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Department of Animal Science, Michigan State University, East Lansing, MI
3To whom correspondence should be addressed. E-mail: hillgre{at}msu.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • metallothionein • pharmacological zinc • phytase • pig
In recent years, Zn supplementation has been used in developing countries to treat diarrheal disease in young children (1,2). In addition, the U.S. swine industry adds 2000–3000 mg Zn/kg as zinc oxide (ZnO) to the diets of newly weaned pigs for the first 14 d postweaning to promote growth (3,4) and fecal consistency (5,6), both of which can be impaired by weaning stress. Feeding pharmacological Zn (3000 mg/kg) for 14 d to newly weaned pigs improves gut morphology by increasing villous height and reducing crypt depth in the duodenum and jejunum, thus potentially increasing the absorptive capacity of the small intestine and consequently improving growth (7).
Since the 1960s, there has been increasing evidence that phytic acid, an organic molecule found in cereal grains, possesses antinutritional properties (8,9). Phytic acid forms insoluble salts with bivalent cations such as calcium (Ca) and Zn, thus affecting their bioavailability at neutral and alkaline pH, leading to increased mineral excretion and reduced mineral absorption (10). Because pigs consume grain diets and lack the ability to produce sufficient phytase endogenously, supplementation of this enzyme in feed has been shown to improve mineral bioavailability and decrease nutrient excretion (11). However, the effect of phytase when pharmacological Zn is fed has not been evaluated.
Metallothionein (MT)4 is a low-molecular-weight (
7 kDa), cysteine-rich protein present in many living organisms (12). This protein has been considered to be an intracellular marker of excess Zn inside cells, based on the increased induction of MT when dietary Zn intakes are well above normal (13). Despite many decades of research, the precise physiologic function of MT has not been elucidated. However, some suggested functions attributed to this protein include detoxification of nonessential heavy metals (cadmium and mercury), homeostasis of Zn and copper (Cu), metal transfer, free-radical scavenger, and metal storage (14,15).
Transcriptional regulation of MT by dietary Zn was demonstrated in rats and cultured cells treated with Actinomycin D (16). Moreover, evidence suggests that transcriptional regulation of the MT gene occurs by activation of metal response elements located in the promoter region of the MT gene, in response to the binding of intracellular Zn to metal transcription factor-1 (MTF-1) (17). The MTF-1 acts as an intracellular Zn sensor able to coordinate several genes involved in Zn homeostasis (18).
The purpose of this experiment was to determine the effects of supplementing pharmacological Zn with or without phytase on liver, kidney, and intestinal mucosa MT mRNA abundance and protein concentration. In previous research in which swine diets contained pharmacological Zn, an interaction between Zn, Cu, and iron (Fe) was observed (5,19). Thus, the dietary effect of pharmacological Zn and phytase on hepatic, renal, and plasma mineral (Cu, Fe, Zn and P) concentration of pigs was also investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Mineral analysis. All glassware used during the analysis was soaked for 12 h in 500 mmol/L nitric acid solution, followed by five rinses with deionized-distilled water. To determine mineral concentrations, the tissue and feed were microwave digested (21). The concentrations of Cu, Fe, and Zn were determined by flame atomic absorption spectrometry (Unicam 989 Atomic Absorption Spectrometer, SOLAAR Series); tissue P concentration was detected by a colorimetric method (22) utilizing a Beckman DU7400 spectrophotometer (Beckman Coulter). Bovine liver was used as standard reference (1577b, U.S. Department of Commerce, National Institute of Standards and Technology) and was analyzed with the tissue samples for instrument standardization and quality control.
Plasma samples were deproteinized with 800 mmol/L trichloroacetic acid solution before the Cu, P, and Zn analyses. Briefly, plasma samples were added 1:4 (v:v), mixed on a vortex, centrifuged at 2000 x g for 15 min, and analyzed for Cu and Zn by flame atomic absorption spectrometry, and P by a colorimetric method (22). The number of plasma samples available for the P analysis varied among the dietary treatments (Zn150, n = 3; Zn150P, n = 2; Zn1000, n = 4; Zn1000P, n = 2; Zn2000, n = 2; Zn2000P,n = 4) due to inadequate sample volume for each pig. Plasma Fe was determined by flame atomic absorption spectrometry (4).
