Sex differences in postprandial responses to different dairy products on lipoprotein subclasses: a randomised controlled cross-over trial

Men have earlier first-time event of CHD and higher postprandial TAG response compared with women. The aim of this exploratory sub-study was to investigate if intake of meals with the same amount of fat from different dairy products affects postprandial lipoprotein subclasses differently in healthy women and men. A total of thirty-three women and fourteen men were recruited to a randomised controlled cross-over study with fourdairy meals consisting of butter,cheese, whippedcream orsourcream, correspondingto 45 g offat (approximately 60 energypercent). Blood samples were taken at 0, 2, 4 and 6 h postprandially. Lipoprotein subclasses were measured using NMR and analysed using a linear mixed model. Sex had a significant impact on the response in M-VLDL ( P = 0 · 04), S-LDL ( P = 0 · 05), XL-HDL ( P = 0 · 009) and L-HDL ( P = 0 · 001) particle concentration (P), with women having an overall smaller increase in M-VLDL-P, a larger decrease in S-LDL-P and a larger increase in XL- and L-HDL-P compared with men, independent of meal. Men showed a decrease in XS-VLDL-P compared with women after intake of sour cream ( P < 0 · 01). In men only, XS-VLDL-P decreased after intake of sour cream compared with all other meals ( v . butter: P = 0 · 001; v . cheese: P = 0 · 04; v . whippedcream: P = 0 · 006). Meals withthe sameamount of fat from differentdairyproducts induce differentpostprandial effects on lipoprotein subclass concentrations in men and women.

mortality (29,30) . For the first time, we recently demonstrated divergent postprandial TAG and HDL-C responses (measured as the 0-6 h incremental AUC (iAUC 0-6h )) between different dairy products in healthy adults (31) , with a borderline significant effect of sex on serum TAG-iAUC 0-6h . We have also shown that dietary fat quality affects the peak time of larger VLDL subclasses postprandially in healthy adults and adults with familial hypercholesterolemia (32) . How intake of different dairy products affects postprandial lipoprotein subclass concentrations in women and men has, to the best of our knowledge, not been reported. Thus, the aim of this sub-study was to investigate if men and women respond differently to high-fat dairy meals, with the same amount of fat from different dairy products, with respect to postprandial lipoprotein subclass concentrations. The primary outcomes for this exploratory sub-study were the total 6 h postprandial particle concentration responses of six VLDL subclasses and four HDL subclasses, and secondary outcomes were the corresponding particle concentration responses of intermediate-density lipoprotein (IDL) and LDL subclasses, all measured as the iAUC 0-6h .

Subjects
A total of forty-seven healthy subjects (thirty-three women and fourteen men) who accomplished at least one visit in a postprandial study at the University of Oslo between September 2016 and April 2017 were included in this exploratory sub-study. A total of twenty-one women and ten men completed all four study visits (Fig. 1). As abdominal obesity is associated with an elevated postprandial TAG response (33) , subjects between 18 and 70 years of age with BMI 18·5-25 kg/m 2 and waist circumference <80 cm for women and <94 cm for men, or BMI ≥25 kg/m 2 and waist Dairy products and lipoprotein subclasses 781 circumference ≥80 cm for women and ≥94 cm for men were recruited to the postprandial study. Complete inclusion and exclusion criteria have been described previously (31) .

Study design
A randomised controlled cross-over study was conducted with four high-fat dairy meals as intervention, as has been described earlier (31) .

Clinical measurements
Weight was measured by the Medical Body Composition Analyzer seca 515/514 (seca, software version 1.1). Blood pressure was measured three consecutive times in a sitting position in the subjects' non-dominant arm by a Dinamap Carescape v100 (GE Medical System) at a screening visit.

Blood sampling and lipoprotein subclass measurement
Serum was collected in silica gel tubes (Becton Dickinson Vacutainer Systems) and kept in room temperature for 30-60 min to ensure complete blood coagulation before 15 min centrifugation at 1500 g (Thermo Fischer Scientific). Serum samples were then stored in a refrigerator until being analysed. Standard blood biochemical measurements were performed at an accredited medical laboratory (Fürst Medical Laboratory) (31) . Plasma was collected in EDTA tubes (Becton Dickinson Vacutainer Systems) and kept on ice for less than 15 min before being centrifuged at 2000 g for 15 min at 4°C (Thermo Fischer Scientific). The samples were distributed into smaller tubes and frozen at −80°C for lipoprotein subclass analysis. Lipoprotein subclass profiling was achieved using a commercial proton NMR metabolomics platform (Nightingale Health Ltd) with the following classifications: extremely large (XXL) VLDL with particle diameters of at least 75 nm (including chylomicrons), five VLDL subclasses (very large (XL), large (L), medium (M), small (S) and very small (XS), with average particle diameters of 64·0, 53·6, 44·5, 36·8 and 31·3 nm, respectively), one IDL subclass with an average particle diameter of 28·6 nm, three LDL subclasses (L, M and S, with average particle diameters of 25·5, 23·0, and 18·7 nm, respectively) and four HDL subclasses (XL, L, M and S, with average particle diameters of 14·3, 12·1, 10·9 and 8·7 nm, respectively). Details about the NMR metabolomics platform have been described previously (34,35) .

