BIOCAR - Bioavailability of Carotenoids – Impact of High Mineral Concentrations

This project is finished.

Background

Dietary consumption of carotenoids, especially in form of fruits and vegetables, has been associated with the prevention of several chronic diseases, including type-2 diabetes, cardiovascular diseases, and some types of cancer. While data with respect to carotenoid intake is frequently available, much less is known on factors impacting their bioavailability, including the release from the food matrix, changes during gastro-intestinal digestion, and uptake by the gastro-intestinal epithelium, among others. In general, carotenoid absorption is low (usually ca. 5-20%). Some dietary factors are known to alter carotenoid uptake and absorption, including the positive impact of dietary lipids, and the negative effects of dietary fibre. A factor that has so far been neglected concerns minerals and trace elements and their impact on carotenoid bioavailability. However, our group has previously shown that higher concentrations of dietary minerals, including calcium, magnesium as macro-minerals, and also iron and zinc, can strongly and negatively impact carotenoid micellarization from spinach in in-vitro test systems, following simulated gastro-intestinal digestion, and also cellular uptake as studied by Caco-2 cells. The underlying mechanisms are not entirely understood, but divalent minerals appear to form poorly soluble soaps in the presence of dietary lipids, the latter being crucial for the emulsification and solubilization of carotenoids. These soaps become insoluble and would not be available for emulsification, and might also occlude carotenoids. In addition, minerals could also react with bile acids, which are also needed in the solubilization process of carotenoids. 

Aims of the study

In the present investigation, we strived to further investigate the negative impact that minerals appear to exert on the micellarization and cellular uptake of carotenoids, both in vitro-and in-vivo, from complete food items and/or isolated carotenoids. 

Materials and methods

For this purpose, we first optimized protocols to study the bioaccessibility from carotenoids (with beta-carotene as a representative compound), including studying the influence of lipid source added, presence of emulsifiers, gastric lipase (R. oryzae) and filtration. In a next step, we investigated the bioaccessibility of both pure (isolated) carotenoids and of carotenoids from several food items (carrot juice, tomato juice, apricot juice, spinach and field salad) in dependence of various divalent minerals at different concentrations, during simulated gastro-intestinal digestion, including magnesium (Mg), calcium (Ca), zinc (Zn), and sodium (Na) (as a positive control). Furthermore, we determined the influence of altered digestion conditions on the bioaccessibility of carotenoids from spinach and carrot juice exposed to different minerals (Ca, Mg) during digestion. Altered digestion conditions included bile conc., pancratin conc., various mineral concentrations, and two lipid sources (canola oil, coffee creamer). Finally, a human postprandial randomized double blinded cross-over trial was conducted with 24 male subjects receiving a portion of spinach (250 g, containing approx. 30 mg of total carotenoids), and a capsule with placebo (0 mg Ca), 500 mg or 100 mg calcium, on 3 different test days. Each subject was therefore his own control. Following test meal ingestion, the area under plasma-time curve (AUC) in the triacylglycerol-rich fraction (representing newly absorbed carotenoids) was determined. 

Results

Regarding the optimization procedures, it was found that oil resulted in higher bioaccessibility than cream milk, while filtration through 20 nm filters reduced bioaccessibility. The addition of the gastric lipase (R. oryzae) did not result in any significant changes in beta-carotene bioaccessibility. Emulsifiers added at onset of digestion improved bioaccessibility, especially from a mixture (lecithin plus monoolein plus plus oleic acid) (5). 
When divalent minerals were present during the gastro-intestinal digestion procedure, a significant reduction of the bioaccessibility of pure lutein, neoxanthin, lycopene and beta-carotene was observed with the addition of zinc, calcium and magnesium. Interestingly, addition of sodium partly enhanced bioaccessibility (6). Mean half maximal inhibitory concentrations were 270, 253 and 420 mg/L for calcium, magnesium, and zinc, respectively. Lower bioaccessibility was significantly correlated with lower viscosity but increased surface tension. The effects on carotenoids from real food matrices were less strong, possibly due the much higher complexity of the system. Similarly as to pure carotenoids, sodium increased the bioaccessibility of carotenoids from food matrices. Interestingly, the presence of divalent minerals at lower concentrations improved the bioaccessibility of carotenoids in some food matrices. At higher concentrations micellarization was hindered, decreasing bioaccessibility, in some cases, up to 100%. Divalent minerals yielded less strong effects of on the bioaccessibility of the xanthophylls (lutein, neoxanthin), from the green leafy matrices, and of the colourless carotenoids (phytoene and phytofluene) from juices. Micelle stability, as determined by the zeta-potential, decreased with increasing divalent mineral concentration. Similar effects were found on cellular uptake employing Caco-2 cell models. Evaluation of the effects of varying enzyme concentrations (pancreatin), as well as bile salt and lipid source for Ca and Mg is not yet finalized, though measurement are all completed. 
The human trial was conducted as planned, plasma TRL-fractions separated, and all time points measured by HPLC. However, final results are still pending, and decoding of the calcium dose will be done once all AUC values have been determined. 
Conclusions: Under all studied conditions in vitro, the negative effect of various divalent minerals (Ca, Zn, Mg), on carotenoid bioaccessibility and cellular uptake was observed, at least for higher concentrations of divalent minerals. These concentrations are reachable in the gut, at least for calcium and magnesium, such as during supplemental intake, while required zinc concentrations would be supraphysiological. Xanthophylls and the colourless carotenoids, being more soluble, appear to be less effected. Carotenoids seem to be more bioaccessible from juices, where solubility of carotenoids is already higher due to industrial processing, then from green leafy matrices. The decrease in viscosity and increase in surface tension resulted from a clear precipitation process initiated by the divalent minerals, which also affected micelle integrity. Interestingly, sodium enhanced micellization/bioaccessibility of all carotenoids slightly (10-20%, though significantly), perhaps aiding in the stabilization of micelles. Neoxanthin clearly survived gastric passage of pH3 (though not violaxanthin) but both were not detected any more in plasma samples, suggesting their reaction to unknown compounds (which were observed in the plasma) or non-bioavailability – further work is needed to follow up on these compounds.