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Hematological and Histopathological Study Effect of Molybdate and Sulfate Alone and in Combination Overloaded Intake on Copper Outcome in Mature Male Rats
Abstract
The object of this study were to evaluate the effect of molybdate and sulfate overload alone or in combination overload intake on copper outcome with regard toxicopathological changes in mature male rats. Twenty eight adult male rats were housed and arbitrarily divided into four equal groups (seven rat/group) and treated as follows for 60 days: control received distilled water, groups T1 received sodium molybdate 50 mg/kg B.W and T2: given orally 500 mg/kg B.W of sodium sulfate and T3 group were given sodium molybdate and sodium sulfate in combination at half dose in comparison with T1 and T2 doses. The blood tests results, which included Red Blood Cell count, hemoglobin concentration and Packed Cell Volume % that conducted a after one month and at the end of the experiment showed a significant reductions in general but were more in T1 group followed by T2 group, while T3 group showed less decline than that recorded in each of T1 and T2 groups. These functional changes were accompanied by structural changes in the hepatic and renal tissues. Histopathological changes following molybdate (50 mg/kg B.W) exposure were manifested by extensive areas of necrosis, hemorrhage, and hyperplasia of bile ductules. Besides focal area of necrosis and suppurative granuloma observed in livers of rats received 500 mg/kg B.W of sodium sulfate, mild infiltrate of mononuclear cell within the hepatic parenchyma, suppurative granuloma and proliferation of kupffer’s cells with hyperplasia of bile ductules seen in liver of rats received combination of them at half dose. While section of kidney from T1 group showed marked fibrous thickening of the capsule, severe cortical hemosiderosis with infiltrate of plasma cell and neutrophils, While in T2 group showed atrophy of glomerular tuft, focal interstitial mononuclear cells infiltration with deposition of hyaline cast. The histological changes revealed that renal damage was also observed in T3 group but at a little degree, depending on the result of this study it can be concluded that molybdate and sulfate alone and in combination succeeded to induce copper deficiency with severe changes in hematological and histopathological parameters in rats.
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Copper is required by all living systems. Cells have a variety of mechanisms to deal with this essential, yet toxic trace element. A recently discovered facet of homeostatic mechanisms is the protein-mediated, intracellular delivery of copper to target proteins. This routing is accomplished by a novel class of proteins, the ‘copper chaperones’. They are a family of conserved proteins present in prokaryotes and eukaryotes, which suggests that copper chaperones are used throughout nature for intracellular copper routing.
Acquired copper deficiency has been recognised as a rare cause of anaemia and neutropenia for over half a century. Copper deficiency myelopathy (CDM) was only described within the last decade, and represents a treatable cause of non-compressive myelopathy which closely mimics subacute combined degeneration due to vitamin B12 deficiency. Here, 55 case reports from the literature are reviewed regarding their demographics, aetiology, haematological and biochemical parameters, spinal imaging, treatment and outcome. The pathophysiology of disorders of copper metabolism is discussed. CDM most frequently presented in the fifth and sixth decades and was more common in women (F:M = 3.6:1). Risk factors included previous upper gastrointestinal surgery, zinc overload and malabsorption syndromes, all of which impair copper absorption in the upper gastrointestinal tract. No aetiology was established in 20% of cases. High zinc levels were detected in some cases not considered to have primary zinc overload, and in this situation the contribution of zinc to the copper deficiency state remained unclear. Cytopenias were found in 78%, particularly anaemia, and a myelodysplastic syndrome may have been falsely diagnosed in the past. Spinal MRI was abnormal in 47% and usually showed high T2 signal in the posterior cervical and thoracic cord. In a clinically compatible case, CDM may be suggested by the presence of one or more risk factors and/or cytopenias. Low serum copper and caeruloplasmin levels confirmed the diagnosis and, in contrast to Wilson's disease, urinary copper levels were typically low. Treatment comprised copper supplementation and modification of any risk factors, and led to haematological normalisation and neurological improvement or stabilisation. Since any neurological recovery was partial and case numbers of CDM will continue to rise with the growing use of bariatric gastrointestinal surgery, clinical vigilance will remain the key to minimising neurological sequelae. Recommendations for treatment and prevention are made.
