ATP and integrity of human red blood cells

In spite of the well known significance of ATP in the energy dependent life processes, the role of ATP in maintaining cellular integrity is poorly understood. A possible model for studying ATP dependent life processes is to monitor the kinetics of changes seen intra/extracellularly during ATP depletion. In our model system anticoagulated human whole blood was incubated at different temperatures to reduce intracellular ATP without addition of any chemicals. The red blood cells in their own plasma were incubated for several days at 4 degrees C or at 37 degrees C, and ATP, glucose, K+, Na+, hemoglobin, water content, mean corpuscular volume (MCV), pH and Ca2+ were analyzed in time-sequences. All the examined parameters remained practically unchanged at 4 degrees C, while at 37 degrees C total ATP and glucose decreased parallel and after a transient increase of MCV, the water content of red blood cells decreased. As the actual ATP fell below 10% of the initial ATP content (at 48 h), the release of potassium sharply increased. Release of hemoglobin started only after 96 hours of incubation. Maximums of changes of the examined parameters were found at different time intervals. The maximal speed of concentration changes for glucose was found at 12-24 hours of incubation and at 24-36 hours for ATP, at 48-60 hours for K+(-)Na+ and after 96 hours for hemoglobin.     

Partial requirements forin vitro survival of human red blood cells

Some of the requirements for survival of human red blood cells were studied in vitro at 25 and 37 degrees C for 1--2 weeks. During the first week at 25 degrees C in Krebs-Ringer bicarbonate medium with glucose, the cells at 2--5% hematocrit (HCT) maintained normal K+, Na+, and water contents with negligible hemolysis. After six days ion gradients decreased, preceded by decline of ATP. With adenosine, ATP was maintained for 1--2 weeks. Sustained in vitro survival of human red blood cells at 25 or 37 degrees C requires constant pHo and sufficient substrates to support a glycolytic carbon flux as well as a nitrogen flux via nucleotide turnover. In Earle's salts buffered with HEPES and supplemented with glucose, Eagle's essential vitamins, albumin, and antibiotics, suspensions at 0.1% HCT exhibited constant pH at 7.39 +/- 0.03 for at least two weeks at 37 degrees C. With glucose alone, ATP declined steadily to negligible levels despite constant pHo, but 0.1 mM adenine supported ATP for one week. Also, several amino acids partially prevented the decline of reduced glutathione during the first week at 37 degrees C. These results and current knowledge of red cell metabolism suggest a new defined medium for experiments requiring long term incubations, and extend the characterization of human red cell in vitro survival to a time period not previously studied.     

Morphology of glutaraldehyde-fixed preserved red blood cells and 24-hr post-transfusion survival

Liquid-stored red blood cells and washed, previously frozen red blood cells were studied to determine whether a correlation existed between morphology and post-transfusion survival. Red cell concentrates were stored at 4 °C in citrate-phosphate-dextrose (CPD) for 21 days or in CPD-adenine (CPDA-1, CPDA-2, or CPDA-3) for as long as 35 days as liquid-preserved red cells. Both nonrejuvenated and rejuvenated red blood cells were frozen with 40%$\text{w}\text{v}$ glycerol at ?80 °C and were washed prior to testing.Samples of fresh, liquid-stored, and washed, previously frozen red blood cells were fixed with a 2% veronal glutaraldehyde solution. Phase, light, and electron microscopy were used to measure the numbers of discocytes, discoechinocytes, echinocytes, echinospherocytes, and spherocytes in each sample. A morphology score was assigned, with 100 representing all discocytes and 500 all spherocytes. In all samples phase and light microscopy gave nearly identical scores (r = 0.94), and phase and electron microscopy gave highly similar scores (r = 0.83).The morphology score proved to be a good indicator of 24-hr post-transfusion survival in liquid-stored red blood cells but not in washed, previously frozen red blood cells. Red blood cells stored in the liquid state at 4 °C in CPD, CPDA-1, CPDA-2, or CPDA-3 showed a significant inverse correlation between morphology and 24-hr post-transfusion survival (r = ?0.611) and a significant correlation between red cell ATP and 24-hr post-transfusion survival (r = 0.742). We saw no significant correlation between morphology scores and 24-hr post-transfusion values or between ATP levels and post-transfusion survival values in nonrejuvenated or rejuvenated washed, previously frozen red blood cells.     

