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Article

Metallothionein-Null Mice Absorb Less Zn from an Egg-White Diet, but a Similar Amount from Solutions, although with Altered Intertissue Zn Distribution

Division of Clinical Biochemistry, Institute of Medical and Veterinary Science, Adelaide, SA 5000 Australia

	ABSTRACT

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The influence of metallothionein (MT) on Zn transfer into non-gut tissues was investigated in MT-null (MT-/-) and normal (MT+/+) mice 4 h after oral gavage of aqueous ⁶⁵ZnSO₄solution at doses of 154, 385, 770 and 1540 nmol Zn per mouse. Zn transfer was not significantly different between MT+/+ and MT-/- mice and was directly proportional to the oral dose (slope = 0.127, r = 0.991; 0.146, r = 0.994, respectively). Blood ⁶⁵Zn and plasma Zn concentrations increased progressively in MT-/- mice at doses >154 nmol Zn, reaching levels of 2.4% of oral dose and 60 µmol/L, respectively, at the 1540 nmol Zn dose. The corresponding values for MT+/+ mice were approximately half, 1.0% and 29 µmol/L. Intergenotypic differences were found in tissue distribution of ⁶⁵Zn within the body; MT-/- mice had higher ⁶⁵Zn levels in muscle, skin, heart and brain, whereas MT+/+ mice retained progressively more Zn in the liver, in conjunction with a linear increase in hepatic MT up to the highest Zn dose. MT induction in the small intestine reached its maximum at an oral dose of 385 nmol Zn and did not differ at higher doses. Absorption of a 770 nmol ⁶⁵Zn dose from a solid egg-white diet was only one fourth (MT+/+) and one eighth (MT-/-) of the Zn absorption from the same dose of ⁶⁵Zn in aqueous solution. MT+/+ mice had greater (P < 0.05) Zn absorption from the egg-white diet than did MT-/- mice, indicating that gut MT confers an absorptive advantage, but only when Zn is incorporated into solid food.

KEY WORDS: • zinc • metallothionein • intestinal absorption • metallothionein-null mice

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Uptake of Zn from the diet is thought to be regulated in the gut by both absorptive and excretory processes, supported by mechanisms that have yet to be defined (Cousins 1985 , Hoadley et al. 1987 , Vallee and Falchuk 1993 ). Zn absorption responds rapidly to alterations in dietary Zn; its efficiency increases at low Zn intakes and decreases when dietary Zn is excessive (Cousins 1985 ). The transcellular transport of Zn from the intestinal lumen to the vasculature is thought to occur via both passive and carrier-mediated processes (Hoadley et al. 1987 and 1988 , Steel and Cousins, 1985 ); although the precise control mechanisms of the facilitated process remain elusive, there is mounting evidence to suggest that recently described Zn transporter proteins (ZnT-1 to ZnT-4)³are involved in transmembrane Zn movements (Davis et al. 1998 , McMahon and Cousins 1998 , Palmiter and Findley 1995 , Palmiter et al. 1996 ).

There are conflicting viewpoints regarding the influence of intestinal metallothionein (MT) on Zn absorption. An inverse relationship between MT-bound Zn and Zn absorption has been demonstrated by some workers but not by others (reviewed by Lönnerdal 1989 ). It has been proposed that mucosal MT sequesters Zn in the intestinal wall, thereby reducing absorption and enabling Zn transfer back into the gut lumen. MT synthesis in mucosal cells is triggered by high luminal Zn concentrations but is not significantly induced at normal dietary Zn intakes, leading to the view that the influence of MT on Zn absorption is minimal (Cousins 1985 , Lönnerdal 1989 , Vallee and Falchuk 1993 ). Cysteine-rich intestinal peptide (CRIP) may be involved in intracellular Zn transport, and a model based on the relative concentrations and binding constants of MT and CRIP was proposed (Hempe and Cousins 1992 ). CRIP is not induced by dietary Zn and has a lower binding affinity for Zn than MT. The model predicted that when Zn intake is inadequate, MT expression in the mucosa would be low, allowing CRIP to bind more Zn and resulting in a higher rate of absorption. When dietary Zn is adequate or in excess, MT synthesis is induced; subsequently, this sequesters Zn that would otherwise be bound by CRIP, thus reducing the proportion of dietary Zn absorbed.

