Elimination of mercury from amalgam in rats.

The aim of this study was to measure the urinary mercury excretion in rats exposed to amalgam over a two months period. Animals were either exposed to mercury from 4 dental amalgams or fed the diet containing powdered amalgams. The results showed significantly higher mercury amount in urine of both exposed groups than in control. Even two months after the amalgam had been placed in rats teeth, the amount of mercury in the urine remained 4-5 times higher than in control, and 4 times higher than in rats exposed to diet containing powdered amalgam. The elevated urinary Hg amount was accompanied by an increased level of total protein in urine. In the same exposure period the excretion of total protein in urine of rats with amalgam fillings was 2 times higher than in control and 1.5 times higher than in rats exposed to amalgam through diet. Concentrations of mercury in the sera of all groups were below the detection limit of the method. The results show that amount of mercury and protein in the urine of rats were related to the mercury release from dental malgam.     

Biomonitoring of DNA damage in peripheral blood lymphocytes of subjects with dental restorative fillings

Dental fillings provide a major iatrogenic exposure to xenobiotic compounds due to the high prevalence of surface restorations in developed countries. Experimental data suggest that both amalgams, which contain mercury, and resin-based dental materials cause an impairment of the cellular pro- and anti-oxidant redox balance. The aim of this study was to assess the potential genotoxicity of dental restorative compounds in peripheral blood lymphocytes of young exposed subjects compared with controls. The study examined, by use of the comet assay, 68 carefully selected subjects taking into account the major known confounding factors. In the 44 exposed subjects, the mean numbers of restored surfaces was 3.0 and 3.8 in males and females, respectively. Tail length, percentage of DNA in the tail, tail moment or Olive tail moment were twofold higher in the exposed group than in unexposed controls, with significant differences. No significant difference was observed between amalgam and composite fillings. Furthermore, as shown by multivariate analysis, the association between dental fillings and DNA damage was enhanced by the number of fillings and by the exposure time. Among the lifestyle variables, a moderate physical activity showed a protective effect, being inversely correlated to the DNA damage parameters evaluated. On the whole, the use of DNA-migration allowed us to detect for the first time the potential adverse impact on human health of both kinds of dental filling constituents, the amalgams and the methacrylates. The main mechanism underlying the genotoxicity of dental restorative materials of various nature may be ascribed to the ability of both amalgams and methacrylates to trigger the generation of cellular reactive oxygen species, able to cause oxidative DNA lesions.     

Biomonitoring of DNA damage in peripheral blood lymphocytes of subjects with dental restorative fillings

Dental fillings provide a major iatrogenic exposure to xenobiotic compounds due to the high prevalence of surface restorations in developed countries. Experimental data suggest that both amalgams, which contain mercury, and resin-based dental materials cause an impairment of the cellular pro- and anti-oxidant redox balance. The aim of this study was to assess the potential genotoxicity of dental restorative compounds in peripheral blood lymphocytes of young exposed subjects compared with controls. The study examined, by use of the comet assay, 68 carefully selected subjects taking into account the major known confounding factors. In the 44 exposed subjects, the mean numbers of restored surfaces was 3.0 and 3.8 in males and females, respectively. Tail length, percentage of DNA in the tail, tail moment or Olive tail moment were twofold higher in the exposed group than in unexposed controls, with significant differences. No significant difference was observed between amalgam and composite fillings. Furthermore, as shown by multivariate analysis, the association between dental fillings and DNA damage was enhanced by the number of fillings and by the exposure time. Among the lifestyle variables, a moderate physical activity showed a protective effect, being inversely correlated to the DNA damage parameters evaluated. On the whole, the use of DNA-migration allowed us to detect for the first time the potential adverse impact on human health of both kinds of dental filling constituents, the amalgams and the methacrylates. The main mechanism underlying the genotoxicity of dental restorative materials of various nature may be ascribed to the ability of both amalgams and methacrylates to trigger the generation of cellular reactive oxygen species, able to cause oxidative DNA lesions.     

