Description and analysis of metabolic connectivity and dynamics in the human red blood cell

The human red blood cell (hRBC) metabolic network is relatively simple compared with other whole cell metabolic networks, yet too complicated to study without the aid of a computer model. Systems science techniques can be used to uncover the key dynamic features of hRBC metabolism. Herein, we have studied a full dynamic hRBC metabolic model and developed several approaches to identify metabolic pools of metabolites. In particular, we have used phase planes, temporal decomposition, and statistical analysis to show hRBC metabolism is characterized by the formation of pseudoequilibrium concentration states. Such equilibria identify metabolic "pools" or aggregates of concentration variables. We proceed to define physiologically meaningful pools, characterize them within the hRBC, and compare them with those derived from systems engineering techniques. In conclusion, systems science methods can decipher detailed information about individual enzymes and metabolites within metabolic networks and provide further understanding of complex biological networks.     

Membrane rafts of the human red blood cell

The cell type of election for the study of cell membranes, the mammalian non-nucleated erythrocyte, has been scarcely considered in the research of membrane rafts of the plasma membrane. However, detergent-resistant-membranes (DRM) were actually first described in human erythrocytes, as a fraction resisting solubilization by the nonionic detergent Triton X-100. These DRMs were insoluble entities of high density, easily pelleted by centrifugation, as opposed to the now accepted concept of lipid raft-like membrane fractions as material floating in low-density regions of sucrose gradients. The present article reviews the available literature on membrane rafts/DRMs in human erythrocytes from an historical point of view, describing the experiments that provided the solution to the above described discrepancy and suggesting possible avenue of research in the field of membrane rafts that, moving from the most studied model of living cell membrane, the erythrocyte’s, could be relevant also for other cell types.     

Dynamic simulation of red blood cell metabolism and its application to the analysis of a pathological condition

Background  

Cell simulation, which aims to predict the complex and dynamic behavior of living cells, is becoming a valuable tool. In silico models of human red blood cell (RBC) metabolism have been developed by several laboratories. An RBC model using the E-Cell simulation system has been developed. This prototype model consists of three major metabolic pathways, namely, the glycolytic pathway, the pentose phosphate pathway and the nucleotide metabolic pathway. Like the previous model by Joshi and Palsson, it also models physical effects such as osmotic balance. This model was used here to reconstruct the pathology arising from hereditary glucose-6-phosphate dehydrogenase (G6PD) deficiency, which is the most common deficiency in human RBC.     

Elasticity of the human red blood cell skeleton

We have measured by optical tweezers micromanipulations the area expansion and the shear moduli of spectrin skeletons freshly extracted from human red blood cells, in different controlled salinity conditions. At medium osmolarity (150 mOsm/kg), we measure KC=9.7+/-3.4 microN/m, muC=5.7+/-2.3 microN/m, KC/muC=2.1+/-0.7. When decreasing the osmolarity, both KC and muC decrease, while KC/muC is nearly constant and equal to about 2. This result is consistent with the predictions made when modeling the spectrin skeleton by a two-dimensional triangular lattice of springs. From the measured elastic moduli we estimate the persistence length of a spectrin filament: xi approximately 2.5 nm at 150 mOsm/kg.     

Permeability of the human red blood cell tomeso-erythritol

Summary Using¹⁴C-erythritol, we measured net as well as unidirectional erythritol fluxes. Up to near saturation, net and unidirectional fluxes were virtually identical and linearly related to the erythritol concentration in the medium (isotonic saline). No saturation of the transfer system was observed. At 20°C, a maximum of 60 to 70% of the erythritol flux could be inhibited by glucose, phlorizin, or a combination of both substances. Dinitrofluorobenzene and HgCl₂ also reduce erythritol permeability. These findings confirm the earlier conclusion of F. Bowyer and W. F. Widdas that the glucose transport system is involved in erythritol permeation. Glycerol partially inhibits the glucose-phlorizin-sensitive component of erythritol flux, but not the glucose-phlorizin-insensitive component. Apparently glycerol has a slight affinity to that portion of the glucose transport system which is involved in erythritol transfer, whereas the glucosephlorizin-insensitive fraction of erythritol movements is not identical with the glycerol system. This latter inference is supported by the observation that, in contrast to glycerol permeability, erythritol permeability is insensitive to variations of pH or to the addition of copper. The apparent activation energy of the glucose-phlorizin-sensitive and-insensitive fractions of erythritol permeation are 22.2 and 20.7 kcal/mole, respectively. These values are not significantly different from one another.     