Evidence conflicting with the CRIP/MT model was reported by Fleet et al. (1993) from studies in human adenocarcinoma (Caco-2) cell monolayers. Transepithelial Zn transport was shown to occur through a mechanism that is both vitamin D sensitive and lysosomal mediated, with the latter accounting for ~68% of the Zn transport. In addition, activation of the vitamin D–stimulated Zn transport pathway was observed to coincide with greater MT gene expression and diminished CRIP mRNA levels, a finding in opposition to that predicted by the CRIP/MT model. The incorporation of LIM motif sequences, the existence of CRIP in a range of other tissues and its high expression in immune cells, in particular, also suggest that CRIP has a more general function (e.g., proliferation, differentiation or cellular repair) than transcellular Zn transport in the enterocyte (Hallquist et al. 1996 , Khoo et al. 1996 and 1997 ).

Mice lacking gene expression for MT-I and MT-II have been valuable in confirming the contribution of hepatic MT to Zn homeostasis in vivo and in vitro (Coyle et al. 1995 , Kelly et al. 1996 , Philcox et al. 1995 , Rofe et al. 1996 ). Only recently have MT-/- mice been used to examine intestinal Zn transport. Davis and co-workers (1998) reported that serum Zn concentrations were 2.3 times higher in MT-null mice than in normal mice 2 h after the administration of a single 0.5 mmol/kg oral Zn dose by gastric gavage, supporting the view that MT is inhibitory to Zn absorption. In seeming contradiction to the proposed role of MT in sequestering Zn in the mucosa, however, the MT-null mice were found to accumulate more Zn in the intestine than normal control mice. Davis argued that intestinal MT may provide a labile Zn pool for maintaining a mucosal-to-luminal Zn flux rather than act as a Zn sequestrant.

In this paper, we investigated the dynamics of Zn absorption and body Zn distribution in normal and MT-null mice using four incremental oral doses of ⁶⁵Zn in the physiologically relevant range from 0.006 to 0.06 mmol Zn/kg. The interplay of gut and liver MT synthesis in the control of plasma Zn has been highlighted. A further study, in which 0.03 mmol Zn/kg was administered in an appropriate amount of egg-white diet, showed that the gavage of simple aqueous Zn solutions yields data that overestimate Zn transfer and underestimate the influence of MT on dietary Zn absorption.

	MATERIALS AND METHODS

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MT-null (MT-/- ) mice were F3 derivatives of the interbreeding of normal (MT+/+ ) C57BL6 (Animal Resources Centre, Canning Vale, Western Australia) and MT-/- mice produced at the Murdoch Institute, Royal Children's Hospital, Victoria (Michalska and Choo 1993 ). The latter were of mixed genetic background of OLA129 and C57BL6 strains. The genotypes of the F2 mice were established by DNA analysis of tail-tip samples using the technique described by Michalska and Choo (1993) . Mice were housed on sawdust in plastic cages in an animal house at 22°C with a 14-h light:10-h dark cycle and were given free access to water and a commercial nonpurified diet (Milling Industries, Adelaide, Australia) described in a previous report (Coyle et al. 1993 ). The concentrations of Zn, Cu and Fe in this diet were 100, 5 and 66 mg/kg respectively (Zn and Fe are more concentrated than reported in 1993). When the mice were deprived of food, coprophagy was prevented by housing them in plastic cages with stainless steel wire floors: 45 cm x 16 cm, 7.5-mm mesh, 1-mm wire diameter.

⁶⁵Zn absorption in MT+/+ and MT-/- mice: dose response to aqueous Zn.

