Sharon Mumby

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 537u2005U·mL−1, interquartile range (IQR) 383–1,119), as compared to ventilated controls (median 255u2005U·mL−1, IQR 83–338) and normal individuals (median 215u2005U·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.

Variation in Iron Homeostasis Genes Between Patients With ARDS and Healthy Control Subjects

BACKGROUNDnAbnormal plasma and lung iron mobilization is associated with the onset and progression of ARDS and is detectable in specific at-risk populations. Patients with ARDS also have pronounced oxidative and nitrosative stress that can be catalyzed and thereby aggravated by the bioavailability of redox active iron. ARDS of pulmonary and extrapulmonary origin may differ pathophysiologically and require different ventilatory strategies. Evidence suggests that genetic predisposition is relevant to the pathogenesis of ARDS. We therefore explored the hypothesis that polymorphisms from a panel of genes encoding iron-metabolizing proteins determine susceptibility to ARDS.nnnMETHODSnRetrospective case-control study conducted at the adult ICUs of two university hospitals. Patients with ARDS (n = 122) and healthy control subjects (n = 193) were genotyped. Sequence-specific primer polymerase chain reaction was used to genotype selected biallelic single-nucleotide polymorphisms. An audit of the patient database was conducted, and 104 of the 122 ARDS patients were eligible for the final data analysis.nnnRESULTSnPreliminary analysis indicated differences between ARDS and healthy control subjects in the incidence of polymorphism of the gene encoding ferritin light chain. Subgroup analysis indicated the prevalence of ferritin light-chain gene -3381GG homozygotes was increased in patients with ARDS of extrapulmonary origin compared to healthy control subjects. Secondly, a common haplotype in the heme oxygenase 2 gene was reduced in patients with ARDS compared to healthy control subjects and was more evident in those with ARDS of direct or pulmonary etiology.nnnCONCLUSIONSnThese results provide preliminary evidence to suggest a distinction in the genetic background of the subpopulations studied, inferring that the ferritin light-chain gene genotype confers susceptibility to ARDS, while the heme oxygenase 2 haplotype is protective against the onset of the syndrome. Such data support further previous findings that suggest abnormalities in iron handling resulting in redox imbalance are implicated in the pathogenesis of ARDS.

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.

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 4u2005h after LPS. Ferritin mRNA levels (light and heavy chains) remained unaltered. Protein TfR levels were significantly upregulated in lungs and downregulated in hearts 4u2005h 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.

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.

Is bleomycin-detectable iron present in the plasma of patients with septic shock?

Objective: To assess plasma iron status in critically ill patients with septic shock. Design: Observational, prospective study. Setting: Adult intensive care units in teaching and tertiary referral hospitals. Patients and participants: Fifteen adult patients with established septic shock. Normal control subjects (n = 10) were also investigated. Data from patients and controls were compared with previously published iron values in critical care patients. Measurements and results: The indices investigated and correlated with clinical scores of illness severity included bleomycin-detectable iron, non-haem iron; transferrin and its percentage iron saturation, and the iron binding (anti-oxidant) activity of transferrin. Bleomycin-detectable iron was not present in the plasma of patients with septic shock whilst the plasma transferrin remained unsaturated with iron. One patient in multi-organ failure displayed bleomycin-detectable iron in plasma (1.16 μmol/l) and had 100 % iron-saturation of transferrin. The plasma non-haem iron levels (7.84 ± 1.82 μmol/l) were the lowest of all critical care patient groups studied by us. The plasma transferrin levels were also low but resulted in a near normal percentage saturation of transferrin with iron (34.6 ± 6.5 %). The scores of clinical severity correlated with changes in plasma iron chemistry. Conclusions: Patients with septic shock rarely have iron saturated transferrin in their plasma leading to the presence of bleomycin-detectable iron.

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.

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.

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.