The study, approved by the Program Advisory Committee at the Malaysian Palm Oil Board, Bangi, Malaysia, included informed signed consent by the human volunteers. The 3 dietary fats were compared using a crossover design with 4 wk for each fat. Specifically, diets were identified as A, B, or C, and subjects were assigned to each in random order until all three had been consumed by each person. Thus, all three diets were available during each 4 wk treatment period, but individual assignment to each was randomized to help negate possible carryover effects. Subjects were advised to maintain their usual food intake for all test rotations, which were identical in all aspects except for the cooking fat. An electronic weighing balance in the food service area was used to weigh food portions. Blood samples were collected prior to beginning a test period and twice during the last week (averaged as the terminal value).
Within each 4 wk period, an identical subgroup of 19 subjects participated in an acute postprandial challenge with their respective test fat after 2 wk into each diet rotation. Thus, volunteers were preconditioned with the test fat during the 14 d prior to the postprandial challenge. After an overnight fast of at least 12 h, volunteers reported to the laboratory on the 15th morning. Body weight was recorded and baseline fasted blood was drawn. Volunteers then consumed a standardized test meal comprising weighed portions of fried rice, fried potatoes, a slice of papaya and tea. This test breakfast contributed a total of 53 g test fat, and consumption of this meal was completed within 20 minutes of the first (baseline, 0 h) blood sampling. Blood samples were then taken sequentially at 2, 4, 5, 6 and 8 h after the test meal was consumed. Volunteers abstained from consuming any food during this 8 h period, except bottled mineral water. They also refrained from any strenuous activity during this interval. Following the 8 h sample, volunteers received a fully cooked meal.
Eligibility criteria included absence of family history for atherosclerosis or hypertension, as well as no use of tobacco or alcohol or adherence to any weight loss program or prescribed medication. A total of 11 healthy men and 21 women (ave age 30 ± 8 y) were initially recruited by advertisement among the 450 staff members at the research facilities of the Palm Oil Board. One subject was unable to comply with the dietary protocol and subsequently was dropped from the study, while another did not complete the third rotation due to acute appendicitis. The baseline demographics of the remaining 30 fasting subjects who were recruited after initial screening and who successfully completed the study were as follows (mean ± SD): BMI 22 ± 4; body wt 56 ± 10 kg; blood lipids (total cholesterol, 5.05 ± 0.53 mmol/L; LDL-C, 3.17 ± 0.51 mmol/L; HDL-C mmol/L, 1.48 ± 0.25 mmol/L; TG, 0.89 ± 0.30 mmol/L) and plasma glucose concentrations (5.43 ± 0.29 mmol/L).
The 3 test fats (Table ) included palm olein, as a natural dietary fat (POL); a partially hydrogenated soybean oil (PHSO); and an interesterified fat (IE) prepared in a 2-step procedure. Palm olein is the liquid to semisolid oil fraction obtained when palm oil is melted, followed by rapid cooling to separate the upper liquid palm olein layer (approximately 70%) from the lower solid stearin fraction (approximately 30%) by a physical fat modification process termed fractionation. POL contains more monounsaturated cis18:1 and less16:0 and 18:0 saturates relative to original palm oil, which has more saturated 16:0 than monounsaturated cis18:1. Both of the chemically modified fats, namely PHSO and IE, typically have less PUFA and cis18:1 content compared to native soybean oil (SBO), but have higher solid fat content to confer more plasticity and less susceptibility to rancidity. These three fats each provided a TG structure and plasticity that renders them suitable for use in common solid fat-containing food formulations.
Fatty acid profiles for individual test fats and overall FA profile of test diets
To prepare the IE fat, a first-step full hydrogenation of refined SBO converted all unsaturated C18 fatty acids to 18:0, which was then blended with refined SBO and POL that yielded the target fatty acid composition. The blend was then subjected to chemical interesterification. To prepare the PHSO, refined SBO was first partially hydrogenated using standard catalytic hydrogenation to obtain a targeted amount of TFA in the oil. For the final PHSO composition, 40% of this partially hydrogenated SBO was blended with 30% refined SBO and 30% POL.
