Detection of a ferrylhemoglobin intermediate in an endothelial cell model after hypoxia-reoxygenation.
(9/606)
A cell culture model of bovine aortic endothelial cells attached to microcarrier beads was used to study the interaction of diaspirin cross-linked hemoglobin (an oxygen-carrying blood substitute) with hypoxia-reoxygenation. Hemoglobin (200 microM) and hypoxia-volume restriction (3-5 h), together and separately, caused toxicity in this model, as measured by decreased cellular replating efficiency. Hemoglobin (60 microM) caused a reduction in hydrogen peroxide concentration and an increase in lipid peroxidation above that induced by hypoxia alone. Incubation of hemoglobin with endothelial cells caused transient oxidation of hemoglobin to its highly reactive and toxic ferryl species after >/=3 h of hypoxia, followed by 1 h of reoxygenation. Lipid peroxidation, which may occur in the presence of ferrylhemoglobin, also occurred after 1 h of reoxygenation. Hemoglobin caused a dose-dependent decrease in intracellular glutathione concentration, suggesting that it caused an oxidative stress to the cells. However, addition of ascorbate, alpha-tocopherol, or trolox did not decrease hemoglobin oxidation in the presence of normal or hypoxic cells. It is concluded that diaspirin cross-linked hemoglobin forms a ferryl intermediate in the absence of any exogenously added oxidant and contributes to the oxidative burden experienced by endothelial cells after hypoxia-reoxygenation, a condition that is likely to be encountered during trauma and surgery when hemoglobin solutions are used as perfusion agents. (+info)
Effects of S-nitrosation on oxygen binding by normal and sickle cell hemoglobin.
(10/606)
S-Nitrosated hemoglobin (SNO-Hb) is of interest because of the allosteric control of NO delivery from SNO-Hb made possible by the conformational differences between the R- and T-states of Hb. To better understand SNO-Hb, the oxygen binding properties of S-nitrosated forms of normal and sickle cell Hb were investigated. Spectral assays and electrospray ionization mass spectrometry were used to quantify the degree of S-nitrosation. Hb A(0) and unpolymerized Hb S exhibit similar shifts toward their R-state conformations in response to S-nitrosation, with increased oxygen affinity and decreased cooperativity. Responses to 2, 3-diphosphoglycerate were unaltered, indicating regional changes in the deoxy structure of SNO-Hb that accommodate NO adduction. A cycle of deoxygenation/reoxygenation does not cause loss of NO or appreciable heme oxidation. There is, however, appreciable loss of NO and heme oxidation when oxygen-binding experiments are carried out in the presence of glutathione. These results indicate that the in vivo stability of SNO-Hb and its associated vasoactivity depend on the abundance of thiols and other factors that influence transnitrosation reactions. The increased oxygen affinity and R-state character that result from S-nitrosation of Hb S would be expected to decrease its polymerization and thereby lessen the associated symptoms of sickle cell disease. (+info)
Longitudinal study of Plasmodium falciparum infection and immune responses in infants with or without the sickle cell trait.
(11/606)
BACKGROUND: Individuals may be homozygous (SS) or heterozygous (AS) sickle cell gene carriers or have normal adult haemoglobin (AA). Haemoglobin S could have a protective role against malaria but evidence is sparse and the operating mechanisms are poorly known. METHODS: We followed two cohorts of children. The first was enrolled at birth (156 newborn babies) and the second at 24-36 months old (84 children). Both cohorts were followed for 30 months; monthly for parasitological data and half yearly for immunological data. RESULTS: In the first cohort, 22%, and in the second 13% of children were AS. Whatever their age parasite prevalence rates were similar in AA and AS individuals. Mean parasite densities increased less rapidly with age in AS than in AA children, and were significantly lower in AS than in AA children >48 months old. The AA children tended to be more often admitted to hospital than AS children (22% versus 11%, NS). Both anti-Plasmodium falciparum and anti-Pfl55/RESA antibody rates increased more rapidly in AA than in AS children. Conversely, the prevalence rate of cellular responders to the Pfl55/RESA antigen was similar in AA and AS children during the first 2 years of life, then it was higher in AS than in AA children. CONCLUSIONS: Sickle cell trait related antimalarial protection varies with age. The role of the modifications of the specific immune response to P. falciparum in explaining the protection of AS children against malaria is discussed. (+info)
Inhaled nitric oxide augments nitric oxide transport on sickle cell hemoglobin without affecting oxygen affinity.
(12/606)
Nitric oxide (NO) inhalation has been reported to increase the oxygen affinity of sickle cell erythrocytes. Also, proposed allosteric mechanisms for hemoglobin, based on S-nitrosation of beta-chain cysteine 93, raise the possibility of altering the pathophysiology of sickle cell disease by inhibiting polymerization or by increasing NO delivery to the tissue. We studied the effects of a 2-hour treatment, using varying concentrations of inhaled NO. Oxygen affinity, as measured by P(50), did not respond to inhaled NO, either in controls or in individuals with sickle cell disease. At baseline, the arterial and venous levels of nitrosylated hemoglobin were not significantly different, but NO inhalation led to a dose-dependent increase in mean nitrosylated hemoglobin, and at the highest dosage, a significant arterial-venous difference emerged. The levels of nitrosylated hemoglobin are too low to affect overall hemoglobin oxygen affinity, but augmented NO transport to the microvasculature seems a promising strategy for improving microvascular perfusion. (+info)
UV resonance raman spectra of ligand binding intermediates of sol-gel encapsulated hemoglobin.