Lactate interferes with ATP release from red blood cells

Upon exposure to low Po(2), the red blood cells of most species, including humans, release increased amounts of ATP that ultimately serves as a regulator of vascular tone matching oxygen supply with demand. In pathological conditions such as malaria and sepsis, a maldistribution of perfusion exists with its severity often correlated with the extent of elevation of serum lactate frequently in the absence of an alteration in pH. We hypothesized that the increased levels of lactate might impair the ability of red blood cells to appropriately respond to conditions of low Po(2), thus preventing its important blood flow regulatory role. Using an in vitro system and rabbit red blood cells, we evaluated the capacity of cells incubated with lactate to release increased amounts of ATP in response to acute exposure to low Po(2). We found that in the presence of lactate, the red blood cells did not release ATP. However, when sodium dichloroacetate, a drug used clinically to lower blood lactate levels, was added, ATP release was restored to levels that were not different from that of control cells (no lactate), even though intracellular levels of ATP were not. These results support the presence of a distinct flow regulatory pool of ATP within the red blood cell that can be independently regulated, and that lactate interferes with the ATP production within this pool, thereby diminishing the amount of ATP available for release on exposure to low Po(2). Therefore, if lactate levels can be reduced, the vascular regulatory capacity of the red blood cell should be restored, thus enabling the appropriate matching of oxygen supply with oxygen demand.     

Membrane protein organization in ATP-depleted and irreversibly sickled red cells

It has been proposed that the spectrin-actin submembrane network participates in control of red cell shape and deformability. We have examined ATP- and calcium-dependent changes in organization of spectrin in the membrane employing cross-linking of the nearest membrane protein neighbors by spontaneous or catalyzed (CuSO₄, O-phenanthroline) intermolecular disulfide couplings and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Cross-linking of fresh red cells resulted in the formation of spectrin and actin dimers and tetramers. ATP-depleted red cells differed from fresh cells in the presence of an additional reducible polymer of MW > 1 × 10⁶ selectively enriched in spectrin. This polymer formed spontaneously when red cells were depleted of ATP under aerobic conditions. After anaerobic ATP depletion, the polymer formed in ghosts after cross-linking by catalytic oxidation. Polymerization was prevented by maintenance of ATP and coincided with an ATP-dependent discocyte-echinocyte transformation. This suggests that, in ATP-depleted red cells, spectrin is rearranged to establish closer contacts, and that this may contribute to the discocyte-echinocyte transformation. The introduction of greater than 0.5 mM Ca⁺⁺ into ghosts by inclusion in hemolysis buffer or into fresh red cells (but not ATP-depleted red cells) by treatment with ionophore A23187 spontaneously produced a nonreducible polymer which others have attributed to transamidative cross-linking of spectrin, band 3, and other proteins. Spontaneous formation of both polymer types (reducible in aerobically ATP-depleted red cells and nonreducible in fresh, Ca⁺⁺ enriched red cells) resulted in stabilization (“autocatalytic fixation”) of spheroechinocytic shape. Irreversibly sickled cells, which have increased calcium and decreased ATP, and exhibit a permanent membrane deformation, failed to form any of the above polymers. This suggests that in contrast to normal cells depleted of ATP in vitro, fixation of ISC shape in vivo is not related to Ca- and ATP-dependent membrane protein polymerization. However, ISCs had an increased propensity to form the reducible, spectrin-rich polymer during a subsequent metabolic depletion in vitro. This was associated with transformation of ISCs into spheroechinocytes. Similar echinocytic ISCs were found to constitute 5–10% of the densest fractions of freshly separated ISCs. ISCs then exhibit sphero-echniocyte transformation, both in vitro and in vivo. We propose that this is due to spectrin reorganization that presumably results from the progressively increasing calcium and decreasing ATP of ISCs. These data provide evidence of altered spectrin organization in membranes of ATP-depleted, calcium-enriched red cells in vitro and in vivo.     

Interaction of ruthenium red with isolated sarcolemma

Summary The stain ruthenium red binds very strongly to isolated sarcolemma and the maximal binding is about 125 nmoles/mg protein, decreasing slightly in lipid-extracted membranes. The binding is half maximal when the free stain concentration is about 1.0 M, for both intact and lipid-depleted material. The nucleotide, ATP, reduces markedly the binding and the apparent affinity of the membranes for the stain. Minute concentrations of ruthenium red (10 M) inhibit by 80 to 90% the Ca⁺⁺-binding by sarcolemma. The inhibition does not depend on the Ca⁺⁺ concentration and is similar in both intact and lipid-extracted preparations. Ruthenium red inhibits the ATPase activity of sarcolemma. The inhibition is decreased by increasing the ATP concentration in the medium.     

Effect of anoxia on ATP levels and ion transport rates in red beet

The ATP content of disks of storage tissue of red beet is reduced under N₂ atmosphere to between 10 and 25% of its value in air. Plasmalemma fluxes of K⁺ and Cl⁻ are inhibited within 1 minute or less following removal of O₂, and the extent of this inhibition is entirely attributable to the depletion of ATP. The ATP content remains approximately constant or decreases slightly for up to 1 hour under N₂. On return to air, the ATP content recovers, reaching 75% of the aerobic level within 45 second.     

