Multi-omic analysis of the effects of low frequency ventilation during cardiopulmonary bypass surgery.

BACKGROUND: Heart surgery with cardio-pulmonary bypass (CPB) is associated with lung ischemia leading to injury and inflammation. It has been suggested this is a result of the lungs being kept deflated throughout the duration of CPB. Low frequency ventilation (LFV) during CPB has been proposed to reduce lung dysfunction. METHODS: We used a semi-biased multi-omic approach to analyse lung biopsies taken before and after CPB from 37 patients undergoing coronary artery bypass surgery randomised to both lungs left collapsed or using LFV for the duration of CPB. We also examined inflammatory and oxidative stress markers from blood samples from the same patients. RESULTS: 30 genes were induced when the lungs were left collapsed and 80 by LFV. Post-surgery 26 genes were significantly higher in the LFV vs. lungs left collapsed, including genes associated with inflammation (e.g. IL6 and IL8) and hypoxia/ischemia (e.g. HIF1A, IER3 and FOS). Relatively few changes in protein levels were detected, perhaps reflecting the early time point or the importance of post-translational modifications. However, pathway analysis of proteomic data indicated that LFV was associated with increased "cellular component morphogenesis" and a decrease in "blood circulation". Lipidomic analysis did not identify any lipids significantly altered by either intervention. DISCUSSION: Taken together these data indicate the keeping both lungs collapsed during CPB significantly induces lung damage, oxidative stress and inflammation. LFV during CPB increases these deleterious effects, potentially through prolonged surgery time, further decreasing blood flow to the lungs and enhancing hypoxia/ischemia.

Pro-oxidant iron in exhaled breath condensate: a potential excretory mechanism.

AIMS: Pro-oxidant iron provides a potential measure of iron-catalysed oxidative stress in biological fluids. This study aimed, to investigate if the Bleomycin technique for measurement of pro-oxidant iron in biological fluids could be utilised for determinations in exhaled breath condensate (EBC). Secondly, to measure levels of pro-oxidant iron in EBC from asthmatics after exposure to polluting city environments. METHODS: Retrospective analysis of samples of EBC and bronchoalveolar lavage fluid (BALF). Pro-oxidant iron levels were determined by the Bleomycin method. Transferrin levels were determined by radial diffusion immunoassay and lactoferrin by ELISA. SUBJECTS: Patients undergoing surgery necessitating cardiopulmonary bypass, normal healthy controls, "healthy" smokers, and asthmatics (mild and moderate). RESULTS: Pro-oxidant iron was significantly decreased (p<0.05) post cardiac surgery in both EBC and BALF. In smokers levels of pro-oxidant iron in EBC were significantly (p<0.05) increased verses healthy controls. In asthmatics with more severe disease, there were significant increases in EBC pro-oxidant iron content post exposure to city environments (p<0.001), with levels most elevated after exposure to the most polluted setting. CONCLUSION: Similar patterns in the levels of pro-oxidant iron detectable in EBC and paired BALF from patients undergoing cardiopulmonary bypass (pre and post surgery) suggest a potential for EBC determinations. Significantly elevated levels in EBC from smokers relative to control subjects provide further support for this technique. In asthma disease severity and environmental exposure influenced levels of pro-oxidant iron measured in EBC indicating a potential for enhanced iron-catalysed oxidative stress.

KL-6 levels are elevated in plasma from patients with acute respiratory distress syndrome.

