European Journal of Clinical Nutrition (1999) 53, 597±605 ß 1999 Stockton Press. All rights reserved 0954±3007/99 $12.00 http://www.stockton-press.co.uk/ejcn

Effect of synthetic of myristic, palmitic, and on

JT Snook1*, S Park1, G Williams1, Y-H Tsai1 and N Lee1

1Department of and Management, The Ohio State University, 1787 Neil Avenue, Columbus, Ohio, 43210 USA

Objectives: To determine relative effects of diets high in synthetic sources of myristic (14:0), palmitic (16:0) or stearic (18:0) acid on concentrations and metabolism of serum . Design: Eighteen healthy women participated in a three-way cross-over study for ®ve week periods separated by seven week washout periods, diets were assigned in random order. Subjects: Premenopausal women, not on medication, were from three races (Caucasian, African-American, Asian) and four E phenotype groups (3=3, 3=2, 4=3, and 4=2). Intervention: During the ®rst week the subjects consumed a baseline diet providing 11 energy (en)% saturated , 10 en% polyunsaturated fat and 14 en% monounsaturated fat. Followed by test diets with 19 en% (including 14 en% test saturated ), 3 en% polyunsaturated fat, and 14 en% monounsaturated fat for four weeks. Synthetic (, , and tristearin) were used in blends with natural fats and . Results: Mean concentrations of serum total, esteri®ed and LDL were signi®cantly lower after 18:0 than after 16:0 (n ˆ 16 ± 18, P < 0.01 for treatment effect). Myristic acid (14:0) had an intermediate effect. Receptor-mediated degradation of 125I-LDL in mononuclear cells obtained from the subjects was lower after 16:0 than after 14:0 and 18:0 (n ˆ 16 ± 18, P ˆ 0.05 for treatment effect). Differences in the digestibilities of the fats were not a major factor in the results. Strong cholesterolemic responses to the 16:0 diet were partly explained by apoE phenotype. Conclusions: As noted previously, stearic acid was neutral compared to 14:0 and 16:0. In contrast to studies involving natural fats, 14:0, fed as a synthetic , was less cholesterolemic than 16:0 in a majority of subjects. ApoE phenotype in¯uenced the cholesterolemic response particularly when diets high in 16:0 were eaten. Sponsorship: Funded by United States Department of Agriculture Grant 93-37200-8978. Descriptors: ; ; stearic acid; LDL-cholesterol; LDL metabolism

Introduction and 16:0 but not 18:0 are the hypercholesterolemic fatty acids. There has also been an indication that 16:0, under Structured triglycerides are molecules having fatty acids certain conditions, is not as detrimental as 12:0 and 14:0 rearranged on the molecule in a random or (Sundrum et al, 1994). However, we (Wardlaw et al, 1995) purposeful fashion (Haumann, 1997). Currently, there is reported that a structured made up of theoretically considerable interest in development of structured trigly- neutral medium chain fatty acids (8:0 ‡ 10:0) and a poorly cerides which may have unique physiological properties digested long chain saturated fatty acid (, 22:0) and special uses in patient populations (Bell et al, 1997). had an effect on serum low lipoprotein cholesterol Formulation and development of speci®c structured trigly- (LDL-C) concentration similar to that of natural fats high in ceride for special purposes requires knowledge of the myristic and palmitic () and lauric, myristic and metabolic properties of individual fatty acids. These prop- palmitic acids ( ‡ ) suggesting that erties may or may not be extrapolated from studies of the current concepts about the relative cholesterolemic proper- effects of the fatty acids provided in natural fats or oils. ties of the saturated fatty acids are more complicated than From data obtained from studies of natural fats and oils, previously believed. Hegsted et al (1965) developed multiple regression equa- Myristic acid, often considered the most cholesterolemic tions predicting that myristic acid (14:0) is more hyperch- saturated fatty acid in both human and animal studies olesterolemic than lauric (12:0) and palmitic acids (16:0) (Hegsted et al, 1995; Hayes & Khosla, 1992), is not the while stearic acid (18:0) and medium chain fatty acids, major saturated fatty acid in any commonly consumed similar to , a monounsaturated fatty acid (18:1), natural fat or oil (USDA, 1979). One must feed a synthetic are essentially neutral. Subsequent human studies with triglyceride to obtain a diet in which myristate is the major natural sources of triglyceride (Kris-Etherton et al, 1993; saturated fatty acid. Results of human feeding studies Tholstrup et al, 1994a) supported the idea that 12:0 ‡ 14:0 involving a high myristic or palmitic acid synthetic fat suggested that myristic acid either was not more cholester- olemic than palmitic acid (Tholstrup et al, 1994a) or *Correspondence: Dr JT Snook, 1787 Neil Ave, Columbus, OH, 43210 produced, on average, about 4% higher levels of LDL-C USA (Zock et al, 1994). A synthetic high oil raised E-mail: [email protected] Received 4 August 1998; revised 1 February 1999; accepted plasma LDL cholesterol concentrations less than did a 16 February 1999 natural source of palmitic acid (Denke & Grundy, 1992). Effect of synthetic triglycerides on serum lipoprotein metabolism JT Snook et al 598 A diet high in a synthetic source of stearic acid reduced Committee. Two subjects left the study at the end of the plasma total cholesterol 14% compared to a diet high in a ®rst period; one of these was replaced by her sister, who had natural source of