Caeruloplasmin Activity Research Articles

A Marked Increase in Free Copper Levels in the Plasma and Liver of LEC Rats: An Animal Model for Wilson Disease and Liver Cancer

Most of copper present in rat plasma and liver binds to caeruloplasmin and metallothionein, respectively, and is not redox active. However, free forms of copper including loosely bound forms to other molecules are redox active. We assessed the free copper in Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease and liver cancer. Compared to those of control rats, the liver and plasma of LEC rats showed a marked elevation of free copper, especially at the stage of acute hepatitis, in parallel with an increase of total copper levels in the livers and a decrease of plasma caeruloplasmin (ferroxidase I) activity. At the onset of jaundice, the total copper levels, however, decreased in liver, but increased in plasma, while free copper levels in both liver and plasma remained higher. Free iron levels in both liver and plasma were also determined and did not change significantly, except for the case of plasma in jaundiced rats. The data are consistent with a proposal in which increased levels of redox active free copper in the liver of LEC rats catalyze Fenton-type reactions, producing a large flux of hydroxyl radicals that would play an important role in the observed liver dysfunction, leading to acute hepatitis, and, finally, hepatocarcinoma. This is the first demonstration that the free copper may participate in the pathophysiology of the LEC rats and Wilson disease.

Carbamylation Inhibits the Ferroxidase Activity of Ceruloplasmin

The ferroxidase activity of caeruloplasmin (EC 1.16.3.1) is an important antioxidant defence mechanism in man. In chronic renal failure proteins become carbamylated as a result of reactions with urea-derived cyanate. We have therefore investigated the effects of carbamylation on the ferroxidase activity of caeruloplasmin. Increasing degrees of carbamylation produce a progressive impairment of ferroxidase activity measured using o-dianisidine as substrate, and the ability of caeruloplasmin to load ferrous iron onto ovotransferrin is reduced. Carbamylation of caeruloplasmin may contribute to reduced antioxidant capacity in patients with renal failure.

High iron intake depresses hepatic copper content in goats

Earlier studies with ruminants point to a depressant effect of dietary iron on the copper status. To verify this we determined hepatic copper concentrations in dry, non-pregnant goats subjected to a 56 x 56-days cross-over trial with adequate copper rations containing either 269 or 2380 mg iron/kg dry matter. High iron intake reduced the group mean plasma copper (by 18%) and caeruloplasmin activity (by 13%) and produced a significant decrease (27%) in hepatic copper concentrations. Hepatic iron concentrations were raised (by 56%) after feeding the high iron ration. It is concluded that high dietary levels of iron, within the range of their fluctuation in silage and forage, can impair the copper status of ruminants, especially when concurrent intakes of copper are low.

Vitamin C at concentrations observed in premature babies inhibits the ferroxidase activity of caeruloplasmin.

High concentrations of total vitamin C have been measured in the plasma of premature infants. At these concentrations ascorbic acid inhibited the ferroxidase activity of caeruloplasmin measured directly in vitro. The degree of inhibition was dependent on the ratio of ascorbic acid: caeruloplasmin. Values for the ratio of vitamin C: caeruloplasmin measured in premature babies would be predicted to inhibit ferroxidase activity by up to at least 80%. Ferroxidase activity measured in the plasma of premature babies increased from birth but was significantly lower than in plasma collected from adults (< 0.001). Plasma ferroxidase activity was correlated with plasma caeruloplasmin concentration and, in premature babies only, showed a negative correlation with the ratio of vitamin C to caeruloplasmin. High levels of vitamin C in premature babies may compromise antioxidant mechanisms and exacerbate oxidant damage.

High plasma vitamin C concentrations at birth associated with low antioxidant status and poor outcome in premature infants.

Concentrations of circulating antioxidants may be important in the aetiology of disease in premature infants. Blood samples were taken from 49 premature infants within two hours of birth. Plasma ascorbic acid, caeruloplasmin, and the ability of plasma to prevent lipid peroxidation in vitro were measured. Plasma ascorbic acid concentrations at birth ranged from 26.3 to 185.4 mumol/l. Plasma antioxidant activity at birth (microliter plasma required to inhibit lipid peroxidation) showed a strong negative correlation with plasma ascorbic acid and the plasma ascorbic acid to caeruloplasmin ratio. After correcting for gestational age and birth weight, plasma antioxidant activity at birth remained a significant predictor of mortality. Such plasma antioxidant activity is partly determined by the ferroxidase activity of caeruloplasmin. This may be modulated by high concentrations of ascorbic acid which may inhibit the antioxidant activity of plasma and thereby influence outcome.

Monoclonal antibody against the active site of caeruloplasmin and the ELISA system detecting active caeruloplasmin.

