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THE USE OF 65ZINC FOR THE ESTABLISHMENT OF THE MECHANISM OF ZINC HOMEOSTASIS

D. Oberleas, Texas Tech University (Emeritus), Lubbock, Texas, 79423, USA
B.F. Harland, Howard University, Washington, D.C. 20059 USA

Abstract

The mechanism for the maintenance of zinc homeostasis has been established. Phytate, a natural compound in plant seeds, has been shown to be the important component in the alteration of that equilibrium for monogastric species. Since phytate is a component of virtually all diets of humans throughout the world, its presence dictates consideration in the study of zinc homeostasis. The pancreas is an important organ in the maintenance of zinc homeostasis. The pancreas, by virtue of its location, is a difficult organ to study. Thus it is important to understand the endogenous flux of this organ to establish a mechanism for zinc homeostasis. 65Zinc was appropriate to establish this mechanism in an animal model however a stable isotope could be used for the verification in humans. Three important factors must be considered to establish an appropriate model. First, the animal or human subject must be slightly depleted of zinc to establish space in the acinar cells of the pancreas for the injected zinc to equilibrate within the acinar cells. Second, it requires 5 to 7 days to establish equilibrium within the acinar cells so that a differential can be established. For this reason, a 10 to 14 day fecal collection is necessary to determine the size of the endogenous pool. Third, dietary phytate may need to be present in at least half of the diets at a molar ratio greater than 10, with respect to zinc, to observe and quantify the endogenous contribution to the fecal excretion. It is due to the lack of these factors that a valid and appropriate computer model has not as yet been established for humans.

Introduction

Several experimental models are available with animals for the production of zinc deficiency. The most popular diet utilizes egg albumin (ovalbumin) as a protein source. While experimentally it may have some merit, on a practical scale as a human model it has no merit. No human population is consuming this type of diet. This diet is almost devoid of zinc and thus depends upon depletion of the body zinc stores by elimination without dietary replacement. The absolute replacement of zinc in the human body requires about 5 mg zinc per day [1]. This requirement has been derived by several investigators and is easily determined. The average diet has been determined to contain 9 to 12 mg zinc per day [2]. In southern Iran, where clinical zinc deficiency was first described, dietary zinc intake was estimated between 22 and 30 mg per day [3]. Since zinc deficiency is obviously the most prevalent nutritional deficiency worldwide, it seems unlikely that zinc deficiency is as simple as the lack of dietary intake.

Almost every diet worldwide contains phytate, a natural component of mature plant seeds. Phytate is contained in the bran and germ of cereal grains and is complexed to proteins in legumes. In many developing countries, foods of plant seed origin constitute the major portion of the diet. Chemically, phytate has been shown to complex with several di- or multivalent elements [4]. This complexation is dependent on the molar concentration, pH and atomic density. Phytate has been shown to affect the absorption of iron, ferric greater than ferrous. This effect on iron must be on the dietary pool because iron is not recycled through the gastrointestinal tract. Calcium, magnesium and zinc are the divalent cations that have been shown to be recycled through the pancreas [5].

The purpose of this presentation is to point out the necessary precautions that must be considered in the study and establishment of a homeostasis model for zinc in monogastric species.

Experimental

Male Sprague-Dawley rats (60-80 or 120-130 g starting weight) were used as the experimental subjects. In these experiments, each animal was injected intravenously or intraperitoneally with 370 kBq of 65zinc per animal. The animals were placed in individual metabolic cages, fed balanced diets with one group receiving casein as the non-phytate containing protein source and the other group isolated soybean protein as the phytate containing protein source. Dietary calcium levels of 8 or 16 g/kg were also experimental variables. Elevated calcium enhances the complexation of zinc by phytate and accentuates the effect on zinc homeostasis [6]. Distilled water was available at all times.

