Phase separation in aqueous solutions of lens gamma-crystallins: special role of gamma s.

We have studied liquid-liquid phase separation in aqueous ternary solutions of calf lens gamma-crystallin proteins. Specifically, we have examined two ternary systems containing gamma s--namely, gamma IVa with gamma s in water and gamma II with gamma s in water. For each system, the phase-separation temperatures (Tph (phi)) alpha as a function of the overall protein volume fraction phi at various fixed compositions alpha (the "cloud-point curves") were measured. For the gamma IVa, gamma s, and water ternary solution, a binodal curve composed of pairs of coexisting points, (phi I, alpha 1) and (phi II, alpha II), at a fixed temperature (20 degrees C) was also determined. We observe that on the cloud-point curve the critical point is at a higher volume fraction than the maximum phase-separation temperature point. We also find that typically the difference in composition between the coexisting phases is at least as significant as the difference in volume fraction. We show that the asymmetric shape of the cloud-point curve is a consequence of this significant composition difference. Our observation that the phase-separation temperature of the mixtures in the high volume fraction region is strongly suppressed suggests that gamma s-crystallin may play an important role in maintaining the transparency of the lens.     

Counterflow centrifugation of red cell populations: a cell age related separation technique

S ummary . Red cell populations were fractionated on the basis of differences in density by a centrifugation method described by Murphy and on the basis of differences in mean cell volume (MCV) by counterflow centrifugation. By ⁵⁹Fe-incorporation and determination of the HbA_1c content, both methods were studied for their ability to separate red cell populations into fractions of different mean cell age. It can be concluded that separation on the basis of differences in cell volume results in a linear separation according to age whereas separation on the basis of density only results in an accumulation of very young cells in the top fractions. A gradual decrease in cell volume with age, combined with a constant haemoglobin concentration in the cells, indicates release of haemoglobin from the red cells during their lifespan.     

Phase separation in lens cytoplasm is genetically linked to cataract formation in the Philly mouse.

The variation of the phase-separation temperature, Tc, in lenses was studied during the postnatal development of three genetically different mouse strains: Swiss-Webster, Philly, and the (Swiss-Webster x Philly)F1 hybrid. The general behavior of Tc during early postnatal development has two stages: in stage I, Tc increased to a maximum and then, in stage II, Tc decreased. Philly mice are a strain that develops hereditary cataracts about 36 days following birth. In F1 hybrids of Philly and Swiss-Webster mice, cataracts appeared about 49 days following birth, approximately equal to 13 days later in development than in the Philly mice. In the Philly and hybrid mice, stage I and stage II were followed by stage III in which Tc reached a minimum value and then increased toward body temperature. The values of Tc at birth, the slope of the increase during stage I, and the maximum Tc were characteristic for each mouse strain. These results establish that the behavior of the temperature of the phase separation Tc in mouse lens is linked to the genetic strain of the mice and that the value of Tc at birth is an early indicator of lenses that will develop cataracts and lenses that will develop normally.     

Binary-liquid phase separation of lens protein solutions.

We have determined the coexistence curves (plots of phase-separation temperature T versus protein concentration C) for aqueous solutions of purified calf lens proteins. The proteins studied, calf gamma IIIa-, gamma IIIb-, and gamma IVa-crystallin, have very similar amino acid sequences and three-dimensional structures. Both ascending and descending limbs of the coexistence curves were measured. We find that the coexistence curves for each of these proteins and for gamma II-crystallin can be fit, near the critical point, to the function /(Cc-C)/Cc/ = A [(Tc - T)/Tc]beta, where beta = 0.325, Cc is the critical protein concentration in mg/ml, Tc is the critical temperature for phase separation in K, and A is a parameter that characterizes the width of the coexistence curve. We find that A and Cc are approximately the same for all four coexistence curves (A = 2.6 +/- 0.1, Cc = 289 +/- 20 mg/ml), but that Tc is not the same. For gamma II- and gamma IIIb-crystallin, Tc approximately 5 degrees C, whereas for gamma IIIa- and gamma IVa-crystallin, Tc approximately 38 degrees C. By comparing the published protein sequences for calf, rat, and human gamma-crystallins, we postulate that a few key amino acid residues account for the division of gamma-crystallins into low-Tc and high-Tc groups.     

