CVD and CVD-risk markers

In 2017, CVD were the leading cause of diet-related deaths globally⁽⁸⁾. For many years the ‘Diet-heart hypothesis’ has associated SFA consumption with increased LDL cholesterol leading to CVD and CHD mortality^{(Reference Antoni and Griffin9,Reference Dubroff and De Lorgeril10)} . Many have investigated the association between nutrients in dairy, such as SFA and calcium, and mortality from CVD in meta-analyses of observational studies, and continue to do so^{(Reference Mazidi, Mikhailidis and Sattar11,Reference Naghshi, Naemi and Sadeghi12)} . Nevertheless, newer research which takes into account the food source of the nutrient has questioned this association because not all dairy products increase LDL-cholesterol^{(Reference Hjerpsted, Leedo and Tholstrup13,Reference Feeney, Barron and Dible14)} . Thus, a potential causal link between SFA intake and CVD/CHD mortality may be weak. A recent review by Givens^{(Reference Givens15)} provides an overview of these investigations including the food source as an important factor. Here, associations between dairy intake and the incidence or mortality of all-causes/CVD/CHD/stroke or intermediate risk-markers such as cholesterols, TAG and blood pressure are presented^{(Reference Givens15)}. Dairy intake has been investigated as total dairy intake^{(Reference Schwingshackl, Hoffmann and Iqbal7,Reference Elwood, Pickering and Givens16–Reference Godos, Tieri and Ghelfi19)} and specific dairy products such as butter^{(Reference Godos, Tieri and Ghelfi19)}, milk^{(Reference Elwood, Pickering and Givens16,Reference Soedamah-Muthu, Ding and Al-Delaimy17,Reference Godos, Tieri and Ghelfi19)} , cheese^{(Reference Godos, Tieri and Ghelfi19)}, yoghurt^{(Reference Godos, Tieri and Ghelfi19)} or in categories related to fat content^{(Reference Soedamah-Muthu, Ding and Al-Delaimy17)} and fermentation^{(Reference Companys, Pla-Pagà and Calderen-Pérez20)}. Overall, these observational data, especially those using more recent statistical approaches, provide no consistent evidence of negative health associations between consumption of dairy and hard endpoint or intermediate risk markers related to CVD, despite the majority of dairy products having a high content of SFA and these finding therefore challenge the ‘Diet-heart hypothesis’. The results from these observational studies have been reviewed, discussed and summarised by many^{(Reference Poppitt2,Reference Yu and Hu21,Reference Hirahatake, Astrup and Hill22)} especially because the results are not reflected in all (national) dietary guidelines. When these results are interpreted, it has to be remembered that foods vary in the type of SFA^{(Reference Li, Lei and Jiang23)} and that the replacement nutrient or food also has an impact on the outcome, an impact which is seldom neutral^{(Reference Astrup, Bertram and Bonjour24–Reference Mente, Dehghan and Rangarajan26)}.

The LDL-cholesterol concentration has been used as a risk markers for prediction of disease and mortality for many year. Today, the use of LDL-cholesterol as a CVD-risk marker is questioned and other risk markers (e.g. non- high density lipoprotein (HDL) cholesterol and Apo (Apo) B) are discussed^{(Reference Aggarwal, Kathariya and Verma27)}. Furthermore, recent results from the prospective PURE study^{(Reference Mente, Dehghan and Rangarajan26)} is of interest. Based on fasting blood samples they observed that the ratio between ApoB and ApoA1 was a better predictor of CVD risk than LDL-cholesterol alone, probably because this ratio reflects presence of small dense LDL particles, which seem to be more atherogenic than large LDL particles^{(Reference Mente, Dehghan and Rangarajan26)}. However, this newly suggested risk marker needs to be validated by others. Another relevant discussion is the use of fasting v. postprandial measurement^{(Reference Kolovou and Ooi28,Reference Bansal, Buring and Rifai29)} , which will be raised later.