Sodium movements in the human red blood cell

Measurements were made of the sodium outflux rate constant, ^o k _Na, and sodium influx rate constant, ⁱ k _Na, at varying concentrations of extracellular (Na_o) and intracellular (Na_c) sodium. ^o k _Na increases with increasing [Na_o] in the presence of extracellular potassium (K_o) and in solutions containing ouabain. In K-free solutions which do not contain ouabain, ^o k _Na falls as [Na_o] rises from 0 to 6 mM; above 6 mM, ^o k _Na increases with increasing [Na_o]. Part of the Na outflux which occurs in solutions free of Na and K disappears when the cells are starved or when the measurements are made in solutions containing ouabain. As [Na_o] increases from 0 to 6 mM, ⁱ k _Na decreases, suggesting that sites involved in the sodium influx are becoming saturated. As [Na_c] increases, ^o k _Na at first increases and then decreases; this relation between ^o k _Na and [Na_c] is found when the measurements are made in high Na, high K solutions; high Na, K-free solutions; and in (Na + K)-free solutions. The relation may be the consequence of the requirement that more than one Na ion must react with the transport mechanism at the inner surface of the membrane before transport occurs. Further evidence has been obtained that the ouabain-inhibited Na outflux and Na influx in K-free solutions represent an exchange of Na_c for Na_o via the Na-K pump mechanism.     

Network-level analysis of metabolic regulation in the human red blood cell using random sampling and singular value decomposition

Background  

Extreme pathways (ExPas) have been shown to be valuable for studying the functions and capabilities of metabolic networks through characterization of the null space of the stoichiometric matrix (S). Singular value decomposition (SVD) of the ExPa matrix P has previously been used to characterize the metabolic regulatory problem in the human red blood cell (hRBC) from a network perspective. The calculation of ExPas is NP-hard, and for genome-scale networks the computation of ExPas has proven to be infeasible. Therefore an alternative approach is needed to reveal regulatory properties of steady state solution spaces of genome-scale stoichiometric matrices.     

The human red blood cell proteome and interactome

The red blood cell or erythrocyte is easily purified, readily available, and has a relatively simple structure. Therefore, it has become a very well studied cell in terms of protein composition and function. RBC proteomic studies performed over the last five years, by several laboratories, have identified 751 proteins within the human erythrocyte. As RBCs contain few internal structures, the proteome will contain far fewer proteins than nucleated cells. In this minireview, we summarize the current knowledge of the RBC proteome, discuss alterations in this partial proteome in varied human disease states, and demonstrate how in silico studies of the RBC interactome can lead to considerable insight into disease diagnosis, severity, and drug or gene therapy response. To make these latter points we focus on what is known concerning changes in the RBC proteome in Sickle Cell Disease.     

NMR water-proton spin-lattice relaxation time of human red blood cells and red blood cell suspensions

NMR water-proton spin-lattice relaxation times were studied as probes of water structure in human red blood cells and red blood cell suspensions. Normal saline had a relaxation time of about 3000 ms while packed red blood cells had a relaxation time of about 500 ms. The relaxation time of a red cell suspension at 50% hematocrit was about 750 ms showing that surface charges and polar groups of the red cell membrane effectively structure extracellular water. Incubation of red cells in hypotonic saline increases relaxation time whereas hypertonic saline decreases relaxation time. Relaxation times varied independently of mean corpuscular volume and mean corpuscular hemoglobin concentration in a sample population. Studies with lysates and resealed membrane ghosts show that hemoglobin is very effective in lowering water-proton relaxation time whereas resealed membrane ghosts in the absence of hemoglobin are less effective than intact red cells.     

Extreme pathway analysis of human red blood cell metabolism

The development of high-throughput technologies and the resulting large-scale data sets have necessitated a systems approach to the analysis of metabolic networks. One way to approach the issue of complex metabolic function is through the calculation and interpretation of extreme pathways. Extreme pathways are a mathematically defined set of generating vectors that describe the conical steady-state solution space for flux distributions through an entire metabolic network. Herein, the extreme pathways of the well-characterized human red blood cell metabolic network were calculated and interpreted in a biochemical and physiological context. These extreme pathways were divided into groups based on such criteria as their cofactor and by-product production, and carbon inputs including those that 1) convert glucose to pyruvate; 2) interchange pyruvate and lactate; 3) produce 2,3-diphosphoglycerate that binds to hemoglobin; 4) convert inosine to pyruvate; 5) induce a change in the total adenosine pool; and 6) dissipate ATP. Additionally, results from a full kinetic model of red blood cell metabolism were predicted based solely on an interpretation of the extreme pathway structure. The extreme pathways for the red blood cell thus give a concise representation of red blood cell metabolism and a way to interpret its metabolic physiology.     

