Abstract
The aim of this study was to determine short-term effects of trans fatty acid (TFA) intake from ruminant and industrial sources on surrogate markers of cardiovascular risk in the context of a balanced diet with 30–36% of daily energy from fat.
Prospective, randomized, double-blind, parallel-design study.
In this study, 142 healthy volunteers aged 45 to 69 years were randomly allocated to three different diets: either a diet enriched with 2% of daily energy intake from ruminant TFA (rTFA) or with industrial TFA (iTFA), or a diet without TFA (wTFA), for a duration of four weeks. The primary outcome parameter was endothelial function measured by brachial artery flow mediated dilation (FMD). Secondary outcome parameters included biomarkers for inflammation, coagulation and endothelial function and lipid profiles. One hundred and twenty-nine participants completed the study.
Neither alpine butter with TFA from ruminant source nor margarine with industrially produced TFA showed significant effects on brachial artery FMD (FMD% differences: rTFA vs. iTFA 0.04 (95% confidence interval 0.91 to 0.98), rTFA vs. wTFA –0.98 (–2.00 to 0.04) and iTFA vs. wTFA –1.04 (–2.38 to 0.30). With rTFA, there was a small but significant increase of total cholesterol: rTFA over wTFA 1.04 (1.00 to 1.07 mmol/l) and LDL-cholesterol: rTFA over wTFA 1.08 (1.03 to 1.14 mmol/l) without concomitant increase of biomarkers for inflammation or coagulation.
Short-term intake of TFA at 2% of total daily energy intake from neither ruminant nor industrially produced sources does not have any negative impact on brachial artery FMD, inflammation and coagulation markers in healthy subjects.
Introduction
Deleterious effects of intake of high trans fatty acids (TFA) on cardiovascular risk factors and risk of coronary heart disease have been found in several epidemiological studies.1–4 The 2016 European Guidelines on cardiovascular disease prevention in clinical practice recommend to derive <1% of total fat intake from TFA – the less the better. A meta-analysis of prospective cohort studies has shown that a 2% increase of energy intake from TFA was associated with a 23% increase in the incidence of coronary heart disease.2 In this context, differential effects of TFA on cardiovascular risk factors have been postulated according to their origin.5 Industrial TFA that are generated by partial hydrogenation of vegetable oils seem to be more harmful in terms of cardiovascular risk compared with ruminant TFA that are produced through bacterial hydrogenation of unsaturated fatty acids in the rumen of ruminants.1,5-8 The diverse associations are likely to be explained by lower intake levels of ruminant compared with industrial TFA and by different isomer distributions.1 In contrast to previous findings, results from the Ludwigshafen Risk and Cardiovascular Healthy Study9 found that low concentrations of total TFA were even inversely associated with adverse cardiac outcomes; while the natural occurring TFA C16:1n-7t was associated with reduced risk, no increased risk was found for industrially produced TFA.
In vitro studies support the concept of a differential effect between different TFA isomers on molecular and cellular pathways mediating endothelial dysfunction through inflammation and reduced nitric oxide production in endothelial cells.10 Endothelial dysfunction, as measured by flow-mediated dilation (FMD), is considered a surrogate for nitric oxide bioavailability and an early marker of atherosclerosis.11,12 Studies on the effects of TFA intake on brachial artery FMD are scarce and provide conflicting results.13,14 In a study with high doses of TFA (9.2 energy%) from partially hydrogenated soy and vegetable oils, de Roos et al.13 reported impaired FMD of the brachial artery after a period of four weeks. Although inflammatory processes are likely to impact on endothelial cell function,15-17 the precise mechanisms by which TFA cause endothelial dysfunction are not fully clear yet.
Currently there is a lack of studies investigating the effects of TFA from different sources on cardiovascular risk in the context of a balanced diet. We evaluated the short-term impact of diets with alpine butter rich in ruminant TFA (rTFA) or with margarine rich in industrial TFA (iTFA) or with margarine without TFA (wTFA) on surrogate markers of cardiovascular risk including brachial artery FMD, lipid levels and pro-inflammatory and anticoagulative biomarkers in healthy humans. Our primary aim was to show that a diet with an alpine butter rich in TFA from ruminant sources is not inferior to a diet with margarine without TFA. In the case of non-inferiority being shown, we aimed to determine whether the diet with alpine butter is superior to a diet with margarine rich in iTFA.
Methods
Study design and participants
In this randomized, controlled, double-blind, parallel-group trial, healthy volunteers of both sexes, between 45 and 69 years of age and with a body mass index (BMI) between 20 and 30 kg/m2 were included. We recruited study participants between October 2009 and May 2013 through newspaper advertisements, the hospital website and flyers distributed at national nutrition meetings. The upper age limit for study inclusion was changed from 65 to 69 years in May 2009 to facilitate participant recruitment. Exclusion criteria were known cardiovascular disease, smoking, hypertension, diabetes, abnormal liver and kidney function, existing signs of inflammation, anaemia, electrolyte abnormalities (Na, K, Ca), intake of medication (including supplements such as vitamins and minerals) and allergies to milk products.
After validation of eligibility criteria, participants were randomly assigned to three different diets: a diet enriched with rTFA (rTFA diet); a diet enriched with iTFA (iTFA diet); or a diet without TFA (wTFA diet, control group). Randomization was based on a randomly permuted block design (block sizes of three, six and nine study participants) in 3:2:3 ratios stratified by sex and age-group (45–55 years; 55–69 years). The allocation sequence was generated beforehand by CTU Bern, Switzerland using Stata 12.1 (Stata Corporation, College Station, Texas, USA). A person at Agroscope, Switzerland who was not otherwise involved in the trial coded the study products according to the randomization list. Products were identical in appearance and could not be distinguished by either study participants or investigators. Participants, study team members and the FMD echocardiographer were all blinded to treatment assignment. All participants provided written informed consent. Ethical approval was obtained from the cantonal ethical committee of Bern, Switzerland. The trial was registered with ClinicalTrials.gov (NCT00933322).
Procedures
After a two-week run-in period, during which all subjects followed the wTFA diet, participants continued with iTFA, rTFA or wTFA diets according to their randomization. Subjects were provided with specific dietary fats to use at home accounting for an estimated 33–36% of total energy intake. The remainder of their energy need for maintaining normal weight was covered by a variety of very low fat foods. Subjects received regular dietary advice and their dietary intake was meticulously checked to ensure compliance with fatty acid intake specifications. Details on dietary procedures and study products are available in the Supplementary Material and Table S1 and Table S2 online. We collected blood samples from participants, and performed brachial artery FMD imaging, at screening (week 0), at treatment start (week 2, post run-in) and at treatment end (week 6). Brachial artery FMD imaging was performed according to standardized protocols.18
Statistical analysis
The primary idea was to see whether a diet rich in rTFA is better than a diet rich in TFA from partially hydrogenated vegetable oil as stated in clinicaltrials.gov. This is reflected in the superior test. However, we first needed to establish safety of the new diet, which is reflected in the non-inferiority test.
Sample size calculations were performed using a hierarchical approach, with the first stage being a non-inferiority test of rTFA versus wTFA diets, and, in the case of non-inferiority being shown, a second stage with a superiority test of rTFA versus iTFA diets in which we hypothesized that a rTFA diet results in larger FMD changes than iTFA. The standard deviation for FMD in all three groups was assumed to be 2%. The non-inferiority margin, that is, the maximal acceptable difference between rTFA and wTFA, was set at 0.8% and the minimally clinically relevant difference between rTFA and iTFA was set at 1.5%. Power for both tests was set at 80% and alpha was fixed at 0.05 (one-sided for non-inferiority and two-sided for the superiority test). With a randomization ratio of 3:2:3 we aimed at recruiting 77:55:77 participants for the rTFA:iTFA:wTFA groups respectively (209 participants altogether).
We analysed the primary endpoint using linear models including FMD (%) at the treatment end visit (week 6) as the dependent variable, and treatment group as the independent variable. Analysis was performed following the intention-to-treat principle (ITT), and sensitivity analysis included per-protocol data. Further details on analysis are provided in the online Supplementary Material. All analyses were performed using Stata version 12.1 (Stata Corporation, College Station, Texas, USA).
Results
Neither alpine butter with rTFA nor margarine with iTFA showed significant effects on important cardiovascular risk markers in the context of a balanced diet with 30–36% of daily energy from fat.
The trial was prematurely stopped after four years of study recruitment. We found only minimal FMD changes after four weeks of intervention in all three intervention groups and it was evident that even with a larger sample size, neither non-inferiority nor superiority margins could be reached. The stringent inclusion criteria meant that out of more than 1500 interested participants, only 154 participants met eligibility criteria and were invited to the laboratory. Upon screening, 12 participants failed to meet the inclusion criteria (Figure 1). One hundred and forty-two subjects performed all measurements at the screening visit and were randomized to the different study groups. Thirteen participants resigned during the run-in phase and per definition were not included in the ITT protocol. Thus, the ITT analysis included 129 participants. Four participants had FMD data of insufficient quality and were therefore not included in the per protocol analysis.