Observation of liquid-liquid phase separation for eye lens gammaS-crystallin

gammaS-crystallin (gammaS) is an important human and bovine eye lens protein involved in maintaining the transparency of the eye. By adding small amounts of polyethylene glycol (PEG) to the binary aqueous bovine gammaS solutions, we have observed liquid-liquid phase separation (LLPS) at -8 degrees C and revealed that, in the binary gammaS-water system, this phase transition would occur at -28 degrees C. We have measured both the effect of PEG concentration on the LLPS temperature and proteinPEG partitioning between the two liquid coexisting phases. We use our measurements of proteinPEG partitioning to determine the nature and the magnitude of the gammaS-PEG interactions and to quantitatively assess the effectiveness of PEG as a crystallizing agent for gammaS. We use our measurements of LLPS temperature as a function of protein and PEG concentration to successfully determine the location of the critical point for the binary gammaS-water system. This phase transition cannot be observed in the absence of PEG because it is inaccessible due to the freezing of the system. Our findings indicate that the effective interactions between gammaS molecules in the binary gammaS-water system are attractive. We compare the magnitude of the attraction found for gammaS with the results obtained for the other gamma-crystallins for which the critical temperature is located above the freezing point of the system. This work suggests that PEG can be used to reveal the existence of LLPS for a much wider range of binary protein-water systems than known previously.     

Phase separation and rotor self-assembly in active particle suspensions

Adding a nonadsorbing polymer to passive colloids induces an attraction between the particles via the "depletion" mechanism. High enough polymer concentrations lead to phase separation. We combine experiments, theory, and simulations to demonstrate that using active colloids (such as motile bacteria) dramatically changes the physics of such mixtures. First, significantly stronger interparticle attraction is needed to cause phase separation. Secondly, the finite size aggregates formed at lower interparticle attraction show unidirectional rotation. These micro-rotors demonstrate the self-assembly of functional structures using active particles. The angular speed of the rotating clusters scales approximately as the inverse of their size, which may be understood theoretically by assuming that the torques exerted by the outermost bacteria in a cluster add up randomly. Our simulations suggest that both the suppression of phase separation and the self-assembly of rotors are generic features of aggregating swimmers and should therefore occur in a variety of biological and synthetic active particle systems.     

Phase separation in fluids with many interacting components

Fluids in natural systems, like the cytoplasm of a cell, often contain thousands of molecular species that are organized into multiple coexisting phases that enable diverse and specific functions. How interactions between numerous molecular species encode for various emergent phases is not well understood. Here, we leverage approaches from random-matrix theory and statistical physics to describe the emergent phase behavior of fluid mixtures with many species whose interactions are drawn randomly from an underlying distribution. Through numerical simulation and stability analyses, we show that these mixtures exhibit staged phase-separation kinetics and are characterized by multiple coexisting phases at steady state with distinct compositions. Random-matrix theory predicts the number of coexisting phases, validated by simulations with diverse component numbers and interaction parameters. Surprisingly, this model predicts an upper bound on the number of phases, derived from dynamical considerations, that is much lower than the limit from the Gibbs phase rule, which is obtained from equilibrium thermodynamic constraints. We design ensembles that encode either linear or nonmonotonic scaling relationships between the number of components and coexisting phases, which we validate through simulation and theory. Finally, inspired by parallels in biological systems, we show that including nonequilibrium turnover of components through chemical reactions can tunably modulate the number of coexisting phases at steady state without changing overall fluid composition. Together, our study provides a model framework that describes the emergent dynamical and steady-state phase behavior of liquid-like mixtures with many interacting constituents.

