Effect of calcium on fatty acid digestibility and weight development in mice

Background: High intake of saturated fatty acids (SFA) has consistently been shown to raise low-density lipoprotein cholesterol, a known risk factor of cardiovascular disease (CVD). One of the most significant contributors of SFAs to the adult diet is cheese. While containing high amounts of SFAs, cheese itself does not seem to increase the risk of disease. The high calcium (Ca) content in cheese has been proposed to explain this paradox through limiting the uptake of long-chain SFAs by forming insoluble soaps. Also, other abilities of the cheese have been suggested to affect SFA uptake through a so-called matrix effect. Aim: We aimed to determine whether fatty acid (FA) digestibility, weight development and changes in body fat mass was affected by Ca and type of matrix (cheese versus butter) in mice. Furthermore, we wanted to investigate whether Ca and differences in food matrix affected intestinal health. Finally, we wanted to assess the accuracy of a nuclear magnetic resonance (NMR) instrument from Bruker on determining body fat in live mice. Methods: Male mice (C57BL/6JRj) were fed a high-fat (HF) diet with FAs either from cheese (n=20) or butter (n=30) and received varying Ca levels [low-Ca (2g/kg feed), medium-Ca (7g/kg feed) and high-Ca (20g/kg feed)] for 5 weeks. A control group received a low-fat chow diet with a standard Ca content of 5g/kg (n=10). Food consumption and weight were registered weekly. Furthermore, fat mass was measured weekly using a body composition analyser (Minispec LF50, Bruker) and faeces were collected every day during week 4. A method was developed to quantify the FA content in faeces and food which was used to calculate % digestibility = (FA consumed – FA excreted) * (FA consumed)-1 x 100. Upon termination of the animals, the mucosal tissues were harvested from the small intestines, and the expression of genes related to inflammation and intestinal barrier function were analysed using real-time polymerase chain reaction (RT-PCR). NMR fat mass measurements were compared to the weight of dissected large fat depots in the same mice to evaluate the accuracy of the instrument. Results: Our results show that Ca impacts FA digestibility in mice receiving HF diets, in a dose dependent manner. Mice on a high-Ca/HF diet had 2.5% lower uptake of Palmitic acid (16:0) and 6% lower uptake of Stearic acid (18:0) compared to mice fed a low-Ca/HF-based diet. Ca content also affected weight gain in the mice that received HF butter-based diets with a lower weight gain (-9%) in the high-Ca group compared to the low-Ca group. No effects of Ca were found on fat mass development. Furthermore, no matrix effect was observed on 16:0 and 18:0 digestibility, but there was a trend that the matrix affected weight development of the mice. Overall, no effects of Ca were seen on the expression of inflammation markers in the small intestine. However, the tight junction gene ZO-1 had the highest expression in HF fed mice with low Ca levels (butter-based HF diet) and medium Ca levels (cheese-based HF diet). In addition, the matrix was found to significantly influence the expression of ZO-1, with a higher expression being observed in the cheese-based diets. Finally, the NMR instrument emerged as a reliable and good predictor of body fat mass in mice. Conclusion: We found Ca to impact SFA digestibility, but to a lesser extent than shown in the literature. The reduction in FA digestibility cannot explain the difference in weight gain between the groups, which might be due different metabolic activity in the mice or an effect of the matrix. Expression of the tight junction gene ZO-1 was found to be influenced by Ca and matrix. Further research is needed to elucidate the interplay between Ca and fat, and how this affects disease development