Search

Reliable measurements using modern techniques and consensus in experimental design have enabled the assessment of novel data sets for normal maternal and foetal respiratory physiology at term. These data sets include (a) principal factors affecting placental gas transfer, e.g., maternal blood flow through the intervillous space (IVS) (500 mL/min) and foeto-placental blood flow (480 mL/min), and (b) O 2 , CO 2 and pH levels in the materno-placental and foeto-placental circulation. According to these data, the foetus is adapted to hypoxaemic hypoxia. Despite flat oxygen partial pressure (pO 2 ) gradients between the blood of the IVS and the umbilical arteries of the foetus, adequate O 2 delivery to the foetus is maintained by the higher O 2 affinity of the foetal blood, high foetal haemoglobin (HbF) concentrations, the Bohr effect, the double-Bohr effect, and high foeto-placental (=umbilical) blood flow. Again, despite flat gradients, adequate CO 2 removal from the foetus is maintained by a high diffusion capacity, high foeto-placental blood flow, the Haldane effect, and the double-Haldane effect. Placental respiratory gas exchange is perfusion-limited, rather than diffusion-limited, i.e., O 2 uptake depends on O 2 delivery., (© 2022. Springer Nature Switzerland AG.)

COVID-19 infection is less common in children (with higher fetal hemoglobin levels). In our preliminary study, we also observed a low prevalence and fatality of COVID-19 in countries with high rate of hemoglobinopathy carries. Given these two facts, the hemoglobin structure can play a role in the physiopathology of COVID-19 disease. Several drugs are known to increase fetal hemoglobin in adults. Adding these drugs to COVID-19 clinical trials may improve the patients' outcomes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The clinical severity of sickle cell disease and β-thalassemia, the major disorders of β-globin, can be ameliorated by increased production of fetal hemoglobin (HbF). Here, we provide a brief overview of the fetal-to-adult hemoglobin switch that occurs in humans shortly after birth and review our current understanding of one of the most potent known regulators of this switching process, the multiple zinc finger-containing transcription factor BCL11A. Originally identified in genome-wide association studies, multiple orthogonal lines of evidence have validated BCL11A as a key regulator of hemoglobin switching and as a promising therapeutic target for HbF induction. We discuss recent studies that have highlighted its importance in silencing the HbF-encoding genes and discuss opportunities that exist to further understand the regulation of BCL11A and its mechanism of action, which could provide new insight into opportunities to induce HbF for therapeutic purposes., (© 2016 New York Academy of Sciences.)

The term sickle cell disease embraces a group of genetic conditions in which pathology results from the inheritance of the sickle cell gene either homozygously or as a double heterozygote with another interacting gene. The spectrum of resulting conditions is therefore influenced by the geography of individual hemoglobin genes, but in most populations, the commonest genotype at birth is homozygous sickle cell (SS) disease. Because this genotype generally manifests a greater mortality, the relative proportion of sickle cell genotypes is influenced by age as well as the geographical distribution of individual genes.

Background: HbF-Monserrato-Sassari is a newly discovered abnormal fetal hemoglobin observed in an apparently normal newborn baby during a hemoglobinopathies survey at birth in North Sardinian population., Methods: Electrophoretic analysis of the cord blood lysate evidenced for an abnormal tetramer due to a mutated fetal globin chain. Electrospray ionisation-mass spectrometry and gene sequencing were used to identify the mutation. Oxygen binding ability of the variant Hb was determined., Results: Sequencing of the γ globin genes revealed the TGT→CGT transition at codon 93 in one of the two (G)γ genes, which leads to the Arg for Cys amino acid replacement at position 9 of the F α-helix. The amino acid substitution was confirmed by mass spectrometric analysis of the globin chains. Since modifications or substitutions at position β93 are known to affect the arrangement of a salt bridge at the α1β2 sliding contacts that are crucial for subunit cooperativity, the functional properties of the variant were studied to evaluate the effect of the replacement at the same position in the γ globin chain. With respect to normal HbF, the variant showed a significant increase in oxygen affinity and a slight decrease of both Bohr effect and cooperativity., General Significance: Result indicates a key role of the Cys γ93 residue for subunit cooperativity in the T→R transition of the HbF tetramer. Substitutions at the F9 position of the (G)γ globin may result in stabilization of the high affinity R-state of the Hb tetramer. Because of the loss of Cys γ93 residue, this variant is considered to be potentially compromised in nitric oxide transport., (2011 Elsevier B.V. All rights reserved.)