The causal link between dietary consumption and outcomes can only be confirmed by randomised controlled trials (RCTs). However, the disadvantage of RCTs is the duration, which is seldom long enough to study hard endpoints. Therefore, intermediate markers (e.g. TAG and cholesterol) have been used instead. Like other studies, RCTs have often focused more on individual nutrients and less on whole foods. However, more than 10 years ago Hjerpsted et al.^{(Reference Hjerpsted, Leedo and Tholstrup13)} suggested that the 6 weeks' consumption of cheese had beneficial effects on total and LDL-cholesterol compared to butter. In 2017, a working group discussed the individual nutrient v. whole food research approach^{(Reference Thorning, Bertram and Bonjour30)} and today, the whole food approach has gained attention because there may be (dairy) matrix effects which are lost when focus is only on individual nutrients. This thought, that health effects cannot be predicted from the nutritional content itself, is gaining greater acceptance – also because humans consume foods and drinks – not individual nutrients^{(Reference Poppitt2,Reference Weaver4)} . Subsequently, Raziani et al.^{(Reference Raziani, Tholstrup and Kristensen31)} investigated if consumption of a regular fat cheese compared to a reduced fat cheese affected the lipid profile, and it was found that it did not. Recently, Feeney et al.^{(Reference Feeney, Barron and Dible14)} confirmed that 6 weeks' consumption of fat from a cheese matrix compared to butter had beneficial effects on cholesterol levels and that these effects were not explained by differences in calcium and protein content of the diet, highlighting that the food (matrix) rather than the individual nutrients are important to consider when health outcomes are evaluated.

For production of new food products with beneficial health effects, it is important to consider any healthy and less healthy properties of the food matrix. These properties may arise from interactions between nutrients within the food matrix and have the potential to alter the bioactive properties and affect nutrient absorption^{(Reference Michalski, Genot and Gayet32)}. However, the key problem is that investigating effects of the matrix per se is highly complicated because for most products varying the food matrix will result in differences in nutritional content. Thus, using existing (dairy) products on the market to study matrix effect per see is more or less impossible, because none of these have the exact same nutritional content. In case existing (dairy) products on the marked is used, the full diet to be studied needs to be supplemented with lacking nutrients and this approach is more reflecting effects of nutrients being inside or outside a food matrix, rather than the effect of the matrix per se. Another issue is that of study matrix effects in RCTs over a longer period because strictly controlled conditions are needed to exclude effects from variations in overall nutrient content of diets. Thus, the best suited study for the effect of the matrix per see may be a strictly controlled (i.e. short-term) trial where all food is provided and consumed within a laboratory-setting.

Postprandial effects of dairy products

Effects of strictly controlled diets/foods are most often assessed in short-term settings such as a meal test conducted over some hours. After consumption of a meal, the biological markers related to fat intake that change are TAG and NEFA, whereas cholesterol concentrations are relatively constant^{(Reference Kolovou and Ooi28)}, even when a very energy-rich meal with high fat content is consumed^{(Reference Kjølbæk, Schmidt and Rouy33)}. Tholstrup and colleagues have conducted several postprandial meal tests with dairy products^{(Reference Tholstrup, Høy and Andersen34,Reference Kjølbaek, Raziani and Tholstrup35)} . Likewise, Hansson et al.^{(Reference Hansson, Holven and Øyri36)} conducted a meal study with dairy products (Table 1). Common for these three studies^{(Reference Tholstrup, Høy and Andersen34–Reference Hansson, Holven and Øyri36)} was that the test meals were not matched for either fat, energy or calcium content. Tholstrup et al.^{(Reference Tholstrup, Høy and Andersen34)} compared effects of butter, cheese and whole milk and except a little higher HDL₂ concentration at 8 h after intake of cheese compared to butter and whole milk, no time-meal interactions were observed for total and LDL-cholesterol, NEFA and TAG. Although they adjusted for lactose and milk protein content, large differences (1769–1979 mg/10 MJ) in calcium content between the test meals were observed. In another trial by the Tholstrup group^{(Reference Kjølbaek, Raziani and Tholstrup35)}, regular and reduced fat cheeses were compared. Here, the regular fat cheese increased TAG compared to the reduced fat cheese, but these meals were not iso-energetic and not matched on fat intake. Hansson et al.^{(Reference Hansson, Holven and Øyri36)} compared butter, cheese, whipped cream and sour cream and observed that sour cream increased the TAG incremental area under the curve (iAUC) compared to all other products and increased HDL iAUC compared to cheese. No effects on NEFA, total and LDL cholesterols were observed. In this study, the meals were matched for fat content, but they were not iso-energetic (e.g. the cheese meal provided 255 kJ and 18⋅8 g protein more than the sour cream meal).