palmitic acid (Bonanome & Grundy, an identical apoE phenotype (3=2) and very similar baseline 1988). In a stable isotope study with ethyl of lipoprotein concentrations. The other subject was not palmitic, myristic and stearic acid, which were fed to ®ve replaced, and her data were not used. An alternate, added men, Hughes et al (1996) concluded that the three saturated during the last two periods, followed the rotation designated fatty acids are metabolized differently in the postprandial for the subject who left. Two other subjects left the study at state. It is important to investigate the effects of different the end of the second period and were not replaced. fatty acids on various aspects of lipoprotein metabolism in humans as well as animal models. Design The objective of this study was to investigate the The eighteen subjects were randomized into three groups cholesterolemic and metabolic effects of two saturated using a three-period and six-sequence design (363 cross- fatty acids, 14:0 and 16:0 (believed to be cholesterolemic over design) except that only 17 subjects consumed the by most if not all researchers) and 18:0 (generally believed myristic acid diet and only 16 consumed the palmitic acid to be neutral). A high (13 ± 14 en%) level of one of the three diet for reasons described above. For standardization pur- saturated fatty acids was fed in each test diet while levels of poses the subjects consumed a baseline diet higher in other individual saturated fatty acids were held below polyunsaturated fat and lower in saturated fat (Table 1) 3 en%. A large portion of each test saturated fatty acids for the ®rst week of each period. According to results of a was furnished by structured triglycerides that provided the previous study, serum lipoprotein concentrations respond test fatty acid in all positions on the glycerol molecule. strongly to substitution of a high polyunsaturated fat diet for a saturated diet or subjects' usual diet within one week (Snook et al, 1985). One of the high saturated fat diets was Methods given during the subsequent four weeks. There were Subjects washout periods of seven weeks between treatments so Eighteen healthy premenopausal women were initially that blood samples at baseline and at the end of the enrolled in this three-period cross-over study, the minimum experimental period were always obtained at the same number needed to detect a mean difference for serum stage of each subject's menstrual cycle. lipoprotein levels between treatments of about 10% with Blood samples were obtained by venipuncture after an a standard deviation of approximately 8% and a power of overnight fast two times during the last three days of the 0.9. The subjects were not taking medication known to baseline period and twice during the last three days of each in¯uence lipoprotein metabolism, including oral contra- feeding period; data were averaged. Depending on the ceptives. Subjects who completed at least two study periods requirements of the procedure, analyses commenced imme- diately or serum, obtained by centrifugation (5006g for were 19 ± 43 y of age (meanÆ s.e.m. ˆ 28 Æ 1.5) and  weighed between 50 and 77 kg. The subjects' weight 20 min), was frozen at 7 80 C for later analysis. (paired-t analysis) did not change signi®cantly during the study. According to results of a 7 d activity recall (Blair et Diets al, 1985) daily energy expenditure was between 7.7 and The diets were prepared in a metabolic kitchen in the 12.9 MJ. MeanÆ s.e.m. serum cholesterol concentration at Department of Human Nutrition and Food Management. screening was 4.62Æ 0.25 mmoles L71, range ˆ 3.18 ± On weekdays subjects ate breakfast and dinner in the 6.54 mmoles L71. Ethnic background was Caucasian, 12; metabolic dining room and took out a sack lunch. Except African-American, 2; and Asian, 4. The subjects had the for Saturday night dinner, which was served in the meta- following phenotypes: apoE 3=3, 10; bolic unit, subjects took out weekend meals. The baseline apoE 3=2, 4, apoE 4=3, 3 and apoE 4=2, 1. Prior to starting diet provided 40 en% total fat, 10 en% 18:2, 14 en% 18:1, this study, subjects signed a consent form approved by the 11 en% saturated fat, and about 327 mg cholesterol per day Ohio State University Biomedical Human Studies (Table 1). The three experimental diets were formulated to

Table 1 Daily energy and nutrient intake on three saturated fat diets and baseline diet (meanÆ s.e.m.)

Baseline Myristic acid Palmitic acid Stearic acid Dietary variable n ˆ 51 n ˆ 17 n ˆ 16 n ˆ 18

Energy (MJ)a 8.3Æ 0.1 8.5Æ 0.2 8.2Æ 0.1 8.3Æ 0.1 (g)a 74Æ 1 (15) 74Æ 1 (15) 73Æ 1 (15) 74Æ 1 (15) Carbo. (g)a 226Æ 2 (46) 229Æ 4 (45) 225Æ 3 (46) 227Æ 3 (46) Total fat (g)a 87Æ 2 (40) 91Æ 3 (40) 87Æ 2 (40) 89Æ 2 (40) Fatty acids (g)b 8:0 ‡ 10:0 1.8Æ 0.0 (1) 1.3Æ 0.0 (1) 1.5Æ 0.0 (1) 0.8Æ 0.0 ( < 1) 12:0 3.6Æ 0.1 (2) 2.7Æ 0.1 (1) 3.2Æ 0.1 (2) 2.4Æ 0.0 (1) 14:0 2.4Æ 0.0 (1) 31.1Æ 1.3 (14) 2.1Æ 0.0 (1) 1.1Æ 0.0 (1) 16:0 8.4Æ 0.1 (4) 6.3Æ 0.2 (3) 27.8Æ 0.7 (13) 5.7Æ 0.1 (3) 18:0 7.3Æ 0.1 (3) 3.5Æ 0.4 (2) 4.0Æ 0.1 (2) 28.2Æ 0.6 (13) 18:1 31.0Æ 0.7 (14) 31.0Æ 0.9 (14) 32.8Æ 0.7 (15) 35.3Æ 0.7 (16) 18:2 23.1Æ 0.4 (10) 7.6Æ 0.2 (3) 7.9Æ 0.2 (4) 7.5Æ 0.1 (3) Chol. (mg)a 327Æ 2 319Æ 2 328Æ 2 308Æ 2