Background: In Africa, infant susceptibility to Plasmodium falciparum malaria increases substantially as fetal hemoglobin (HbF) and maternal immune IgG disappear from circulation. During the first few months of life, however, resistance to malaria is evidenced by extremely low parasitemias, the absence of fever, and the almost complete lack of severe disease. This resistance has previously been attributed in part to poor parasite growth in HbF-containing red blood cells (RBCs). A specific role for maternal immune IgG in infant resistance to malaria has been hypothesized but not yet identified., Methods and Findings: We found that P. falciparum parasites invade and develop normally in fetal (cord blood, CB) RBCs, which contain up to 95% HbF. However, these parasitized CB RBCs are impaired in their binding to human microvascular endothelial cells (MVECs), monocytes, and nonparasitized RBCs--cytoadherence interactions that have been implicated in the development of high parasite densities and the symptoms of malaria. Abnormal display of the parasite's cytoadherence antigen P. falciparum erythrocyte membrane protein-1 (PfEMP-1) on CB RBCs accounts for these findings and is reminiscent of that on HbC and HbS RBCs. IgG purified from the plasma of immune Malian adults almost completely abolishes the adherence of parasitized CB RBCs to MVECs., Conclusions: Our data suggest a model of malaria protection in which HbF and maternal IgG act cooperatively to impair the cytoadherence of parasitized RBCs in the first few months of life. In highly malarious areas of Africa, an infant's contemporaneous expression of HbC or HbS and development of an immune IgG repertoire may effectively reconstitute the waning protective effects of HbF and maternal immune IgG, thereby extending the malaria resistance of infancy into early childhood.

While supportive care remains the best option for most well children with sickle cell disease (SCD), increasing awareness of early signs of chronic organ damage in childhood has focused attention on therapy which modulates the natural history of the disease. Since cure by stem cell transplantation is only feasible for a minority and gene therapy remains developmental, pharmacological modification by Haemoglobin F (HbF)-inducers, is the most widely used approach in SCD. Currently, the only HbF modulator with a clear place in the management of childhood SCD is hydroxycarbamide for which the main indications are frequent painful crises and recurrent acute chest syndrome. In the majority of SCD children treated with hydroxycarbamide there is clear evidence of clinical benefit and the drug is well tolerated. The main disadvantages are the need for frequent monitoring and uncertainity about long-term risks of carcinogenicity and impaired fertility, although these risks appear to be very low. The role of hydroxycarbamide in sickle-associated central nervous system disease remains to be established. Decitabine and butyrate derivatives show some promise although robust data in children with SCD are lacking. A number of other drugs are currently under investigation for their effects on HbF production including thalidomide and lenolidamide.

Increased levels of red cell fetal hemogloblin, whether due to hereditary persistence of expression or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease (SCD). Therefore, we developed erythroid-specific, gamma-globin lentiviral vectors for hematopoietic stem cell (HSC)-targeted gene therapy with the goal of permanently increasing fetal hemoglobin (HbF) production in sickle red cells. We evaluated two different gamma-globin lentiviral vectors for therapeutic efficacy in the BERK sickle cell mouse model. The first vector, V5, contained the gamma-globin gene driven by 3.1 kb of beta-globin regulatory sequences and a 130-bp beta-globin promoter. The second vector, V5m3, was identical except that the gamma-globin 3'-untranslated region (3'-UTR) was replaced with the beta-globin 3'-UTR. Adult erythroid cells have beta-globin mRNA 3'-UTR-binding proteins that enhance beta-globin mRNA stability and we postulated this design might enhance gamma-globin expression. Stem cell gene transfer was efficient and nearly all red cells in transplanted mice expressed human gamma-globin. Both vectors demonstrated efficacy in disease correction, with the V5m3 vector producing a higher level of gamma-globin mRNA which was associated with high-level correction of anemia and secondary organ pathology. These data support the rationale for a gene therapy approach to SCD by permanently enhancing HbF using a gamma-globin lentiviral vector.

In human beta-thalassemia, the imbalance between alpha- and non-alpha-globin chains causes ineffective erythropoiesis, hemolysis, and anemia: this condition is effectively treated by an enhanced level of fetal hemoglobin (HbF). In spite of extensive studies on pharmacologic induction of HbF synthesis, clinical trials based on HbF reactivation in beta-thalassemia produced inconsistent results. Here, we investigated the in vitro response of beta-thalassemic erythroid progenitors to kit ligand (KL) in terms of HbF reactivation, stimulation of effective erythropoiesis, and inhibition of apoptosis. In unilineage erythroid cultures of 20 patients with intermedia or major beta-thalassemia, addition of KL, alone or combined with dexamethasone (Dex), remarkably stimulated cell proliferation (3-4 logs more than control cultures), while decreasing the percentage of apoptotic and dyserythropoietic cells (<5%). More important, in both thalassemic groups, addition of KL or KL plus Dex induced a marked increase of gamma-globin synthesis, thus reaching HbF levels 3-fold higher than in con-trol cultures (eg, from 27% to 75% or 81%, respectively, in beta-thalassemia major). These studies indicate that in beta-thalassemia, KL, alone or combined with Dex, induces an expansion of effective erythropoiesis and the reactivation of gamma-globin genes up to fetal levels and may hence be considered as a potential therapeutic agent for this disease.

