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Article

Zn-Depleted Mice Absorb More of an Intragastric Zn Solution by a Metallothionein-Enhanced Process Than Do Zn-Replete Mice

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

¹To whom correspondence should be addressed.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The influence of metallothionein (MT)² on Zn absorption was investigated in MT-null (MT-/-) and normal (MT+/+) mice fed Zn-depleted (ZnD) diets for 7 d and compared with those fed Zn-replete (ZnR) diets in a previous study. Mice were starved for 20 h, then administered an oral gavage of aqueous ⁶⁵ZnSO₄ solution at doses of 154, 770 or 1540 nmol of Zn, and the amount transferred into nongut tissues was determined 4 h later. ⁶⁵Zn transfer did not differ between genotypes in ZnR mice. However ZnD MT+/+ mice had a 30–40% greater transfer from the 154 and 770 Zn doses compared to ZnR MT+/+ mice. This was not observed in MT-/- mice. In MT+/+ mice, Zn depletion enhanced the induction of MT by Zn in the intestine and pancreas. ⁶⁵Zn uptakes in the liver and pancreas were greater in MT+/+ than MT-/- mice, and this was greater (50%) at the 154 and 770 doses in mice fed ZnD diets. Plasma Zn concentrations were raised to a similar extent in ZnR and ZnD MT-/- mice. ZnR MT+/+ mice had significantly lower plasma Zn levels than MT-/-mice; this difference was less marked in the ZnD mice. We conclude that a MT-facilitated enhancement in Zn absorption occurs in response to dietary Zn deficiency.

KEY WORDS: • Zinc • metallothionein • intestinal absorption • metallothionein-null mice • Zn deficiency

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Zn absorption is primarily influenced by the amount ingested, physicochemical interaction of binding ligands in liquid and food, gut regional pH, retention by the glycocalyx, gastric emptying and intestinal transit rate (Whitehead et al. 1996 ). Part of the complex gut dynamics are underlying paracellular and transcellular Zn-transport processes that are thought to take place primarily in the upper small intestine (Lönnerdal 1989 ). There is substantial evidence supporting the view that Zn absorption is in part facilitated by carrier-mediated processes (Cousins 1985 , Lönnerdal 1989 ). Despite intensive research, however, the interrelationships between the ligands that facilitate the uptake, secretion and transport of Zn across the intestinal wall are only now being unraveled. In yeast, high- and low-affinity Zn transporters have been identified which have concentrations regulated by extracellular Zn, both by transcriptional regulation of the transporter protein genes and by control of the rate of degradation of the protein itself (Gitan et al. 1998 ). Another family of Zn transporters (ZnT-1 to ZnT-4)² has been described in rodents, providing compelling evidence for carrier-mediated Zn transport in eukaryotes (Huang and Gitschier 1997 , Palmiter et al. 1996a ,b , , Palmiter and Findley 1995 ). ZnT-1 in particular, has been located in the basolateral membrane of the small intestine; its mRNA has been shown to be increased by high dietary Zn, and it appears to be essential for Zn efflux from enterocytes (McMahon and Cousins 1998 ). Transporters controlling the influx of Zn, however, remain elusive. Recently, an iron transporter protein, namely divalent cation transporter –1, with a broad specificity for other metals including Zn, has been characterized (Gunshin et al. 1997 ). DCT-1 mRNA level has been found to be highest in duodenal crypts, and its expression is increased in iron deficiency. Whether the expression of DCT-1 is influenced by deficiency of other divalent cations such as Zn remains to be demonstrated.

Mucosal-to-luminal secretion of endogenous Zn may occur simultaneously with Zn absorption and also contribute to luminal Zn concentration and hence bioavailability (Evans et al. 1979 , Flanagan et al. 1983 , Hoadley et al. 1988 ). Many studies have demonstrated enhanced efficiency of Zn absorption in ZnD animals (Flanagan et al. 1983 , Hoadley et al. 1988 , Jackson et al. 1981 , Smith and Cousins 1980 , Weigand and Kirchgessner 1980 ). It has been argued that this may result from increased absorption at low Zn intakes, decreased secretion of endogenous Zn or a balance between the two (Lönnerdal 1989 ). Control of Zn homeostasis appears to species-specific, with absorption being regulated more than secretion in rats but just the opposite in mice (Flanagan et al. 1983 ).