Daily fecal collections were made for each animal at about the same time each day and were counted for 5 minutes. Animals that were in homeostasis at the time of zinc injection, did not provide equilibrium within the pancreas within the 14-day collection period (D Oberleas, unpublished data 1981). Therefore, the counts were not different between the experimental groups. In subsequent experiments, the animals were placed on a zinc deficient diet for 1 or 4 weeks prior to allotment to experimental groups and injection with 65zinc. A 14-day collection period with individual daily counting followed.

Results

The mean weight of each rat was recorded on a weekly basis to monitor the progress of zinc deficiency. Figure 1 shows the growth curve for the first experiment in which the induction of a deficiency preceded the injection of 65zinc. A daily record for a 14-day collection period followed intraperitoneal injection of radioactive zinc. The diet utilized in each case was the isolated soybean-protein diet, high or low calcium, appropriate for that experiment. The effect of zinc deficiency upon growth rate is more apparent for the high calcium diet. Though less obvious for the low calcium diet, growth stimulation was observed when casein replaced the isolated soybean-protein.

Figure 1. Growth curve for rats depleted for 4 weeks with a 20% isolated soybean protein
diet prior to injection of 65zinc. Dietary calcium levels were 8 and 16 g/kg. The same isolated
soybean protein diets and similar casein diets were fed during the collection period.

Figure 2 shows the daily fecal radioactivity counts per minute per animal for the above animals. What should be noticed was the artifacts that occurred during the early days of fecal collection. Thus, since the endogenous zinc was via the pancreas, the equilibrium within the pancreas did not occur until 5 to 7 days following the injection. Thus a collection period greater than 7 days would be necessary to detect differences if they existed. To date collections beyond 14 days have not been reported.


Figure 2. The daily fecal excretion of radioactive zinc as affected by protein source
and dietary calcium level following 4 weeks of dietary zinc depletion.

If there were no effect of phytate on the endogenously secreted zinc, the ratios of counts (isolated soybean protein/casein protein) would have been equal to 1. Thus Figure 3 shows only those ratios for soy-assay protein diets to that of casein-based diets above 1. Under the circumstances of this experiment, the ratios for days 7 through 14 show clearly the effect of phytate on the endogenously secreted zinc. This is equivalent to the previous estimates of pancreatic secretion being 2 to 4 times that of the dietary zinc [5].

Figure 3. The mean ratio above 1 of 65zinc excreted by rats following intraperitoneal injection
as affected by dietary protein source and dietary calcium level.

To study the effect of a shorter depletion period on the equilibrium and endogenous secretion of zinc, Figure 4 shows the growth parameters of a one-week depletion followed by 65zinc injection and a 14-day collection period. The rats had a starting weight greater than the previous experiment. The effects of dietary variables were clearly evident in these data.

Figure 4. Growth curves for rats depleted of zinc for 1 week with a 20% isolated soybean protein
diet prior to intraperitoneal injection with 65zinc. Dietary calcium levels were 8 and 16 g/kg.
The same isolated soybean protein diets and similar casein protein diets were
fed during the collection period.

Though the secretions had somewhat fewer artifacts in these animals, a clear pattern of secretion, Figure 5, indicates that the secretion pattern was not stabilized until the 5th day post-injection. Again the secretion pattern indicated that a short-term experiment would not provide adequate equilibrium for reliable measurements of endogenous secretion.

Figure 5. The daily fecal excretion of radioactive zinc as affected by protein source and
dietary calcium level following 1 week of zinc depletion.

Figure 6 again shows that in this short-term depletion, ratios of pancreatic zinc secretion between the dietary variables were less obvious and were not apparent on both series until day 9 of the collection period.

Figure 6. The mean ratio above 1 of 65zinc excreted by rats following 1 week of
dietary zinc depletion, intraperitoneal injection and as affected by dietary
protein source and dietary calcium level.


