Total Liver Copper Research Articles

The Protective Effect of Soybean and Thyme on Iron Deficiency Anemia in Rats

Objective: The present study is carried out to investigate the protective effect and antioxidant activity of soybean and thyme on iron deficiency anemia. Material and Methods: Thirty five male albino rats were divided into five groups (7rats each). The first group fed on basal diet, iron sufficient (35 mg Fe / kg), and served as control. Rats of other groups (second - fifth) were induced anemic by placing them on diet containing 3mg Fe / kg for 21 days, then divided to four dietary groups. The second group (anemic) stayed on basal diet with Fe-deficiency. The third group fed on basal diet with sufficient iron in the form of ferrous sulphate. The fourth group fed modified basal diet free from iron and supplemented with soybean. The fifth group fed basal diet free from iron and supplemented with thyme. All three iron sources provide 35 mg iron/kg diet. At the end of experiment (49 days), rats were anesthetized, whole blood was used for determination of hemoglobin (Hb), hematocrit (HCt) and reduced glutathione (GSH) levels. Serum was used for determination of iron and lipid profile as well as lipid peroxidation as malondialdehyde (MDA). The liver was used for determination of iron and copper concentrations. Results: The present results indicated that Fe-deficiency caused many adverse effects reflected the significant decrease of Hb, HCt, serum iron, liver iron, GSH and high density lipoprotein-cholesterol (HDL-C). Fe-deficiency also caused significant increase in total iron binding capacity (TIBC), liver copper, MDA, triacylglycerols (TG) and total cholesterol (TC). In contrary, administration of ferrous sulphate (FeSO4), soybean or thyme induced a significant increase of serum and liver iron profile. Conclusion: Soybean and thyme could able to provide iron to correct dietary irondeficiency anemia and powerful antioxidant effect of soybean or thyme was reflected on marked decrease of MDA and increase of GSH and HDL-C.

Elements in autopsy liver tissue samples from Greenlandic Inuit and Danes. IV. Copper measured by X-ray fluorescence spectrometry

Objective: To measure the content of copper (Cu) in liver tissue samples from Greenlandic Inuit and compare the results with those obtained in Caucasian Danes. Materials and methods: Normal liver tissue samples were obtained at autopsy from 50 Inuit (27 men, 23 women) with a median age of 61 years (range 23 - 83) and from 74 Danes (44 men, 30 women) with a median age of 60 years (range 15 - 87). Total liver copper content was measured by X-ray fluorescence spectrometry. Results: The content of copper given as median and 5 - 95 percentile was in Inuit 0.2982 mmol/kg dry liver (0.1810 -0.6531), and in Danes 0.3777 mmol/kg dry liver (0.1889 - 0.7550) (p = 0.02). This difference was due to a higher median liver copper content in Danish men compared with Danish women (p = 0.04), Inuit men (p = 0.005) and Inuit women (p = 0.05), and was based solely on the 3 highest liver copper values in Danish men. Inuit displayed no gender difference concerning liver copper content. There was no correlation between liver copper content and age either in Inuit or in Danes. In Inuit, the median hepatic copper index (liver copper content divided by age) was 0.0045 in men and 0.0066 in women (p = 0. 14). There was an inverse correlation between hepatic copper index and age in the entire Inuit series (r s = -0.63, p < 0.0001). In Danes, the median hepatic copper index was 0.0082 in men and 0.0046 in women (p = 0.005). There was an inverse correlation between hepatic copper index and age in the entire Danish series (r s = -0.76, p < 0.0001). Conclusion: There is no evidence to suggest that the prevalence of copper deficiency or copper overload in adults differ in Inuit and Danes. Inuit have a hepatic copper content, which is similar to that found in Caucasian Danes and other populations from the Western societies.

The effects of supplementation of the diet of young steers with Mo and S on the intracellular distribution of copper in liver and on copper fractions in blood

Young Jersey steers weighing between 100 and 200 kg were fed a basic diet supplemented with Mo, 35 ppm, and S, 0·3% of dry matter intake, for 24 weeks. Total liver copper levels, assessed from biopsy material, fell more rapidly than those of a control group fed the basic diet. While the copper levels declined in all the subcellular fractions examined, that is nuclear, mitochondrial and cytosol; the decrease was most marked in the nuclear fraction. Moderate supplementation (5 ppm Cu) of the controls increased liver copper values, particularly in the nuclear fraction. Total plasma copper levels rose initially in the Mo and S supplemented animals, but the most striking feature was the appearance of a TCA-insoluble copper fraction in plasma. Levels of TCA-insoluble copper were highest in the first month of supplementation, afterwards levels declined. Caeruloplasmin activities were related to the levels of TCA-soluble Cu in plasma. In the Mo and S supplemented animals activities fell whereas in the control animals caeruloplasmin was unaffected. There was some correlation between the copper in the TCA-soluble plasma fraction and total liver copper ( r = 0·7928) and a better correlation ( r = 0·8448) between the TCA-insoluble plasma fraction and the copper of the nuclear fraction in liver. Regression equations were derived from the data which relate the total copper in liver and in the nuclear fraction to copper levels in these plasma fractions.