Induction of metallothionein (MT) synthesis in mucosal cells is triggered by both fasting and high luminal zinc concentrations but is not significantly induced at normal dietary zinc intakes (Cousins 1985 , Tran et al. 1998 , 1999 ). Additionally, pancreatic Zn secretion is an important component of Zn homeostasis, and we have recently shown that MT-/- mice sequester less Zn in the pancreas and secrete more into the intestinal lumen (Rofe et al. 1999 ). There was clear demonstration of increased pancreatic secretion in MT-/- mice, although increased mucosal-to-luminal secretion could not be ruled out.

The relative contribution of intestinal MT to Zn absorption and secretory processes is contentious (Evans et al. 1979 , Hoadley et al. 1988 , Starcher et al. 1980 ). Evans and co-workers (1979) did not find a role for MT in Zn absorption, whereas others have found that MT can facilitate Zn absorption when dietary Zn is limited (Hoadley et al. 1988 ). It has also been argued that when there is excess Zn in the diet, MT sequesters Zn in the intestinal wall, thereby transiently reducing the absorption of Zn and favoring Zn transfer back into the gut lumen (Cousins 1985 , Lönnerdal 1989 ). More recently Davis and coworkers (1998) found that MT knockout mice had higher intestinal Zn content than mice which overexpress MT after being given intragastric Zn, indicating that MT does not reduce Zn absorption solely by sequestering Zn in the mucosa.

Recently we found that Zn absorption was the same in normal and MT-/- mice fed a ZnR diet and given an intragastric dose of Zn in aqueous solution (Coyle et al. 1999 ). The presence of MT, however, conferred an absorptive advantage when Zn was given in solid food. This may indicate that intestinal MT aids in sequestering Zn when it is present in the lumen either attached to food-ligands or when Zn supply is limited. Whether MT plays a role in Zn absorption in ZnD mice has not been examined. In this report, we examine the influence of MT on Zn absorption in ZnD mice by determining the uptake of ⁶⁵Zn in normal and MT-/- mice fed ZnD diets for 7 d.

	MATERIALS AND METHODS

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MT-/- mice were F3 derivatives of the interbreeding of MT+/+ C57BL6 (Animal Resources Center, Canning Vale, Western Australia) and MT-/- mice produced at the Murdoch Institute, Royal Children’s Hospital, Victoria, Australia (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 in plastic cages with stainless steel wire floors: 45 cm x 16 cm, 7.5 mm mesh, 1 mm wire diameter to prevent coprophagy, in an animal house at 22°C with a 14-h light/10-h dark cycle. Weight and sex matched mice from each genotype were housed together for 7 d in groups of eight and given free access to water and a ZnD purified diet (Coyle et al. 1999 ). In this study, we compare the results on ZnD mice with our previously published study on ZnR mice (Coyle et al. 1999 ). The ZnD and ZnR studies were conducted within a 6-mo period with several overlapping experiments. Mice used in both studies were from cohorts derived from the same parents. The ⁶⁵Zn, carrier-Zn and experimental protocols were the same in both studies. The Zn concentrations of the ZnR and ZnD diets as determined by atomic absorption spectrometry were 100 and 0.8 Zn mg/kg, respectively. After 7 d of consuming these diets, plasma Zn concentrations were 11.5 ± 0.7 and 13.0 ± 1.4 µmol/L in four ZnR MT+/+ and MT-/- mice, respectively, and 7.7 ± 0.3 and 10.7 ± 0.3 in five mice fed ZnD diets.

⁶⁵Zn Absorption in MT+/+ and MT-/- mice.

