Metal Supplementation Research Articles

Zinc as an insulin replacement in hybridoma cultures

There are many advantages to the use of protein-free media for biologics production, including a reduced risk of viral contamination from animal-derived proteins and simplification of downstream purification. In the course of developing protein-free media for hybridoma and myeloma cells, zinc was found to be an effective replacement for insulin, with no negative impact on viable cell density and antibody production. Transcript profiling using DNA microarrays indicated no major change in the global expression profile between the insulin and zinc-supplemented cultures, which is consistent with their similar growth and metabolic characteristics. Both DNA microarray and quantitative RT-PCR analysis showed increase in insulin receptor substrate 1 (Irs1) expression in zinc-supplemented cultures, while several key genes downstream of Irs1 in the insulin-signaling pathway, such as protein kinase B (PKB/Akt) and 3-phosphoinositide dependent protein kinase 1 (Pdpk1) did not show significant differences at the transcript level. Comparison of transcript profiles from cultures with low versus optimal zinc supplementation implicated the involvement of the insulin-related genes Pax6 and Phas1. Subtle differences were also observed between insulin and zinc in the serine-473 phosphorylation of Akt. Zinc increased serine-473 phosphorylation of Akt, but to a lesser extent than insulin. The phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, totally blocked the effect of both zinc and insulin on Akt activation, indicating the involvement of PI3K in the activation of Akt by zinc, rather than zinc acting on Akt directly. Our results highlight the impact of trace metal supplementation as protein-free media formulations move towards greater chemical definition.

Emission of heavy metals from animal carcass incinerators in Taiwan

The metal emissions from three incinerators burning different feedstock in Taiwan were characterized in this study. It was found that the Incinerators A and B, treating pig carcasses and animal (including pigs) carcasses, respectively, had much higher metal concentrations in stack flue gases than Incinerator C that combusted medical wastes. However, Incinerator A obtained relative lower metal contents in fly ash and bottom ash than the other two incinerators, mainly because the former used a much lower feedstock rate (although burning at a lower temperature) than the latter. For all the incinerators, (1) Fe, Ni, Pb, and Zn were dominant in both the fly ash and bottom ash while most of the Cd and Pb (more volatile) were present in the fly ash; (2) Fe emission factor was the highest and Zn/Pb/Ni/Cr emission factors were greater than those of Mn/Cd/Cu; (3) the Cu emission factors in bottom ash were relatively higher in comparison with those in fly ash; and (4) indicatory metals were the same (Fe, Zn, Pb, and Cu). The metal emission factors obtained from the livestock incinerators were much higher than those reported from MSW incinerators. Likewise, crematories that burn human cadavers must create similar pollution issues since metal supplements are part of human's normal diets. This causes an environmental concern and this work has important ramifications both in technical and regulatory decisions.

Effect of initial COD concentration, nutrient addition, temperature and microbial acclimation on anaerobic treatability of broiler and cattle manure

In this study, anaerobic treatability and biogas generation potential of broiler and cattle manure were investigated. For this purpose, seven sets of anaerobic batch reactor experiments were performed using broiler and cattle manure and their mixtures in five different ratios (100% broiler; 75% broiler, 25% cattle; 50% broiler, 50% cattle; 25% broiler, 75% cattle; 100% cattle). These manure mixtures had two different initial chemical oxygen demand (COD) (12,000 and 53,500 mg/l) concentrations. The effects of initial COD concentration, nutrient and trace metal supplementation, microbial acclimation and digestion temperature were investigated. Results revealed that the efficiency of total COD removal was 32.0–43.3% and 37.9–50% for initial COD concentrations of 12,000 and 53,500 mg/l, respectively. The biogas yields observed for initial COD concentrations of 12,000 and 53,500 mg/l were 180–270 and 223–368 ml gas/g COD added, respectively. A decrease in biogas yield was observed as the fraction of broiler manure increased in mixture of broiler and cattle manure at initial COD values of 53,500 mg/l.

