Early and advanced glycation end-products are increased in dietary copper deficiency

The hypothesis that nonenzymatic glycosylation of proteins (glycation) contributes to damage associated with dietary copper deficiency has depended largely on indirect evidence. Thus far, the observation of an elevated percentage of glycated hemoglobin in copper-deficient rats has provided the only direct evidence of an increase in glycation. We sought further direct evidence of increased glycation in copper deficiency. Male weanling rats were fed a copper-adequate (CuA, 6.4 mg Cu/kg diet) or copper-deficient diet (CuD, 0.4 mg Cu/kg diet) for 5 weeks. Rats fed the CuD diet were copper deficient as judged by depressed organ copper concentrations and a variety of indirect indices. Measurements of hemoglobin A(1) and serum fructosamine (both early glycation end-products) as well as serum pentosidine (an advanced glycation end-product) indicated that all three compounds were elevated in CuD rats relative to CuA rats. This finding further supports the view that glycation is enhanced and thus may contribute to defects associated with dietary copper deficiency.     

Rat brain iron concentration is lower following perinatal copper deficiency

Experiments performed with Holtzman rats demonstrated that brain iron (Fe) was lower by postnatal day 13 (P13) in pups born and nursed by dams that began copper-deficient (-Cu) treatment at embryonic day 7. Transcardial perfusion of P24-P26 males and females to remove blood Fe contamination revealed that brain Fe was still 20% lower in -Cu than +Cu rats. Estimated blood content of brain for -Cu rats was greater than for +Cu rats; for all groups, values ranged between 0.43 and 1.03%. Using group-specific data and regression analyses, r = 0.99, relating blood Fe to hemoglobin, brain Fe in non-perfused rats in a replicate study was lower by 33% at P13 and 39% at P24 in -Cu rats. Brain extracts from these rats and from P50 rats from a post-weaning model were compared by immunoblotting for transferrin receptor (TfR1). P24 brain -Cu/+Cu TfR1 was 3.08, suggesting that brains of -Cu rats were indeed Fe deficient. This ratio in P13 rats was 1.44, p < 0.05. No change in P50 -Cu rat brain TfR1 or Fe content was detected despite a 50% reduction in plasma Fe. The results suggest that brain Fe accumulation depends on adequate Cu nutriture during perinatal development.     

Cardiovascular effects of dietary copper deficiency

Dietary copper deficiency may impair cardiovascular health by contributing to high blood pressure, enhancement of inflammation, anemia, reduced blood clotting and arteriosclerosis. The purpose of this review is to compile information on the numerous changes of the heart, blood and blood vessels that may contribute to these cardiovascular defects. These alterations include weakened structural integrity of the heart and blood vessels, impairment of the use of energy by the heart, reduced ability of the heart to contract, altered ability of blood vessels to control their diameter and to grow, and altered structure and function of circulating blood cells. The fundamental causes of these changes rest largely on reduced effectiveness of enzymes that depend on copper for their activity.     

DC-SIGN and influenza hemagglutinin dynamics in plasma membrane microdomains are markedly different

DC-SIGN, a Ca²⁺-dependent transmembrane lectin, is found assembled in microdomains on the plasma membranes of dendritic cells. These microdomains bind a large variety of pathogens and facilitate their uptake for subsequent antigen presentation. In this study, DC-SIGN dynamics in microdomains were explored with several fluorescence microscopy methods and compared with dynamics for influenza hemagglutinin (HA), which is also found in plasma membrane microdomains. Fluorescence imaging indicated that DC-SIGN microdomains may contain other C-type lectins and that the DC-SIGN cytoplasmic region is not required for microdomain formation. Fluorescence recovery after photobleaching measurements showed that neither full-length nor cytoplasmically truncated DC-SIGN in microdomains appreciably exchanged with like molecules in other microdomains and the membrane surround, whereas HA in microdomains exchanged almost completely. Line-scan fluorescence correlation spectroscopy indicated an essentially undetectable lateral mobility for DC-SIGN but an appreciable mobility for HA within their respective domains. Single-particle tracking with defined-valency quantum dots confirmed that HA has significant mobility within microdomains, whereas DC-SIGN does not. By contrast, fluorescence recovery after photobleaching indicated that inner leaflet lipids are able to move through DC-SIGN microdomains. The surprising stability of DC-SIGN microdomains may reflect structural features that enhance pathogen uptake either by providing high-avidity platforms and/or by protecting against rapid microdomain endocytosis.     

