Elemental composition of anatomically distinct regions of rat liver.

Experiments were conducted to test the commonly held assumption that analysis of a portion of rat liver is representative of the elemental concentration of the whole organ. Male Sprague-Dawley rats (initial body weight approximately 250 g) fed a chow diet or weanling male Long-Evans rats (initial body weight approximately 50 g) fed a semipurified diet with or without copper in the mineral premix were sacrificed after 4 wk on their respective diets and livers were dissected into seven portions representing major anatomical divisions of this organ. Elemental analyses by atomic absorption spectroscopy (calcium, magnesium, iron, zinc, copper, manganese), atomic emission spectroscopy (sodium, potassium), or colorimetric assay (phosphorus) demonstrated no statistically significant differences in composition of these nine elements among anatomical regions of liver. Dietary copper deficiency led to equivalently reduced copper concentration in all portions of rat liver and did not cause any other significant alterations in liver composition of these nine elements within the 4 wk of these studies. These results confirm the validity of the common assumption that analysis of a portion of rat liver can be representative of the elemental composition of the whole organ. This conclusion will allow more analyses to be performed on fewer animals, thereby reducing animal use and reagent costs without sacrificing analytical accuracy.

Magnesium status and excretion in age-matched subjects with normal and elevated blood pressures.

Plasma, erythrocyte, and leucocyte magnesium (Mg) concentration, as well as urinary Mg, calcium (Ca), and phosphorus (P) excretion were determined in male subjects with elevated diastolic blood pressure (DBP) > 90 mmHg. These parameters were compared to those in normotensive age-matched (DBP < 85 mmHg) and drug-treated hypertensive subjects. Mg and Ca were determined by atomic absorption spectroscopy and P was determined by a colorimetric method. Urinary excretion was expressed in terms of creatinine (Cr) excretion. There were no significant differences in plasma, erythrocyte, or leucocyte Mg concentrations. Both urinary Ca and Mg were significantly decreased in the group with elevated blood pressure (BP) and the drug-treated group compared to the normotensive group. Urinary P was elevated in the drug-treated hypertensive group. Of all the variables studied, urinary Mg was the only one that was significantly inversely correlated with both systolic (SBP) and DBP. These data suggest that decreased urinary Mg, which may be indicative of a decreased Mg intake, is associated with hypertension.

The relationship between the rate of chelator-induced zinc efflux from erythrocytes and zinc status.

The rate of zinc (Zn) release from rat erythrocytes incubated in buffers containing a variety of chelators was measured. Only o-phenanthroline, 8-hydroxyquinoline-5-sulfonate, and EDTA caused detectable Zn release. The relationship between the rate of this release in the presence of o-phenanthroline and Zn status was determined in rats. Rats were fed one of the following: a modified AIN-76 diet providing 46 mumol (3 mg) Zn per kg of diet, a pair-fed diet providing 459 mumol (30 mg)/kg, or the previous diet fed ad lib. Animals were sacrificed at 2-wk intervals for 12 wk, and the Zn efflux rate, plasma, liver, and femur Zn concentrations were determined. The efflux rate was lower in erythrocytes taken from the rats fed the low-Zn diet. The efflux rate was also well correlated with femur Zn (r = 0.509, n = 98, p < 0.0001). A poorer correlation was observed with plasma Zn in the rats. Correlations also were determined between efflux rates and plasma Zn levels in human subjects. There was a significant correlation only in the males. In was concluded that the Zn efflux rate from erythrocytes incubated in the presence of o-phenanthroline is related to Zn status but is not sensitive enough to be a useful index of this status.

Indices of iron and copper status during experimentally induced, marginal zinc deficiency in humans.

