Purification, crystallization and preliminary X-ray analysis of haemoglobin I from the armoured catfish Liposarcus anisitsi

Liposarcus anisitsi is an armoured catfish that presents accessorial air oxygenation through a modified stomach, which allows this species to survive in waters with very low oxygen content. Analysis of its haemolysate has shown the presence of four haemoglobins; this work focuses on the main component, haemoglobin I. It has been crystallized in two different forms and X-ray diffraction data have been collected to 2.77 and 2.86 Angstrom resolution using synchrotron radiation. Crystals were determined to belong to the space groups C2 and P2(1) and preliminary structural analysis revealed the presence of one tetramer in the asymmetric unit in both crystal forms. The structure was determined using a standard molecular-replacement technique.

Crystallization, preliminary X-ray analysis and molecular-replacement solution of the carboxy form of haemoglobin I from the fish Brycon cephalus

Haemoglobin, the 'honorary enzyme' [Brunori (1999), Trends Biochem. Sci. 24, 158-161], constitutes a prime prototype for allosteric models. Here, the crystallization and preliminary X-ray analysis of haemoglobin I from the South American fish Brycon cephalus are reported. X-ray diffraction data have been collected to 2.5 Angstrom resolution using synchrotron radiation (LNLS). Crystals were determined to belong to the space group P6(1)22 and preliminary structural analysis revealed the presence of one dimer (alpha beta) in the asymmetric unit. The structure was determined using standard molecular-replacement techniques.

Structure of the haptoglobin-haemoglobin complex

Red cell haemoglobin is the fundamental oxygen-transporting molecule in blood, but also a potentially tissue-damaging compound owing to its highly reactive haem groups. During intravascular haemolysis, such as in malaria and haemoglobinopathies(1), haemoglobin is released into the plasma, where it is captured by the protective acute-phase protein haptoglobin. This leads to formation of the haptoglobin-haemoglobin complex, which represents a virtually irreversible non-covalent protein-protein interaction(2). Here we present the crystal structure of the dimeric porcine haptoglobin-haemoglobin complex determined at 2.9 angstrom resolution. This structure reveals that haptoglobin molecules dimerize through an unexpected beta-strand swap between two complement control protein (CCP) domains, defining a new fusion CCP domain structure. The haptoglobin serine protease domain forms extensive interactions with both the alpha- and beta-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the alpha beta dimer is highly overlapping with the interface between the two alpha beta dimers that constitute the native haemoglobin tetramer. Several haemoglobin residues prone to oxidative modification after exposure to haem-induced reactive oxygen species are buried in the haptoglobin-haemoglobin interface, thus showing a direct protective role of haptoglobin. The haptoglobin loop previously shown to be essential for binding of haptoglobin-haemoglobin to the macrophage scavenger receptor CD163 (ref. 3) protrudes from the surface of the distal end of the complex, adjacent to the associated haemoglobin alpha-subunit. Small-angle X-ray scattering measurements of human haptoglobin-haemoglobin bound to the ligand-binding fragment of CD163 confirm receptor binding in this area, and show that the rigid dimeric complex can bind two receptors. Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of multimeric haptoglobin-haemoglobin for CD163 (ref. 4).

Erythropoietin treatment elevates haemoglobin concentration by increasing red cell volume and depressing plasma volume

[EN] Erythropoietin (Epo) has been suggested to affect plasma volume, and would thereby possess a mechanism apart from erythropoiesis to increase arterial oxygen content. This, and potential underlying mechanisms, were tested in eight healthy subjects receiving 5000 IU recombinant human Epo (rHuEpo) for 15 weeks at a dose frequency aimed to increase and maintain haematocrit at approximately 50%. Red blood cell volume was increased from 2933 +/- 402 ml before rHuEpo treatment to 3210 +/- 356 (P < 0.01), 3117 +/- 554 (P < 0.05), and 3172 +/- 561 ml (P < 0.01) after 5, 11 and 13 weeks, respectively. This was accompanied by a decrease in plasma volume from 3645 +/- 538 ml before rHuEpo treatment to 3267 +/- 333 (P < 0.01), 3119 +/- 499 (P < 0.05), and 3323 +/- 521 ml (P < 0.01) after 5, 11 and 13 weeks, respectively. Concomitantly, plasma renin activity and aldosterone concentration were reduced. This maintained blood volume relatively unchanged, with a slight transient decrease at week 11, such that blood volume was 6578 +/- 839 ml before rHuEpo treatment, and 6477 +/- 573 (NS), 6236 +/- 908 (P < 0.05), and 6495 +/- 935 ml (NS), after 5, 11 and 13 weeks of treatment. We conclude that Epo treatment in healthy humans induces an elevation in haemoglobin concentration by two mechanisms: (i) an increase in red cell volume; and (ii) a decrease in plasma volume, which is probably mediated by a downregulation of the rennin-angiotensin-aldosterone axis. Since the relative contribution of plasma volume changes to the increments in arterial oxygen content was between 37.9 and 53.9% during the study period, this mechanism seems as important for increasing arterial oxygen content as the well-known erythropoietic effect of Epo.