We describe a woman with severe neutropenia and dependency on red blood cell transfusions who had previously undergone Billroth II surgery and whose bone marrow (BM) showed morphologic characteristics typical of myelodysplastic syndrome (MDS) with ringed sideroblasts. She had transient reversal of anemia and severe neutropenia after therapy with erythropoietin and granulocyte colony-stimulating factor. Because of relapse while receiving growth factors, the patient was referred for allogeneic BM transplantation. A pretransplantation nutritional evaluation revealed severe copper deficiency, and her hematologic abnormalities resolved fully with copper therapy. This case shows that copper deficiency should be an integral part of the differential diagnosis of sideroblastic MDS, even in patients not requiring parenteral nutrition.
The importance of molybdenum for plant growth is disproportionate with respect to the absolute amounts required by most plants. Apart from Cu, Mo is the least abundant essential micronutrient found in most plant tissues and is often set as the base from which all other nutrients are compared and measured. Molybdenum is utilized by selected enzymes to carry out redox reactions. Enzymes that require molybdenum for activity include nitrate reductase, xanthine dehydrogenase, aldehyde oxidase and sulfite oxidase.
Loss of Mo-dependent enzyme activity (directly or indirectly through low internal molybdenum levels) impacts upon plant development, in particular, those processes involving nitrogen metabolism and the synthesis of the phytohormones abscisic acid and indole-3 butyric acid. Currently, there is little information on how plants access molybdate from the soil solution and redistribute it within the plant. In this review, the role of molybdenum in plants is discussed, focusing on its current constraints in some agricultural situations and where increased molybdenum nutrition may aid in agricultural plant development and yields.
Molybdenum deficiencies are considered rare in most agricultural cropping areas; however, the phenotype is often misdiagnosed and attributed to other downstream effects associated with its role in various enzymatic redox reactions. Molybdenum fertilization through foliar sprays can effectively supplement internal molybdenum deficiencies and rescue the activity of molybdoenzymes. The current understanding on how plants access molybdate from the soil solution or later redistribute it once in the plant is still unclear; however, plants have similar physiological molybdenum transport phenotypes to those found in prokaryotic systems. Thus, careful analysis of existing prokaryotic molybdate transport mechanisms, as well as a re-examination of know anion transport mechanisms present in plants, will help to resolve how this important trace element is accumulated.
Electrospray tandem mass spectrometry was used to determine steady-state serum and urinary inorganic sulfate and sulfate ester kinetic profiles of nine normal men after intravenous injection of the stable isotope sodium [34S]sulfate. Sulfate ester appearance was traced by eliminating inorganic sulfate from samples, followed by hydrolysis of sulfate esters to inorganic sulfate for analysis. Whole body inorganic sulfate turnover in steady state was calculated using standard tracer techniques. Rate of appearance and disappearance of inorganic sulfate was 841 +/- 49 micromol/h. Average urinary inorganic sulfate excretion was 609 +/- 41 micromol/h, and the whole body sulfation rate (total rate of disappearance minus rate of urinary excretion) was 232 +/- 36 micromol/h. Tracer-labeled sulfate esters appeared in serum and urine within 1 h of tracer injection. The kinetics of inorganic sulfate and sulfate esters were linked by means of a compartmental model. The appearance and excretion of sulfate esters accounted for approximately 50% of the total sulfation rate. These results indicate that human whole body sulfation accounts for approximately 27% of inorganic sulfate turnover and that extracellular inorganic sulfate is an important pool for intracellular sulfation. A substantial fraction of newly synthesized sulfate esters promptly enters the extracellular space for excretion in the urine.