Weight- and sex-matched mice from each genotype, age 10–13 wk (n = 72), were housed together for 7 d in groups of eight and fed a purified egg-white diet (EWD) (see Table 1 )containing 100 mg Zn/kg. They were then deprived of food for 20 h [body weight (BW) = 23.0 ± 0.3 g after fasting] before oral gavage with 0.1 mL of ⁶⁵Zn (37 kBq; NEN Life Science Products, Boston, MA) containing 154, 385, 770 or 1540 nmol (10, 25, 50 or 100 µg, respectively) of Zn as ZnSO₄. Four hours later, 1 mL of blood was taken from each mouse by cardiac puncture under light halothane anesthesia before mice were killed by cervical luxation. Where possible, urine was collected by direct needle aspiration from the bladder. The carcass was carefully dissected, staged to avoid cross-contamination with ⁶⁵Zn. A 4-cm²sample of skin and a 200-mg portion of abdominal muscle were taken first. The gastrointestinal tract from stomach to colon was removed and divided into six segments as follows: stomach, proximal, middle and distal thirds of the small intestine, cecum and colon. The three segments of small intestine were immediately subjected to gamma counting. The lumens were then flushed with normal saline to remove the contents and the washed segments counted again (within 20 min of the initial counting) to determine the ⁶⁵Zn uptake by the gut wall. The heart, liver, kidneys, spleen, testes and brain were excised. Radioactivity was determined in the tissue samples by using a cassette-fed Packard Auto-Gamma Counter, model 5650 (Canberra-Packard, Melbourne, Australia).

The fractions of body weight used were 0.33 for muscle, 0.13 for skin, 0.07 for whole blood and 0.035 for plasma. Liver, pancreas and small intestine were chilled on crushed ice, counted without delay and then homogenized in 10 mmol/L Tris buffer, pH 7.4, with the use of an Ultra-Turrax homogenizer; MT was determined by a Cd/Heme assay (modified from Eaton and Toal 1982 ). Instead of commercial dried heme, which we found to give poor linearity between serial dilutions of high MT liver samples, we use red cell hemolysate from fresh human blood. With the use of a 1:400 wt/v (wet liver/total reaction volume) dilution, the detection limit is 2–3 nmol Cd bound/g, equivalent to ~0.3–0.4 nmol/g MT, assuming that seven gram atoms of Zn per mole MT are displaced by Cd in the assay. When livers from fed, unstressed normal and MT-null mice were assayed within the same batch, the measured "MT" concentrations of all MT-null mice (2.2 ± 0.2 nmol Cd bound/g, n = 20) were less than the lowest MT concentrations from normal mice (6.6 ± 0.9 nmol Cd bound/g, n = 9). After intraperitoneal injection with lipopolysaccharide, normal mice had liver MT concentrations of 80–200 nmol Cd bound/g, whereas the "MT" values in MT-null mice remained identical to those of the uninjected mice. MT-null mice never have measured MT concentrations above the lowest obtained in nonstimulated normal mice within the same assay batch. We consider liver MT levels in MT-null mice to represent the noise threshold of the assay. Serum Zn concentrations were determined by atomic absorption spectrometry (AAS).

⁶⁵Zn Absorption in MT+/+ and MT-/- mice: uptake from food.

Eight male MT+/+ and eight male MT-/- mice, age 10 wk, were housed on sawdust in plastic cages and given free access to the nonpurified diet. All mice were then deprived of food for 20 h, with free access to water. After food deprivation, MT+/+ mice weighed 20.4 ± 0.3 g and MT-/- mice 20.7 ± 0.3 g. The mice were fed a Zn-dosed meal based on the diet described in Table 1 , containing ⁶⁵Zn (37 kBq) and 770 nmol of Zn as ZnSO₄. The radiolabeled diet was initially made as a slurry, air dried to 12% moisture content and then molded into spherical pellets of 0.50 g containing the specified dose. The mice were caged separately and given free access to the food pellet and water; consumption of both was monitored. The time taken to eat the pellet was 82.5 ± 7.3 min for MT+/+ mice and 87.5 ± 7.3 min for MT-/- mice. Over this period, they consumed 0.62 ± 0.07 and 0.63 ± 0.07 mL of water, respectively. Exactly 4 h after completion of the meal, the mice were killed and the tissues sampled as described above. Small particles of food remained uneaten by some mice, and there was also a minor amount of ⁶⁵Zn in feces. The ⁶⁵Zn counts for each of these components were recorded for individual mice. Nevertheless, the recoveries of ⁶⁵Zn from the gut and tissues were similar between genotypes (MT+/+, 84.1 ± 5.1%; MT-/-, 85.7 ± 3.4%). In calculating Zn transfer in individual mice, correction was made for the ⁶⁵Zn remaining in uneaten food and excreted in the feces.