Whole-body imaging of the distribution of mercury released from dental fillings into monkey tissues

The fate of mercury (Hg) released from dental "silver" amalgam tooth fillings into human mouth air is uncertain. A previous report about sheep revealed uptake routes and distribution of amalgam Hg among body tissues. The present investigation demonstrates the bodily distribution of amalgam Hg in a monkey whose dentition, diet, feeding regimen, and chewing pattern closely resemble those of humans. When amalgam fillings, which normally contain 50% Hg, are made with a tracer of radioactive 203Hg and then placed into monkey teeth, the isotope appears in high concentration in various organs and tissues within 4 wk. Whole-body images of the monkey revealed that the highest levels of Hg were located in the kidney, gastrointestinal tract, and jaw. The dental profession's advocacy of silver amalgam as a stable tooth restorative material is not supported by these findings.     

Levels and distribution of zinc,copper, magnesium,and calcium in rats fed different levels of dietary zinc

Effects of altered dietary zinc on levels of zinc, copper, magnesium, and calcium in organ and peripheral tissues were studied. When rats fed a zinc-deficient diet (1.3 μg Zn/g) for 28 d were compared with rats fed a control diet (37.5 μg Zn/g), levels of zinc were slightly lower in plasma, hair, and skin and 50% lower in femur and pancreas, whereas the levels of copper were higher in all tissue except plasma. Magnesium levels were higher than controls in the heart and lower in the spleen, whereas the calcium levels were lower in plasma, lung, spleen, kidney, and skin and strikingly higher in brain, hair, and femur. When rats fed a zinc-supplemented diet (1.0 mg Zn/g) were compared to the same conrols, levels of zinc in these were higher in all organs and peripheral tissues studied, except heart, lung, and liver; copper levels were higher in liver, kidney, and spleen; magnesium levels were significantly higher in the spleen, but were little affected in other tissues, although calcium levels were higher in pancreas, spleen, kidney, and skin and lower in plasma and hair. These data indicate that overall copper organ and peripheral tissue levels are affected inversely, and zinc and calcium levels directly, by zinc nutriture.     

The effects of age and sex on seven elements of Sprague-Dawley rat organs

This study reports age-related changes in 7 element (iron, copper, zinc, manganese, mercury, cadmium and lead) concentrations in the liver, kidney and brain of male and female Sprague-Dawley rats from 1 to 364 days of age. Atomic absorption spectrometry was used for the measurements. Copper, mercury and cadmium in the male and female kidneys increased from weaning until 127 days of age, as did iron concentrations in the female liver and kidney. After 127 days, especially, the copper concentration in the female kidney and cadmium concentration in the male and female kidney increased further. Consistent and statistically significant (P less than 0.05) sex differences in element concentrations were found for three elements (iron, copper and zinc). Except for the zinc concentration in the liver from 50 to 72 days, iron (in liver and kidney), zinc (in kidney) and copper (in liver, kidney and brain) concentrations in female rats during the adult stage, were all higher than those of male rats. Isolated differences for other elements (manganese, mercury and cadmium) were also found. The data will be helpful when setting up long-term animal investigations of the biological effect of elements.     

Mercury from maternal “silver” tooth fillings in sheep and human breast milk

Neonatal uptake of mercury (Hg) from milk was examined in a pregnant sheep model, where radioactive mercury (Hg²⁰³)/silver tooth fillings (amalgam), were newly placed. A crossover experimental design was used in which lactating ewes nursed foster lambs. In a parallel study, the relationship between dental history and, breast milk concentration of Hg was also examined in 33 lactating women. Results from the animal studies showed that, during pregnancy, a primary fetal site of amalgam Hg concentration is the liver, and, after delivery, the neonatal lamb kidney receives additional amalgam Hg from mother's milk. In lactating women with aged, amalgam fillings, increased Hg excretion in breast milk and urine correlated with the number of fillings or Hg vapor concentration levels in mouth air. It was concluded that Hg originating from maternal amalgam tooth fillings transfers across the placenta to the fetus, across the mammary gland into milk ingested by the newborn, and ultimately into neonatal body tissues. Comparisons are made to the U.S. minimal risk level recently established for adult Hg exposure. These findings suggest, that placement and removal of “silver” tooth fillings in pregnant and lactating humans will subject the fetus and neonate to unnecessary risk of Hg exposure.     