Values in parentheses are calculated values for percent of food energy provided by the nutrient. aAnalyzed from daily dietary records using Food Processor II Nutrition Analyses Software (ESHA Research, Salem, OR). bAs determined by gas liquid chromatography. Effect of synthetic triglycerides on serum lipoprotein metabolism JT Snook et al 599 provide 40 en% total fat, 19 en% saturated fat (the test fats LDL receptor activity furnished over two thirds of this), 14 ± 16 en% monounsa- LDL binding to and degradation via LDL receptors in turated fat, 3 ± 4 en% 18:2, and 320 mg cholesterol=d (Table freshly isolated mononuclear cells (MNC) was determined 1). Fats and oils were blended after taking into account the using methods described in detail previously (Ho et al, fatty acid composition of the in the diet (56 g lean 1976; Kuo et al, 1989; Park & Snook, 1995; Park et al, beef and 56 g turkey breast or turkey ham). The types and 1996). Brie¯y, iodinated LDL (McFarlane, 1958) was amounts (grams) of fat in the blends consumed by the prepared within 3 d prior to each major blood draw using typical subject were as follows: Baseline (butter fatty acids, pooled blood obtained from a constant group of donors who 7; saf¯ower oil, 17; high oleic saf¯ower oil, 18; coconut consumed self-selected diets. We assumed the composition oil, 6; and sheanut oil, 7); High Myristic (butter fatty acids, of the donor LDL remained constant throughout the study, 5; trimyristin, 27; high oleic saf¯ower oil, 28; , but this was not speci®cally assessed. For measurement of 5); High Palmitic (coconut oil, 6; butter fatty acids, 7; LDL degradation and binding, MNC were isolated by tripalmitin 18; high oleic saf¯ower oil, 27; , 5); and centrifugation (8006g for 20 min at room temperature) High Stearic (coconut oil, 4; tristearin, 14; high oleic from saline-diluted whole blood anticoagulated with saf¯ower oil, 21; sheanut oil, 24). The smaller amounts sodium citrate and overlayered with NycoprepTM washed of tristearin and tripalmitin compared to trimyristin used in and counted. Degradation of 125I-LDL by MNC was the fat blends re¯ected the higher availability of these fatty assessed by incubating cell samples with 125I-LDL acids in natural oils and the dif®culties we encountered in (20 mgmL71) in the presence and absence of 20-fold preparing acceptable food products with these structured excess of unlabeled LDL at 37C for 5 h, a period pre- fats. At least 20% of each test saturated fatty acid was viously shown (Park & Snook, 1995) to be suitable for provided in the sn-2 position of the triglyceride. demonstrating differences among treatments. Nonspeci®c The fat blends were incorporated into cakes and cookies, and speci®c degradation represented, respectively, degra- used as fat spreads on bread, and added to the main dishes dation in the presence of 20-fold excess unlabeled LDL and at dinner. A basal menu providing 8.3 MJ (about 2000 kcal) the difference between the latter and degradation in the was given initially. Subjects requiring more calories to absence of excess unlabeled LDL. For determination of maintain body weight (as determined by daily weighing) speci®c and nonspeci®c binding of LDL, MNC were were given extra cakes and cookies made up of fat blends incubated for one hour at 4C with 5, 10, 20 and 40 and with the same proportion of fatty acids as the overall diet. 80 mgmL71 125I-LDL in the presence and absence of Adjustments over 1.2 MJ were met by altering menus to excess unlabeled LDL. furnish additional protein, fat, and . Daily records of the subjects' food intakes were kept and analyzed using Food Processor II software (ESHA Cholesterol biosynthesis in MNC Research, Salem, OR) with added data on fatty acid The rate of cholesterol biosynthesis by MNC was deter- composition of the in the menu (Table 1). Compo- mined as described by McNamara et al (1985). Brie¯y, sites of the rotating four day menus were made and MNC were incubated with 2.5 mM (2-14C) (DuPont analyzed for speci®c fatty acid composition by gas liquid NEN) and autologous serum obtained at the time of the test chromatography (Table 1). at 37C for 4 h in a metabolic shaker. The reaction was terminated by adding 5 M KOH. An internal standard Digestibility of dietary fatty acids (1,2-3H cholesterol) and the reaction mixture were saponi- To assess apparent digestibility of dietary fatty acids, we ®ed by heating. Nonsaponifed were extracted with administered 50 mg Brilliant Blue before breakfast and then and applied to a column of aluminum oxide. The again three days later during the fourth week of each eluted (:) sterol fraction was reduced in period. Feces collected between the appearance of the volume and counted in a scintillation counter. two markers were composited, mixed thoroughly with a known amount of water, and stored at 7 20C until fatty acid analysis. Endogenous cholesterol esteri®cation and transfer The isotopic method of Channon et al (1990) was used to Serum lipoprotein concentrations measure endogenous cholesterol esteri®cation (- Previously frozen serum samples were analyzed for total cholesterol acyl transferase (LCAT) activity) and transfer cholesterol (TC) and free cholesterol by an enzymatic assay (cholesterol transfer protein (CETP) activity) in (Allain et al, 1974). Total HDL-, HDL2- and HDL3-C were serum. Brie¯y, for determination of esteri®cation rate, determined by the same enzymatic procedure after preci- fresh serum was preincubated at 4C for 1 h with 3H- pitation of apoprotein B-containing lipoproteins with dex- cholesterol-albumin emulsion; after equilibration of added 2 ‡ tran sulfate-Mg solution and then removal of HDL2 with and endogenous