Effects of Butter Oil Blends with Increased Concentrations of Stearic, Oleic and Linolenic Acid on Blood Lipids in Young Adults

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Effects of butter oil blends with increased concentrations of stearic, oleic and linolenic acid on blood lipids in young adults

CC Becker1, P Lund1, G Hùlmer1*, H Jensen2 and B SandstroÈm2

1Department of Biochemistry and Nutrition, Center for Food Research, Technical University of Denmark, Denmark; and 2Research Department of Human Nutrition, Center for Food Research, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark

Objective: The aim of this present project was to evaluate a more satisfactory effect on plasma lipoprotein pro®le of spreads based on dairy fat. Design: This study was designed as a randomised cross-over experiment with a three-week treatment separated by a three-week wash-out period. Sixty ®ve grams of the fat content of the habitual diets was replaced by either butter=grapeseed oil (90 : 10) (BG); butter oil and low erucic rapeseed oil (65 : 35) (BR) or butter blended in a 1 : 1 ratio with a interesteri®ed mixture of rapeseed oil and fully hydrogenated rapeseed oil (70 : 30) (BS). Subjects: Thirteen healthy free-living young men (age 21±26 y) ful®lled the study. Interventions: At the beginning and end of each diet period two venous blood samples were collected. Triacylglycerol and cholesterol concentrations in total plasma and VLDL, LDL, IDL and HDL fractions were measured, as were apo A-1 and apo B concentrations. Fatty acid composition of plasma phospholipids, plasma cholesterol ester and platelets was also determined. Results: Signi®cantly (P < 0.05) lower total and LDL-cholesterol concentrations were observed after the BR and BS period, compared to BG. The effect of BR and BS did not differ. BG and BR resulted in equal concentrations of HDL-C, but signi®cantly higher than BS. Consequently, a signi®cantly lower LDL-C=HDL-C ratio was seen after the BR treatment compared to BG and BS. Apo A-1 concentrations were not signi®cantly different, but Apo B was signi®cantly increased after BG. Conclusions: Partially replacing milk fat with rapeseed oil seems to yield a more healthy spread. Stearic acid had a HDL-C lowering effect compared to milk fat, but did not affect LDL-C signi®cantly. The addition of stearic acid did not improve the plasma lipoprotein pro®le for young men with low cholesterol levels. Sponsorship: Danish Food Research Programme (FéTEK I) and Danish Dairy Research Foundation. Descriptors: butter oil; rapeseed oil; cholesterol; apoproteins; platelets; plasma phospholipids; plasma cholesterol esters; stearic acid; oleic acid; linolenic acid

Introduction saturated fatty acid, seems to have an effect on plasma lipids that more closely resembles that of oleic acid than the The effect of individual fatty acids on blood lipids and other principal saturated fatty acids. The reason for the lipoproteins and their potential atherogenicity depends on observed neutrality of stearic acid is unknown, but it has chain length and degree of unsaturation (Mensink & Katan, been suggested that it is poorly absorbed (Grande et al, 1992; Yu et al, 1995; Howell et al, 1997). Fatty acids with 1970), or quickly desaturated to oleic acid (Bonanome & fewer than twelve carbon atoms do not seem to in¯uence Grundy, 1989). It seems, however, that desaturation is not plasma cholesterol concentrations more than carbohydrate extensive enough to explain the non-cholesterolemic effect does (Hill et al, 1990), whereas saturated fatty acid with of this fatty acid in humans (Rhee et al, 1997; Pai & Yeh, chain lengths of 12, 14, and 16 carbon atoms have shown a 1997). hypercholesterolemic effect in humans (Hegsted et al, Generally, oleic acid and linoleic acid consumption 1965; Keys et al, 1965; Yu et al, 1995). Early studies leads to a lowering of plasma cholesterol (C) and LDL-C (Ahrens et al, 1957; Hegsted et al, 1965; Keys et al, 1965), concentrations when replacing saturates with 12±16 car- as well as more recent ones (Kris-Etherton et al, 1993; Yu bons (SFA) iso-energetically (Hegsted et al, 1965; Mensink et al, 1995) have shown that stearic acid, although a & Katan, 1992; Yu et al, 1995). In a meta-analysis of 27 clinical studies, Mensink & Katan (Mensink & Katan, 1992) concluded that polyunsaturated fatty acids (mostly *Correspondence: Prof G Hùlmer, Department of Biochemistry and linoleic acid) and