HEMOGLOBIN: NORMAL VALUES

Hematologists are not in agreement as to the “normal” amount of hemoglobin in the blood, nor is there agreement as to what amount of hemoglobin can be considered “a hemoglobin value of 100 per cent.” Different hospitals base reports of hemoglobin on different standards, which obviously can be misleading.By biometric study of the great mass of data on hemoglobin content that has become available as a result of the blood procurement program, it should be possible to determine what “normal” values are and to provide a basis for uniformity in reporting.     

Limited synthesis of labile hemoglobin A1 in vivo and in vitro by the preexisting hemoglobin A1 in diabetic subjects

The synthesis of labile hemoglobin A1 in vivo was studied in subjects with non-insulin dependent diabetes mellitus, impaired and normal glucose tolerance. The labile hemoglobin A1 index defined as delta labile hemoglobin A1 divided by delta plasma glucose at 30 min after oral glucose load, representing the rate of labile hemoglobin A1 synthesis in vivo, was low in diabetic subjects and high in normal subjects, showing an inverse correlation with the amount of preexisting hemoglobin A1. The study on the synthesis of labile hemoglobin A1 in vitro showed a lower initial rate of synthesis and a smaller increase in labile hemoglobin A1 at saturation in red blood cells from diabetic subjects with a relatively large amount of preexisting hemoglobin A1, as opposed to red blood cells from normal subjects. Although the further study is necessary in which delta plasma glucose levels are kept relatively constant in each of 3 groups by glucose-clamp methods, our data suggest that the synthesis of labile hemoglobin A1 is limited in vivo and in vitro in diabetic subjects by the preexisting hemoglobin A1 due to the saturability of its synthesis.     

ANEMIA—Differentiating Between Thalassemia Minor and Iron Deficiency

Many of the conditions noted in examination of the blood of patients with thalassemia minor are much like those observed in patients with iron deficiency anemia. A study was made of similarities and contrasts between blood and bone marrow features in both conditions for purposes of differential diagnosis. A salient distinction is that bone marrow hemosiderin is present in normal amount in patients with thalassemia minor, but not in those with iron deficiency anemia. If therapy with iron does not restore hemoglobin values to normal, thalassemia minor is strongly suspect. Even in the latter disease, however, there may be small fluctuations in hemoglobin values, particularly in pregnancy. One must be alert to this possibility lest a slight, fleeting increase in hemoglobin be mistakenly ascribed to iron therapy.     

Anemia,differentiating between thalassemia minor and iron deficiency

Many of the conditions noted in examination of the blood of patients with thalassemia minor are much like those observed in patients with iron deficiency anemia. A study was made of similarities and contrasts between blood and bone marrow features in both conditions for purposes of differential diagnosis. A salient distinction is that bone marrow hemosiderin is present in normal amount in patients with thalassemia minor, but not in those with iron deficiency anemia. If therapy with iron does not restore hemoglobin values to normal, thalassemia minor is strongly suspect. Even in the latter disease, however, there may be small fluctuations in hemoglobin values, particularly in pregnancy. One must be alert to this possibility lest a slight, fleeting increase in hemoglobin be mistakenly ascribed to iron therapy.     

Acute upper gastrointestinal tract bleeding and recent alcohol ingestion

Alcohol ingestion is a reason for hospital admission for what may be termed trivial or even spurious gastrointestinal bleeding. This clinical entity can be recognized and hospital admission avoided. It is suggested that in the case of men under the age of 40 who present with a history of hematemesis and who are intoxicated to a variable degree but without a history of recent salicylate ingestion or previous dyspepsia, and in whom the hemoglobin level is above 13.0 g/dl it is acceptable management to send them home with instructions to seek help only if there is fresh, authenticated bleeding. This attitude is further supported by a normal blood volume measurement and the absence of blood from a gastric aspirate.     

High-resolution proton nuclear magnetic resonance studies of sickle cell hemoglobin.