Flow chart of participant recruitment and study enrolment.
rTFA: diet enriched with ruminant trans fatty acids; iTFA: diet enriched with industrial trans fatty acids; wTFA: diet without trans fatty acids; ITT: intention to treat
Participant baseline characteristics and FMD data (treatment start week 2) are shown in Table 1. Baseline blood biomarkers are shown in the Supplementary Material (Table S3). There were no significant and clinical relevant differences of baseline characteristics between the three groups. Evaluation of food diaries and feedback of the dietician indicate that participants complied well with the study protocol in regard to eating habits. They also kept stable physical activity levels during the study period, determined with the International Physical Activity Questionnaire short form. Additionally, participants’ adherence to the study diet has been evaluated by fatty acid analysis in serum cholesteryl esters in a subgroup of study participants and results confirmed good compliance (Table S2 online).
Study participants’ characteristics at baseline after the run-in phase.
| Variable | wTFA | iTFA | rTFA |
|---|---|---|---|
| N = 48 | N = 31 | N = 50 | |
| Age, years | 54.5 (50.0–59.8) | 54.0 (48.0–61.0) | 55.0 (48.8–58.0) |
| Gender, % male | 20 (42) | 14 (45) | 21 (42) |
| Height, m | 1.7 (1.6–1.8) | 1.7 (1.6–1.8) | 1.7 (1.7–1.8) |
| Weight, kg | 72.3 (63.4–81.1) | 71.3 (63.6–83.6) | 72.4 (61.5–79.7) |
| BMI, kg/m2 | 24.4 (22.5–26.9) | 25.0 (22.6–28.4) | 25.0 (22.7–26.4) |
| Waist circumference, cm | 89.0 (80.0–96.0) | 90.0 (83.0–98.0) | 86.0 (80.0–91.5) |
| Systolic BP, mmHg | 119.0 (110.0–129.5) | 118.0 (108.0–125.0) | 119.0 (111.5–25.3) |
| Diastolic BP, mmHg | 75.5 (70.0–80.0) | 76.0 (70.0–84.0) | 78.0 (73.5–80.5) |
| Insulin, mU/l | 5.0 (3.5–6.2) | 4.7 (3.4–6.7) | 3.9 (2.9–5.6) |
| Glucose, mmol/l | 4.9 (4.6–5.2) | 5.1 (4.7–5.3) | 4.9 (4.7–5.2) |
| Vascular function | |||
| BAD, baseline, mm | 4.0 (3.5–4.8) | 4.2 (3.7–4.9) | 4.1 (3.7–4.5) |
| BAD, maximum, mm | 4.3 (3.7–5.1) | 4.3 (3.9–5.1) | 4.2 (3.8–4.7) |
| Blood velocity, baseline, m/s | 0.2 (0.2–0.3) | 0.2 (0.1–0.3) | 0.2 (0.2–0.3) |
| Time-to-peak diameter, s | 46.9 (36.0–56.3) | 53.0 (35.0–81.6) | 47.1 (33.0–60.9) |
| Shear rate, 1/s | 226.0 (146.3–311.8) | 196.0 (144.0–273.0) | 210.0 (147.0–278.0) |
| Shear rate AUC ttp, 1/s | 32,360 (24,266–45,750) | 31,017 (20,810–39,469) | 29,640 (23,520–47,197) |
| FMD, % | 4.3 (3.1–7.2) | 4.7 (3.7–6.8) | 5.0 (3.2–7.1) |
| NMD, % | 15.5 (12.7–18.1) | 13.4 (11.4–17.6) | 14.9 (12.1–17.3) |
| FMD/NMD ratio | 0.3 (0.2–0.5) | 0.3 (0.3–0.5) | 0.3 (0.2–0.5) |
| Variable | wTFA | iTFA | rTFA |
|---|---|---|---|
| N = 48 | N = 31 | N = 50 | |
| Age, years | 54.5 (50.0–59.8) | 54.0 (48.0–61.0) | 55.0 (48.8–58.0) |
| Gender, % male | 20 (42) | 14 (45) | 21 (42) |
| Height, m | 1.7 (1.6–1.8) | 1.7 (1.6–1.8) | 1.7 (1.7–1.8) |
| Weight, kg | 72.3 (63.4–81.1) | 71.3 (63.6–83.6) | 72.4 (61.5–79.7) |
| BMI, kg/m2 | 24.4 (22.5–26.9) | 25.0 (22.6–28.4) | 25.0 (22.7–26.4) |
| Waist circumference, cm | 89.0 (80.0–96.0) | 90.0 (83.0–98.0) | 86.0 (80.0–91.5) |
| Systolic BP, mmHg | 119.0 (110.0–129.5) | 118.0 (108.0–125.0) | 119.0 (111.5–25.3) |
| Diastolic BP, mmHg | 75.5 (70.0–80.0) | 76.0 (70.0–84.0) | 78.0 (73.5–80.5) |
| Insulin, mU/l | 5.0 (3.5–6.2) | 4.7 (3.4–6.7) | 3.9 (2.9–5.6) |
| Glucose, mmol/l | 4.9 (4.6–5.2) | 5.1 (4.7–5.3) | 4.9 (4.7–5.2) |
| Vascular function | |||
| BAD, baseline, mm | 4.0 (3.5–4.8) | 4.2 (3.7–4.9) | 4.1 (3.7–4.5) |
| BAD, maximum, mm | 4.3 (3.7–5.1) | 4.3 (3.9–5.1) | 4.2 (3.8–4.7) |
| Blood velocity, baseline, m/s | 0.2 (0.2–0.3) | 0.2 (0.1–0.3) | 0.2 (0.2–0.3) |
| Time-to-peak diameter, s | 46.9 (36.0–56.3) | 53.0 (35.0–81.6) | 47.1 (33.0–60.9) |
| Shear rate, 1/s | 226.0 (146.3–311.8) | 196.0 (144.0–273.0) | 210.0 (147.0–278.0) |
| Shear rate AUC ttp, 1/s | 32,360 (24,266–45,750) | 31,017 (20,810–39,469) | 29,640 (23,520–47,197) |
| FMD, % | 4.3 (3.1–7.2) | 4.7 (3.7–6.8) | 5.0 (3.2–7.1) |
| NMD, % | 15.5 (12.7–18.1) | 13.4 (11.4–17.6) | 14.9 (12.1–17.3) |
| FMD/NMD ratio | 0.3 (0.2–0.5) | 0.3 (0.3–0.5) | 0.3 (0.2–0.5) |
Values are median (interquartile range) for continuous variables, and number (%) for count variables.
AUC ttp: area under the curve until peak vasodilation; BAD: brachial artery diameter; BMI: body mass index; BP: blood pressure; FMD: flow-mediated dilation; iTFA: diet enriched with industrial trans fatty acids; NMD: nitroglycerin-mediated dilation; rTFA: diet enriched with ruminant trans fatty acids; wTFA: diet without trans fatty acids
Study participants’ characteristics at baseline after the run-in phase.