To study the effect of the food matrix it is important that all intervention products/meals are highly similar in their nutritional content; otherwise, the observed effect(s) might not be exclusively caused by the food matrix and the potential impact of the nutrients on the results should be carefully considered. Few RCTs aiming to investigate postprandial effects of the dairy matrix have been conducted. These studies have not been able to completely match the nutritional content in their dairy products, however the studies had minor adjustments and therefore served highly similar meals. The minor adjustments to bread included in the test meal were addition of sodium caseinate in one study^{(Reference Drouin-Chartier, Tremblay and Maltais-Giguère37)}, whereas we added salt, lactose and/or potato starch to some of the breads in our own study^{(Reference Kjølbæk, Schmidt and Rouy33)}. In the study by Drouin-Chartier et al.^{(Reference Drouin-Chartier, Tremblay and Maltais-Giguère37)}, the effects of cream cheese, cheddar cheese and butter were investigated. Effects on TAG, ApoB48 and NEFA were observed. Cream cheese increased the 2 h TAG concentration compared to all other products, but it also decreased the 6 h TAG concentration compared to cheese resulting in no difference in (i)AUC. The cream cheese reduced the ApoB48 concentration at 4 h and 6 h compared to cheddar cheese resulting in a lower ApoB48 iAUC compered to cheddar. For NEFA, cream cheese lowered the 2 h concentration compared to cheddar cheese. The biggest nutritional difference between the dairy products was the protein and calcium contents (highest in the cheddar cheese and lowest in the butter). The protein difference was adjusted in the bread served as part of the meal whereas the calcium content varied from 74 to 141 mg/MJ serving. These results did not indicate any dose-response effect related to protein and calcium contents of the dairy products, but to a large extent the effects seemed to be explained by the dairy matrix. To address these issues the authors and their collaborators developed dairy products that were matched not only on fat content, but also on protein and calcium contents to avoid speculation on the effects of nutrients being in- or outside the matrix^{(Reference Kjølbæk, Schmidt and Rouy33,Reference Schmidt, Kjølbæk and Jensen38)} . The authors also designed a gelled dairy matrix^{(Reference Schmidt, Kjølbæk and Jensen38)}, however, this resulted in a different carbohydrate content that was adjusted by adding lactose and potato starch (and salt) to the bread served with the meals. In our study^{(Reference Kjølbæk, Schmidt and Rouy33)}, we observed that the gelled product (a micellar casein isolate (MCI) gel) increased the TAG concentration resulting in a higher iAUC compared to cheddar cheese and a homogenised cheddar cheese, without any differences in ApoB48 concentration. For NEFA, both the commercial and the homogenised cheddar lowered NEFA concentrations at 1, 1⋅5 and 2 h, but it did not increase incremental area over the curve (iAOC) compared to other products.

Based on these highly controlled and nutritionally similar test meals there are very good indications of dairy matrix effects. The disadvantage with the postprandial studies is the translation of the results into longer-term effects as few postprandial markers are accepted and used in the diagnoses/assessment of cardiometabolic health. However, it is debated^{(Reference Kolovou and Ooi28,Reference Bansal, Buring and Rifai29)} if postprandial markers are more predictive of health and disease risk than the usual and most commonly used fasting values in the majority of both observational and long-term experimental studies. This warrants further investigations as well as inclusion and validation of the newer proposed risk markers from the PURE study mentioned earlier.

Proposed mechanism – matrix effects

To summarise, the acute postprandial lipid responses after consumption of dairy products showed that cheese and butter could not be described as a healthy or less healthy choice, respectively, as could have been expected from fasting lipid profile after 6 weeks intake in RCTs. In postprandial studies, the dairy products that differentiated from others were products with a semi-solid structure, i.e. sour cream, cream cheese and a gelled product, which all increased the postprandial TAG response. Potential mechanisms are discussed below.

In the gastrointestinal tract, calcium can form insoluble calcium-soaps and precipitate bile acids, which reduce fat absorption and bile acids recycling and increase faecal excretion^{(Reference Kjølbæk, Lorenzen and Larsen39)}. From postprandial trials using iso-energetic meals^{(Reference Tholstrup, Høy and Andersen34,Reference Drouin-Chartier, Tremblay and Maltais-Giguère37)} one possible explanation of different lipid responses might be related to the content of calcium. In both cases, faecal fat excretion was not investigated and a possible difference on lipid concentrations between butter which has a low calcium content and hard cheese which has a high calcium content was not observed. Recently, Feeney et al.^{(Reference Feeney, Daly and Dunne40)} investigated the effect of calcium being inside and outside the cheese matrix i.e. total calcium intake was similar. Here, intake of cheese with high calcium content did not increase faecal fat excretion compared to intake of cheese with a reduced calcium content + calcium supplementation, but due to the very small population (n = 7 completers) a type 2 error cannot be excluded. This theory warrants further trials; however, we have previously suggested that other minerals and also dietary fibre may affect faecal fat excretion^{(Reference Kjølbæk, Lorenzen and Larsen39)}. Another speculation is that an effect of calcium might depend on the location of nutrients within the food matrix as the likelihood of interaction between nutrients (in this case fat and calcium) may depend on how close there are located especially during digestion in the gastrointestinal tract. In our postprandial study, we provided products and meals with highly similar calcium contents (dairy products range: 1327–1463 mg, total meal range: 1415–1524 mg) and thus any calcium-driven effect on the TAG concentration between the MCI gel and other products might be due to the placement of nutrients within the food matrix, rather than the calcium content.

