N. T. Davies

The effects of phytate on intestinal absorption and secretion of zinc, and whole-body retention of Zn, copper, iron and manganese in rats

1. The inclusion of phytate (10 g/kg) in a purified diet containing zinc (15 mg/kg) fed to young male rats significantly reduced growth rate and food intake, and promoted a cyclic pattern of food intake characteristic of an uncomplicated Zn deficiency. The decreased growth rate could be accounted for by the reduced food consumption. 2. Rats maintained on a Zn-deficient diet (0.5 mg Zn/kg) were found to have a cyclic pattern of food intake and a very slight weight gain. The addition of phytate (10 g/kg) to the Zn-deficient diet promoted a net loss of mean body-weight. 3. Rats maintained on the Zn-supplemented diet containing phytate excreted significantly more Zn in their faeces than either pair-fed or ad lib.-fed control rats. Rats given the Zn-deficient diet supplemented with phytate excreted more Zn in their faeces than Zn-deficient control rats. 4. Dietary phytate significantly reduced the average daily accumulation (mug/d) and whole-body retention (relative to dietary intake) of iron, copper, manganese and Zn, whether or not the diet was supplemented with Zn. 5. The addition of phytate to the lumen fluid of ligated loops of rat duodenum maintained in situ significantly inhibited 65Zn absorption, compared with the control systems without added phytate. 6. Other studies using ligated duodenal and ileal loops indicated that Zn is secreted into the gut lumen and approximately one-third of this is normally reabsorbed. Recycling of endogenous Zn may be a significant process in the over-all body economy of this trace element. 7. The absorption of 65Zn added to the diet was significantly reduced by dietary phytate. Dietary phytate also reduced the biological half-life of body 65Zn from 61 to 211 h post-administration, possibly by inhibiting reabsorption of endogenous 65Zn and thus promoting a more rapid loss from the body.

Studies on the phytate: zinc molar contents in diets as a determinant of Zn availability to young rats.

1. Studies were carried out in vitro to examine the effects of phytate on the solubility of the trace elements zinc, copper and manganese. Appropriate volumes of a solution of sodium phytate were added to a mineral solution to achieve phytate: Zn values of from 0 : 1 to 45 : 1. In a second series the same values for phytate: Zn were achieved by varying the amount of added Zn at a fixed phytate concentration. 2. In both experiments greater than 85% of the Zn was rendered insoluble at pH 6.5 even at the lowest value for phytate: Zn (5 : 1). The effect of phytate on Zn solubility was greater than effects on Cu or Mn. 3. In a dietary study, rats were offered a semi-synthetic egg-albumin-based diet with added phytate. Two series of diets were prepared, the first had a constant Zn content (18.5 mg Zn/kg) and the amount of sodium phytate varied so as to achieve values for phytate: Zn of from 0 : 1 to 40 : 1 (series 1). In the second series, the same values for phytate: Zn were achieved by adding a fixed amount of phytate (7.4 g phytic acid/kg) while the amount of Zn was varied (series 2). 4. Dietary phytate caused significant reductions in growth rates, plasma Zn concentrations and hair Zn concentrations and greying of the coat at values for phytate: Zn of 15 : 1, 10 : 1, 15 : 1 and 15 : 1, respectively. 5. While phytate was apparently slightly more effective in reducing Zn status when phytate: Zn values were achieved at the lower absolute levels of phytate and zn (series 1 diets), the differences at equivalent phytate: Zn values were small. It was concluded that phytate: Zn values can be used as an indicator of Zn availability from phytate-rich diets. 6. Rats offered three diets containing soya-bean-based textured-vegetable-protein (TVP) exhibited low rates of weight gain compared with rats offered an egg-albumen-based diet of similar Zn content (14.5 mg Zn/kg). Additional Zn supplied in drinking-water (25 mg Zn//l) was without effect on rats consuming the egg-albumin diet but significantly improved the weight gain of rats on the TVP diets. 7. It was concluded that phytate naturally present in TVP behaves similarly to phytate added to an otherwise phytate-free diet and that the reduced availability of Zn in TVP diets can be accounted for entirely by their phytate contents.

The effect of pregnancy and lactation on the absorption of zinc and lysine by the rat duodenum in situ

1. The absorption of zinc by the duodenum of the rat was greatly enhanced at late stages of pregnancy and during lactation. 2. During pregnancy no increase in lysine uptake could be demonstrated, but during lactation, when further increases in Zn absorption occurred, uptake of lysine was increased. 3. The increased absorption of Zn at different stages of pregnancy and lactation appeared to be related to the demand made by the developing foetuses and post-natal offspring.