High-resoluiton proton nuclear magnetic resonance spectroscopy at 250 MHz has been used to investigate sickle cell hemoglobin. The hyperfine shifted, the ring-current shifted, and the exchangeable proton resonances suggest that the heme environment and the subunit interfaces of the sickle cell hemoglobin molecule are normal. These results suggest that the low oxygen affinity in sickle cell blood is not due to conformational alterations in the heme environment or the subunit interfaces. The C-2 proton resonances of certain histidyl residues can serve as structural probes for the surface conformation of the hemoglobin molecule. Several sharp resonances in sickle cell hemoglobin are shifted upfield from their positions in normal adult hemoglobin. These upfield shifts, which are observed in both oxy and deoxy forms of the molecule under various experimental conditions, suggest that some of the surface residues of sickle cell hemoglobin are altered and they may be in a more hydrophobic environment as compared with that of normal human adult hemoglobin. These differences in surface conformation are pH and ionic strength specific. In particular, upon the addition of organic phosphates to normal and sickle cell hemoglobin samples, the differences in their aromatic proton resonances diminish. These changes in the surface conformation may, in part, be responsible for the abnormal properties of sickle cell hemoglobin.     

Alteration of T-state binding properties of naturally glycated hemoglobin, HbA1c

The thermodynamic and kinetic properties of the most abundant glycated hemoglobin in human blood, HbA1c, have been studied in detail. They display significant differences as compared to normal hemoglobin, HbA0, in that (1) the shape of the oxygen binding curve of HbA1c in the Hill plot is markedly asymmetrical, with a lower asymptote extending up to approximately 40% oxygen saturation, and the oxygen affinity of the T state being tenfold higher than in HbA0; (2) oxygen pulse experiments on HbA1c show a slower rate of ligand dissociation (k = 25 s-1) even at low levels of oxygen saturation, where the T state is largely predominant; (3) kinetics of CO combination to deoxy HbA1c followed by means of stopped-flow experiments reveal the presence of a quickly reacting component, whose fraction increases upon dilution of hemoglobin. These results show that in contrast to what has been stated by other authors, HbA1c displays functional properties markedly different from HbA0. Analysis indicates that glycation of human hemoglobin affects the T quaternary structure, bringing about a more "relaxed" T state and leading to preferential binding to one type of chain (which is unaffected by chloride ions).     

Hemoglobin-mediated nitric oxide signaling

The rate that hemoglobin reacts with nitric oxide (NO) is limited by how fast NO can diffuse into the heme pocket. The reaction is as fast as any ligand/protein reaction can be and the result, when hemoglobin is in its oxygenated form, is formation of nitrate in what is known as the dioxygenation reaction. As nitrate, at the concentrations made through the dioxygenation reaction, is biologically inert, the only role hemoglobin was once thought to play in NO signaling was to inhibit it. However, there are now several mechanisms that have been discovered by which hemoglobin may preserve, control, and even create NO activity. These mechanisms involve compartmentalization of reacting species and conversion of NO from or into other species such as nitrosothiols or nitrite which could transport NO activity. Despite the tremendous amount of work devoted to this field, major questions concerning precise mechanisms of NO activity preservation as well as if and how Hb creates NO activity remain unanswered.     

Structure and expression of genes involved in transport and storage of iron in red-blooded and hemoglobin-less antarctic notothenioids

Antarctic notothenioids are characterized by a drastic reduction of the hemoglobin content, a condition that reaches its extreme in icefish that, following a gene deletion event, are completely devoid of hemoglobin. To answer the question on what type of adaptive changes occurred in icefish to prevent accumulation of potentially dangerous ferrous iron, we investigated the genes of four proteins known to play a key role in iron metabolism. For this purpose, we cloned and sequenced the cDNAs encoding ceruloplasmin, transferrin, ferritin and divalent metal transporter 1. While the inferred amino acid sequences of transferrin from different Antarctic fish species showed a high level of similarity with the homologous proteins from other species, ceruloplasmin sequence featured amino acid substitutions affecting a copper binding site. Another peculiarity was the presence in subunit H of the icefish ferritin of the two sets of sites involved in iron oxidation and iron mineralization, which in mammals are located on two distinct ferritin subunits. Significant differences in the expression levels of the four genes were found between hemoglobinless and red-blooded notothenioids. An increased expression of ceruloplasmin mRNA in icefish was interpreted as a compensatory mechanism to prevent accumulation of ferrous iron in hemoglobinless fish. In icefish, the amounts of ferritin H-chain mRNA measured in liver, blood and head kidney were lower than in the same organs of the red-blooded fish. In the spleen of both fishes, the expression levels of ferritin H-chain were significantly lower than in the spleen of a "pink-blooded" notothenioid with an intermediate hemoglobin content. Finally, the amount of divalent metal transporter mRNA measured in the head-kidney was lower in the icefish than in the same organ of its red-blooded counterpart. These results indicate that the loss of hemoglobin in icefish is accompanied by remodulation of the iron metabolism.     