Effect of age and dietary protein level on tissue mineral levels in female rats

Mineral (phosphorus, sulfur, potassium, calcium, magnesium, iron, zinc, copper, and manganese) concentrations were measured in plasma, and several tissues from female Wistar rats (young: 3-wk-old; mature: 6-mo-old) were fed on a dietary regimen designed to study the combined or singular effects of age and dietary protein on mineral status. Three diets, respectively, contained 5, 15, and 20% of bovine milk casein. Nephrocalcinosis chemically diagnosed by increased calcium and phosphorus in kidney was prevented in rats fed a 5% protein diet. Renal calcium and phosphorus were more accumulated in young rats than mature rats. A 5% protein diet decreased hemoglobin and blood iron. The hepatic and splenic iron was increased by a 5% protein diet in mature rats but was not altered in young rats. Mature rats had higher iron in brain, lung, heart, liver, spleen, kidney, muscle, and tibia than young rats. A 5% protein diet decreased zinc in plasma and liver. Zinc in tibia was increased with dietary protein level in young rats but was not changed in mature rats. A 5% protein diet decreased copper concentration in plasma of young rats but not in mature rats. Mature rats had higher copper in plasma, blood, brain, lung, heart, liver, spleen, and kidney than young rats. With age, manganese concentration was increased in brain but decreased in lung, heart, liver, kidney, and muscle. These results suggest that the response to dietary protein regarding mineral status varies with age.     

Dental amalgam affects urinary selenium excretion

Selenium may have a protective effect against mercury toxicity. The aim of the present study was to investigate if selenium excretion in urine was affected in persons with dental amalgam fillings. The reason for this study is that dental amalgam is the most important source of inorganic mercury exposure in the general population, although the potential toxic effects of this exposure remain a subject for debate. The chelating agent 2,3 dimercaptopropane-1-sulfonate (DMPS) was injected intravenously (2 mg/kg) to provoke metal excretion. Urine samples were subsequently collected at intervals over a 24-h period. Selenium concentration was determined by hydride-generation atomic absorption spectrometry. The study was comprised of 20 persons who claimed symptoms from dental amalgam and 21 healthy persons with amalgam fillings. There were two control groups without amalgam. One control group had amalgam replaced because of concern about illness resulting from mercury release (n=20), whereas the other control group never had amalgam (n=19). Individuals with amalgam excreted less selenium (36.4 μg, median value) over 24 hours than those without amalgam (47.5 μg) (p=0.016). There was no difference in selenium excretion between groups with (42.4 μg) and without (39.4 μg) amalgam-related symptoms (p=0.15). The findings indicate that individuals exposed to low levels of elemental mercury from dental amalgam excrete less selenium to urine than unexposed individuals.     

A significant relationship between mercury exposure from dental amalgams and urinary porphyrins: a further assessment of the Casa Pia children’s dental amalgam trial

Previous studies noted specific changes in urinary porphyrin excretion patterns associated with exposure to mercury (Hg) in animals and humans. In our study, urinary porphyrin concentrations were examined in normal children 8–18 years-old from a reanalysis of data provided from a randomized, prospective clinical trial that was designed to evaluate the potential health consequences of prolonged exposure to Hg from dental amalgam fillings (the parent study). Our analysis examined dose-dependent correlations between increasing Hg exposure from dental amalgams and urinary porphyrins utilizing statistical models with adjustments for the baseline level (i.e. study year 1) of the following variables: urinary Hg, each urinary porphyrin measure, gender, race, and the level of lead (Pb) in each subject’s blood. Significant dose-dependent correlations between cumulative exposure to Hg from dental amalgams and urinary porphyrins associated with Hg body-burden (pentacarboxyporphyrin, precoproporphyrin, and coproporphyrin) were observed. Overall, 5–10% increases in Hg-associated porphyrins for subjects receiving an average number of dental amalgam fillings in comparison to subjects receiving only composite fillings were observed over the 8-year course of the study. In contrast, no significant correlations were observed between cumulative exposure to Hg from dental amalgams and urinary porphyrins not associated with Hg body-burden (uroporphyrin, heptacarboxyporphyrin, and hexacarboxyporphyrin). In conclusion, our study, in contrast to the no-effect results published from the parent study, further establishes the sensitivity and specificity of specific urinary porphyrins as a biomarker for low-level Hg body-burden, and also reveals that dental amalgams are a significant chronic contributor to Hg body-burden.     