The final test fats had the following slip melting points (POL, 24.0; PHSO, 38.5; IE, 43.0°C). At 20°C the % solids equaled 20, 20 and 43 for POL, PHSO and IE, respectively. The final test diet supplying POL provided 12.0 %en as 16:0, while the PHSO fat yielded a TFA content of about 10% by wt, but contributing 3.2 %en as TFA plus 6.5 %en as 16:0 when incorporated into the final diet. The zero-trans, IE-fat diet provided 12.5 %en as 18:0 (Table ).
Diets were prepared by a caterer who received detailed instruction from the research dietitian about the menu plan, portion size, and procedures for incorporating the test fats. A uniform menu was utilized for all 3-diet periods, which differed only in the type of test fat incorporated. Three meals a day, comprising breakfast, lunch and high tea were provided for the 4-wk period of each fat rotation. The menu, which was used to prepare the meals from Monday through Saturday noon, was constructed according to a fixed meal plan: For breakfast, a rice or noodle dish and a snack item cooked with the test fat was served with either coffee or tea. Lunch included either fish or chicken and 2 vegetables cooked with the test fat and accompanied by rice and fruits along with either tea or coffee. For high tea, a fried snack item incorporated the test fat, which was served with either tea or coffee. Because the subjects consumed their off-campus evening meal and Sunday meals with their families at home, they were provided with the appropriate cooking fat to incorporate into home meal preparations during each dietary period.
Fat and protein content of weighed food portions were determined by established AOAC methods (22). Energy value of the diets was determined with an automated bomb calorimeter [C5000 IKA-Calorimeter system, IKA® WERKE, KG, Germany]. Diets contained about 31 %en from fat, 3.5–7 %en as linoleic acid, with each test fat providing more than 70% of the total fat under controlled conditions. Dietary carbohydrate provided approximately 54 %en, and protein approximately15 %en. Carbohydrate content was indirectly estimated by difference of total protein and total lipid mass from the dried mass of the food samples. In addition, the use of the cooking fat in homes (including Sundays) was recorded in a diary to serve as an additional compliance marker. Overall, subjects were blind to the test fat fed during each test rotation.
Twenty-ml blood samples were collected at baseline and on days 28 and 29 of each test-fat rotation after overnight fasting. For the postprandial study 12-ml blood samples were collected (fasting baseline, and 2,4,5,6 and 8 h). Vaccutainer® tubes [Becton Dickinson Vaccutainer, NJ, USA] with or without EDTA (0.117 ml of 15% EDTA) were used for plasma and serum preparations, respectively. For serum preparation, blood was allowed to clot at room temperature for exactly 2 h. The Vaccutainer tubes were then centrifuged at 3000 × g for 20 min at 4°C to separate the serum or plasma, which was aliquoted for various lipid and lipoprotein analyses, snap-frozen with liquid nitrogen and stored at -80°C for subsequent analyses. To reduce intra-assay variation, samples from all 3 arms of dietary treatment were analyzed in a single batch.
