Amino acid modulation of in vivo intestinal zinc absorption in freshwater rainbow trout
1 Division of Life Sciences, Kings College, London, 150 Stamford Street, London SE1 9NN, UK and
2 T. H. Morgan School of Biological Sciences, University of Kentucky, Lexington, Kentucky, 40506-0225, USA
*Author for correspondence (e-mail: chris.glover{at}kcl.ac.uk)
Accepted 19 October 2001
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Summary |
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Key words: zinc, uptake, intestine, dietary metal, zinc-binding ligand, transport, histidine, cysteine, taurine, amino acid, fish, rainbow trout, Oncorhynchus mykiss.
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Introduction |
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The accompanying paper (Glover and Hogstrand, 2002) characterises the in vivo uptake of zinc in freshwater rainbow trout using an intestinal perfusion system. This method allowed kinetic characterisation of zinc uptake and highlighted important differences between intestinal uptake in freshwater fish and studies in mammals and marine teleosts. This technique has been used in this study to investigate the roles of amino acids in modifying the uptake and absorption of zinc. In fish the introduction of an amino acidzinc chelate to the diet has been reported to increase zinc concentrations in the body of rainbow trout, compared to inorganic zinc treatments (Hardy et al., 1987
; Paripatananont and Lovell, 1995
). This has potential benefits for the aquaculture industry where increased Zn(II) absorption is associated with improved fish growth and health (Watanabe et al., 1997
).
Facilitatory effects of amino acids on zinc absorption have been documented in a number of mammalian and invertebrate tissues (e.g. Wapnir et al., 1983; Ackland and McArdle, 1990
; Bobilya et al., 1993
; Buxani-Rice et al., 1994
; Horn et al., 1995
; Vercauteren and Blust, 1996
). Histidine and cysteine have high binding affinities for zinc, with dissociation constant (Kd) values of 12.1 and 18.2, respectively, for Zn(II)-(amino acid)2 species (Martell and Smith, 1974
). Amino acids may increase bioavailability by removing chelated zinc from dietary zinc-binding constituents such as phytic acid. Evidence of such an action has been presented in studies of zinc absorption in fish where the beneficial effects of adding zinc as an amino acid chelate were greater in diets that contained high levels of phytic acid (Paripatananont and Lovell, 1995
). This increase in bioavailability may be achieved in one of two ways. Amino acids may act to shuttle zinc from dietary components with low zinc binding affinity to uptake surfaces with higher affinity (Wapnir et al., 1985
; Ackland and McArdle, 1990
; Bobilya et al., 1993
). Alternatively, the formation of an amino acidzinc chelate may create a substrate for transport across the epithelial surface (Wapnir et al., 1983
; Ashmead et al., 1985
).
Experimental evidence regarding the exact mechanism of the enhancement effect of amino acids upon zinc uptake is scant. Physiological investigations have yet to yield a candidate transporter. Although the basic amino acid carrier y+ was a candidate for mediating the uptake process in human erythrocytes, subsequent experiments suggest this is not the case (Horn et al., 1995). In lobster hepatopancreas epithelia, it was proposed that zinc may combine with L-proline to form a more readily transportable complex, in addition to acting at an allosteric binding site to stimulate the absorption of L-proline (Monteilh-Zoller et al., 1999
).
The role of amino acids in altering the uptake, fate and metabolism of intestinally introduced zinc was the focus of the present study. Histidine and cysteine were chosen as amino acids that are known to have biological relevance as zinc chelators. Concentrations were tested that altered free zinc ion activity and the nature of amino acidzinc chelates. Taurine was included as an amino acid of biological importance, but without known chelation effects. The results demonstrated the complex nature of in vivo intestinal zinc uptake, including an L-histidine-mediated uptake pathway, and specific actions of D-histidine and L-cysteine upon qualitative zinc absorption. The results suggested that dietary constituents could play a vital role in modulating intestinal zinc uptake, with potential implications for aquaculture and environmental toxicity.
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Materials and methods |
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Experimental procedure
The in vivo intestinal cannulation procedure was identical to that described by Glover and Hogstrand (2002). Experimental perfusion solutions consisted of 65Zn(II) (as ZnCl2: approx. 4 kBq ml1, New England Nuclear) in a 77 mmol l1 NaCl saline, with Zn(II) added as ZnSO4·7H2O to a final concentration of 50 µmol l1. Amino acids (L- or D-histidine, L- or D-cysteine, taurine) were included individually in perfusates with Zn(II) at the concentrations stated (2 or 100 mmol l1). These two concentrations resulted in dramatically different Zn(II)amino acid chelate speciation (Table 1), and allowed the testing of hypotheses regarding potentially important uptake moieties. Solutions were made fresh from stock on the day of experiment. The pH of perfused solutions was neither adjusted, nor buffered and ranged from 6.06.4. Experimental perfusions were of 3 h duration.