monounsaturated cis fatty acids (mostly Nutrition, Bldg. 224, Technical University of Denmark, DK-2800 Lyngby, oleic acid) have similar effects on the concentration of Denmark. Contributors: G Hùlmer and B SandstroÈm initiated the project, which was HDL-C. Another meta-analysis of 18 studies by Yu et al, part of the PhD study of CC Becker. All the authors were involved in 1995, found that monounsaturated cis fatty acids raised carrying out the experimental work. The food registration was conducted HDL-C three times as much as polyunsaturated fatty acids. by H Jensen and B SandstroÈm. CC Becker made the statistical analysis. CC A technological advantage of oleic acid over linoleic acid is Becker, P Lund and G Hùlmer wrote the article and B SandstroÈm took part less susceptibility to oxidation, giving products with a in its ®nalisation. Guarantor: G Hùlmer. Received Received 30 November 1998; revised 4 February 1999; accepted longer shelf life. Furthermore, the susceptibility to oxida- 13 February 1999 tion and the atherogenicity of plasma lipoproteins decreases Blood lipids and milk fat based spreads CC Becker et al 536 with increasing saturation of its constituent fatty acids, old, and weighed between 61 and 88 kg at the outset of this which re¯ect the diet (Aviram & Eias, 1993). study. The hypercholesterolemic effect of dairy products and The study protocol was reviewed and approved by the butter oil, has been reported often in both clinical (Wardlaw regional ethical committee (Frederiksberg) and was in & Snook, 1990; Kris-Etherton et al, 1993) and epidemio- accordance with the Helsinki declaration of 1983. Informed logical studies (Renaud et al, 1991). Butter fat usually written consent was obtained from each participant. contains about 60% saturates (by weight) of which approximately 10±12% is stearic acid and another 12% are short Test fats chain fatty acids. Three test fats were prepared by Aarhus Oil, Aarhus, The purpose of this study was to investigate the effects Denmark. They were BG: butter=grapeseed oil (90 : 10); of enriching butter with oleic and linolenic acids (from BR: butter and low-erucic rapeseed oil (65 : 35); and BS: rapeseed oil) or with stearic acid (from fully hydrogenated butter blended in a 1 : 1 ratio with an interesteri®ed mixture rapeseed oil) on the plasma concentrations of lipids, and of rapeseed oil and fully hydrogenated rapeseed oil apolipoproteins. The re¯ections of dietary fatty acids on (70 : 30). All fats were made to contain about 8.5% fatty acid distribution in blood lipids as well as platelets (w=w) linoleic acid, to ensure an adequate supply thereof. were also examined. The fatty acid compositions of the dietary test fats are Through dilution of the butter fat with vegetable oils, a given in Table 1. Regiospeci®c analyses were carried out product, similar to butter in appearance and taste, but with according to (Becker et al, 1993). A single lot of each test less ability to raise cholesterol concentrations in the blood fat was prepared to cover the needs of the entire study. stream, can be obtained. Rapeseed oil was selected for this Fatty acid composition of the test fats was determined by study, as it is a rich source of both oleic, and linolenic the gas chromatographic (GC) analysis of fatty acid methyl acids. Linolenic acid seems to have an indirect inhibitory esters, obtained by KOH catalysed transmethylation effect on platelet aggregation (Renaud, 1990) and throm- according to (Christofferson & Glass, 1969). Fatty acid bosis as n-3 fatty acids give rise to other prostanoids than methyl ester analyses were carried out on Hewlett-Packard do n-6 fatty acids. 5880A gas chromatograph with a 30 m60.32 mm fused There is however a limit for the addition of vegetable oil silica capillary column coated with a 0.2 mm ®lm of SP2380 if a solid spread, at room temperature, should be obtained. (Supelco Inc., Bellefonte, PA, USA). Initial oven tempera- Increasing the content of stearic acid might be a bene®cial ture was maintained at 35C for 3 min before rising to way of solving this problem instead of using partially 155C at a rate of 20C=min. After 2 min the temperature hydrogenated oils, which may have unfavourable effects was increased at a rate of 10C=min to 225C and main- on blood lipids (Aro et al, 1997). tained there for 7 min.

