Ferritin-bound Iron Research Articles

Chasing shadows: What determines DTI metrics in gray matter regions? An in vitro and in vivo study

To characterize the relationship between superparamagnetic ferritin-bound iron and diffusion tensor scalars in vitro, and validate the results in vivo. The in vitro model consisted of a series of 40-mL 1.1% agarose gels doped with ferritin covering and exceeding those concentrations normally found within healthy human gray matter. Additionally, regions of interest were placed in the caudate, putamen, and globus pallidus of 29 healthy volunteer subjects 19-80 years of age. Carr-Purcell-Meiboom-Gill sequence (CPMG) and diffusion tensor imaging (DTI) data were collected at 1.5 Tesla (T) and 3T in vitro, and at 1.5T in vivo. In vitro, linear relationships were observed between ferritin-bound iron concentration, R2 (1/T2 ) and 1/SNR. Eigenvalue repulsion with increasing R2 (decreasing SNR) was reflected in an artifactual increase of fractional anisotropy. In vivo, similar relationships were observed, with mean diffusivity also decreasing linearly with increasing R2 . Lambda 3 showed the strongest correlation with R2 both in vitro and in vivo. The observation that DTI metrics correlate with ferritin-bound iron is an important consideration in the design and interpretation of studies exploring the diffusion characteristics of gray matter regions, especially in studies focused on adolescence as well as diseases associated with altered brain-iron load such as pantothenate kinase-associated neurodegeneration, Huntington disease and multiple system atrophy.

Iron deficiency in parkinsonism: region-specific iron dysregulation in Parkinson's disease and multiple system atrophy.

Alpha synuclein pathology is widespread and found in diverse cell types in multiple system atrophy (MSA) as compared to Parkinson's disease (PD). The reason for this differential distribution is unknown. Regional differences in the distribution of iron are associated with neurodegenerative diseases, and here we characterize the relationship between iron homeostasis proteins and regional concentration, distribution and form of iron in MSA and PD. In PD substantia nigra, tissue iron and expression of the iron export protein ferroportin increased, while the iron storage protein ferritin expression was unchanged. In the basis pontis of MSA cases, increased total iron concentration coupled with a disproportionate increase in ferritin in dysmorphic microglia and a reduction in ferroportin expression. This is supported by isothermal remanent magnetisation evidence consistent with elevated concentrations of ferritin-bound iron in MSA basis pontis. Conventional opinion holds that excess iron is involved in neurodegeneration. Our data support that this may be the case in PD. While region-specific changes in iron are evident in both PD and MSA, the mechanisms of iron dysregulation appear quite distinct, with a failure to export iron from the MSA basis pontis coupling with significant intracellular accumulation of ferritin iron. This pattern also occurs, to a lesser extent, in the MSA putamen. Despite the excess tissue iron, the manner of iron dysregulation in MSA is reminiscent of changes in anemia of chronic disease, and our preliminary data, coupled with the widespread pathology and involvement of multiple cell types, may evidence a deficit in bioavailabile iron.

G10 Correlation between relaxometry and diffusion tensor imaging in the globus pallidus of Huntington disease patients

Backround Huntington9s disease (HD) is an inherited autosomal dominant neurodegenerative disorder with progressive impairment of motor and cognitive functions and behavioural abnormalities. The clinical features of HD are closely related to the degeneration of the basal ganglia, predominantly the striatum. The main striatal output structure, the globus pallidus, strongly accumulates ferritin-bound iron, which is believed to change the diffusion tensor in a systematic way. Aim, subject and methods To test the hypothesis that this same effect exists in the iron-rich basal ganglia of HD patients, we examined changes in the globus pallidus using a DTI WIP sequence and a T2 relaxometry CPMG sequence. Quantitative MR, clinical data and genetic data (the number of CAG repeats) were obtained from 14 HD patients. Coregistered MR parameters such as the T2 relaxation rate (RR), fractional anisotrophy (FA) and mean diffusivity (MD) were analysed by two independent readers in FSL and further processed in GraphPad. Bonferroni9s correction was used for multiple statistical comparisons. Results Significant correlations between RR and FA (R2=0.84), between RR and MD (R2=0.66), between FA and MD (R2=0.85) and between CAG and RR (R2=0.59) were found. A trend towards a significant correlation between CAG and FA (R2=0.44) was noted. No significant correlation between CAG and MD or between MR and clinical parameters was found. We also found that there were no significant hemispheric differences in the MR parameters and no significant difference between HD patients and age-matched healthy controls. Conclusion These results indicate that FA in the globus pallidus may have the potential to be used as a biomarker of iron accumulation in future studies of HD patients.