MT assay. The MT protein concentration was determined by a modification of the silver saturation assay (23). A RBC hemolysate was prepared from fresh porcine blood (24). Reagents were prepared daily. Tissue (0.2–0.5 g) was homogenized (Janke & Kunkel Ultramax T25, Tekmar Company) in 250 mmol/L sucrose buffer (1:4) and centrifuged at 18,000 x g for 20 min. For renal and intestinal samples from pigs fed Zn150 and Zn150P, two additional repetitions of the hemolysate step were performed.
RNA isolation. Total RNA was isolated from kidney, liver, and intestinal mucosa cells using TRIzol Reagent (GIBCO BRL, Life Technologies) according to the manufacturer’s protocol. RNA concentrations were determined by absorption at 260 nm (A260) using a Beckman DU650 spectrophotometer (Beckman Coulter). RNA quality and integrity were determined by calculating the A260/280 ratio and by agarose gel electrophoresis, respectively.
Dot blot and northern blot analysis. Duplicate dot blots were prepared as previously described (25) by spotting 1, 3, and 5 µg of denatured kidney, liver, or intestinal mucosa RNA (n = 6) onto nylon membranes (Hybond-XL, Amersham Biosciences). Total RNA samples were also subjected to Northern blot analysis as previously described (25) to determine the number and size of MT transcripts. The dot and Northern blots were hybridized with a mouse MT-1 cDNA probe (provided by J. Carrasco and J. Hidalgo of the Universidad Autónoma de Barcelona, Barcelona, Spain) using modifications of standard procedures (26,27). Blots were also probed with a porcine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe for normalization (28). Probes were synthesized using 32P-dCTP (Perkin Elmer, Life Sciences) and the Multiprime DNA Labeling System (RPN 1601Z, Amersham Biosciences). Hybridizations were done at 65°C for 16–22 h. Membranes were rinsed as previously described (25) and exposed to Kodak BioMax film (Eastman Kodak) in the presence of intensifying screens at -80°C for 1–4 d. Quantification of MT and GAPDH relative mRNA abundance was achieved by densitometry scan of autoradiographs using a Fluor-S MultiImager (Bio-Rad).
Statistical analysis. The data were analyzed using regular analysis of variance procedures based on the MIXED procedure of SAS (29). Factors of interest always included zinc, phytase, and their interaction. The experimental unit in the growth performance analysis was the pen, whereas for plasma and tissue minerals, and MT protein concentration, the individual pigs served as experimental units. For MT mRNA abundance, the blot by treatment (zinc x phytase) interaction was modeled additionally along with pig as random effects to define the experimental units for this analysis, with Satterthwaite’s approximation used to determine the error df for test. Furthermore, GAPDH mRNA abundance was modeled as a covariate for a regression-based normalization of MT mRNA abundance in accordance with recent recommendations by Poehlman (30).
With the exception of Fe values, a logarithmic transformation was required to make the data more normally distributed. Thus, back-transformed means and their respective 95% CI (lower and upper limits) are provided as point and interval estimates, respectively. Scheffé’s test was used to determine the presence of linearity of response to treatments.
The Fe concentrations and remaining log-transformed values were used for a residual correlation analysis between plasma and tissue mineral concentrations, MT relative mRNA abundance, and protein concentrations. Each pair of traits was jointly modeled using a series of bivariate mixed effects models that each included the specified between-trait residual correlation to be estimated. SAS PROC MIXED was used to do this in a way that is very similar to what was also recently considered by Thiebaut et al. (31). The residual correlation is an adjusted or partial correlation. That is, after the mixed model effects (e.g., zinc, phytase, zinc x phytase, blot, blot x zinc x phytase, pig) are accounted or adjusted for, the correlation for whatever is left over (hence the term residual correlation) between responses of interest is estimated. Residual correlations were considered to be different from 0 when P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Feeding pharmacological Zn (1000 or 2000 mg Zn/kg) with or without phytase to the nursery pigs did not affect plasma Cu (Table 2). However, a significant interaction of phytase and dietary Zn on renal Cu was obtained (P < 0.05). The dietary treatments did not significantly affect hepatic Cu or Fe, or plasma or renal Fe concentrations (data not shown). Plasma P concentration was higher (P < 0.05) in the pigs fed the phytase-supplemented diets, except in the pigs fed Zn2000P. In this study, the pigs’ renal P concentration was not affected by the dietary treatments. However, both dietary Zn (P < 0.05) and supplemental phytase (P < 0.05) increased hepatic P (Table 2).