Fluids composed of many components with multiple coexisting phases are widespread in living and soft matter systems. A striking example occurs in the eukaryotic cell, where distinct biochemical pathways are compartmentalized into membraneless organelles called biomolecular condensates, which often form through liquid–liquid phase separation (1–3). Unlike two-phase oil–water mixtures, the cellular milieu is organized into tens of coexisting phases, each of which is enriched in specific biomolecules (1, 2, 4–8). Other prominent examples include microbial ecosystems that organize into fluid-like communities (9–11), self-assembling colloidal systems (12, 13), and synthetic multiphase materials derived from biomolecules (14, 15). Despite their extensive prevalence, our understanding of how microscopic interaction networks between individual constituents encode emergent multiphase behavior remains limited.Delineating the coexisting phases of a heterogeneous mixture is a problem with a rich history (16)—determined by constraints of chemical, mechanical, and thermal equilibrium. In mixtures with few components (fewer than five), a combination of theory, simulation, and experiment has enabled extensive characterization of phase-separation kinetics and equilibrium coexistence (17–23) and the interplay between phase separation and chemical reactions (19, 24, 25). In the biological context, recent studies have begun to connect biomolecular features to their macroscopic phase behavior in binary or ternary mixtures (7, 26, 27). However, as the number of components increases, determining the emergent phase behavior from the underlying constraints becomes unwieldy and intractable—from both analytical and numerical standpoints, except for very particular systems such as polydisperse blends of a single species (28). An alternate approach, originally proposed by Sear and Cuesta (29), aims to characterize the phase behavior of mixtures that contain many components whose pairwise interactions are drawn from a random distribution. By building on results on properties of random matrices, originally identified by Wigner (30) and subsequently applied in various contexts (31–33), they relate the initial direction of phase separation to properties of the interaction distribution, subsequently confirmed independently by simulation (34). These results, however, are limited to describing only the initial direction of phase separation for marginally stable fluid mixtures (i.e., coinciding exactly at the spinodal). Consequently, little is known about the overall phase behavior of fluid mixtures that spontaneously demix (i.e., within the spinodal)—including kinetics beyond the initial direction of phase separation or the number and composition of coexisting phases at equilibrium. More generally, the emergent phase behavior of fluid mixtures with many randomly interacting components is not well understood. This lack of understanding, in turn, limits our ability to rationally program fluid mixtures with different macroscopic properties.Here, we develop a dynamic model of phase separation in fluid mixtures with many randomly interacting components. Through simulation of the model, we demonstrate that fluid mixtures with many components exhibit characteristic similarities in phase-separation kinetics and in the number and compositional features of coexisting phases at steady state, even when the underlying interactions are random. We propose a simple model, combining insights from random-matrix theory and dynamical systems analyses, that predicts dynamical and steady-state characteristics of the emergent phase behavior. We subsequently discuss two distinct ensembles (or component design strategies) that encode either linear or nonmonotic scaling (i.e., with an optima) between the number of coexisting phases and components. Finally, we extend our framework to incorporate chemical reactions and show that active turnover of components can tunably modulate the number of coexisting phases at steady state even without altering overall fluid composition. Overall, our model provides a framework to predict and design emergent multiphase kinetics, compositions, and steady-state properties in fluid mixtures with many interacting components.     