cholesterol, the mixture was incubated at a stronger dextran sulfate-Mg2 ‡ solution (Warnick et al, 37C for 3 h. Lipids were extracted, separated by thin layer 1982). LDL-cholesterol (in mg dL71) was calculated as TC chromatography, and radioactivity of cholesterol ester and minus (HDL-C plus 0.2 triglycerides). Results for the two free cholesterol spots counted by liquid scintillation. For pre- and post-period analyses were averaged and converted cholesterol transfer activity, transfer of radiolabeled cho- to mmoles L71. Serum from a previously frozen sample lesterol ester into VLDL and LDL was measured ®rst by pool was analyzed with each analysis for TC. The precision precipitating the two apoB-containing lipoproteins from the (CV) of the sequenced runs of this pool was 2.7%. A incubate with dextran sulfate. Then, total radioactivity in turbimetric procedure (Raichem, San Diego, California, the incubate, in the HDL-containing supernatant, and in the USA) was used to assay serum A-1 and apoprotein B-containing lipids was measured by liquid B in one pre- and one post-period blood sample that had scintillation counting. These data were used to calculate been stored at 7 80C. the rate of transfer of the cholesterol ester. Effect of synthetic triglycerides on serum lipoprotein metabolism JT Snook et al 600 Apolipoprotein E phenotype Table 2 Percent excretion of fatty acids in experimental dietsa Apo E phenotype was determined by isoelectrofocusing of Myristic acid diet Palmitic acid diet Stearic acid diet delipidated plasma followed by immunoblotting using Fatty acid n ˆ 17 n ˆ 14 n ˆ 16 polyclonal goat anti-human apo E antiserum as ®rst anti- body and rabbit antiserum to goat IgG as second antibody 14:0 2.4Æ 0.5b,c 1.5Æ 0.3c 9.2Æ 1.6d according to the method of Kamboh et al (1988) and as 16:0 5.7Æ 1.3 5.3Æ 1.0 8.2Æ 1.2 18:0 11.1Æ 1.6 11.3Æ 2.1 14.1Æ 2.3 adapted for use in our laboratory by Park et al (1996) and 18:1 1.0Æ 0.1 0.8Æ 0.2 0.8Æ 0.3 Tso et al (1998). 18:2 1.4Æ 0.3 1.2Æ 0.6 1.1Æ 0.3

Fatty acid analysis aMeanÆ s.e.m. bFecal samples were collected for 3 d during the ®nal week of each diet Fatty acid composition of food composites, feces, and red period. Samples from subjects with incomplete collections or in whom the blood cell membranes, isolated by the method of Fraga et al 3 d collection period could not be clearly delineated were not analyzed. (1990) was determined as described previously (Sukija & c,dValues within a row are different (P ˆ 0.000). Palmquist, 1988). acid diet was excreted while the other dietary fatty acids Statistical analyses usually were well absorbed ( < 3% excreted). However, the The baseline and end of period values and mean differences 9.2Æ 1.6% excretion of 14:0 was higher (P < 0.05) on the between the beginning and the end of each period were stearic acid diet than on the myristic acid and palmitic acid analyzed statistically. Differences between baseline and diets. experimental values were analyzed by paired-t test in which individual subjects served as their own controls. Serum lipoprotein concentrations Differences among the three experimental diets, of which There was a signi®cant (P < 0.05) independent effect of the the main differences were the three saturated fatty acid saturated fat diets on serum concentrations of TC and LDL- combinations, were analyzed using a linear model which C and on their changes from baseline values. At the end of contained terms for the subjects, periods, direct treatments the four week treatment periods, the 14:0 and 16:0 diets and carry-over effects and adjusted for missing data. The raised (P < 0.05) mean serum total cholesterol concentra- models were ®tted using PROC MIXED in the Statistical tions above baseline values whereas 18:0 had no effect Analysis System (SAS) program, Cary, North Carolina, (Table 3). Postperiod values for total cholesterol were USA. Signi®cance level was set at a ˆ 0.05. Means which were signi®cantly different among diets were separated in Table 3 Serum lipoprotein concentrations (meanÆ s.e.m.) on three the linear model by PROC MIXED. Treatment effects were saturated fatty acid mixtures adjusted for any signi®cant effects of other variables in the Myristic acid Palmitic acid Stearic acid model including carry over effects and period effects. Serum parameter n ˆ 17 n ˆ 16 n ˆ 18 Relationships among dependent variables were determined Total cholesterol (mmole6L71) by correlation analysis. Preperiod 3.84Æ 0.20 3.79Æ 0.23 4.00Æ 0.23 Postperiod 4.10Æ 0.17a 4.21Æ 0.23a 3.82Æ 0.19b a a b Results Difference 0.26Æ 0.11* 0.42Æ 0.13* 7 0.18Æ 0.12 LDL-cholesterol (mmole6L71) Diet composition and apparent availability of dietary Preperiod 2.21Æ 0.22 2.21Æ 0.20 2.37Æ 0.21 Postperiod 2.32Æ 0.16ab 2.55Æ 0.20a 2.20Æ 0.15b fatty acids ab a b The experimental saturated fatty acid diets provided similar Difference 0.11Æ 0.11 0.33Æ 0.13* 7 0.17Æ 0.10 71 amounts of dietary cholesterol and poly- and mono-unsa- HDL cholesterol (mmole6L ) Preperiod 1.37Æ 0.08 1.25Æ 0.07 1.35Æ 0.06 turated fat (Table 1), differing only in amount of 14:0, 16:0 Postperiod 1.44Æ 0.09 1.30Æ 0.08 1.32Æ 0.08 or 18:0. Each experimental diet contained approximately Difference 0.07Æ 0.06 0.05Æ 0.06 7 0.03Æ 0.04 equal amounts of test saturated fatty acid (28 ± 31 g) and 71 HDL2 cholesterol (mmole6L ) 18:1 (31 ± 35 g). The baseline diet provided three times as Preperiod 0.56Æ 0.08 0.45Æ 0.08 0.48Æ 0.05 much 18:2 as the experimental diets