| Variable | wTFA | iTFA | rTFA |
|---|---|---|---|
| N = 48 | N = 31 | N = 50 | |
| Age, years | 54.5 (50.0–59.8) | 54.0 (48.0–61.0) | 55.0 (48.8–58.0) |
| Gender, % male | 20 (42) | 14 (45) | 21 (42) |
| Height, m | 1.7 (1.6–1.8) | 1.7 (1.6–1.8) | 1.7 (1.7–1.8) |
| Weight, kg | 72.3 (63.4–81.1) | 71.3 (63.6–83.6) | 72.4 (61.5–79.7) |
| BMI, kg/m2 | 24.4 (22.5–26.9) | 25.0 (22.6–28.4) | 25.0 (22.7–26.4) |
| Waist circumference, cm | 89.0 (80.0–96.0) | 90.0 (83.0–98.0) | 86.0 (80.0–91.5) |
| Systolic BP, mmHg | 119.0 (110.0–129.5) | 118.0 (108.0–125.0) | 119.0 (111.5–25.3) |
| Diastolic BP, mmHg | 75.5 (70.0–80.0) | 76.0 (70.0–84.0) | 78.0 (73.5–80.5) |
| Insulin, mU/l | 5.0 (3.5–6.2) | 4.7 (3.4–6.7) | 3.9 (2.9–5.6) |
| Glucose, mmol/l | 4.9 (4.6–5.2) | 5.1 (4.7–5.3) | 4.9 (4.7–5.2) |
| Vascular function | |||
| BAD, baseline, mm | 4.0 (3.5–4.8) | 4.2 (3.7–4.9) | 4.1 (3.7–4.5) |
| BAD, maximum, mm | 4.3 (3.7–5.1) | 4.3 (3.9–5.1) | 4.2 (3.8–4.7) |
| Blood velocity, baseline, m/s | 0.2 (0.2–0.3) | 0.2 (0.1–0.3) | 0.2 (0.2–0.3) |
| Time-to-peak diameter, s | 46.9 (36.0–56.3) | 53.0 (35.0–81.6) | 47.1 (33.0–60.9) |
| Shear rate, 1/s | 226.0 (146.3–311.8) | 196.0 (144.0–273.0) | 210.0 (147.0–278.0) |
| Shear rate AUC ttp, 1/s | 32,360 (24,266–45,750) | 31,017 (20,810–39,469) | 29,640 (23,520–47,197) |
| FMD, % | 4.3 (3.1–7.2) | 4.7 (3.7–6.8) | 5.0 (3.2–7.1) |
| NMD, % | 15.5 (12.7–18.1) | 13.4 (11.4–17.6) | 14.9 (12.1–17.3) |
| FMD/NMD ratio | 0.3 (0.2–0.5) | 0.3 (0.3–0.5) | 0.3 (0.2–0.5) |
| Variable | wTFA | iTFA | rTFA |
|---|---|---|---|
| N = 48 | N = 31 | N = 50 | |
| Age, years | 54.5 (50.0–59.8) | 54.0 (48.0–61.0) | 55.0 (48.8–58.0) |
| Gender, % male | 20 (42) | 14 (45) | 21 (42) |
| Height, m | 1.7 (1.6–1.8) | 1.7 (1.6–1.8) | 1.7 (1.7–1.8) |
| Weight, kg | 72.3 (63.4–81.1) | 71.3 (63.6–83.6) | 72.4 (61.5–79.7) |
| BMI, kg/m2 | 24.4 (22.5–26.9) | 25.0 (22.6–28.4) | 25.0 (22.7–26.4) |
| Waist circumference, cm | 89.0 (80.0–96.0) | 90.0 (83.0–98.0) | 86.0 (80.0–91.5) |
| Systolic BP, mmHg | 119.0 (110.0–129.5) | 118.0 (108.0–125.0) | 119.0 (111.5–25.3) |
| Diastolic BP, mmHg | 75.5 (70.0–80.0) | 76.0 (70.0–84.0) | 78.0 (73.5–80.5) |
| Insulin, mU/l | 5.0 (3.5–6.2) | 4.7 (3.4–6.7) | 3.9 (2.9–5.6) |
| Glucose, mmol/l | 4.9 (4.6–5.2) | 5.1 (4.7–5.3) | 4.9 (4.7–5.2) |
| Vascular function | |||
| BAD, baseline, mm | 4.0 (3.5–4.8) | 4.2 (3.7–4.9) | 4.1 (3.7–4.5) |
| BAD, maximum, mm | 4.3 (3.7–5.1) | 4.3 (3.9–5.1) | 4.2 (3.8–4.7) |
| Blood velocity, baseline, m/s | 0.2 (0.2–0.3) | 0.2 (0.1–0.3) | 0.2 (0.2–0.3) |
| Time-to-peak diameter, s | 46.9 (36.0–56.3) | 53.0 (35.0–81.6) | 47.1 (33.0–60.9) |
| Shear rate, 1/s | 226.0 (146.3–311.8) | 196.0 (144.0–273.0) | 210.0 (147.0–278.0) |
| Shear rate AUC ttp, 1/s | 32,360 (24,266–45,750) | 31,017 (20,810–39,469) | 29,640 (23,520–47,197) |
| FMD, % | 4.3 (3.1–7.2) | 4.7 (3.7–6.8) | 5.0 (3.2–7.1) |
| NMD, % | 15.5 (12.7–18.1) | 13.4 (11.4–17.6) | 14.9 (12.1–17.3) |
| FMD/NMD ratio | 0.3 (0.2–0.5) | 0.3 (0.3–0.5) | 0.3 (0.2–0.5) |
Values are median (interquartile range) for continuous variables, and number (%) for count variables.
AUC ttp: area under the curve until peak vasodilation; BAD: brachial artery diameter; BMI: body mass index; BP: blood pressure; FMD: flow-mediated dilation; iTFA: diet enriched with industrial trans fatty acids; NMD: nitroglycerin-mediated dilation; rTFA: diet enriched with ruminant trans fatty acids; wTFA: diet without trans fatty acids
With all three diets, FMD changes were minimal after four weeks. We could not confirm non-inferiority of the rTFA group over the wTFA group for FMD (%), as the confidence intervals (CIs) included the pre-defined clinically relevant difference of –0.8% (estimated difference: –0.98, 90% CI –2.00 to 0.04; Figure 2).
Difference in brachial artery FMD (%) among the three study groups at treatment start (pre, week 2) and treatment end (post, week 6). Data are means and 95% confidence interval by: Shown are individual data points as well as mean values with 95% confidence intervals.
FMD: flow mediated dilation; wTFA: diet without trans fatty acids; iTFA: diet enriched with industrial trans fatty acids; rTFA: diet enriched with ruminant trans fatty acids
We found a trend for different diet effects according to gender in the superiority test, with rTFA tending to be superior to iTFA in males, but not in females (males estimate 1.06 95% CI –0.24 to 2.35; females estimate –0.79 95% CI –2.13 to 0.55). For a complete account of the comparisons see Table 2. In the superiority test comparing the rTFA group with the iTFA group, we found a significant interaction between diet and BMI. Participants in the high BMI group (25–30 kg/m2) allocated to the rTFA group had significantly increased FMD values, possibly showing clinical relevance (defined as 1.5% increase in FMD measurement); for participants in the normal BMI range (20–24.9 kg/m2) this effect was not found (estimate –1.20, 95% CI –2.84 to 0.54).
Differences in brachial artery flow mediated dilation (FMD, %) comparing the different diets.
| rTFA vs. iTFA | rTFA vs. wTFA | iTFA vs. wTFA | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Model | Analysis | N | Estimated difference (95% CI) | p for interaction | N | Estimated difference (95% CI) | p for interaction | N | Estimated difference (95% CI) | p for interaction |
| Primary modela | ||||||||||
| Intention-to-treat | 81 | 0.04 (–0.91 to 0.98) | 98 | –0.98 (–2.00 to 0.04) | 79 | –1.04 (–2.38 to 0.30) | ||||
| Per protocol | 77 | 0.06 (–0.89 to 1.00) | 94 | –0.96 (–1.98 to 0.07) | 77 | –1.04 (–2.39 to 0.32) | ||||
| Adjusted for stratification factorsb | ||||||||||
| Intention-to-treat | 76 | 0.05 (–0.92 to 1.02) | 94 | –1.00 (–2.02 to 0.01) | 76 | –1.04 (–2.36 to 0.28) | ||||
| Analyses of interactions and stratified analysesa | ||||||||||
| Gender | 0.06 | 0.08 | 0.97 | |||||||
| Female | 44 | –0.79 (–2.13 to 0.55) | 55 | –1.72 (–3.28, –0.15) | 43 | –1.01 (–3.17 to 1.15) | ||||
| Male | 33 | 1.06 (–0.24 to 2.35) | 39 | 0.15 (–0.97 to 1.26) | 34 | –0.97 (–2.43 to 0.48) | ||||
| BMI | 0.02 | 0.95 | 0.08 | |||||||
| 20–24 kg/m2 | 33 | –1.20 (–2.94 to 0.54) | 41 | –0.99 (–2.62 to 0.64) | 34 | 0.29 (–1.96 to 2.53) | ||||
| 25–30 kg/m2 | 44 | 1.06 (0.03 to 2.09) | 52 | –1.05 (–2.40 to 0.31) | 42 | –2.09 (–3.84, –0.34) | ||||
| Age group | 0.69 | 0.61 | 0.95 | |||||||
| 45–55 years | 41 | –0.11 (–1.54 to 1.32) | 50 | –1.26 (–2.61 to 0.09) | 39 | –1.14 (–3.15 to 0.86) | ||||
| 56–69 years | 35 | 0.34 (–0.98 to 1.67) | 44 | –0.74 (–2.31 to 0.82) | 37 | –1.13 (–3.10 to 0.84) |
| rTFA vs. iTFA | rTFA vs. wTFA | iTFA vs. wTFA | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Model | Analysis | N | Estimated difference (95% CI) | p for interaction | N | Estimated difference (95% CI) | p for interaction | N | Estimated difference (95% CI) | p for interaction |
| Primary modela | ||||||||||
| Intention-to-treat | 81 | 0.04 (–0.91 to 0.98) | 98 | –0.98 (–2.00 to 0.04) | 79 | –1.04 (–2.38 to 0.30) | ||||
| Per protocol | 77 | 0.06 (–0.89 to 1.00) | 94 | –0.96 (–1.98 to 0.07) | 77 | –1.04 (–2.39 to 0.32) | ||||
| Adjusted for stratification factorsb | ||||||||||
| Intention-to-treat | 76 | 0.05 (–0.92 to 1.02) | 94 | –1.00 (–2.02 to 0.01) | 76 | –1.04 (–2.36 to 0.28) | ||||
| Analyses of interactions and stratified analysesa | ||||||||||
| Gender | 0.06 | 0.08 | 0.97 | |||||||
| Female | 44 | –0.79 (–2.13 to 0.55) | 55 | –1.72 (–3.28, –0.15) | 43 | –1.01 (–3.17 to 1.15) | ||||
| Male | 33 | 1.06 (–0.24 to 2.35) | 39 | 0.15 (–0.97 to 1.26) | 34 | –0.97 (–2.43 to 0.48) | ||||
| BMI | 0.02 | 0.95 | 0.08 | |||||||
| 20–24 kg/m2 | 33 | –1.20 (–2.94 to 0.54) | 41 | –0.99 (–2.62 to 0.64) | 34 | 0.29 (–1.96 to 2.53) | ||||
| 25–30 kg/m2 | 44 | 1.06 (0.03 to 2.09) | 52 | –1.05 (–2.40 to 0.31) | 42 | –2.09 (–3.84, –0.34) | ||||
| Age group | 0.69 | 0.61 | 0.95 | |||||||
| 45–55 years | 41 | –0.11 (–1.54 to 1.32) | 50 | –1.26 (–2.61 to 0.09) | 39 | –1.14 (–3.15 to 0.86) | ||||
| 56–69 years | 35 | 0.34 (–0.98 to 1.67) | 44 | –0.74 (–2.31 to 0.82) | 37 | –1.13 (–3.10 to 0.84) |
As non-inferiority in the comparison rTFA vs. wTFA was not shown, all confidence intervals and p-values should be considered exploratory.