Transport of sugars into human red blood cell ghosts

It has been found that human red cell ghosts react differently in the presence of various sugars in the medium. The stability of spheric ghosts is preserved in solutions of sugars entering red cells by means of the common carrier. In media of other sugars the ghosts' shapes change to shrunken, crenated forms and between the microscope slides to discoid ones. Under the conditions employed it was further observed that the incubation of fructose- or rhamnose-containing ghosts in solutions of sugars sharing the carriers led to an equilibration of sugars between the medium and the ghosts. The impermeability of ghosts for sugars not sharing the carriers was supported by the finding that fructose could be washed out to a much less extent than glucose. These results suggest that sugars without affinity to the carriers may move in the erythrocyte membrane through nonspecific sites (pores, channels).     

Temperature dependence of anion transport in the human red blood cell

Arrhenius plots of chloride and bromide transport yield two regions with different activation energies (Ea). Below 15 or 25 degrees C (for Cl- and Br-, respectively), Ea is about 32.5 kcal/mol; above these temperatures, about 22.5 kcal/mol (Brahm, J. (1977) J. Gen. Physiol. 70, 283-306). For the temperature dependence of SO4(2-) transport up to 37 degrees C, no such break could be observed. We were able to show that the temperature coefficient for the rate of SO4(2-) transport is higher than that for the rate of denaturation of the band 3 protein (as measured by NMR) or the destruction of the permeability barrier in the red cell membrane. It was possible, therefore, to extend the range of flux measurements up to 60 degrees C and to show that, even for the slowly permeating SO4(2-) in the Arrhenius plot, there appears a break, which is located somewhere between 30 and 37 degrees C and where Ea changes from 32.5 to 24.1 kcal/mol. At the break, the turnover number is approx. 6.9 ions/band 3 per s. Using 35Cl- -NMR (Falke, Pace and Chan (1984) J. Biol. Chem. 259, 6472-6480), we also determined the temperature dependence of Cl- -binding. We found no significant change over the entire range from 0 to 57 degrees C, regardless of whether the measurements were performed in the absence or presence of competing SO4(2-). We conclude that the enthalpy changes associated with Cl- - or SO4(2-)-binding are negligible as compared to the Ea values observed. It was possible, therefore, to calculate the thermodynamic parameters defined by transition-state theory for the transition of the anion-loaded transport protein to the activated state for Cl-, Br- and SO4(2-) below and above the temperatures at which the breaks in the Arrhenius plots are seen. We found in both regions a high positive activation entropy, resulting in a low free enthalpy of activation. Thus the internal energy required for carrying the complex between anion and transport protein over the rate-limiting energy barrier is largely compensated for by an increase of randomness in the protein and/or its aqueous environment.     

Kinetics of bicarbonate-chloride exchange across the human red blood cell membrane

The kinetics of bicarbonate-chloride exchange across the human red cell membrane was studied by following the time course of extracellular pH in a stopped-flow rapid-reaction apparatus during transfer of H+ into the cell by the CO2 hydration-dehydration cycle, under conditions where the rate of the process was determined by HCO3--Cl- exchange flux across the membrane. The flux of bicarbonate increased linearly with [HCO3-] gradient from 0.6 to 20 mM across the red cell membrane at both 37 degrees C and 2 degrees C, and decreased as transmembrane potential was increased by decreasing extracellular [Cl-]. An Arrhenius plot of the rate constants for the exchange indicates that the Q10 is strongly dependent on temperature, being about 1.7 between 24 degrees C and 42 degrees C and about 7 between 2 degrees C and 12 degrees C. These data agree well with the published values for Q10 of 1.2 between 24 degrees C and 40 degrees C and of 8 between 0 degrees C and 10 degrees C. The results suggest that different processes may determine the rate of HCO3- -Cl- exchange at low vs. physiological temperatures, and that the functional (and/or structural) properties of the red cell membrane vary markedly with temperature.     

Mutagenicity of some heterocyclic amines in Salmonella typhimurium with metabolic activation by human red blood cell cytosol.

Purified human red blood cell cytosol was used to activate the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) into mutagenic intermediate(s) in the Salmonella test. The liquid preincubation method in the presence of strain TA98 was utilized. In order to understand the mechanism involved in this metabolic activation, some modulators were incorporated in the medium. The results suggest that an oxygenated hemoprotein, probably oxyhemoglobin, is involved in the activation into genotoxic intermediate(s).     