Diets Subjects and methods The habitual dietary intake was assessed by a seven-day weighed food record. This assessment was repeated for Design seven days in each dietary period. Energy and nutrient Thirteen young men participated serving as their own intake were calculated with the use of a national food controls in this intervention trial. A three-period three- database `Dankost-2' (Mùller, 1989). During the test per- treatment cross-over design was used to minimise the iods the subjects were given 65 g of test fat per day. Thirty effect of variation between subjects. The study consisted grams of test fat was delivered as spread and the remaining of three three-week dietary periods interrupted by a three- week wash-out period. The subjects were randomly assigned to one of the three treatment groups in the ®rst Table 1 Fatty acid composition of the triacylglycerols (TG) and 2- period and subsequently randomly assigned to one of the monoacylglycerols (2-MG) of the test fats. (wt%) remaining two treatments. No intercurrent illnesses BG BR BS occurred and the concentrations of C-reactive proteins were below 5 mg=L throughout the experiment, except in TG 2-MG TG 2-MG TG 2-MG two cases where it was 12 and 40 mg=L. Values below 10 C4:0 5.0 0.0 3.7 0.0 2.8 0.0 were considered normal so 12 mg=L is a little greater than C6:0 2.6 0.0 1.9 0.0 1.5 0.0 normal, while 40 mg=L is elevated and indicative of a C8:0 1.4 0.0 1.1 0.0 0.8 0.0 minor infection (Kessler et al, 1994). However, the lipid C10:0 3.0 0.8 2.2 0.7 1.8 0.8 C12:0 3.6 4.2 2.6 2.5 2.0 2.6 values for these persons were normal, and they were not C14:0 10.4 18.9 7.6 13.2 6.0 11.9 excluded. C14:1 n-5 1.4 2.3 1.1 1.6 0.8 1.4 C15:0 1.1 1.6 0.8 1.1 0.6 1.0 C16:0 26.1 34.8 20.0 25.5 16.8 23.7 Subjects C16:1 n-7 0.9 2.7 1.3 2.0 1.0 1.8 Participants were adult male volunteers in good health C17:0 0.7 0.5 0.4 0.4 0.4 0.3 recruited among students from the University campus. C18:0 9.9 5.0 7.3 3.9 19.2 14.5 Applicants were selected according to age, the results of C18:1 n-9a 23.0 18.5 34.3 26.5 30.9 26.4 C18:1 n-7 0.6 0.3 1.5 0.4 1.5 1.5 a blood test and an interview. Eligible were non-smoking C18:2 n-6 8.7 8.9 8.5 13.6 8.2 8.4 male subjects in their twenties who had total serum cho- C18:2 conj 1.0 0.6 1.2 0.4 1.0 0.4 lesterol concentrations greater than 4.0 mmol=L, and who C18:3 n-3 0.5 0.5 4.3 7.9 3.9 4.5 exercised moderately or not at all. Candidates who were C20:0 0.1 0.1 0.4 0.2 0.6 0.6 diabetic or taking medication for any reason were elimi- BG: 90% butter oil 10% grapeseed oil; BR: 65% butter oil 35% nated. All participants agreed to maintain their pre-study rapeseed oil; BS: 50% butter oil 50% interesteri®ed (70% rapeseed weight and level of physical activity. They were 21±26 y oil 30% fully hydrogenated rapeseed oil). aIncluding trans 18:1 isomers. Blood lipids and milk fat based spreads CC Becker et al 537 in the form of two daily snacks (a cake and a bun) prepared at chloroform=methanol, separated by thin layer chromatogra- the Department. The test fats were weighed in individual daily phy, isolated and methylated (transmethylation catalysed by portions and delivered weekly. Individual dietary instruc- BF3) as previously described (Hùy & Hùlmer, 1988). tions, based on the food records, and aimed to reduce habitual The fatty acid composition was determined using a fat intake by an amount similar to the provided test fat, were HP5880A gas chromatograph with split injection, FID given by a dietician. All visible fat in the form of spreads was and a 30 m60.32 mm fused silica column with a 0.2 mm excluded as well as cheese. High fat dairy products were ®lm of SP2380 (Supelco Inc. Bellefonte, PA). The oven replaced by low-fat products. Low-fat meat cuts, marmalade temperature was programmed to rise at a rate of 2C=min and honey were delivered free from the Department in from 140±160C where it was maintained for 2 min before amounts requested by the subjects for bread meals, which rising again at 3C=min to 200C. comprise a relatively large part of the Danish diet. The subjects were also instructed to keep their food habits as Platelets similar as possible in the three test periods and to avoid Platelet-rich plasma (separated by centrifugation 2006g excessive food and alcohol intake. Duplicate portions of the for 10 min) was centrifuged at 10006g for 20 min. After test foods and fats were directly analysed to check their the removal of platelet-poor plasma, the platelet pellet was conformity with the planned fat intake. washed twice with aqueous NaCl (0.15 mol=L). The platelets