This study examined the effect of diet-induced, marginal zinc deficiency for 7 wks in 15 men (aged 25.3 +/- 3.3 yrs; mean +/- SD) on selected indices of iron and copper status. The regimen involved low-zinc diets based on egg albumin and soy protein with added phytate and calcium such that mean [phytate]/[Zn] and [phytate] X [Ca]/[Zn] molar ratios were 209 and 4116, respectively, for 1 wk, followed by 70 and 2000, respectively, for 6 wks. Subjects were then repleted with 30 mg Zn/d for 2 wks. Plasma copper, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) activity in plasma and red blood cells (RBC), hemoglobin, hematocrit, and serum ferritin were determined weekly on fasting blood samples. Significant reductions (p less than 0.05) after 7 wks in RBC Cu,Zn-superoxide dismutase (49.5 +/- 7.2 vs 33.6 +/- 6.3 U/mg Hb) and serum ferritin (69.2 +/- 38.7 vs 53.8 +/- 33.7 micrograms/L) occurred; no comparable decline was noted for plasma Cu, hemoglobin, or hematocrit. Significant (p less than 0.05) but less consistent changes were also observed in plasma superoxide dismutase activity. None of the changes were associated with the decreases in plasma, urinary and hair zinc concentrations, and alkaline phosphatase activity in RBC membranes. Results indicate that the biochemical iron and copper status of the subjects was marginally impaired, probably from the dietary regimen that induced marginal zinc deficiency.

Effects of low copper and high zinc intakes and related changes in Cu,Zn-superoxide dismutase activity on DMBA-induced mammary tumorigenesis.

The effect of low copper and high zinc intakes on Cu,Zn-superoxide dismutase (Cu,Zn-SOD) activity and mammary tumorigenesis induced by 9,10-dimethyl-1,2-benzanthracene (DMBA) was investigated. Groups of 40 weanling female Sprague-Dawley rats were fed a modified AIN-76 diet containing the following (/kg diet): 1 mg Cu (0.016 mmol) and 30 mg Zn (0.459 mmol); 6 mg Cu (0.094 mmol) and 30 mg Zn (0.459 mmol) (control); or 6 mg Cu (0.094 mmol) and 150 mg Zn (2.295 mmol) for 21 wk. At 5 wk, 30 rats/group were given 4 mg (15.6 mumol) DMBA in corn oil intragastrically, and controls (10/group) received corn oil alone. Erythrocyte Cu,Zn-SOD activity was measured at 3, 5 (just before DMBA), 9, 13, 17, and 21 wk. The group fed the high-Zn diet had a slightly lower weight gain and food consumption. DMBA treatment had no effect on these parameters. Plasma and liver Cu concentration decreased in the low-Cu group. Femur zinc was significantly elevated in the high-Zn group. Erythrocyte Cu,Zn-SOD activity was decreased in the low-Cu group from 3 to 21 wk and was significantly elevated in the high-Zn group at 3 and 5 wk. In the low-Cu group, there were 5 nonmalignant adenomas and 3 malignant adenocarcinomas; in the control group, there were 4 adenomas and 3 adenocarcinomas; in the high-Zn group, there were 5 adenomas and 3 adenocarcinomas. No relationship between Cu,Zn-SOD activity and the presence of tumors could be found.

An evaluation of plasma and erythrocyte magnesium concentration and the activities of alkaline phosphatase and creatine kinase as indicators of magnesium status.

Rats were fed diets containing magnesium at concentrations ranging from 3.3 to 26.7 mmol/kg of diet (80 to 650 mg/kg). The magnesium concentration of their plasma and erythrocytes, and the activities of plasma alkaline phosphatase and creatine kinase were investigated to determine their usefulness as indices of magnesium status. All the indices increased with increasing dietary magnesium levels. The best correlations were observed between dietary intake and plasma concentration of magnesium (r = 0.846, p less than 0.001) and between intake and femur concentration (r = 0.811, p less than 0.001). There was an extremely high correlation between plasma concentration and femur concentration (r = 0.930, p less than 0.001). Although significant, the correlations between intake and the enzyme activities were not strong. It is concluded that plasma magnesium concentration is the most useful indicator of magnesium status and that the activities of the two magnesium-requiring enzymes can only be used for the purpose of diagnosing severely deficient magnesium status.

Effects of dietary selenium on DMBA-induced carcinogenesis in rats fed a diet high in mixed fats.