Endogenous Zn in the lumen of the small intestine.

Six female MT+/+ mice and six female MT-/- mice, age 10–13 wk, were housed together and fed the purified diet (Table 1) containing 100 mg Zn/kg for 7 d; mice were then deprived of food for 20 h before oral gavage with 0.1 mL distilled water. Exactly 4 h after gavage, blood was taken by cardiac puncture under light halothane anesthesia. The mice were then killed and the small intestine and pancreas excised. The small intestine was cut into three segments of equal length, comprising the duodenum-jejunum, jejunum-ileum and distal ileum. The lumen of each segment was flushed with water and the extruded contents collected. The apportioned contents, the pancreas and the entire small intestine from each mouse were dried for 96 h at 70°C before nitric acid digestion for Zn analysis.

Statistical analyses.

Repeated measures ANOVA using the general linear model on Minitab (Minitab, State College, PA) was used for comparison of differences between several means. Because absorption data had already been normalized by expression as percentage uptake, no further transformation of data was undertaken before ANOVA. Variability is expressed as the root mean square error (RMSE), an estimate of the standard deviation across treatments. When interactions were significant, Tukey's post-hoc test was used to distinguish differences between specific means (Tukey 1949 ). Where appropriate, data are expressed as the mean ± SEM and differences assessed by two-tailed unequal variance Student's t test. Differences were considered significant at P < 0.05. Text references state the highest P-value obtained. This work was approved by the Institute of Medical and Veterinary Science Animal Ethics Committee.

	RESULTS

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Zn absorption in MT+/+ and MT-/- mice.

The body distribution of ⁶⁵Zn, 4 h after intragastric gavage with the defined doses of Zn, is presented in Table 2 .The recovery of the oral dose, including that in the gastrointestinal tract, was 97.1 ± 1.2 % (n = 72). The transferred dose of ⁶⁵Zn is defined here as the percentage of the oral dose retained by the body, excluding that in the gastrointestinal tract. In mice of both genotypes, ⁶⁵Zn transfer was similar and directly proportional to the oral Zn dose (MT-/-, Zn transferred = 0.146 x oral Zn dose, r = 0.994; MT+/+, Zn transferred = 0.127 x oral Zn dose, r = 0.991). MT-/- mice had whole-blood ⁶⁵Zn levels approximately double those of MT+/+ mice gavaged with 154–1540 nmol of Zn. The plasma Zn concentrations (Fig. 1 )increased with Zn dose in both genotypes but were much higher in MT-/- at all doses. There were significant differences in the intertissue ⁶⁵Zn distribution pattern between genotypes (Table 2) . MT-/- mice retained more ⁶⁵Zn in muscle, skin, brain and heart than MT+/+ mice given the oral Zn dose of 1540 nmol (Table 2) . In MT+/+ mice, however, the liver retained significantly more ⁶⁵Zn at all Zn doses 385 nmol. At the highest dose, livers of MT+/+ mice retained 21 nmol more Zn from the gavaged dose than those of MT-/- mice. The percentage of the Zn dose transferred declined with increasing dose. The uptake by muscle was constant at all Zn doses in MT-/- mice but fell by almost one third in MT+/+ mice. In liver, this fall in Zn uptake was much greater in MT-/- mice.