Aging is an organ-specific process: changes in homeostasis of iron and redox proteins in the rat

Organ-specific changes of iron- and redox-related proteins occur with age in the rat. Ferritin, the major iron storage and detoxifying protein, as well as the proteins of the methionine-centered redox cycle (MCRC) were examined in old and young animals, and showed organ-dependent changes. In spleens and livers of aged rats, ferritin (protein) levels were greater than in young ones, and their iron saturation increased, rendering higher ferritin-bound iron (FtBI). Iron saturation of the ferritin molecule in the tongues and sternohyoids of old rats was lower but ferritin level was higher than in young rats, resulting in increased FtBI with age. Ferritin level in the esophagus of older rats was lower than in young rats but its molecular iron content higher thus the total FtBI remained the same. In the larynx, both ferritin and its iron content were the same in young and old animals. MCRC proteins were measured in livers and spleens only. With aging, methionine sulfoxide reductase A and B (MsrA and MsrB) levels in livers and spleens decreased. Thioredoxin1 (Trx) and Trx-reductase1 were elevated in old spleens, but reduced in livers. Aged spleens showed reduced Msr isozyme activity; but in the liver, its activity increased. mRNA changes with age were monitored and found to be organ specific. These organ-specific changes could reflect the different challenges and the selective pathways of each organ and its resultant capacity to cope with aging.

Iron homeostasis and methionine-centred redox cycle in nasal polyposis

Nasal polyposis is a multifactorial disease with a strong inflammatory component. Its pathogenesis is often associated with ROS production catalysed by redox-active iron. This study aimed to characterize the roles of iron homeostasis and redox status in the pathogenesis of polyposis. Nasal polyps (NP) from asthmatics and non-asthmatics and turbinates from controls and NP-patients were analysed for ferritin, ferritin-bound iron (FBI) and levels of methionine-centred redox cycle proteins. The ferritin content in both NPs was significantly higher than in adjacent turbinates. No differences in FBI were observed between both NP groups and both turbinates groups, while in NPs it was significantly higher. In NP-turbinates the highest levels of redox proteins were observed. In conclusion, re-distribution of iron occurs upon the development of NP. While FBI is elevated in NPs, the adjacent turbinate remain iron-poor and low-inflammatory, suggesting the formation of virtual boundary between these tissues.

Superoxide-dependent reduction of free Fe3+ and release of Fe2+ from ferritin by the physiologically-occurring Cu(I)–glutathione complex

The intracellularly-occurring Cu(I)-glutathione complex (Cu(I)-[GSH](2)) has the ability to reduce molecular oxygen into superoxide radicals (O2·-). Based on such ability, we addressed the potential of this complex to generate the redox-active Fe(2+) species, during its interaction with free Fe(3+) and with ferritin-bound iron. Results show that: (i) the complex reduces free Fe(3+) through a reaction that totally depends on its O2·--generating capacity; (ii) during its interaction with ferritin, the complex reduces and subsequently releases iron through a largely (77%) SOD-inhibitable reaction; the remaining fraction is accounted for by a direct effect of GSH molecules contained within the complex. The O2·--dependent iron-releasing efficiency of the complex was half that of its iron-reducing efficiency; (iii) the ability of the complex to release ferritin-bound iron was increased, concentration-dependently, by the addition of GSH and totally prevented by SOD; (iv) in the presence of added H(2)O(2), the Fe(2+) ions generated through (i) or (ii) were able to catalyze the generation of hydroxyl radicals. Thus, the present study demonstrates the ability of the Cu(I)-[GSH](2) complex to generate the redox-active Fe(2+) species and suggest that by favouring the occurrence of superoxide-driven Fenton reactions, its pro-oxidant potential could be increased beyond its initial O2·--generating capacity.