The effects of selenium intake on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis were examined in rats fed a diet high in mixed fats and representative of that consumed in North America. Six groups of 20 rats were fed an AIN-76 diet modified to contain 20% fat from lard:corn oil (3:1 wt/wt) and various amounts of selenium (0.1, 0.035, 0.1, 1.0, 2.0 or 4.0 mg Se/kg diet). At wk 5, animals in groups 2-6 were dosed with 4.32 mg of DMBA. Serum clinical parameters and the activities of plasma selenium-dependent and total glutathione peroxidase (GSHPx), erythrocyte GSHPx and superoxide dismutase (SOD) were determined every 4 wk for 25 wk. The extent of lipid peroxidation was determined by measuring urinary malondialdehyde during wk 13 and 24, and erythrocyte malondialdehyde at wk 25. Erythrocyte GSHPx was found to be a better indicator of selenium status than plasma activity, while SOD did not vary with dietary selenium. The group of animals fed 4.0 mg Se/kg diet had reduced numbers of tumors (P less than 0.01), but this reduction was associated with evidence of chronic selenium toxicity. Variations in GSHPx activity with dietary selenium did not result in differences in tumor incidence, nor in changes in lipid peroxidation in the other groups. Thus, nontoxic levels of selenium do not appear to offer any protective effect during carcinogenesis in rats fed a casein-based diet similar in fat content to that consumed by North Americans.

Changes in selenium and antioxidant status during DMBA-induced mammary carcinogenesis in rats.

Female weanling Sprague-Dawley rats were used to examine the changes that occurred in selenium and antioxidant status during the development of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors. Animals were fed an AIN-76 diet, modified to contain 20% fat (3:1 wt/wt, lard:corn oil) and 0.1, 0.035, 0.1, 1.0, 2.0 or 4.0 mg Se/kg diet. At wk 5, rats in groups 2-6 were administered by intragastric tube 4.32 mg of DMBA dissolved in corn oil. Control rats received corn oil only. A blood sample was removed from the tail vein and analyzed for selenium-dependent glutathione peroxidase (Se-GSHPx) and superoxide dismutase (SOD) activity at wk 5, and every 4 wk until wk 25. At the end of the experiment, rats were classified by tumor status, and each diet group was subdivided into two groups: those rats remaining free of tumors for 25 wk and those with tumors. DMBA treatment caused an initial decrease in erythrocyte SeGSHPx and SOD activity compared to untreated control rats. SeGSHPx activity in rats with tumors remained lower than controls, while SeGSHPX activity increased in rats with no tumors. These changes, however, were not associated with any changes in lipid peroxidation.

Effect of dietary selenium and tumor status on the retention of 75Se by tissues and mammary tumors of DMBA-treated rats.

The effects of the presence of mammary tumors on 75Se retention was examined in DMBA-treated rats. Tumor bearing rats fed varying amounts of Se exhibited an inverse linear dose response between dietary Se intake and tissue retention of 75Se in whole body, heart, lungs, ovaries, adrenals, spleen, and muscle. Tumor 75Se retention, however, was independent of the dietary intake of Se. Tumor bearing rats excreted more 75 Se label in the urine compared to both control rats fed the same amount of Se and DMBA-treated animals that remained tumor free. In the short term, no significant differences were seen in tissue retention of 75Se. By 7 d, the increased urinary excretion of the label resulted in significantly decreased retention of 75Se in blood, spleen, liver, lungs, and kidneys of tumor-bearing rats compared to tumor-free animals. The presence of tumors, however, did not affect the liver distribution of the label among cytosolic proteins. These results suggest that tumor bearing animals have an accelerated urinary excretion of Se compared to animals without tumors and that tumors either have a very slow turnover of Se or a low priority for the element.

Effect of dietary iodine on autoimmune thyroiditis in the BB Wistar rats.

Diabetes-prone BB Wistar rats were fed a modified AIN-76 diet providing the following amounts of iodine for 10 wk: 0.2 mg/kg diet (recommended amount); 1.0 mg/kg; 2.0 mg/kg; or 3.0 mg/kg. The thyroids were examined for gross and microscopic changes and sera were assayed for antibodies to triiodothyronine (T3), thyroxine (T4), and thyroglobulin (Tg). The body weights and food consumption of the rats fed 0.2 mg of iodine/kg were significantly lower than those of the animals fed higher amounts. Urinary iodine excretion reflected dietary intakes. The thyroids from animals fed 2.0 and 3.0 mg/kg were significantly (P less than 0.01) larger than those from animals fed 0.2 mg/kg. One rat fed 0.2 mg/kg and 2 rats in each group fed 2.0 and 3.0 mg/kg had extensive lymphocytic thyroiditis. Three rats fed 1.0 mg/kg, 6 fed 2.0 mg/kg and 6 fed 3.0 mg/kg had enlarged thyroids. Two rats fed 0.2 mg/kg, 2 fed 2.0 mg/kg and 6 fed 3.0 mg/kg had detectable Tg antibodies. These data suggest that high iodine intakes increase Tg antibodies, which may be associated with an increase in autoimmune thyroiditis in these animals.