Effect of blood haemoglobin concentration on V(O2,max) and cardiovascular function in lowlanders acclimatised to 5260 m

[EN] The principal aim of this investigation was to determine the influence of blood haemoglobin concentration ([Hb]) on maximal exercise capacity and maximal O(2) consumption (V(O(2),max)) in healthy subjects acclimatised to high altitude. Secondarily, we examined the effects of [Hb] on the regulation of cardiac output (CO), blood pressure and muscular blood flow (LBF) during exercise. Eight Danish lowlanders (three females and five males; 24 +/- 0.6 years, mean +/- S.E.M.) performed submaximal and maximal exercise on a cycle ergometer after 9 weeks at an altitude of 5260 m (Mt Chacaltaya, Bolivia). This was done first with the high [Hb] resulting from acclimatisation and again 2-4 days later, 1 h after isovolaemic haemodilution with Dextran 70 to near sea level [Hb]. After measurements at maximal exercise while breathing air at each [Hb], subjects were switched to hyperoxia (55 % O(2) in N(2)) and the measurements were repeated, increasing the work rate as tolerated. Hyperoxia increased maximal power output and leg V(O(2),max), showing that breathing ambient air at 5260 m, V(O(2),max) is limited by the availability of O(2) rather than by muscular oxidative capacity. Altitude increased [Hb] by 36 % from 136 +/- 5 to 185 +/- 5 g l(-1) (P < 0.001), while haemodilution (replacing 1 l of blood with 1 l of 6 % Dextran) lowered [Hb] by 24 % to 142 +/- 6 g l(-1) (P < 0.001). Haemodilution had no effect on maximal pulmonary or leg V(O(2),max), or power output. Despite higher LBF, leg O(2) delivery was reduced and maximal V(O(2)) was thus maintained by higher O(2) extraction. While CO increased linearly with work rate irrespective of [Hb] or inspired oxygen fraction (F(I,O(2))), both LBF and leg vascular conductance were systematically higher when [Hb] was low. Close and significant relationships were seen between LBF (and CO) and both plasma noradrenaline and K(+) concentrations, independently of [Hb] and F(I,O(2)). In summary, under conditions where O(2) supply limits maximal exercise, the increase in [Hb] with altitude acclimatisation does not improve maximal exercise capacity or V(O(2),max), and does not alter peak CO. However, LBF and vascular conductance are higher at altitude when [Hb] is lowered to sea level values, with both relating closely to catecholamine and potassium concentrations. This suggests that the lack of effect of [Hb] on V(O(2),max) may involve reciprocal changes in LBF via local metabolic control of the muscle vasculature.

Fetal haemoglobin levels in adult type 1 (insulin-dependent) diabetic patients

Glycated haemoglobin levels (HbA1 and HbA1c) are established parameters of long-term glycaemic control in diabetic patients. Depending on the method used, fetal haemoglobin interferes with the assays for glycated haemoglobin. If present in high amounts, fetal haemoglobin may lead to overestimation of glycated haemoglobin levels, and therefore, of average blood glucose concentration in diabetic patients. Glycated (HbA1c) and fetal haemoglobin levels were measured by high pressure liquid chromatography in 60 (30 female) adult Type 1 (insulin-dependent) diabetic patients of Swiss descent, and were compared with levels obtained from 60 normal, non-diabetic control subjects matched for age and sex. Fetal haemoglobin levels were significantly higher in the diabetic patients (0.6 +/- 0.1%, mean +/- SEM; range: 0-3.6%) than in the control subjects (0.4 +/- 0.1%, p < 0.001). Elevated fetal haemoglobin levels (> or = 0.6%) were found in 23 of 60 diabetic patients (38%) compared to 9 of 60 control subjects (15%; chi 2 = 8.35, p < 0.01). In addition, fetal haemoglobin levels in diabetic patients are weakly correlated with glycated haemoglobin (HbA1c) (r = 0.38, p < 0.01). Fetal haemoglobin results were confirmed with the alkali denaturation procedure, and by immunocytochemistry using a polyclonal rabbit anti-fetal haemoglobin antibody. A significant proportion of adult patients with Type 1 diabetes has elevated fetal haemoglobin levels. In certain patients this may lead to a substantial over-estimation of glycated haemoglobin levels, and consequently of estimated, average blood glucose levels. The reason for this increased prevalence of elevated fetal haemoglobin remains unclear, but it may be associated with poor glycaemic control.