Limited effects of combined dietary copper deficiency/iron overload on oxidative stress parameters in rat liver and plasma

Copper (Cu) deficiency decreases the activity of Cu-dependent antioxidant enzymes such as Cu,zinc-superoxide dismutase (Cu,Zn-SOD) and may be associated with increased susceptibility to oxidative stress. Iron (Fe) overload represents a dietary oxidative stress relevant to overuse of Fe-containing supplements and to hereditary hemochromatosis. In a study to investigate oxidative stress interactions of dietary Cu deficiency with Fe overload, weanling male Long–Evans rats were fed one of four sucrose-based modified AIN-93G diets formulated to differ in Cu (adequate 6 mg/kg diet vs. deficient 0.5 mg/kg) and Fe (adequate 35 mg/kg vs. overloaded 1500 mg/kg) in a 2×2 factorial design for 4 weeks prior to necropsy. Care was taken to minimize oxidation of the diets prior to feeding to the rats. Liver and plasma Cu content and liver Cu,Zn-SOD activity declined with Cu deficiency and liver Fe increased with Fe overload, confirming the experimental dietary model. Liver thiobarbituric acid reactive substances were significantly elevated with Fe overload (pooled across Cu treatments, 0.80±0.14 vs. 0.54±0.08 nmol/mg protein; P<.0001) and not affected by Cu deficiency. Liver cytosolic protein carbonyl content and the concentrations of several oxidized cholesterol species in liver tissue did not change with these dietary treatments. Plasma protein carbonyl content decreased in Cu-deficient rats and was not influenced by dietary Fe overload. The various substrates (lipid, protein and cholesterol) appeared to differ in their susceptibility to the in vivo oxidative stress induced by dietary Fe overload, but these differences were not exacerbated by Cu deficiency.     

Molecular localization of copper and zinc in rat fetal liver in dietary and drug-induced copper deficiency

The teratogenicity of copper deficiency is well known, but underlying mechanisms have not been delineated. One method of studying the biochemical lesions of copper deficiency is the use of chelating drugs with different chemical characteristics. The teratogenicity of a copper deficient diet and of diets containing either D-penicillamine or triethylenetetramine is quite different, although all three diets result in decreased fetal liver copper levels. Feeding D-penicillamine can result in decreased fetal liver zinc, while feeding triethylenetetramine can result in increased fetal liver zinc. The effect of these three diets on fetal liver copper and zinc molecular localization was determined. Gel filtration showed that fetal liver copper and zinc in controls was localized in 3 fractions with MWs of > 50,000 (H), 30,000 (I) and 8–10,000 (L). Independent of dietary treatment, as liver copper diminished, copper was missing first from the L peak, then the I peak and with severe deficiency, from the H peak. Drug induced increases and decreases in fetal liver zinc were reflected in the L peak. These data suggest that the absolute levels of copper in the liver of the term fetus determines the distribution of the element among its binding ligands.     

Development of glutathione peroxidase activity during dietary and genetic copper deficiency

Copper deficiency was produced in developing rodents to study a possible interaction between copper and the selenoenzyme, glutathione peroxidase (GSH-Px). Dietary copper deficiency was investigated in Sprague-Dawley rats and in three mouse strains (C57BL, C3H/HeJ, C58); genetic copper deficiency was studied in two of the mouse strains, C57BL and C3H/HeJ, using brindled mice. In certain cases it appeared that copper deficiency was associated with depressed liver GSH-Px activity; values from copper-deficient livers were 40–70% of control values. However, the decrease in liver GSH-Px in both rats and mice was only observed when body weight was also depressed and did not necessarily correlate with copper deficiency signs, such as lower serum ceruloplasmin or liver cytochrome oxidase activities. In weanling rats, serum GSH-Px activity was normal despite a 60% reduction in liver activity. Mouse liver GSH-Px activity rose fourfold during the first 3 weeks of life to 75% of the adult level. Rat liver GSH-Px also increased during the suckling period. When perinatal copper deficiency, nutritional or genetic, was severe enough to retard growth, liver GSH-Px activity was depressed. Unlike rats, adult murine liver GSH-Px was equivalent in males and females.     