Transferrin Receptor 1: A Ferritin Receptor as Well?

Transferrin Receptor 1: A Ferritin Receptor as Well?

Quantification of Ferritin-Bound Iron in Plant Samples by Isotope Tagging and Species-Specific Isotope Dilution Mass Spectrometry

Ferritin is nature's predominant iron storage protein. The molecule consists of a hollow protein shell composed of 24 subunits which is capable of storing up to 4500 iron atoms per molecule. Recently, this protein has been identified as a target molecule for increasing iron content in plant staple foods in order to combat dietary iron deficiency, a major public health problem in developing countries. Here, we present a novel technique for quantification of ferritin-bound iron in edible plant seeds using species-specific isotope dilution mass spectrometry (IDMS) by means of a biosynthetically produced (57)Fe-labeled ferritin spike and negative thermal ionization mass spectrometry (NTIMS). Native plant ferritin and added spike ferritin were extracted in 20 mM Tris buffer (pH 7.4) and separated by anion exchange chromatography (DEAE Sepharose), followed by isotopic analysis by thermal ionization mass spectrometry. The chosen IDMS approach was critically evaluated by assessing the (i) efficiency of analyte extraction, (ii) identical behavior of spike and analyte, and (iii) potential iron isotope exchange with natural iron. Repeatabilities that can be achieved are on the order of <5% RSD for quintuplicate analyses at an absolute detection limit of 60 ng of ferritin-bound iron for plant seeds. Studies in six different legumes revealed ferritin-iron contents ranging from 15% of total iron in red kidney beans up to 69% in lentils.

Soybean ferritin: implications for iron status of vegetarians

Meeting the requirement for absorbed iron is difficult for vegetarians, and their iron status often is lower than that of nonvegetarians. Beans contain ferritin in low concentrations, but it is possible to enhance this content by plant breeding or by inserting the gene for ferritin into plants, eg, soybeans. Because each ferritin molecule can bind to thousands of iron atoms, this may be a sustainable means to increase the iron contents of plants. Before such efforts are launched, it is important to determine whether iron in ferritin is bioavailable. This has been assessed in vitro by using human intestinal (Caco-2) cells and in vivo by using radiolabeled ferritin and whole-body counting in human subjects. Dietary factors affecting iron absorption, eg, ascorbic acid, phytate, and calcium, had limited effect on iron uptake from intact ferritin by Caco-2 cells, which suggests that ferritin-bound iron is absorbed via a mechanism different from that of nonheme iron. In an in vitro digestion system, ferritin was shown to be relatively resistant to proteolytic enzymes. Binding of ferritin to Caco-2 cells was shown to be saturable, and the kinetics for binding were characteristic of a receptor-mediated process. In human subjects, iron from purified soybean ferritin given in a meal was as well absorbed as iron from ferrous sulfate. In conclusion, iron is well absorbed from ferritin and may represent a means of biofortification of staple foods such as soybeans.

Alteration in Iron Metabolism during Retinal Degeneration in rd10 Mouse

Altered iron metabolism was implicated in retinal and macular degeneration. This study was designed to further elucidate iron homeostasis during the course of retinal degeneration in mice. Retinal mRNA and protein expression of transferrin, transferrin receptor, and ceruloplasmin were evaluated during retinal degeneration in rd10 mice and chemokine receptor 2 (ccr2)-deficient mice. Retinal ferritin protein levels, ferritin-bound iron, and total iron were evaluated in rd10 mice. Transferrin and ceruloplasmin mRNA levels increased between 2- and 12-fold during the course of retinal degeneration in rd10 mice compared with same-age controls (P < 0.01), whereas transferrin receptor mRNA levels increased only at the late stages of degeneration in rd10 mice (2.7-fold; P = 0.005). Transferrin mRNA also increased in retinas of aged ccr2-deficient mice (1.5-fold; P = 0.05). Transferrin and ceruloplasmin protein levels corroborated with mRNA levels changes in rd10 mice albeit at a lower magnitude. Retinal ferritin protein levels increased between 1.5-fold and 2-fold (P < 0.03) in rd10 mice, and ferritin-bound iron levels increased 1.6-fold in 3-week-old rd10 mice (P = 0.03). Three-week-old rd10 mice also had a 1.4-fold increase in total retinal iron level (P = 0.05). Combined with previous reports, these data suggest that retinal degenerations are associated with altered iron homeostasis regardless of the primary insult. Given the potential of iron to generate oxidative injury, its role as a therapeutic target in retinal and macular degenerations should be evaluated.