Effect of ionophores on the accumulation of copper in the livers of sheep

ABSTRACTLambs were allocated to three treatments: control (C), monensin (M) and lasalocid sodium (L) in a concentrate diet containing 36 mg copper per kg food. After 2 months deaths from copper toxicity occurred and the trial was terminated. All lambs were slaughtered. The mean liver copper concentration (mg/kg dry matter) of 1907 in the M treatment was higher than the mean concentrations of 1342 and 1229 in the C and L groups respectively (P &lt; 0·01). Total liver copper was 234, 260 and 230 mg and the retention of dietary copper in the liver was 93·5, 108·5 and 93·3 g/kg for the C, M and L treatments respectively. These differences were not significant. From reduced liver size (as a proportion of body weight) and high copper levels in the kidneys, it was concluded that the group given monensin had accumulated copper to a level closer to the toxicity threshold than the other two groups.

The lysosomal copper concentration in the liver in primary biliary cirrhosis.

The copper concentration in the lysosomes of periportal hepatocytes of nine patients with primary biliary cirrhosis was determined by X-ray microanalysis in ultrathin sections, the chlorine concentration of the embedding medium being taken as internal standard. A positive correlation was found between the lysosomal and the total liver copper concentration. No correlation was found between the lysosomal copper concentration and the grade of orcein staining for lysosomal copper-associated protein in paraffin sections. It is concluded that in contrast to the grade of orcein staining, the lysosomal copper concentration is a reliable measure of the total liver copper concentration in primary biliary cirrhosis.

Copper metabolism during acute inflammation: studies on liver and serum copper concentrations in normal and inflamed rats.

1 The concentration of copper in serum and liver was determined by atomic absorption spectrophotometry in a study performed on normal rats of either sex and in female rats with carrageenan-induced pleurisy. 2. In the normal animal, total serum copper concentration is significantly higher in female rats, and appears to be higher in mature animals in females. 3 In normal rats of either sex, liver copper concentration undergoes daily variations which are inversely related to the weight of the organ and which leave constant the total amount of metal in the liver. Moreover a day to day non-cyclic variability of liver copper concentration and liver weight was observed. 4 This first set of data showed that comparison with time control was essential. 5 In the inflamed rat, a significant rise of total serum copper at 22, 48 and 72 h after the induction of inflammation was observed. From 96 h up to 240 h post-injection no significant differences were evident. 6 Total liver copper content did not change in the inflamed rats. 7 During acute inflammation in the rat, the copper needed for the increased synthesis of caeruloplasmin is supplied without depletion of liver copper stores.

The effect of molybdenum and sulfur on the kinetics of copper metabolism in sheep

Sheep were given 0, 5, 10, 15 or 25 mg/day molybdenum with or without 1.1 g/day sulfur orally for 1 week, and the nature of the interaction between these elements and copper was then investigated by injecting 64Cu intravenously, and analysing the disappearance of 64Cu from the blood by compartmental analysis. Molybdenum treatment decreased the rate of disappearance of 64Cu from the blood, and this decrease was enhanced by the concurrent administration of sulfur. Analysis of the disappearance curves in terms of a three-compartment catenary model showed that calculated total liver copper pool size (mean�s.d. (n = 4)) was decreased from 37.5�6.4 mg (no treatment) to 6.3 � 1.9 mg (50 mg/day molybdenum plus sulfur), whereas the liver copper pool size (determined by biopsy) remained unchanged. The reduction in calculated pool size was proportional to the molybdenum dose and was similar in magnitude to the reduction in actual copper pool size obtained following long-term (6 months) dosing with molybdenum and sulfur. These changes were most likely due to alterations in the kinetic parameters responsible for the initial phase of removal of 64Cu from the blood, i.e. K21 (the rate of transfer between blood copper (Q1)and liver copper (Q1 + Q2)), and bloodcopper. Consequently, therelationship between K21,various blood copper fractions and molybdenum dose rate was investigated using the results of this experiment and data obtained from the literature; it was found that a reduction in the amount of copper in the blood which exchanged freely with the 64Cu was the most likely explanation for the decrease in the rate of disappearance of 64Cu from the blood following molybdenum and sulfur treatment.