Another proposed effect of the food matrix is related to digestion, where the theory is that increasing the complexity of the food structure increases the time needed for digestion (i.e. complexity slow down the digestion rate). This results in, either a slower digestion which can give a skewed absorption pattern, but not necessarily a smaller absorption of nutrients, or a slow digestion which can increase the faecal excretion of nutrients because the nutrients are not released from the food matrix at the time/place where they would have otherwise been absorbed. Drouin-Chartier et al.^{(Reference Drouin-Chartier, Tremblay and Maltais-Giguère37)} observed that a cream cheese skewed the TAG response compared to cheddar cheese and butter, but an effect on the total TAG response (i.e. AUC) was not found. They discussed their results in relation to the size of the lipid droplets and suggested that the small homogenised droplets in the soft semi-solid protein gel of the cream cheese was rapidly digested and absorbed. This rapid digestion may be explained by the larger surface that appear when droplets become smaller in diameter. A related structural part of the dairy matrix is the MFGM. The presence or absence of an intact MFGM or a re-structuring of it is particularly interesting in the discussion of the influence of the dairy matrix. Beals et al.^{(Reference Beals, Kamita and Sacchi41)} investigated the effect of presence/absence of the MFGM in participants with metabolic syndrome. Their dairy intervention products (smoothies) had very similar nutritional content i.e. less than 0⋅2 % variation in macronutrients across test products, thus increasing the likelihood that any observed effects were caused by the presence or absence of the MFGM. They found no effect on postprandial lipid response, however, effects on inflammation and insulin metabolism were observed (Table 1). In support of this, none of the postprandial trials comparing cheese and butter found differences in lipid response. It is worth noting that butter is a product where most of the MFGM is removed with the buttermilk when the cream is churned. Thus, effects on postprandial lipid responses of dairy products do not seem to be caused by the presence or absence of the MFGM. In our study, we homogenised the commercial cheddar cheese with water, which resulted in a semi-solid structure where the fat droplets were reduced and the structure and composition of the original MFGM was altered^{(Reference Schmidt, Kjølbæk and Jensen38)}. We did also not observe differences between the cheddar cheese and the homogenised cheddar cheese^{(Reference Kjølbæk, Schmidt and Rouy33)}. Thus, smaller fat droplets and alteration of the MFGM did not affect the postprandial lipid response in our study. However, it remains to be investigated in detail how processing of the intervention products used in these RCTs not only affect fat droplet size, but also how the subsequent alteration of the MFGM affects the fat droplet surface composition e.g. by reorganisation of casein which might affect digestion^{(Reference Lopez, Cauty and Guyomarc'h5)}. Thus, a very detailed characterisation of the investigated dairy products is needed.

Consumption of solid products slow down the rate of digestion and absorption in the gastrointestinal tract, compared to liquids. Theoretically, this might affect the lipid response and results from non-dairy trials investigating effects of the food matrix have also shown that a liquid product (drink) increased TAG concentrations compared to a nutritionally similar solid product (cookie)^{(Reference Dias, Zhu and Thompson42)}. However, liquid dairy products e.g. milk, which could be regarded as products with a fast digestion related to a fast gastric emptying, seem to behave differently. Tholstrup et al.^{(Reference Tholstrup, Høy and Andersen34)} discussed the potential precipitation of casein in the stomach based on their results and in a review by Mulet-Cabero et al.^{(Reference Mulet-Cabero, Mackie and Brodkorb43)} the matter of gastric digestion is discussed in details. Additionally, Sanggard et al.^{(Reference Sanggaard, Holst and Rehfeld44)} showed that fast gastric emptying after milk intake did not lead to a fast rise in TAG concentration compared to a semi-solid fermented milk product (A38). From in vitro studies on our intervention products^{(Reference Schmidt, Kjølbæk and Jensen38)}, we observed that the liquid MCI drink coagulated in the ‘stomach’ which increased the viscosity to the same level as our two semi-solid products. In our in vivo study^{(Reference Kjølbæk, Schmidt and Rouy33)}, there was no indications of faster gastric emptying causing faster digestion and absorption rate for the liquid product compared to semi-solid and solid products.