Additive effects of lupin protein and phytic acid on aortic calcification in ApoE deficient mice

Abstract

Lupin proteins have repeatedly been shown to exhibit lipid lowering properties and reduce aortic calcification in atherosclerosis models. Despite many efforts on its identification, the component which is responsible for the observed effects is still under debate. Phytic acid which is generally associated with lupin protein isolates has currently been described as bioactive plant compound. The objective of the study was to determine the role of associated phytic acid for the described lupin protein effects. A two-factorial study with ApoE knockout mice was conducted in which mice received lupin protein isolate or casein with or without phytase. Phytic acid was added to the casein diets to a final concentration identical to the lupin protein diets. Here we show that the serum concentrations of cholesterol, lathosterol and desmosterol were lower and the faecal bile acid excretion was higher in the groups fed lupin proteins than in the groups fed casein (p < 0.05). Mice that received the lupin protein diet containing phytic acid were characterized by a lower aortic calcification than mice of the other three groups (p < 0.05). In conclusion, our results show that the cholesterol lowering properties of lupin protein isolate were not caused by phytic acid. However, the hypocalcific action of lupin proteins appears to depend on the combination of lupin proteins and phytic acid.

Keywords: 2WA, two-way ANOVA; ApoE KO mice; ApoE, apolipoprotein E; Atherosclerosis; C, casein; CD68, cluster of differentiation 68; Faecal sterols; KO, knockout; L, lupin protein isolate; Lupin proteins; PA, phytic acid; Phytic acid; Plasma cholesterol.

Figure 1

Effects of dietary protein and phytic acid on serum sterols and faecal bile acid contents. The serum samples were analyzed for cholesterol (A), lathosterol (B) and desmosterol (C) concentrations and also the faecal bile acid content (D) was analyzed. Quantification was performed as described in the materials and methods section. Values are means ± SEM of 12 mice per group for serum parameters and 6 faeces samples per group for bile acid content. 2WA = two-way ANOVA, *p < 0.05.

Figure 2

The impact of proteins and phytic acid on aortic calcification. The relative calcified area (A) and the number of spots per section (B) were analyzed in aortic root sections after von-Kossa silver staining. (C) Representative pictures of von-Kossa stained aortic root section of each group. The black staining shows aortic calcification. Values are means ± SEM of 12 mice per group. 2WA = two-way ANOVA, **p < 0.001, *p < 0.05.

Figure 3

Effects of dietary protein and phytic acid on lipid content (A), plaque area (B) and CD68 expression, which is a marker protein of macrophages (C). Quantification was performed as described in the materials and methods section. Values are means ± SEM of 12 mice per group for all parameters. 2WA = two-way ANOVA, *p < 0.05.

Figure 4

The impact of proteins and phytic acid on lumen size and necrotic area. The relative lumen size area (A) and the necrotic area (B) were analyzed in aortic root sections after Movat staining. (C) Representative pictures of Movat stained aortic root section of each group. The black staining shows aortic calcification. Values are means ± SEM of 12 mice per group. 2WA = two-way ANOVA, *p < 0.05.