Human embryonic stem cells are a promising tool to study events associated with the earliest ontogenetic stages of hematopoiesis. We describe the generation of erythroid cells from hES (H1) by subsequent processing of cells present at early and late stages of embryoid body (EB) differentiation. Kinetics of hematopoietic marker emergence suggest that CD45+ hematopoiesis peaks at late D14EB differentiation stages, although low-level CD45- erythroid differentiation can be seen before that stage. By morphologic criteria, hES-derived erythroid cells were of definitive type, but these cells both at mRNA and protein levels coexpressed high levels of embryonic (epsilon) and fetal (gamma) globins, with little or no adult globin (beta). This globin expression pattern was not altered by the presence or absence of fetal bovine serum, vascular endothelial growth factor, Flt3-L, or coculture with OP-9 during erythroid differentiation and was not culture time dependent. The coexpression of both embryonic and fetal globins by definitive-type erythroid cells does not faithfully mimic either yolk sac embryonic or their fetal liver counterparts. Nevertheless, the high frequency of erythroid cells coexpressing embryonic and fetal globin generated from embryonic stem cells can serve as an invaluable tool to further explore molecular mechanisms.

Extensive studies during the last 30 years have led to considerable understanding of cellular and molecular control of hemoglobin switching. Cell biology studies in the 1970s defined the control of globin genes during erythroid differentiation and led to development of therapies for sickle cell disease. Molecular investigations of the last 20 years have delineated the two basic mechanisms that control globin gene activity during development--autonomous silencing and gene competition. Studies of hemoglobin switching have provided major insights on the control of gene loci by remote regulatory elements. Research in this field has an impact on understanding regulatory mechanisms in general and is of particular importance for eventual development of molecular cures for sickle cell disease and beta thalassemia.

Glucose 6-phosphate dehydrogenase (G6PD) (EC 1.1.1.42) is an essential enzyme for the rapid production of NADPH, as required on exposure to oxidative stress. Mouse embryonic stem (ES) cells can produce all embryonic and fetal/adult cell types. By studying the in vitro differentiation of embryoid bodies produced from G6pdDelta ES cells that are totally unable to produce G6PD protein, we found that these cells are able to differentiate into mesodermal cells, cardiomyocytes, hepatocytes, and primitive erythroid cells. However, we show here that, after the hemoglobin switch has taken place, definitive erythrocytes die by apoptosis. This apoptotic death is delayed by reducing agents and by a caspase inhibitor, but it is prevented only by the restoration of G6PD activity. Thus, G6PD proves indispensable for definitive erythropoiesis.

Haemoglobins from mammals of sub-Arctic and Arctic species, as well as fetal human Hb, are all characterized by a significantly lower Delta H of oxygenation compared with the majority of mammalian haemoglobins from temperate species (exceptions are represented by some cold-resistant species, such as cow, horse and pig). This has been interpreted as an adaptive mechanism of great importance from a physiological point of view. To date, the molecular basis of this thermodynamic characteristic is still not known. In the present study, we show that binding of extra chloride (with respect to adult human Hb) ions to Hb would significantly contribute to lowering the overall heat of oxygenation, thus providing a molecular basis for the low effect of temperature on the oxygenation-deoxygenation cycle. To this aim, the oxygen binding properties of bovine Hb, bear (Ursus arctos) Hb and horse Hb, which are representative of this series of haemoglobins, have been studied with special regard to the effect of heterotropic ligands, such as organic phosphates (namely 2,3-diphosphoglycerate) and chloride. Functional results are consistent with a mechanism for ligand binding that involves an additional binding site for chloride ion. Analysis of computational chemistry results, obtained by the GRID program, further confirm the hypothesis that the reason for the lower Delta H of oxygenation is mainly due to an increase in the number of the oxygen-linked chloride-binding sites.

In order to provide the appropriate level of oxygen transport to respiring tissues, we need to produce a molecular oxygen transporting system to supplement oxygen diffusion and solubility. This supplementation is provided by hemoglobin. The role of hemoglobin in providing oxygen transport from lung to tissues in the adult is well-documented and functional characteristics of the fetal hemoglobin, which provide placental oxygen exchange, are also well understood. However the characteristics of the three embryonic hemoglobins, which provide oxygen transport during the first three months of gestation, are not well recognized. This review seeks to describe the state of our understanding of the temporal control of the expression of these proteins and the oxygen binding characteristics of the individual protein molecules. The modulation of the oxygen binding properties of these proteins, by the various allosteric effectors, is described and the structural origins of these characteristics are probed., (Copyright 2002 Elsevier Science Ltd.)