Adjusted for FMD at treatment start.
Adjusted for FMD at treatment start, gender and age group.
BMI: body mass index; CI: confidence interval; FMD: flow mediated dilation; iTFA: diet enriched with industrial trans fatty acids; rTFA: diet enriched with ruminant trans fatty acids; wTFA: diet without trans fatty acids.
Differences in brachial artery flow mediated dilation (FMD, %) comparing the different diets.
| rTFA vs. iTFA | rTFA vs. wTFA | iTFA vs. wTFA | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Model | Analysis | N | Estimated difference (95% CI) | p for interaction | N | Estimated difference (95% CI) | p for interaction | N | Estimated difference (95% CI) | p for interaction |
| Primary modela | ||||||||||
| Intention-to-treat | 81 | 0.04 (–0.91 to 0.98) | 98 | –0.98 (–2.00 to 0.04) | 79 | –1.04 (–2.38 to 0.30) | ||||
| Per protocol | 77 | 0.06 (–0.89 to 1.00) | 94 | –0.96 (–1.98 to 0.07) | 77 | –1.04 (–2.39 to 0.32) | ||||
| Adjusted for stratification factorsb | ||||||||||
| Intention-to-treat | 76 | 0.05 (–0.92 to 1.02) | 94 | –1.00 (–2.02 to 0.01) | 76 | –1.04 (–2.36 to 0.28) | ||||
| Analyses of interactions and stratified analysesa | ||||||||||
| Gender | 0.06 | 0.08 | 0.97 | |||||||
| Female | 44 | –0.79 (–2.13 to 0.55) | 55 | –1.72 (–3.28, –0.15) | 43 | –1.01 (–3.17 to 1.15) | ||||
| Male | 33 | 1.06 (–0.24 to 2.35) | 39 | 0.15 (–0.97 to 1.26) | 34 | –0.97 (–2.43 to 0.48) | ||||
| BMI | 0.02 | 0.95 | 0.08 | |||||||
| 20–24 kg/m2 | 33 | –1.20 (–2.94 to 0.54) | 41 | –0.99 (–2.62 to 0.64) | 34 | 0.29 (–1.96 to 2.53) | ||||
| 25–30 kg/m2 | 44 | 1.06 (0.03 to 2.09) | 52 | –1.05 (–2.40 to 0.31) | 42 | –2.09 (–3.84, –0.34) | ||||
| Age group | 0.69 | 0.61 | 0.95 | |||||||
| 45–55 years | 41 | –0.11 (–1.54 to 1.32) | 50 | –1.26 (–2.61 to 0.09) | 39 | –1.14 (–3.15 to 0.86) | ||||
| 56–69 years | 35 | 0.34 (–0.98 to 1.67) | 44 | –0.74 (–2.31 to 0.82) | 37 | –1.13 (–3.10 to 0.84) |
| rTFA vs. iTFA | rTFA vs. wTFA | iTFA vs. wTFA | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Model | Analysis | N | Estimated difference (95% CI) | p for interaction | N | Estimated difference (95% CI) | p for interaction | N | Estimated difference (95% CI) | p for interaction |
| Primary modela | ||||||||||
| Intention-to-treat | 81 | 0.04 (–0.91 to 0.98) | 98 | –0.98 (–2.00 to 0.04) | 79 | –1.04 (–2.38 to 0.30) | ||||
| Per protocol | 77 | 0.06 (–0.89 to 1.00) | 94 | –0.96 (–1.98 to 0.07) | 77 | –1.04 (–2.39 to 0.32) | ||||
| Adjusted for stratification factorsb | ||||||||||
| Intention-to-treat | 76 | 0.05 (–0.92 to 1.02) | 94 | –1.00 (–2.02 to 0.01) | 76 | –1.04 (–2.36 to 0.28) | ||||
| Analyses of interactions and stratified analysesa | ||||||||||
| Gender | 0.06 | 0.08 | 0.97 | |||||||
| Female | 44 | –0.79 (–2.13 to 0.55) | 55 | –1.72 (–3.28, –0.15) | 43 | –1.01 (–3.17 to 1.15) | ||||
| Male | 33 | 1.06 (–0.24 to 2.35) | 39 | 0.15 (–0.97 to 1.26) | 34 | –0.97 (–2.43 to 0.48) | ||||
| BMI | 0.02 | 0.95 | 0.08 | |||||||
| 20–24 kg/m2 | 33 | –1.20 (–2.94 to 0.54) | 41 | –0.99 (–2.62 to 0.64) | 34 | 0.29 (–1.96 to 2.53) | ||||
| 25–30 kg/m2 | 44 | 1.06 (0.03 to 2.09) | 52 | –1.05 (–2.40 to 0.31) | 42 | –2.09 (–3.84, –0.34) | ||||
| Age group | 0.69 | 0.61 | 0.95 | |||||||
| 45–55 years | 41 | –0.11 (–1.54 to 1.32) | 50 | –1.26 (–2.61 to 0.09) | 39 | –1.14 (–3.15 to 0.86) | ||||
| 56–69 years | 35 | 0.34 (–0.98 to 1.67) | 44 | –0.74 (–2.31 to 0.82) | 37 | –1.13 (–3.10 to 0.84) |
As non-inferiority in the comparison rTFA vs. wTFA was not shown, all confidence intervals and p-values should be considered exploratory.
Adjusted for FMD at treatment start.
Adjusted for FMD at treatment start, gender and age group.
BMI: body mass index; CI: confidence interval; FMD: flow mediated dilation; iTFA: diet enriched with industrial trans fatty acids; rTFA: diet enriched with ruminant trans fatty acids; wTFA: diet without trans fatty acids.
The exploratory analyses suggested that an alpine butter diet (rTFA) results in a greater increase in blood lipids (total cholesterol and low density lipoprotein cholesterol (LDL)) compared with the wTFA and iTFA groups. In addition, there was a greater increase in the LDL/high density lipoprotein cholesterol ratio in the rTFA compared with the wTFA group. No consistent changes in inflammatory and coagulative biomarkers were observed at the end of the intervention (Table 3).
Summary and comparison of secondary outcomes among the treatment groups.
| Variable | N missing | wTFA | iTFA | rTFA | rTFA over wTFA Ratio (95% CI) | rTFA over iTFA Ratio (95% CI) |
|---|---|---|---|---|---|---|
| Vascular function | ||||||
| BAD, baseline, mm | 2 | 3.96 (3.46–4.72) | 4.26 (3.69–4.90) | 3.96 (3.58–4.59) | 1.003 (0.984, 1.022) | 0.998 (0.973, 1.023) |
| BAD, maximum, mm | 5 | 4.13 (3.75–5.10) | 4.45 (3.84–5.05) | 4.18 (3.76–4.80) | 0.994 (0.974, 1.014) | 1.008 (0.990, 1.027) |
| Blood velocity, baseline, m/s | 2 | 0.21 (0.16–0.34) | 0.23 (0.17– 0.32) | 0.23 (0.17–0.33) | 1.098 (0.960, 1.256) | 0.963 (0.816, 1.136) |
| Time-to-peak diameter, s | 6 | 47.00 (32.80–56.30) | 47.70 (41.40–67.57) | 46.10 (39.17–66.88) | 1.046 (0.902, 1.213) | 0.938 (0.802, 1.097) |
| Shear rate, 1/s | 2 | 217.50 (149.50–302.50) | 226.00 (169.0–319.00) | 226.00 (176.00–341.00) | 1.098 (0.957, 1.260) | 0.963 (0.807, 1.150) |
| Shear rate AUC ttp, 1/s | 12 | 29576 (20765– 39319) | 29822(25371–49163) | 32934 (22960–44086) | 1.162 (0.996, 1.355) | 0.941 (0.786, 1.127) |
| NMD, % | 10 | 15.64 (12.97–18.64) | 14.22 (11.53–17.94) | 15.31 (10.77–18.74) | 0.977 (0.852, 1.121) | 1.006 (0.856, 1.181) |
| FMD/NMD ratio | 14 | 0.36 (0.26–0.55) | 0.31 (0.19– 0.45) | 0.35 (0.23–0.52) | 0.972 (0.769, 1.227) | 1.159 (0.914, 1.469) |
| Blood lipids | ||||||
| Total cholesterol, mmol/l | 3 | 5.01 (4.57–5.64) | 4.76 (4.21–5.33) | 5.30 (4.57–5.94) | 1.041 (1.005, 1.078) | 1.041 (1.001, 1.082) |