were stored at 7 40C under methanol containing Blood collection and analysis 0.01% BHT to avoid fatty acid oxidation. The lipids were At the beginning and end of each diet period two venous later extracted with chloroform=methanol (1 : 1 v=v) and blood samples were collected on consecutive days after freed from protein and water by ®ltration through a layer of 10 min supine rest in the morning with minimal stasis anhydrous Na2SO4. After evaporating the solvent the lipid (Davis et al, 1990). Subjects were fasting ( > 12 h) and was dissolved in chloroform=methanol (95 : 5) containing had abstained from drinking alcohol ( > 12 h) and heavy 0.005% BHT. Fatty acid methyl esters were prepared by physical activity ( > 48 h). Twelve millilitre samples were BF3 catalysed transmethylation and analysed by gas chro- drawn into siliconised evacuated tubes containing EDTA matography using a HP5890 gas chromatograph with a FID (plasma analysis) or citrate (platelet analysis). Plasma was and on-column injection. The column was a separated by centrifugation (10006g for 15 min) within the 50 m60.25 mm fused silica capillary coated with 0.2 mm hour, and aliquots were isolated for the immediate analysis CP-Sil 88 (Chrompack, Middleburg, the Netherlands). The of apoproteins A-1 and B, and for plasma C, HDL-C and temperature was programmed to rise from 90±160Cata TG measurement. VLDL, LDL, IDL and HDL fractions rate of 40C=min, and then to 200Cat4C=min, and were isolated by ultracentrifugation (L7-55, Beckman ®nally after 10 min to 220C at a rate of 4C=min. Instruments, Palo Alto, CA) of fresh plasma in a 50.4 Ti rotor (Beckman Instruments, Palo Alto, CA). Statistical analysis VLDL was isolated by adjusting the density of 3 mL Statistical analyses were performed using Statgraphics plasma to 1.006 g=L and centrifugation for 16 h at statistical software (Manugistics Inc., Rockville, MD) or 150 0006g. The tubes were sliced 30 mm from the a validated spreadsheet template (Excel). Tests were car- bottom. The top VLDL fraction and the bottom fraction ried out at the 5% level of signi®cance, unless otherwise containing IDL, LDL and HDL were each brought to a total noted. A two tailed test with a signi®cance level of 1% was volume of 5 mL. Likewise IDL and VLDL were isolated by used in tables of fatty acid compositions. adjusting the density of 3 mL plasma to 1.019 g=L and While the results are tabulated as the means of multiple centrifugation for 16 h at 150 0006g (top fraction: subjects, the actual statistical tests were carried out on IDL VLDL; bottom fraction: LDL HDL) and HDL individual differences using Students t-test for paired was isolated by adjusting the density of 3 mL plasma to observations. Therefore, each subject served as his own 1.063 g=L and centrifugation for 18 h at 170 0006g (top control and the variation between individuals was mini- fraction: VLDL IDL LDL; bottom fraction: HDL). mised to make the effects between diets more easily Enzymatic methods were used to assess concentrations detectable. The values shown in the tables are combined of cholesterol and triacylglycerols in plasma and lipopro- data from all subjects because statistical analysis showed tein subfractions of fresh plasma (Boehringer Mannheim no signi®cant effect (P < 0.05) of time or sequence in GmbH, Mannheim, Germany). Measurements were per- feeding the diets. formed on a robotic analyser (Cobas Mira, Hoffmann la Roche, Basle, Switzerland). Serum HDL-cholesterol was Results enzymatically determined after the precipitation of apo B containing lipoproteins by phosphor-wolfram acid-MgCl2 Subjects reagent. The concentration of serum C-reactive protein was Of the 15 selected participants, 13 completed the protocol, determined by an immunochemical method (Orion Diag- and two people left the study for personal reasons. The nostica, Espoo, Finland). The concentrations of apolipo- body mass of the participants did not change signi®cantly proteins A-1 and B were determined manually on fresh throughout the experiment. Participants used checklists to plasma. Assay kits and reference standards were purchased keep daily records of all food eaten extramurally and these from Boehringer Mannheim (Mannheim, Germany). The were discussed during informal interviews. The key data average coef®cient of variation (CV) for total C, HDL-C are summarised in Table 2. Average cholesterol intake was and TG determinations was less than 2%. The mean CV for 9% higher