Phase separation in the outer membrane of Escherichia coli

Gram-negative bacteria are surrounded by a protective outer membrane (OM) with phospholipids in its inner leaflet and lipopolysaccharides (LPS) in its outer leaflet. The OM is also populated with many β-barrel outer-membrane proteins (OMPs), some of which have been shown to cluster into supramolecular assemblies. However, it remains unknown how abundant OMPs are organized across the entire bacterial surface and how this relates to the lipids in the membrane. Here, we reveal how the OM is organized from molecular to cellular length scales, using atomic force microscopy to visualize the OM of live bacteria, including engineered Escherichia coli strains and complemented by specific labeling of abundant OMPs. We find that a predominant OMP in the E. coli OM, the porin OmpF, forms a near-static network across the surface, which is interspersed with barren patches of LPS that grow and merge with other patches during cell elongation. Embedded within the porin network is OmpA, which forms noncovalent interactions to the underlying cell wall. When the OM is destabilized by mislocalization of phospholipids to the outer leaflet, a new phase appears, correlating with bacterial sensitivity to harsh environments. We conclude that the OM is a mosaic of phase-separated LPS-rich and OMP-rich regions, the maintenance of which is essential to the integrity of the membrane and hence to the lifestyle of a gram-negative bacterium.

Diderm bacteria, such as Escherichia coli, are surrounded by an outer membrane (OM) that protects cells against the immune systems of plants and animals, contributes to the mechanical stability of the cell, and excludes many classes of antibiotics, thereby contributing to antimicrobial resistance (1, 2). The OM is comprised of an asymmetric bilayer of phospholipids in the inner leaflet, lipopolysaccharides (LPS) in the outer leaflet, and many outer-membrane proteins (OMPs). OMPs are hugely diverse β-barrel proteins that can be present at hundreds to hundreds of thousands of copies per cell (3). They have been shown to be relatively static (4), probably due to promiscuous protein–protein interactions and binding of LPS that exists in a slow-moving, liquid-crystalline state (5, 6). Using fluorescent labels, some OMPs have been shown to cluster into supramolecular islands of ∼0.3- to 0.5-μm sizes (4, 7–9). However, it remains unknown how abundant OMPs are organized across the entire bacterial surface and how this relates to the lipids in the membrane.To address this fundamental question, we have imaged the entire surface of live and metabolically active bacteria at nanometer resolution using atomic force microscopy (AFM). Applying such large-scale, high-resolution imaging on engineered E. coli strains and complementing it by specific labeling of abundant OMPs, we identify large-scale and near-static protein-rich networks interspersed with nanoscale domains that are enriched in LPS. Key components of the protein-rich networks are abundant trimeric porins such as OmpF, in addition to (the monomeric) OmpA, which forms noncovalent interactions to the underlying cell wall (10). By contrast, no significant protein content is detected in the LPS-rich domains, which are also found to grow and merge with other patches during cell elongation. When the LPS–phospholipid asymmetry of the OM is perturbed by mislocalization of phospholipids to the outer leaflet (11), we find deformation of the membrane rather than expansion of LPS patches, indicating the appearance of a new, phospholipid-enriched phase at the bacterial surface.     

Galactokinase activity and red blood cell age

Human erythrocyte galactokinase has been studied during red cell aging. The decay rate of enzyme activity is slower than glucose-6-phosphate dehydrogenase which is used as red cell age marker. The Michaelis constants for galactose and ATPMg²⁻ of galactokinase of young cells are similar to the Km’s of the enzyme of total cells, while the Km increases in old cells. The behaviour of galactokinase during cell aging is similar to that of other erythrocyte enzymes.     

Glycosylated and acetylated hemoglobins in relation to gestational age and red cell age

Cord blood samples were obtained at delivery from women at gestational periods of 30 to 36 weeks (preterm) and from a control group at 40 weeks of gestational age (term). Total glycosylated hemoglobin (GHb) was determined by affinity chromatography and the percentages of the minor Hbs FIa+b and FIc (of total Hb F) were determined by Bio-Rex 70 chromatography in whole blood samples and in red cell populations separated by Dextran 40 density gradient centrifugation. The absolute levels and the increase due to red cell aging of GHb, Hb FIa+b and Hb FIc were significantly reduced in the preterm samples compared to the term samples indicating decreased glycosylation in preterm fetus. When the nonglycosylated Hbs from term and preterm newborns were separated by Bio-Rex 70 chromatography after removal of the GHb by affinity chromatography, the acetylated form of Hb FIc also showed an increase with red cell aging indicating posttranslational enzymatic or nonenzymatic acetylation of Hb F during the entire life span of red cells. Moreover, as with GHb, the acetylated Hb was also decreased in the preterm newborns.     