Serum caeruloplasmin deficiency is a characteristic biochemical abnormality found in patients with Wilson's disease, but the mechanism of this disease is unknown. Although the phenylenediamine oxidase activity of serum caeruloplasmin is markedly low in patients with Wilson's disease, mRNA of caeruloplasmin exists to some extent. To investigate the deficiency of caeruloplasmin oxidase activity in Wilson's disease, we generated 14 monoclonal antibodies (MAbs) and selected ID1, which had the strongest reactivity, and ID2, which had neutralizing ability. We also established a system to measure active caeruloplasmin specifically using these MAbs. These MAbs and the system will be useful tools in analyzing the active site of caeruloplasmin in patients with Wilson's disease.

Peroxynitrite releases copper from caeruloplasmin: implications for atherosclerosis

Peroxynitrite may be formed in the vasculature by the reaction of puperoxide with nitric oxide. When the blue copper-containing protein, caeruloplasmin, is incubated with peroxynitrite, copper is released, and ferroxidase activity and the blue colouration are lost. When plasma from normal subjects is incubated with peroxynitrite, the oxidant reacts with numerous plasma constituents but is still able to release copper from caeruloplasmin. As the ferroxidase activity of caeruloplasmin is lost in plasma in the presence of peroxynitrite, a second ferroxidase activity associated with peroxidised lipids, and not inhibited by azide, is formed.

Ferrous ions detected in cerebrospinal fluid by using bleomycin and DNA damage.

1. During pathological states of iron-overload or oxidant stress, low-molecular-mass iron can become available within extracellular fluids. 2. This iron would be converted to the ferrous state were it not for the protective anti-oxidant protein caeruloplasmin. 3. The ferrous-ion-oxidizing activity of caeruloplasmin rapidly converts ferrous ions back to the less reactive ferric state so that they can bind to available binding sites on transferrin. 4. Cerebrospinal fluids, however, often appear to contain low-molecular-mass iron, high levels of ascorbate and low levels of ferroxidase activity with little or no iron-binding capacity. 5. When iron ions are present in cerebrospinal fluid they are therefore likely to be in the ferrous state. 6. The development and application of an assay to speciate and measure ferrous ions in simple aqueous solution and their redox cycling activity in biological fluids is described.

Plasma ascorbate levels and inhibition of the antioxidant activity of caeruloplasmin

1. The copper-containing protein caeruloplasmin has several oxidase activities. 2. Its ability to catalyse the oxidation of ferrous ions to the ferric state (ferroxidase activity) makes it an important antioxidant in vivo. 3. Recent reports have suggested that oral supplementation with vitamin C can inhibit the oxidase activities of caeruloplasmin. 4. As expected, damage to DNA and membrane lipids was stimulated by mixtures of iron salt and ascorbate, and this damage could be inhibited by caeruloplasmin provided the molar ratio of ascorbate to caeruloplasmin was kept sufficiently low. 5. When the molar ratio of ascorbate to caeruloplasmin was greater than 200 substantial loss of ferroxidase antioxidant activity occurred. 6. It is unlikely, however, that oral supplementation with vitamin C can raise plasma levels sufficiently to inhibit caeruloplasmin activity in vivo.

The effects of trinitrotoluene toxicity on zinc and copper metabolism

Wistar rats were exposed to trinitrotoluene (TNT, 200 mg/kg/d × 6 d/wk) for 6 weeks, and a 2-week recovery period was continued after treatment. After initiation of TNT exposure, zinc and copper concentrations in rat testes, liver and serum, and serum caeruloplasmin activity were assayed every 2 weeks. Testicular size and zinc concentrations were decreased throughout the exposure and recovery periods. After 6 weeks of TNT exposure, rat testicular copper and serum zinc concentrations were significantly decreased and increased, respectively. The activities of caeruloplasmin were decreased after 4 and 6 weeks of exposure. Liver zinc and copper concentrations and serum copper concentrations had not changed significantly in rats throughout the exposure and recovery periods. Copper concentration in hair samples from TNTexposed workers was lower than that in control workers, but zinc content was significantly increased compared with that in control workers.

Effects of dietary copper deficiency on male offspring of heterozygous brindled mice.

Female C57BL mice heterozygous for the brindled gene were mated to normal males and fed on a purified diet low in copper throughout gestation and lactation with (+Cu) or without (-Cu) Cu-supplemented drinking water. Male offspring of two genotypes (control, +/y and brindled, Mobr/y) were compared when 10-12 d old. Brindled mice from dams on the -Cu treatment were smaller and had lower packed cell volumes than brindled mice from dams on the +Cu treatment. The -Cu brindled mice were smaller than their littermate brothers (+/y) but had equivalent biochemical features consistent with severe Cu deficiency. Compared with control mice from dams on the +Cu treatment, caeruloplasmin (EC 1.16.3.1) activity was lower in offspring of all three other groups including Mobr/y mice who were not anaemic. Iron levels were similar in organs and bone marrow from all four groups of offspring. When dietary Cu is limiting in brindled mice a more severe Cu deficiency ensues. Thus, appropriate Cu nutriture is important to the management of Menkes' disease in humans, a genetic analogue of the brindled mouse.