Ethics
The study was approved by the Regional Committees for Medical and Health Research Ethics (2016/418/REK sør-øst B) and conducted according to the principles of the Declaration of Helsinki. Written informed consents were obtained from all subjects. The study was registered at www.clinicaltrials.gov as NCT02836106.

Statistics
The primary outcome of the original study was the serum TAG-iAUC 0-6h , and the sample size calculation has been described previously (31) . In this exploratory sub-study, differences in characteristics between women and men at baseline were analysed by the Mann-Whitney U test using IBM SPSS Statistics for Windows 24.0 (IBM Corp.). Baseline characteristics are presented as medians (25th-75th percentiles). Data from the postprandial measurements were analysed with a linear mixed model using Stata Special Edition 15.1 (StataCorp LLC). All subjects who completed at least one test day were included in the analysis. The response variable was the iAUC 0-6h , calculated from the different time points using the trapezoid method (36,37) , and the model included the variables meal, visit number, age, BMI, sex and a meal×sex interaction as fixed effects in addition to a random intercept at subject level. Response differences between meals were stratified by sex when the meal×sex interaction was significant. Non-significant meal×sex interactions were excluded from the model, and as the meal×sex interaction represents sex-specific meal differences, response differences between meals were calculated for the whole study group independent of sex in these cases. All results beyond baseline characteristics originate from this model. The significance level was set to α=0·05. To adjust for multiple testing when performing comparisons on each lipoprotein subclass, the Bonferroni correction was applied (i.e. all P values for differences between meals were multiplied by the number of meal comparisons, and all P values for meal response differences between sexes were multiplied by the number of meals). Only Bonferroni-corrected P values are presented in the text and the figures. Pairwise meal comparisons within each sex were performed by combining the appropriate regression coefficients from the linear mixed model. All data that were analysed by the linear mixed model are shown as mean values with standard errors in the figures.

Postprandial differences between sexes
The meal by sex interaction, representing sex-specific meal differences, was only statistically significant for XS-VLDL-P (P=0·008). Significant sex effects on other lipoprotein subclass responses were independent of meal. VLDL subclasses. After intake of each of the four test meals, men tended to have larger iAUC 0-6h particle concentrations for all VLDL subclasses, except XS-VLDL, compared with women. However, a significant sex effect, independent of meal, was only found for M-VLDL (P=0·04), whereas a borderline significant sex effect was found for XXL-VLDL (P=0·05) and S-VLDL (P=0·06) (Fig. 2, online Supplementary Table 1). In contrast, men showed a decrease in XS-VLDL-P, whereas women showed a small increase after intake of sour cream (P=0·01) (Fig. 2, online Supplementary Tables 1 and 2). HDL subclasses. Sex had a significant impact on the increase in XL-HDL-P (P=0·009) and L-HDL-P (P=0·001) with larger increases observed in women compared with men, independent of meal (Fig. 3, online Supplementary Table 1).
Intermediate-density lipoprotein and LDL subclasses. Sex had a significant impact on the decrease in S-LDL-P (P=0·05) with overall larger decreases observed in women compared with men, independent of meal ( Fig. 4, online Supplementary Table 1).