Phase separation and liquid crystallization of complementary sequences in mixtures of nanoDNA oligomers

Using optical microscopy, we have studied the phase behavior of mixtures of 12- to 22-bp-long nanoDNA oligomers. The mixtures are chosen such that only a fraction of the sample is composed of mutually complementary sequences, and hence the solutions are effectively mixtures of single-stranded and double-stranded (duplex) oligomers. When the concentrations are large enough, such mixtures phase-separate via the nucleation of duplex-rich liquid crystalline domains from an isotropic background rich in single strands. We find that the phase separation is approximately complete, thus corresponding to a spontaneous purification of duplexes from the single-strand oligos. We interpret this behavior as the combined result of the energy gain from the end-to-end stacking of duplexes and of depletion-type attractive interactions favoring the segregation of the more rigid duplexes from the flexible single strands. This form of spontaneous partitioning of complementary nDNA offers a route to purification of short duplex oligomers and, if in the presence of ligation, could provide a mode of positive feedback for the preferential synthesis of longer complementary oligomers, a mechanism of possible relevance in prebiotic environments.     

Functional compartmentalization and metabolic separation in a prokaryotic cell

The prokaryotic cell is traditionally seen as a “bag of enzymes,” yet its organization is much more complex than in this simplified view. By now, various microcompartments encapsulating metabolic enzymes or pathways are known for Bacteria. These microcompartments are usually small, encapsulating and concentrating only a few enzymes, thus protecting the cell from toxic intermediates or preventing unwanted side reactions. The hyperthermophilic, strictly anaerobic Crenarchaeon Ignicoccus hospitalis is an extraordinary organism possessing two membranes, an inner and an energized outer membrane. The outer membrane (termed here outer cytoplasmic membrane) harbors enzymes involved in proton gradient generation and ATP synthesis. These two membranes are separated by an intermembrane compartment, whose function is unknown. Major information processes like DNA replication, RNA synthesis, and protein biosynthesis are located inside the “cytoplasm” or central cytoplasmic compartment. Here, we show by immunogold labeling of ultrathin sections that enzymes involved in autotrophic CO₂ assimilation are located in the intermembrane compartment that we name (now) a peripheric cytoplasmic compartment. This separation may protect DNA and RNA from reactive aldehydes arising in the I. hospitalis carbon metabolism. This compartmentalization of metabolic pathways and information processes is unprecedented in the prokaryotic world, representing a unique example of spatiofunctional compartmentalization in the second domain of life.