Discussion

When studying zinc homeostasis in animals or humans, there are three major compartments that provide the most influence. The dietary pool provides the initial input or replacement of zinc to the body. The pancreatic zinc secretion has been studied in several species: dogs [7, 8, mice 9], pig [10], ruminants [5], and rats [11]. The pancreas secretes back into the duodenum 2 to 4 times the dietary intake for a day thus representing milligram quantities of zinc secretion each day. Fecal output is the major excretory pathway for zinc. In each of these species, except mice, and humans the quantity of zinc in these three compartments is expressed in milligrams whereas urinary excretion in humans is 400 to 600 micrograms per day. Other insensible losses have only a minor effect on total zinc homeostasis. For humans these compartments have been estimated as follows: dietary intake, 9-12 mg per day [12-14], pancreatic secretion has been estimated between 20 and 35 mg per day [2]. About 10% of the pancreatic zinc secretion is associated with carboxypeptidase A and B and possibly other large proteins that represent obligatory losses; the remainder of the zinc may be secreted in a fragile protein matrix that becomes inorganic zinc in the duodenum [15]. Fecal excretion is 5.1 to 10.3 mg per day, dependent on the phytate intake [16]. This provides a duodenal content of 30 to 40 mg of zinc each day. Obviously a major portion of this duodenal zinc, of dietary and pancreatic origin, must be absorbed and reabsorbed to sustain homeostasis.

Of the endogenous pancreatic divalent cation secretions only zinc is a trace element. These complexations are pH dependent and concentration sensitive. Again zinc, at equal concentrations to other pancreatic cations, forms complexes with phytate that are least soluble at pH 6, the approximate pH of the duodenum, compared to other endogenous cations [4].

In creating a computer model for humans, it is necessary to be able to estimate accurately the fluxes of each of these compartments. The dietary pool and the fecal pool can be readily sampled and quantified by several standardized means. Other zinc fluxes have been estimated by the injection and/or oral gavage of either radioactive or stable zinc isotopes [17-22]. The compartment most difficult to study has been the pancreatic endogenous secretion. The pancreas is the zinc storage organ in the body [23]. The pancreatic zinc stores are in dynamic equilibrium with other body compartments but mainly via secretion into the duodenum and reabsorption to supply zinc to various organs and tissues in the body. The above results demonstrate that the uptake of zinc from the bloodstream by the pancreas is not a simple, straightforward process. The pancreatic zinc appears to have a first-in; first-out mechanism. The pancreas also has two zinc pools, a stable pool represented by that zinc that is tightly bound to large proteins such as carboxypeptidase A and B, and a labile pool that represents the recycled and reabsorbable zinc that is more readily depleted. The data show the necessity of depleting the pancreatic pool, probably the labile pool, in order for equilibrium to be formed between the stable intracellular zinc and either radioactively labeled or enriched stable isotopes of zinc. By studying the data between Figures 3 and 6, the greater the depletion of the animals before the injection of 65zinc, the greater is the precision with which pancreatic secretion can be determined.

Conclusions

The problem of creating an appropriate and accurate computer model for the flux of zinc within the body becomes how to accurately measure the secretion and the effect of phytate on the endogenously secreted zinc. The pancreas is hidden beneath the liver, thus with the injection of 65zinc and the use of external counters, it is not possible to differentiate between liver zinc and pancreatic zinc. To study the effect of phytate that is necessary to establish a homeostasis model, the radioactive zinc secreted into the duodenum can be studied by feeding differential diets, one containing phytate and one that is phytate-free. Thus two circumstances are necessary to obtain reliable results from pancreatic secretion. The first is to precede the injection of 65zinc with the induction of a deficiency of zinc. In rats, the depletion phase required is up to 4 weeks to provide a representative equilibrium. The greater the depletion, the better is the estimate of the dynamic flux of zinc back into the duodenum. The second is the collection period sufficient to observe the appropriate equilibrium in the pancreas. Due to the lack of consideration of these necessary factors, a valid and appropriate computer model has not as yet been established for humans.

References

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