Sixty Angus (n = 29) and Angus-Sim-mental cross (n = 31) steers, averaging 9 mo of age and 277 kg of initial BW, were used in a 148-d study to determine the bioavailability of copper glycinate (CuGly) relative to feed-grade copper sulfate (CuSO(4)) when supplemented to diets high in S and Mo. Steers were blocked by weight within breed and randomly assigned to 1 of 5 treatments: 1) control (no supplemental Cu), 2) 5 mg of Cu/kg of DM from CuSO(4), 3) 10 mg of Cu/kg of DM from CuSO(4), 4) 5 mg of Cu/kg of DM from CuGly, and 5) 10 mg of Cu/kg of DM from CuGly. Steers were individually fed a corn silage-based diet (analyzed 8.2 mg of Cu/kg of DM), and supplemented with 2 mg of Mo/kg of diet DM and 0.15% S for 120 d (phase 1). Steers were then supplemented with 6 mg of Mo/kg of diet DM and 0.15% S for an additional 28 d (phase 2). Average daily gain and G:F were improved by Cu supplementation regardless of source (P = 0.01). Final ceruloplasmin, plasma Cu, and liver Cu values were greater (P < 0.05) in steers fed supplemental Cu compared with controls. Plasma Cu, liver Cu, and ceruloplasmin values were greater (P < 0.05) in steers supplemented with 10 mg of Cu/kg of DM vs. those supplemented with 5 mg of Cu/kg of DM. Based on multiple linear regression of final plasma Cu, liver Cu, and ceruloplasmin values on dietary Cu intake in phase 1 (2 mg of Mo/kg of DM), bioavailability of Cu from CuGly relative to CuSO(4) (100%) was 140 (P = 0.10), 131 (P = 0.12), and 140% (P = 0.01), respectively. Relative bio-availability of Cu from CuGly was greater than from CuSO(4) (P = 0.01; 144, 150, and 157%, based on plasma Cu, liver Cu, and ceruloplasmin, respectively) after supplementation of 6 mg of Mo/kg of DM for 28 d. Results of this study suggest that Cu from CuGly may be more available than CuSO(4) when supplemented to diets high in S and Mo.
In this review, we discuss genetic disorders involving altered coppermetabolism, particularly in relation to Menkes disease (MD), occipital horn syndrome (OHS), and Wilson’s disease (WD). The responsible genes for MD and WD are ATP7A and ATP7B, respectively. Both proteins encoded by these genes are responsible for transporting copper from the cytosol to the Golgi apparatus. However, the pathology of MD is completely different from that of WD, that is, MD is characterized by a copper deficiency while WD is caused by a toxic excess of copper. The reason for this difference is related to the particular cell types in which the ATP7A and ATP7B proteins are expressed. ATP7A is expressed in almost all cell types except hepatocytes, whereas ATP7B is mainly expressed in hepatocytes. MD and OHS are X-linked recessive disorders characterized by copper deficiency. Typical features of MD, such as neurological disturbances, connective tissue disorders, and hair abnormalities, can be explained by the abnormally low activity of copper-dependent enzymes. The current standard-of-care treatment for MD is parenteral administrations of copper–histidine. When the treatment is initiated in newborn babies prior to two months of age, the neurological degeneration may be prevented, but delayed treatment is considerably less effective. Moreover, copper–histidine treatment does not improve symptoms of the connective tissue disorders. As such, systems for mass screening of neonates for MD should be implemented. At the same time, novel treatments targeting connective tissue disorders need to be developed. OHS is a milder form of MD and is characterized by connective tissue abnormalities. Although formal trials have not been conducted for OHS, OHS patients are typically treated in a similar manner to those with MD. WD is an autosomal recessive disorder characterized by the toxic effects of chronic exposure to high levels of copper. The hepatic and nervous systems are typically most severely affected. Numerous other symptoms can also be observed, however, making an early diagnosis difficult. Chelating agents and zinc are effective for the treatment of WD, but they are ineffective for the patients with fulminant hepatic failure. Some patients with neurological diseases show poor response to chelating agents; here again, early diagnosis and treatment are critical. Screening of newborn babies or infants for WD can help lead to timely diagnosis and treatment. Patients with WD may have a risk of hepatocellular carcinoma despite receiving treatment. An understanding of the relation between WD and hepatocellular carcinoma will provide clues to help prevent hepatocellular carcinoma in patients with WD.
Copper is an element that is both essential and toxic. It is a required micronutrient for energy production in aerobic eukaryotes, from unicellular yeast to plants and mammals. Copper is also required for the acquisition and systemic distribution of the essential metal iron, and so copper deficiency results in iron deficiency. Copper enzymes have been identified that explain the wide variety of symptoms suffered by copper deficient subjects. The cloning of the genes encoding transport proteins responsible for copper-related Menkes and Wilson diseases inspired and coincided with the discovery of copper chaperones that stimulated the copper homeostasis field. Copper continues to be implicated in new array of proteins, notably those involved in a variety of neurodegenerative diseases. Here we will describe the cadre of important historical copper proteins and survey the major metallochaperones and transporters responsible for mobilization and sequestration of copper in yeast, mammals and plants.