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For all treatments a 65Zn(II) budget was constructed. For some experiments it was noted that not all 65Zn(II) could be accounted for. This was likely a consequence of Zn(II) binding to experimental apparatus. To correct for this, parameters were adjusted based on the percentage of recovered radioactivity (Hardy et al., 1987). For calculation of epithelial, subepithelial and post-intestinal accumulation rates, data were standardised to account for differences in the retained Zn(II) fraction that otherwise may have distorted patterns of accumulation. Retained and absorbed Zn(II) fractions are quantitative measures of the amount of Zn(II) entering the animal. Standardised accumulation parameters therefore represent the qualitative change in Zn(II) distribution caused by the amino acid treatments. All calculations were based on equations described in the accompanying paper (Glover and Hogstrand, 2002
).
Data are expressed as means ± S.E.M. Significant effects (P<0.05) of treatments were tested using analysis of variance (ANOVA), unless otherwise stated.
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Results |
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Plots of the appearance of 65Zn(II) from the efferent cannulae (Fig. 1) showed that the slopes of the linear portion of recovered 65Zn(II) differed in the presence of L-histidine and L-cysteine at 100 mmol l1. These treatments also reached steady state more rapidly than the control. Slopes obtained in taurine experiments and for 2 mmol l1 cysteine and histidine treatment groups were unchanged from the controls [Zn(II) alone]. This effect was stereospecific, as neither D-histidine nor D-cysteine exhibited a significantly altered slope of recovered 65Zn(II) (data not shown).
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Amino acids had little impact upon epithelial (mucus and epithelial cells) Zn(II) accumulation (Fig. 3AC). The only effect of note was a significant stimulation of epithelial accumulation with 100 mmol l1 D-histidine when compared with the corresponding 2 mmol l1 treatment.
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Post-intestinal Zn(II) accumulation was analysed to determine if the effects of taurine and cysteine at 100 mmol l1 were compartment-specific. Taurine was found to decrease both blood and body Zn(II) accumulation, whereas the stereospecific effect of L-cysteine was solely on the plasma and erythrocyte Zn(II) accumulation rate (Fig. 4).
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Discussion |
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A specific effect of histidine upon Zn(II) uptake has been observed previously in a wide range of species and tissues. There are two theories to explain these actions. Histidine may act as a donor molecule facilitating the release of Zn(II) to a membrane transport entity (Wapnir et al., 1985; Ackland and McArdle, 1990
; Bobilya et al., 1993
). Alternatively the L-histidinezinc chelate may be transported intact across the epithelium (Wapnir et al., 1983
; Ashmead et al., 1985
). Both these models of transport account for the stereospecificity of the observed response. Either the Zn(L-His)2 complex had a competitive advantage in gaining access to the transport moiety, or the transporter itself exhibited enantiomer dependence. From the experiments described here it is not possible to discern the nature of L-histidine-mediated Zn(II) uptake. Significantly different slopes of Zn(II) recovered from the outflow cannula for L-histidine compared to control and D-histidine also lend support to a stereospecific mechanism of Zn(II) uptake, a mechanism that also differed from the uptake of unchelated Zn(II).
The histidine effect upon intestinal Zn(II) uptake in rainbow trout was mediated by a Zn(His)2 species. There is compelling evidence from studies in mammalian erythrocytes that passage into cells via a bis-histidine Zn(II) complex is responsible for the stimulatory effects of histidine (Horn et al., 1995). Vercauteren and Blust (1996
) showed a facilitatatory effect of histidine upon Zn(II) in the mussel, Mytilus edulis, correlated with the mono-histidine species. Uptake of Zn(II) across the bloodbrain barrier of rats was also believed to be mediated by Zn(His)+ (Buxani-Rice et al., 1994
). Where there is evidence for histidine-mediated Zn(II) uptake in mammalian intestine, Zn(His)2 is the species implicated (Wapnir et al., 1983
). While the nature of the uptake species remains controversial there is increasing evidence across a wide range of organisms, tissues and experimental systems that Zn(II) uptake can be achieved by a histidine-mediated process.
The lack of a stimulatory action of amino acids upon quantitative Zn(II) uptake in the present study is not surprising. Dietary supplementation of low molecular mass Zn(II)-binding ligands such as cysteine and histidine enhance uptake by chelating Zn(II), removing it from other dietary constituents. Consequently Zn(II) bioavailability is increased. In the present experiments competing ligands were absent and therefore enhancement of quantitative Zn(II) uptake was not observed. Lack of any effect at this level does not necessarily equate with a lack of biological effect. Aquacultural studies have shown that Znmethionine complexes can more easily meet piscine dietary Zn(II) requirements than Zn(II) in inorganic complexes (Paripatananont and Lovell, 1995), without altering the amount of Zn(II) absorbed (Wekell et al., 1983
). The beneficial effects of organic Zn(II) chelates may be explained by altered patterns of accumulation, such as those observed by Hardy et al. (1987
) and below.