| LDL-C, mmol/l | 3 | 3.18 (2.84–3.73) | 3.00 (2.77–3.49) | 3.41 (2.91–4.06) | 1.089 (1.036, 1.144) | 1.082 (1.032, 1.135) |
| HDL-C, mmol/l | 3 | 1.57 (1.15–2.02) | 1.49 (1.15–1.65) | 1.48 (1.29–1.76) | 0.991 (0.949, 1.034) | 1.020 (0.969, 1.073) |
| Total cholesterol/HDL ratio | 3 | 3.42 (2.66–4.12) | 3.28 (2.75–4.28) | 3.61 (2.77–4.08) | 1.053 (1.001, 1.107) | 1.016 (0.962, 1.073) |
| LDL/HDL ratio | 3 | 2.25 (1.62–2.84) | 2.16 (1.69–2.72) | 2.40 (1.66–2.91) | 1.112 (1.044, 1.185) | 1.063 (0.999, 1.130) |
| Triglycerides, mmol/l | 3 | 0.80 (0.63–1.10) | 0.83 (0.66–1.33) | 0.93 (0.65–1.17) | 1.016 (0.881, 1.172) | 0.909 (0.791, 1.044) |
| Lp-a, mg/l | 3 | 0.15 (0.07–0.44) | 0.14 (0.07–0.26) | 0.14 (0.06–0.42) | 0.887 (0.725, 1.086) | 0.863 (0.672, 1.109) |
| Apo A1, g/l | 3 | 1.96 (1.52–2.63) | 1.87 (1.42–2.15) | 1.77 (1.30–2.31) | 0.939 (0.810, 1.088) | 0.897 (0.703, 1.144) |
| Apo B, g/l | 3 | 0.91 (0.52–1.35) | 0.87 (0.49–1.39) | 0.96 (0.62–1.40) | 1.031 (0.848, 1.252) | 0.899 (0.732, 1.105) |
| ox-LDL Ab, mU/ml | 5 | 404.00 (204.00–1194.00) | 702.00 (349.00–1624.00) | 669.00 (191.00–1451.00) | 1.123 (0.885, 1.427) | 0.887 (0.695, 1.132) |
| Biomarkers of inflammation | ||||||
| CRP hs, mg/l | 3 | 0.67 (0.38–1.53) | 0.72 (0.50–1.45) | 0.85 (0.38–1.20) | 1.139 (0.591, 2.195) | 1.585 (0.807, 3.111) |
| IL-6, pg/ml | 5 | 0.79 (0.46–1.05) | 0.64 (0.44–0.92) | 0.70 (0.46–0.96) | 0.987 (0.778, 1.251) | 1.038 (0.792, 1.361) |
| TNFα, pg/ml | 5 | 4.41 (2.47–6.79) | 4.62 (1.95–7.55) | 3.80 (2.60–5.84) | 0.998 (0.831, 1.199) | 0.991 (0.775, 1.268) |
| TNF-receptor, pg/ml | 5 | 1070.00 (701.00–1265.00) | 945.00 (724.00– 1160.00) | 865.00 (685.00– 1156.00) | 0.867 (0.754, 0.997) | 0.930 (0.817, 1.059) |
| TNF-receptor 2, pg/ml | 5 | 3530.00 (2729.00– 5415.00) | 4887.00 (3095.00– 654.00) | 4110.00 (2984.00–6076.00) | 1.190 (1.060, 1.336) | 0.984 (0.852, 1.135) |
| Biomarkers of coagulation | ||||||
| PAI-1, ng/ml | 3 | 30.70 (17.50–40.80) | 28.13 (20.30–45.10) | 29.90 (22.40–42.70) | 0.961 (0.817, 1.130) | 0.952 (0.799, 1.134) |
| Fibrinogen, g/l | 2 | 2.98 (2.56–3.50) | 2.59 (2.41–3.02) | 2.92 (2.55–3.32) | 1.012 (0.951, 1.076) | 1.093 (1.030, 1.160) |
| Biomarkers of endothelium | ||||||
| ELAM-1, ng/ml | 5 | 0.72 (0.10–3.26) | 0.16 (0.08–2.69) | 0.20 (0.13–2.19) | 0.929 (0.582, 1.484) | 0.715 (0.471, 1.086) |
| ICAM, ng/ml | 5 | 12.90 (10.60–20.50) | 11.40 (7.10–19.20) | 12.80 (8.70–16.40) | 0.985 (0.640, 1.516) | 1.002 (0.617, 1.626) |
| VCAM, ng/ml | 5 | 138.00 (96.00– 45.00) | 120.20 (72.90–183.60) | 164.80 (119.90–271.00) | 1.403 (0.893, 2.204) | 2.138 (1.207, 3.790) |
| Endothelin, pg/ml | 5 | 2.02 (1.56–2.76) | 2.17 (1.48–2.62) | 1.94 (1.60–2.84) | 1.155 (0.881, 1.516) | 0.899 (0.613, 1.320) |
| vWF/Rco, % | 2 | 100.00 (72.00–145.00) | 98.00 (83.00–132.00) | 100.00 (74.00–127.00) | 0.975 (0.895, 1.062) | 1.000 (0.927, 1.079) |
| vWF/Ag, % | 2 | 90.00 (72.00–137.00) | 105.00 (70.00–130.00) | 101.00 (71.00–132.00) | 1.030 (0.958, 1.108) | 0.993 (0.925, 1.067) |
| Variable | N missing | wTFA | iTFA | rTFA | rTFA over wTFA Ratio (95% CI) | rTFA over iTFA Ratio (95% CI) |
|---|---|---|---|---|---|---|
| Vascular function | ||||||
| BAD, baseline, mm | 2 | 3.96 (3.46–4.72) | 4.26 (3.69–4.90) | 3.96 (3.58–4.59) | 1.003 (0.984, 1.022) | 0.998 (0.973, 1.023) |
| BAD, maximum, mm | 5 | 4.13 (3.75–5.10) | 4.45 (3.84–5.05) | 4.18 (3.76–4.80) | 0.994 (0.974, 1.014) | 1.008 (0.990, 1.027) |
| Blood velocity, baseline, m/s | 2 | 0.21 (0.16–0.34) | 0.23 (0.17– 0.32) | 0.23 (0.17–0.33) | 1.098 (0.960, 1.256) | 0.963 (0.816, 1.136) |
| Time-to-peak diameter, s | 6 | 47.00 (32.80–56.30) | 47.70 (41.40–67.57) | 46.10 (39.17–66.88) | 1.046 (0.902, 1.213) | 0.938 (0.802, 1.097) |
| Shear rate, 1/s | 2 | 217.50 (149.50–302.50) | 226.00 (169.0–319.00) | 226.00 (176.00–341.00) | 1.098 (0.957, 1.260) | 0.963 (0.807, 1.150) |
| Shear rate AUC ttp, 1/s | 12 | 29576 (20765– 39319) | 29822(25371–49163) | 32934 (22960–44086) | 1.162 (0.996, 1.355) | 0.941 (0.786, 1.127) |
| NMD, % | 10 | 15.64 (12.97–18.64) | 14.22 (11.53–17.94) | 15.31 (10.77–18.74) | 0.977 (0.852, 1.121) | 1.006 (0.856, 1.181) |
| FMD/NMD ratio | 14 | 0.36 (0.26–0.55) | 0.31 (0.19– 0.45) | 0.35 (0.23–0.52) | 0.972 (0.769, 1.227) | 1.159 (0.914, 1.469) |
| Blood lipids | ||||||
| Total cholesterol, mmol/l | 3 | 5.01 (4.57–5.64) | 4.76 (4.21–5.33) | 5.30 (4.57–5.94) | 1.041 (1.005, 1.078) | 1.041 (1.001, 1.082) |
| LDL-C, mmol/l | 3 | 3.18 (2.84–3.73) | 3.00 (2.77–3.49) | 3.41 (2.91–4.06) | 1.089 (1.036, 1.144) | 1.082 (1.032, 1.135) |
| HDL-C, mmol/l | 3 | 1.57 (1.15–2.02) | 1.49 (1.15–1.65) | 1.48 (1.29–1.76) | 0.991 (0.949, 1.034) | 1.020 (0.969, 1.073) |
| Total cholesterol/HDL ratio | 3 | 3.42 (2.66–4.12) | 3.28 (2.75–4.28) | 3.61 (2.77–4.08) | 1.053 (1.001, 1.107) | 1.016 (0.962, 1.073) |
| LDL/HDL ratio | 3 | 2.25 (1.62–2.84) | 2.16 (1.69–2.72) | 2.40 (1.66–2.91) | 1.112 (1.044, 1.185) | 1.063 (0.999, 1.130) |
| Triglycerides, mmol/l | 3 | 0.80 (0.63–1.10) | 0.83 (0.66–1.33) | 0.93 (0.65–1.17) | 1.016 (0.881, 1.172) | 0.909 (0.791, 1.044) |
| Lp-a, mg/l | 3 | 0.15 (0.07–0.44) | 0.14 (0.07–0.26) | 0.14 (0.06–0.42) | 0.887 (0.725, 1.086) | 0.863 (0.672, 1.109) |
| Apo A1, g/l | 3 | 1.96 (1.52–2.63) | 1.87 (1.42–2.15) | 1.77 (1.30–2.31) | 0.939 (0.810, 1.088) | 0.897 (0.703, 1.144) |
| Apo B, g/l | 3 | 0.91 (0.52–1.35) | 0.87 (0.49–1.39) | 0.96 (0.62–1.40) | 1.031 (0.848, 1.252) | 0.899 (0.732, 1.105) |
| ox-LDL Ab, mU/ml | 5 | 404.00 (204.00–1194.00) | 702.00 (349.00–1624.00) | 669.00 (191.00–1451.00) | 1.123 (0.885, 1.427) | 0.887 (0.695, 1.132) |
| Biomarkers of inflammation | ||||||
| CRP hs, mg/l | 3 | 0.67 (0.38–1.53) | 0.72 (0.50–1.45) | 0.85 (0.38–1.20) | 1.139 (0.591, 2.195) | 1.585 (0.807, 3.111) |
| IL-6, pg/ml | 5 | 0.79 (0.46–1.05) | 0.64 (0.44–0.92) | 0.70 (0.46–0.96) | 0.987 (0.778, 1.251) | 1.038 (0.792, 1.361) |
| TNFα, pg/ml | 5 | 4.41 (2.47–6.79) | 4.62 (1.95–7.55) | 3.80 (2.60–5.84) | 0.998 (0.831, 1.199) | 0.991 (0.775, 1.268) |