during the BG period compared to the BR apoprotein measurements was less than 3%. period. Other variables differed little. There was no indica- The fatty acid pro®les of plasma phospholipids (PL) and tion of a preference for any one of the three test fats among cholesterol esters (CE) were determined at the end of each the subjects and an independent taste panel later found the dietary period. The plasma lipids were extracted into test fats to be equally palatable (unpublished results). Blood lipids and milk fat based spreads CC Becker et al 538 Table 2 Nutrient and energy content of the diets consumed during Plasma lipid and lipoprotein changes experimental periodsa Table 3 presents the concentrations of plasma C, plasma BG BR BS TG, LDL-C (calculated according to Friedewald (Friede- wald et al, 1972)), HDL-C (precipitated) and apoproteins Energy (MJ) 13.1Æ 2.2 12.9Æ 1.8 13.2Æ 2.3 Protein (g) 88.5Æ 20.6 90.5Æ 18.6 93.8Æ 24.4 A-1 and B, as well as the concentrations of C and TG in Fat (g) 104.5Æ 15.8 105.5Æ 15.8 107.4Æ 16.6 VLDL, LDL, IDL and HDL fractions isolated by ultracen- Saturated fatty acids (g) 50.1Æ 12.1 39.7Æ 5.3 44.1Æ 7.4 trifugation. To convert cholesterol or triacylglycerol values Monounsat. fatty acids (g) 30.6Æ 10.2 37.0Æ 5.4 34.2Æ 5.6 to mg=dL, multiply by 38.61 or 88.5. Polyunsat. fatty acids (g) 14.7Æ 4.4 15.9Æ 3.0 15.3Æ 2.5 A signi®cant (P < 0.009) decrease of 0.32 and Cholesterol (mg) 448Æ 91 411Æ 82 427Æ 121 Carbohydrates (g) 412.7Æ 88.2 400.5Æ 79.5 408.6Æ 90.1 0.30 mmol=L (7%) occurred in total plasma cholesterol Fibre (g) 30.8Æ 6.2 29.5Æ 4.6 28.8Æ 7.5 concentrations (total C) when BG was replaced with BR Vitamin C (mg) 93.6Æ 105.7 61.6Æ 44.7 46.6Æ 38.4 or BS. This reduction was almost entirely due to decreased Calcium (mg) 1025Æ 443 948Æ 367 1045Æ 411 concentrations of LDL-C. Estimated (Friedewald et al, Iron (mg) 13.7Æ 2.3 13.7Æ 3.1 14.0Æ 3.0 Zinc (mg) 12.5Æ 2.9 12.5Æ 2.2 12.8Æ 3.1 1972) concentrations of LDL-C fell by 0.31 (12%, Protein (% of energy) 11Æ 212Æ 212Æ 2 P < 0.019) and 0.21 mmol=L (8%, P < 0.002), respectively, Fat (% of energy) 31Æ 432Æ 431Æ 4 while LDL-C isolated by ultracentrifugation was decreased Carbohydrates (% of energy) 54Æ 654Æ 552Æ 5 by 0.41 (16%, P < 0.014) and 0.27 mmol=L (11%, Alcohol (% of energy) 4Æ 33Æ 24Æ 3 P < 0.005). BG and BR gave similar HDL-C values, BG: 90% butter oil 10% grapeseed oil, n 13; BR: 65% butter which were 0.10±0.13 mmol=L greater than the HDL-C oil 35% rapeseed oil, n 12; BS: 50% butter oil 50% interesteri®ed concentration after BS (P < 0.045). These differences were (70% rapeseed oil 30% fully hydrogenated rapeseed oil), n 13. highly signi®cant for the results obtained by ultracentrifu- a Calculated from weighed food records and analysis of test fats. Values gation (P < 0.005). Hence, the total C=HDL-C ratio and the are meansÆ s.d. LDL-C=HDL-C ratios determined either after precipitation or ultracentrifugation were signi®cantly lower (8%, 15% and 17% respectively) after BR. Test fat composition The test fats resulted in apo A-1 concentrations which The total, as well as the regiospeci®c, compositions of the were not signi®cantly different. BR and BS gave concen- test fats are given in Table 1. The concentration of stearic trations of apo B which were not signi®cantly different, but acid in BS, was 1.9 times greater than in BG and 2.6 times were signi®cantly lower (9%, P < 0.05) than the apo B greater than in BR. BR contained more oleic and linolenic concentration after BG. Following BR and BS intake, the acid than BG, due to the incorporation of rapeseed oil, but ratios of apo B to apo A-1 were not signi®cantly different was comparable to BS in that respect. BG had a greater but were signi®cantly lower (P < 0.05) than the BG value. concentration of the saturated fatty acids with 16 or fewer Triacylglycerol concentrations were unaffected by the carbon atoms than the other test fats. The sn-2 position of experimental diets except for a decrease in LDL-TG after the test fats did not contain any of the characteristic short BS compared to BG. and medium chain fatty acids found in dairy fats, but was, as expected, enriched in myristic and palmitic acids. All the test fats contained approximately 8.5% linoleic acid. More Fatty acid composition of plasma lipids and platelets than half of the linoleic acid (C18:2 n-6) and linolenic acid The fatty acid composition of the plasma PL and CE (Table (C18:2 n-3) of BR was concentrated in the sn-2 position, 4) was only slightly affected by the differences between the while these were more evenly distributed in the other fats. test fats. This might be due to dilution by the lipid that was