Qualitative Zn(II) absorption is modulated by luminal amino acids
Examination of qualitative changes in Zn(II) absorption provides further evidence of specific effects of amino acids on metal metabolism. Histidine stereoisomers stimulated the passage of Zn(II) into the subepithelium (intestinal tissue remaining after mucosal scraping). In contrast to the L-enantiomer effect upon quantitative Zn(II) absorption, this effect was only statistically significant in the presence of D-histidine. Interestingly Aiken and colleagues noted a similar scenario. In rat erythrocytes the effect of histidine upon uptake of Zn(II) was stereospecific (Aiken et al., 1992a); however, histidine also acted to influence body distribution of Zn(II) in vivo in an enantiomer-independent manner (Aiken et al., 1992b
).
Such results are difficult to explain. In mammalian systems the actions of a transporter in mediating an altered tissue distribution can be eliminated, owing to strict stereospecificity of such entities (Aiken et al., 1992b). However, intestinal amino acid transport in fish is thought to be more promiscuous than that of adult mammals. For at least some amino acids, D- and L-isomers are able to share the same transporter (Huang and Chen, 1975
). It is possible, therefore, that the actions of D-histidine could be, in part, mediated by a transporter of D-histidinezinc complexes.
The effects of cysteine upon intestinal Zn(II) absorption are distinct from those of histidine. Snedeker and Greger (1983) described a similar situation in rats. Cysteine had an L-enantiomer-dependent stimulatory action upon post-intestinal Zn(II) absorption. Time-to-steady-state Zn(II) flux for L-cysteine was also greater than for the D-isomer, indicating a mechanistic difference at the uptake surface. Such a difference may again be explained by a specific transport entity or may represent a competitive advantage for L-cysteine to donate Zn(II) to a membrane moiety for uptake. The experiments described here do not allow for a distinction between these scenarios.
Cysteine infusion in rats was found to increase plasma Zn(II) levels (Abu-Hamden et al., 1981). The increase in post-intestinal Zn(II) levels in the present study was the result of enhanced Zn(II) accumulation specifically in the blood compartments. Body Zn(II) accumulation rate was unchanged, despite the fact that this compartment has reserve capacity for accumulation (Glover and Hogstrand, 2002
). In the study of Abu-Hamdan (1981
), the rise in plasma Zn(II) levels was concomitant with an increase in Zn(II) excretion. It is not known whether a similar effect exists in the present investigation. Enhanced post-intestinal Zn(II) may be a result of increased plasma cysteine levels, drawing Zn(II) from the epithelium into the blood where it is retained. Alternatively the passage of a cysteineZn(II) chelate into the blood is possible (Ashmead et al., 1985
).
Effects of taurine: evidence for reciprocal regulation of Zn(II) uptake pathways
The marked effect of taurine upon Zn(II) absorption was unexpected. Taurine was included in the study as a control, being an amino acid with low affinity for Zn(II) (Sakurai and Takeshima, 1983). Harraki and colleagues (1994
) reported taurine stimulation of Zn(II) absorption in human fibroblasts. To our knowledge this report and the present study are unique in exhibiting an influence of taurine upon Zn(II) uptake. While rarely reported, such an association is not without biological relevance. Both Zn(II) and taurine are found in high concentrations in the tapetum lucidum of the retina (Sturman, 1983
), mossy fibre boutons of the cerebral cortex and hippocampus (Crawford, 1983
; Wu et al., 1985
), and in combination both enhance membrane stability (Gaull et al., 1985
).
Taurine is the only modulator of Zn(II) metabolism thus far tested in the in vivo perfused trout intestine that has effects on both subepithelial and post-intestinal Zn(II) accumulation. Taurine uptake was shown to be Na+-dependent in flounder intestine (King et al., 1986), and the replacement of Na+ with choline did not affect the pattern of Zn(II) accumulation with taurine in rainbow trout intestine (C. N. Glover and C. Hogstrand, unpublished observations). The effect noted is therefore likely to be unrelated to taurine uptake, assuming that taurine transport is conserved between species. Taurine is known to alter the membrane binding and transport of Ca2+ (Huxtable, 1992
). Reports have shown interactions between Ca2+ and Zn(II) in fish gill and gut absorption (e.g. Hogstrand et al., 1995
, 1996
) (C. N. Glover and C. Hogstrand, in preparation). The taurine-induced response may be a secondary effect due to alteration of Ca2+ metabolism, or an action of taurine specifically upon transport or membrane interactions of Zn(II). The effect of taurine is intriguing given it is the most abundant free amino acid in intestinal mucosa of fish (Auerswald et al., 1997
).
Data presented here suggest the presence of several parallel uptake pathways for Zn(II), including an amino acid-mediated process and an uptake route for inorganic Zn(II). While of obvious value, in vitro methods such as gut bags and isolated membrane vesicles may not discern the complex nature of intestinal Zn(II) absorption, highlighting the importance of the in vivo approach that we have used.
Evidence from this investigation and others (e.g. Kramer et al., 1997) suggests that interactions between amino acids and metals in the intestine are likely to have important ramifications upon the absorption of both these dietary constituents. Luminal composition therefore has the potential to modulate the oral toxicity of metals, and will also have implications for nutrition and aquaculture. The ability to maintain the uptake of essential nutrients while regulating the accumulation of potential toxicants is key to preserving life in contaminated environments.
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Acknowledgments |
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