Induction of metallothionein in rat tissues following subchronic exposure to mercury shown by radioimmunoassay

Although the analysis of metallothionein (MT) by radioimmunoassay (RIA) is not a common technique, its use is preferred over other methods since it offers the advantages of sensitivity and specificity. In this paper we present data on the basal levels of MT in rat tissues and physiological fluids of female Sprague-Dawley rats. The mean basal MT concentrations of the following organs and fluids were determined by RIA to be: liver (9.8 μg/g), kidney (68 μ/g), brain (0.8 μg/g), spleen (1.0 μg/g), heart (5.4 μg/g), plasma (11 ng/ml), and urine (200–300 μg/g creatinine). Following subcutaneous exposure to inorganic mercury (0.2 μmol/kg/d, 5 d a week for up to 4 wk), the metal accumulated primarily in the kidney. There was also a simultaneous accumulation of zinc in the liver and of zinc and copper in the kidney. Induction of MT did take place in liver, kidney, brain, and spleen. No increases in the MT contents of blood and urine were noted. The excess zinc and copper in the kidney of exposed animals were found to be associated predominantly with MT. No overt signs of mercury toxicity were noted in these animals and the incidence of proteinurea was nil. The data are discussed with reference to methods of MT determination in animal tissues and in relation to mercury metabolism and toxicity.     

Urinary mercury after administration of 2,3-dimercaptopropane-1-sulfonic acid: correlation with dental amalgam score.

There is considerable controversy as to whether dental amalgams may cause systemic health effects in humans because they liberate elemental mercury. Most such amalgams contain as much as 50% metallic mercury. To determine the influence of dental amalgams on the mercury body burden of humans, we have given volunteers, with and without amalgams in their mouth, the sodium salt of 2,3-dimercaptopropane-1-sulfonic acid (DMPS), a chelating agent safely used in the Soviet Union and West Germany for a number of years. The diameters of dental amalgams of the subjects were determined to obtain the amalgam score. Administration of 300 mg DMPS by mouth increased the mean urinary mercury excretion of the amalgam group from 0.70 to 17.2 micrograms and that of the nonamalgam group from 0.27 to 5.1 micrograms over a 9-h period. Two-thirds of the mercury excreted in the urine of those with dental amalgams appears to be derived originally from the mercury vapor released from their amalgams. Linear regression analysis indicated a highly significant positive correlation between the mercury excreted in the urine 2 h after DMPS administration and the dental amalgam scores. DMPS can be used to increase the urinary excretion of mercury and thus increase the significance and reliability of this measure of mercury exposure or burden, especially in cases of micromercurialism.     

Chronological changes in tissue copper, zinc and iron in the toxic milk mouse and effects of copper loading

The toxic milk (tx) mouse is a rodent model for Wilson disease, an inherited disorder of copper overload. Here we assessed the effect of copper accumulation in the tx mouse on zinc and iron metabolism. Copper, zinc and iron concentrations were determined in the liver, kidney, spleen and brain of control and copper-loaded animals by atomic absorption spectroscopy. Copper concentration increased dramatically in the liver, and was also significantly higher in the spleen, kidney and brain of control tx mice in the first few months of life compared with normal DL mice. Hepatic zinc was increased with age in the tx mouse, but zinc concentrations in the other organs were normal. Liver and kidney iron concentrations were significantly lower at birth in tx mice, but increased quickly to be comparable with control mice by 2 months of age. Iron concentration in the spleen was significantly higher in tx mice, but was lower in 5 day old tx pups. Copper-loading studies showed that normal DL mice ingesting 300 mg/l copper in their diet for 3 months maintained normal liver, kidney and brain copper, zinc and iron levels. Copper-loading of tx mice did not increase the already high liver copper concentrations, but spleen and brain copper concentrations were increased. Despite a significant elevation of copper in the brain of the copper-loaded tx mice no behavioural changes were observed. The livers of copper-loaded tx mice had a lower zinc concentration than control tx mice, whilst the kidney had double the concentration of iron suggesting that there was increased erythrocyte hemolysis in the copper-loaded mutants.     