Table 3 Concentrations of cholesterol and triacylglycerol in plasma, VLDL, IDL, LDL, HDL and apoproteins A-1 and Ba

BG BR BS

Total cholesterol, mmol=L 4.23Æ 0.18 3.91Æ 0.19{{{ 3.93Æ 0.15{{ VLDL cholesterol, mmol=Lb 0.18Æ 0.02 0.17Æ 0.03 0.22Æ 0.03 LDL cholesterol (Friedewald) mmol=Lc 2.61Æ 0.19 2.30Æ 0.18{{ 2.40Æ 0.13{{{ LDL cholesterol, mmol=Lb 2.55Æ 0.17 2.14Æ 0.18{{ 2.28Æ 0.11{{{ IDL cholesterol, mmol=Lb 0.07Æ 0.01 0.08Æ 0.01{ 0.14Æ 0.02{{ HDL cholesterol (precipitated) mmol=L 1.27Æ 0.07 1.28Æ 0.05§ 1.18Æ 0.07{{{{ HDL cholesterol, mmol=Lb 1.43Æ 0.06 1.41Æ 0.06§§§ 1.30Æ 0.06{{{{{{ Plasma triacylglycerol, mmol=L 0.77Æ 0.05 0.73Æ 0.06 0.77Æ 0.05 VLDL triacylglycerol, mmol=Lb 0.48Æ 0.05 0.42Æ 0.06 0.50Æ 0.05 LDL triacylglyerol, mmol=Lb 0.13Æ 0.01 0.12Æ 0.01 0.11Æ 0.01{ IDL triacylglycerol, mmol=Lb 0.04Æ 0.00 0.04Æ 0.00 0.04Æ 0.00 HDL triacylglycerol (precipitated) mmol=L 0.15Æ 0.01 0.16Æ 0.01 0.15Æ 0.01 HDL triacylglycerol, mmol=Lb 0.13Æ 0.01 0.13Æ 0.01 0.12Æ 0.01 Apo A-1, mg=L 1374Æ 22 1417Æ 46 1364Æ 32 Apo B, mg=L 1034Æ 63 957Æ 53{ 930Æ 44{ C=HDL-C (precipitated) 3.33Æ 0.24 3.06Æ 0.18{{§§§ 3.33Æ 0.20{{{ Apo B=Apo A-1 0.76Æ 0.05 0.68Æ 0.04{ 0.69Æ 0.04{ LDL-C=HDL-Cd 2.17Æ 0.22 1.83Æ 0.16{{§§§ 2.13Æ 0.18{{{ LDL-C=HDL-Cb 1.84Æ 0.16 1.52Æ 0.12{§§ 1.81Æ 0.12{{

BG: 90% butter oil 10% grapeseed oil; BR: 65% butter oil 35% rapeseed oil; BS: 50% butter oil 50% interesteri®ed (70% rapeseed oil 30% fully hydrogenated rapeseed oil). aMeanÆ s.e.m. n 13. bIsolated by ultracentrifugation. cCalculated according to the formula: LDL-C TC 7 (HDL- C Tot.TG=2.2). dLDL (Friedewald) and precipitated HDL. {Differs from BG. {(P < 0.05); {(P < 0.02); {{(P < 0.005). {Differs from BR. {(P < 0.05); {{(P < 0.02); {{{(P < 0.005). §Differs from BS. §(P < 0.05); §§(P < 0.02); §§§(P < 0.005). Blood lipids and milk fat based spreads CC Becker et al 539 Table 4 Major fatty acids in plasma phospholipid (PL) and cholesterol ester (CE). (wt%)a

PL CE

BG BR BS BG BR BS

C14:0 0.6Æ 0.1 0.7Æ 0.1 0.7Æ 0.1 1.1Æ 0.1 1.0Æ 0.0 0.9Æ 0.1 C16:0 26.1Æ 0.2§ 25.7Æ 0.2§ 24.6Æ 0.3{{ 11.4Æ 0.2§ 11.4Æ 0.2§ 10.5Æ 0.1{{ C16:1 n-7 0.5Æ 0.0 0.5Æ 0.0 0.5Æ 0.0 2.2Æ 0.2 2.1Æ 0.2 2.0Æ 0.2 C18:0 13.2Æ 0.3 12.8Æ 0.4 13.8Æ 0.2 1.7Æ 0.1 1.7Æ 0.1 1.6Æ 0.1 C18:1 n-9 9.0Æ 0.2{ 9.8Æ 0.2{ 9.6Æ 0.3 16.2Æ 0.4{ 17.4Æ 0.4{ 17.3Æ 0.4 C18:1 n-7 1.4Æ 0.0{ 1.6Æ 0.1{§ 1.4Æ 0.1{ 1.1Æ 0.0{ 1.3Æ 0.0{§ 1.1Æ 0.0{ C18:2 n-6 22.7Æ 0.6 21.8Æ 0.5 22.5Æ 0.4 49.4Æ 0.8 47.8Æ 0.9 48.9Æ 0.9 C18:3 n-3 0.2Æ 0.0{ 0.4Æ 0.0{ 0.4Æ 0.0{ 0.7Æ 0.0{§ 1.1Æ 0.0{ 1.1Æ 0.0{ C20:3 n-6 0.5Æ 0.1 0.3Æ 0.1 0.4Æ 0.1 0.7Æ 0.0 0.6Æ 0.0 0.6Æ 0.0 C20:4 n-6 8.7Æ 0.3 9.0Æ 0.3 8.5Æ 0.2 5.2Æ 0.2 5.3Æ 0.3 5.2Æ 0.2 C20:5 n-3 1.4Æ 0.2 1.6Æ 0.2 1.5Æ 0.2 0.9Æ 0.2 0.9Æ 0.1 1.0Æ 0.1 C22:6 n-3 4.7Æ 0.3 4.5Æ 0.3 4.1Æ 0.3 0.6Æ 0.1 0.6Æ 0.0 0.6Æ 0.1

BG: 90% butter oil 10% grapeseed oil; BR: 65% butter oil 35% rapeseed oil; BS: 50% butter oil 50% interesteri®ed (70% rapeseed oil 30% fully hydrogenated rapeseed oil). aMeanÆ s.e.m. n 13. {Signi®cantly different from BG (P < 0.01). {Signi®cantly different from BR (P < 0.01). §Signi®cantly different from BS (P < 0.01). in the self selected part of the diets. However, compliance difference. There was no signi®cant difference between the was con®rmed by the higher amount of linolenic acid in effects of the experimental fats on the linoleic acid com- both PL and CE after the rapeseed oil containing test fats. position of the platelets, probably because all test fats Oleic and palmitic acid concentrations also varied in a contained the same amount of C18:2 n-6. dose-dependent manner, with the lowest concentrations of palmitic acid after BS (P < 0.003). The concentration of oleic acid was higher after BR than BG with the value for Discussion BS being intermediate and insigni®cantly different from the The subjects went through the study without signi®cant two. changes in the intake of cholesterol, total fat or energy, or The fatty acid pro®le of the platelets (Table 5) largely any other micro or macro nutrient (Table 2). Each treatment re¯ected the composition of the test fats. The concentration period lasted three weeks. Previous experiments have of stearic acid was signi®cantly higher (P < 0.01) after BS shown that this duration is suf®cient to achieve steady compared