In MT+/+ mice (Fig. 2 ),liver MT was directly proportional to the oral Zn dose [y = 0.022x + 14.067; r = 0.9926, where y = MT (nmol Cd bound/g wet weight) and x = Zn dose (nmol)]. The difference in liver MT between MT+/+ mice receiving the 154 and 1540 nmol Zn oral doses was sufficient to incorporate an additional 27.5 nmol Zn in the liver (liver weight, 0.95 ± 0.02 g , n = 46). MT synthesis could thus account for the greater amount of Zn retained in the liver (21 nmol) in the MT+/+ mice administered 1540 nmol of Zn, compared with their MT-/- counterparts. In the small intestine, on the other hand, MT concentrations of MT+/+ mice were near maximum at a dose of 385 nmol Zn, with a slight further increase at the highest dose (Fig. 3 ).The difference in MT in the small intestine between the lowest and highest doses of oral Zn represented binding of only 8 nmol of Zn (small intestine weight, 0.83 ± 0.01 g, n = 46). Pancreatic MT concentrations were 129 ± 7.6 (nmol Cd bound/g wet tissue) at 154 nmol Zn, rising to 175 ± 7.3 nmol/g at 385 nmol Zn and remaining constant (165 ± 9.4 nmol/g) up to 1540 nmol Zn. MT in the small intestine and liver was measured in MT-/- mice and was 2.1 ± 0.1 nmol/g (n = 13) and 3.0 ± 0.1 nmol/g (n = 4), respectively, at an oral Zn dose of 770 nmol. These values were considered to represent nonspecific binding within the assay, not MT.

View larger version (12K): [in this window] [in a new window]   Figure 2. Liver metallothionein concentrations in MT+/+ mice after gastric gavage with varying amounts of Zn. Points represent the means and SEM, n = 5–14. Mice were deprived of food for 20 h then gavaged with 65Zn in selected amounts of ZnSO4 (154–1540 nmol) (see Materials and Methods). Mice were killed 4 h later and liver MT was determined by the Cd-hemoglobin affinity assay. Differences between means were analyzed by Tukey's post-hoc test by using the mean square error derived from ANOVA. aSignificantly different from mice gavaged with 154 nmol Zn, P < 0.05. bSignificantly different from mice gavaged with 385 nmol Zn, P < 0.05. cSignificantly different from mice gavaged with 770 nmol Zn, P < 0.05.

View larger version (13K): [in this window] [in a new window]   Figure 3. Small intestinal metallothionein concentrations in MT+/+ mice after gastric gavage with varying amounts of Zn. Points represent the means and SEM, n = 5–14. Mice were deprived of food for 20 h then gavaged with 65Zn in selected amounts of ZnSO4 (154–1540 nmol) (see Materials and Methods). Mice were killed 4 h later and MT was determined by the Cd-heme affinity assay on washed small intestine. Differences between means were analyzed by Tukey's post-hoc test by using the mean square error derived from ANOVA. aSignificantly different from mice gavaged with 154 nmol Zn, P < 0.05. bSignificantly different from mice gavaged with 385 nmol Zn, P < 0.05. cSignificantly different from mice gavaged with 770 nmol Zn, P < 0.05.

Endogenous Zn in the lumen of the small intestine (potential for variation caused by differences in isotope dilution between genotypes).

Absorption differences of ⁶⁵Zn between genotypes could be caused by inequalities in the endogenous Zn present in the small intestinal lumen, which alter the specific activity of the orally administered ⁶⁵Zn. The endogenous Zn present in the luminal wall and intestinal contents of both MT+/+ and MT-/- mice was determined 4 h after oral gavage with water. The total Zn (nmol) in the luminal contents of the duodenum-jejunum (MT+/+, 8.0 ± 2.0; MT-/-, 6.0 ± 1.2) and jejunum-ileum (MT+/+, 9.3 ± 1.7; MT-/-, 8.1 ± 1.0) were not significantly different between MT+/+ and MT-/- mice. The distal ileal Zn contents (MT+/+, 35.4 ± 11.6; MT-/-, 6.3 ± 2.4), particularly in the MT+/+ mice, covered a much broader range of values, but not at concentrations high enough to seriously influence the specific activities of the Zn doses given in the other experiments. Furthermore, the increment in ⁶⁵Zn in blood (Table 2) at each oral dose corresponded closely to that in plasma Zn determined by AAS, indicating that there were no significant isotope-dilution–related affects.

⁶⁵Zn absorption in MT+/+ and MT-/- mice: uptake from food.