The acute respiratory distress syndrome (ARDS) is an extreme form of lung injury characterised by disruption to the alveolar epithelium. KL-6 is a mucin-like glycoprotein expressed on type II pneumocytes. Circulating levels of KL-6 have diagnostic and prognostic significance in a number of interstitial lung diseases, and when elevated are thought to indicate disruption of the alveolar epithelial lining. In this study, the authors sought to determine whether plasma KL-6 levels were elevated in patients with ARDS and whether these were associated with aetiology, disease severity, outcome or ventilatory strategy. Plasma samples were collected from 28 patients with ARDS, nine ventilated controls of matched illness severity and 10 healthy individuals. KL-6 concentrations were measured by enzyme-linked immunosorbent assay. Patients with ARDS had higher plasma levels of KL-6 (median 537 U x mL(-1), interquartile range (IQR) 383-1,119), as compared to ventilated controls (median 255 U x mL(-1), IQR 83-338) and normal individuals (median 215 U x mL(-1), IQR 149-307). In patients with ARDS, plasma KL-6 levels were higher in nonsurvivors than survivors, and correlated positively with oxygenation index and negatively with arterial oxygen tension:inspiratory oxygen fraction ratio. There were also significant positive correlations with mean and peak airway pressures. Elevated levels of plasma KL-6 may provide a useful marker for acute respiratory distress syndrome in ventilated patients and have possible prognostic significance. Alveolar epithelial cell damage may be influenced by the nature of mechanical ventilatory support.

Variable tissue expression of transferrin receptors: relevance to acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is associated with altered plasma and lung iron chemistry. Iron can promote microbial virulence and catalyse pro-oxidant reactions, thereby contributing to the oxidative stress that characterises the syndrome. Therefore, the expression of ferritin and transferrin receptors (TfR) were sought in the lungs and hearts of rodents treated with lipopolysaccharide (LPS), and measurements of TfR and ferritin protein expression were taken from lung biopsy specimens from patients with ARDS and appropriate controls. TfR messenger ribonucleic acid (mRNA) was significantly upregulated in the lungs and significantly downregulated in the hearts of rats 4 h after LPS. Ferritin mRNA levels (light and heavy chains) remained unaltered. Protein TfR levels were significantly upregulated in lungs and downregulated in hearts 4 h post-LPS. Ferritin protein levels were significantly downregulated in lungs compared to baseline values but were unaltered in hearts. Nonhaem iron levels were increased in lungs and decreased in hearts, and iron-regulatory-protein activity increased in hearts but not lungs. TfR protein levels were significantly increased in lung biopsies from patients with ARDS compared to controls. Transferrin receptors are upregulated in rodent lungs during inflammation but are downregulated in the heart. Transferrin receptor protein levels were significantly increased in the lungs in clinical acute respiratory distress syndrome. These findings have implications for the pathogenesis of acute respiratory distress syndrome, especially in relation to the role of iron as a mediator of oxidative stress.

Risk of iron overload is decreased in beating heart coronary artery surgery compared to conventional bypass.

Conventional cardiopulmonary bypass surgery (CCPB) increases the iron loading of plasma transferrin often to a state of plasma iron overload, with the presence of low molecular mass iron. Such iron is a potential risk factor for oxidative stress and microbial virulence. Here we assess 'off-pump' coronary artery surgery on the beating heart for changes in plasma iron chemistry. Seventeen patients undergoing cardiac surgery using the 'Octopus' myocardial wall stabilisation device were monitored at five time points for changes in plasma iron chemistry. This group was further divided into those (n=9) who had one- or two- (n=8) vessel grafts, and compared with eight patients undergoing conventional coronary artery surgery. Patients undergoing beating heart surgery had significantly lower levels of total plasma non-haem iron, and a decreased percentage saturation of their transferrin at all time points compared to conventional bypass patients. Plasma iron overload occurred in only one patient undergoing CCPB. Beating heart surgery appears to decrease red blood cell haemolysis, and tissue damage during the operative procedures and thereby significantly decreases the risk of plasma iron overload associated with conventional bypass.

Plasma membrane estrogen receptors are coupled to endothelial nitric-oxide synthase through Galpha(i).

