Ex Vivo and in Vivo Study of Sucrosomial® Iron Intestinal Absorption and Bioavailability

Abstract

The present study aimed to demonstrate that Sideral® RM (SRM, Sucrosomial® Raw Material Iron) is transported across the excised intestine via a biological mechanism, and to investigate the effect that this transport route may produce on oral iron absorption, which is expected to reduce the gastrointestinal (GI) side effects caused by the bioavailability of non-absorbed iron. Excised rat intestine was exposed to fluorescein isothiocyanate (FITC)-labeled SRM in Ussing chambers followed by confocal laser scanning microscopy to look for the presence of fluorescein-tagged vesicles of the FITC-labeled SRM. To identify FITC-labeled SRM internalizing cells, an immunofluorescence analysis for macrophages and M cells was performed using specific antibodies. Microscopy analysis revealed the presence of fluorescein positive particulate structures in tissues treated with FITC-labeled SRM. These structures do not disintegrate during transit, and concentrate in macrophage cells. Iron bioavailability was assessed by determining the time-course of Fe3+ plasma levels. As references, iron contents in liver, spleen, and bone marrow were determined in healthy rats treated by gavage with SRM or ferric pyrophosphate salt (FP). SRM significantly increased both area under the curve (AUC) and clearance maxima (Cmax) compared to FP, thus increasing iron bioavailability (AUCrel = 1.8). This led to increased iron availability in the bone marrow at 5 h after single dose gavage.

Keywords: iron bioavailability; iron storage; lecithin; self-assembled vesicles; sucrester.

Conflict of interest statement

The authors declare that Elisa Brilli and Germano Tarantino are employed by the company Pharmanutra S.p.A. which financed this research. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, nor in the writing of the manuscript.

Figures

Figure 1

Representative images of rat gut sections after 0.5 h, 1 h and 2 h incubation in Ussing chamber. TM: tunica mucosa; TS: tunica serosa; V: intestinal villus. (A) Hematoxylin and eosin stain. Scale bars 50 µm. (B–E) Immunofluorescence confocal microscopy; individual fluorescent channels: green (SRM, Sucrosomial® Raw Material Iron) and blue (nuclei). (B) Bidimensional image of the maximum intensity projection. Scale bar 50 µm. (C) Blow-up of the squares shows higher magnification of the three-dimensional pictures with the respective orthogonal projections. Scale bar 50 µm. (D) Images acquired in one Z-plane (31st of 48, 46th of 75, and 33rd of 65, incubated for 0.5, 1 and 2 h respectively). Scale bar 50 µm. (E) Images acquired in one Z-plane (28th of 40, 15th of 46, 28th of 47, incubated for 0.5, 1 and 2 h, respectively) at a magnification of different sections higher than that of (B–D). Scale bar 10 µm.

Figure 2

Confocal laser scanning microscopy: representative images of rat gut sections after 1 and 2 h incubation in an Ussing chamber. Asterisks indicate enterocytes; arrows indicate connective cells. TM: tunica mucosa. Individual fluorescent channels: green (SRM) and blue (nuclei). Scale bar 10 µm.

Figure 3

Confocal laser scanning microscopy: representative images of rat gut sections after 0.5 and 1 h incubation in Ussing chamber. Negative controls obtained omitting primary antibodies are shown in double line squares. Individual fluorescent channels: green (SRM), red (CD68 + cells) and blue (nuclei). (A) Bidimensional image of the maximum intensity projection. Scale bar 25 µm. (B) Images acquired in one Z-plane (29th of 47 and 26th of 40, incubated 0.5 and 1 h, respectively) at a magnification of different sections higher than that of (A). Scale bar 10 µm.

Figure 4

Confocal laser scanning microscopy: representative images of rat gut sections after 0.5 and 1 h incubation in Ussing chamber. Negative controls obtained omitting primary antibodies are shown in double line squares. Individual fluorescent channels: green (SRM), red (GP2 + cells) and blue (nuclei). (A) bidimensional image of the maximum intensity projection. Scale bar 25 µm. (B) Images acquired in one Z-plane (38th of 59, 22nd of 64, incubated 0.5 h and 1 h, respectively) at a higher magnification. Scale bar 10 µm.

Figure 5

Plasma Fe3+ concentration vs time plots following administration of 400 μL of a suspension (5 mg/kg of iron) of SRM and ferric pyrophosphate salt (FP, ferric pyrophosphate salt) (control) compared to Fe3+ concentration in untreated animals (basal). Means ± SD of at least six values obtained with different animals.

Figure 6

Quantity (μg) of Fe3+ per gram of organ withdrawn from rats sacrificed after 3 or 5 h from the gavage. * p < 005. Means ± SD of at least six values obtained with different animals.

Figure 7

Comparison between plasma concentration vs time profiles following administration of SRM or FITC-labeled SRM. In case of SRM plasma was analyzed for Fe3+; in case of FITC-labeled SRM plasma was analyzed for FITC. Means ± SD of three values obtained with different animals.