The effect of breed of sire on the accumulation of copper in lambs, with particular reference to copper toxicity

ABSTRACTLambs, 9 weeks of age, the offspring of sires of the Scottish Blackface, East Friesland, Finnish Landrace, Suffolk and Texel breeds mated to Scottish Blackface females, as a common maternal breed, were given diets containing 12 or 20 mg copper per kg dry matter for 13 weeks. Concentrations of liver copper and of copper and aspartate amino-transferase in the plasma were measured regularly, and the concentrations of copper in the kidney, mandible and cheek muscle were also measured terminally. Livers were examined for histological symptoms of copper toxicity.Wide breed variation was apparent in liver copper concentration after 6 weeks and in plasma aspartate aminotransferase concentration after 9 weeks. At slaughter, total liver copper and kidney copper concentration showed significant breed variation. For all traits, the Texel-cross lambs had the highest concentrations, followed by the Suffolk crosses, and the Blackface had the lowest. The proportion of ingested copper retained in the liver of the Texel-cross lambs was twice that of the Blackface lambs (0·137 v. 0·056), with the other breed crosses intermediate.There was a significant difference between diets for liver, kidney and mandible copper concentrations, plasma aspartate amino-transferase concentration, and for total liver copper but not for cheek muscle. Both diets produced elevated plasma aspartate amino-transferase concentration and histological symptoms of copper toxicity in the livers of some lambs.The results show large breed differences in the susceptibility to copper poisoning, some breeds being at risk when given diets containing 12mg copper per kg dry matter for long periods. There was no suggestion that breeds differed in resistance to poisoning other than through different retentions of copper consumed

Human fetal liver contains both zinc- and copper-rich forms of metallothionein.

Metallothionein was purified under anaerobic conditions from livers of human fetuses ranging from 19 weeks gestational age to term. Homogeneous metallothionein obtained in the absence of reducing agents from the soluble fraction of the tissue which contained 24% and 85% of the total liver copper and zinc, respectively, had less than 1 g atom of copper and about 3.1 g atoms of zinc/mol. Extraction with 1% 2-mercaptoethanol of the insoluble fraction of the tissue (containing 76% and 15% of the liver copper and zinc, respectively) yielded a metallothionein with approximately 2.5 g atoms of copper and 1.3 g atoms of zinc. When the whole tissue was extracted similarly by the procedure of Rydén and Deutsch ((1978) J. Biol. Chem. 253, 519), nearly equal proportions of the two metals were obtained in the soluble fraction from which metallothionein, with about 3.0 g atoms of zinc and 1.5 g atoms of copper, was purified. Thus, zinc- and copper-rich forms of metallothionein are differently distributed between the soluble and insoluble fractions of fetal liver. Nevertheless, the predominant metal in metallothionein of human fetal liver is zinc as is the case in the adult; significant amounts of copper are also present, consistent with the elevated quantity of this metal in the fetal tissue relative to the adult. Cadmium was not detected. Zinc is much more easily removed from the protein than is copper.

A kinetic model of copper metabolism in sheep

A three-compartment model was developed to provide a simple kinetic description of the metabolism of copper in six sheep with liver copper concentrations of 433�51 ppm ( mean � SD dry weight). This was achieved by measuring the radioactivity in samples of blood, liver, urine and bile as a function of time after the intravenous administration of c. 1.4 mCi of 64Cu. Sizes of compartments, flow rates and rate constants were evaluated and validated experimentally by measuring the excretion of copper in urine and bile and its accumulation in the liver. The model postulates two separate mechanisms for the handling of copper by the liver, and biliary copper excretion was not related to total liver copper content. The model was tested in sheep with a wider range of liver copper concentrations, and the copper in the two compartments (C2 and C3), attributed to liver, corresponded to the actual liver copper content when this was between 30 and 70 mg. The rate constants also responded consistently to increased liver copper status. Within the 'normal' liver copper range of 50-70 mg, the three-compartment model was closed but outside this range, the undefined parameter K03 was no longer zero. This response suggests that copper is moving from C2 and C3 to either supply tissue requirements or be redistributed in an additional storage compartment.

The effect of lupinosis on liver copper, selenium and zinc concentrations in merino sheep.