Ferritin functions as a proinflammatory cytokine via iron‐independent protein kinase C zeta/nuclear factor kappaB–regulated signaling in rat hepatic stellate cells†

Circulating ferritin levels reflect body iron stores and are elevated with inflammation in chronic liver injury. H-ferritin exhibits a number of extrahepatic immunomodulatory properties, although its role in hepatic inflammation and fibrogenesis is unknown. Hepatic stellate cells respond to liver injury through production of proinflammatory mediators that drive fibrogenesis. A specific receptor for ferritin has been demonstrated on activated hepatic stellate cells, although its identity and its role in stellate cell activation is unclear. We propose that ferritin acts as a cytokine regulating proinflammatory function via nuclear factor kappaB (NF-kappaB)-regulated signaling in hepatic stellate cell biology. Hepatic stellate cells were treated with tissue ferritin and iron-free apoferritin, recombinant H-ferritins and L-ferritins, to assess the role of ferritin versus ferritin-bound iron in the production of proinflammatory mediators of fibrogenesis, and to determine whether signaling pathways act via a proposed H-ferritin endocytosis receptor, T cell immunoglobulin-domain and mucin-domain 2 (Tim-2). This study demonstrated that ferritin activates an iron-independent signaling cascade, involving Tim-2 independent phosphoinositide 3 (PI3)-kinase phosphorylation, protein kinase C zeta (PKCzeta) and p44/p42-mitogen-activated protein kinase, resulting in p50/p65-NF-kappaB activation and markedly enhanced expression of hepatic proinflammatory mediators interleukin-1beta (IL-1beta), inducible nitric oxide synthase (iNOS), regulated on activation normal T cell expressed and secreted (RANTES), inhibitor of kappa Balpha (IkappaBalpha), and intercellular adhesion molecule 1 (ICAM1). This study has defined the role of ferritin as a proinflammatory mediator of hepatic stellate cell biology acting through the NF-kappaB signaling pathway, and suggests a potential role in the inflammatory processes associated with hepatic fibrogenesis.

Heart protection by ischemic preconditioning: A novel pathway initiated by iron and mediated by ferritin

Ischemic preconditioning is a well-known procedure transiently protecting the heart against injury associated with prolonged ischemia, through mechanism/s only partly understood. The aim of this study was to test whether preconditioning-induced protection of the heart involves an iron-based mechanism, including the generation of an iron signal followed by accumulation of ferritin. In isolated rat hearts perfused in the Langendorff configuration, we measured heart contractility, ferritin levels, ferritin-iron content, and mRNA levels of ferritin subunits. Ischemic preconditioning caused rapid accumulation of ferritin, reaching 359% of the baseline value (set at 100%). This was accompanied by a parallel decline in ferritin-bound iron: from 2191 ± 548 down to 760 ± 34 Fe atoms/ferritin molecule, p < 0.05. Ferritin levels remained high during the subsequent period of prolonged ischemia, and returned to nearly the baseline value during the reperfusion phase. Selective iron chelators (acetyl hydroxamate or Zn-desferrioxamine) abrogated the functional protection and suppressed ferritin accumulation, thus demonstrating the essentiality of an iron signal in the preconditioning-induced protective mechanism. Moreover, introduction of an iron-containing ternary complex, known to import iron into cells, caused a three-fold accumulation of ferritin and simulated the preconditioning-induced functional protection against prolonged myocardial ischemia. The ischemic preconditioning-and-ischemia-induced increase in ferritin levels correlated well with the accumulation of ferritin L-subunit mRNA: 5.44 ± 0.47 vs 1.23 ± 0.15 (units) in the baseline, p < 0.05, suggesting that transcriptional control of ferritin L-subunit synthesis had been activated. Ischemic preconditioning initiates de novo synthesis of ferritin in the heart; the extra ferritin is proposed to serve a ‘sink’ for redox-active iron, thus protecting the heart from iron-mediated oxidative damage associated with ischemia–reperfusion injury. The present results substantiate a novel iron-based mechanism of ischemic preconditioning and could pave the way for the development of new modalities of heart protection.