| TNF-receptor, pg/ml | 5 | 1070.00 (701.00–1265.00) | 945.00 (724.00– 1160.00) | 865.00 (685.00– 1156.00) | 0.867 (0.754, 0.997) | 0.930 (0.817, 1.059) |
| TNF-receptor 2, pg/ml | 5 | 3530.00 (2729.00– 5415.00) | 4887.00 (3095.00– 654.00) | 4110.00 (2984.00–6076.00) | 1.190 (1.060, 1.336) | 0.984 (0.852, 1.135) |
| Biomarkers of coagulation | ||||||
| PAI-1, ng/ml | 3 | 30.70 (17.50–40.80) | 28.13 (20.30–45.10) | 29.90 (22.40–42.70) | 0.961 (0.817, 1.130) | 0.952 (0.799, 1.134) |
| Fibrinogen, g/l | 2 | 2.98 (2.56–3.50) | 2.59 (2.41–3.02) | 2.92 (2.55–3.32) | 1.012 (0.951, 1.076) | 1.093 (1.030, 1.160) |
| Biomarkers of endothelium | ||||||
| ELAM-1, ng/ml | 5 | 0.72 (0.10–3.26) | 0.16 (0.08–2.69) | 0.20 (0.13–2.19) | 0.929 (0.582, 1.484) | 0.715 (0.471, 1.086) |
| ICAM, ng/ml | 5 | 12.90 (10.60–20.50) | 11.40 (7.10–19.20) | 12.80 (8.70–16.40) | 0.985 (0.640, 1.516) | 1.002 (0.617, 1.626) |
| VCAM, ng/ml | 5 | 138.00 (96.00– 45.00) | 120.20 (72.90–183.60) | 164.80 (119.90–271.00) | 1.403 (0.893, 2.204) | 2.138 (1.207, 3.790) |
| Endothelin, pg/ml | 5 | 2.02 (1.56–2.76) | 2.17 (1.48–2.62) | 1.94 (1.60–2.84) | 1.155 (0.881, 1.516) | 0.899 (0.613, 1.320) |
| vWF/Rco, % | 2 | 100.00 (72.00–145.00) | 98.00 (83.00–132.00) | 100.00 (74.00–127.00) | 0.975 (0.895, 1.062) | 1.000 (0.927, 1.079) |
| vWF/Ag, % | 2 | 90.00 (72.00–137.00) | 105.00 (70.00–130.00) | 101.00 (71.00–132.00) | 1.030 (0.958, 1.108) | 0.993 (0.925, 1.067) |
Results show the ratio of estimated medians. Analysis was done on the per protocol subjects. Statistical comparisons adjusting for baseline measurements were done using Poisson regression with sandwich estimators due to the multitude of variables with strongly skewed distributions.
Apo A1: apolipoprotein A1; Apo B: apolipoprotein B; AUC ttp: area under the curve until time-to-peak vasodilation; BAD: brachial artery diameter; CI: confidence interval; CRP hs: high-sensitivity C-reactive protein; FMD: flow-mediated dilation; ELAM-1: endothelial leucocyte adhesion molecule; HDL-C: high-density lipoprotein cholesterol; ICAM: intercellular adhesion molecule; iTFA: diet enriched with industrial trans fatty acids; IL-6: interleukin-6; LDL-C: low-density lipoprotein cholesterol; Lp-a: lipoprotein a; NMD: nitroglycerin-mediated dilation; ox-LDL Ab: antibodies of oxidized low-density lipoprotein; PAI-1: plasminogen-activator inhibitor 1; rTFA: diet enriched with ruminant trans fatty acids; TNF: tumor necrosis factor; VCAM: vascular cellular adhesion molecule; vWF/Ag: von Willebrand antigen; vWF/Rco: von Willebrand factor ristocetin cofactor; wTFA: diet without trans fatty acids.
Summary and comparison of secondary outcomes among the treatment groups.
| Variable | N missing | wTFA | iTFA | rTFA | rTFA over wTFA Ratio (95% CI) | rTFA over iTFA Ratio (95% CI) |
|---|---|---|---|---|---|---|
| Vascular function | ||||||
| BAD, baseline, mm | 2 | 3.96 (3.46–4.72) | 4.26 (3.69–4.90) | 3.96 (3.58–4.59) | 1.003 (0.984, 1.022) | 0.998 (0.973, 1.023) |
| BAD, maximum, mm | 5 | 4.13 (3.75–5.10) | 4.45 (3.84–5.05) | 4.18 (3.76–4.80) | 0.994 (0.974, 1.014) | 1.008 (0.990, 1.027) |
| Blood velocity, baseline, m/s | 2 | 0.21 (0.16–0.34) | 0.23 (0.17– 0.32) | 0.23 (0.17–0.33) | 1.098 (0.960, 1.256) | 0.963 (0.816, 1.136) |
| Time-to-peak diameter, s | 6 | 47.00 (32.80–56.30) | 47.70 (41.40–67.57) | 46.10 (39.17–66.88) | 1.046 (0.902, 1.213) | 0.938 (0.802, 1.097) |
| Shear rate, 1/s | 2 | 217.50 (149.50–302.50) | 226.00 (169.0–319.00) | 226.00 (176.00–341.00) | 1.098 (0.957, 1.260) | 0.963 (0.807, 1.150) |
| Shear rate AUC ttp, 1/s | 12 | 29576 (20765– 39319) | 29822(25371–49163) | 32934 (22960–44086) | 1.162 (0.996, 1.355) | 0.941 (0.786, 1.127) |
| NMD, % | 10 | 15.64 (12.97–18.64) | 14.22 (11.53–17.94) | 15.31 (10.77–18.74) | 0.977 (0.852, 1.121) | 1.006 (0.856, 1.181) |
| FMD/NMD ratio | 14 | 0.36 (0.26–0.55) | 0.31 (0.19– 0.45) | 0.35 (0.23–0.52) | 0.972 (0.769, 1.227) | 1.159 (0.914, 1.469) |
| Blood lipids | ||||||
| Total cholesterol, mmol/l | 3 | 5.01 (4.57–5.64) | 4.76 (4.21–5.33) | 5.30 (4.57–5.94) | 1.041 (1.005, 1.078) | 1.041 (1.001, 1.082) |
| LDL-C, mmol/l | 3 | 3.18 (2.84–3.73) | 3.00 (2.77–3.49) | 3.41 (2.91–4.06) | 1.089 (1.036, 1.144) | 1.082 (1.032, 1.135) |
| HDL-C, mmol/l | 3 | 1.57 (1.15–2.02) | 1.49 (1.15–1.65) | 1.48 (1.29–1.76) | 0.991 (0.949, 1.034) | 1.020 (0.969, 1.073) |
| Total cholesterol/HDL ratio | 3 | 3.42 (2.66–4.12) | 3.28 (2.75–4.28) | 3.61 (2.77–4.08) | 1.053 (1.001, 1.107) | 1.016 (0.962, 1.073) |
| LDL/HDL ratio | 3 | 2.25 (1.62–2.84) | 2.16 (1.69–2.72) | 2.40 (1.66–2.91) | 1.112 (1.044, 1.185) | 1.063 (0.999, 1.130) |
| Triglycerides, mmol/l | 3 | 0.80 (0.63–1.10) | 0.83 (0.66–1.33) | 0.93 (0.65–1.17) | 1.016 (0.881, 1.172) | 0.909 (0.791, 1.044) |
| Lp-a, mg/l | 3 | 0.15 (0.07–0.44) | 0.14 (0.07–0.26) | 0.14 (0.06–0.42) | 0.887 (0.725, 1.086) | 0.863 (0.672, 1.109) |
| Apo A1, g/l | 3 | 1.96 (1.52–2.63) | 1.87 (1.42–2.15) | 1.77 (1.30–2.31) | 0.939 (0.810, 1.088) | 0.897 (0.703, 1.144) |
| Apo B, g/l | 3 | 0.91 (0.52–1.35) | 0.87 (0.49–1.39) | 0.96 (0.62–1.40) | 1.031 (0.848, 1.252) | 0.899 (0.732, 1.105) |
| ox-LDL Ab, mU/ml | 5 | 404.00 (204.00–1194.00) | 702.00 (349.00–1624.00) | 669.00 (191.00–1451.00) | 1.123 (0.885, 1.427) | 0.887 (0.695, 1.132) |
| Biomarkers of inflammation | ||||||
| CRP hs, mg/l | 3 | 0.67 (0.38–1.53) | 0.72 (0.50–1.45) | 0.85 (0.38–1.20) | 1.139 (0.591, 2.195) | 1.585 (0.807, 3.111) |
| IL-6, pg/ml | 5 | 0.79 (0.46–1.05) | 0.64 (0.44–0.92) | 0.70 (0.46–0.96) | 0.987 (0.778, 1.251) | 1.038 (0.792, 1.361) |
| TNFα, pg/ml | 5 | 4.41 (2.47–6.79) | 4.62 (1.95–7.55) | 3.80 (2.60–5.84) | 0.998 (0.831, 1.199) | 0.991 (0.775, 1.268) |
| TNF-receptor, pg/ml | 5 | 1070.00 (701.00–1265.00) | 945.00 (724.00– 1160.00) | 865.00 (685.00– 1156.00) | 0.867 (0.754, 0.997) | 0.930 (0.817, 1.059) |