We have determined whether derivated fetal haemoglobin (dHbF, consisting of glycated and acetylated HbF) can be used as a cell age marker for fetal red blood cells (RBCs). Cord blood was obtained between 19 and 39 weeks of gestation from 28 alloimmunised anaemic fetuses (23 RhD+ and 5 Kell) and from 20 non-anaemic fetuses and newborns (controls). Density gradient centrifugation was applied to 36 samples (20 RhD+, 15 controls and 1 Kell) to obtain fractions of increasing cell age. Blood samples were used for measurements of mean cellular volume (MCV), mean cell haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), pyruvate kinase activity (PK) and derivated fetal haemoglobin (dHbF) by cation-exchange HPLC. Reticulocytes were counted only in the whole blood samples. In all density gradient separated RBC fractions, the values for MCV, MCH and PK activity decreased and those of MCHC and dHbF increased with increasing density (equivalent to increasing cell age). The mean density was lower for RBCs of the anaemic RHD group (1.072+/-0.007 g/ml) than for the non-anaemic controls (1.077+/-0.005 g/ml) (p<0.05) The RBC density of the Kell sensitised fetus did not differ from those of the controls. In the control group, the values of the cell age markers in whole blood changed significantly with the gestational age, showing an increase of mean age of the erythrocyte population. The best linear relationship was found for dHbF (y=6.28+0.17*weeks; r=0.84; p<0.001). In the anaemic RhD+ fetuses, the RBC age markers did not change with gestational age; the dHbF percentages were lower, and the MCV, MCH, PK values and the reticulocyte counts were higher than in the controls (0.05

The relative proportions of the two gamma chain species (G gamma and A gamma) have been reinvestigated in newborns, during the physiological switch from foetal to adult haemoglobin, and in adults with some persistent expression of HbF. In newborns, with about 80% HbF, the G gamma percentage was close to 70% while in adult RBC, with less than 0.5% HbF, the G gamma chain was almost non-detectable and may reflect the completion of the foetal to adult switch. Conversely, in adult patients with HbF above 0.6%, usually accompanying some degree of marrow stimulation, the relative ratio of G gamma varied between 40 and 60%, independently of HbF level. This ratio corresponds to what has been described in the literature as being the adult type of HbF. In all the cases where we found higher levels of G gamma, the results could be explained by the presence of a specific genetic background such as the Senegalese haplotype in sickle cell disease.

Embryonic- and adult-stage globin subunits assemble into haemoglobin (Hb) heterotetramers that are expressed at low levels throughout human intrauterine development. These haemoglobins differ from adult Hb A (alpha2beta2) by the substitution of embryonic zeta for adult alpha globin (Hb zeta2beta2), or embryonic epsilon for adult beta globin (Hb alpha2epsilon2). Several key physiological properties of these 'semiembryonic' haemoglobins remain undefined, as ethical and methodological considerations have limited their availability from both human sources and conventional expression systems. The current study attempts to estimate how the physiological properties of semiembryonic and adult haemoglobins may differ, by determining whether the O2-binding characteristics of hybrid human/mouse haemoglobins change when human alpha- or beta-globin subunits are replaced by human embryonic zeta- or epsilon-globin subunits respectively. Each of the four human globins is expressed in transgenic mice that are nullizygous for either the endogenous mouse alpha- or beta-globin genes, resulting in the high-level expression of haemoglobins that can be studied either in situ in intact erythrocytes or in vitro. We showed that the exchange of human zeta-globin for human alpha-globin chains increased haemoglobin O2 affinity, both in the presence and in the absence of 2, 3-bisphosphoglycerate (2,3-BPG), and reduced the pH-dependent shift in its oxygen equilibrium curve (Bohr effect). By comparison, hybrid haemoglobins containing either human epsilon-globin or human beta-globin exhibited nearly identical O2-binding properties, both in situ and in vitro, regardless of 2,3-BPG levels or ambient pH. Neither the zeta-for-alpha nor the epsilon-for-beta substitutions substantially altered binding affinity for 2,3-BPG or cooperativity between globin subunits. These studies suggest that semiembryonic haemoglobins that assemble entirely from human subunits may exhibit properties that are similar to those of human Hb A.