On the kinetics of erythroid cell differentiation in fetal mice. I. Microspectrophotometric determination of the hemoglobin content in erythroid cells during gestation

In a microspectrophotometric study, photographic emulsions and a computer are used for measuring the hemoglobin content of a large number (about 50,000) of erythroid cells in fetal mice. Histograms of the hemoglobin content in erythroid cells illustrate the kinetics of erythropoiesis in yolk sac derived nucleated cells in the fetal peripheral blood, in fetal liver, and in fetal spleen. After the occasional extrusion of their nucleus, yolk sac derived erythrocytes remain as “macrocytes” in fetal circulation two or three days longer than the nucleated yolk sac derived erythrocytes do. Erythrocytes in fetal liver have a constant hemoglobin content of 28 pg ² until day 17 of gestation. During further erythropoiesis in liver and then in the spleen, this amount is gradually adapted to the normal hemoglobin content in red blood cells of 16 pg.     

Global allostery model of hemoglobin. Modulation of O(2) affinity,cooperativity, and Bohr effect by heterotropic allosteric effectors

The O(2) equilibria of human adult hemoglobin have been measured in a wide range of solution conditions in the presence and absence of various allosteric effectors in order to determine how far hemoglobin can modulate its O(2) affinity. The O(2) affinity, cooperative behavior, and the Bohr effect of hemoglobin are modulated principally by tertiary structural changes, which are induced by its interactions with heterotropic allosteric effectors. In their absence, hemoglobin is a high affinity, moderately cooperative O(2) carrier of limited functional flexibility, the behaviors of which are regulated by the homotropic, O(2)-linked T/R quaternary structural transition of the Monod-Wyman-Changeux/Perutz model. However, the interactions with allosteric effectors provide such "inert" hemoglobin unprecedented magnitudes of functional diversities not only of physiological relevance but also of extreme nature, by which hemoglobin can behave energetically beyond what can be explained by the Monod-Wyman-Changeux/Perutz model. Thus, the heterotropic effector-linked tertiary structural changes rather than the homotropic ligation-linked T/R quaternary structural transition are energetically more significant and primarily responsible for modulation of functions of hemoglobin.     

Estimation of nitric oxide concentration in blood for different rates of generation. Evidence that intravascular nitric oxide levels are too low to exert physiological effects

Endothelium-derived nitric oxide (NO) is a potent vasodilator in the cardiovascular system. Several lines of experimental evidence suggest that NO or NO equivalents may also be generated in the blood. However, blood contains a large amount of hemoglobin (Hb) in red blood cells (RBCs). The RBC-encapsulated Hb can react very quickly with NO, which is only limited by the rate of NO diffusion into the RBCs. It is unclear what the possible NO concentration levels in blood are and how the NO diffusion coefficient (D) and the permeability (Pm) of RBC membrane to NO affect the level of NO concentration. In this study, a steady-state concentration experimental method combined with a spherical diffusion model are presented for determining D and Pm and examining the effect of NO generation rate (V0) and hematocrit (Hct) on NO concentration. It was determined that Pm is 4.5 +/- 1.5 cm/s and D is 3410 +/- 50 microm2/s at 37 degrees C. Simulations based on experimental parameters show that, when the rate of NO formation is as high as 100 nm/s, the maximal NO concentration in blood is below 0.012 nM at Pm = 4.5 cm/s and Hct = 45%. Thus, it is unlikely that NO is directly exported or generated from the RBC as an intravascular signaling molecule, because its concentration would be too low to exert a physiological role. Furthermore, our results suggest that, if RBCs export NO bioactivity, this would be through NO-derived species that can release or form NO rather than NO itself.     