Normal mice deprived of food for 20 h immediately before being fed 0.5 g of a solid egg-white diet containing 770 nmol ⁶⁵Zn had Zn transfer values at 4 h of only 2.97% of the administered dose, representing a Zn bioavailability that was 24% that of aqueous solutions (see Table 4 ).Interestingly, the corresponding value (1.66 %) for MT-/- mice was significantly lower at 13%. Plasma Zn concentrations remained within the normal range for all mice fed 770 nmol Zn in EWD, and ⁶⁵Zn in the blood was 18% (MT+/+) and 7% (MT-/-) that obtained from 770 nmol Zn gavaged in aqueous solution. The aqueous 154 nmol Zn dose gave a quantitatively similar Zn transfer to the 770 nmol solid diet. However, the former was associated with a more pronounced elevation in plasma Zn over baseline concentrations, especially in MT-/- mice.

	DISCUSSION

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In MT-/- mice, at intakes of 154–1540 nmol Zn per mouse, the transferred dose was directly proportional to the oral Zn dose with no evidence for saturation of a transport mechanism. Although the existence of a carrier-mediated process is not necessarily excluded because saturation may occur at a higher Zn dose than those used, it nonetheless appears that, without MT, the chief determinant of small intestinal absorption from Zn-containing solutions is luminal Zn concentration.

A similar Zn dose-dependent trend was seen inMT+/+ mice that was not significantly different from that in MT-/- mice by two-way ANOVA. Nonetheless, when the highest Zn dose was taken in isolation, Zn transfer in MT+/+ mice was 15% lower (P < 0.05, two-tailed Student's t test ) than in the MT-/- mice; thus we cannot exclude inhibition of Zn transfer by intestinal MT at higher Zn intakes. The mean difference in small intestinal MT between MT+/+ mice gavaged with the lowest and highest oral Zn doses (i.e., MT induced by the highest dose) represents the stoichiometric incorporation of only 8 nmol of Zn, much lower than the 36 nmol Zn difference in transferred doses between MT+/+ and MT-/- mice gavaged with 1540 nmol. If intestinal MT inhibits Zn absorption at high Zn intakes, then it would have to be a dynamic process in which Zn incorporated into MT is rapidly transferred to other ligands within the mucosal cells, or more likely, to one or more of the recently described cation diffusion facilitators that function as Zn transporters (ZnT-1 to ZnT-4) (Davis et al. 1998 , McMahon and Cousins 1998 , Palmiter and Findley 1995 , Palmiter et al. 1996 ). ZnT-1 would appear to be the most likely candidate because it serves as a Zn luminal effluxer and has been shown to have greater expression in response to high Zn intake (Davis et al. 1998 ).

Plasma Zn concentrations were above the normal level in all mice and significantly higher in MT-/- thanMT+/+ mice at oral aqueous doses from 154 nmol Zn, reaching 60.1± 2.3 and 29.3± 2.5 µmol/L, respectively, at the 1540 nmol Zn dose. This could result from a lower rate of Zn excretion in MT-/- mice, as suggested by Davis et al.(1998) ; they reported plasma Zn levels >200 µmol/L inMT-/- mice, 2.3 times the corresponding levels in normal mice, 2 h after an oral Zn dose equivalent to 12,500 nmol for a 25-g mouse. However, some doubt is cast on this explanation by our results from a preliminary study in which MT+/+ and MT-/- mice were injected subcutaneously with ⁶⁵Zn. After 6 h, the incorporated radioactivity, although similar in the gut wall in each genotype, was 20% higher in the luminal contents of MT-/- mice (A. M. Rofe et al., unpublished). A more probable explanation is that, in mice lacking MT expression, a less restricted flow of Zn from the intestinal lumen into the plasma compartment, in tandem with a lower rate of hepatic Zn clearance, forces plasma Zn to higher concentrations. It appears therefore, that MT-/- mice excrete more endogenous Zn than MT+/+ mice and are also able to absorb more exogenous Zn from the gut lumen from large aqueous doses, indicating that intestinal MT can inhibit Zn flux between the intestinal lumen and vascular compartment, in either direction.