To correlate amino acid sequence changes with hemoglobin function we are carrying out a detailed recombinant analysis of the adult hemoglobin/fetal hemoglobin (HbA/HbF) systems. The important physiological differences between these two tetramers lie at unspecified sites in the 39 sequence substitutions of the 146 amino acids in their beta and gamma chains. In this paper, significant differences in the tetramer-dimer dissociation constants (referred to as tetramer "strength" or "stability") of adult (HbA) and fetal (HbF) hemoglobin tetramers have been used to probe the relationship between the allosteric, sliding interface and the effects of the allosteric regulator, 2,3-DPG, in promoting oxygen release. The single amino acid difference at the allosteric interfaces of these two hemoglobins, Glu-43(beta) --> Asp-43(gamma), which is not near the DPG binding site, leads to a significantly lower DPG response, approaching that of HbF. The results are inconsistent with the long-held idea that the replacement of His-143(beta) in HbA to Ser-143(gamma) in HbF is solely responsible for the lowered DPG response in HbF. On the other hand, the Val-1(beta) --> Gly-1(gamma) replacement near the DPG binding site has no effect on the DPG response. The replacement of His-116(beta) by the hydrophobic Ile-116(gamma) at the rigid alpha(1)beta(1) interface has a marginal yet detectable effect on the allosteric alpha(1)beta(2) interface. The results, overall, are interpreted using a model involving electrostatic coupling between certain side chains and extend the concept of a long-range relationship between some distant regions of the tetramer that are likely mediated through the central cavity.

Hemoglobin plays an important physiologic role, one that begins early in fetal development. The focus of this article is hemoglobin physiology. Developmental erythropoiesis, developmental stages of hemoglobin, and postnatal erythropoiesis and hemoglobin production are discussed. The function of hemoglobin, its affinity for oxygen, and the clinical significance of the oxyhemoglobin dissociation curve are also explored.

Studies in vitro by Pasvol et al (Nature, 270:171, 1977) have indicated that the growth of Plasmodium falciparum in cells containing fetal hemoglobin (HbF = alpha2gamma2) is retarded, but invasion is increased, at least in newborn cells. Normal neonates switch from about 80% HbF at birth to a few percent at the end of the first year of life. Carriers of beta-thalassemia trait exhibit a delay in the normal HbF switch-off, which might partially explain the protection observed in populations with this gene. To study this hypothesis in vivo, we used transgenic (gamma) mice expressing human Agamma and Ggamma chains resulting in 40% to 60% alpha2Mgamma2 hemoglobin, infected with rodent malaria. Two species of rodent malaria were studied. P chabaudi adami causes a nonlethal infection, mainly in mature red blood cells (RBC). P yoelii 17XNL is a nonlethal infection, invading primarily reticulocytes, whereas P yoelii 17XL is a lethal variant of P yoelii 17XNL and causes death of mice in approximately 1 to 2 weeks. Data indicate that this strain may cause a syndrome resembling cerebral malaria caused by P falciparum (Am J Trop Med Hyg, 50:512, 1994). In gamma transgenic mice infected with P chabaudi adami, the parasitemia rose more quickly (in agreement with Pasvol) than in control mice, but was cleared more rapidly. In mice infected with P yoelii 17XNL, a clear reduction in parasitemia was observed. Interestingly, splenectomy before this infection, did not reverse protection. The most striking effect was in lethal P yoelii 17XL infection. Control mice died between 11 to 13 days, whereas gamma mice cleared the infection by day 22 and survived, a phenomenon also observed in splenectomized animals. These results suggest that HbF does indeed have a protective effect in vivo, which is not mediated by the spleen. In terms of mechanisms, light microscopy showed that intraerythrocytic parasites develop slowly in HbF erythrocytes, and electron microscopy showed that hemozoin formation was defective in transgenic mice. Finally, digestion studies of HbF by recombinant plasmepsin II demonstrated that HbF is digested only half as well as hemoglobin A (HbA). We conclude that HbF provides protection from P falciparum malaria by the retardation of parasite growth. The mechanism involves resistance to digestion by malarial hemoglobinases based on the data presented and with the well-known properties of HbF as a super stable tetramer. In addition, the resistance of normal neonates for malaria can now be explained by a double mechanism: increased malaria invasion rates, reported in neonatal RBC, will direct parasites to fetal cells, as well as F cells, and less to the approximately 20% of HbA containing RBC, amplifying the antimalarial effects of HbF.