Effects of Dietary Restriction and Metal Supplementation on the Accumulation of Iron-Laden Glial Inclusions in the Aging Rat Hippocampus

The mechanisms responsible for the pathological deposition of iron and other redox-active metals in the aging and degenerating mammalian CNS remain poorly understood. We previously demonstrated that normal aging and pharmacological (oxidative) stressors promote the transformation of astroglial mitochondria to iron-laden, diaminobenzidine (DAB)-positive cytoplasmic inclusions in sub-cortical regions of the rat brain. In the current study, we demonstrate that (1) numbers of DAB-positive glial granules in the rat dorsal hippocampus, an area implicated in learning and memory, progressively increase between 3, 12 and 22 months of age; (2) dietary restriction (40%), a manipulation that attenuates many mammalian aging processes, has no effect on the age-related accumulation of these gliosomes in the rat hippocampus; and (3) the latter can be accelerated by dietary supplementation of iron and copper. Our data support the view that dietary exposure to iron and/or copper in adult life can impact the sequestration of redox-active metals in aging hippocampal astroglia.

Hen Mineral Nutrition Impacts Progeny Livability

SUMMARY A concern of nutritionists and live production personnel is to improve early flock livability and growth rate of broilers. Improvements in progeny performance through trace metal supplementation in the hen’s diet may be a method to accomplish this. The focus of this experiment was to evaluate live performance and carcass parameters of progeny from broiler breeders fed diets containing different levels and forms of Zn and Mn. Cobb 500 breeders received a control diet or diets containing supplemental Zn and Mn from inorganic sources, amino acid complexes, or a combination of the two. Body weight gain, feed conversion ratio, livability, percentage carcass without giblets, percentage abdominal fat, and percentage breast meat were measured on the progeny of 37-wk-old breeders. Feeding broiler breeders supplemental Zn and Mn from amino acid complexes improved livability of progeny without affecting growth or carcass characteristics. In conclusion, future research should address progeny carryover of Zn and Mn to improve livability during stress or disease conditions.

Hydrolysis and acidogenesis of particulate organic material in mesophilic and thermophilic anaerobic digestion

The purpose of this study was to evaluate the effect of pH and inorganic nutrient supplementations for anaerobic hydrolysis and addogenesis of paniculate organic materials at both mesophilic (35 °C) and thermophilic (55 °C) temperatures. Hydrolysis and addogenesis of a synthetic sludge was observed in batch operation for the evaluation of the pH effect. pH was uncontrolled in one reactor and controlled at 4.5, 5.5, and 6.5 in the other three reactors at both temperatures. The greatest degree of hydrolysis and addogenesis occurred when the pH was controlled at 6.5. The pH of the uncontrolled reactor dropped to 3.4 at both temperatures severely retarding hydrolysis and addogenesis. Concentrations of acetic and n‐butyric adds predominated with lower concentrations of propionic add at both temperatures in all reactors. Lactic add was produced as the earliest intermediate but as the reaction proceeded, short chain VF As were produced as final end products with a decrease in lactic add. The higher the pH, the earlier this trend was observed. For the controlled reactors at pH 6.5, the soluble COD production and the VSS reduction peaked in 4 days at 55 °C whereas it took about 11 days at 35 °C to obtain the same result. During the linear SCOD production period at a pH of 6.5 the hydrolysis rate of the thermophilic reactor was greater than that for mesophilic. Thermophilic conditions appeared to be more sensitive to pH than mesophilic ones for both hydrolysis and addogenesis. Additional experiments were conducted to establish the effect of inorganic nutrient (Ca, Fe, Co, and Ni) supplementation on hydrolysis and addogenesis at both temperatures. It has, prior to this, been assumed that only methanogenesis benefited from trace metal supplementation. However, the results demonstrated the importance of inorganic nutrient supplementation to optimize hydrolysis and addogenesis at both temperatures.

Effects of various nutritional supplements on biosurfactant production by a strain of Bacillus subtilis at 45°C

AbstractEffects of various factors on growth and biosurfactant production by Bacillus subtilis MTCC 2423 were studied. Sucrose (2%) and potassium nitrate (0.3%) were the best carbon and nitrogen sources. The addition of various metal supplements (magnesium, calcium, iron, and trace elements) greatly affected growth and biosurfactant production. The effect of the metal cations, used together, is greater than when they are used individually. The biosurfactant production increased considerably (almost double) by addition of metal supplements. Very high concentrations of metal supplements, however, inhibited biosurfactant production. Amino acids such as aspartic acid, asparagine, glutamic acid, valine, and lysine increased the final yield of biosurfactant by about 60%. The organism could produce biosurfactant at 45°C and within the pH range of 4.5–10.5. The biosurfactant was thermostable and pH stable (from 4.0 to 12.0). The capability of the organism to produce biosurfactant under thermophilic, alkaliphilic, and halophilic conditions makes it a suitable candidate for field applications. Infrared, nuclear magnetic resonance, and mass spectroscopy studies showed the surfactant to be identical to surfactin.