Discussion
The two major findings from the present study are that men and women have different postprandial lipoprotein subclass responses after intake of similar meals, and that different dairy products cause different responses within men and women, respectively. To the best of our knowledge, this is the first study to investigate sex differences in postprandial lipoprotein subclass responses to high-fat meals with different dairy products.
Regarding the postprandial iAUC 0-6h particle concentrations of the HDL subclasses, sex had a significant effect on the response in XL-HDL-P and L-HLD-P with a consistently larger response seen in women compared with men. Intake of sour cream induced the largest increase in XL-, L-and M-HDL-P, which is in accordance with our previous findings from the same study where sour cream induced the largest postprandial increase in HDL-C (31) . This could be due to sour cream being Dairy products and lipoprotein subclasses 785 a homogenised dairy product, which means that sour cream consists of more and smaller fat droplets that generate increased initial lipid digestion in the gastrointestinal tract (38,39) . This increase in lipolysis may potentially lead to escalated formation of nascent pre-β HDL particles in the enterocytes, which could partly explain the increase in HDL particles after intake of sour cream (40) . Interestingly, even though there was no significant meal by sex interaction for any of the HDL subclasses, the figures indicate that the strong increase in XL-, L-and M-HDL-P after intake of sour cream mainly occurred in the women. Holmes et al. found that the particle concentrations of non-fasting XL-, L-HDL and M-HDL were inversely associated with myocardial infarction in Chinese adults, whereas S-HDL-P had neutral effect on myocardial infarction but was positively associated with ischaemic stroke. Furthermore, the correlations between HDL subclasses and coronary artery calcification have been studied in both women and men with or without type 1 diabetes (41) , showing an inverse association between large HDL subclasses (corresponding to M-, L-and XL-HDL in the present study) and coronary artery calcification in both women and men without diabetes. Thus, it could be that intake of sour cream generates a more favourable postprandial HDL profile than butter, cheese and whipped cream, especially in healthy women. This provides one possible explanation for the neutral and sometimes positive epidemiological associations between intake of fermented dairy products and cardiovascular health. However, the potential health benefits of increased postprandial HDL particle concentrations remain to be investigated. Regarding the postprandial iAUC 0-6h particle concentrations of the VLDL subclasses, men showed an overall larger response in M-VLDL-P and a borderline larger response in XXL-VLDL-P and S-VLDL-P compared with women. These findings are in line with the observed borderline significant effect of sex on the serum TAG response in our previous publication (31) . Based on the response patterns in Fig. 2, the lack of significant sex effects could potentially be due to the lower number of men compared with women in the study. Intake of cheese induced the largest iAUC 0-6h for XXL-, XL-and L-VLDL-P, which is a deviating finding from our previous result showing a significantly larger TAG-iAUC 0-6h from intake of sour cream (31) . This could be explained by the different methods of measurements. Firstly, the NMR technology only measures the TAG content inside the lipoproteins, which gives a somewhat incomplete picture of the total postprandial TAG concentration, as the early postprandial phase is characterised by high lipoprotein lipase activity and, thus, a substantial amount of hydrolysed or partly hydrolysed TAG in the circulation, which are not captured with NMR. Secondly, the NMR technology has difficulties measuring very large and TAG-rich chylomicrons that can be present after a high-fat meal. It may therefore be that the sour cream generated more of these very large TAG-rich chylomicrons. This has been supported by Vors et al. who found that emulsified fat (similar to homogenised fat droplets) induced a larger increase in chylomicrons/apo-B48 and fatty acid spillover (42) . The most apparent deviation in this sub-study, though, may be the observed decrease in XS-VLDL-P in men, but not in women, induced by intake of sour cream. This lipoprotein has an average diameter close to the one defining IDL and is therefore more of a remnant particle than a TAG-rich particle, with an assumed atherogenic capacity to enter the arterial wall (43) . Non-fasting XS-VLDL-P has been linked to increased odds for myocardial infarction and ischaemic stroke in a nested case-control study (14) . In addition, findings from the JUPITER (Justification for the Use of Statins in Prevention) trial showed reduced residual risk for CVD when lowering the fasting concentration of small-sized VLDL particles (with diameters corresponding to XS-VLDL measured in the present study) in statin-treated subjects (15,16) . Interestingly, lowering the concentrations of medium and large-sized VLDL particles did not result in further risk reduction in that study. Applying these findings to our study indicates that sour cream may generate a more favourable postprandial VLDL profile than butter, cheese and whipped cream in men, potentially through a higher clearance rate of XS-VLDL particles.
Regarding the postprandial iAUC 0-6h particle concentrations of IDL and the LDL subclasses, men showed an overall larger response in S-LDL-P compared with women, which is one of the lipoprotein subclasses considered to be the most atherogenic (14,44) . Intake of sour cream induced significantly larger decreases in L-, M-and S-LDL-P compared with whipped cream. The LDL-cholesterol concentration is expected to temporarily decrease 5-10 % in the postprandial state due to the competition of lipoprotein lipase between intestinally derived chylomicrons and hepatic VLDL particles, where chylomicrons are the preferred lipoproteins (45)(46)(47) . As whipped cream induced overall smaller increases in the largest VLDL subclasses (and thus possibly less chylomicrons) compared with sour cream, this may possibly partly explain the smaller decrease in L-, M-and S-LDL-P after intake of whipped cream.
We found that men and women responded differently to the dairy meals for some lipoprotein subclasses, with women showing a pattern of less increased VLDL-P and more increased HDL-P compared with men. These findings have support in the literature (7,8,48,49) and one proposed mechanism is a higher lipoprotein lipase capacity in women, generating more lipolysis (7) . This could explain both the partly lower VLDL-P responses and the partly higher HDL-P responses in women, as increased lipolysis stimulates the production of HDL particles by releasing more surface components to be incorporated into new HDL (40) .
Our study has several strengths including a randomised controlled cross-over design with four dairy meals containing dairy products with different matrices. The meals were equal in fat content and not adjusted for differing nutrient contents since we were interested in the effect of the fat from dairy products as whole foods. This also means that we did not adjust the amount of fat based on body mass, and differences in responses due to variations in the amount of fat ingested can therefore be ruled out. The limitations of the study are what we cannot ensure absolute standardisation of the evening meal and amount of physical activity the day before each test day, even though the participants received guidelines and reminders before each visit. The male group had higher BMI than the female group; however, the median was within normal range for both groups, and BMI was adjusted for in the analysis. The Bonferroni method was applied to the meal and meal×sex comparisons to reduce the risk of false-positive findings. Since this was an exploratory sub-study, the number of parameters was not adjusted for.
In conclusion, the present study shows that intake of meals with the same amount of fat from different dairy products induces different postprandial effects on lipoprotein subclass concentrations, with sour cream potentially being the most healthy option. We also show that women and men respond differently to high-fat dairy meals in general, but also to certain dairy products in particularly, and that women seem to respond more beneficially to high-fat dairy meals than men.