Biochemical and immunological changes in mice following postweaning copper deficiency

Weanling albino male mice rapidly develop biochemical signs of copper deficiency when fed a purified diet containing 0.5 mg Cu/kg. Plasma ceruloplasmin activity of copper-deficient (-Cu) mice was 5% of that of copper-adequate (+Cu) control mice after only 3 d on the diet. More gradual loss of organ (liver, spleen, and thymus) cytochrome c oxidase activity was observed during the next 4 wk. Body weight was equivalent between +Cu and -Cu mice, but thymus weight dropped faster in -Cu mice than +Cu mice. The number of antibody producing cells to sheep erythrocytes was lower in -Cu mice compared to +Cu mice after 17 d on the diet. Spleen cytochrome oxidase activity of -Cu mice was 50% of that of +Cu mice by 10 d on the diet. Mitogenic response of splenic and thymic lymphocytes to concanavalin A (con A) was not greatly different between +Cu and -Cu mice. Splenocytes from -Cu mice had a 3-fold higher thymidine incorporation rate in the absence of mitogen compared to +Cu mice. The depressed antibody and high mitogenic background responses of -Cu mice were similar to previous work with another strain (C58) of mice that had been started on copper-deficient treatment from birth. However, the normal proliferative response to con A stimulation in postweaning copper deficiency differs from the previous model. Mice of both studies were very copper-deficient as judged by liver copper levels. Timing of the copper-deficient treatment influences the manner in which copper deficiency alters the immune response.     

Changes in copper and zinc status and response to dietary copper deficiency in metallothionein-overexpressing transgenic mouse heart

Previous studies have shown that cardiac-specific overexpression of metallothionein (MT) inhibits progression of dietary copper restriction-induced cardiac hypertrophy. Because copper and zinc are critically involved in myocardial response to dietary copper restriction, the present study was undertaken to understand the effect of MT on the status of copper and zinc in the heart and the subsequent response to dietary copper restriction. Dams of cardiac-specific MT-transgenic (MT-TG) mouse pups and wild-type (WT) littermates were fed copper-adequate (CuA) or copper-deficient (CuD) diet starting on the fourth day post delivery, and the weanling mice were continued on the same diet until they were sacrificed. Zinc and copper concentrations were significantly elevated in MT-TG mouse heart, but the extent of zinc elevation was much more than that of copper. Dietary copper restriction significantly decreased copper concentrations to the same extent in both MT-TG and WT mouse hearts, and decreased zinc concentrations along with a decrease in MT concentrations in the MT-TG mouse heart. Copper deficiency-induced heart hypertrophy was significantly inhibited, but copper deficiency-induced suppression of serum ceruloplasmin or hepatic Cu,Zn-SOD activities was not inhibited in the MT-TG mice. These results suggest that elevation in zinc but not in copper in the heart may be involved in the MT inhibition of copper deficiency-induced cardiac hypertrophy.     

Role of copper in thermal stability of human ceruloplasmin

Human ceruloplasmin (CP) is a multicopper oxidase essential for normal iron homeostasis. The protein has six domains with one type-1 copper in each of domains 2, 4, and 6; the remaining coppers form a catalytic trinuclear cluster at the interface between domains 1 and 6. To assess the role of the coppers in CP thermal stability, we have probed the thermal unfolding process as a function of scan rate of holo- and apo-forms using several detection methods (circular dichroism, aromatic and 8-anilino-naphthalene-1-sulfonic acid fluorescence, visible absorption, activity, and differential scanning calorimetry). Both species of CP undergo irreversible thermal reactions to denatured states with significant residual structure. For identical scan rates, the thermal midpoint appears at temperatures 15-20° higher for the holo- as compared with the apo- form. The thermal data for both forms were fit by a mechanistic model involving two consecutive, irreversible steps (N → I → D). The holo-intermediate, I, has lost one oxidized type-1 copper and secondary structure in at least one domain; however, the trinuclear copper cluster remains intact as it is functional in oxidase activity. The activation parameters obtained from the fits to the thermal transitions were used to assess the kinetic stability of apo- and holo-CP at physiological temperatures (i.e., at 37°C). It emerges that native CP (i.e., with six coppers) is rather unstable and converts to I in <1 day at 37°C. Nonetheless, this form remains intact for more than 2 weeks and may thus be a biologically relevant state of CP in vivo. In contrast, apo-CP unfolds rapidly: the denatured state is reached in <2 days at 37°C.     

Neointima formation in the rat carotid artery is exacerbated by dietary copper deficiency

Dietary copper is an essential trace element with roles in both functional and structural aspects of the cardiovascular system. In particular, the vascular response to inflammatory stimuli is known to be significantly augmented in copper-deficient rats. The current study was designed to quantify the extent of injury-induced neointimal proliferation and stenosis in rats fed diets either adequate or deficient in copper. Male, weanling Sprague-Dawley rats were fed purified diets that were either adequate (CuA; 5.6 microg Cu/g) or deficient (CuD; 0.3 microg Cu/g) in copper for 4 weeks. Balloon injury was induced in the left external carotid arteries. Fourteen days after injury, histomorphometric analysis of cross-sections from carotid arteries showed increased neointimal formation in the CuD group compared with the CuA controls (neointima/media ratio: 4.55 +/- 0.93 vs 1.45 +/- 0.2, respectively). These results correspond with data indicating that the activity of Cu/Zn-superoxide dismutase (SOD) is depressed in rats fed this CuD diet. Because superoxide anion and redox status are known to play a key role in the extent of neointimal formation in response to injury, we propose that the exaggerated neointimal proliferation seen in the CuD group is the result of the diminished Cu/Zn-SOD activity.     