Temperature dependence of anion transport in the human red blood cell

Arrhenius plots of chloride and bromide transport yield two regions with different activation energies (E_a). Below 15 or 25°C (for Cl⁻ and Br⁻, respectively), E_a is about 32.5 kcal/mol; above these temperatures, about 22.5 kcal/mol (Brahm, J. (1977) J. Gen. Physiol. 70, 283–306). For the temperature dependence of SO₄²⁻ transport up to 37°C, no such break could be observed. We were able to show that the temperature coefficient for the rate of SO₄²⁻ transport is higher than that for the rate of denaturation of the band 3 protein (as measured by NMR) or the destruction of the permeability barrier in the red cell membrane. It was possible, therefore, to extend the range of flux measurements up to 60°C and to show that, even for the slowly permeating SO₄²⁻ in the Arrhenius plot, there appears a break, which is located somewhere between 30 and 37°C and where E_a changes from 32.5 to 24.1 kcal/mol. At the break, the turnover number is approx. 6.9 ions/band 3 per s. Using ³⁵Cl⁻-NMR (Falke, Pace and Chan (1984) J. Biol. Chem. 259, 6472–6480), we also determined the temperature dependence of Cl⁻-binding. We found no significant change over the entire range from 0 to 57°C, regardless of whether the measurements were performed in the absence or presence of competing SO₄²⁻. We conclude that the enthalpy changes associated with Cl⁻-or SO₄²⁻-binding are negligible as compared to the E_a values observed. It was possible, therefore, to calculate the thermodynamic parameters defined by transition-state theory for the transition of the anion-loaded transport protein to the activated state for Cl⁻, Br⁻ and SO₄²⁻ below and above the temperatures at which the breaks in the Arrhenius plots are seen. We found in both regions a high positive activation entropy, resulting in a low free enthalpy of activation. Thus the internal energy required for carrying the complex between anion and transport protein over the rate-limiting energy barrier is largely compensated for by an increase of randomness in the protein and/or its aqueous environment.     

Dynamic simulations on the arachidonic acid metabolic network

Drug molecules not only interact with specific targets, but also alter the state and function of the associated biological network. How to design drugs and evaluate their functions at the systems level becomes a key issue in highly efficient and low–side-effect drug design. The arachidonic acid metabolic network is the network that produces inflammatory mediators, in which several enzymes, including cyclooxygenase-2 (COX-2), have been used as targets for anti-inflammatory drugs. However, neither the century-old nonsteriodal anti-inflammatory drugs nor the recently revocatory Vioxx have provided completely successful anti-inflammatory treatment. To gain more insights into the anti-inflammatory drug design, the authors have studied the dynamic properties of arachidonic acid (AA) metabolic network in human polymorphous leukocytes. Metabolic flux, exogenous AA effects, and drug efficacy have been analyzed using ordinary differential equations. The flux balance in the AA network was found to be important for efficient and safe drug design. When only the 5-lipoxygenase (5-LOX) inhibitor was used, the flux of the COX-2 pathway was increased significantly, showing that a single functional inhibitor cannot effectively control the production of inflammatory mediators. When both COX-2 and 5-LOX were blocked, the production of inflammatory mediators could be completely shut off. The authors have also investigated the differences between a dual-functional COX-2 and 5-LOX inhibitor and a mixture of these two types of inhibitors. Their work provides an example for the integration of systems biology and drug discovery.     

Pyruvate kinase-catalyzed ATP-formation in human red blood cell membranes

Previous studies on the linkage between enzymatically catalyzed ATP-generating reactions in the red blood cell membrane and the sodium and potassium transport in the control of overall glycolysis of human erythrocytes were controversial. In this study a significant amount of pyruvate kinase activity is shown to be localized within the membrane. Membrane fragments produce 20.5 mumol of ATP per 10(10) membranes per hour from phosphoenolpyruvate and ADP. The kinetics of the membrane-localized pyruvate kinase do not differ from those of the enzyme from hemolysates. The results clearly document the presence of the second ATP-generating enzyme of glycolysis, pyruvate kinase, in human red blood cell membranes. The main fraction of the enzyme is deeply hidden in the lipid layers of the membrane. It can be demasked by mechanical desintegration of membranes at high levels of activity. It is suggested that the amount of the membrane-localized fraction of pyruvate kinase is related to the clinical severity of the hemolytic process in pyruvate kinase deficiency.