Anaerobic biotransformation and methane generation potential of cheese whey in batch and UASB reactors

Anaerobic treatability and methane generation potential of cheese whey were determined in batch reactors. Furthermore, the effect of nutrient and trace metal supplementation on the batch anaerobic treatment, and the high-rate anaerobic treatability of cheese whey in upflow anaerobic sludge blanket (UASB) reactors were investigated. To this purpose biochemical methane potential experiments were conducted and single- and two-stage UASB reactors with granular cultures were operated. In UASB experiments significance of process staging, operational parameters such as hydraulic retention time (HRT), influent chemical oxygen demand (COD) concentration and loading rate were also investigated. The results revealed that nutrient and trace metal supplementation is vital for the anaerobic treatment of cheese whey; the anaerobic methane generation for the cheese whey studied was found to be 424 ml CH 4/g COD (23.4 l CH 4/l cheese whey); undiluted cheese whey could be treated anaerobically at relatively short HRT values (2.06–4.95 days) without any significant stability problems; HRT values as low as 2–3 days can be used for the anaerobic treatment of cheese whey, with a COD removal efficiency of 95–97% at influent COD concentration of 42 700±141–55 100±283 mg/l.

Clonal differences in copper and zinc tolerance of birch in metal-supplemented soils

Metal tolerance of a range of birch clones ( Betula pendula and Betula pubescens) originating from metal-contaminated sites in England, Wales, Belgium and Finland were tested in soils supplemented with several concentrations of copper (Cu) or zinc (Zn) (500, 2000, 5000 mg kg −1 dry wt. soil of CuSO 4·5H 2O or ZnSO 4·7H 2O) for 4 months and with sub-toxic metal supplements (500 mg CuSO 4, 2000 mg ZnSO 4) for 6 months. When grown at high concentrations of metals, severe toxicity symptoms (growth inhibition, chlorosis, necrosis) and clear evidence for differences in tolerance to this toxicity were found in a subset of the clones. When all clones were grown at a much lower, sub-toxic level of metal, again significant differences could be found between some of the clones. Clones derived from the same population varied greatly in their tolerance. However, the overall pattern of metal specificity varied in agreement with the type of soil contamination at the site of origin. The growth of the clones from Harjavalta Cu/nickel smelter area was 19% better in Cu than in Zn-supplemented soil, on average. The growth of clones from Maatheide Zn smelter are was 19% poorer in Cu- than in Zn-supplemented soil. Sensitive clones accumulated more Cu and Zn to the above-ground parts. Some birch clones were able to survive at about 20-fold higher than typical total background Cu or Zn concentrations, whereas most clones were able to grow without serious toxic symptoms at about 10-fold concentrations.

Prooxidant and antioxidant activities of macrophages in metal-mediated LDL oxidation: the importance of metal sequestration.

Murine macrophages incubated in metal-supplemented RPMI could block or promote oxidation of low-density lipoprotein (LDL) depending on the degree of metal supplementation. Only at high concentrations of Cu (1 micromol/L) and Fe (30 micromol/L) were cells prooxidant, leading to an accelerated rate of LDL oxidation over that measured in comparable cell-free media. At lower concentrations of Cu and Fe in RPMI, LDL oxidation in the presence of macrophages was inhibited relative to the cell-free condition. This appeared to be dependent on a stable modification of the culture medium, because preconditioning of media by incubation with macrophages could also decrease their capacity to sustain subsequent cell-free LDL oxidation. This was due, in part, to a removal of metal from the media during preconditioning. However, resupplementation of media with metals did not fully restore oxidative capacity, indicating that other cell-dependent antioxidant modifications occurred. This did not involve significant alterations to the thiol content of the media. This study highlights the complexity of the role that cells such as macrophages have with regards to LDL oxidation in vitro and demonstrate that there are both antioxidative and prooxidative components.