and only about half as Postperiod 0.60Æ 0.08 0.50Æ 0.06 0.53Æ 0.06 much saturated fatty acid (Table 1). The composition of the Difference 0.04Æ 0.06 0.05Æ 0.05 0.06Æ 0.04 71 baseline diet and the experimental diets was not typical of HDL3 cholesterol (mmole6L ) Preperiod 0.81Æ 0.05 0.80Æ 0.04 0.87Æ 0.87 fatty acid availability in the USA food supply. The baseline Postperiod 0.84Æ 0.05 0.80Æ 0.03 0.79Æ 0.05 diet was higher in polyunsaturated fat and lower in satu- Difference 0.02Æ 0.04 0.00Æ 0.03 7 0.08Æ 0.03* rated fat than typical USA diets (Marston & Raper, 1987) Triglycerides (mmole6L71) while the myristic acid diet furnished about 20 times more Preperiod 0.79Æ 0.45 0.80Æ 0.15 0.66Æ 0.09 14:0, the palmitic acid diet about four times more 16:0 and Postperiod 0.74Æ 0.43 0.80Æ 0.13 0.64Æ 0.10 the stearic acid diet about eight times more 18:0 acid than Difference 7 0.05Æ 0.07 0.00Æ 0.08 7 0.01Æ 0.07 typically consumed (Cresanta et al, 1988). Apolipoprotein A-1 (g6L71) Apparent absorption of the test fatty acids depended on Preperiod 1.59Æ 0.07 1.58Æ 0.08 1.57Æ 0.06 Postperiod 1.62Æ 0.06 1.58Æ 0.05 1.53Æ 0.10 chain length and saturation. The meanÆ s.e.m. apparent Difference 0.05Æ 0.05 0.00Æ 0.07 7 0.04Æ 0.08 excretion of 18:0 by subjects fed the stearic acid diet was (g6L71) 14.1Æ 2.3% (Table 2) although one subject excreted Preperiod 0.68Æ 0.06 0.76Æ 0.07 0.72Æ 0.07 33.5%; one, 29%; and ®ve others, about 20%. The other Postperiod 0.71Æ 0.04 0.76Æ 0.08 0.67Æ 0.06 subjects excreted less than 10% of the ingested dose. The Difference 0.03Æ 0.05 7 0.01Æ 0.03 7 0.05Æ 0.05 18:0 was not well absorbed (11.1Æ 1.6% and 11.3Æ 2.1% *Signi®cant difference between pre- and postperiods values (P < 0.05). excretion) from the 14:0 and 16:0 diets, respectively. On Values with different superscripts within a row are signi®cantly different average, only about 5.3Æ 1.0% of the 16:0 in the palmitic (P < 0.05). Effect of synthetic triglycerides on serum lipoprotein metabolism JT Snook et al 601 higher for 14:0 and 16:0 than 18:0 (P ˆ 0.001 for treatment Table 4 Saturated fat and rates of cholesterol esteri®cation and transfer effect), but effects produced by 14:0 and 16:0 were not in serum different (P ˆ 0.36). Myristic acid Palmitic acid Stearic acid Mean concentrations of serum LDL-C were higher on Serum variable n ˆ 17 n ˆ 16 n ˆ 18 16:0 than on 18:0 (P ˆ 0.002) at the end of the treatment periods. Individuals varied considerably in their response to Free cholesterol (mmole6L71) Preperiod 1.14Æ 0.06 1.23Æ 0.08 1.26Æ 0.08 the three high saturated fat diets (Figure 1). For example, Postperiod 1.32Æ 0.07 1.27Æ 0.09 1.21Æ 0.07 when 14:0 replaced the baseline diet, three subjects had Difference 0.18Æ 0.06* 0.04Æ 0.06 7 0.05Æ 0.06 relatively large increases in LDL-C, while the remaining 14 Cholesterol ester (mmole6L71) subjects had modest increases or decreases. On the 16:0 Preperiod 2.70Æ 0.17 2.56Æ 0.17 2.75Æ 0.17 diet, six subjects had strong increases in LDL-C while the Postperiod 2.77Æ 0.14ab 2.94Æ 0.16a 2.61Æ 0.15b other 10 subjects who ate this diet had only small changes Difference 0.09Æ 0.08a 0.38Æ 0.13* b 7 0.13Æ 0.14a (Figure 1). Nine subjects had modest increases in LDL-C Rate of cholesterol esteri®cation (mmole6L71 h71) on the stearic acid diet while the other nine had decreases in Preperiod 49.3Æ 4.2 49.8Æ 3.2 49.1Æ 2.1 Postperiod 52.6Æ 4.8 50.9Æ 4.4 43.9Æ 2.7 LDL-C (Figure 1). All four of the subjects with an apo E4 Difference 3.3Æ 5.3 1.1Æ 3.2 7 5.3Æ 2.3* allele had strong responses to the high 16:0 diet, but apo E Rate of cholesterol ester transfer (mmole L71 h71); phenotype was not a factor in the response to the 14:0 and Preperiod 17.9Æ 1.4 19.3Æ 2.2 18.7Æ 1.6 18:0 diets in this study. Furthermore, subjects who were Postperiod 24.7Æ 3.3 26.8Æ 2.9 22.4Æ 1.4 strong responders to one diet were not necessarily strong Difference 6.8Æ 3.7** 7.5Æ 3.1* 3.7Æ 1.6* responders to the other experimental diets. MeanÆ s.e.m There was no independent effect of dietary treatment on *Signi®cant difference from preperiod (P < 0.05). serum concentrations of total triglycerides, HDL-C and **P ˆ 0.086. Values within a row with different superscripts are different (P < 0.05). HDL2-C, but HDL3-C decreased (P < 0.05) when 18:0 was fed (Table 3). Mean serum concentrations of apolipo- protein B and A-1 were not altered by the saturated fat diets 18:0 diet compared to baseline but was not altered sig- although changes in apoB and LDL-C were weakly corre- ni®cantly by 14:0 and 16:0. The rate of cholesterol ester lated (r ˆ 0.3, P ˆ 0.03) while changes in apo A-1 and transfer (CETP activity) from HDL to triglyceride-rich lipoproteins increased on all of the experimental diets. In HDL3-C were also weakly associated (r ˆ 0.29, P ˆ 0.037). the data set analyzed as a whole, changes in LDL-C and Changes in variables related to cholesterol esteri®cation CETP activity were weakly correlated (r ˆ 0.29, P ˆ 0.038) and transfer in serum as were postperiod values for CETP activity and LDL-C As with serum LDL-C, there was a signi®cant (P ˆ 0.025) concentrations (r ˆ 0.46, P ˆ 0.0006) and total HDL-C independent effect of dietary treatment on concentrations of (r ˆ 7 0.34, P ˆ 0.015). cholesterol ester, which were lower after four weeks on the 18:0 diet than on the 16:0 diet. Consumption of high 16:0 Changes in cholesterol biosynthesis and LDL receptor but not 14:0 or 18:0 also produced a signi®cant increase in activity in MNC cholesterol