It has become clear that serum "free" copper (the copper not bound to ceruloplasmin in the blood) is the copper causing copper toxicity in Wilson's disease. But up until now, free copper has not been closely followed during initiation of anticopper therapy in neurologically presenting patients. During this period of initial therapy, the future fate of these patients hangs in the balance-if they worsen neurologically as often happens with penicillamine or trientine therapy, many never recover. We hypothesize that free copper levels are a biological marker of clinical outcome in these patients. In this article, we evaluate the control of free copper in 3 studies of initial anticopper treatment in neurologically presenting Wilson's disease patients. The first (study 1) is a 55-patient open-label trial of tetrathiomolybdate, the second (study 2) is a 48-patient double-blind trial comparing tetrathiomolybdate and trientine, and the third (study 3) is a 40-patient double-blind comparison of 2 disease regimens of tetrathiomolybdate. Free copper levels were determined by subtracting ceruloplasmin and tetrathiomolybdate bound copper from total serum copper. Tetrathiomolybdate showed very strong control of free copper levels over the 8 weeks of treatment in the 55-patient open-label study (study 1), reducing it to a mean value of about one fourth, or less, of baseline. In the tetrathiomolybdate/trientine double blind (study 2), tetrathiomolybdate again showed good control of free copper levels over 8 weeks of treatment, which is significantly better than trientine. In the trientine arm of study 2, mean free copper levels actually went up during trientine therapy. The 5 patients who neurologically worsened on trientine therapy over 8 weeks of treatment showed significant spikes in serum free copper levels associated in time with their neurologic worsening. Patients who did not worsen neurologically generally did not show significant spikes in free copper. Tetrathiomolybdate controlled copper less well in the dose regimen study (study 3) than in the previous 2 studies of tetrathiomolybdate treatment, probably because of a change in the way "away from food" tetrathiomolybdate was given.
The molybdenum cofactor (Moco) is an essential component of a large family of enzymes involved in important transformations in carbon, nitrogen and sulfur metabolism. The Moco biosynthetic pathway is evolutionarily conserved and found in archaea, eubacteria and eukaryotes. In humans, genetic deficiencies of enzymes involved in this pathway trigger an autosomal recessive and usually deadly disease with severe neurological symptoms. The MoaC protein, together with the MoaA protein, is involved in the first step of Moco biosynthesis.
MoaC from Escherichia coli has been expressed and purified to homogeneity and its crystal structure determined at 2 A resolution. The enzyme is organized into a tightly packed hexamer with 32 symmetry. The monomer consists of an antiparallel, four-stranded beta sheet packed against two long alpha helices, and its fold belongs to the ferredoxin-like family. Analysis of structural and biochemical data strongly suggests that the active site is located at the interface of two monomers in a pocket that contains several strictly conserved residues.
Asp128 in the putative active site appears to be important for catalysis as its replacement with alanine almost completely abolishes protein activity. The structure of the Asp128-->Ala variant reveals substantial conformational changes in an adjacent loop. In the human MoaC ortholog, substitution of Thr182 with proline causes Moco deficiency, and the corresponding substitution in MoaC severely compromises activity. This residue is located near the N-terminal end of helix alpha4 at an interface between two monomers. The MoaC structure provides a framework for the analysis of additional dysfunctional mutations in the corresponding human gene.
Plasma total homocysteine (tHcy) level is increased in patients with renal disease, parallel to serum creatinine concentration. In renal failure, the final product of sulfated amino acid metabolism, sulfate, also accumulates as renal function declines. We hypothesized that the elevation in sulfate level could cause hyperhomocysteinemia and tested the relation between tHcy level and both urinary excretion and plasma levels of sulfate.