Merino wether weaners were exposed to toxic lupin stubbles for periods of one, two and six to nine weeks, and the effect on their liver copper, selenium and zinc concentrations studied. After the one week period there was a slight decrease in liver copper and selenium concentrations in sheep severely affected with lupinosis. This was attributed to loss of these elements from necrotic cells, and greatly increased quantities of fat in the liver. In the same period liver zinc concentrations declined and were negatively correlated with the degree of liver injury. After two weeks or more of exposure to toxic lupins a positive correlation existed between both the liver copper and selenium concentrations, and the degree of liver injury. Furthermore, total liver copper and selenium levels were also positively correlated with the degree of liver injury. Liver zinc concentrations were negatively correlated with the degree of liver injury. It is suggested that when the liver is under the influence of the toxins causing lupinosis for more than two weeks, it stores copper and selenium, and loses zinc.

The distribution of soluble copper- and zinc-binding proteins in goat liver.

The distribution of copper and zinc in the soluble proteins from goat liver after gel filtration were determined. Samples from normal slaughtered animals as well as from experimentally copper-loaded goats were tested. The copper- and zinc-binding proteins were mainly separated into 3 different fractions. The approximate molecular weights were greater than 65,000, 35,000 and 10,000. Varying amounts of zinc were eluted in a fourth fraction of molecular weight less than 2000. The results indicate that the distribution of copper and zinc among soluble metal-binding proteins are highly dependent on the total liver zinc concentration. In the majority of samples, with zinc levels within 20 to 45 microgram/g wet weight, the dominating copper- and zinc-binding protein fraction was the high-molecular weight fraction. This did not seem to be influenced by the total liver copper level. In the samples with high total zinc levels (greater than 60 microgram/g w.w.) there was a shift in protein distribution of both metals toward the low-molecular weight fractions.

Hair as a biopsy material V. Hair metal as an index of hepatic metal in rats: copper and zinc

Hair copper in 500 day-old adult rats was found to correlate directly with copper in whole liver (P < 0.001) and the subcellular liver nuclear (P < 0.022), and cytosol fractions (P < 0.001). An inverse correlation was found between copper in liver microsomes and plasma cholesterol (P < 0.044). Similar data analysis for zinc showed no significant correlations (P > 0.05). The determination of copper in hair may be useful for assessing total liver copper content in human beings. Copper in liver microsomes may participate in the control of plasma cholesterol.

Controlling Factors in the Development of Ceruloplasmin in Pigs during the Neonatal Growth Period

The sequence of ceruloplasmin development and limiting factors controlling this process in neonatal piglets was studied in a randomized block design. Guided by the change in serum ceruloplasmin levels, the liver copper concentrations of piglets fed copper-deficient and copper-supplemented diets were compared in three different periods. The plasma of piglets was devoid of both apo- and holo-ceruloplasmin at birth. The system responsible for the synthesis of apoceruloplasmin developed shortly after birth as indicated by a steady increase in serum ceruloplasmin activity during the first week without being affected by the diet. Copper apparently was not the limiting factor at this stage since very high concentrations of copper were found in the livers of newborn piglets. However, the liver copper was depleted quickly within 1 to 2 weeks and became the limiting factor if the diet was deficient in copper. The livers of these piglets were fractionated by differential centrifugation to compare the liver copper distribution in the two dietary groups with and without copper supplementation. The treatments did not affect the copper distribution on a percentage of total liver copper basis. However, fractionation study revealed that, unlike adult pig livers, about 70% to 80% of copper was present in the heavier particle fractions of piglet livers.

Localization and uptake of copper into chromatin

Analysis of total liver, liver nuclei, and nuclear fractions obtained from mice given either deionized water (control) or deionized water containing cupric chloride (experimental) revealed the presence of copper in all samples tested including control nuclei and control heterochromatin and euchromatin. Copper content in control euchromatin appear to remain constant at approximately 0.3 μg/mg DNA, while the concentration in control heterochromatin seem to be subject to wider variations. An elevation in total liver copper followed prolonged treatment with metal ( in vivo) and was accompanied by an increase in total nuclear copper. A preferential uptake of copper by heterochromatin was suggested by the majority of metal binding to this fraction; there was no significant metal accumulation in euchromatin. Dialysis equilibrium experiments were carried out at 25°C, pH 6.0 to determine in vitro binding of the two nuclear fractions. Under physiologic ionic strength (154 m m) heterochromatin binds considerable copper, whereas euchromatin fails to bind appreciable amounts. This biological specificity is lost under conditions of low ionic strength (<2 m m NaCl).