Collectively sickle cell disease and beta-thalassaemia are the most commonly inherited single-gene defects world-wide and were the first group of diseases for which DNA-based detection strategies were utilized. Although genotypically distinct, these two groups of diseases exhibit several common clinical features: moderate-to-severe haemolytic anaemia, acute and progressive tissue damage, disease- or treatment-related organ failure and premature death. Within the last two decades, a striking improvement in life expectancy in the two patient populations has been observed, by dint of primary and secondary prevention strategies. However, apart from bone marrow transplantation, a generally applicable, specific and non-toxic form of treatment remains unavailable for these disorders. Nonetheless, a greater appreciation of the developmental control of human globin gene expression coupled with observations of the effects of certain classes of agents to 'reverse' erythroid cellular phenotype in in vitro and animal models have led to pharmacological trials to obtain meaningful increases in haemoglobin F production in patients affected by these two severe beta-globin disorders. Contemporary understanding of the quantitative relationship between the abnormal molecules in the red cells (aggregates of sickle haemoglobin) in the sickle cell syndromes and aggregated alpha-globin polypeptides in the beta-thalassemia syndromes, and the extent of the red cell and/or organ involvement, has now enabled investigators to predict how much inhibition of these intracellular pathogenic processes might be necessary to achieve partial or total abrogation of disease manifestations. The results of the Multicenter Study of Hydroxyurea and other controlled trials now bear out these predictions.

In the present review the role and regulation of foetal haemoglobin (HbF) in sickle-cell disease as well as the clinical results of therapy aimed at augmenting HbF synthesis are briefly discussed. Enhanced levels of HbF have an inhibitory effect on sickling and ameliorate the clinical course of sickle-cell disease. This knowledge has incited a search for pharmacological agents capable of stimulating HbF production. Cytostatic drugs, in particular hydroxyurea, but also other agents such as erythropoietin, butyrate and its analogues have been shown to induce HbF production. The mechanisms of action and the clinical effects of these agents are summarized. Only hydroxyurea has shown significant clinical effects in terms of reduction of pain-crises, chest syndrome and transfusions in sickle-cell patients. However, not all patients experience clinical amelioration and it appears difficult to predict which patient will gain from hydroxyurea therapy. Further studies need to expand the clinical experience with hydroxyurea, other HbF-inducing agents and combinations of these drugs. Moreover, they will hopefully provide criteria to enable appropriate selection of those patients with sickle-cell anaemia who will benefit clinically from HbF stimulation, also taking into account the risks and costs of long-term pharmacological therapy.

Fetal hemoglobin, the predominant hemoglobin of the fetus, is good for sickle cell anemia. This hemoglobin inhibits the polymerization of sickle hemoglobin. Clinical studies have shown that at any level of fetal hemoglobin, the more that is present, the better off is the patient. We are now able to increase fetal hemoglobin levels by pharmacologic means. We should know shortly if this is associated with clinical benefit.

Considering the variety of species that depend on hemoglobin for oxygen transport, these molecules must execute their primary function under extreme environmental conditions. Hence, a thermodynamic analysis of oxygen binding with hemoglobins from different species reveals a series of adaptive mechanisms which are based on the thermodynamic connection between the binding of heterotropic effectors and the reaction with oxygen. The examples reported, from fishes to human fetus, illustrate how evolution can alter the structural basis of the heterotropic interactions to optimize the oxygenation-deoxygenation cycle in dependence of the physiological needs of the particular organisms. Moreover they show that a thermodynamic analysis of the reaction with oxygen overcomes the meaning of a detailed structural and functional characterization going deeper into the physiology of the specific organism.

The concentration of haemoglobins F and A2 in 132 Hb SS children aged 6 months to 16 years and 50 age- and sex-matched controls has been determined. The mean HbF concentration in Hb SS patients and controls were 9.5 per cent (range 1.7-24.5 per cent) and 1.5 per cent (range 0.5-14.4 per cent) respectively while HbA2 concentrations were 2.7 per cent (range 1.6-3.7 per cent) and 2.4 per cent (range 1.3-3.9 per cent) respectively. For 18 Hb SS patients seen in the stable state and during vaso-occlusive crisis, there was no significant difference in HbF concentration between the two states.

This chapter reviews general aspects of the normal human haemoglobins which include those predominantly present in the embryo, the fetus and newborn baby, and in the normal adult. Special emphasis is given to factors which affect the levels of fetal haemoglobin in the adult because increased percentages of Hb F can be of great benefit to adults with certain haemoglobinopathies such as sickle cell anaemia and beta-thalassaemia. A review of the numerous Hb variants published since the discovery of Hb S in 1959 reveals a steady stream of newly detected abnormalities; most of these are the result of single-point mutations in the alpha-, beta-, gamma-, or delta-globin genes. Of the more than 600 variants listed in a repository, some 200 have clinical significance because of a decreased stability, a change in functional properties, among others. Methodology developed for the detection and quantitation of normal and abnormal Hb components has been greatly modified during the past 30 years; isoelectrofocusing and different fast developing micro chromatographic procedures are the methods of choice. Analyses of DNA isolated from the white cells has become most useful for the final characterization of the variants; this methodology consists of amplification of a desired segment of DNA and identification of a mutation with labelled oligonucleotide probes. Additional methods include sequence determination of this amplified DNA and identification of known mutations with an allele specific amplification procedure.