The similarity between alkaline phosphatase ( EC 3.1.3.1) and phytase ( EC 3.1.3.8) activities in rat intestine and their importance in phytate-induced zinc deficiency

1. The activities of alkaline phosphatase (EC 3.1.3.1) and phytase (EC 3.1.3.8) were similarly distributed in the small intestine of rats. Regional differences in activity were reflected by similar differences in the capacity of ligated intestinal segments to hydrolyse phytate in vivo. Activities were greatest in the duodenum and lowest in the terminal ileum. 2. Specific activities of both enzymes were tenfold greater in the brush border fraction of duodenal mucosa compared with entire mucosal homogenates. 3. Brush-border alkaline phosphatase and phytase activities required both magnesium and zinc ions for maximal activity. 4. Zn deficiency induced by feeding a diet low in Zn (0.5 mg Zn/kg) caused similar reductions in activity of both enzymes. 5. Zn deficiency induced by feeding diets marginally adequate in Zn (12 mg/kg) and phytate (10 g/kg) caused reductions in alkaline phosphatase, phytase activities and phytate hydrolysis in vivo. 6. It is suggested that phytase activity is a manifestation of alkaline phosphatase and the significance of this in relation to phytate-induced in Zn deficiency is discussed.

Studies on the appearance of a hepatic copper-binding protein in normal and zinc-deficient rats

1. A study has been made by gel-filtration techniques of the soluble copper- and zinc-binding proteins in rat liver after both intraperitoneal injection of Cu and dietary Cu supplementation. 2. Liver Cu and Zn concentrations increased after injection of Cu, both metals accumulating in the cytosol, mainly in a fraction with an apparent molecular weight of about 12 000. 3. When Zn-deficient rats were injected with Cu, there was little change in liver Zn concentration and the occurence of Cu in the low-molecular-weight form (about 12 000) was more transient. At most periods after injection, Cu accumulated mainly in a fraction with a molecular weight greater than 65 000. 4. When the rats were Cu-loaded by dietary supplementation, virtually no Cu or Zn was found in the low-molecular-weight form in Zn-deficient rats, although they were found in the Zn-supplemented animals. 5. The results suggest that Zn is essential for the accumulation of Cu in this form, but not for Cu to stimulate production of the metal-binding protein by a process requiring active protein protein synthesis.

Effect of dietary copper deficiency. in the rat on fatty acid composition of adipose tissue and desaturase activity of liver microsomes.

1. Male rats were maintained from wearing to between 4 and 16 weeks of age on a semisynthetic diet which was deficient in copper. 2. Methyl esters of fatty acids from adipose tissue of the rats were analysed by gas-liquid chromatography and the desaturase activity of liver microsomes, with [1-14C]=stearic acid as the substrate, was determined. Liver and plasma Cu concentration, cytohrome c oxidas (EC 1.9.3.I) activity and caeruloplasmin activity were determined as indices of Cu status. 3. Cu deficiency was associated with decreased mono-unsaturated:saturated ratios for C16 and C18 fatty acids from subcutaneous adipose tissue and decreased desaturase activity for liver microsomes. When Cu-deficient rats were given free access to the Cu-adequate diet or were injected intraperitoneally with an aqueous solution of CuSO4, that is, when the animals were related with Cu, the indices of Cu status, and desaturase activity for liver microsomes returned to values found in control animals. 4. When Cu or a Cu-chelator (Neocuproine) was added to microsomes, there was no effect on the activity of the desaturase enzyme system; the stability of the desaturase was not affected by Cu. 5. These results are indicative of an involvement of Cu in the desaturase reaction. It is suggested that the site of this involvement could be the terminal component of the microsomal electron transport chain.

Studies on the effects of dietary zinc dose on 65Zn absorption in vivo and on the effects of Zn status on 65Zn absorption and body loss in young rats.