The effect of copper loading on the distribution of copper in rat liver cytosol

Male Sprague-Dawley rats were given daily injections of copper (2.5 mg Cu 2+/kg body wt as copper sulfate in 0.9% NaCl) for 1–10 days. Rats were sacrificed at various time intervals and the perfused livers were fractionated. Copper in the total liver and cytosol were measured and the cytosol was further examined after chromatography on Sephadex G-75. Copper accumulated predominantly in the particulate fractions of the liver after ip injection of copper; whereas 46% of the copper added in vitro was found in the cytosol. The distribution of copper within the cytosol was different when copper was added in vivo or in vitro. In the former case, as much as 60% of the copper co-chromatographed with proteins of molecular weight 11,500–14,000 (C-peak proteins) whereas in the latter case the added copper was bound to high molecular-weight proteins (>100,000). Copper loading favored the binding of copper to the C-peak proteins in rat liver cytosol. The copper in the C-peak decreased exponentially when the copper injections were terminated. Acrylamide gel electrophoresis was used to determine the protein content of cooper-containing fractions prepared using Sephadex G-75. A protein has been identified which may act as a transport protein and whose copper content depends on the degree of copper balance in the rat; i.e., it varies with the total liver copper. The nature of the copper-binding to the C-peak proteins was also discussed.

Clinically silent hypocuprosis and the effect of molybdenum loading on beef calves in Gippsland, Victoria

Whole blood and liver copper concentrations of less than 0.5 µg per ml and 6 p.p.m. dry weight respectively were found in healthy beef calves from an acid soil region at Koonwarra in Victoria. Pasture samples collected each month for a year contained 2.4–6.7 p.p.m. dry weight of copper, 2.1–9.2 p.p.m, dry weight of molybdenum, and 0.07–0.47% dry weight of sulphate. The copper/molybdenum ratio was usually well below 2.0.Changes in the copper status of the cattle were related to seasonal fluctuations in the copper and molybdenum contents of the pastures. A macrocytic hypochromic anaemia was found in hypocuprotic calves. However, pallor of the mucous membranes was not observed and there was no relationship between coat colour and copper concentration of either hair or whole blood. The subcutaneous injection of 120 mg copper as glycinate raised the concentration of copper in the whole blood and liver of calves to c. 0.8 µg/ml and up to 50 p.p.m. dry weight respectively. Growth rates of calves given copper improved significantly and their carcass weights were 8.4% heavier than controls. In this environment the effects of a single treatment with copper glycinate lasted only 5 months. Although liver copper contents ranged from below 5 to above 70 p.p.m. dry weight, the copper contents of other tissues examined, such as kidney, spleen, heart, and bones, were similar whether the animals had received additional copper or molybdenum or neither. At the levels of molybdenum found in the pastures (2–9 p.p.m. dry weight) the synthesis of ceruloplasmin was readily decreased, whereas erythrocyte copper decreased more slowly. Therefore the estimation of ceruloplasmin oxidase activity in plasma is suggested as a reliable and convenient means of assessing bovine hypocuprosis. The regression analyses showed a strongly positive correlation (r = 0.97) of plasma copper with ceruloplasmin oxidase activity as determined with p-phenylenediamine as substrate at the optimal pH. Special molybdenum pellets given to the calves augmented the tissue levels of molybdenum tenfold, but copper depletion did not occur to the point where clinical signs of copper deficiency or of molybdenosis appeared. In contrast to calves with large copper reserves, the mitochondria1 fraction of liver cells from hypocuprotic calves was found to contain more and the microsomal fraction less of the total liver copper. Because of the complex relationships between soil, pasture, and animals, it is concluded that a general recommendation for the minimum copper requirement of grazing cattle cannot reasonably be made without reference to soil and pasture concentrations of copper, molybdenum, and sulphate.

Effects of liming and various forms of oral copper supplementation on the copper status of grazing sheep.

AbstractSheep grazing limed pasture for periods extending to 14 months had consistently lower mean blood copper concentrations than similar sheep on comparable untreated pasture. Liver copper concentrations and total liver copper contents were also significantly lower for the sheep on the limed herbage. Liming did not affect the herbage copper concentration. Herbage copper concentrations of 8‐9 ppm appeared to be insufficient to maintain the blood and liver copper status of sheep grazing limed pasture at adequate levels.Prolonged oral supplementation providing 70 mg Cu per week as the sulphate, glycinate or EDTA salt increased blood and liver copper concentrations to similar degrees and with no apparent risk of toxicity.

Effect of malnutrition on liver copper in the rat.

SummaryMalnourished rats have a lower total liver copper than normal rats. However there is no significant change in copper content per 100 g liver or per g body weight.