Lipoprotein Oxidation and Induction of Ferroxidase Activity in Stored Human Extracellular Fluids

It was observed that during the storage of human extracellular fluids at -20 degrees C the azide-inhibitable ferroxidase activity of caeruloplasmin declined, whilst a new azide-resistant ferroxidase activity (ARFA) developed. The literature suggested that storage-induced ARFA might be due to either a poorly defined enzymatic activity of a low density lipoprotein (LDL) or to lipid peroxides formed within the different lipoprotein fractions. To study this further, the major lipoprotein classes were separated from human serum by density gradient centrifugation. After storage of the lipoprotein fractions, it was found that the LDL fraction had the highest specific activity of ARFA and the highest content of lipid peroxidation products, as assessed by diene conjugates. The ARFA of LDL correlated with its content of diene conjugates and TBA reactive material, which initially suggested that the Fe(II) oxidising activity of peroxidase LDL arose from the reduction of peroxides by Fe(II) in the classical reaction between the metal ion and free radical reduction of lipid peroxides. However, steady state kinetic analysis indicated an enzymic role of LDL in Fe(II) oxidation, with lipid peroxides acting as a substrate for the enzyme. These results indicate that LDL may contain a peroxidase activity, catalysing the oxidation of Fe(II) by lipid peroxides, as well as a ferrous oxidase activity where O2 is the oxidising substrate.

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.

An automated method for the kinetic measurement of ferroxidase activity.

The oxidase activity of caeruloplasmin towards Fe II (ferroxidase) appears to be of physiological significance both in iron metabolism and as a serum anti-oxidant. We have developed an automated assay for ferroxidase using a centrifugal analyser. The method is quick and precise, and is not subject to interference by haemolysis, jaundice or lipaemia. Ferroxidase activities correlate significantly with caeruloplasmin and copper concentrations. Normal reference ranges have been measured.

The effect of zinc supplements on lipoproteins and copper status

As part of a study to determine the effect of 150 mg zinc/day on plasma lipoproteins, healthy young female (n = 26; mean age 27 years) and male (n = 21; mean age 28 years) volunteers took part in a double-blind cross-over trial lasting 12 weeks. During 6 weeks of supplementation, plasma Zn rose significantly in both groups, indicating compliance. Plasma total cholesterol remained unchanged in both males and females. However, mean LDL-cholesterol decreased from 2.38 to 2.17 mmol/1 in females and there was a trend for total HDL-cholesterol to be redistributed in that HDL 2 rose and HDL 3 fell slightly. In parallel with these changes in females, Zn supplements reduced the ferroxidase activity of serum caeruloplasmin (from 13.0 to 11.3 U/ml) and the antioxidant activity of erythrocyte superoxide dismutase (E-SOD) (from 4557 to 3638 U/g Hb) and CuZn E-SOD (from 2184 to 1672 U/g Hb). Plasma Cu and haematocrit were unaffected. No such changes were seen in males in either lipoproteins or these indicators of Cu status. Since the females were lighter than the males but received the same dose, a dose-response effect rather than a sex difference cannot be ruled out. Overall, Zn supplements significantly decrease a major risk factor for CHD in females but reduced their Cu status.

Use of soluble glass rumen boluses to provide a supplement of copper for suckled calves

SummaryAn experiment was carried out on a site where the copper content of herbage was relatively low (4·7–6·2 mg/kg d.m.) to examine the value of copper contained in soluble glass rumen boluses as a means of providing supplementary copper to calves.Forty-five spring-born suckled calves (mean live weight 91 kg) were divided into three groups on the basis of body weight. Animals in group GB were given two boluses initially, those in group CI each received copper injections at approximately monthly intervals and those in group C remained as controls throughout. Group C calves became progressively hypocupraemic (minimum mean plasma copper 8·5 μmol/1) compared with those in groups GB and CI which showed an increase in plasma copper concentration. In addition measurements of plasma caeruloplasmin activity, blood haemoglobin concentration and erythrocyte superoxide dismutase activity all showed significant increases due to treatment. No response in live-weight gain was observed but overall live-weight gain was approximately 1·05 kg/day. Although the initial response in plasma copper was quicker with copper injections, measurements of erythrocyte superoxide dismutase activity and haemoglobin showed advantages of glass boluses not shared by copper injections. This possibly resulted from the slow release nature of copper in the glass. Under the conditions of the experiment where herbage molybdenum values were relatively low (1·2–1·9 mg/kg d.m.), copper contained in soluble glass boluses appeared to prevent hypocupraemia in calves as effectively as regular copper injections.