Compartmentalization is one of the distinguishing features of eukaryotic cells, which contain membrane-bound organelles in order to perform their specific functions more efficiently, like photosynthesis and CO₂ fixation in plants. Here, autotrophic CO₂ assimilation proceeds via the Calvin–Benson cycle in the stroma of chloroplasts. Evolutionary ancestors of chloroplasts, the cyanobacteria, contain the key carboxylating enzyme of this cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), in specifically insulated proteinaceous microcompartments, carboxysomes, to enrich both the carboxylase and its substrate CO₂ and to exclude competing O₂ at the active site of RubisCO (1). Other enzymes involved in autotrophy are located in the cytoplasm. Besides the Calvin–Benson cycle, autotrophic prokaryotes have developed at least six alternative ways of CO₂ fixation (2–4). The recently discovered autotrophic cycle, the dicarboxylate/4-hydroxybutyrate (DC/HB) cycle (Fig. 1A), functions in the anaerobic hyperthermophilic Crenarchaeota of the orders Desulfurococcales and Thermoproteales, for example, Ignicoccus hospitalis, Thermoproteus neutrophilus, and Pyrolobus fumarii (2, 5–9). In the carbon fixation phase of this pathway, one molecule acetyl-CoA, one molecule CO₂, and one molecule bicarbonate are converted to oxaloacetate via pyruvate synthase and phosphoenolpyruvate (PEP) carboxylase reactions. In the reduction phase, oxaloacetate is reduced to succinyl-CoA and then to 4-hydroxybutyrate, a name-giving intermediate of the cycle. Finally, in the acetyl-CoA regeneration phase of the cycle, 4-hydroxybutyrate is activated to the corresponding CoA-ester, dehydrated to crotonyl-CoA, and converted to two molecules of acetyl-CoA via β-oxidation reactions.Open in a separate windowFig. 1.The DC/HB cycle (adapted from ref. 3) (A), ultrastructure (B), and 3D model (C) of I. hospitalis. Enzymes: 1) pyruvate synthase, 2) pyruvate:water dikinase, 3) PEP carboxylase, 4) malate dehydrogenase, 5) fumarate hydratase, 6) fumarate reductase (natural electron donor is not known), 7) succinyl-CoA synthetase, 8) succinyl-CoA reductase (natural electron acceptor is not known), 9) succinic semialdehyde reductase, 10) 4-hydroxybutyrate-CoA ligase, 11) 4-hydroxybutyryl-CoA dehydratase, 12) crotonyl-CoA hydratase, 13) (S)-3-hydroxybutyryl-CoA dehydrogenase, and 14) acetoacetyl-CoA β-ketothiolase. Fd, ferredoxin; MV, methyl viologen. The enzymes studied in this work are highlighted in red. Ultrathin section (B) and 3D model of a semithin section (C) of a cryo-fixed, freeze-substituted, Epon-embedded cell. CCC (central cytoplasmic compartment), PCC (peripheric cytoplasmic compartment), ICM (inner cytoplasmic membrane), TN (tubular network), OCM (outer cytoplasmic membrane), and Neq (Nanoarchaeum equitans). 3D model highlights the TN originating from the CCC. (Scale bars, 500 nm.)The DC/HB cycle has first been discovered in the hyperthermophilic autotrophic sulfur-reducing archaeon Ignicoccus hospitalis (6) that grows under anaerobic conditions with molecular hydrogen and sulfur at T = 73 to 98 °C (T_opt 90 °C) (10). Apart from the unusual carbon assimilation pathway, Ignicoccus cells exhibit an extraordinary two-membrane ultrastructure (Fig. 1 B and C). The outer cellular membrane (not to be confused with the outer membrane of gram-negative bacteria) encases the intermembrane compartment, while the inner membrane surrounds a modified cytoplasm. Here, we suggest naming these compartments peripheric and central cytoplasmic compartment (CC), respectively, that are enclosed by outer and inner cytoplasmic membranes (CM). The peripheric CC contains a complex tubular network, derived from the central CC (10–13). The spatial compartmentalization goes along with a functional compartmentalization, as energy-conserving ATP synthase and H₂:sulfur oxidoreductase are located in the outer CM, while the major energy-consuming anabolic steps, DNA replication, RNA synthesis, and translation, take place in the central CC (14). Here, we present the subcellular localization of four enzymes of the DC/HB cycle and the biochemical characterization of three of them. Our study shows that autotrophic CO₂ assimilation in I. hospitalis proceeds in the peripheric CC, thus demonstrating another unique feature of I. hospitalis: the spatial separation of major anabolic processes in I. hospitalis, namely DNA replication, RNA synthesis, and protein biosynthesis from inorganic carbon fixation.     

Phase separation in hydrogen–helium mixtures at Mbar pressures

The properties of hydrogen–helium mixtures at Mbar pressures and intermediate temperatures (4000 to 10000 K) are calculated with first-principles molecular dynamics simulations. We determine the equation of state as a function of density, temperature, and composition and, using thermodynamic integration, we estimate the Gibbs free energy of mixing, thereby determining the temperature, at a given pressure, when helium becomes insoluble in dense metallic hydrogen. These results are directly relevant to models of the interior structure and evolution of Jovian planets. We find that the temperatures for the demixing of helium and hydrogen are sufficiently high to cross the planetary adiabat of Saturn at pressures ≈5 Mbar; helium is partially miscible throughout a significant portion of the interior of Saturn, and to a lesser extent in Jupiter.     