Receptor-mediated uptake of ferritin-bound iron by human intestinal Caco-2 cells

Ferritin (Ft) is a large iron (Fe)-binding protein (∼450 kDa) that is found in plant and animal cells and can sequester up to 4500 Fe atoms per Ft molecule. Our previous studies on intestinal Caco-2 cells have shown that dietary factors affect the uptake of Fe from Ft in a manner different from that of Fe from FeSO 4, suggesting a different mechanism for cellular uptake. The objective of this study was to determine the mechanism for Ft–Fe uptake using Caco-2 cells. Binding of 59Fe-labeled Ft at 4°C showed saturable kinetics, and Scatchard analysis resulted in a K d of 1.6 μM, strongly indicating a receptor-mediated process. Competitive binding studies with excess unlabelled Ft significantly reduced binding, and uptake studies at 37°C showed saturation after 4 h. Enhancing and blocking endocytosis using Mas-7 (a G-protein activator) and hypertonic medium (0.5 M sucrose), respectively, demonstrated that Ft–Fe uptake by Mas-7-treated cells was 140% of control cells, whereas sucrose treatment resulted in a statistically significant reduction in Ft–Fe uptake by 70% as compared to controls. Inhibition of macropinocytosis with 5-( N, N-dimethyl)-amiloride (Na +/H + antiport blocker) resulted in a decrease (by ∼20%) in Ft–Fe uptake at high concentrations of Ft, suggesting that enterocytes can use more than one Ft uptake mechanism in a concentration-dependent manner. These results suggest that Ft uptake by enterocytes is carried out via endocytosis when Ft levels are within a physiological range, whereas Ft at higher concentrations may be absorbed using the additional mechanism of macropinocytosis.

Ischemic preconditioning of the rat retina: Protective role of ferritin

Ischemic preconditioning (IPC) of the retina, accomplished by ischemia of short duration, is highly effective in preventing subsequent severe injury caused by iron-dependent free radical burst after prolonged ischemia. To investigate the mechanistic basis for IPC rescue, we examined changes in the levels of the retinal redox-active and labile iron pool, ferritin, and ferritin-bound iron. Prolonged ischemia severely impaired retinal function, with total loss of the full-field electroretinographic response. IPC provided marked protection against such injury. Histological examination revealed that ischemia-associated structural damage and loss of cells in the outer and inner nuclear layers were largely prevented by IPC. Ferritin levels decreased after prolonged ischemia but remained close to normal when the ischemic episode was preceded by IPC. The protective effect of IPC on retinal function and ferritin was blocked by a zinc–desferrioxamine complex known to interfere with iron signaling. The results suggest a mechanism whereby IPC activates an iron signaling pathway leading to a marked increase in ferritin levels, which mediates resistance to prolonged ischemia.

Biosynthesis, isolation and characterization of 57Fe-enriched Phaseolus vulgaris ferritin after heterologous expression in Escherichia coli