Estrogen causes rapid endothelial nitric oxide (NO) production because of the activation of plasma membrane-associated estrogen receptors (ER) coupled to endothelial NO synthase (eNOS). In the present study, we determined the role of G proteins in eNOS activation by estrogen. Estradiol-17beta (E(2), 10(-8) m) and acetylcholine (10(-5) m) caused comparable increases in NOS activity (15 min) in intact endothelial cells that were fully blocked by pertussis toxin (Ptox). In addition, exogenous guanosine 5'-O-(2- thiodiphosphate) inhibited E(2)-mediated eNOS stimulation in isolated endothelial plasma membranes, and Ptox prevented enzyme activation by E(2) in COS-7 cells expressing ERalpha and eNOS. Coimmunoprecipitation studies of plasma membranes from COS-7 cells transfected with ERalpha and specific Galpha proteins demonstrated E(2)-stimulated interaction between ERalpha and Galpha(i) but not between ERalpha and either Galpha(q) or Galpha(s); the observed ERalpha-Galpha(i) interaction was blocked by the ER antagonist ICI 182,780 and by Ptox. E(2)-stimulated ERalpha-Galpha(i) interaction was also demonstrable in endothelial cell plasma membranes. Cotransfection of Galpha(i) into COS-7 cells expressing ERalpha and eNOS yielded a 3-fold increase in E(2)-mediated eNOS stimulation, whereas cotransfection with a protein regulator of G protein signaling, RGS4, inhibited the E(2) response. These findings indicate that eNOS stimulation by E(2) requires plasma membrane ERalpha coupling to Galpha(i) and that activated Galpha(i) mediates the requisite downstream signaling events. Thus, novel G protein coupling enables a subpopulation of ERalpha to initiate signal transduction at the cell surface. Similar mechanisms may underly the nongenomic actions of other steroid hormones.

Antioxidant binding of caeruloplasmin to myeloperoxidase: myeloperoxidase is inhibited, but oxidase, peroxidase and immunoreactive properties of caeruloplasmin remain intact.

The neutrophil enzyme myeloperoxidase (MPO) purposefully makes hypochlorous acid (HOCl) as part of the cells defence against microbial infections. During cell lysis, however, MPO will be released into the extracellular environment where production of HOCl, a powerful oxidant, will lead to molecular damage. Extracellular MPO binds to the copper-containing protein caeruloplasmin (Cp) and prevents MPO making HOCl. Cp has several important antioxidant functions in extracellular fluids associated with its ability to catalyse oxidation of ferrous ions and to remove peroxides. The binding of MPO to Cp did not inhibit these important extracellular antioxidant activities of Cp, but in so doing it provided additional antioxidant protection against formation of HOCl.

Acute respiratory distress syndrome secondary to cardiopulmonary bypass: do compromised plasma iron-binding anti-oxidant protection and thiol levels influence outcome?

OBJECTIVES: Cardiopulmonary bypass (CPB) surgery is often associated with mild lung injury and in some patients leads to acute lung injury and acute respiratory distress syndrome (ARDS). Aberrant plasma iron chemistry (increased iron loading of transferrin and/or the presence of redox-active low molecular mass iron) and increased plasma thiol levels are features of this type of surgery and represent a potential pro-oxidant risk for oxidative damage. Oxidative damage is a feature of ARDS, and we hypothesized that pro-oxidant forces may contribute to the onset and progression of ARDS. DESIGN: Prospective, single center, observational study. SETTING: University-affiliated tertiary referral cardiothoracic center. PATIENTS: A total of 19 patients with ARDS secondary to CPB surgery and 64 patients with ARDS secondary to a variety of other predisposing causes. INTERVENTIONS: Supportive techniques appropriate to the treatment of ARDS. MEASUREMENTS AND MAIN RESULTS: Blood samples were collected into lithium heparin tubes for all patient groups on the first day of the admission of patients to the intensive care unit immediately after the diagnosis of ARDS. Plasma was immediately assayed for thiol content and total protein and albumin levels. Plasma from patients with ARDS secondary to CPB surgery was also assayed for changes in iron chemistry. Nonsurviving patients with ARDS secondary to CPB surgery displayed significantly greater levels of aberrant iron chemistry (elevated levels of iron saturation of transferrin) with decreased iron-binding antioxidant protection and elevated plasma thiol levels than did survivors. Plasma thiol levels in patients with ARDS secondary to other predisposing causes were (with the exception of lung-surgery patients) significantly elevated in survivors compared with those in nonsurvivors of the syndrome. CONCLUSIONS: Increased levels of plasma thiol appear to be associated with mortality in patients with ARDS secondary to CPB surgery.