Forty patients with renal disease were divided into three groups: patients without renal failure (nRF; creatinine clearance [CCr] > or = 80 mL/min/1.73 m2 [> or =1.33 mL/s/1.73 m2]), patients with mild renal failure (mRF; 80 > CCr > or = 25 mL/min/1.73 m2 [1.33 > CCr >/ or 0.42 mL/s/1.73 m2]), and patients with severe renal failure (sRF; CCr < 25 mL/min/1.73 m2 [<0.42 mL/s/1.73 m2]). Daily urinary excretion and plasma levels of tHcy, total cysteine (tCys), and sulfate were measured. A healthy control (HC) group also was tested. Serum methionine, taurine, vitamin B12, and folate levels also were determined in patients with renal disease.
Plasma tHcy and sulfate concentrations in the groups with mRF and sRF were greater than in the HC group. Plasma tCys concentrations in the mRF and sRF groups were greater than in the nRF group. Daily urinary Hcy and Cys excretion did not differ among the four groups. Daily urine sulfate and urea nitrogen excretion in the sRF group were significantly less than in the HC and nRF groups. Multiple regression analyses showed that plasma creatinine (beta = 0.40) and sulfate (beta = 0.43) levels were independently associated with plasma Hcy level; among urine parameters, only daily urine sulfate excretion (beta = -0.52) was independently associated with plasma Hcy level.
The elevated plasma sulfate level, in accordance with renal function, is associated with plasma tHcy level. Decreased sulfate excretion, which might parallel the intake of sulfated amino acid or protein, may increase tHcy levels.
The transition element molybdenum (Mo) is of essential importance for (nearly) all biological systems as it is required by enzymes catalyzing diverse key reactions in the global carbon, sulfur and nitrogen metabolism. The metal itself is biologically inactive unless it is complexed by a special cofactor. With the exception of bacterial nitrogenase, where Mo is a constituent of the FeMo-cofactor, Mo is bound to a pterin, thus forming the molybdenum cofactor (Moco) which is the active compound at the catalytic site of all other Mo-enzymes. In eukaryotes, the most prominent Mo-enzymes are (1) sulfite oxidase, which catalyzes the final step in the degradation of sulfur-containing amino acids and is involved in detoxifying excess sulfite, (2) xanthine dehydrogenase, which is involved in purine catabolism and reactive oxygen production, (3) aldehyde oxidase, which oxidizes a variety of aldehydes and is essential for the biosynthesis of the phytohormone abscisic acid, and in autotrophic organisms also (4) nitrate reductase, which catalyzes the key step in inorganic nitrogen assimilation. All Mo-enzymes, except plant sulfite oxidase, need at least one more redox active center, many of them involving iron in electron transfer. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also includes iron as well as copper in an indispensable way. Moco as released after synthesis is likely to be distributed to the apoproteins of Mo-enzymes by putative Moco-carrier proteins. Xanthine dehydrogenase and aldehyde oxidase, but not sulfite oxidase and nitrate reductase, require the post-translational sulfuration of their Mo-site for becoming active. This final maturation step is catalyzed by a Moco-sulfurase enzyme, which mobilizes sulfur from l-cysteine in a pyridoxal phosphate-dependent manner as typical for cysteine desulfurases.
Copper deficiency is an established cause of hematological abnormalities but is frequently misdiagnosed. Copper deficiency can present as a combination of hematological and neurological abnormalities and it may masquerade as a myelodysplastic syndrome. We reviewed the records of patients with hypocupremia and hematologic abnormalities identified between 1970 and 2005. Patients with hypocupremia unrelated to copper deficiency (e.g. Wilson's disease) were excluded. Forty patients with copper deficiency and hematological abnormalities were identified. Ten patients (25%) had undergone bariatric (weight reduction) surgery and an additional 14 patients (35%) had undergone surgery on the gastrointestinal tract, most commonly gastric resection. In 12 cases, no cause for copper deficiency was identified. Anemia and neutropenia were the most common hematologic abnormalities identified and the majority of the patients also had neurologic findings, most commonly due to myeloneuropathy. Abnormalities observed on bone marrow examination including vacuoles in myeloid precursors, iron-containing plasma cells, a decrease in granulocyte precursors and ring sideroblasts may be valuable clues to the diagnosis. Copper deficiency is an uncommon but very treatable cause of hematologic abnormalities.
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