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Bioplex® Cu

Understanding organic mineral uptake mechanisms: experiments with Bioplex® Cu

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The term ‘organic minerals’ is misleading in that minerals themselves are inorganic, meaning that they do not contain carbon. However, the term has been adopted to refer to mineral supplements containing inorganic minerals bound to organic substrates. An example of this would be copper (Cu) proteinate, which would be classified as organic, as opposed to Cu sulfate, which would be classified as inorganic.

In general, it has been assumed that minerals dissociate from their organic or inorganic carriers in the intestinal lumen and are then absorbed via a passive and/or active route into enterocytes lining the gastrointestinal tract. Therefore, reported differences in the bioavailability of organic and inorganic minerals have been attributed to differences in dissociation rates of the mineral from the organic or inorganic substrate to which they are bound, or to differences in solubility. However, as will be discussed below, it may be possible for some organic minerals to be absorbed while still attached to organic carriers.


Passive, active, and paracellular transport: role of peptide transporters


One of the major challenges when discussing or investigating mineral absorption is that there is very little known about the mechanisms by which minerals are absorbed, and while data exist, they are predominantly from rodent studies. This is particularly true when discussing trace minerals. For example, in Present Knowledge of Nutrition copper absorption is described as: “The metal likely enters small intestine epithelial cells by an undefined facilitated process that involves specific copper transporters or nonspecific divalent metal ion transporters on the brush border membrane surface” (Bowman and Russell, 2001). This exceedingly vague description of copper absorption is common for many minerals, although some progress is being made. So, before potential differences between organic and inorganic mineral supplements can be discussed it is important to first consider the potential routes of mineral absorption and where regulation of those routes is likely to occur (Figure 1).

Minerals can potentially be absorbed passively via simple diffusion or through an ion channel, both of which are either driven by an electrochemical gradient or a concentration gradient. Active transport can be accomplished through specific or non-specific ion transport mechanisms that may require the cotransport of an ion down its concentration gradient, which is directly or indirectly established by the basolateral Na/K ATPase. Finally, some mineral absorption may occur through a paracellular route resulting from the loosening of tight junctions between adjacent enterocytes.


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Figure 1. Potential routes of mineral absorption from the gastrointestinal tract.


Passive transport is dependent upon either a concentration gradient or an electrochemical gradient, and therefore there is limited regulation of transport rate. However, the movement of the mineral out of the cell across the basolateral membrane or the sequestration of the mineral in cytosolic vacuoles will result in lower cytosolic mineral concentrations and thereby increase the inward concentration gradient of the mineral. Active transport is more complicated and can be regulated in numerous locations including: transcription, translation and post-translational modifications of the transporter, movement of the transporter in and/or out of the brush border membrane, and through regulation of the co-transporter ion gradient. Passive and active uptake of minerals may also require the transport of the mineral through the cell via a transport protein. Finally, paracellular transport is probably less understood but appears to be ion gradient dependent, and relatively non-specific. In general, if present, paracellular transport is important when luminal mineral concentrations are high.

If we use copper as an example, it has been proposed that copper can diffuse across the brush border membrane, and that this passive diffusion dominates at high dietary copper concentrations (Wapnir, 1998). However, as the level of copper in the diet decreases, a saturable, active transport mechanism is involved in copper transport. Presumably, copper from ‘inorganic’ or ‘organic’ sources is absorbed in a similar manner via passive diffusion or active uptake, once copper has dissociated from the inorganic or organic chelate. Therefore, to date, differences in copper bioavailability between mineral sources have been attributed to differences in dissociation rates and mineral-chelate solubilities.

However, recent work from our laboratory suggests that, in addition to these differences, copper bound as a Cu-proteinate may be absorbed attached to a di- or tri-peptide through the PepT1 transporter. PepT1, a brush border membrane bound di- and tri-peptide transporter, has been identified in all major livestock species in addition to humans and small rodents. This transporter is not overly specific, as molecules other than di- and tri-peptides can be transported through it. For instance, it has been demonstrated that PepT1 is capable of transporting certain ß-lactam antibiotics (Terada et al., 1998).


Experiments with Bioplex® Cu

Our laboratory specializes in studying nutrient uptake by the gastrointestinal tract using modified Ussing chambers (Figure 2). Intestinal tissues are removed immediately following euthanasia, stripped of the outermost serosal layer, and mounted between two buffer-filled chambers. The mucosa faces one chamber and the basolateral or serosal side faces the other. Both sides of the tissue are osmotically balanced, and a voltage clamp is then placed across the tissue in order to ‘short-circuit’ the tissue and reduce the external potential difference to zero. This allows for the measurement of ion transport in the absence of electrochemical and osmotic gradients. Electrogenic ion transport across the tissue is measured as the short circuit current (Isc), or the amount of external current necessary to nullify the transmural potential difference. The change in Isc (ΔIsc) represents the difference between the basal Isc conditions and the Isc conditions following a nutrient challenge.