Mice (weight after food deprivation: ZnR, 22.9 ± 0.3 g; ZnD, 21.5 ± 0.2 g) were starved for 20 h prior to oral gavage with 0.1 mL of ⁶⁵Zn (37 kBq; NEN Life Science Products, Boston, MA) containing 154, 770 or 1540 nmol [10, 50 or 100 µg, respectively,] of Zn as ZnSO₄. Four hours later, blood was taken from each mouse by cardiac puncture under light halothane anesthesia before killing by cervical luxation. The carcass was carefully dissected and staged to avoid cross-contamination with ⁶⁵Zn. A 4-cm² sample 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 viz: 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 using a cassette-fed Packard Auto-Gamma Counter, model 5650 (Canberra-Packard, Melbourne, Australia).

The term "Zn transfer" is defined here as the fraction of ⁶⁵Zn absorbed from the gavaged dose into all nongut tissues. It is the sum of the ⁶⁵Zn uptakes of nongut tissues expressed as a percentage of the oral ⁶⁵Zn dose. The total ⁶⁵Zn associated with whole blood, plasma, skin and muscle was calculated as follows:

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 of Tris buffer pH 7.4 using an Ultra-Turrax homogenizer, and MT was determined by a Cd/Heme assay [modified from Eaton and Toal (1982) ]. Plasma Zn concentrations were determined by atomic absorption spectrometry.

Statistical analyses.

Three-way ANOVA using the general linear model on Minitab (Minitab State College, PA) was used for comparison of differences between means for factors of genotype (MT+/+ or MT-/-), diets (ZnR or ZnD) and oral Zn dose (154, 770 or 1540 nmol). 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, an estimate of the SD across treatments. When interactions were significant, the post-hoc test of Tukey 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.

Plasma Zn increased in proportion to the oral Zn dose and in MT-/- mice was approximately double that in MT+/+ mice gavaged with the highest Zn dose. Plasma Zn levels were significantly higher in MT+/+ mice fed the ZnD than those fed the ZnR diet when gavaged the two highest Zn doses and in MT-/- mice fed ZnD, gavaged with 770 nmol of Zn. In order to determine the body distribution of Zn, the uptake of ⁶⁵Zn was determined in various tissues 4 h after intragastric gavage, and the results are presented in Table 1 . The recovery of the oral dose, including that in the gastrointestinal tract, was 96.3 ± 0.9% (n = 131).

The uptake of ⁶⁵Zn and the relative effects of both genotype and diet varied between tissues. Diet and genotype affected blood ⁶⁵Zn levels. MT-/- mice had blood ⁶⁵Zn levels on average 90% higher than MT+/+ mice for all oral Zn doses. Blood ⁶⁵Zn uptakes were greater in MT+/+ (at 770 nmol) and MT-/- mice (at 154 nmol) fed ZnD than those fed ZnR.

In muscle and skin, ⁶⁵Zn uptakes were higher in MT-/- than MT+/+ mice gavaged with 1540 nmol of Zn. In muscle, ⁶⁵Zn levels were one-third greater in mice of both genotypes fed ZnD than ZnR at a Zn dose of 154 nmol and in MT+/+ mice gavaged with 770 nmol Zn. The uptake of ⁶⁵Zn by skin was also higher in MT-/- mice fed ZnD than ZnR at the lowest oral Zn dose. In heart, ⁶⁵Zn levels were 38 and 53% higher in MT-/- mice than in MT+/+ mice fed the ZnR diet for doses of 770 and 1540 nmol of Zn, respectively, and were greater in MT-/- mice fed the ZnD diet compared with those fed the ZnR diet at 154 nmol of Zn. Likewise in brain, MT-/- mice had higher uptakes than MT+/+ mice when gavaged with 154 nmol of Zn regardless of diet, or when gavaged with 1540 nmol of Zn and fed the ZnR diet. Brain ⁶⁵Zn levels were greater in mice of both genotypes fed ZnD than ZnR diet and gavaged with 154 nmol Zn, and in MT+/+ mice gavaged with the highest Zn dose.

