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Institute of Nutrition Physiology, Department of Food and Nutrition, Technical University of Munich, D-85350 Freising-Weihenstephan, Germany
1To whom correspondence should be addressed. E-mail: roth_p{at}wzw.tum.de.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • zinc deficiency • dietary protein • protein synthesis • rats
The first characteristic signs of alimentary Zn deficiency in growing rats include lack of appetite, decreased feed intake and consequently depressed growth (1,2). The exact cause of the low feed intake in Zn deficiency is not known. Zn-depleted rats fail to grow because they do not eat enough. When rats are fed a Zn-deficient diet in physiologic amounts artificially by gavage, they become very sick within a few days. The more food that is administered daily, the earlier the rats become ill and the worse the external signs of clinical deficiency (3). The reason for this is thought to be an altered protein metabolism because increasing the dietary protein level also causes animals to become ill more quickly. Zn-deficient rats, given a choice between a high protein and a low protein diet, will choose the low protein diet (4,5). Zn-deficient rats are thus unable to utilize a more plentiful and protein-rich diet as energy for growth at an equivalent rate. Growth restrictions due to a reduction in feed intake must therefore be considered a useful adjustment to an inadequate Zn supply because this response at least delays the emergence of clinically apparent signs of deficiency. Zn-depleted rats who consumed feed ad libitum, resulting in a considerably lower feed and protein intake, survive for several weeks longer than force-fed Zn-depleted rats. The most significant growth-restricting factor in Zn deficiency is therefore thought to be the protein metabolism. In this study, we administered isoenergetic Zn-deficient diets with different protein levels to growing rats to determine whether changing the dietary protein content could influence or improve the Zn status of rats.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1 mg Zn/kg dry matter. The protein level of the Zn-deficient diets was increased continuously for these seven groups from 2 to 5, 8, 10, 15, 20 and 25% casein. All diets were isoenergetic, containing 16.5 kJ metabolizable energy (ME)/g diet. The energy differential due to the different protein levels was compensated by a corresponding modification of the carbohydrate content of the diets (Table 2). For the purpose of calculating the physiologic energy value of the diets, an energy content of 16.7 kJ/g was assumed for protein (casein) and carbohydrates and of 37.7 kJ/g for fat (soybean oil).
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The rats were kept in a controlled-environment rat chamber at 23°C, 60% relative humidity, with a 12-h light:dark cycle, at three rats per cage in metal-free macrolon cages. The rats had access to deionized drinking water in nipple bottles, which was adjusted to the mean osmolarity of tap water by adding 0.14 g/L reagent grade NaCl. The rats were weighed daily and then moved to freshly cleaned cages that had been washed out with deionized water. On d 21 of the trial, all rats were deprived of food for 10 h and then decapitated under ether anesthesia. Femur, quadriceps and liver were removed immediately from the exsanguinated rats, shock-frozen in liquid nitrogen at -196°C and kept at -80°C until needed for analytical processing. Blood was collected in Eppendorf reaction vessels, centrifuged for 5 min at 8000 x g for serum extraction and frozen in portions at -80°C. A further portion of blood was transferred to Sarstedt reaction vessels, which had been treated with lithium heparinate as anticoagulant and were immediately placed in an ice bath. After 30 min, the plasma was separated by centrifugation (8000 x g for 5 min) and the ammonium concentration determined with an enzyme diagnostic kit (Sigma-Aldrich, Deisenhofen, Germany).
The Zn status of the rats was estimated by measuring the activity of alkaline phosphatase in serum and the Zn concentrations in serum, bone, muscle and liver. Serum (200 µL) was diluted 1:5 with double-deionized water and the Zn concentration measured directly in the flame of an atomic absorption spectrometer (Perkin Elmer, Model 5100, Norwalk, CT). Femur, liver and muscle samples were dry-ashed for 48 h at 480°C in platinum crucibles in a muffle furnace, and the residue brought into solution with HCl (0.6 mol/L). The tissue samples were brought to volume and the Zn concentration was determined by atomic absorption spectrometry as described above for serum. The activity of alkaline phosphatase (EC 3.1.3.1) in serum was determined with an enzyme diagnostic kit (Sigma-Aldrich, Deisenhofen, Germany). The urea concentration in serum, the activity of alanine aminotransferase (EC 2.6.1.2) in plasma and liver and of glutamate dehydrogenase (EC 1.4.1.2) in liver were determined using commercially available enzymatic diagnostic kits (Roche, Mannheim, Germany). The activity of carbamoylphosphate synthetase (EC 6.3.4.16) in liver was measured by the method of Nuzum and Snodgrass (7). The enzyme units obtained were expressed with reference to the protein concentration of the sample material. The determination of the protein concentrations in the tissue homogenates was performed with bicinchonic acid according to Smith et al. (8).