Iron overload in paediatrics undergoing cardiopulmonary bypass.

Pathological changes in iron status are known to occur during bypass and will be superimposed upon physiological abnormalities in iron distribution, characteristic of the neonatal period. We have sought to define the severity of iron overload in these patients. Plasma samples from 65 paediatric patients undergoing cardiopulmonary bypass (CPB) were analysed for non-haem iron, total iron binding capacity, transferrin and bleomycin-detectable iron. Patients were divided into four age groups for analysis. Within each age group, patients who were in iron overload at any time point were statistically compared to those who were not. The most significant changes in iron chemistry were seen in the plasma of neonates, with 25% in a state of plasma iron overload. 18.5% of infants and 14.3% of children at 1-5 years were also in iron overload at some time point during CPB. No children over 5 years, however, went into iron overload. Increased iron saturation of transferrin eliminates its ability to bind reactive forms of iron and to act as an antioxidant. When transferrin is fully saturated with iron, reactive forms of iron are present in the plasma which can stimulate iron-driven oxidative reactions. Our data suggest that paediatric patients are at greater risk of iron overload during CPB, and that some form of iron chelation therapy may be advantageous to decrease oxidative stress.

Haem oxygenase shows pro-oxidant activity in microsomal and cellular systems: implications for the release of low-molecular-mass iron.

Haem oxygenase-1 (HO-1) is a highly inducible stress protein that removes haem from cells with the release of biliverdin, carbon monoxide and low-molecular-mass iron (LMrFe). Several antioxidant functions have been ascribed to HO; its induction is considered to be a protective event. However, LMrFe produced during haem catabolism might elicit a pro-oxidant response, with deleterious consequences. We therefore investigated the delicate balance between pro-oxidant and antioxidant events with the use of a microsomal lipid peroxidation (LPO) system. By using microsomal-bound HO in an NADPH-dependent LPO system, we assessed the pro-oxidant nature of the released LMrFe and the antioxidant effect of the released bilirubin. Hb, a biologically relevant substrate for HO, was included with the microsomes to supplement the source of haem iron and to promote LPO. We found significant increases in microsomal LPO, by using the thiobarbituric acid (TBA) test, after incubation with Hb. This Hb-stimulated peroxidation was inhibited by HO inhibitors and by iron chelators, suggesting a HO-driven, iron-dependent mechanism. GLC-MS was employed to measure the specific LPO product 4-hydroxy-2-nonenal and to confirm our TBA test results. A HO inhibitor attenuated an increase in intracellular LMrFe that occurred after treatment of rat pulmonary artery smooth-muscle cells with Hb. Additionally, exogenously added bilirubin at an equimolar concentration to the LMrFe present in both microsomal and liposomal systems was unable to prevent the pro-oxidant effect of the iron. Under certain circumstances HO can act as a pro-oxidant and seems to have a role in stimulating microsomal LPO.

Acute right ventricular restrictive physiology after repair of tetralogy of Fallot: association with myocardial injury and oxidative stress.