| TNF-receptor 2, pg/ml | 5 | 3530.00 (2729.00– 5415.00) | 4887.00 (3095.00– 654.00) | 4110.00 (2984.00–6076.00) | 1.190 (1.060, 1.336) | 0.984 (0.852, 1.135) |
| Biomarkers of coagulation | ||||||
| PAI-1, ng/ml | 3 | 30.70 (17.50–40.80) | 28.13 (20.30–45.10) | 29.90 (22.40–42.70) | 0.961 (0.817, 1.130) | 0.952 (0.799, 1.134) |
| Fibrinogen, g/l | 2 | 2.98 (2.56–3.50) | 2.59 (2.41–3.02) | 2.92 (2.55–3.32) | 1.012 (0.951, 1.076) | 1.093 (1.030, 1.160) |
| Biomarkers of endothelium | ||||||
| ELAM-1, ng/ml | 5 | 0.72 (0.10–3.26) | 0.16 (0.08–2.69) | 0.20 (0.13–2.19) | 0.929 (0.582, 1.484) | 0.715 (0.471, 1.086) |
| ICAM, ng/ml | 5 | 12.90 (10.60–20.50) | 11.40 (7.10–19.20) | 12.80 (8.70–16.40) | 0.985 (0.640, 1.516) | 1.002 (0.617, 1.626) |
| VCAM, ng/ml | 5 | 138.00 (96.00– 45.00) | 120.20 (72.90–183.60) | 164.80 (119.90–271.00) | 1.403 (0.893, 2.204) | 2.138 (1.207, 3.790) |
| Endothelin, pg/ml | 5 | 2.02 (1.56–2.76) | 2.17 (1.48–2.62) | 1.94 (1.60–2.84) | 1.155 (0.881, 1.516) | 0.899 (0.613, 1.320) |
| vWF/Rco, % | 2 | 100.00 (72.00–145.00) | 98.00 (83.00–132.00) | 100.00 (74.00–127.00) | 0.975 (0.895, 1.062) | 1.000 (0.927, 1.079) |
| vWF/Ag, % | 2 | 90.00 (72.00–137.00) | 105.00 (70.00–130.00) | 101.00 (71.00–132.00) | 1.030 (0.958, 1.108) | 0.993 (0.925, 1.067) |
| Variable | N missing | wTFA | iTFA | rTFA | rTFA over wTFA Ratio (95% CI) | rTFA over iTFA Ratio (95% CI) |
|---|---|---|---|---|---|---|
| Vascular function | ||||||
| BAD, baseline, mm | 2 | 3.96 (3.46–4.72) | 4.26 (3.69–4.90) | 3.96 (3.58–4.59) | 1.003 (0.984, 1.022) | 0.998 (0.973, 1.023) |
| BAD, maximum, mm | 5 | 4.13 (3.75–5.10) | 4.45 (3.84–5.05) | 4.18 (3.76–4.80) | 0.994 (0.974, 1.014) | 1.008 (0.990, 1.027) |
| Blood velocity, baseline, m/s | 2 | 0.21 (0.16–0.34) | 0.23 (0.17– 0.32) | 0.23 (0.17–0.33) | 1.098 (0.960, 1.256) | 0.963 (0.816, 1.136) |
| Time-to-peak diameter, s | 6 | 47.00 (32.80–56.30) | 47.70 (41.40–67.57) | 46.10 (39.17–66.88) | 1.046 (0.902, 1.213) | 0.938 (0.802, 1.097) |
| Shear rate, 1/s | 2 | 217.50 (149.50–302.50) | 226.00 (169.0–319.00) | 226.00 (176.00–341.00) | 1.098 (0.957, 1.260) | 0.963 (0.807, 1.150) |
| Shear rate AUC ttp, 1/s | 12 | 29576 (20765– 39319) | 29822(25371–49163) | 32934 (22960–44086) | 1.162 (0.996, 1.355) | 0.941 (0.786, 1.127) |
| NMD, % | 10 | 15.64 (12.97–18.64) | 14.22 (11.53–17.94) | 15.31 (10.77–18.74) | 0.977 (0.852, 1.121) | 1.006 (0.856, 1.181) |
| FMD/NMD ratio | 14 | 0.36 (0.26–0.55) | 0.31 (0.19– 0.45) | 0.35 (0.23–0.52) | 0.972 (0.769, 1.227) | 1.159 (0.914, 1.469) |
| Blood lipids | ||||||
| Total cholesterol, mmol/l | 3 | 5.01 (4.57–5.64) | 4.76 (4.21–5.33) | 5.30 (4.57–5.94) | 1.041 (1.005, 1.078) | 1.041 (1.001, 1.082) |
| LDL-C, mmol/l | 3 | 3.18 (2.84–3.73) | 3.00 (2.77–3.49) | 3.41 (2.91–4.06) | 1.089 (1.036, 1.144) | 1.082 (1.032, 1.135) |
| HDL-C, mmol/l | 3 | 1.57 (1.15–2.02) | 1.49 (1.15–1.65) | 1.48 (1.29–1.76) | 0.991 (0.949, 1.034) | 1.020 (0.969, 1.073) |
| Total cholesterol/HDL ratio | 3 | 3.42 (2.66–4.12) | 3.28 (2.75–4.28) | 3.61 (2.77–4.08) | 1.053 (1.001, 1.107) | 1.016 (0.962, 1.073) |
| LDL/HDL ratio | 3 | 2.25 (1.62–2.84) | 2.16 (1.69–2.72) | 2.40 (1.66–2.91) | 1.112 (1.044, 1.185) | 1.063 (0.999, 1.130) |
| Triglycerides, mmol/l | 3 | 0.80 (0.63–1.10) | 0.83 (0.66–1.33) | 0.93 (0.65–1.17) | 1.016 (0.881, 1.172) | 0.909 (0.791, 1.044) |
| Lp-a, mg/l | 3 | 0.15 (0.07–0.44) | 0.14 (0.07–0.26) | 0.14 (0.06–0.42) | 0.887 (0.725, 1.086) | 0.863 (0.672, 1.109) |
| Apo A1, g/l | 3 | 1.96 (1.52–2.63) | 1.87 (1.42–2.15) | 1.77 (1.30–2.31) | 0.939 (0.810, 1.088) | 0.897 (0.703, 1.144) |
| Apo B, g/l | 3 | 0.91 (0.52–1.35) | 0.87 (0.49–1.39) | 0.96 (0.62–1.40) | 1.031 (0.848, 1.252) | 0.899 (0.732, 1.105) |
| ox-LDL Ab, mU/ml | 5 | 404.00 (204.00–1194.00) | 702.00 (349.00–1624.00) | 669.00 (191.00–1451.00) | 1.123 (0.885, 1.427) | 0.887 (0.695, 1.132) |
| Biomarkers of inflammation | ||||||
| CRP hs, mg/l | 3 | 0.67 (0.38–1.53) | 0.72 (0.50–1.45) | 0.85 (0.38–1.20) | 1.139 (0.591, 2.195) | 1.585 (0.807, 3.111) |
| IL-6, pg/ml | 5 | 0.79 (0.46–1.05) | 0.64 (0.44–0.92) | 0.70 (0.46–0.96) | 0.987 (0.778, 1.251) | 1.038 (0.792, 1.361) |
| TNFα, pg/ml | 5 | 4.41 (2.47–6.79) | 4.62 (1.95–7.55) | 3.80 (2.60–5.84) | 0.998 (0.831, 1.199) | 0.991 (0.775, 1.268) |
| TNF-receptor, pg/ml | 5 | 1070.00 (701.00–1265.00) | 945.00 (724.00– 1160.00) | 865.00 (685.00– 1156.00) | 0.867 (0.754, 0.997) | 0.930 (0.817, 1.059) |
| TNF-receptor 2, pg/ml | 5 | 3530.00 (2729.00– 5415.00) | 4887.00 (3095.00– 654.00) | 4110.00 (2984.00–6076.00) | 1.190 (1.060, 1.336) | 0.984 (0.852, 1.135) |
| Biomarkers of coagulation | ||||||
| PAI-1, ng/ml | 3 | 30.70 (17.50–40.80) | 28.13 (20.30–45.10) | 29.90 (22.40–42.70) | 0.961 (0.817, 1.130) | 0.952 (0.799, 1.134) |
| Fibrinogen, g/l | 2 | 2.98 (2.56–3.50) | 2.59 (2.41–3.02) | 2.92 (2.55–3.32) | 1.012 (0.951, 1.076) | 1.093 (1.030, 1.160) |
| Biomarkers of endothelium | ||||||
| ELAM-1, ng/ml | 5 | 0.72 (0.10–3.26) | 0.16 (0.08–2.69) | 0.20 (0.13–2.19) | 0.929 (0.582, 1.484) | 0.715 (0.471, 1.086) |
| ICAM, ng/ml | 5 | 12.90 (10.60–20.50) | 11.40 (7.10–19.20) | 12.80 (8.70–16.40) | 0.985 (0.640, 1.516) | 1.002 (0.617, 1.626) |
| VCAM, ng/ml | 5 | 138.00 (96.00– 45.00) | 120.20 (72.90–183.60) | 164.80 (119.90–271.00) | 1.403 (0.893, 2.204) | 2.138 (1.207, 3.790) |
| Endothelin, pg/ml | 5 | 2.02 (1.56–2.76) | 2.17 (1.48–2.62) | 1.94 (1.60–2.84) | 1.155 (0.881, 1.516) | 0.899 (0.613, 1.320) |
| vWF/Rco, % | 2 | 100.00 (72.00–145.00) | 98.00 (83.00–132.00) | 100.00 (74.00–127.00) | 0.975 (0.895, 1.062) | 1.000 (0.927, 1.079) |
| vWF/Ag, % | 2 | 90.00 (72.00–137.00) | 105.00 (70.00–130.00) | 101.00 (71.00–132.00) | 1.030 (0.958, 1.108) | 0.993 (0.925, 1.067) |
Results show the ratio of estimated medians. Analysis was done on the per protocol subjects. Statistical comparisons adjusting for baseline measurements were done using Poisson regression with sandwich estimators due to the multitude of variables with strongly skewed distributions.