Effects of concurrent copper deficiency and gastro-intestinal nematodiasis on circulating copper and protein levels, liver copper and bodyweight in sheep

Sheep were rendered hypocupraemic using parenteral ammonium tetrathiomolybdate (ATM). Fifteen thousand third stage larvae of Trichostrongylus axei and T. colubriformis in the ratio 1:1 were administered three times per week for six weeks, starting four weeks after cessation of ATM treatment. The changes in liver and plasma copper (Cu), caeruloplasmin activity, serum proteins, faecal nematode egg counts and total nematode counts were measured in the sheep for 10 weeks after infection. Decreases in liver Cu, plasma Cu and caeruloplasmin activities were detected soon after infection. There was a significant ( P<0.05) interaction of the effects of Cu deficiency and nematode infection on these changes. Hypoproteinaemia, attributed to serum albumin loss, was demonstrated sevem weeks after infection, but this was not associated with the interaction of Cu deficiency and nematode infection. No changes in serum globulins were detected. Although the results support the contention that gastro-intestinal nematodiasis can significantly exacerbate an existing Cu deficiency in sheep, there was no evidence that hypocuprosis would predispose sheep to higher nematode burdens.

The effects of metal ions on the oxidase activity of caeruloplasmin

The effects of metal ions on the oxidase activity of caeruloplasmin

Effect of soluble-glass boluses on the copper, cobalt and selenium status of sheep

A soluble glass which has the trace elements copper, cobalt and selenium as part of its structure has been devised by Telfer et al. (1983). This soluble glass is manufactured by Pilkington Brothers and marketed by the Wellcome Foundation under the registered tradename of COSECURE. The COSECURE soluble glass is designed so that it can be given orally to ruminant animals such that when swallowed by the animal, the glass bolus will lodge within the reticulo-rumen of the animal and dissolve in a controlled fashion, thus making the trace elements copper, cobalt and selenium available to the animal. These glasses have been designed for use in both sheep and cattle. The cattle and adult sheep glasses will be effective for I year while the bolus for lambs is designed to be effective for up to 6 months. The trial reported here was carried out on Swaledale lambs, each lamb being given a single lamb-sized bolus of COSECURE at 7-8 weeks after birth. The effect of the COSECURE treatment was monitored by measuring several biochemical variables in the blood of the lambs and comparing these values with a group of untreated lambs. The liveweight gain of the lambs was also measured. The results of this experiment are given in Table 1 and show the response in the lambs blood on the copper status (plasma copper, plasma caeruloplasmin, and erythrocyte superoxide dismutase activity, cobalt status (serum vitamin B, ?) and selenium status (erythrocyte glutathione peroxidase activity). The differences between the treated and control lambs were significant at up to 150 days after treatment except for erythrocyte superoxide dismutase at 150 days. The liveweight gains of the treated lambs were also significantly greater than those of the controls ( 1 2.4 kg in treated as against 8.6kg in control, P<O.OOI). The use of soluble glasses containing trace elements as implants in animals has been reported by Allen et al. (1979) to be an example of a controlled-release system with a possible biological use. Work by Telfer & Zervas ( 1 982) and Telfer et al. (1984) has shown that soluble glasses in the form of rumen boluses can also be used in ruminant animals. The development of a suitable glass for the administration of several trace elements in one bolus by Telfer et al. (1983) has produced a product which will prevent or cure deficiencies of trace elements in cattle and sheep. The results reported here have shown that in the control lambs produced on one farm there were indications of marginal copper status, marginal selenium status and vitamin B , * deficiency at the various times of sampling. The effect of the treatment with the soluble-glass COSECURE was such as to elevate the blood status of the treated lambs in respect of copper, cobalt and selenium such that at 57 and 150 days after treatment these lambs could be taken as having a normal copper, cobalt and selenium status. Therefore soluble glass in the form of a rumen bolus will provide a controlled release of trace elements over a long period of time.

Serum copper and caeruloplasmin activity in experimental malignancies

Serum copper content and caeruloplasmin activity were measured in 595 Strain A male mice. The mice carried either benzo(a)pyrene induced fibrosarcoma, transplanted sarcoma-180, Dalton's lymphoma, or Ehrlich carcinoma. The serum copper concentration was raised in all the tumours when compared with normal controls. The observed fall in activity of caeruloplasmin during malignancy may be related to defective iron mobilization.