Analyses of lipids and lipoproteins
Cholesterol and TG concentrations in plasma were analyzed by the enzymatic procedures of Allain et al [13
] and Nägele et al. [14
], respectively. HDL-C determination was based on a 2-step methodology, which required first precipitating LDL and VLDL from plasma with dextran and magnesium sulfate and then assaying the supernatant containing the HDL for cholesterol [15
]. All assays were performed using a Ciba-Corning 550 Express Autoanalyzer [Ciba-Corning Diagnostics Corp., Oberlin, Ohio, USA]. All reagents, calibrators and controls were supplied by Bayer Corp. (Tarrytown, NY, USA), HDL-C precipitant by Chiron Diagnostics Corp. [E. Walpole, MA, USA/Bayer Corp., Tarrytown, NY, USA]. LDL-C and VLDL-C were calculated using Friedewald's equations [18
TG-MS composition was determined by reverse-phase HPLC. The fat blends were used directly and not subjected to further purification prior to the analysis. The mobile phase was acetone/acetonitrile (Merck) at a gradient composition beginning with 65% acetone and increasing to 85% acetone in 30 min. The mobile phase flow rate was 1.5 mL/min. Two commercially packed Genesis C18 HPLC columns (15 cm length × 4.6 mm i.d.) of 4 um particle size (Jones Chromatography, Mid Glamorgan, United Kingdom) were used to separate the TG-MS. The TG-MS were detected by an Evaporative Light Scatter Detector, ELSD. Individual peaks were identified by comparing the retention times with those of pure TG-MS standards and common vegetable oils of known TG-MS composition [17
Determination of plasma glucose, serum insulin and C-peptide
Plasma glucose was assessed from frozen samples by the glucose oxidase method, a 2-step enzymatic procedure [19
]. A reagent kit for the enzymatic procedure, SERA-PAK®
Glucose (which was fully Bayer Corporation, Tarrytown, NY, USA) was used in the analysis, which was automated using the Ciba-Corning 550 Express Autoanalyzer (Ciba-Corning Diagnostics Corporation, Ohio, USA). Serum insulin was measured [20
] for 0 wk, 4 wk, and postprandial samples by automated immunoassay with an IMMULITE 1000 Analyzer (Euro/DPC Limited Diagnostics Products Corporation, Los Angeles, Ca.) and expressed as uU/ml. Circulating C-peptide was measured in postprandial serum as an index of insulin secretion [21
]. Samples were diluted (1:4) prior to immunoassay with the IMMULITE 1000 Analyzer. All components (chemiluminescent substrate, controls, standards and diluent) for the insulin and C-peptide immunoassays were supplied in IMMULITE kits. Results are expressed as pmol/L.
General compliance to diet was assessed by body weight changes and the comparison between the fatty acid composition of diets eaten and plasma TG. In the first instance, weight was recorded for every subject at baseline and at weekly intervals for each test rotation. This ensured that food intake was adequate and that weight fluctuations were minimized between the test-fat rotations.
Plasma TG fatty acid composition of was determined for all subjects at the end of each test rotation. Total lipids were extracted from plasma and subjected to thin-layer chromatography to separate TG and cholesteryl esters [22
]. In addition, fatty acids from diets as eaten were assessed following Soxhlet extraction and conversion of extracted lipids into fatty acid methyl esters for analysis by gas chromatography [Perkin-Elmer Autosystem, Perkin-Elmer, Norwalk, CT]. Test fats themselves were assayed directly after conversion to methyl esters [22
]. Results from gas chromatography of fatty acids were obtained as percentage composition (by weight), calculated by reference to standards. The fatty acid profile of meals consumed by the subjects served to check whether the research diets achieved fatty acid targets, while the plasma TG fatty acids served to check compliance with the research protocol.
The crossover design enabled each subject to serve as his/her own control. The Statistical Package for Social Sciences, SPSS®
for Windows™ application (Version 11.0, SPSS Inc., Chicago, USA) was used for all required statistical analyses. The mean of values for days 28 and 29 was treated as the end of the study period. Univariate analysis was performed for linearly independent pair-wise comparisons between baseline and end values for plasma lipids, lipoproteins, glucose, and insulin, as well as for the percent change in the measured parameters for each dietary treatment. Multivariate analyses for repeated measures (ANOVA), using the general linear model, was performed for all time × diet interactions for blood glucose, insulin and C-peptide parameters following each postprandial test fat challenge. Corrected models used against baseline values were taken as true measures of change occurring during the post-absorptive period resulting from dietary treatment. Univariate analysis was performed for linearly independent pair-wise comparisons of incremental area-under-the-curve (IAUC) data for the 8-h postprandial period calculated by the trapezoidal rule [25
]. Post-hoc analyses included Bonferroni's adjustment for multiple paired comparisons of estimated marginal means as well as Duncan's test for homogeneity of the effects generated by the test fat treatments. Significance was set at P
< 0.05 for all evaluated measures.