Liver ⁶⁵Zn levels were unaffected by diet. ⁶⁵Zn uptake in the liver of MT+/+ mice was 47 and 119% higher than MT-/- mice when fed the ZnD diet and gavaged with 154 and 770 nmol of Zn, respectively. Likewise, the pancreatic uptake of ⁶⁵Zn was unaffected by diet but was different between genotypes. Pancreatic uptakes in MT+/+ mice were 50–140% higher than MT-/- mice, gavaged with 154 nmol of Zn, and 55% higher in those mice fed the ZnD diet and gavaged with 770 nmol of Zn. The uptake of ⁶⁵Zn by kidneys, spleen and lungs (the latter two not shown) were unaffected by diet or genotype.

In summary, at the lower Zn doses, ⁶⁵Zn uptake was generally higher in blood, muscle, skin, heart and brain of MT+/+ and MT-/- mice fed the ZnD compared with the ZnR diets. In addition, MT-/- mice had higher ⁶⁵Zn uptakes in those individual tissues than their MT+/+ counterparts. In contrast, liver and pancreas had higher ⁶⁵Zn uptakes in MT+/+ mice regardless of diet. The transferred dose, which is an estimate of the combined intertissue changes, showed higher ⁶⁵Zn uptakes in MT+/+ mice fed the ZnD diet primarily because of the increased levels in liver and pancreas.

The radioactivity remaining in the gut wall and contents 4 h after intragastric dosing with ⁶⁵Zn was determined (Table 2 ). Very little radioactivity was found in the stomach, and this did not differ due to diet, genotype or oral Zn doses. The fraction of the dose retained in the upper two-thirds of the small intestine was inversely proportional to the oral Zn dose but not affected by diet or genotype. The counts retained in the ileum were mainly greater in MT-/- (36.3 ± 2.5% of ⁶⁵Zn dose) than MT+/+ mice (17.0 ± 1.7% of ⁶⁵Zn dose), and there was a correspondingly lower ⁶⁵Zn level in the cecum and colon in the former. On average, the washed ileal wall retained ~50% of the counts in the combined wall and contents, and reflected the same effect of genotype, with higher ⁶⁵Zn levels found in MT-/- than MT+/+ mice. The counts in the ileum + cecum + colon were 67.1 ± 1.2% of the oral dose, and were the same for both diet and genotype. In summary, the findings indicate that approximately the same bolus of ⁶⁵Zn reaches the distal small intestine, cecum and colon regardless of genotype, but MT-/- mice had proportionally more counts in the ileum than in the large bowel, suggesting that they have a slower intestinal transit than MT+/+ mice. Nonetheless, at 4 h, the majority of the oral dose had passed the main absorptive region of the small intestine in both genotypes.

View larger version (16K): [in this window] [in a new window]   Figure 1. Metallothionein (MT) concentrations in liver (panel A), small intestine (panel B) and pancreas (panel C) of MT+/+ mice after gastric gavage with varying amounts of Zn. Points represent the means and SEM, n = 5–16. Mice were fed for 7 d on a purified egg-white diet that was either Zn-replete (100 mg of Zn/kg) or Zn-depleted (0.8 mg of Zn/kg). They were then deprived of food for 20 h and gavaged with 65Zn in selected amounts of ZnSO4 (154–1540 nmol) (see the Materials and Methods section). Mice were killed 4 h later, and liver MT was determined by the Cd-hemoglobin affinity assay. MT concentrations in liver and small intestine of Zn-replete mice have previously been published (Coyle et al. 1999). Differences between means were analyzed by the post-hoc Tukey test using the mean square error derived from ANOVA. * Significantly different from mice fed Zn-replete diets at the same oral Zn dose, P < 0.05.

Pancreatic MT concentrations were proportional to the oral Zn dose [ZnR, y = 0.026x + 119.5; r = 0.905, ZnD; y = 0.103x + 62.2; r = 0.989, where y = MT (nmol Cd bound/g wet pancreas) and x = Zn dose (nmol)] (Fig. 1C ). MT levels in the pancreas of mice fed ZnD diets were more responsive to the oral Zn dose than those fed ZnR diets. In the ZnD group, MT values were 33% lower than their ZnR counterparts when gavaged with 154 nmol of Zn and 38% higher with the highest oral dose.