The statistical analysis of the results was done by two-way ANOVA with subsequent Tukey’s test for comparison of zinc effects within protein levels and differences among protein levels. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Surprisingly, the rats fed a Zn-deficient diet with extremely low protein levels of 2 or 5% had no visible signs of Zn deficiency throughout the 3-wk trial. However, with increasing dietary protein levels, the incidence of Zn deficiency signs rose in the Zn-depleted rats toward the end of the trial, starting with slight redness of the skin and hair loss (8–10% dietary protein) and advancing to severe skin lesions and deep fissures on the snout and the extremities, with total alopecia in some cases (15–25% dietary protein). Moreover, the higher dietary protein levels of 15 to 25% led to an increasingly cyclical feed intake pattern in the Zn-deficient rats in a rhythm of 3–3.5 d.
Sensitive indices measured to assess the Zn status of rats included the Zn concentration and the activity of a Zn-metalloenzyme-like alkaline phosphatase in the serum. At dietary protein levels of 2 and 5%, no differences in the serum activity of alkaline phosphatase were observed between the Zn-deficient group and the two control groups (Fig. 3a). However, at a dietary protein level of 8%, activity of alkaline phosphatase in the serum of the Zn-depleted rats declined sharply (>50%) compared with the control rats. At increasing dietary protein levels(10–25%), the activity of alkaline phosphatase in the serum of the Zn-deficient rats was one third that of the control rats. At a dietary protein level of 2%, the Zn concentration in the serum, like the activity of alkaline phosphatase earlier, did not differ between Zn-deficient rats and control rats (Fig. 3b). At a dietary protein level of 5%, the Zn concentration in the serum of the Zn-depleted rats was half that in the two control groups, but was still sufficient to fully maintain the activity of alkaline phosphatase in serum. From dietary protein levels of 8% upward, the Zn concentration in the serum of Zn-deficient rats declined sharply to values ≤20% that of the two corresponding control groups. Both indices thus confirmed the presence of Zn deficiency in the zinc-depleted rats at dietary protein levels of 8% or higher.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The protein requirement of growing laboratory rats as stated by the NRC is 15% (24). In the study described here, the feed intake of the control rats peaked at a dietary protein level of only 8% and remained at that level up to 25% dietary protein. The feed intake of the control rats was thus not increased at a dietary protein level of 8 and 10% to compensate for the protein deficit relative to the rats with an adequate protein supply. As a consequence, the body weight of these rats was significantly lower at the end of the experiment than that of the rats fed 15, 20 and 25% protein in the diet (Fig. 1b). This means that the protein requirement of these rats is likely somewhere in the region of 10–15% dietary protein, which is in close agreement with the results of Du et al. (25) who estimated a requirement of 12.5% dietary protein. The feed intake of the Zn-depleted rats rose only slightly from 8% dietary protein upward (Fig. 1a) and remained steady up to a dietary protein level of 25%, as did the their body weight (Fig. 1b). On the other hand, clinical signs of Zn deficiency appeared earlier and became more severe with rising protein levels in the Zn-depleted rats, and an increasingly cyclical feed intake pattern developed, as previously described by other authors in Zn-deficient rats with dietary protein levels of 20% (26–30). The pair-fed control rats had significantly higher body weights from 10% dietary protein upward, despite an identical feed intake as dictated by the experimental design. This confirms previous reports of reduced digestibility and decreased intermediate utilization of food (10,12) and malabsorption of nutrients in alimentary Zn deficiency (31).
In the rats with extremely low dietary protein levels of 2 and 5%, neither feed intake nor body weight were influenced by Zn deficiency (Fig. 1a,b). The zinc-depleted rats also did not exhibit any Zn deficiency signs. The activity of alkaline phosphatase, a Zn metalloenzyme, and the Zn concentration in the serum, both of which are considered sensitive indices for estimating the Zn status, did not differ between Zn-depleted rats and control rats at the lowest protein level of 2%. At 5% dietary protein, the alkaline phosphatase activity in the Zn-depleted rats was still unchanged in relation to both control groups, whereas the serum Zn concentration was already reduced by half (Fig. 3b). The osseous system, which contains 30% of the body’s total zinc content, had a significant decrease in the Zn concentration compared with both control groups even at 2 and 5% dietary protein. This means that these rats, who showed no growth due to the low feed intake and the resulting shortage of energy and protein, and hence achieved no or minimal net protein synthesis, had a very low Zn requirement. In the case of the Zn-depleted rats, the mobilization of zinc from the osseous system was therefore sufficient at these two low protein levels to meet the low Zn requirement for most biochemical functions; thus, neither signs of Zn deficiency nor a cyclical feeding pattern developed throughout the trial. With increasing dietary protein levels, feed intake and growth of the Zn-deficient rats also increased slightly, leading to a higher Zn requirement and hence greater mobilization of zinc from the osseous system. As the Zn requirement increased, the mobilization of zinc from bone became progressively insufficient to maintain all vital functions, resulting in a higher incidence of clinical Zn deficiency signs and lower feed intakes. If less food is eaten, the body compensates by increasing the breakdown of endogenous protein through catabolic processes. This gives rise to ammonia, which has to be detoxified; this in turn requires energy and causes the feed intake to rise, leading to the familiar cyclical pattern of feed intake by Zn-depleted rats in a 3- to 3.5-d rhythm. The dietary protein level is the key factor for the cyclical feed intake. Reducing the protein level of the Zn-deficient diet to <8% causes this pattern of cyclical feed intake to disappear in the rats, as was observed also by other authors (4).