Effect of dietary copper and zinc levels on tissue copper,zinc, and iron in male rats

The interaction between dietary copper and zinc as determined by tissue concentrations of trace elements was investigated in male Sprague-Dawley rats. Animals were fed diets in a factorial design with two levels of copper (0.5, 5 μg/g) and five levels of zinc (1, 4.5, 10, 100, 1000 μg/g) for 42 d. In rats fed the low copper diet, as dietary zinc concentration increased, the level of copper decreased in brain, testis, spleen, heart, liver, and intestine. There was no significant effect of dietary copper on tissue zinc levels. In the zinc-deficient groups, the level of iron was higher in most tissues than in tissues from controls (5 μg Cu, 100 μg Zn/g diet). In the copper-deficient groups, iron concentration was higher than control values only in the liver. These data show that dietary zinc affected tissue copper levels primarily when dietary copper was deficient, that dietary copper had no effect on tissue zinc, and that both zinc deficiency and copper deficiency affected tissue iron levels.     

Effect of dietary iron deficiency on mineral levels in tissues of rats

To clarify the influence of iron deficiency on mineral status, the following two synthetic diets were fed to male Wistar rats: a control diet containing 128 micrograms iron/g, and an iron-deficient diet containing 5.9 micrograms iron/g. The rats fed the iron-deficient diet showed pale red conjunctiva and less reactiveness than the rats fed the control diet. The hemoglobin concentration and hematocrit of the rats fed the iron-deficient diet were markedly less than the rats fed the control diet. The changes of mineral concentrations observed in tissues of the rats fed the iron-deficient diet, as compared with the rats fed the control diet, are summarized as follows: . Iron concentrations in blood, brain, lung, heart, liver, spleen, kidney, testis, femoral muscle, and tibia decreased; . Calcium concentrations in blood and liver increased; calcium concentration in lung decreased; . Magnesium concentration in blood increased; . Copper concentrations in blood, liver, spleen and tibia increased; copper concentration in femoral muscle decreased; . Zinc concentration in blood decreased; . Manganese concentrations in brain, heart, kidney, testis, femoral muscle and tibia increased. These results suggest that iron deficiency affects mineral status (iron, calcium, magnesium, copper, zinc, and manganese) in rats.     

Increased mercury emissions from modern dental amalgams

All types of dental amalgams contain mercury, which partly is emitted as mercury vapor. All types of dental amalgams corrode after being placed in the oral cavity. Modern high copper amalgams exhibit two new traits of increased instability. Firstly, when subjected to wear/polishing, droplets rich in mercury are formed on the surface, showing that mercury is not being strongly bonded to the base or alloy metals. Secondly, high copper amalgams emit substantially larger amounts of mercury vapor than the low copper amalgams used before the 1970s. High copper amalgams has been developed with focus on mechanical strength and corrosion resistance, but has been sub-optimized in other aspects, resulting in increased instability and higher emission of mercury vapor. This has not been presented to policy makers and scientists. Both low and high copper amalgams undergo a transformation process for several years after placement, resulting in a substantial reduction in mercury content, but there exist no limit for maximum allowed emission of mercury from dental amalgams. These modern high copper amalgams are nowadays totally dominating the European, US and other markets, resulting in significant emissions of mercury, not considered when judging their suitability for dental restoration.     

Expression of metallothionein in the liver and kidney of rats is influenced by excess dietary histidine

It is well known that excess dietary histidine induces the metabolic changes in copper and zinc. Therefore, this study was carried out to clarify whether excess dietary histidine alters the gene expressions of metallothionein-1 and metallothionein-2 in the liver and kidney. Male rats were fed the control (ad libitum and pair-fed) or histidine-excess (50 g of L-histidine per kg of diet) diet for 0, 1 and 3 days. The levels of liver metallothionein-1 and metallothionein-2 mRNA were markedly lower in the rats fed the histidine-excess diet as compared to those of the control (ad libitum and pair-fed) diet, when fed for 1 or 3 days. The levels of renal metallothionein-1 and metallothionein-2 mRNA in the rats fed the histidine-excess diet were higher or tended to be higher as compared with the rats fed the control (ad libitum and pair-fed) diet when fed for 1 or 3 days, respectively. At the same time, hepatic copper content was decreased and renal zinc content was increased by dietary histidine. It thus appears, that such a response on the level of liver metallothionein mRNA might be related to the contents of liver copper, but of kidney metallothionein mRNA might be due to the content of zinc.     