Sulfide-hemoglobin interactions in the sulfide-tolerant salt marsh resident,the California killifish Fundulus parvipinnis

Summary Sulfide can potentially damage hemoglobin or be detoxified by hemoglobin. In the sulfide-tolerant California killifish neither seems to be the case at environmentally realistic (micromolar) and physiologically relevant (millimolar) sulfide concentrations. An 8-h exposure of killifish to 5 and 8 mmol sulfide · 1^-1 results in 50–100% mortality, but not due to sulfhemoglobin (where sulfide covalently binds to the porphyrin) nor ferric hemoglobin (Hb⁺), both dysfunctional hemoglobin derivatives. Killifish hemoglobin converts to sulfhemoglobin in vitro only in the presence of 1–5 mmol sulfide · 1^-1. The amount of sulfhemoglobin formed increases with time and heme concentration but decreases with pH. Hb⁺ binds sulfide as ferric hemoglobin sulfide (Hb⁺S, an unstable complex where sulfide ligates to the iron), and also as sulfhemoglobin. Killifish blood does not catalyze the oxidation of 10–500 mol sulfide · 1^-1 to any appreciable extent. Radiolabeled sulfide incubated with oxyhemoglobin or whole blood disappears at rates greater than in buffers, but only minimal amounts of thiosulfate and no sulfate nor sulfite are formed (elemental sulfur and bound sulfide not quantified). Sulfide disappearance rates increase linearly with initial sulfide concentration. Hb⁺ does catalyze the oxidation of sulfide to thiosulfate in vitro. Similar experiments on another sulfide-tolerant species, the long-jawed mudsucker Gillichthys mirabilis, produced similar results.Abbreviations ANOVA analysis of variance - BV benzyl viologen - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HPLC high-pressure liquid chromatography - RBC red blood cells - SHb sulfhemoglobin     

Cytotoxic action of polymorphonuclear leukocytes on tumor and normal cells during ascite tumor development in vitro and in vivo

A vast number of studies, including the authors' own research, support the important role polymorphonuclear leukocytes (PMNL) in the development of ascite tumors. The method of luminol-dependent chemiluminescence (CL) was used to show the presence of two functionally different PMNL pools in a tumor-bearing organism: 1) "primed" PMNL, which circulate in the blood stream, and 2) "activated" PMNL, which are accumulated in the tumor zone and are capable of spontaneous CL. The purpose of the present investigation was to compare cytotoxic effects of primed and activated PMNL on tumor cells (ascite Ehrlich carcinoma (AEC), ascite Zajdel hepatoma) upon co-cultivation, as well as on normal cells of the organism, erythrocytes in vitro and in vivo. Upon stimulation with phorbol myristate acetate (PMA), PMNL effectively damaged AEC cells within the first 24 h until PMNL apoptosis occurred. Upon further co-cultivation, the tumor cells grew in number, which suggest the participation of PMNL in tumor protection. When stimulated with PMNL, pools suppressed tumor growth in vitro, since in this case the cytotoxicity was due to both reactive oxygen species and proteolytic enzymes. As it has been shown earlier by the authors, the functional potential of PMNL increases many times during tumor growth, and we suggested that not only tumor but also normal cells could be damaged. In this connection, we have studied the cytotoxic effect of primed and activated PMNL on rat erythrocytes in vitro on their co-cultivation. On stimulation with PMA, the rate of lysis of erythrocytes by primed PMNL increase many times compared to the norm. The fMLP-stimulated cytotoxity was 1.5-2.0 times higher than in the norm. Activated PMNL without stimulation are capable of producing only a partial lysis of erythrocytes (5-7 %). In order to assess the cytotoxic action of PMNL on erythrocytes in vivo, the hemoglobin content in erythrocytes and blood plasm of rats was measured in the course of tumor growth. The hemoglobin content in erythocytes during growth tumor decreased from 135 +/- 10 to 85 +/- 5 g/l, whereas in the blood plasm the hemoglobin content gradually increased by almost two times. The results enable us to suggest that one of death causes of tumor-bearing organisms may be the cytotoxic action of PMNL on normal cells of the organism caused by hyperproduction of ROS.     