(13/606)
We report for the first time specific conformational changes for a homogeneous population of ligand-bound adult deoxy human hemoglobin A (HbA) generated by introducing CO into a sample of deoxy-HbA with the effector, inositol hexaphosphate, encapsulated in a porous sol-gel. The preparation of ligand-bound deoxy-HbA results from the speed of ligand diffusion relative to globin conformational dynamics within the sol-gel (1). The ultraviolet resonance Raman (UVRR) difference spectra obtained reveal that E helix motion is initiated upon ligand binding, as signaled by the appearance of an alpha14beta15 Trp W3 band difference at 1559 cm(-1). The subsequent appearance of Tyr (Y8a and Y9a) and W3 (1549 cm(-1)) UVRR difference bands suggest conformational shifts for the penultimate Tyralpha140 on the F helix, the "switch" region Tyralpha42, and the "hinge" region Trpbeta37. The UVRR results expose a sequence of conformational steps leading up to the ligation-induced T to R quaternary structure transition as opposed to a single, concerted switch. More generally, this report demonstrates that sol-gel encapsulation of proteins can be used to study a sequence of specific conformational events triggered by substrate binding because the traditional limitation of substrate diffusion times is overcome. (+info)
Particulate air pollution and the blood.
(14/606)
BACKGROUND: Particulate air pollution has been associated with excess deaths from, and increases in hospital admissions for, cardiovascular disease among older people. A study was undertaken to determine whether this may be a consequence of alterations in the blood, secondary to pulmonary inflammation caused by the action of fine particles on alveolar cells, by repeatedly measuring haematological factors in older people and relating them to measurements of exposure to airborne particles. METHODS: One hundred and twelve individuals aged 60+ years in two UK cities provided repeated blood samples over 18 months, 108 providing the maximum of 12 samples. Estimates of individual exposure to particles of less than 10 microm diameter (PM(10)), derived from a mathematical model based on activity diaries and comparative measurements of PM(10) at multiple sites and during a variety of activities, were made for each three day period prior to blood sampling. The relationships between blood values and estimates of both personal exposure and city centre measurements of PM(10) were investigated by analysis of covariance, adjusting for city, season, temperature, and repeated individual measurements. RESULTS: Estimated personal exposure to PM(10) over the previous three days showed negative correlations with haemoglobin concentration, packed cell volume (PCV), and red blood cell count (p<0.001), and with platelets and factor VII levels (p<0.05). The changes in red cell indices persisted after adjustment for plasma albumin in a sample of 60 of the subjects. City centre PM(10) measurements over three days also showed negative correlations with haemoglobin and red cell count (p<0.001) and with PCV and fibrinogen (p<0.05), the relationship with haemoglobin persisting after adjustment for albumin. C reactive protein levels showed a positive association with city centre measurements of PM(10) (p<0.01). Based on a linear relationship, the estimated change in haemoglobin associated with an alteration in particle concentration of 100 microg/m(3) is estimated to have been 0.44 g/dl (95% CI 0.62 to 0.26) for personal PM(10) and 0.73 g/dl (95% CI 1.11 to 0.36) for city centre PM(10) measurements. CONCLUSIONS: This investigation is the first to estimate personal exposures to PM(10) and to demonstrate associations between haematological indices and air pollution. The changes in haemoglobin adjusted for albumin suggest that inhalation of some component of PM(10) may cause sequestration of red cells in the circulation. We propose that an action of such particles either on lung endothelial cells or on erythrocytes themselves may be responsible for changing red cell adhesiveness. Peripheral sequestration of red cells offers an explanation for the observed cardiovascular effects of particulate air pollution. (+info)
Hb Ube-2 in a diabetic case with an abnormally low HbA1C value.
(15/606)
A 69-year-old male diabetic patient had an abnormally low HbA1C value of 2.8%, which was inconsistent with his elevated fasting plasma glucose of 8.2 mmol/l. Hb analysis disclosed that the abnormal Hb was Hb Ube-2 [alpha68 (E17) Asn --> Asp] and it accounted for 21.5% of the total Hb. Since the glycated abnormal Hb emerged at the same position as did HbF on high performance liquid chromatography, the HbA1C value was falsely low. The present case demonstrates that Hb Ube-2 is one of the abnormal Hbs in which caution should be exercised when monitoring diabetic control. (+info)
The role of amino acid alpha38 in the control of oxygen binding to human adult and embryonic haemoglobin Portland.
(16/606)
The role of the amino acid at position alpha(38) in haemoglobin has been probed using site-directed mutagenesis. When the Thr residue at position alpha(38) (which is totally conserved in all mammals) is changed to a Gln, the equilibrium properties of the protein are significantly altered. Equilibrium and kinetic data show that the R-state properties of the protein are essentially unaffected by the mutation whilst the allosteric equilibrium and T-state properties are changed. Mutation of the naturally occurring Gln(38) of the human embryonic haemoglobin zeta-chain (the only known non-Thr containing globin) to a Thr residue shows the converse change in properties produced by the adult mutation, although in this case the situation is complicated by significant chain heterogeneity in the T state. An extension of the two-state model of co-operativity is presented to describe quantitatively the equilibrium ligand binding in the presence of T-state chain heterogeneity. A molecular model is described in which the putative interaction of alphaGln(38) and betaTyr(145) is identified which make a significant contribution to the previously reported unusual ligand-binding properties of the zeta-chain containing human embryonic haemoglobins. (+info)