	DISCUSSION

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Discussion of Zn absorption in MT+/+ and MT-/- mice fed ZnR diets has been presented in a previous report (Coyle et al. 1999 ). Maintenance of Zn homeostasis during periods of Zn depletion by regulating the efficiency of Zn absorption has been questioned. Many earlier studies using orally administered ⁶⁵Zn to demonstrate regulation of Zn absorption have been criticized because of poor experimental design. In particular, it has been argued that endogenous Zn present in the lumen of the small intestine may have affected the specific activity of the ⁶⁵Zn, resulting in the wrong conclusions being drawn as to the amount of Zn absorption (Flanagan et al. 1983 ). These investigators controlled for endogenous Zn levels in the small intestine and found that in rats but not in mice, intestinal Zn absorption increased in response to dietary Zn deficiency. Our findings with mice do not support theirs and demonstrate that, like rats, ZnD mice absorb proportionally more Zn from an intragastric dose than ZnR mice, which is consistent with the premise that Zn homeostasis is in part maintained by regulation of Zn absorption.

The endogenous Zn levels in the absorptive region of the small intestinal lumen were determined in mice fed ZnR or ZnD diets for 7 d, starved for 20 h, but gavaged with water rather than radioactive Zn. The total Zn (nmol) in the luminal contents in the duodenum-jejunum (ZnR: MT+/+, 8.0 ± 2.0, MT-/-, 6.0 ± 1.2. ZnD: MT+/+, 7.4 ± 0.9, MT-/- 13.2 ± 2.3) and jejunum-ileum (ZnR: MT+/+, 9.3 ± 1.7, MT-/-, 8.1 ± 1.0. ZnD: MT+/+, 11.1 ± 2.9, MT-/-, 13.2 ± 2.0) were not significantly different among groups. Thus, differences in the specific activity of ⁶⁵Zn between ZnR and ZnD are too small to influence comparisons, particularly at the 770 nmol Zn dose. It could be argued that Flanagan and coworkers (1983) used a lower oral Zn dose of 100 nmol, rendering their absorption measurements more susceptible to differences in endogenous Zn.

In the present study, ⁶⁵Zn transfer into nongut tissues was 30 and 40% higher in ZnD MT+/+ mice than in their ZnR counterparts given the low and normal Zn doses. While the increased efficiency of Zn absorption was significant only in MT+/+ mice, there was nonetheless, no significant difference between genotypes fed the ZnD diet. MT-/- mice fed the ZnD diet and gavaged with the lowest Zn dose had significantly higher plasma Zn concentrations and greater Zn uptakes in blood, muscle, skin, heart and brain than those fed ZnR diets. These findings indicate that Zn absorption may be augmented by an MT independent process in ZnD mice at lower dietary Zn intakes. While mechanisms for Zn uptake await elucidation in eukaryotes, a saturable high- affinity transporter that is active in Zn limited cells and a low-affinity transporter which works in ZnR cells have been characterized in yeast (Gitan et al. 1998 ). These transporter proteins are both transcriptionally and post-translationally regulated by Zn. In eukaryotes, a saturable iron transporter protein, DCT-1, is increased in iron deficiency and has broad specificity for other metals including Zn (Gunshin et al. 1997 ). A Zn-specific transporter that is upregulated in ZnD states remains to be found.