Effect of acute administration of mercuric chloride on the disposition of copper, zinc, and iron in the rat

The present study was designed to investigate the effect of mercuric chloride administration on copper, zinc, and iron concentrations in the liver, kidney, lung, heart, spleen, and muscle of rats. The results showed that after dose and time exposure to mercuric chloride, the concentration of mercury in the six tissues was significantly elevated. Data showed that there were no interaction between mercury and tissue iron. There was a considerable elevation of the content of copper in the kidney and liver. The most significant changes in the copper concentration took place in the kidneys. About a twofold increase in the copper content of the kidney was noted after exposure to mercuric chloride (3 mg and 5 mg/kg). Only slight elevations in the copper content occurred in the liver, especially in high dose and longer exposure time. In the remaining organs, the copper content was not changed significantly (p>0.05). The most significant changes in the zinc concentration took place in liver, kidney, lung, and heart (5 mg/kg). Marked changes in kidney zinc concentrations were observed at any of the specified doses. Zinc concentrations were significantly increased in kidney of rats sacrificed 9–48 h after sc injection of HgCl₂ (5 mg/kg); in liver obtained from rats at 18, 24, or 48 h after injection; and in lung after 24 or 48 h of treatment. The heart and spleen zinc concentrations were elevated at 24 and 48 h after injection of HgCl₂ (5 mg/kg), respectively. The results of this study implicate that effects on copper and zinc concentrations of the target tissues of mercury may play an important role in the pathogenesis of acute mercuric chloride intoxication.     

Cadmium, copper and zinc in tissues of deceased copper smelter workers

Workers at a copper and lead smelter in northern Sweden have a multifactorial exposure to a number of heavy metals. The concentrations of cadmium, copper and zinc in liver, lung, kidney and brain tissues have been determined by atomic absorption spectrometry in 32 deceased long-term exposed male lead smelter workers, and compared with those of 10 male controls. Furthermore, copper and zinc levels in hair and nails were determined by energy-dispersive X-ray fluorescence.

The highest cadmium concentrations among both workers and controls were observed in kidney, followed in order by liver, lung and brain. The levels in kidney, liver and lung were all significantly higher in the workers than in the controls (p < 0.03). Among the workers relatively strong positive correlations (p < 0.03) were observed between cadmium concentrations in liver and lung, liver and kidney, liver and brain, and lung and brain. In the exposed workers a positive correlation was observed between cadmium and zinc concentrations in the kidney (r_s = 0.38; p = 0.034). This is probably mainly due to the protein metallothionein, which is stored in the kidney, binding equimolar amounts of these two metals.

The highest concentrations of copper were found in hair and nails among both workers and controls, followed in order by liver, brain, kidney and lung. The tissue concentrations of copper in brain, lung and kidney were all significantly higher among the smelter workers than in the controls (p ≤ 0.036). Copper levels in lung and age at time of death were positively correlated among the exposed workers (r_s = 0.39; p = 0.029). In the same group, positive correlations between copper and zinc concentrations in kidney (r_s = 0.45; p = 0.009) and nails (r_s = 0.68; p < 0.001) were also observed, reflecting possible biological interactions between these two metals.

Among both workers and controls, the highest zinc concentrations were found in hair, followed in order by nails, liver, kidney, brain and lung. Significantly higher tissue concentrations among the workers as compared with the reference group were noted in kidney, liver and brain (p ≤ 0.033).

Neither copper nor zinc concentrations in hair and nails seemed to provide a useful measure of the trace element status of the smelter workers.     

Effect of low dietary rubidium on plasma biochemical parameters and mineral levels in rats

The effects of low dietary rubidium on plasma biochemical parameters and mineral levels in tissues in rats were studied. Eighteen male Wistar rats, weighing about 40 g, were divided into two groups and fed the diets with or without supplemental rubidium (0.54 vs 8.12 mg/kg diet) for 11 wk. Compared to the rats fed the diet with supplemental rubidium, the animals fed the diet without rubidium supplementation had higher urea nitrogen in plasma; lower rubidium concentration in tissues; lower sodium in muscle; higher potassium in plasma, kidney and tibia, and lower potassium in testis; lower phosphorus in heart and spleen; lower calcium in spleen; higher magnesium in muscle and tibia; higher iron in muscle; lower zinc in plasma and testis; and lower copper in heart, liver, and spleen, and higher copper in kidney. These results suggest that rubidium concentration in tissues reflects rubidium intake, and that rubidium depletion affects mineral (sodium, potassium, phosphorus, calcium, magnesium, iron, zinc, and copper) status.