In vitro incorporation of different transition metal ions into a cambialistic superoxide dismutase from Propionibacterium shermanii

The superoxide dismutase (SOD) of Propionibacterium shermanii can form enzymatically active complexes in vivo with iron or manganese as well as inactive complexes with copper or cobalt, depending on the metal supplementation to the growth medium. In vitro, the superoxide dismutase apoprotein build complexes with different metal ions, preferably in the 1+/2+ valence state. Metal ions with a charge 3+ or higher were unable to form complexes with the protein. Only iron- or manganese-ions in the valence state 2+ led to an active enzyme. The different metal substituted proteins showed identical UV- and CD-spectra in the tryptophan region and nearly identical CD-spectra in the UV region related to secondary structure, suggesting that metal binding to the active site had only a minor effect, if at all, on the protein structure. Differences were obtained in the visible spectra likely due to the absorption of the metal ions.

Capillary zone electrophoresis with UV‐scanning detection for the analysis of metal species and their organic binding partners in biological systems

AbstractA capillary zone electrophoretic method with UV‐scanning detection for the separation and identification of both free ligands and metal species is presented. The electrophoretic behavior of naturally occurring binding partners such as organic acids and amino acids was studied and compared with their metal‐complexes. Copper(II) complexes of citrate and amino acids showed decreased electrophoretic mobilities and altered UV spectra. The optimized method was validated on ultrafiltered cow's milk and human milk samples. In cow's milk six low molecular weight substances, including citrate, orotate, and hippurate, could be separated. Metal supplementation with Cu(II), Ni(II), or Zn(II) decreased not only the citrate peak but also the orotate signal and in the case of copper(II) supplementation a new signal for the copper(II)‐citrate complex appeared. In human milk samples various amino acids such as glutamate, phenylalanine, tyrosine, and tryptophan could be identified besides citrate. The electrophoretic mobilites and peak areas of the amino acids were also influenced by the metal supplementation.

Copper-dependent metabolism of Cu,Zn-superoxide dismutase in human K562 cells. Lack of specific transcriptional activation and accumulation of a partially inactivated enzyme.

The regulation of Cu,Zn-superoxide dismutase by copper was investigated in human K562 cells. Copper ions caused a dose- and time-dependent increase, up to 3-fold, of the steady-state level of Cu,Zu-superoxide dismutase mRNA. A comparable increase was also observed for actin and ribosomal protein L32 mRNAs, but not for metallothionein mRNA which was augmented more than 50-fold and showed a different induction pattern. The copper-induced mRNAs were actively translated as judged from their enhanced loading on polysomes, the concomitantly increased cellular protein levels and an augmented incorporation of [3H]lysine into acid-precipitable material. Cu,Zn-superoxide dismutase protein followed this general trend, as demonstrated by dose- and time-dependent increases in immunoreactive and enzymically active protein. However, a specific accumulation of Cu,Zn-superoxide dismutase was noticed in cells grown in the presence of copper, that was not detectable for other proteins. Purification of the enzyme demonstrated that Cu,Zn-superoxide dismutase was present as a reconstitutable, copper-deficient protein with high specific activity (kcat./Cu = 0.89 x 10(9) M-1.s-1) in untreated K562 cells and as a fully metallated protein with low specific activity (kcat./Cu = 0.54 x 10(9) M-1.s-1) in copper-treated cells. Pulse-chase experiments using [3H]lysine indicated that turnover rates of Cu,Zn-superoxide dismutase in K562 cells were not affected by growth in copper-enriched medium, whereas turnover of total protein was significantly enhanced as a function of metal supplementation. From these results we conclude that: (i) unlike in yeast [Carrì, Galiazzo, Ciriolo and Rotilio (1991) FEBS Lett. 278, 263-266] Cu,Zn-superoxide dismutase is not specifically regulated by copper at the transcriptional level in human K562 cells, suggesting that this type of regulation has not been conserved during the evolution of higher eukaryotes; (ii) copper ions cause an inactivation of the enzyme in intact K562 cells; and (iii) the metabolic stability of Cu,Zn-superoxide dismutase results in its relative accumulation under conditions that lead to increased protein turnover.

In vivo incorporation of cobalt into Propionibacterium shermanii superoxide dismutase

Propionibacterium shermanii, an aerotolerant anaerobic bacterium, has already been shown to incorporate, depending on the metal supplementation to the medium, either iron or manganese or copper into the same superoxide dismutase protein. The in vivo incorporation of cobalt in the same superoxide dismutase was obtained in an iron-, manganese- and copper-depleted medium. The protein was isolated and characterized by NMR which offers the possibility to identify the amino acid residues at the active site exploiting isotropically shifted proton resonance.

