Postprandial lipid and vascular responses following consumption of a commercially-relevant interesterified palmitic acid-rich spread in comparison to functionally-equivalent non-interesterified spread and spreadable butter: a randomised controlled trial in healthy adults

Background: Interesterification is an industrial processing technique used widely where hard fats are essential for functionality and consumer acceptability, e.g. margarines and lower fat spreads. Objective: The aim of this study was to compare acute cardiovascular effects of functionally equivalent spreads (similar solid fat content) made with interesterified (IE) or non-IE palm-based fats, or spreadable butter. Methods: A randomised, controlled, 4-armed crossover, double-blind study (25 men, 25 women; 35–75 years; healthy; mean BMI 24.5, SD 3.8), compared effects of mixed nutrient meals containing 50 g fat from functionally equivalent products [IE spread, non-IE spread and spreadable butter (SB), with rapeseed oil (RO) as a reference treatment: with 16.7%, 27.9%, 19.3% and 4% palmitic acid, respectively] on 8 h postprandial changes in plasma triacylglycerol (TAG) and endothelial dysfunction (flow-mediated dilatation; FMD). Circulating reactive oxygen species (estimated using a neutrophil oxidative burst assay), glucose, insulin, NEFA, lipoprotein particle profiles, inflammatory markers (glycoprotein acetylation (Glyc-A) and IL-6), and biomarkers of endotoxemia were measured. Results: Postprandial plasma TAG concentrations after test meals were similar. However following RO versus the 3 spreads, there were significantly higher postprandial apolipoprotein B concentrations, and small HDL and LDL particle concentrations, and lower postprandial extra-large, large, and medium HDL particle concentrations, as well as smaller average HDL and LDL particle sizes. There were no differences following IE compared to the other spreads. Postprandial FMD% did not decrease after high-fat test meals, and there were no differences between treatments. Postprandial serum IL-6 increased similarly after test meals, but RO provoked a greater increase in postprandial concentrations of glycoprotein acetyls (GlycA), as well as 8 h sCD14, an endotoxemia marker. All other postprandial outcomes were not different between treatments. Conclusions: In healthy adults, a commercially-available IE-based spread did not evoke a different postprandial triacylglycerol, lipoprotein subclass, oxidative stress, inflammatory or endotoxemic response to functionally-equivalent, but compositionally-distinct alternative spreads. Clinical trial registry number: NCT03438084 (https://ClinicalTrials.gov).

Electronic Supplementary Material (ESI) for Food & Function.This journal is © The Royal Society of Chemistry 2024 and spreadable butter (SB), with a reference rapeseed oil (RO).Supplementary Figure 3. Lipolysis rates of emulsions prepared with IE spread, a non-IE spread, a spreadable butter (SB) and rapeseed oil (RO).