Although the transferred dose was the same in MT+/+ and MT-/- mice fed ZnD diets, there were significant intergenotype differences in ⁶⁵Zn distribution among tissues. In general, at one or more of the oral Zn doses, ⁶⁵Zn uptakes were greater in blood, muscle, skin and brain in MT-/- than MT+/+ mice. In liver and pancreas, however, the relative uptakes were reversed with greater uptakes in MT+/+ than MT-/- mice. Similar differential uptakes in tissues between genotypes have followed the systemic injection of ⁶⁵Zn (Rofe et al. 1999 ). Increased Zn uptake by liver and pancreas in MT+/+ mice paralleled increased MT accumulation in these tissues. In liver, MT accumulation in MT+/+ mice fed ZnD and given 154 or 770 nmol of Zn represents the stoichiometric incorporation of ~22 and 32 nmol of Zn/g (liver wt 0.96 ± 0.02 g, n = 29), respectively. This accumulation of MT is sufficient to account for all of the extra Zn (2.6% of 154 nmol and 3.9% of the 770 nmol, oral Zn dose) found in the liver of MT+/+ mice compared to that in MT-/- mice fed the ZnD diet. In the pancreas, there was only a small increase in MT accumulation in MT+/+ mice fed ZnR diets but a greater increase in those fed ZnD diets. In the latter, MT levels were ~200 nmol/g, which represents the stoichiometric incorporation of ~40 nmol of Zn (pancreas wt 0.20 ± 0.01 g, n = 29). Thus, the dose-related increase of MT in the pancreas of mice fed ZnD diets was sufficient to account for the increase in Zn uptake (1–2% of lowest two oral Zn doses) compared to that in MT-/- mice fed the same diet.

This study also demonstrates that, in the small intestine, pancreas and liver, MT synthesis is proportional to the oral Zn load. It is further shown that MT induction can occur within the postprandial period, suggesting that MT is responsive to fluctuations in dietary Zn. ZnD mice had MT inductions 2.5- and 4-fold greater than ZnR mice in the intestine and pancreas. It is a commonly held view that MT synthesis in the small intestine is associated with a reduction in Zn absorption (Cousins 1985 ). However, enhanced MT synthesis occurred concurrently with increased Zn absorption in the small intestine of ZnD mice when given the oral dose of 770 nmol of Zn. Moreover, in our previous study we showed that MT+/+ mice absorbed 80% more Zn (dose of 770 nmol incorporated into an egg-white meal) than MT-/- mice, despite the presence of intestinal MT (Coyle et al. 1999 ). Thus, MT appears to enhance Zn absorption in ZnD mice fed intragastric Zn and in ZnR mice fed Zn in a meal. It is possible that the enhanced MT synthesis could reflect a higher flux of Zn through the enterocyte as a result of an upregulation of Zn transport under conditions in which Zn supply is limited. Under such a circumstance, MT could capture Zn in the enteroctye to be later processed for absorption or secretion, or it might facilitate Zn transfer to other ligands within the mucosal cells. Zinc-transporter protein-1 may be important in this regard because it is the Zn transporter which is localized on the basolateral membrane and appears to mediate the efflux of Zn from the enterocyte to the serosa (McMahon and Cousins 1998 , Palmiter and Findley 1995 ).

The responsiveness of pancreatic MT synthesis to Zn in the ZnD mice needs to be considered in light of the role of the pancreas in Zn secretion (Lönnerdal 1989 ). We have previously demonstrated that MT-null mice secrete more ⁶⁵Zn from a systemically injected dose and that a major contributor to this extra Zn loss was the pancreas (Rofe et al. 1999 ). The remaining loss was via intestinal mucosal-to-luminal transfer. It can be postulated that the increased responsiveness of MT induction in the pancreas in the ZnD mice may reflect the ability to limit Zn loss by this route. Another interpretation of the increased MT in the pancreas in ZnD mice is that sequestration of Zn for Zn-dependent digestive enzymes is of central importance when Zn intake is limited. The responsiveness of intestinal MT to ZnD can be considered in the light of limiting serosal-to-luminal Zn loss.