The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash

Recent studies surprisingly show that dietary inorganic nitrate, abundant in vegetables, can be metabolized in vivo to form nitrite and then bioactive nitric oxide. A reduction in blood pressure was recently noted in healthy volunteers after dietary supplementation with nitrate; an effect consistent with formation of vasodilatory nitric oxide. Oral bacteria have been suggested to play a role in bioactivation of nitrate by first reducing it to the more reactive anion nitrite. In a cross-over designed study in seven healthy volunteers we examined the effects of a commercially available chlorhexidine-containing antibacterial mouthwash on salivary and plasma levels of nitrite measured after an oral intake of sodium nitrate (10 mg/kg dissolved in water). In the control situation the salivary and plasma levels of nitrate and nitrite increased greatly after the nitrate load. Rinsing the mouth with the antibacterial mouthwash prior to the nitrate load had no effect on nitrate accumulation in saliva or plasma but abolished its conversion to nitrite in saliva and markedly attenuated the rise in plasma nitrite. We conclude that the acute increase in plasma nitrite seen after a nitrate load is critically dependent on nitrate reduction in the oral cavity by commensal bacteria. The removal of these bacteria with an antibacterial mouthwash will very likely attenuate the NO-dependent biological effects of dietary nitrate.     

Effect of high dietary zinc on plasma ceruloplasmin and erythrocyte superoxide dismutase activities in Copper-depleted and repleted rats

The effect of moderately high dietary zinc (Zn) on the activities of plasma (PL) ceruloplasmin (CP), and PL and erythrocyte (RBC) copper (Cu), Zn superoxide dismutase (SOD) was determined in weanling rats fed Cu-deficient (DEF; <1 mg Cu/kg), marginal (MAR; 2 mg Cu/kg), or control (CON; 5 mg Cu/kg) copper diets containing normal or high Zn (HZn; 60 mg/kg) for 4 wk and supplemented with oral Cu (CuS; 5 mg/L) in drinking water for 0, 1, 3, or 7 d. PL Cu decreased (67% compared to CON;p≤0.05) in the DEF and increased to control level after 3 d of CuS; increased in the MAR group after 1 d of CuS. HZn reduced overall PL Cu by 27% in all groups, but did not alter the linear increase in PL Cu between 0 and 3 d of Cu S. PL CP activity altered concomitantly with PL Cu levels: The time course of increase in CP activity after 0–3 d of CuS was not influenced by HZn in the diet and CP declined in the DEF group by 92%. There was no correlation between dietary Cu level and PL CP. PL SOD activity decreased by 46% (p≤.05) in the DEF group, increased to control activity after 1 d of CuS and declined slighty after 7 d; MAR diet did not alter PL SOD. HZn diet increased PL SOD activity in all groups by 150%, reduced activity in the DEF and MAR groups by 65 and 37% and delayed the recovery of PL SOD after CuS. RBC SOD declined in the DEF and MAR groups by 56 and 33% (p≤0.05) and did not respond to CuS; HZn diet did not influence RBC SOD activity. These data indicate that moderately high Zn in the diet reduces PL Cu, but not PL CP activity or the recovery of PL Cu or CP activity after oral CuS of Cu-deficient rats, modifies the response of PL SOD to dietary Cu, but does not influence RBC SOD activity.     

Phospholipid metabolism and protein kinase C mediated protein phosphorylation in dietary protein deficiency in rat lung