Supplementary method for analysis of neutrophil NADPH oxidase activity
Neutrophil NADPH oxidase activity was measured by the neutrophil oxidative burst assay and flow cytometry.Full details are in Supplementary material.All reagents were purchased from Sigma-Aldrich (Gillingham, UK) unless stated otherwise.Dihydrorhodamine123 (DHR123) was dissolved in dimethyl sulfoxide (DMSO) (2.5 mg/mL).Phorbol 12-myristate 13-acetate (PMA) was diluted in DMSO (100 µg/mL).
These were then further diluted with phosphate buffered saline with azide (PBA) (with 2.5 % bovine serum albumin and 0.2 % sodium azide) yielding the final concentration of the working dilution of DHR123 at 15 µg/mL and PMA at 300 ng/mL.Stock ammonium chloride solution (Pharm Lyse, BD Biosciences) was diluted 1 in 10 in deionised water to give a working solution.Cells were stained with anti-human CD45 Krome Orange antibody (BioLedgend, London, UK) and fixed in 1% v/v formaldehyde in PBA.
Heparinized peripheral venous blood samples were obtained at 0, 4, and 6 h and tested immediately after the time of collection.Three fluorescence-activated cell sorting (FACS) tubes were labelled as (1) blood only, (2) DHR resting, and (3) PMA stimulated.To all the tubes, 100 µL of blood was diluted 1:10 with PBA.Immediately, 25 µL of DHR123 was added to tube 2 and 3 and all tubes were incubated in a warm-water bath (37°C).After 15 minutes, 100 µL of PMA solution was added to tube 3 to stimulate neutrophils to produce superoxide and undergo the oxidative burst.All tubes were incubated at 37°C for another 15 minutes during which time DHR123 is thereby oxidised to rhodamine (RHO).This releases a green fluorescent signal at 585 nm when excited by a 488 nm laser which can be measured by flow cytometry.Following the incubation, the samples were centrifuged and washed.The samples were then stained with 5 µL of anti-human CD45 antibody and lysed with ammonium chloride for 15 minutes in the dark.Finally, the samples were centrifuged and washed again, and fixed in 1% formalin, before analysis of reagent 'blood only', 'DHR resting', and 'PMA stimulated' FACS tubes on a CytoFLEX Flow Cytometer (Beckman Coulter, Miami, FL), recording 10 000 neutrophil events.Data were subsequently analysed using CytExpert Software (Beckman Coulter, Miami, FL) Supplementary Table 1 Incremental area under the curve (0-8 h) for postprandial serum lipoprotein subclass particle concentrations and lipoprotein size following an interesterified (IE) spread, non-IE spread, and spreadable butter (SB), with a reference rapeseed oil (RO).Post hoc Bonferroni-adjusted pairwise comparisons were not statistically significant between treatments for L-LDL particle concentrations.S-LDL particle concentrations were significantly higher postprandially following RO compared with non-IE and SB (post hoc Bonferroni-adjusted pairwise comparison P <0.005 and <0.05 respectively).Average LDL particle size was significantly smaller following RO compared with IE, non-IE and SB (post hoc Bonferroniadjusted pairwise comparison P <0.001), and following SB compared with non-IE (post hoc Bonferroni-adjusted pairwise comparison P <0.05).
. The typical location of the neutrophil population was identified by the scattergram of CD45 expression vs. side scatter and selected by gating.Gating of the neutrophils in this CD45 positive region excludes debris and other cells present in the samples.A histogram of rhodamine fluorescence was obtained for the cells in the gated region of the flow scatterplot for each of the samples.Fluorescence is quantified by mean peak channel fluorescence; a weak level or no fluorescence at all indicates a reduced level of ROS.The NADPH oxidase activity is expressed as an oxidative index of neutrophils which is the ratio of mean fluorescence of PMA stimulated to mean fluorescence of DHR resting sample.

h Treatment effect P value Time 1 P value Treatment x Time P value ApoB, mg/mL 2 RO
<0.05).Average HDL particle size was significantly smaller following RO compared with IE, non-IE and SB (post hoc Bonferroni-adjusted pairwise comparison P <0.001)

Emulsion droplet size distribution of emulsions prepared with IE spread, a non-IE spread, a spreadable butter (SB) and rapeseed oil (RO).
). -fraction palm oil, palm kernel oil, and rapeseed oil; and the other a spreadable butter made with butter and rapeseed oil) relative to a reference rapeseed oil.Data are means with standard errors.Comparison of test fats by linear mixed-model analysis (dependent variable postprandial values, fixed factors of treatment, time, period, sex, treatment × time interaction, treatment × period interaction; treatment × sex; random effect participant; covariate baseline) showed no significant treatment or time effects.There was a significant sex effect (P =0.031) for sCD14, but no treatment × sex interactions; there was a tendency for sCD14 to decrease postprandially in males and increase in females.Females: IE n=19, non-IE n=19, SB n=20, RO n=16.Males: IE n=21, non-IE n=19, SB n=18, RO n=17.