The Zn concentration in soft tissues such as muscle, brain, liver and skin is not available for mobilization to support important metabolic functions (32). This means that the muscle tissue, which accounts for 60% of the body’s total zinc content, or the liver itself, could not be drawn upon for mobilization of zinc in severe alimentary Zn deficiency (Fig. 4b,c). By contrast, the Zn concentration of the femur in the experimental Zn deficiency study described here, as well as in previous studies (9,33,34), declined by up to 70%. Zn concentration in the bones can therefore be considered the method of choice for estimating the Zn status of experimental animals under standardized conditions.
It can be presumed that in the rats with extremely low dietary protein levels of 2 or 5%, large amounts of endogenous protein were metabolized, causing the rats in all three treatment groups to be in a catabolic state. The likely consequences of this protein catabolism include raised concentrations of urea in the serum (Fig. 5a). The greatest proportion of amino acids is normally utilized for protein synthesis, whereas only a small proportion is involved in transamination and deamination reactions. With increasing dietary protein levels (8–10%), the amino acids transported to the liver were derived mainly from the breakdown of dietary protein in the gastrointestinal tract rather than degradation of endogenous protein, causing the urea concentration in the serum to fall. When the body is in nitrogen balance, urea synthesis is minimal and a direct function of protein intake. From 15% dietary protein upwards, the serum urea concentration rose once again (Fig. 5a), indicating that the protein requirement of these rats was between 10 and 15%. The concentrations of urea and ammonia in serum and plasma were not different between Zn-depleted and control rats (Fig. 5a,b), as was also observed in an earlier study (35). When the level of dietary protein rose above the requirement, the proportion of amino acids for transamination and deamination reactions increased, as demonstrated by the increased urea concentration in the serum. The significant increase in the activity of alanine aminotransferase in the serum of Zn-depleted rats relative to both control groups and in the liver relative to the control group at protein levels of 10 and 15% or higher (Fig. 6a,b) indicated damage to the liver cells and hence a disturbance of the protein metabolism in Zn deficiency, as was also observed by Eder and Kirchgessner (36).
There are several ways in which the highly toxic ammonia can be fixed in metabolism; urea synthesis by the liver is quantitatively by far the most important. The key enzyme for the synthesis of urea and the first enzyme in the urea cycle is carbamoylphosphate synthetase. The activity of this enzyme was significantly increased in the Zn-depleted rats with dietary protein levels of 10–25% compared with the two control groups (Fig. 8). Because feed intake and hence the dietary protein supply were identical in the Zn-depleted rats and the pair-fed controls, it can be assumed that the increased activity of carbamoylphosphate synthetase was the result of excessive protein degradation and disturbed protein synthesis. Another route for detoxifying the ammonia produced in metabolism is via glutamate dehydrogenase in the liver because the equilibrium constant favors the formation of glutamate over ammonia. It was shown in a previous study that Zn deficiency increases the activity of glutamate dehydrogenase in rat liver (35), as was the case here at dietary protein levels ≥15% (Fig. 7). This indicates increased elimination of ammonia in Zn-deficient rats if their diets contain protein concentrations in excess of the requirement. The greater nitrogen excretion in the urine of Zn-depleted rats fed a high protein diet (10) supports this statement and is thought to be due to a defective protein metabolism in these rats. Other authors have also shown conclusively that zinc stimulates protein synthesis (37,38) and that Zn deficiency weakens protein synthesis (20), making zinc an essential factor for protein synthesis.
In summary, we conclude that growth restrictions due to a reduction in feed intake can be considered a useful adjustment to an inadequate zinc supply because this response at least delays the emergence of clinically apparent signs of Zn deficiency. If the protein intake is so low that it supports only minimal net protein synthesis, then the rat’s Zn requirement is correspondingly low and mobilization of zinc from the skeleton is sufficient to prevent the occurrence of deficiency signs for a considerable time. If the dietary protein level meets or slightly exceeds the requirement of growing rats, then the rat’s mobilizable Zn stores are fairly rapidly depleted to cover the increased Zn requirement for protein synthesis. Once this happens, feed intake decreases and takes on a cyclical pattern, accompanied by distinct clinical Zn deficiency signs. The reasons for reduced feed intakes and decreased growth of Zn-depleted rats with high dietary protein levels compared with control rats are thought to be due to disturbed protein synthesis, as demonstrated by the increased activities of alanine aminotransferase, glutamate dehydrogenase and carbamoylphosphate synthetase in the liver. Zinc, as an essential component of the diet, is thus vital for the efficient utilization of dietary protein.
Manuscript received 16 January 2003. Initial review completed 7 February 2003. Revision accepted 10 April 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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