Ferritin is the major iron storage protein in the biosphere. Iron stores of an organism are commonly assessed by measuring the concentration of the protein shell of the molecule in fluids and tissues. The amount of ferritin-bound iron, the more desirable information, still remains inaccessible owing to the lack of suitable techniques. Iron saturation of ferritin is highly variable, with a maximum capacity of 4,500 iron atoms per molecule. This study describes the direct isotopic labeling of a complex metalloprotein in vivo by biosynthesis, in order to measure ferritin-bound iron by isotope dilution mass spectrometry. [(57)Fe]ferritin was produced by cloning and overexpressing the Phaseolus vulgaris ferritin gene pfe in Escherichia coli in the presence of (57)FeCl(2). Recombinant ferritin was purified in a fully assembled form and contained approximately 1,000 iron atoms per molecule at an isotopic enrichment of more than 95% (57)Fe. We did not find any evidence of species conversion of the isotopic label for at least 5 months of storage at -20 degrees C. Transfer efficiency of enriched iron into [(57)Fe]ferritin of 20% was sufficient to be economically feasible. Negligible amounts of non-ferritin-bound iron in the purified [(57)Fe]ferritin solution allows for use of this spike for quantification of ferritin-bound iron by isotope dilution mass spectrometry.

MR relaxometry in Huntington's disease: Correlation between imaging, genetic and clinical parameters

Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disease. It has been hypothesized that changes of iron content in the brain may be involved in the pathogenesis of HD. To ascertain the hypothesis, we investigated the relationship between T2 relaxation time (T2), the number of cytosine-adenine-guanine triplet repeats (CAG) and clinical status in patients suffering from HD. 34 HD patients (mean age 50.1+/-11.8 standard deviation (SD) years) and 34 control subjects (49.6+/-13.3) were scanned using a 1.5 tesla magnetic resonance (MR) scanner and the patients underwent clinical and genetic testing. A multiple echo sequence was employed for T2 measurements. T2 from healthy volunteers matched previous studies. A T2 shortening was found in the pallidum of HD patients compared to controls (65.4+/-6.4 ms vs. 71.8+/-3.6 ms, P<0.00001). A correlation between the number of CAG and T2 was found for the left pallidum (decrease in T2, P<0.05) and an inverse correlation for the left caudate (increase in T2, P<0.05). In HD patients, alterations in iron levels may be caused by an alteration in its axonal transport. The observed T2/CAG covariations may reflect changes in levels and forms of iron: this suggests that HD patients with a higher genetic load have more ferritin-bound ("safe form") iron in the pallidum and/or more low-molecular ("toxic") iron in the caudate. An increase in "toxic" iron in the caudate may enable oxidative stress and thus underlie progression of the disease.

The Importance and Bioavailability of Phytoferritin-Bound Iron in Cereals and Legume Foods

Ferritin is present in several types of plants in low concentrations, but it is possible to enhance this content by plant breeding, or by inserting the gene for ferritin into staple foods. Since each ferritin molecule can bind thousands of iron atoms, this may be a sustainable means to increase the iron content of plants. Before launching such efforts it is important to determine whether ferritin-bound iron is bioavailable. We assessed this in vitro using Caco-2 cells and in vivo using radiolabeled ferritin and whole body counting in human subjects. In Caco-2 cells, we found that dietary factors affecting iron absorption, such as ascorbic acid, phytate, and calcium, had very limited effect on iron uptake from intact ferritin, suggesting that ferritin-bound iron is absorbed via a mechanism different from that of non-heme iron. Using in vitro digestion, we found that ferritin was relatively resistant against proteolytic enzymes. Binding of ferritin to Caco-2 cells was found to be saturable and the kinetics for binding characteristic for a receptor-mediated process. In human subjects, we found that iron absorption from animal ferritin was similar to that from ferrous sulfate, suggesting that iron is well absorbed from ferritin. We did not find any significant difference between iron absorption from ferritin reconstituted with high-phosphate (plant-type) and low-phosphate (animal-type) ferritin mineral, suggesting that plant ferritin-iron is bioavailable. In a subsequent human study we also found that iron from purified soybean ferritin given in a meal was as well absorbed as ferrous iron. In conclusion, iron is well absorbed from phytoferritin and may represent a means of biofortification of staple foods.