Although we were unable to clearly demonstrate intergenotype differences in Zn transfer from aqueous solutions, an influence of MT on Zn distribution within the body was evident. In particular, blood ⁶⁵Zn concentration, in concert with the previously noted plasma Zn levels, was much higher in MT-/- mice at all Zn doses. At 1540 nmol Zn, ⁶⁵Zn levels were also greater in the muscle, skin, heart and brain of MT-/- mice. These elevations would be due at least in part to higher ⁶⁵Zn in the extracellular fluid compartment of MT-/- mice, in line with the plasma ⁶⁵Zn concentration. The liver, and to a lesser extent the pancreas, were the only tissues we examined that contained significantly more ⁶⁵Zn in MT+/+ than MT-/- mice. This difference in the liver was equivalent to a theoretical 21 nmol (40% more) in mice gavaged with 1540 nmol ⁶⁵Zn, an amount that approximated the 27.5 nmol difference in MT between MT+/+ mice dosed at 1540 and 154 nmol Zn. MT in the liver was found to increase in direct proportion to oral Zn dose over the range from 154 to 1540 nmol, in contrast to MT in the small intestine and pancreas, which had no further increase in response to doses >385 nmol Zn.

It could be argued that a higher rate of Zn MT synthesis in the liver augments Zn transfer from the intestine by lowering plasma Zn, hence steepening the Zn concentration gradient between the gut lumen and vascular compartment (via the mucosal and serosal surfaces of the enterocytes). On the other hand, plasma Zn concentrations in all mice gavaged with aqueous Zn were much higher than we have ever observed in fed or fasted mice, including those that consumed 770 nmol Zn mixed in EWD. The rise in liver MT is therefore more likely to have been caused by the gross increase in plasma Zn resulting from Zn administration in aqueous solution; this is the liver's adaptive response to an unphysiologically high Zn concentration in the blood rather than part of the normal Zn absorption process and is similar in nature to the rise in hepatic MT after intraperitoneal Zn injection (Coyle et al. 1995 ). This does not exclude a role for hepatic MT in Zn absorption during inflammation or pregnancy in which MT synthesis is triggered by cytokines and hormones rather than a rise in plasma Zn. Results from studies using aqueous Zn gavage must therefore be interpreted with caution. We would not agree that the oral Zn dose of 0.5 mmol/kg used by Davis (Davis et al. 1998 ) was, as claimed, "nutritionally relevant" when in our studies, ZnSO₄ solutions in doses as low as 0.006 mmol/kg (154 nmol per mouse) raised the mean plasma Zn concentration of MT-/- mice to 28.4±1.0 µmol/L, 10 µmol/L above our normal range for fed MT-/- mice. MT+/+ mice given the same Zn solution had plasma Zn concentrations of 20.4 ± 2.1 µmol/L, significantly lower than their MT-/- counterparts, demonstrating that the altered intestinal Zn processing in MT-/- mice described by Davis is demonstrable at Zn doses two orders of magnitude lower than the dose they employed.

The 770 nmol Zn dose, when consumed in food, however, gave no increase in plasma Zn concentrations at 4 h in either genotype. Uptake of Zn by tissues was therefore able to keep up with absorption of Zn into the bloodstream. However, there was a significantly higher Zn transfer in MT+/+ than MT-/- mice (P < 0.03) that was essentially uniform across tissues, irrespective of their MT status, e.g.,170% in kidneys and skin, 175% in muscle, 195% in liver and 180% overall. This suggests that gut MT is the major determinant of Zn transfer and confers an absorptive advantage only when Zn is incorporated into the normal dietary milieu.

The percentage of the Zn dose transferred from the EWD in mice appears low against the common perception that Zn absorption exceeds 20% in rodents. It must be emphasized, however, that the term Zn absorption used by others, does not fit the definition of Zn transfer that is used here; this transfer is strictly limited to the amount of Zn taken from the dietary supply into non-gut tissues and fluids, 4 h postingestion of the Zn dose. Various descriptions of Zn absorption may also include the Zn remaining in the gut mucosa and be measured after a different time interval. An earlier study by Flanagan et al. (1983) used similar definitions and produced findings in mice that are consistent with those reported here.

Zn balance studies, using profoundly Zn-deficient diets and metabolic cages, have demonstrated an obligatory loss from endogenous Zn reserves via gut and kidneys of ~5 µg/d in our mice (J. C. Philcox et al., unpublished data), although Zn losses through hair shedding, skin secretions and desquamation were not measured. A 3% transfer from 0.5 g of a 50 mg Zn/kg diet represents a transfer to tissues of 5.3 µg/d, assuming a normal food consumption of 3.5 g/d.