The most studied pharmacological intervention in sickle cell anemia aiming at elevating HbF expression is the use of hydroxyurea. At the present time the experience has been that after 1 year of treatment with maximum tolerated doses (MTD) all patients showed increases of percent HbF, with a mean of 15% HbF, without apparent side effects besides the reversible ones observed during the process of attaining the MTD. The question of efficacy is presently being investigated by a multicenter placebo controlled double blind clinical trial that involves more than 20 sites. The goal of the study is to determine if hydroxyurea can decrease the incidence of painful crises by 50%. Results of this study are not expected before the end of 1993.

Present concepts of the mechanism of reactivation of synthesis of fetal hemoglobin (HbF) in the adult under conditions of erythropoietic stress are briefly reviewed. Since HbF can be considered an effective natural antisickling agent, the reactivation of its synthesis in patients with sickle cell anemia as a desirable therapeutic goal has been extensively explored since the discovery in 1982 that 5-azacytidine increases HbF levels in the baboon. Hydroxyurea (HU) has become the most widely used agent, although its effectiveness in increasing HbF levels and the number of F cells is highly variable. Recent investigations are cited showing that other agents such as butyrate, and the addition of recombinant hemopoietic growth factors, such as erythropoietin and stem cell factor, especially in combination with HU, offer important therapeutic possibilities. Transacting nuclear proteins are briefly discussed as possibly having a future role in the efforts of stimulating gamma-chain synthesis.

This report describes a mathematical model of the countercurrent shunting (CCS) of O2 in the intestinal villus. The anatomic basis for the model is the close proximity of the arteriole and venule between which O2 is free to diffuse. The model divides the villus into four segments from base to tip. Steady-state equations describe the convective and diffusive fluxes of O2 in the arteriolar, capillary, and tissue compartments within each segment. Longitudinal diffusion along the length of the villus is assumed to be negligible. Simulations with the model led to the following observations: 1) CCS shifted the VO2 vs. blood flow curve down and to the right, slightly impairing VO2 at a given blood flow; 2) the base-to-tip PO2 gradient caused by the tissue O2 consumption was reduced by CCS; 3) when blood flow was reduced, the base-to-tip PO2 gradient increased until the tip PO2 fell to zero and then fell with further flow reductions; 4) lowering blood flow initially caused slight increases in shunting but further decreases in flow reduced shunting; 5) in the blood flow range in which VO2 was flow independent, increasing the O2 demand or decreasing the intervascular distance increased shunting because of the greater arteriole-to-capillary O2 concentration gradient and the decreased diffusion distance, respectively; and 6) lowering the hemoglobin's P50 to simulate fetal blood caused slight reductions in shunting and reduced VO2 at a given flow. In summary, the model confirms the potentially deleterious effects of CCS on intestinal oxygenation, and, in contrast to assertions in the literature, it shows that a base-to-tip PO2 gradient is not prima facie evidence of counter-current shunting.

In premature infants there is a temporal and causal relation between the change in the erythropoiesis after birth and the manifestation of retinopathy. The physiological substitution of the fetal erythrocytes can be controlled by an analysis of the fetal hemoglobin. In premature infants in which the erythropoiesis has not yet been converted there is an interval of time where there is a chronic hypoxemia of the retina and in which neovascularisation occurs. The oxygen deficit arises because fetal blood - in comparison with adult blood - exhibits a higher affinity to oxygen. If high values of HbF are ascertained in premature infants the chronic hypoxemia of the retina can be avoided by replacing the erythrocytes. In this way, a secondary prevention of the illness is possible.

This review describes three biological processes in which there is evidence for single cells being able to measure elapsed time. We describe the work that has led to this view, and review more recent work that has provided new insights into possible mechanisms for the measurement of time.

With the help of diverse factors, definitive erythropoiesis is induced by the junction of a pluripotential stem-cell with a specialized macrophage. In the normal adult marrow the latter becomes the reticular cell forming the core of an 'erythroblastic island'. It stays in close contact with all the erythroid cells descending from the stem-cell until their full maturation. While this contact lasts, only alpha and beta (delta) globin chains are produced. The production of gamma chains is inhibited. Stress forces the reticular cell to let the erythroid cells go. The stronger the stress, the earlier the separation. When free from the reticular cell, the erythroid cells stop producing beta chains and switch to form gamma ones. Increase in the volume of blood circulation is a potent stress. Since it is relatively enormous in the embryo and the fetus, most hemoglobin produced at these periods will be fatal. The important quality of fetal hemoglobin is its high oxidizability. It permits the senescent fetal red cell to be hemolysed intravascularly before any membrane alterations appear. The heme is dealt with by the placenta. Since no erythrophagocytosis has occurred, no bilirubin is formed. This process prevents bilirubin, which is toxic to the fetal liver, to reach it.