BACKGROUND: Acute right ventricular (RV) restrictive physiology after tetralogy of Fallot repair results in low cardiac output and a prolonged stay in the intensive care unit (ICU). However, its mechanism remains uncertain. METHODS AND RESULTS: In the first 24 hours after tetralogy of Fallot repair (n=11 patients), serial prospective measurements were performed of cardiac troponin T, indexes of NO production (NO(2)(-) and NO(3)(-) combined as NOx), and iron metabolism and antioxidants. RV diastolic function was assessed by transthoracic Doppler echocardiography. Patients who had a long stay in the ICU were characterized by restrictive RV physiology (nonrestrictive group [n=7]: 3.0+/-0.6 days [mean+/-SD]; restrictive group [n=4]: 10.7+/-3.1 days). Troponin T peak concentration and the area under its concentration-time curve (AUC) were higher in the restrictive RV group (peak: restrictive group 17. 0+/-2.8 microg/L, nonrestrictive group 10.4+/-4.6 microg/L, P<0.03; AUC: restrictive group 268.8+/-73.6 microg. h(-1). L(-1), nonrestrictive group 136.2+/-48.3 microg. h(-1). L(-1), P<0.03). Plasma NOx/creatinine concentrations were higher in the restrictive group than the nonrestrictive group at 2 hours after bypass (restrictive group 1.3+/-0.4, nonrestrictive group 0.8+/-0.2; P=0. 04) but were similar by 24 hours. Iron loading peaked 2 to 10 hours after bypass and was more severe in the restrictive group (peak transferrin saturation: restrictive group 83.9+/-13.0%, nonrestrictive group 58.3+/-16.2%, P=0.05; minimum total iron-binding capacity: restrictive group 0.59+/-0.21%, nonrestrictive group 0.76+/-0.06%, P=0.04; minimum iron-binding antioxidant activity to oxyorganic radicals: restrictive group 9. 5+/-22.4%, nonrestrictive group 50.6+/-11.4%, P=0.01). CONCLUSIONS: After tetralogy of Fallot repair, acute restrictive RV physiology is associated with greater intraoperative myocardial injury and postoperative oxidative stress with severe iron loading of transferrin.

Antioxidant protection against iron toxicity: plasma changes during cardiopulmonary bypass in neonates, infants, and children.

Cardiopulmonary bypass surgery is associated with the release of low molecular mass iron, which increases the saturation of plasma transferrin to over 50% in all adult patients treated. In a significant minority, however plasma transferrin becomes 100% iron saturated and non-transferrin bound iron can be detected in the plasma. An iron-saturated transferrin is also a common physiological finding in normal term and pre-term infants at a time when their plasma antioxidants, which protect against iron toxicity and radical scavenging, are profoundly different from those seen in adults. This study was conducted to assess the extent to which antioxidants, which protect against iron toxicity, are altered in neonates, infants, and children undergoing cardiopulmonary bypass surgery.

Alternate coupling of receptors to Gs and Gi in pancreatic and submandibular gland cells.

Many Gs-coupled receptors can activate both cAMP and Ca2+ signaling pathways. Three mechanisms for dual activation have been proposed. One is receptor coupling to both Gs and G15 (a Gq class heterotrimeric G protein) to initiate independent signaling cascades that elevate intracellular levels of cAMP and Ca+2, respectively. The other two mechanisms involve cAMP-dependent protein kinase-mediated activation of phospholipase Cbeta either directly or by switching receptor coupling from Gs to Gi. These mechanisms were primarily inferred from studies with transfected cell lines. In native cells we found that two Gs-coupled receptors (the vasoactive intestinal peptide and beta-adrenergic receptors) in pancreatic acinar and submandibular gland duct cells, respectively, evoke a Ca2+ signal by a mechanism involving both Gs and Gi. This inference was based on the inhibitory action of antibodies specific for Galphas, Galphai, and phosphatidylinositol 4,5-bisphosphate, pertussis toxin, RGS4, a fragment of beta-adrenergic receptor kinase and inhibitors of cAMP-dependent protein kinase. By contrast, Ca2+ signaling evoked by Gs-coupled receptor agonists was not blocked by Gq class-specific antibodies and was unaffected in Galpha15 -/- knockout mice. We conclude that sequential activation of Gs and Gi, mediated by cAMP-dependent protein kinase, may represent a general mechanism in native cells for dual stimulation of signaling pathways by Gs-coupled receptors.