It has been argued that changes in plasma Zn concentrations can be used as an indicator of Zn absorption (Davis et al. 1998 ). In ZnD mice fed an intragastric Zn dose, plasma Zn levels were on average 13% higher in MT-/- mice and 23% higher in MT+/+ mice than in their ZnR counterparts. However, the greatest difference in plasma Zn concentrations was between genotypes with levels in MT-/- mice, being nearly double those in MT+/+ controls. These findings are in agreement with those of Davis and coworkers (1998) who reported concentrations in MT-/- mice 2.3 times those in controls, 2 h after receiving an oral gavage. Our studies indicate that the plasma Zn concentration is determined as much by MT-facilitated uptake in certain tissues (e.g., liver and pancreas) as by intestinal Zn absorption. MT-/- mice lack this means of Zn removal from the plasma, and hence show a strong association (P < 0.001) between plasma Zn and the amount of Zn absorbed, [ZnR; y = 0.1566x + 22.548, r = 0.900, ZnD; y = 0.2437x + 18.358, r = 0.921, where y = plasma Zn (µmol/L) and x = Zn absorbed (nmol)]. Only when MT+/+ mice were fed the ZnD diet did plasma Zn correlate with Zn absorbed: (y = 0.1503x + 13.434, r = 0.812, P < 0.001). No association was found in ZnR MT+/+ mice (y = 0.0388x + 21.04, r = 0.305), indicating that plasma Zn concentrations are determined by Zn absorption in MT+/+ mice fed ZnD but not in those fed ZnR diets. In the latter case, MT synthesis in various tissues was less responsive to Zn dose than in the ZnD mice, but still sufficient to prevent the plasma Zn from reaching the levels in MT-/- mice. We believe that, in the absence of MT, there is less impediment to Zn transfer from the small intestine into the plasma which, when coupled with less sequestration of Zn by liver and pancreas, causes plasma Zn concentrations to be higher. Other studies in which MT-/- and MT+/+ mice were given either intraperitoneal (Coyle et al. 1995 ) or subcutaneous (Rofe et al. 1999 ) injections of Zn have shown that MT-/- mice maintained elevated plasma Zn concentrations longer and excreted more endogenous Zn than controls.

The distribution of nonabsorbed radioactivity along the gut of mice fed ZnR or ZnD diets was similar, indicating that the Zn content does not greatly influence gut transit. After 4 h, the majority of the oral Zn dose had moved past the major Zn absorptive region of the upper small intestine, and the radioactivity was found mainly in the ileum, cecum and colon. There was an effect of genotype on the distribution of ⁶⁵Zn. MT-/- mice had slower gut transit with more radiolabel found in the ileal wall and contents and less in the cecum and colon than MT+/+ mice. It is possible that the transferred dose may be an underestimate of the optimal absorption in MT-/- mice, as more ileal Zn could be absorbed had the experiment been carried out for a period longer than 4 h. In a previous study, however, we determined ⁶⁵Zn uptake in MT-/- mice 6 h after administration of oral Zn dose and found that the transferred dose was the same as that at 4 h, despite nearly all of the radioactivity having passed into the cecum and colon. Zn appears to be absorbed mainly in the upper small intestine, with the ileum contributing little (Coyle et al. 1999 ).

In general, regardless of treatment, nearly 50% of the radioactivity retained in the ileum was associated with the washed ileal wall, and this percentage was even higher in proximal small intestine that was largely devoid of contents. There were no genotypic differences in ⁶⁵Zn uptake in the wall of the proximal intestine but MT-/- mice, on average, had higher ileal uptakes than MT+/+ mice. This trend occurred despite a marked dose-related MT accumulation in the small intestine that was most apparent in MT+/+ mice fed ZnD diets. These findings indicate that the amount of Zn incorporated into the wall of the small intestine is independent of MT accumulation. In previous studies, mice and rats fed varying amounts of Zn in their diets for 7 d had a dose-related increase in mucosal Zn levels, ~80% of which appeared to be associated with membranes and structural protein rather than the cytosolic fraction of the mucosal cells (Tran et al. 1998 , 1999 ). Although intestinal MT increased in proportion with dietary Zn, it represented only a small fraction of Zn associated with the enterocytes. These studies indicate that Zn is sequestered from the luminal contents and is largely bound to ligands on the external surface of the enterocyte. That which passes into the enterocyte at normal Zn intakes is either involved in metabolism or is absorbed or secreted in response to whole body Zn status. MT would appear not to accumulate until excessive amounts of Zn are transferred through the enterocyte.

	FOOTNOTES

 
² Abbreviations used: MT, metallothionein; MT-null, MT-/-; MT-normal, MT+/+; ZnD, zinc-depleted; ZnR, zinc-replete.

Manuscript received July 29, 1999. Initial review completed September 2, 1999. Revision accepted November 30, 1999.

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