Nutritional deprivation of proteins decreases the protein kinase C (PKC) activity in rat lung. The activity of (PKC) is influenced by lipid metabolism. Changes in PKC activity may influence phosphorylation of its substrate proteins in the tissues. Therefore, alterations in phospholipid metabolism and PKC mediated protein phosphorylation in dietary protein deficiency in rat lung were envisaged. The study was conducted on rats fed on three different types of diet viz., casein (20% protein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threoning). Feeding of protein deficient diet caused reduction in incorporation of [3H] myo-inositol in the total phosphoinositides in lungs and an increase in total inositol phosphate pool. There was a significant reduction in the contents and turnover rate of phosphatidyl inositol and phosphatidyl inositol monophosphate. Supplementation of diet with L-lysine and DL-threonine had a reversing effect on total pool of phosphoinositides and, the metabolism of phosphatidyl inositol bisphosphate and phosphatidyl inositol. In phosphatidyl choline metabolism, the dietary protein deficiency led to a decrease in incorporation of [14C-methyl] choline-chloride in total phospholipids. In contrast, its incorporation increased in phosphatidyl choline pool. The contents of phosphatidyl choline and residue, incorporation of [14C-methyl] choline-chloride in them and their turnover rate also increased. Supplementation of diet had a reversal effect on most of these parameters. Phosphorylation of proteins of 84, 47, 35 and 16 kDa was identified to be mediated by PKC. In dietary protein deficiency, phosphorylation of all these proteins, except that of 47 kDa, increased. Supplementation of diet reversed the pattern except that of 84 kDa. The findings suggest that changes in phospholipid metabolism in dietary protein deficiency may effect the activity of PKC thereby influencing the phosphorylation of its substrate proteins and hence associated functions that may lead to pathophysiology of lung.     

Oxidase activity of ceruloplasmin and concentrations of copper and zinc in serum in chronic arterial occlusion of the lower limbs.

The presence of ischaemic tissue excites an inflammatory reaction and synthesis of acute phase proteins (APhPs). Ceruloplasmin (Cp) protein binds 90% of the copper in plasma and it is one of the positive APhPs, and its concentration increases in infection, inflammation or necrosis. The study presents the relationship of the oxidase activity of Cp and concentrations of Cu and Zn in serum of men with different degrees of ischaemia of the lower limbs. The subjects were 32 men with chronic arterial occlusion (AO) of the lower limbs. The oxidase activity of Cp was measured in serum with o-dianisidine as a substrate. Concentrations of Cu and Zn were determined by using atomic absorption spectrometry. The mean activity of Cp in serum in AO (173 +/- 69.2 U/l) was higher as compared with the control group (123.7 +/- 28.6 U/l), and in men with critical ischaemia (> or = 194.8 U/l) than in men with a moderate level of ischaemia (109.3 +/- 31.6 U/l). The mean concentrations of Cu and Zn in serum were found to be higher in AO (22.2 +/- 4.2 and 19.1 +/- 6.9 mumol/l, respectively) than in the control group (16.3 +/- 1.8 and 15.2 +/- 2.3 mumol/l), and in men with critical ischaemia (> or = 22.2 and 19.1 mumol/l) than in men with a moderate level of ischaemia (18.5 +/- 3.3 and 14.5 +/- 4.3 mumol/l). Significant positive correlation coefficients were calculated for the activities of Cp and concentrations of Cu in the control group (r = 0.86) and the AO group (r = 0.76), and low, but significant, correlations for Cp and Zn in the AO group (r = 0.66). The increase in the oxidase activity of Cp and concentration of Cu in serum in ischaemia is caused by the acute phase response. The relationship of Zn concentration and Cp activity in ischaemia is indirect and needs further study.     

Multiple mechanisms account for lower plasma iron in young copper deficient rats

Copper deficiency lowers brain copper and iron during development. The reduced iron content could be due to hypoferremia. Experiments were conducted to evaluate plasma iron and “ferroxidase” hypotheses by determining copper and iron status of Holtzman albino rats following gestational/lactational copper deficiency. Copper deficient (Cu−) dams on treatment for 5 weeks, two of gestation and three of lactation, had markedly lower copper content of milk and mammary tissue, and lower milk iron. Newborn pups from Cu− dams had lower copper and iron concentrations. Compared to Cu+ pups, Cu− pups, analyzed between postnatal age (P) 0 and P26, were smaller, anemic, had lower plasma iron, cardiac hypertrophy, and near zero ceruloplasmin activity. Liver copper in Cu+ pups increased then decreased during development and major reductions were evident in Cu− pups. Liver iron in Cu+ pups decreased with age while nursing but increased after eating solid food. Liver iron was lower in Cu− pups at P0 and P13 and normal at P20 and P26. Small intestinal copper decreased with age in Cu+ pups and was lower in Cu− pups. Intestinal iron levels in Cu− pups were higher than Cu+ pups postweaning in some experiments. Reduction in plasma iron in Cu− pups is likely due to a decreased “ferroxidase” function leading to lower placental iron transport, a lower milk iron diet, and partial block in iron uptake from intestine but is not due to failure to mobilize hepatic iron, in contrast to older rats eating diet with adequate iron.