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Northern blot analysis was performed to determine the number and size of MT transcripts in kidney, liver, and intestinal mucosa. The analysis revealed the presence of a single MT transcript (0.5 kb) in all three tissues, although no transcript was detected in the intestinal mucosa of pigs fed Zn150 (data not shown). The blots were stripped and reprobed with GAPDH and a single transcript (1.2 kb) was detected in all of the samples (data not shown).
Dot blot analysis was performed to determine relative MT mRNA abundance in liver (Fig. 2), kidney, and intestinal mucosa of individual pigs. The pigs fed Zn1000P, Zn2000, or Zn2000P had greater MT mRNA abundance than pigs fed adequate Zn (Zn150, Zn150P) or 1000 mg Zn/kg (Zn1000). To correct for loading differences, all dot blots were stripped and reprobed with porcine GAPDH (data not shown).
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The results of the mineral residual correlation (Table 3) demonstrated that in the liver and kidney, Cu and Fe were significantly correlated. Moreover, in the kidney, Zn was correlated with kidney Cu (P < 0.01) and Fe (P < 0.02). However, residual correlations between plasma mineral concentrations and tissue minerals, relative MT mRNA abundance, and protein were not significant (data not shown).
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Growth performance.
Daily weight gains differed during wk 1 of the study. Pigs fed supplemental phytase gained weight faster (203.49 ± 33.20 vs. 238.76 ± 34.55 g/d, P < 0.03). For the remainder of the experiment, daily body weight gain, feed intake, and feed efficiency did not differ among the groups (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Proposed mechanisms for enhanced growth due to pharmacological Zn include increased metallothionein, a protein that plays a key role in Zn homeostasis by sequestering Zn in the intestine, and improved gut morphology (7). In agreement with studies of rats and mice that examined dietary Zn regulation of the MT gene (16,34), in this study we confirmed that pharmacological Zn and phytase supplementation increase the abundance of MT mRNA in the liver, kidney, and intestinal mucosa cells of nursery pigs. Northern blot analyses of liver, kidney, and intestinal mucosa RNA revealed the presence of an 0.5-kb MT transcript, which is consistent with a rat transcript size previously published for MT (35).
Liver, the tissue with the highest concentrations of Zn and MT, was the most responsive tissue, similar to the findings of Carlson et al. (4). Pigs fed 1000 mg Zn/kg with no phytase had liver MT mRNA abundance and protein concentration similar to those of pigs fed adequate Zn, regardless of phytase inclusion. However, when phytase was added at 1000 mg Zn/kg, MT mRNA and protein concentration increased and were similar to those of pigs fed 2000 mg Zn/kg with or without phytase. Interestingly, this dietary treatment effect also occurred with Zn concentrations in the liver, suggesting that zinc and phytase supplementation increases the amount of Zn absorbed, and this is shown by an overall greater MT mRNA abundance, MT protein, and Zn concentration.
In renal tissue, the response pattern for MT mRNA abundance and protein differed from that obtained in the liver. A significant interaction of zinc and phytase on MT mRNA abundance was obtained. Metallothionein protein concentration increased with dietary Zn, whereas phytase did not have a synergistic effect. Nevertheless, MT mRNA abundance, protein, and Zn concentrations were positively correlated (P < 0.01), similar to the findings of Blalock et al. (36) in which the kidney MT mRNA abundance in rats fed 180 mg Zn/kg was linearly correlated with total MT protein. Blalock et al. also observed a tissue difference in the induction of both MT mRNA and protein.
The amount of intestinal mucosa MT differed from that in the liver and kidney, emphasizing tissue specificity. The amount induced in pigs fed Zn150, Zn150P, or Zn1000 was below the detection limits of the MT mRNA abundance and protein assays; hence no data are reported. Metallothionein mRNA abundance and protein concentrations from pigs fed Zn2000P were higher than in pigs fed Zn1000P or Zn2000. This suggests that in the upper intestine, MT is induced to a greater extent when exposed to high dietary Zn, 10 to 20 times the requirement. The effect is increased by cosupplementation of phytase with pharmacological Zn (2000 mg Zn/kg), an effect not seen with other tissues. In addition, the early work of Richards and Cousins (16) showed that dietary Zn regulates MT, which plays a key role in Zn absorption by controlling the amount of Zn entering the body and transferred into portal circulation.