Weanling male rats were maintained on diets containing 5, 10, 20, 40, 80 or 160 mg zinc/kg for 14 d. On day 15 they received 65Zn either by intraperitoneal injection or in a test meal containing 20 mg Zn/kg. After dosing, the rats were again maintained on the diets they had received previously. Whole-body 65Zn retention was measured immediately after dosing and daily for a further 9 d. From regression analysis of the semi-logarithmic plots of 65Zn retention from 0 to 192 h after 65Zn administration, the true extent of 65Zn absorption and the biological half-life (t1/2) of body 65Zn stores were calculated. At the end of the experiment, the rats were killed and the entire small intestines of some rats from each group were rapidly flushed out to remove food and faecal residues, frozen in liquid nitrogen and stored under an atmosphere of N2 at -20 degrees before separation of cytosolic Zn-binding fractions by gel filtration on Sephadex G-75. The results suggest that rats which received diets that were either deficient (5 mg Zn/kg), marginal (10 mg Zn/kg) or adequate (20-80 mg Zn/kg) in Zn achieved homeostatic regulation of body Zn by changes in both the extent of Zn absorption and excretion. However, when Zn supply was excessive, increasing from 80 to 160 mg Zn/kg, no further changes were seen in Zn absorption, and homeostatic control appeared to be effected entirely by changes in rates of body Zn loss. Gel chromatography of intestinal cytosol on Sephadex G-75 revealed that Zn was associated with two major fractions. The first (peak 1) had a molecular weight (MW) greater than 75 kdaltons and the second (peak 2), a MW of approximately 10 kdaltons and was assumed to be metallothionein. There was no obvious relation between the amount of Zn bound to peak 1 and dietary Zn content. In contrast, the amount of Zn recovered in peak 2 increased linearly with increasing dietary Zn content. Comparisons between the effect of dietary Zn content on Zn bound to peak 2 and 65Zn retention may, depending on the range of Zn intakes, indicate possible roles for intestinal metallothionein in the control of Zn absorption or excretion. A study of the effects of dietary dose of 65Zn on the extent of 65Zn absorption in rats of normal Zn status indicated a possible biphasic relation. At low doses (5-40 mg Zn/kg) 65Zn absorption appeared to exhibit a curvilinear response to increasing 65Zn dose, indicating possibly a saturable process.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of pregnancy and lactation on copper and zinc retention in the rat

1. The accretion of copper and zinc in maternal tissues, conception products and postnatal offspring of the rat was determined at different stages of pregnancy and lactation. 2. Equations relating the weight of the bodies of the developing young and of the amounts of Cu and Zn in them to the time that has elapsed since conception showed that, early in pregnancy, the specific rates of accretion of Cu or Zn were greater than that of weight, but declined more rapidly as development continued. The instantaneous rates of accretion of both metals rose throughout pregnancy but only that of Cu continued to increase during lactation. 3. The amount of Cu in the maternal body rose significantly during pregnancy and declined thereafter, but relatively small changes in its Zn content occurred. 4. The results are discussed in relation to the relative demands for these metals during pregnancy and lactation.

Studies on the roles of apotransferrin and caeruloplasmin (EC 1.16.3.1) on iron absorption in copper-deficient rats using an isolated vascularly- and luminally-perfused intestinal preparation

1. Studies have been made on the effects of dietary copper on the iron and Cu distribution in rats and on the metabolic activity and absorptive capacity of intestines perfused both vascularly and luminally. 2. Rats maintained for 4-5 weeks on a Cu-deficient diet (0.4 microgram Cu/kg) had significantly lower plasma, liver and intestinal Cu concentrations and significantly reduced plasma caeruloplasmin and liver cytochrome c oxidase (EC 1.9.3.1) activity compared with controls receiving a Cu-supplemented diet (5 micrograms Cu/kg). Disturbances in Fe metabolism in Cu-deficient rats were evident as shown by a mild anaemia, significantly elevated hepatic Fe concentrations and hypoferraemia. 3. Intestinal glucose uptake from both the luminal perfusion medium (LPM) and vascular perfusion medium (VPM) was unaffected by Cu deficiency despite a significant (25-30%) reduction in oxygen consumption. This was associated with a 40% decline in mucosal cytochrome c oxidase activity. 4. In studies of Fe absorption, Fe uptake from the LPM was unaffected by Cu deficiency while transfer of Fe to VPM was significantly reduced (50%) compared with control preparations. Addition of apotransferrin (1 g/l) to the VPM was without effect in preparations from control rats but significantly increased the transfer of Fe to the VPM in preparations from Cu-deficient rats without affecting Fe uptake from the LPM. 5. The addition of either human or porcine caeruloplasmin (together with apotransferrin) to the VPM, such that the resultant ferroxidase (EC 1.16.3.1) activity of the VPM supernatant fraction was four to five times that of normal rat plasma, was without effect on either Fe uptake, tissue retention or Fe transfer to the VPM by preparations from either Cu-deficient or control rats. 6. These findings offer no evidence in support of the proposed role for caeruloplasmin with its associated ferroxidase activity in Fe absorption in the rat.

The susceptibility of suckling lambs to zinc toxicity

1. Suckling lambs maintained for 4 weeks on a milk-substitute diet containing 407 g Toprina yeast/kg dry matter of diet exhibited poor growth, low appetite and extensive renal damage. 2. The batch of yeast used contained 2065 mg zinc/kg. 3. The kidneys of lambs offered a milk diet supplemented with the same Zn content as the yeast-fed diet (32-1 mg Zn/1000 kJ) were similarly affected.