To investigate copper absorption from various sources, we conducted a series of experiments using intestines from 3-week old broiler chickens and weanling pigs. For preliminary evaluation of copper absorption, a total of 36 duodenal sections were mounted in modified Ussing chambers. All tissues were challenged with phosphorus, glutamine and carbachol to serve as controls. In addition, tissue samples were challenged with copper from Bioplex® Cu (Alltech Inc.), copper sulfate, or copper chloride in concentrations ranging from 0.1 to 3 mM. Copper chloride was used initially, and caused a net decrease in short circuit current. Therefore, for the remaining tissue, Bioplex® Cu or copper sulfate were used.

A greater change in short circuit current was observed when Bioplex® Cu was added, compared to copper sulfate. Similar findings were observed in subsequent work with weanling pig jejunal tissue (Figure 3). However, changes in short-circuit current are indicative of total ion flux. Therefore, increased changes in short-circuit current when Bioplex® Cu was added compared to copper sulfate could be indicative of active copper absorption, amino acid/peptide movement, or both. Based on a small number of samples (n=12-17/trt), an approximately 3-fold higher concentration of copper was found in the serosal fluid of tissues challenged with Bioplex® Cu compared to copper sulfate (Figure 4), indicating that a larger proportion of copper from Bioplex® Cu crossed the apical and basolateral membranes compared to copper sulfate.


Figure 2. Tissues mounted in modified Ussing chambers. Intestinal tissue is removed immediately following euthanasia, stripped of the outermost serosal layer (A), opened along the mesenteric border, and mounted on a slide insert (B). This insert is the placed between two buffer-filled chambers (C) with the mucosal surface facing one chamber and the serosal or basolateral surface facing the other chamber.



Figure 3. Change in short circuit current (Isc) following a mucosal challenge with Bioplex® Cu or copper sulfate to swine jejunum.



Figure 4. Copper concentration in serosal buffer after a mucosal challenge with Bioplex® Cu or copper sulfate.


In addition to determining active nutrient uptake in the chambers, passive uptake can be estimated by determining changes in the resistance (Ω/cm2) across the tissue. Based on preliminary data, there is a greater change in resistance when copper sulfate is added to the mucosal chamber compared to Bioplex® Cu. This indicates a greater passive ion flux with copper sulfate than Bioplex® Cu. Changes in resistance are indicative of total passive ion flux, and must be considered within that context. However, based on these preliminary data, it does appear that copper uptake may occur through different routes between the two sources. At least the amount of copper absorbed through passive and active routes may be different. It also appears that the total amount absorbed may be different, although this needs to be confirmed by further analysis of mucosal and serosal buffers following a copper challenge.


Conclusions

Based on these preliminary data, we hypothesized that copper bound as a proteinate might be absorbed through the di- and tri-peptide transporter, PepT1. To test this hypothesis we blocked PepT1 by adding valacyclovir to the mucosal chamber prior to the addition of copper as has been reported previously (Sun et al., 2001). These data will be reported at the ASAS meetings this summer (Aldridge et al., 2007), and indicates that blocking PepT1 results in a reduced absorption of copper from Bioplex® Cu, but not from copper sulfate (Figure 5). Therefore, it appears that organic copper in the form of Bioplex® Cu proteinate can dissociate and be absorbed by passive diffusion, active transport, or can be absorbed while still attached to a di- or tri-peptide through the PepT1 transporter. The physiological significance of this is still unclear, but may explain differences observed in bioavailability for Bioplex® Cu compared to copper sulfate.



Figure 5. Copper concentration (ppm) in mucosal buffer after a mucosal challenge with Bioplex® Cu or copper with or without peptide inhibitors Gly-Sar or Valcyclovir. The inhibition of copper uptake by Gly-Sar is probably due to the formation of insoluble Cu-Gly-Sar salts as opposed to blockage of the peptide transporter, PepT1 (abMeans differ, P<0.05).


References


Aldridge, B.E., K.L. Saddoris and J.S. Radcliffe. 2007. Copper can be absorbed as a Cu-peptide chelate through the PepT1 transporter in the jejunum of weanling pigs. J. Anim. Sci. (Abstr., Accepted).

Bowman, B.A. and R.M. Russell. 2001. Present Knowledge in Nutrition. ILSI Press, Washington, DC.

Sun, D., C.P. Landowski, X. Chu, R. Wallsten, T.E. Komorowski, D. Fleisher and G.L. Amidon. 2001. Drug inhibition of Gly-Sar uptake and hPepT1 localization using hPepT1-GFP fusion protein. AAPS Pharmsci. 3:1-9.

Terada, T., H. Sanito and K. Inui. 1998. Interaction of ß-lactam antibiotics with histidine residue of rat H+/peptide cotransporters, PEPT1 and PEPT2. J. Biol. Chem. 273(10):5582-5585.

Wapnir, R.A. 1998. Copper absorption and bioavailability. Am. J. Clin. Nutr. 67:1054S- 1060S.



Authors: J.S. RADCLIFFE, B.E. ALDRIDGE and K.L. SADDORIS
Purdue University, West Lafayette, Indiana, USA
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