Effects of dietary magnesium on sodium-potassium pump action in the heart of rats.

Sprague-Dawley rats were fed a basal AIN-76 diet containing 80, 200, 350, 500 or 650 mg of magnesium per kilogram of diet for 6 wk. Ventricular slices, as well as microsomal fractions, were prepared from the hearts and were used to determine sodium-potassium pump activity. Sodium-potassium pump activity was assessed in the microsomal membranes by determining the ouabain-inhibitable Na+, K+-ATPase activity and [3H]ouabain binding, and in the ventricular slices, by determining ouabain-sensitive 86Rb uptake under K+-free conditions. The ATPase activity increased with increasing dietary magnesium, so that in the hearts of those animals that were fed 500 and 650 mg of magnesium/kg diet, it was significantly greater than the activity in the hearts of the animals fed 80 and 200 mg/kg diet. Similarly, 86Rb uptake by heart slices from rats fed 500 and 650 mg of magnesium/kg diet was significantly greater than the uptake by heart slices from animals fed 80 and 200 mg/kg diet. [3H]Ouabain binding did not change with increasing dietary magnesium. Thus, magnesium deficiency appears to have no effect on the number of sodium-potassium pump sites, but does decrease the activity of the pump. It is suggested that this leads to an increase in intracellular Na+, resulting in a change in the membrane potential, and may contribute to the arrhythmias associated with magnesium deficiency.

Colorimetric determination of total iodine in foods by iodide-catalyzed reduction of Ce+4.

An earlier acid digestion determination of iodine in foods was modified to provide an improved detection limit and to allow for the analysis of a greater variety and larger amounts of foods. The organic material in the sample was oxidized overnight by concentrated nitric acid, followed by digestion in a mixture of concentrated sulfuric and 70% perchloric acid. The iodine was determined by an automated colorimetric method based on the iodide-catalyzed reduction of Ce+4 by As+3. The method had an average relative standard deviation of 3.1% for the samples analyzed, and a detection limit of 0.1 ng/mL in the digested solution and 5 ng/g in a 2 g sample prior to digestion. The recovery of added iodine ranged from 90.3 to 101.3%, using external standards. Samples analyzed included NBS Standard Reference Material 1549, and composites of a variety of dairy products, meat, eggs and fish, cereals, and potatoes. The iodine detected in these samples ranged from 9 ng/g for the potato group to 3360 ng/g for the standard reference material.

An automated method for the determination of Cu,Zn-superoxide dismutase in plasma and erythrocytes using an ABA-200 discrete analyzer.

An automated enzymatic method is described for the determination of Cu,Zn-superoxide dismutase (SOD) in plasma or erythrocytes using the xanthine-xanthine oxidase and cytochrome C coupled assay. This method was adapted to an Abbott ABA-200 discrete analyzer. Coefficients of variation for within-run and day-to-day analyses were less than 5%. Only 2.5 muL of serum or erythrocyte extract is required so a capillary tube sample of blood (70 muL) is sufficient for the assay. Recovery of added SOD ranged from 92 to 101%. The method reported here is practical for use in a clinical chemistry laboratory for monitoring changes in this enzyme, which is a sensitive early indicator of alterations in copper status.

Effect of zinc supplementation on copper status in adult man.

The effects of zinc supplementation on the copper status of healthy adult men, as assessed by the activities of the copper-metalloenzymes, plasma ferroxidase (ceruloplasmin), and erythrocyte Cu,Zn-superoxide dismutase, were determined. The subjects were given either two daily doses of 25 mg zinc or placebo for 6 wk. No significant differences in the plasma copper levels or the ferroxidase activities between the supplemented and control groups could be detected at 2, 4, or 6 wk. Plasma zinc increased and erythrocyte Cu,Zn-superoxide dismutase decreased in the supplemented group, the difference between the groups becoming significant at 6 wk (p less than 0.05). This suggested that the zinc supplements decreased the copper status of the experimental group.