ester compared to baseline (Table 4). Serum Freshly isolated mononuclear cells (MNC) obtained from samples from the subjects were incubated in vitro to study the subjects before and after they consumed the treatment rates of cholesterol esteri®cation and transfer to apoB diets were used as a model system to study cholesterol containing lipoproteins. These reactions are part of the biosynthesis and LDL degradation in vitro. The rate of reverse cholesterol transfer process and result in remodel- cholesterol biosynthesis in MNC was not affected ing of HDL and other lipoproteins in serum. The rate of (P > 0.05) by the dietary treatments (Table 5). In the cholesterol esteri®cation (LCAT activity) declined on the statistical analysis of the entire subject group, there was a signi®cant treatment (P ˆ 0.05) effect on the rate of recep- tor mediated 125I-LDL degradation which declined 35% from baseline when subjects were fed 16:0 but did not change signi®cantly compared to baseline in the other experimental groups (Table 5). At the end of the experi- mental period, receptor-mediated degradation was higher on 14:0 than on the 16:0 treatment. Nonspeci®c or non- receptor mediated degradation of 125I-LDL was higher (P ˆ 0.03) on the 16:0 treatment than on 14:0 and 18:0. The effects of the treatments on the ability of LDL to bind to LDL receptors in MNC were inconclusive partly because the preperiod values for receptor mediated LDL binding were the only preperiod values to differ signi®- cantly (P ˆ 0.03) among baseline periods in this study. In general, the postperiod values for binding and the changes in receptor mediated binding from baseline suggested a negative effect of 14:0 and 16:0 and a positive effect of 18:0 (P < 0.06) on the ability of the subjects' MNC LDL to bind donor (not their own) LDL. Results of Scatchard Figure 1 Individual changes from baseline in serum concentrations of LDL-C when subjects consumed the experimental diets. Subject n ˆ 17 for analysis (Table 5) of the binding information were in line the myristic acid diet, 16 for the palmitic acid diet, and 18 for the stearic with LDL-C ®ndings since mean values for Bmax (number acid diet. of receptor binding sites) decreased on 14:0 and 16:0 and Effect of synthetic triglycerides on serum lipoprotein metabolism JT Snook et al 602 Table 5 Cholesterol biosynthesis and LDL receptor activity in vitro in saturated fatty acids, provided largely as synthetic trigly- MNC from subjects consuming three saturated fat diets cerides, were compared in the same human study. Another Myristic Palmitic Stearic unique aspect of this human study is that we investigated acid acid acid several metabolic or genetic variables, in addition to serum MNC variable n ˆ 17 n ˆ 16 n ˆ 18 Probability lipoprotein concentrations, to try to explain some of the metabolic effects of the three fatty acids. Cholesterol biosyn., picomoles million MNC71 h71 Preperiod 7.43Æ 1.00 5.66Æ 0.90 5.27Æ 0.76 0.20 Postperiod 6.91Æ 0.97 6.28Æ 0.79 7.22Æ 0.88 0.75 Effects on serum lipoprotein concentrations Difference 7 0.52Æ 1.00 0.62Æ 0.93 1.96Æ 1.14 0.24 Our results agreed with previous work with natural fats and Receptor-mediated degradation of 125I-LDL (ng million MNC71 h71**) oils (Hegsted et al, 1965; Kris-Etherton et al, 1993; Preperiod 16.54Æ 2.51 18.09Æ 2.72 14.37Æ 1.51 0.76 Tholstrup et al, 1994b) in which diets very high in stearic Postperiod 17.02Æ 1.78a 11.87Æ 1.16b 14.87Æ 1.48ab 0.05 acid ( > 10 en%) produced lower serum concentrations of Difference 0.48Æ 1.90 7 6.22Æ 2.06* 0.48Æ 1.83 0.09 LDL-C than diets high in 12:0 ‡ 14:0, 14:0 ‡ 16:0, or 16:0. 125 71 71 Nonspeci®c degradation of I-LDL (ng million MNC h **) Multiple regression analysis of data from investigations of Preperiod 9.61Æ 1.68 10.21Æ 2.85 7.29Æ 1.54 0.82 Postperiod 6.31Æ 1.63a 11.40Æ 2.42b 7.21Æ 1.72a 0.03 natural fats and oils suggested 14:0 is the most cholester- Difference 7 3.30Æ 1.90 1.19Æ 3.10 7 0.08Æ 2.46 0.47 olemic saturated fatty acid (Hegsted et al, 1965; Mensink & Receptor-mediated 125I-LDL Binding (ng million MNC71**) Katan, 1992), but, despite the statistical associations, myr- Preperiod 23.78Æ 3.42a 17.07Æ 2.24b 16.80Æ 2.75b 0.03 istic acid (14:0) is not the major saturated fatty acid in any Postperiod 16.30Æ 2.15 14.50Æ 1.91 21.88Æ 2.60 0.06 commonly consumed natural products including butter, Difference 7 7.47Æ 3.78***7 2.56Æ 2.45 5.08Æ 4.33 0.08 meat, and tropical oils (USDA, 1979), and diets high in 71 Bmax (ng million MNC ) myristic acid are even higher in lauric or palmitic acid. Preperiod 29.7Æ 3.8 30.7Æ 4.3 29.4Æ 2.5 0.95 Postperiod 27.3Æ 2.6 28.0Æ 2.8 31.9Æ 2.7 0.34 Previous research involving the feeding of a synthetic 14:0 Difference 7 0.2Æ 1.1 7 0.7Æ 0.7 1.0Æ 0.9 0.49 diet indicated that mean LDL-C concentration on 14:0 and 16:0 (fed in synthetic form) were not different (Tholstrup et MeanÆ s.e.m. al, 1994a) or that mean LDL-C was about 4% or *Difference between pre- and post period was signi®cant (P < 0.05). **Results from degradation and binding assay series performed with 20 ug 0.11 mmol=L higher (P < 0.0086) on 14:0 than on 16:0 125I-LDL with or without 20-fold excess of unlabelled LDL. fed as palm oil (Zock et al, 1994). In this study myristic ***P ˆ 0.06 for difference between pre- and postperiod. acid seemed less cholesterolemic in contrast to palmitic Values within a row with different superscripts are different (P < 0.05). acid than in many previous studies. In this