Apo A1: apolipoprotein A1; Apo B: apolipoprotein B; AUC ttp: area under the curve until time-to-peak vasodilation; BAD: brachial artery diameter; CI: confidence interval; CRP hs: high-sensitivity C-reactive protein; FMD: flow-mediated dilation; ELAM-1: endothelial leucocyte adhesion molecule; HDL-C: high-density lipoprotein cholesterol; ICAM: intercellular adhesion molecule; iTFA: diet enriched with industrial trans fatty acids; IL-6: interleukin-6; LDL-C: low-density lipoprotein cholesterol; Lp-a: lipoprotein a; NMD: nitroglycerin-mediated dilation; ox-LDL Ab: antibodies of oxidized low-density lipoprotein; PAI-1: plasminogen-activator inhibitor 1; rTFA: diet enriched with ruminant trans fatty acids; TNF: tumor necrosis factor; VCAM: vascular cellular adhesion molecule; vWF/Ag: von Willebrand antigen; vWF/Rco: von Willebrand factor ristocetin cofactor; wTFA: diet without trans fatty acids.
Discussion
In this prospective, randomized, double-blind parallel intervention study, conducted with healthy participants, neither alpine butter with rTFA nor margarine with iTFA at the amount of 2% of daily energy intake in the context of a balanced diet with 33–36% of daily energy from fat showed strong significant effects on important cardiovascular risk markers in healthy subjects. Furthermore, we could not demonstrate overall superiority of a diet with rTFA over a diet with iTFA.
In general, there is scarcity of data on the effects of short-term TFA intake on vascular endothelial function in humans. Two previous studies investigating the impact of a high TFA intake on vascular endothelial function in humans revealed inconsistent findings.13,14 In a randomized cross-over study, replacement of saturated fatty acids by iTFA at about 9% of daily energy intake significantly impaired brachial artery FMD after four weeks.13 Another study, by Dyerberg et al.,14 found no effect of TFA intake from partially hydrogenated soy oil (6% of daily energy intake) on brachial artery FMD in healthy subjects after eight weeks. Studies suggest that polyunsaturated fatty acids may improve brachial artery FMD.19,20 However, although there are some differences in the content of unsaturated fatty acids between alpine butter and margarine, this does not explain the improvement of FMD which we found after the wTFA diet compared with the iTFA diet since both margarines only marginally differ in fatty acid composition apart from TFA (Table S1 online). Looking at individual FMD changes, this result is most probably due to some large individual FMD responses (Figure 2).
In our study, the rTFA diet resulted in a 4% greater increase in total cholesterol and an 8–9% greater increase in LDL-cholesterol compared with the other two diets. The fact that in this modestly sized group of people one group has raised cholesterol and LDL-cholesterol is of importance as cholesterol is a close marker of the biological mechanism for inducing cardiovascular disease. However, it cannot be ruled out that the slightly higher content of saturated fatty acids in the rTFA diet (48%) compared with the iTFA and wTFA diet (both 42%) in combination with the somewhat lower content of mono- and polyunsaturated fatty acids in the rTFA diet (Table S1 online) may have been responsible for the difference in total and LDL-cholesterol with the rTFA diet.21 However, the daily supplementation of 15–25 g rapeseed oil (high in mono- and polyunsaturated fatty acids) relativizes the fatty acid difference between the three diets. The result corresponds to previous reports of potential adverse effects of rTFA on serum lipid levels, although they were predominantly found at high intake levels.22,23 While we found no short-term changes in apolipoprotein A1, apolipoprotein B, lipoprotein a and antibodies of oxidized LDL with any of the three diets we cannot speculate on potential long-term effects.
Potential deleterious effects of TFA on the vascular endothelium would probably be mediated by an increase in serum lipid levels and a concomitant increase in pro-inflammatory markers.2 In our study, we found no consistent increase in inflammatory and coagulatory markers or biomarkers of endothelial function. The reason for this may have been a lower dosage of TFA compared with other studies which have used TFA intake levels as high as 7–10% of daily energy intake,25–27 while no effects were observed at lower TFA intake levels.23,28–30 The absence of inflammation may explain our findings with regard to brachial artery FMD.
The TFA dosage in our study was chosen to closely reflect the TFA amounts consumed by the vast majority of the European population. In the TRANSFAIR study,30 the estimated TFA intake for subjects aged 50–65 years was about 1% of total energy intake (range 0.1–5.7%). Because iTFA levels in foods have decreased among European countries in recent years31 while TFA concentrations in ruminant foods have remained the same, we can assume that nowadays total dietary TFA intake is even lower and that rTFA constitute the largest part of TFA intake in most industrialized countries. Therefore, an intake of 2 energy% of rTFA as applied in this study was likely above the upper limit of current human consumption of rTFA.
This study has limitations. We did not reach the estimated total sample size of N = 209 participants, which would have allowed additional non-inferiority and superiority testing. This was due to an exhaustive recruitment procedure using stringent inclusion criteria focusing on healthy participants in order to minimize the impact of confounding factors and in particular cardiovascular risk factors on brachial artery FMD (primary endpoint). The lack of strong effects of both diets with TFA from either industrial or ruminant source on FMD could be due to a lack of power. However, due to the fact that with either diet FMD changes have been minimal, this seems most unlikely. As we included only healthy participants, we cannot translate our results to subjects with important cardiovascular risk factors and patients with cardiovascular disease.
The overall nutrient compositions of the diets are not known, because, apart from the fat source, participants consumed individual diets according to their liking. However, since participants’ weight remained stable, energy intake was according to individual requirements. Additionally, if participants complied with dietary specifications, then fat intake ranged between 33% and 36% of daily energy intake and was almost exclusively covered by the study products and the provided rapeseed oil. Compliance with dietary instructions was verified in a subgroup of participants. Comparing concentrations of selected serum cholesteryl fatty acids at week 6 with week 2, the results indicate that participants complied with dietary instructions and consumed the assigned study products (Table S2 online). This study examines short-term effects of TFA on cardiovascular risk.
Although four weeks is a short intervention period, previous studies investigating the effect of diets with different fat sources have found significant effects on blood lipids and FMD in the same or even shorter periods of exposure.13,22,23,32 However, our results do not allow firm conclusions with regard to long-term effects. Moreover, we did not control the timing of FMD measurements for menstrual cycle. However, the majority of female participants (92%) were postmenopausal and a sensitivity analysis excluding the six premenopausal subjects had no significant effect in the statistical model. Nevertheless, there seemed to be a gender difference with a lesser effect of rTFA on FMD in men. Therefore, the possibility of a population effect on results should be considered. Differential gender effects of rTFA intake on blood lipid profiles have been reported previously and may stimulate further research.
In conclusion, in the context of a balanced diet, the intake of TFA at the amount of 2% of daily energy showed no significant effect on important cardiovascular risk markers in healthy subjects regardless of the TFA source. The increase in selected blood lipids was significantly greater after the alpine butter diet compared with the other two diets. However, there was no significant effect of alpine butter on inflammation, coagulation and adhesion molecules short-term.
Author contribution
AT, AS and HS designed the study. AT and TR were responsible for participant recruitment. AT, FD and TR collected all data for this study. TR performed blinded echocardiographic measurements and image analysis. MC performed the statistical analysis. AS, HS, PE, MC, MW and TR interpreted the data. AS, PE and TR wrote the first draft of this manuscript. All authors took part in critical revision of the manuscript.
Acknowledgements
We would like to kindly thank all volunteers for their time and effort dedicated to this study. This trial was registered with ClinicalTrials.gov (NCT00933322).
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Agroscope, Institute for Food Sciences, Bern, Switzerland; Federal Office for Agriculture, Bern, Switzerland; Swiss Heart Foundation, Bern, Switzerland; Johanna Dürmüller-Bol Foundation, Muri, Switzerland and Emmi Ltd, Lucerne, Switzerland. The sponsors of this study had no role in the study design, data collection and analysis, or decision to submit for publication.
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