The effects of high dietary zinc and copper deficiency on the activity of copper-requiring metalloenzymes in the growing rat.

The effects of feeding rats high levels of dietary zinc (240 mg Zn/kg diet) on the activities of the copper-requiring metalloenzymes: ceruloplasmin (Cp), cupro -zinc superoxide dismutase (SOD) and cytochrome c oxidase (CCO) were determined. These were compared with those seen during copper deficiency (induced by feeding 0.6 mg Cu/kg diet). The activities of Cp in serum, Cu-Zn SOD in liver and heart, and CCO in heart were significantly reduced in both the high zinc and copper-deficient groups by 2 weeks, when compared to the activities seen in rats fed the control diet (6 mg Cu, 30 mg Zn/kg diet). In animals fed the high zinc diet, the reduction in heart CCO activity followed the decrease seen in heart copper concentration, whereas in blood and liver, the reductions in Cp and SOD activities, respectively, were greater than the reductions seen in copper concentration. Thus, the results of this experiment demonstrate that with high dietary zinc, the reductions seen in the activity of copper-requiring metalloenzymes were similar to those seen during copper deficiency.

The effects of dietary zinc on the activity of copper-requiring metalloenzymes in the rat.

It has long been known that zinc interferes with copper absorption and metabolism. In the present study, the effects of feeding rats 15, 30, 60, 120 or 240 mg Zn/kg diet on their copper status, as assessed by the activities of cupro -zinc superoxide dismutase (SOD) and cytochrome c oxidase (CCO) in heart and liver, and ceruloplasmin (Cp) in serum were determined. Although Cp activity was not related to zinc intake in a linear fashion, the number of animals with extremely low Cp activity increased with increasing zinc. The level of zinc at which 50% of animals would have abnormally low Cp activity was calculated to be 125-129 ppm zinc. Liver SOD and heart CCO activities decreased linearly with increasing zinc and were significantly reduced compared to controls at 120 and 240 ppm Zn, respectively. Thus, animals fed zinc at levels as low as four times the AIN recommended concentration showed biochemical signs of copper deficiency.

Effects of zinc on mucosal copper binding and on the kinetics of copper absorption.

The kinetics of serosal copper transfer and the distribution of copper between metallothionein (MT) and a high-molecular-weight protein fraction (HMWPF) within the mucosal cells were examined, using isolated duodenal segments from rats fed different amounts of zinc. No difference in the Vmax or Km for serosal transfer could be detected between the different zinc groups, suggesting that zinc did not affect this aspect of copper absorption. When intestinal segments from rats fed the low zinc diet were incubated in high copper media, the majority of the copper was associated with the HMWPF, while those from rats fed the high zinc diet had the largest proportion bound to MT. All the intestines incubated in the low copper media, regardless of rat zinc status, had the majority of copper associated with MT. These results suggested that with the low zinc diet, the limited amount of MT present was saturated, and the excess copper was bound to the HMWPF. With larger intakes of zinc, MT synthesis was induced, and the larger amount of this protein was not as readily saturated, resulting in less copper binding to the HMWPF and more to MT. It is suggested that zinc interferes with copper absorption by inducing MT, which sequesters copper in the mucosal cells, making it unavailable for serosal transfer. The copper bound to the HMWPF is available for transfer.

The effect of dietary zinc on intestinal copper absorption.

Everted duodenal segments, tied into sacs, taken from animals fed different amounts of zinc were used to investigate the antagonistic effect of dietary zinc on copper absorption. The intestinal segments taken from animals fed low amounts of zinc transferred more copper from a nutrient medium across the mucosal cells than did intestines from rats fed high levels of zinc. The mucosal cells from animals fed low amounts of zinc retained less copper than the cells from animals fed high amounts of the element. This retained copper was bound to a protein fraction having a molecular weight similar to that of metallothionein. The data suggest that zinc exerts its antagonistic effect by inducing the synthesis of a copper-binding ligand, probably a thionein, in the mucosal cells which sequesters copper from the nutrient medium, making it unavailable for serosal transfer. This may be a possible mechanism by which dietary zinc decreases copper absorption and leads to a decreased copper status.