How Noninvasive Haemoglobin Measurement with Pulse CO-Oximetry Can Change Your Practice: An Expert Review

Trauma related haemorrhagic anaemia is rarely diagnosed by physical examination alone but typically includes measurement of blood haemoglobin, one of the most frequently ordered laboratory tests. Recently, noninvasive technologies have been developed that allow haemoglobin to be measured immediately without the need for intravenous access or having to take venous, arterial, or capillary blood. Moreover, with these technologies haemoglobin can be continuously measured in patients with active bleeding, to guide the start and stop of blood transfusions and to detect occult bleeding. Recent studies on the accuracy of the devices showed promising results in terms of accuracy of hemoglobin measurement compared to laboratory determination. The present review gives an overview on the technology itself and reviews the current literature on the subject.

Syncytiotrophoblast vesicles show altered micro-RNA and haemoglobin content after ex-vivo perfusion of placentas with haemoglobin to mimic preeclampsia.

BACKGROUND Cell-free foetal haemoglobin (HbF) has been shown to play a role in the pathology of preeclampsia (PE). In the present study, we aimed to further characterize the harmful effects of extracellular free haemoglobin (Hb) on the placenta. In particular, we investigated whether cell-free Hb affects the release of placental syncytiotrophoblast vesicles (STBMs) and their micro-RNA content. METHODS The dual ex-vivo perfusion system was used to perfuse isolated cotyledons from human placenta, with medium alone (control) or supplemented with cell-free Hb. Perfusion medium from the maternal side of the placenta was collected at the end of all perfusion phases. The STBMs were isolated using ultra-centrifugation, at 10,000×g and 150,000×g (referred to as 10K and 150K STBMs). The STBMs were characterized using the nanoparticle tracking analysis, identification of surface markers and transmission electron microscopy. RNA was extracted and nine different micro-RNAs, related to hypoxia, PE and Hb synthesis, were selected for analysis by quantitative PCR. RESULTS All micro-RNAs investigated were present in the STBMs. Mir-517a, mir-141 and mir-517b were down regulated after Hb perfusion in the 10K STBMs. Furthermore, Hb was shown to be carried by the STBMs. CONCLUSION This study showed that Hb perfusion can alter the micro-RNA content of released STBMs. Of particular interest is the alteration of two placenta specific micro-RNAs; mir-517a and mir-517b. We have also seen that STBMs may function as carriers of Hb into the maternal circulation.

A multidisciplinary approach for improving services in primary care: randomised controlled trial of screening for haemoglobin disorders

Objective: To investigate the feasibility of improving screening for carriers of haemoglobin disorders in general practice by using a nurse facilitator to work with primary care teams and the relevant haematology laboratories; to identify problems in communication between all those involved in delivering the service, and to implement solutions.

The association between glycated haemoglobin levels and P100 visual evoked potentials in diabetes mellitus

Diabetes mellitus (DM) is a metabolic disorder which is characterised by hyperglycaemia resulting from defects in insulin secretion, insulin action or both. The long-term specific effects of DM include the development of retinopathy, nephropathy and neuropathy. Cardiac disease, peripheral arterial and cerebrovascular disease are also known to be linked with DM. Type 1 diabetes mellitus (T1DM) accounts for approximately 10% of all individuals with DM, and insulin therapy is the only available treatment. Type 2 diabetes mellitus (T2DM) accounts for 90% of all individuals with DM. Diet, exercise, oral hypoglycaemic agents and occasionally exogenous insulin are used to manage T2DM. The diagnosis of DM is made where the glycated haemoglobin (HbA1c) percentage is greater than 6.5%. Pattern-reversal visual evoked potential (PVEP) testing is an objective means of evaluating impulse conduction along the central nervous pathways. Increased peak time of the visual P100 waveform is an expression of structural damage at the level of myelinated optic nerve fibres. This was an observational cross sectional study. The participants were grouped into two phases. Phase 1, the control group, consisted of 30 healthy non-diabetic participants. Phase 2 comprised of 104 diabetic participants of whom 52 had an HbA1c greater than 10% (poorly controlled DM) and 52 whose HbA1c was 10% and less (moderately controlled DM). The aim of this study was to firstly observe the possible association between glycated haemoglobin levels and P100 peak time of pattern-reversal visual evoked potentials (PVEPs) in DM. Secondly, to assess whether the central nervous system (CNS) and in particular visual function is affected by type and/or duration of DM. The cut-off values to define P100 peak time delay was calculated as the mean P100 peak time plus 2.5 X standard deviations as measured for the non-diabetic control group, and were 110.64 ms for the right eye. The proportion of delayed P100 peak time amounted to 38.5% for both diabetic groups, thus the poorly controlled group (HbA1c > 10%) did not pose an increased risk for delayed P100 peak time, relative to the moderately controlled group (HbA1c ≤ 10%). The P100 PVEP results for this study, do however, reflect significant delay (p < 0.001) of the DM group as compared to the non-diabetic group; thus, subclincal neuropathy of the CNS occurs in 38.5% of cases. The duration of DM and type of DM had no influence on the P100 peak time measurements.

Selective disappearance of individuals with high levels of glycated haemoglobin in a free-living bird

Peer reviewed