FH and [21] statin-treated patients with type 2 diabetes [10], most of the squalene and non-cholesterol sterols were carried by LDL and HDL. The proportion of most non-cholesterol sterols transported by HDL became increased and that transported by LDL was decreased by both spreads according to the changes observed in the respective cholesterol levels. In addition, squalene content in HDL was decreased by the sterol ester spread for unknown reasons. These results suggest that non-cholesterol sterols as concentrations are carried in lipoproteins along with cholesterol, such that LDL carries most of non-cholesterol sterols, and their proportions are decreased when LDL cholesterol level is reduced. However, when expressed as ratios to cholesterol, the baseline results confirmed the earlier findings [10,21] that cholesterol precursors (except desmosterol) become accumulated in triglyceride-rich lipoproteins, which reflect the release of precursors from liver to the circulation. On the contrary, cholesterol absorption markers were most abundant in HDL and low in VLDL, which is in agreement with the findings that HDL participates in the transport of plant sterols from tissues to bile [20]. This distribution of non-cholesterol sterols in lipoproteins was not changed by the spreads, even though large decreases and increases of plant sterols were seen in all lipoproteins during consumption of plant stanol and sterol esters, respectively. The impairment of cholesterol absorption (i.e., ratio of cholestanol to cholesterol) was only moderate, and in some subjects the serum cholestanol ratio may have even increased (Fig. 1). The individual analysis revealed that the non-responders were not the same as the subjects who did not respond to the interventions
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by lowering their LDL cholesterol levels. However, an overall ~15% decrease in LDL cholesterol was achieved by the spreads. Similar or slightly larger reductions from 14% to 20% in LDL cholesterol have been achieved by combination of statin and ezetimibe, a new potent cholesterol absorption inhibitor [31]. Even though the goal of LDL cholesterol level b3.0 mmol/l was attained only by one subject, two out of every three subjects during the spreads instead of one out of three at baseline achieved LDL cholesterol below 4.0 mmol/l. The decrease in LDL cholesterol was highest in those subjects with the highest baseline LDL cholesterol values, suggesting that the subjects with low dose of statin or those responding insufficiently to statin treatment may obtain the most additional benefit from the cholesterol absorption inhibitor. Thus, subjects with both low and high doses of statins seem to benefit from additional plant stanol esters by decreasing LDL cholesterol and/or serum plant sterol levels, respectively. To conclude, consumption of plant stanol and sterol ester spreads for 4 weeks resulted in an additional ~15% decrease in LDL cholesterol in statin-treated FH subjects. The sterol ester spread increased the serum, lipoprotein, and red cell plant sterol ratios to cholesterol, while the plant stanol ester spread decreased the respective values. Concerning the recent data of the association of elevated serum plant sterol levels with CHD, the increments of serum plant sterols of this magnitude in FH subjects could be related to an increased risk for CHD. This further suggests that use of agents, such as plant stanol esters, which lower both LDL cholesterol and serum plant sterols, may be more beneficial especially for atherosclerosis-prone individuals receiving statin treatment.
Acknowledgements This study was financially supported by the Finnish Heart Research Foundation, the Finnish Medical Society Duodecim, the Research Foundation of Orion, the Biomedicum Helsinki Foundation, the Finnish Cultural Foundation, the Ida Montin Foundation, and Helsinki University Central Hospital. The spreads were kindly provided by Raisio Benecol, Raisio, Finland. The excellent technical
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assistance of Ms. Leena Kaipiainen, Anne Honkonen, Pia Hoffstrfm, and Ritva Nissil7 is thankfully acknowledged.
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