study the effects of 14:0 and 16:0 on LDL-C concentrations were not different but the mean change in increased on 18:0. However, the data were variable, the LDL-C produced by 14:0 was 5% above baseline values changes were less than 10%, and there was no signi®cant while 16:0 produced a 15% increase in mean concentration treatment effect. of LDL-C. However, there was considerable variation among participants in the cholesterolemic response to the Red blood fatty acid composition different diets. We analyzed red blood cell samples for fatty acid composi- The concept of responders=nonresponders to hypercho- tion during the last two periods of this study but not the lesterolemic diets is not new (Ginsberg et al, 1981; McNa- ®rst. The only fatty acid to change signi®cantly in red blood mara et al, 1987; Glatz et al, 1993). According to Glatz et cell (RBC) membranes between baseline and the end of the al (1993) variations in response may be partly random in experimental period was myristic acid when the high 14:0 nature and partly due to metabolic differences among diet was fed. Membrane 14:0 increased from 0.72Æ 0.42% subjects. Three of the six subjects with strong responses to 1.39Æ 0.12% of total identi®ed fatty acids, (n ˆ 11, to the 16:0 diet had the apoE 4=3 phenotype; another was P < 0.02) on the myristic acid diet whereas 14:0 in RBC the apoE 4=2 subject. The remaining two were apo E3=3. membranes declined to below 0.5% on the other two diets. ApoE phenotype was not associated with a particular There was an insigni®cant (P > 0.05) increase in 18:1 from cholesterolemic response to the diets high in 14:0 and 14.93Æ 0.62% of total identi®ed fatty acids to 18:0. Previously, we reported that according to multiple 17.17Æ 1.16% (n ˆ 11, P < 0.10) when the high 18:0 diet regression analysis, variations in the LDL-C response to was fed. This increase was not signi®cantly different diets high in synthetic 14:0, 16:0 and 18:0 may be deter- (P > 0.05) from the one observed on diets high in 14:0 mined, at least in part, by apoE polymorphism (Tso et al, (from 15.87Æ 0.59% to 16.62Æ 0.66%) and 16:0 1998). For example, 35% of the variance in changes in (16.9Æ 0.59 to 18.18Æ 1.29%). serum LDL-C concentrations was explained by eating the diet with 18:0 (negative or reducing association, partial R2 ˆ 0.18) and having apoE 4=3 phenotype (positive or Discussion enhancing association, partial R2 ˆ 0.18). We cannot In this comparison of three diets providing large amounts exclude the possibility that myristic acid might be as (14 en%) of commonly consumed saturated fatty acids Ð cholesterolemic as palmitic acid or more so in subject myristic, palmitic or stearic acid Ð we observed that pal- populations lacking the apoE 4 isoform. mitic acid, in contrast to stearic acid, produced higher One difference between this study and previous ones serum concentrations of TC, LDL-C and cholesterol ester involving synthetic fats is we fed trimyristin, tripalmitin or and lower receptor-mediated degradation of LDL in an in tristearin blended with natural sources of unsaturated and vitro assay involving MNC of the human subjects enrolled saturated fat while other investigators fed synthetic fats in the study. In our study design, myristic acid was not high in a speci®c saturated acid interesteri®ed with other more cholesterolemic than palmitic acid. This was the ®rst fatty acids so that the majority of the fat molecules were not time, to our knowledge, that diets high in each of the three consumed in the form of trimyristin, tripalmitin and tris- Effect of synthetic triglycerides on serum lipoprotein metabolism JT Snook et al 603 tearin. However, after fatty acid that enters the activity. This result is consistent with previous reports will usually not be a triglyceride of 14:0, 16:0 indicating the diets high in saturated fat increase CETP or 18:0. Vegetable oils such as coconut or (Tall, 1995; Bruce et al, 1998). In this study, higher CETP provide little 14:0 in the sn-2 position and palm oil activity was weakly associated with lower HDL-C provides little 16:0 in this position (Kuksis, 1992), but (r ˆ 7 0.34, P ˆ 0.02), and, interestingly, with lower animal fats such as butter or lard have considerable 14:0 or HDL3-C (r ˆ 7 0.30, P ˆ 0.03), an HDL form more 16:0, respectively, in this position, and the fatty acids likely to participate in removal of tissue cholesterol. This would be expected to remain in the sn-2 position through- overall effect seems to be atherogenic although there was out the digestive process. Therefore, after the digestive no mean reduction of HDL in the treatment groups ana- process is complete, synthetic triglycerides of 14:0 or 16:0 lyzed as a whole. Stearic acid was unique among the three may be a reasonable model for some animal fats. Tholstrup test fatty acids in that it lowered mean concentrations of et al (1994a) speculated that myristic acid is hypercholes- HDL3-C as well as LCAT activity, ®ndings somewhat terolemic not because it is present in the sn-2 position as in indicative of reduced reverse cholesterol transport. fat but rather when it is fed in diets providing Schwab et al (1996) reported that CETP activity was considerable lauric or palmitic acids such as diets high in 12% higher after 16:0 compared to 18:0, a result consistent coconut oil, palm kernel oil or butter. Our results are with ours. consistent with theory. Changes in CETP activity were weakly correlated with changes in