The chemical form of mitochondrial iron in Friedreich’s ataxia

Friedreich’s ataxia (FRDA) results from cellular damage caused by a deficiency in the mitochondrial matrix protein frataxin. To address the effect of frataxin deficiency on mitochondrial iron chemistry, the heavy mitochondrial fraction (HMF) was isolated from primary fibroblasts from FRDA affected and unaffected individuals. X-ray absorption spectroscopy was used to characterize the chemical form of iron. Near K-edge spectra were fitted with a series of model iron compounds to determine the proportion of each iron species. Most of the iron in both affected and unaffected fibroblasts was ferrihydrite. The iron K-edge from unaffected HMFs were best fitted with poorly organized ferrihydrite modeled by frataxin whereas HMFs from affected cells were best fitted with highly organized ferrihydrite modeled by ferritin. Both had several minor iron species but these did not differ consistently with disease. Since the iron K-edge spectra of ferritin and frataxin are very similar, we present additional evidence for the presence of ferritin-bound iron in HMF. The predominant ferritin subunit in HMFs from affected cells resembled mitochondrial ferritin (MtFt) in size and antigenicity. Western blotting of native gels showed that HMF from affected cells had 3-fold more holoferritin containing stainable iron. We conclude that most of the iron in fibroblast HMF from both affected and unaffected cells is ferrihydrite but only FRDA affected cells mineralize significant iron in mitochondrial ferritin.

Glutamine is highly effective in preventingin vivocobalt-induced oxidative stress in rat liver

To evaluate the in vivo effect of glutamine on cobalt-generated oxidative stress and (HO-1) induction in rat liver. Fasted female Wistar rats received a single injection of cobalt chloride (375 micromol/kg body weight) and then were killed at different times. Lipid peroxidation and soluble and enzymatic antioxidant defense system (reduced glutathione (GSH), catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD)) were measured in liver homogenates. Ferritin and ferritin iron contents as well as heme oxygenase-1 (HO-1) activity and expression were also determined. The antioxidant properties of glutamine (Gln) were also evaluated. Cobalt chloride increased lipid peroxidation (50% over control values) 1 h after treatment. GSH reached a minimum at 3 h (40%) increasing thereafter. Twelve hours after CoCl2 injection, the antioxidant enzymes CAT, GSH-Px and SOD also diminished by about 30%. Heme oxygenase-1 induction was observed 6 h after treatment reaching a maximum value of 14-fold over the controls, 12 h after cobalt treatment. A 1.7-fold increase in ferritin and ferritin-bound iron 24 h after treatment were also obtained. Administration of glutamine (300 mg/kg body weight) by gavage 24 h before CoCl2 treatment entirely prevented the increase in thiobarbituric acid reactive substances (TBARS) content, the decrease in GSH levels, and partially reverted heme oxygenase-1 induction. These results suggested that a natural product such as glutamine prevents glutathione depletion and consequently heme oxygenase induction.

Oxidative stress and haemodialysis: role of inflammation and duration of dialysis treatment.

Oxidative stress has long been demonstrated in haemodialysis patients. However, the factors influencing their oxidative status have not been characterized extensively in these patients. Therefore, the present study was designed to investigate the influence of a large number of factors known to be associated with oxidative stress. In the present cross-sectional study, we determined the plasma levels of lipid and protein oxidation markers in 31 non-smoking haemodialysis patients and 18 non-smoking healthy subjects, together with various components of the antioxidant system at the plasma and erythrocyte level. No influence of age, diabetes or iron overload on oxidative markers and plasma and erythrocyte antioxidant systems was detected in these haemodialysis patients. The lack of an association between iron overload and oxidative status may be related to the lower level of plasma ascorbate in haemodialysis patients, since ascorbate favours the generation of free iron from ferritin-bound iron. Interestingly, plasma C reactive protein (CRP) levels measured by highly sensitive CRP assay were correlated positively with plasma levels of thiobarbituric acid reactive substances (r=0.38, P<0.04) and negatively with plasma alpha-tocopherol levels (r=-0.46, P<0.01). Moreover, significant inverse correlations were observed between duration of dialysis treatment and plasma levels of alpha-tocopherol (r=-0.49, P<0.02) and ubiquinol (r=-0.40, P<0.05). Our results suggest that inflammatory status and duration of dialysis treatment are the most important factors relating to oxidative stress in haemodialysis patients.