to the other test fats. The concentration of state (SandstroÈm et al, 1992). Plasma and platelet lipid palmitic acid, on the other hand was signi®cantly lower (Tables 4 and 5) composition varied with the diet, an (P < 0.01) after BS compared to the other test fats indication of compliance. (P < 0.004). After all diet periods the sum of palmitic and The principal result (Table 3) of the experiment was that stearic acid concentrations were between 31.7 and 32.3%, the rapeseed oil (oleic and linolenic acids) enriched fats which is not statistically different. The concentrations of (BR and BS, respectively) appeared to be equally effective C18:3 n-3 and C18:1 n-9 were lower (P < 0.002) after BG in reducing plasma cholesterol and LDL cholesterol com- than after the other test fats, between which there was no pared to BG. We observed a signi®cant (P < 0.009) decrease of 7% in plasma cholesterol by replacing BG with BR or BS. The reduction was almost entirely due to a Table 5 Major fatty acids in platelet lipids. (wt%) decreased concentrations of LDL-C. BS contained 15% BG BR BS more stearic acid than BR, and correspondingly less milk fat. When no change in total C and LDL-C was seen, it may C14:0 1.5Æ 0.2 1.3Æ 0.2 1.3Æ 0.1 indicate that stearic acid and butter oil fatty acids are C16 A 2.5Æ 0.3 2.0Æ 0.4 2.2Æ 0.3 C16:0 16.7Æ 0.5§ 16.5Æ 0.5§ 15.3Æ 0.3{{ equally cholesterolemic. A separate effect of linoleic acid C16:1 n-7 0.8Æ 0.1 0.8Æ 0.1 0.7Æ 0.0 was ruled out as this fatty acid was present in the same C18:0 A 3.6Æ 0.4 2.7Æ 0.5§ 4.1Æ 0.5{ concentration in all fats. The few studies (Singer et al, C18:1 A 1.0Æ 0.3 1.2Æ 0.3 1.1Æ 0.4 1986; Abbey et al, 1990; Mantzioris et al, 1994) published C18:0 15.1Æ 0.4§ 15.4Æ 0.5§ 16.4Æ 0.3{{ C18:1 n-9 13.1Æ 0.4{§ 14.5Æ 0.4{ 14.7Æ 0.4{ that compare linoleic and linolenic acid, report no differ- C18:1 n-7 2.1Æ 0.4§ 2.2Æ 0.4§ 1.2Æ 0.3{{ ence in effect on lipoproteins. C18:2 n-6 5.8Æ 0.1 5.6Æ 0.1 5.8Æ 0.1 A number of authors have carried out meta-analyses of C20:0 0.8Æ 0.1 0.9Æ 0.1 0.9Æ 0.0 previously published studies and condensed these results {§ { { C18:3 n-3 0.2Æ 0.0 0.3Æ 0.0 0.3Æ 0.0 into equations obtained by multiple regression. Two of C20:1 n-9 0.7Æ 0.0{ 0.9Æ 0.0{§ 0.7Æ 0.0{ C20:3 n-6 1.3Æ 0.1 1.2Æ 0.1 1.3Æ 0.0 these take stearic acid into consideration as an independent C20:4 n-6 20.6Æ 0.6 20.8Æ 0.4 21.4Æ 0.2 variable (Derr et al, 1993; Yu et al, 1995) and predict C20:5 n-3 0.7Æ 0.1 0.7Æ 0.0 0.8Æ 0.1 decreases in total C, and LDL-C concentrations of 0.14± C22:4 n-6 1.8Æ 0.1 2.0Æ 0.2 1.8Æ 0.1 0.18 mmol=L when BG is replaced by BR or BS. We C22:5 n-6 0.2Æ 0.0§ 0.2Æ 0.0 0.1Æ 0.0{ C22:5 n-3 1.5Æ 0.1 1.5Æ 0.1 1.6Æ 0.1 observed reductions in total C and LDL-C of approx. C22:6 n-3 2.1Æ 0.1 2.0Æ 0.1 2.0Æ 0.1 0.30 mmol=L and 0.35 mmol=L. This discrepancy between the observed and the expected change in total C may be due BG: 90% butter oil 10% grapeseed oil; BR: 65% butter oil 35% to the high concentration of myristic and lauric acids in rapeseed oil; BS: 50% butter oil 50% interesteri®ed (70% rapeseed oil 30% fully hydrogenated rapeseed oil). aMeanÆ s.e.m. n 13. milk fat. LDL-C is derived from VLDL, and cleared from {Signi®cantly different from BG (P < 0.01). {Signi®cantly different from the bloodstream mainly by the liver through a receptor BR (P < 0.01). §Signi®cantly different from BS (P < 0.01). A: Aldehyde. dependent mechanism. The rate of clearance depends on Blood lipids and milk fat based spreads CC Becker et al 540 the amount of cholesterol ingested, the type and amount of acids are concentrated in the sn-2 position. Christophe et al fatty acids in the diet as well as the activity of the LDL- (1978) reported that feeding six subjects 85 g of interester- receptors. Myristic acid suppresses the removal of LDL i®ed butter oil, with a lower palmitic acid concentration in cholesterol from the bloodstream, while 2±7 E% (Hayes & the sn-2 position, caused a decrease in plasma cholesterol, Khosla, 1992) linoleic acid has the opposite effect in a compared to native butter oil. In a number of other experi- dose-dependent way. Oleic and stearic acids appear neutral ments, however, interesteri®cation of cocoa butter (Grande in their effect on the LDL receptor (Woollett et al, 1992a, et al, 1970) and high-palm oil blend (Zock et al, 1995; 1992b). The BR and BS diet contained about 5 E% linoleic Nestel et al, 1995) did not elicit a different cholesterolemic and linolenic