Phase separation in mixtures of ionized iron in a hydrogen plasma

The accuracy of the hypernetted chain theory for mixtures of iron nuclei and protons in a charge-neutralizing background is demonstrated by comparison with molecular dynamics calculations. Near critical conditions the Debye-Hückel approximation fails to converge, even with second-order concentration corrections. The critical conditions, determined by assuming a uniform electron gas background, have a critical temperature more than a factor of 2 lower than present estimates of interior solar conditions. Qualitative arguments are made that including the polarization of the electron gas background would not change this result significantly and thus phase separation of iron in the interior of the sun is unlikely.     

Phosphorylation of lens fiber cell membrane proteins.

Two intrinsic membrane proteins of calf lens fiber cells can be phosphorylated by a soluble bovine lens cAMP-dependent protein kinase and rabbit muscle cAMP-dependent protein kinase. After electrophoresis of the phosphorylated membranes, 32P comigrates with the lens main intrinsic protein at 26-27 kDa and with a minor band of protein that migrates at 19-20 kDa. 32P is also found with proteins that, based on the molecular sizes, are likely multimers of the 19-kDa and 26-kDa proteins. Upon boiling in NaDodSO4, all the radioactivity is found at the top of the gel, suggesting that both phosphoproteins are intrinsic membrane proteins. Serine is the only phospho amino acid detected in both proteins regardless of the source of protein kinase. The phosphorylation sites of both proteins are lost upon cleavage with trypsin and chymotrypsin. The smaller phosphoprotein is likely not a crystallin, because antibodies directed against alpha-, beta-, or gamma-crystallins do not cross-react with the 19-kDa protein. The 19-kDa 32P-labeled protein does not migrate coincident with calf alpha-crystallin.     

Mechanism of red blood cell aging: Relationship of cell density and cell age

The human red cell has a life span of 120 days. The mechanism that determines cell removal from the circulation with such precision remains unknown. Most studies of red cell aging have been based on analysis of cells of progressively increasing age separated by density. The relationship between red cell age and density has been recently challenged, and the hypothesis has been put forward that cell death is not the result of a progressive deterioration of essential cell constituents. This theory was based on preliminary observations in transient erythroblastopenia of childhood, which could not later be confirmed. When the relationship between cell aging and increasing density is critically reviewed, it appears to be based on firm experimental evidence, confirmed by in vivo demonstration of decreasing survival of cells of increasing age. Analysis of studies using buoyant density gradients reveals that this technique can easily distinguish the single exponential slope of decline for those cell components that change progressively throughout the red cell life span from the biphasic decline of those that decrease drastically at the reticulocyte-mature red cell transition. The view that the aging of the red cell and its removal from the circulation result from a progressive series of events during the 120 days of its life span appears to be the most consistent with the available data. Density separation, validated by much experimental evidence, remains a most useful technique for the study of the mechanism of aging of the red cell. © 1993 Wiley-Liss, Inc.     

Red cell magnesium as a function of cell age

The variation in the magnesium content of human red cells as a function of cell age has been measured by atomic absorption spectrophotometry. The cell population was split into different age fractions using discontinuous density gradient centrifugation, since it is known that cell density increases with age. A mathematical model relating predicted cell age to cell density has been developed which allows the quantification of the observed fall-off in magnesium content with cell age. This model suggests that cells lose magnesium monoexponentially with age, the half-life being approximately 100 days. A previously proposed hypothesis that magnesium could enter the cells only at erythropoiesis and then decay monoexponentially predicted a half-life of 22.4 days and is therefore seen to be an oversimplification of magnesium kinetics in the red cell. The relevance of the present findings to pathologic conditions with abnormal red cell magnesium concentrations is discussed.