The mice fed the ⁶⁵Zn-EWD also had another 16% of the administered Zn dose in the gut proximal to (and including) the ileum. Much of this Zn would be expected to be in the mucosa rather than in the contents (see Table 3 ). Most of the remaining 80% of the dose was in the cecum, from which further absorption is possible (Gisbert-Gonzalez et al. 1996 ). A Zn transfer of 3% is therefore compatible with the provision for the basic Zn requirements of mice, including an allowance for a certain amount of exchange of endogenous for exogenous Zn, if the potentially transferable Zn remaining in the gut mucosa is taken into account.

A comparison with larger mammalian species would also indicate that mice absorb less Zn than currently thought. For example, mice, with their large surface area to mass ratio and high metabolic rate, consume five times more food per unit of body mass than humans and hence need to absorb only one fifth the percentage of trace elements from a similar diet to obtain an equivalent Zn uptake/body mass. We have observed Zn transfers approaching 40% in mice, but only during late gestation (Rofe et al. 1998 ); over a 5-d period, body mass increases by ~70% and maternal hepatic Zn reserves increase to 2.5 times those of nonpregnant mice (liver doubles in size, Zn MT increases from <3 to 100 nmol Cd bound/L). In addition, the total Zn content of the fetal livers near parturition is equivalent to that of another full-sized liver. There is obviously a need for a very large increase in Zn absorption over basal levels to facilitate this process.

Of note was the pancreatic ⁶⁵Zn in MT+/+ mice, 0.40 ± 0.08%, three times that of MT-/- mice, 0.13 ± 0.01%, suggesting greater retention of ⁶⁵Zn by pancreatic MT in MT+/+ mice but pancreatic secretion of ⁶⁵Zn into the gut of MT-/- mice. This difference, however, is insufficient to account for all of the difference in total ⁶⁵Zn transfer between mouse genotypes.

Zn transfer was approximately four (MT+/+ ) and eight times (MT-/- ) higher using an aqueous preparation. This may result from more efficient transfer of Zn from aqueous solutions to the mucous proteins within the stomach and/or enhanced Zn uptake during subsequent passage along the absorptive region(s) of the small intestine. Zn liganding to unabsorbable food constituents may have accounted in part for its relatively low absorption from EWD; however, gross differences in the physical nature of the Zn dose between the two nutrition regimens were much more likely to have been responsible. In this regard, the Zn dose in food was consumed over 85 min, diluted in 1200 mg of diet (including water), whereas the aqueous dose was gavaged instantaneously in 100 mg of water. Gut dynamics and hormonal response to food would be expected to further influence Zn absorption. A more rapid clearance of aqueous fluids from the stomach, resulting in a more concentrated bolus of Zn passing into the small intestine, may also favor greater Zn absorption from solutions, by simple mass action.

Nevertheless, gavage of Zn in aqueous solution does enable a level of reproducibility in absorption studies that cannot be attained by giving Zn in food, in which large differences in intraluminal Zn sequestration between diets can occur (Davies and Olpin 1979 ). In general, Zn incorporated into food is less well extracted because ligands, particularly phytate, cause retention of divalent cations within the gut lumen (Davies and Olpin 1979 , Whitehead et al. 1996 ). Use of aqueous Zn solutions has further advantages in enabling greater transfer of Zn to the tissues and is more useful in examining Zn regulatory systems in extremis. Liquid diets may also better resemble some therapeutic modes of Zn supplementation (or poisoning).

	FOOTNOTES

 
¹ To whom correspondence should be addressed.

¹ The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ''advertisement'' in accordance with 18 USC section 1734 solely to indicate this fact.

² Abbreviations used: AAS, atomic absorption spectrometry; CRIP, cysteine-rich intestinal peptide; EWD, egg-white diet; MT, metallothionein; ZnT, Zn transporter protein.

Manuscript received July 13, 1998. Initial review completed August 18, 1998. Revision accepted October 21, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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