LDL-C concentrations (r ˆ 0.29, P ˆ 0.038). Stearic acid was not absorbed as well as myristic and This result supported the idea expressed by Fielding palmitic acids, but the lower rate of absorption did not (1997) that LDL-C may increase in responders to dietary seem to account for the neutral cholesterolemic response fat because of increases in CETP activity. However, Bonanome & Grundy (1988) explored two possible expla- because dietary treatment had an independent effect on nations for the failure of 18:0 to increase blood cholesterol LDL-C concentrations, but not on CETP activity, it concentrations compared to 16:0 Ð poor absorption of 18:0 appeared that increases in LDL-C preceded, rather than and conversion of at least some of the 18:0 to 18:1 after followed, changes in CETP activity. Synthesis of CETP absorption. In their study, 18:0 was absorbed as well as may be regulated by the size of free cholesterol pools in 16:0 (over 97%) but the relative percentage of 18:1 in cells, according to Bruce et al (1998). Regulatory pools of plasma triglycerides and cholesteryl esters was about 15% cellular free cholesterol increase when more saturated fat higher on the 18:0 diet. In our study, the changes induced in diet is eaten (Woollet et al, 1992). Possibly, these putative serum LDL-C concentrations by the 14:0, 16:0 and 18:0 regulatory pools of free cholesterol are enlarged in subjects diets in a statistical analysis of the combined data were with a strong positive response to a saturated fat diet and modestly correlated with the amount of 18:0 excreted in this effect may enhance CETP synthesis. feces (r ˆ 7 0.31, P ˆ 0.034). Although malabsorption of 18:0 may have had an impact on the results, it is clear that Receptor-mediated LDL degradation in MNC other variables are involved. Spady et al (1993) have stated that steady state concentra- As regards conversion of 18:0 ± 18:1, Hughes et al tions of LDL-C in blood are largely a function of the rate of (1996) in studying a deuterated stearate ethyl ester did LDL-C production and the activity of the LDL-receptor. not ®nd appreciable evidence of desaturation or elongation Woollett et al (1992) assessed the effect of saturated fatty of stearate. This result was consistent with our data for red acids ranging in chain length from 6:0 ± 18:0 which were blood cell membrane 18:1. provided as synthetic triglycerides on LDL receptor activity in hamsters by performing a primed continuous infusion of Intravascular lipoprotein metabolism 125I-LDL. The majority of the LDL was cleared by The process of reverse cholesterol transport (RCT) involves through receptor-mediated processes. These authors cate- HDL (in particular, a pre-beta migrating apoprotein apo A- gorized the saturated fatty acids Ð 12:0, 14:0, and 16:0 Ð 1 HDL), transfer of free cholesterol from tissues to HDL, together in a group that reduced LDL-receptor activity to esteri®caton by LCAT, and either direct transfer to the liver about the same degree when fed in cholesterol-containing or transfer to apoB-containing lipoproteins by cholesterol diets. Stearic acid did not reduce LDL-receptor activity. ester transfer protein (CETP) (Quintao, 1995). Higher We used an MNC model to study LDL receptor activity in CETP activity associated with lower concentrations of an assay that assessed binding and degradation of 125I-LDL plasma HDL-C, has been noted in patients with angiogra- in vitro. Changes in LDL receptor activity in MNC and in phically detectable (Hibino et al, liver are coordinated, according to a study involving rabbits 1996). Evidence from studies of humans with a genetic (Roach et al, 1993). The LDL used in our human study was de®ciency of CETP and of transgenic mice expressing from a donor pool so its composition was constant and not a CETP suggest both atherogenic and antiatherogenic effects variable in the in vitro LDL receptor assay (Fernandez & for the transfer protein (Tall, 1995; Bruce et al, 1998). McNamara, 1989). Therefore, changes in MNC LDL According to these researchers, CETP activity may result in receptor activity in this study should re¯ect the in¯uence lower serum HDL-C, a potential atherogenic effect. On the of the dietary treatment on MNC receptor binding af®nity other hand, CETP activity may convert HDL to a form that or number and not on the composition of the ligand. is more able to participate in RCT, an antiatherogenic In our model 14:0 and 18:0 did not alter LDL receptor effect. activity in MNC compared to the baseline diet. However, Several changes were noted in intravascular cholesterol receptor-mediated LDL degradation in MNC was 35% remodeling in this study, but it would be dif®cult to lower (P < 0.05) on palmitic acid compared to the poly- speculate whether they were atherogenic or antiathero- unsaturated baseline diet. Receptor-mediated LDL binding genic. The saturated fat diets, in contrast to the polyunsa- tended to be lower on both 14:0 and 16:0 than on 18:0 turated baseline diet, increased cholesterol ester transfer (P < 0.06). The 9% insigni®cant decrease in Bmax sug- Effect of synthetic triglycerides on serum lipoprotein metabolism JT Snook et al 604 gested that the 35% decrease in receptor-mediated LDL Fielding CJ (1997): Response of low density lipoprotein cholesterol levels degradation observed in MNC on the 16:0 diet was a to dietary change: contributions of different mechanisms. Current Opin. 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