Using the Zn chelator, Zinquin, and immunohistochemical analysis, Tran et al. (13) reported that rats fed increasing concentrations of Zn (0, 400 mg Zn/kg) displayed an increased proportion of the Zn attached to the luminal surface of the gut, but when Zn was fed at a pharmacological concentration (1000 mg Zn/kg), more Zn was internalized and sequestered by MT synthesized de novo. Perhaps the unique MT increase observed in the intestinal mucosa in pigs fed Zn2000P prevents additional Zn from entering the circulation, thus preventing higher Zn concentrations in the liver. Moreover, it is possible that the hepatic Zn pool of pigs fed Zn150, Zn150P, or Zn1000, could be processed efficiently by several mechanisms, one of which might be MT. However, when pigs are fed Zn1000P, Zn2000, or Zn2000P, the MT concentrations reached a plateau, indicating that an alternate pathway might be required to metabolize this Zn overload.
In the plasma, Zn concentrations increased linearly (P < 0.05) with dietary Zn. However, there were no significant residual correlations between plasma and tissue mineral concentrations, MT protein, or MT mRNA abundance. Similar results were found in young broilers by Sandoval et al. (37). Thus, the use of plasma Zn as an absorption or mineral status indicator in pigs is of little value.
Feeding the combination of Zn and phytase to pigs increased renal Cu concentration (P < 0.05), which is similar to observations by Carlson et al. (4) when nursery pigs were fed 3000 mg Zn/kg (ZnO) for up to 28 d. This documented effect of Zn on Cu metabolism was observed previously in nursery pigs whose mothers were fed pharmacological Zn (5000 mg Zn/kg) for two parities (19). Hepatic Cu concentrations were depressed in young pigs, an effect that has also been observed in humans and rats. The lack of response in hepatic Cu in this study may be due to the limited time of the dietary intervention.
Even though it has been suggested that the Ca to P ratio can be adjusted to obtain a significant phytase effect (38), our experimental diets were formulated to meet the P requirement as established by the NRC for swine (20). Pigs fed the highest Zn diets as well as those fed phytase had increased (P < 0.05) P concentrations in the liver, in agreement with similar results obtained in our laboratory in which a numerical increase in hepatic P concentration was obtained in pigs fed 2000 mg Zn/kg for 14 d (39). This unique pharmacological Zn effect on P metabolism deserves further investigation. In addition, plasma P was also higher (P < 0.05) in pigs fed supplemental phytase. These results demonstrate an apparent increase in P availability caused primarily by phytase supplementation, which is consistent with previous research demonstrating that phytase supplementation increases overall P retention in pigs (11).
In conclusion, these findings support the hypothesis that pigs fed pharmacological Zn have increased organ Zn characterized by increased MT mRNA abundance and MT protein concentrations. Moreover, supplementing phytase further enhanced these effects when a minimum of 1000 mg Zn/kg was fed. Due to the role of MT in sequestering Zn when excess Zn is fed, we hypothesize that adequate dietary Zn concentrations (150 mg Zn/kg) did not provide sufficient Zn to induce MT significantly, even when phytase was supplemented. However, adding phytase to 1000 mg Zn/kg diet did provide the additional Zn necessary for higher MT mRNA abundance and protein compared with 1000 mg Zn/kg alone. These data suggest that current pharmacological doses of Zn fed to pigs (2000 mg Zn/kg) could be reduced to 1000 mg Zn/kg by adding phytase. This dietary modification could result in the enhanced growth benefits of pharmacological Zn, as well as reduced nutrient excretion, especially Zn, by newly weaned pigs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported in part by an Animal Health Formula Fund Grant from the Michigan Agricultural Experiment Station, East Lansing, Michigan.
4 Abbreviations used: FTU, phytase units; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MT, metallothionein; MTF, metal transcription factor; Z150, 150 mg Zn/kg; Zn150P, 150 mg Zn/kg with phytase; Zn1000, 1000 mg Zn/kg; Zn1000P, 1000 mg Zn/kg with phytase; Zn2000, 2000 mg Zn/kg; Zn2000P, 2000 mg Zn/kg with phytase.
Manuscript received 15 August 2003. Initial review completed 6 October 2003. Revision accepted 1 December 2003.
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