acid. At the same time the dietary intake of response compared to the native oil. Previously we have cholesterol was around 400 mg=d. This is not enough to shown that BR and interesteri®ed BR elicited the same severely depress LDL receptor activity, since the subjects response in a similar group of subjects (to be published). still have low plasma C, but apparently enough to make Therefore, we believe that structure did not play an impor- them sensitive to the kind of fatty acid present in diet. The tant role in the response to the dietary challenges in this equations probably underestimated the effect of the BG to study although it was not addressed speci®cally. BR transition as only few of the studies, upon which they are based, used diets with appreciable amounts of butter, which is a concentrated source of the most cholesterolemic Conclusions fatty acid: myristic acid (Derr et al, 1993; Yu et al, 1995). Dairy spreads enriched in oleic and linolenic acids (BR) or BG and BR had very similar effect on HDL-C, and additionally enriched in stearic acid (BS) gave signi®cant resulted in values which were signi®cantly greater than the improvements compared to BG, when predicted cardiovas- HDL-C concentration after BS. This is in accordance with cular risk is considered, as they gave signi®cantly lower results published earlier by others (Tholstrup et al, 1994; plasma C, LDL-C, apo B, and apo B=apo A-1 values. Yu et al, 1995; Dougherty et al, 1995; Aro et al, 1997), However, if the ratio of plasma C to HDL-C or LDL- who also found that when stearic acid is exchanged for C=HDL-C, are considered as well, adding stearic acid to C12±14 saturates, a moderate decrease in HDL-C was BR did not further improve the nutritional value of the observed. blend. On the contrary BR gave higher HDL-C values than Concentrations of arachidonic, eicosapentaenoic, and BS. docosahexaenoic acids did not change signi®cantly in The 7% reduction in plasma C after BR compared to BG platelets or in plasma PL and CE after BR and BS, relative is noteworthy, as it is commonly assumed that one percent to BG, even though the rapeseed oil containing diets decrease in plasma cholesterol leads to a two percent supplied linolenic acid. This observation could be decrease in CDH risk (Lipid Research Clinics Program, explained by the competition between the n-3 and n-6 1984). This demonstrates the great potential that moderate families for desaturases. All diets supplied substantial changes of a component of the diet may have on indices for amounts of linoleic acid, to which was added the contribu- heart disease risk. It is likely that the effect would have tion of the back-ground diet. The conversion of linolenic been even greater in hypercholesterolemic subjects, who acid into long chain metabolites is strongly dependent on are more sensitive to the dairy fat than healthy young men. the concentration of linoleic acid (Emken et al, 1994). In platelets the sum of palmitic and stearic acids Acknowledgements ÐThis study was a collaboration between the Center remained constant, suggesting that one was substituted for Food Research, The Technical University of Denmark, and the Danish for the other in cell membranes. Replacing butter fatty Dairy Research Foundation and ®nanced as a part of the Danish Food acids (15% of test fat) with stearic acid had little in¯uence Research Programme (FéTEK I). We thank IL Grùnfeldt and the staff of on the fatty acid compositions of platelets and plasma PL the metabolic kitchen and gratefully acknowledge the laboratory assistance and CE. Possibly, this is because of preferential incorpora- of technicians K JoÈrgensen, J Bidstrup, and M Rosenberg. We are grateful tion of stearic acid into other fatty acid pools, such as to L Bandholm of Aarhus Olie A=S (Aarhus, Denmark) who kindly adipose tissue. But stearic acid could also be desaturated to delivered the test fats. oleic acid, chain-shortened or fully catabolised. There has been some discussion regarding the rate of absorption of References stearic acid from the intestine, relative to other fatty acids with lower melting points. BS contains 19% stearic acid, Abbey M, Clifton PM, Kestin M, Belling GB & Nestel PJ (1990): Effect of ®sh oil on lipoproteins, lecithin : cholesterol, acyltransferase, and lipid but due to interesti®cation the amount of tristearin present transfer protein activity in humans. 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