Plasma hypoxanthine levels during crystalloid and blood cardioplegias: warm blood cardioplegia increases hypoxanthine levels with a greater risk of oxidative stress.

BACKGROUND: PATIENTS undergoing cardiopulmonary bypass (CPB) are subjected to severe oxidative stress, and frequently show evidence of acute lung injury post surgery. Associations between acute lung injury, oxidative stress, and aberrant ATP catabolism have been made and prompted us to consider whether the purine metabolites xanthine and hypoxanthine alter significantly during CPB when different types of cardioplegia are used. EXPERIMENTAL DESIGN: retrospective follow up study on stored plasma samples from patients randomly selected to receive either warm blood, cold blood, or crystalloid cardioplegia. SETTING: adult intensive care unit of post graduate teaching hospital. PATIENTS: thirty-eight patients undergoing aortic valve replacement, with or without artery grafting. Operation was carried out by a single surgeon. INTERVENTIONS: all patients received either a homograft aortic valve or a stentless porcine valve. RESULTS: No significant differences in xanthine levels at any time points during CPB, or between the different cardioplegic groups. Hypoxanthine levels were, however, significantly higher in patients receiving warm blood cardioplegia (74.84+/-16.715 microM, p=0.0151), and was most marked at time point 3 when the aortic cross clamp was released. PATIENTS receiving crystalloid cardioplegia showed higher levels of hypoxanthine (44.56+/-10.16 microM) than those receiving cold blood cardioplegia (21.57+/-7.106 microM). CONCLUSIONS: Considering these data together, it suggests that aberrant ATP catabolism, characteristic of ischaemia/reperfusion, is further disturbed during warm blood cardioplegia leading to a marked increase in plasma hypoxanthine levels. This has the potential to further increase oxidative stress during CPB.

Iron overload upregulates haem oxygenase 1 in the lung more rapidly than in other tissues.

Haem oxygenase-1 is upregulated by numerous insults, including oxidative stress, and under such circumstances it is considered to be a protective stratagem. We have measured the haem oxygenase-1 expression in heart, lung and liver tissues of control and iron-overloaded rats. Lung tissue from iron-overloaded rats displayed a significant increase in the haem oxygenase-1 protein but no changes in haem oxygenase-1 mRNA. Conversely, heart tissue showed a significant increase in haem oxygenase-1 mRNA but no changes in haem oxygenase-1 protein. We conclude that during oxidative stress caused by iron overload, lung tissue responds with a rapid upregulation of haem oxygenase-1 levels.

Persistent membrane association of activated and depalmitoylated G protein alpha subunits.

Heterotrimeric signal-transducing G proteins are organized at the inner surface of the plasma membrane, where they are positioned to interact with membrane-spanning receptors and appropriate effectors. G proteins are activated when they bind GTP and inactivated when they hydrolyze the nucleotide to GDP. However, the topological fate of activated G protein alpha subunits is disputed. One model declares that depalmitoylation of alpha, which accompanies activation by a receptor, promotes release of the protein into the cytoplasm. Our data suggest that activation of G protein alpha subunits causes them to concentrate in subdomains of the plasma membrane but not to be released from the membrane. Furthermore, alpha subunits remained bound to the membrane when they were activated with guanosine 5'-(3-O-thio)triphosphate and depalmitoylated with an acyl protein thioesterase. Limitation of alpha subunits to the plasma membrane obviously restricts their mobility and may contribute to the efficiency and specificity of signaling.

Hydrogen peroxide and catalase are inversely related in adult patients undergoing cardiopulmonary bypass: implications for antioxidant protection.