Assessment of Blood Contamination in Biological Fluids Using MALDI-TOF MS

Biological fluid sample collection often includes the risk of blood contamination that may alter the proteomic profile of biological fluid. In proteomics studies, exclusion of contaminated samples is usually based on visual inspection and counting of red blood cells in the sample; analysis of specific blood derived proteins is less used. To fill the gap, we developed a fast and sensitive method for ascertainment of blood contamination in crude biological fluids, based on specific blood-derived protein, hemoglobin detection by MALDI-TOF MS. The MALDI-TOF MS based method allows detection of trace hemoglobin with the detection limit of 0.12 nM. UV-spectrometry, which was used as reference method, was found to be less sensitive. The main advantages of the presented method are that it is fast, effective, sensitive, requires very small sample amount and can be applied for detection of blood contamination in various biological fluids collected for proteomics studies. Method applicability was tested on human cerebrospinal and follicular fluid, which proteomes generally do not contain hemoglobin, however, which possess high risk for blood contamination. Present method successfully detected the blood contamination in 12 % of cerebrospinal fluid and 24 % of follicular fluid samples. High percentage of contaminated samples accentuates the need for initial inspection of proteomic samples to avoid incorrect results from blood proteome overlap.     

A new sensitive assay reveals that hemoglobin is oxidatively modified in vivo

Free radical formation in heme proteins is recognised as a factor in mediating the toxicity of peroxides in oxidative stress. As well as initiating free radical damage, heme proteins damage themselves. Under extreme conditions, where oxidative stress and low pH coincide (e.g., myoglobin in the kidney following rhabdomyolysis and hemoglobin in the CSF subsequent to subarachnoid hemorrhage), peroxide can induce covalent heme to protein cross-linking. In this paper we show that, even at neutral pH, the heme in hemoglobin is covalently modified by oxidation. The product, which we term OxHm, is a "green heme" iron chlorin with a distinct optical spectrum. OxHm formation can be quantitatively prevented by reductants of ferryl iron, e.g., ascorbate. We have developed a simple, robust, and reproducible HPLC assay to study the extent of OxHm formation in the red cell in vivo. We show that hemoglobin is oxidatively damaged even in normal blood; approximately 1 in 2,000 heme groups exist as OxHm in the steady state. We used a simple model (physical exercise) to demonstrate that OxHm increases significantly during acute oxidative stress. The exercise-induced increase is short-lived, suggesting the existence of an active mechanism for repairing or removing the damaged heme proteins.     

What befalls the proteins and water in a living cell when the cell dies?

The solvency of solutes of varying molecular size in the intracellular water of freshly-killed Ehrlich carcinoma cells fits the same theoretical curve that describes the solvency of similar solutes in a 36% solution of native bovine hemoglobin--a protein found only in red blood cells and making up 97.3% of the red cell's total intracellular proteins. The merging of the two sets of data confirms the prediction of the AI Hypothesis that key intracellular protein(s) in dying cells undergo(es) a transition from: (1) one in which the polypeptide NHCO groups assume a fully-extended conformation with relatively strong power of polarizing and orienting the bulk-phase water in multilayers; to (2) one in which most of the polypeptide NHCO groups are engaged in alpha-helical and other "introvert" conformations (see below for definition) with much weaker power in polarizing-orienting multilayers of bulk-phase water. This concordance of the two sets of data also shows that what we now call native hemoglobin--supposedly denoting hemoglobin found in its natural state in living red blood cells--, in fact, more closely resembles the water-polarizing, and -orienting intracellular proteins in dead cells. Although in the dead Ehrlich carcinoma cells as well as in the 36% solution of native hemoglobin, much of the protein's polypeptide NHCO groups are engaged in alpha-helical and other "introvert" conformation (Perutz 1969; Weissbluth 1974), both systems produce a weak but nonetheless pervasive and "long-range" water polarization and orientation. It is suggested that in both the dead Ehrlich carcinoma ascites cells and in the 36% native bovine hemoglobin solution, enough polypeptide NHCO groups assume the fully-extended conformation to produce the weak but far-reaching multilayer water polarization and orientation observed.     

Analysis of cooperativity in hemoglobin. Valency hybrids, oxidation, and methemoglobin replacement reactions.

An allosteric model proposed previously for structure-function relations in hemoglobin is applied to the analysis of low- and high-spin valency hybrids. By assuming that the low-spin oxidized chains have the tertiary structure of oxygenated chains while the high-spin oxidized chains have a tertiary structure intermediate between that of deoxygenated and oxygenated chains, the model parameters associated with the different valency hybrids can be obtained, and their equilibrium properties can be estimated. The hybrid results are used also to provide an interpretation of methemoglobin and its ligand replacement reactions and of the oxidation-reduction equilibrium of normal hemoglobin. For the various systems studied it is found that the effects of pH and 2,3-diphosphoglycerate are in agreement with the model.