The survival of the anaerobic propioni-bacterium in the presence of oxygen is dependent on metal supplementation

The survival of the anaerobic propioni-bacterium in the presence of oxygen is dependent on metal supplementation

Response ofAnabaena doliolum to bimetallic combinations of Cu, Ni and Fe with special reference to sequential addition

This study reports physiological features of a N2-fixing cyanobacteriumAnabaena doliolum in response to metal mixtures. Exposure of the cyanobacterium to Cu, Ni and Fe individually, as well as in combinations (Cu + Ni, Cu + Fe, Ni + Fe), showed marked differences in growth inhibition, nutrient uptake (NH4+ and NO3−), photosynthesis, ATP content, nitrate reductase, glutamine synthetase and urease activities. The response to metal combinations was also dependent upon the order in which the metals were added. The Cu-Ni combination resulted in synergistic interaction, in contrast to the antagonism of Cu-Fe and Ni-Fe. Pre-addition of Fe protected the cyanobacterium against Cu and Ni toxicity. Statistically significant (P < 0.005) inhibition of all the processes following metal supplementation was observed. This study suggests that carbon fixation is the most suitable variable for assessing heavy metal toxicity.

Alterations in plasma copper, zinc, and ceruloplasmin levels in patients with thermal trauma.

This review was undertaken to elucidate the trends in copper, zinc, total serum protein, and ceruloplasmin levels in patients with thermal trauma. Medical records of pediatric and adult patients were retrospectively reviewed. The findings showed a significant linear decline in mean plasma copper, ceruloplasmin, and total serum protein during the first three days postburn, followed in general by a gradual rise in levels; however, patients with burns larger than 60% TBSA maintained copper, zinc, and ceruloplasmin levels significantly below the lower limits of normal during the observation period. Significant decrements in copper, zinc, total serum protein, and ceruloplasmin levels have been demonstrated in these patients. Trace metal determinations should be obtained in patients with significant burns soon after admission and routinely throughout their hospital course. Specific trace metal supplementation should be administered when indicated.

Evaluation of the trace metal supplements for a synthetic low lactose diet.

A trace element supplement used with a synthetic low lactose milk (Galactomins 17 and 18) has been evaluated by means of metabolic balance studies in 4 infants with dissacharide intolerances. The supplement was considered satisfactory for iron and manganese but increases in its zinc and copper content are probably necessary to ensure adequate retentions of these metals.

Some nutritional aspects of trace metals.

A brief outline of the known biochemical roles of manganese and chromium is given before the problems of determining human trace metal status are discussed. The factors predisposing to trace metal deficiency are reviewed but particular emphasis is placed upon those which alter the bioavailability of essential trace metals from natural and synthetic diets. This is illustrated by practical examples which show the importance of investigating the bioavailability of the trace metal content of artificial diets used in paediatric care. The results of such studies highlight the need to assess similarly the adequacy of the trace element composition of the many meat substitutes and extenders available currently. Finally, some aspects of trace metal supplementation of intravenous feeding regimens are described.

Methylmethacrylate stabilization for enhancement of posterior cervical arthrodesis in rheumatoid arthritis.

Stainless-steel wire, titanium mesh, and perforated titanium sheets and methylmethacrylate were used in conjunction with autogenous iliac-bone grafts to provide immediate stability in difficult or long posterior cervical fusions in eleven patients with rheumatoid arthritis, who were followed for three to 12.5 years. Two surgical techniques, using methylmethacrylate and metal supplementation of a traditional posterior cervical arthrodesis, are described. One technique includes the occiput, while the other consists of a long posterior fusion that excludes the occiput. In ten of the eleven patients, permanent radiographic stability was maintained as well as a significant reduction of pain and resolution of neural dysfunction. In the eleventh patient a cervical subluxation developed at the distal end of the rigidly fused segment, and eventually the patient died. Four patients who were seen early in the series had wound dehiscences, two of which were infected. Subsequently the methacrylate-metal composite was reduced in volume, and this problem did not recur. In summary, this technique has provided immediate internal splinting and avoided bulky external-fixation devices. It was most useful in fusions of the occiput to the upper part of the cervical spine, in long posterior cervical arthrodesis, and for the repair of pseudarthroses when traditional arthrodesis techniques had failed.