Adult patients undergoing cardiopulmonary bypass (CPB) surgery are subjected to increased oxidative stress and show a spectrum of lung injury. Increased levels of hydrogen peroxide (H2O2) are often seen during episodes of oxidative stress, such as the use of high FiO2s, and this molecule plays a key role in the formation of highly damaging oxidants such as the hydroxyl radical. Oxidative damage to plasma proteins was assessed by measuring free thiol groups, and antioxidant protection against H2O2 by measuring catalase activity. CPB patients (n = 39) receiving either 100% or 50% oxygen at the end of bypass were studied by measuring levels of H2O2 in breath condensate and levels of catalase in their plasma, and comparing these to pre-bypass levels. Post-bypass, all CPB patients exhaled significantly lower levels of H2O2 (P < 0.0001) at a time when they had significantly increased activity (0.809 +/- 0.11 versus 1.688 +/- 0.18 U/mg protein) of catalase in their plasma. There were no significant differences in these parameters between the 100% and 50% oxygen groups. At a time when oxidative stress is greatest, there appears to be a corresponding plasma increase in the antioxidant catalase. Whether this change is fortuitous or a response to oxidative stress is at present under consideration.

Administration of albumin to patients with sepsis syndrome: a possible beneficial role in plasma thiol repletion.

1. Albumin is often administered intravenously to critically ill patients as a volume expander, to combat hypoalbuminaemia, and to decrease hyperbilirubinaemia. There is, however, an ongoing debate concerning the therapeutic benefit of the former which is an expensive form of treatment.2. Albumin has several biological functions, in particular as a ligand binder. It also acts as an extracellular transition metal ion-binding and radical-scavenging antioxidant. These functions are influenced by the presence of an exposed thiol group (cys 34) on the surface of the albumin molecule. 3. The ability of infused albumin to influence the plasma thiol pool, and hence antioxidant potential, was investigated in patients with sepsis syndrome.4. Plasma thiol levels rose rapidly after albumin infusion and remained elevated even after plasma albumin levels had declined significantly, due to interstitial leakage. Data are suggestive of some form of thiol exchange in the plasma of these patients between albumin and molecules containing oxidized thiol groups.5. Administration of albumin to patients with sepsis syndrome leads to a sustained increase in plasma thiols. Thiols have several important antioxidant functions, and thiol repletion in these patients, who are known to suffer from oxidative stress, may have beneficial antioxidant effects. Antioxidant repletion may represent an important facet of clinically administered albumin.

Iron binding and autoreduction by citrate: are these involved in signalling by iron regulatory protein-1?

Ferric ions bind to citrate and undergo an autoreduction to form a ferrous-citrate complex, greatly increasing the redox activity of the iron complex. Ferrous ions and citrate are also essential for the enzymic activity of aconitase. Aconitase, with its iron-sulphur cluster has a versatile structure which allows it to act as an iron regulatory protein (IRP-1). The purpose of this study was to see whether iron binding, and its autoreduction by citrate, could play a physiological signalling role in iron regulation. Significant amounts of ferrous ions were associated with citrate, when measured using ferrozine, however, these did not appear to activate iron-requiring aconitase.

Reactive iron species in biological fluids activate the iron-sulphur cluster of aconitase.

Low molecular mass iron (LMrFe) can appear in plasma when the transferrin becomes fully iron loaded. Such iron poses a risk factor for oxidative damage, and for microbial virulence. A previous novel approach to the detection and measurement of LMrFe in plasma was the use of the iron-binding properties of the glycopeptide antitumour antibiotic bleomycin and its ability to degrade DNA in the presence of oxygen, bound iron, and an iron reducing agent. Since bleomycin is a non-physiological ligand with iron-binding and redox cycling properties, it has been suggested that it may not be a valid biological model for detecting and measuring LMrFe. To address these concerns we have developed a biological approach to the detection and measurement of LMrFe based on the activation of iron-requiring aconitase. Parallel measurements, in a variety of clinical conditions in which there was a complete saturation of the plasma transferrin, showed that the bleomycin assay and the aconitase assay can give similar results for LMrFe.