Oxygen (O2) transport was assessed through the affinity between O2 and hemoglobin (Hb) in 123 newborns of 28 to 40 week gestational ge, with a minimum of 9 newborns for each gestational age group (see table). In order to assess the O2-Hb affinity, we studied the correlation between the pO2 and the Hb saturation for each gestational age, obtaining estimates of the oxy-hemoglobin dissociation curves corresponding to each gestational age (see fig. 3). The pO2 levels corresponding to the 50% saturation (P50) for each gestational age were estimated from there. All newborns were from single vaginal deliveries with no fetal distress before birth and with an adequate weight for gestational age. The latter was calculated according to the date of the last menstrual period (78% of the cases), echography (10.6% of the cases) or neonatal physical exam (11.4% of the cases). A P50 vs. gestational age linear regression showed a high determination rate (r2 = 0.957, p less than 0.00001) (see fig. 2) which supports the hypothesis of the P50 linear growth; decrease in the Hb-O2 affinity with increasive gestational age (Hb-O2 affinity is different in newborns of different gestational ages). With these results one may conclude that the Hb-O2 uptake varies according to gestational age (P50 changes linearly as gestational age increases) and that a single measurement of pO2 in a newborns, blood does not accurately evaluate the amount of O2 that is transported to the tissues, because the transport capacity depends, among other factors, upon gestational age. The Hb saturation better represents the amount of O2 that can get to the cell level.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased numbers of F cells and large amounts of Hb F/F cell appear to produce clinical benefit in rare variants of sickle cell disease and probably in more commonly encountered patients. Fetal hemoglobin interferes with polymerization of Hb S in vitro, but laboratory studies carried out with homogeneous hemoglobin solutions are inadequate models of events in vivo, because RBCs are heterogeneous in their MCHC and Hb F content. Studies of hemoglobin switching in sheep, in tissue culture, and then in baboons led to use of 5-azacytidine for induction of increased Hb F synthesis in SS patients. Drug trials were successful but the theory that led to them was not. An alternate theory, not without flaws, led to the use of hydroxyurea. Chronic administration of the drug can lead to very impressive increases in Hb F synthesis and apparent clinical benefit. It is not clear that such clinical benefit is real rather than a placebo effect. Nor is it entirely clear that all of the effect of hydroxyurea can be related to increased production of F cells and increased F/F cell. Controlled clinical trials and studies of the properties of RBCs from treated patients may answer those questions. It is also likely that they will not only raise still other questions but probably show that our current understanding of the biology and treatment of sickle cell disease is far from complete.

A new cation-exchange microcolumn employing a borate buffer reportedly separates hemoglobin A1c from other labile glycosylated intermediates in a one step procedure. The effect of commonly occurring abnormal hemoglobins on the hemoglobin A1c (Hb A1c) levels as determined by this new technique has been studied and compared to the values obtained by isoelectric focusing. If a patient has hemoglobin levels in the normal range, or if HbS, HbE, or an elevated HbA2 are present, then hemoglobin A1c levels as estimated by this new column are unaffected or decreased. However, if HbF is elevated, a marked increase in the HbA1c level is observed. The increase is almost directly proportional to the HbF level up to 40 percent. This new column technique is therefore useful in eliminating labile glycosylated hemoglobin intermediates but must be coupled with other techniques if high HbF values are present.

Asphyxia is defined as hypoxia with hypercapnia; the physiology and pathophysiology of the O2-saturation-curve in maternal and fetal blood is given. Recent results about the redistribution of the cardiac output in chronically instrumented fetal lambs under the conditions of asphyxia, hypoxia and acidosis are reported. The life-threatening consequences of ischaemic situations in the regional circulations for liver, gut, kidney and lung are described, particularly in the newborn. It is stressed that in asphyxiated babies which show circulatory and/or cerebral disturbances, further organ functions (e.g. respiration, excretion) may be seriously damaged.

Differences in the local structure of the heme in the isolated alpha-, beta- and gamma-chains of the adult and fetal human hemoglobin are detected by XANES (X-ray absorption near-edge structure) spectroscopy. The ligand bonding angle to the iron ion in the ligated forms and the displacement of the Fe respect to the porphyrin plane in the deoxy forms are found to be different for each chain.