Calcium extrusion by high-density human red blood cells

Normal human red blood cells were separated on arabinogalactan density gradients to provide cell populations comprising a very small percentage of the cells (0.4%-1.8%). These very high-density cells were compared to a low-density, mature reference cell population with respect to their ATP content and their capacity to extrude Ca that had been loaded into the cells using the ionophore A23187. Assay of ATP content of freshly drawn and separated cells suggested a decrease of approximately 40% in the ATP concentration of the most dense 0.5%-1% of the cells, but the second most dense percent or so of the cells showed no ATP deficit. When the cells were loaded with millimolar amounts of 45Ca, high and low-density cell populations extruded Ca at the same rate. It appears that even stringently selected cells comprising the highest-density portion of the total cell population have an intact Ca transport system that can rapidly export Ca from the cell when adequate metabolic support is available.     

In vitro evaluation and study of the survival of erythrocytes preserved in a saline-adenine-glucose-mannitol medium for 35 days

The saline-adenine-glucose-mannitol (SAGM) solution for resuspension of red cells was evaluated on 30 blood units tested over 42 days and compared to 5 red cell concentrates collected on the conventional CPD medium. Total and extra-cellular hemoglobin, potassium, pH, ATP and DPG concentrations, osmotic fragility, schizocyte formation, and red cell antigenicity were studied through the storage period. Chromium survival studies of autologous donated red cells were performed in 10 donors. Red cell concentrates resuspended in SAGM solution showed at the 35th day of conservation at 4 degrees C, a mean storage hemolysis of only 0.66%, an ATP concentration of 67% of the initial value, a schizocyte proportion of less than 1.5%, a mean 24 hour posttransfusion viability of 88.33% and a mean red cell T 1/2 survival of 25 days 10 hours. No alteration of common blood group antigens could be found after storage of red cells for 42 days.     

Modulation of glucose transport in human red blood cells by ATP

Depletion of energy stores of human red cells decreases the maximum transport capacity, $\text{J}{}_{\mathrm{<mtext>m</mtext>}}$, for glucose transport to a value one-third or less of that found in red cells from freshly drawn blood. There is no change in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. Hemolysis and resealing of red cells with ATP or ADP reverses the decrease in $\text{J}{}_{\mathrm{<mtext>m</mtext>}}$. The maximum effect occurs at concentrations of ATP in the normal range for red cells, however, there is little effect from ADP concentrations in its normal range in freshly drawn red cells. Hemolysis and resealing with ATP gives an increase in $\text{J}{}_{\mathrm{<mtext>m</mtext>}}$ and an increase in differential labeling by photolytic labeling with tritiated cytochalasin B. Most of the activation is lost after a second hemolysis-reseal without ATP but about 25% of the activation remains.     

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.     

ATP regulation of the human red cell sugar transporter

Purified human red blood cell sugar transport protein intrinsic tryptophan fluorescence is quenched by D-glucose and 4,6-ethylidene glucose (sugars that bind to the transport), phloretin and cytochalasin B (transport inhibitors), and ATP. Cytochalasin B-induced quenching is a simple saturable phenomenon with Kd app of 0.15 microM and maximum capacity of 0.85 cytochalasin B binding sites per transporter. Sugar-induced quenching consists of two saturable components characterized by low and high Kd app binding parameters. These binding sites appear to correspond to influx and efflux transport sites, respectively, and coexist within the transporter molecule. ATP-induced quenching is also a simple saturable process with Kd app of 50 microM. Indirect estimates suggest that the ratio of ATP-binding sites per transporter is 0.87:1. ATP reduces the low Kd app and increases the high Kd app for sugar-induced fluorescence quenching. This effect is half-maximal at 45 microM ATP. ATP produces a 4-fold reduction in Km and 2.4-fold reduction in Vmax for cytochalasin B-inhibitable D-glucose efflux from inside-out red cell membrane vesicles (IOVs). This effect on transport is half-maximal at 45 microM ATP. AMP, ADP, alpha, beta-methyleneadenosine 5'-triphosphate, and beta, gamma-methyleneadenosine 5'-triphosphate at 1 mM are without effect on efflux of D-glucose from IOVs. ATP modulation of Km for D-glucose efflux from IOVs is immediate in onset and recovery. ATP inhibition of Vmax for D-glucose exit is complete within 5-15 min and is only partly reversed following 30-min incubation in ATP-free medium. These findings suggest that the human red cell sugar transport protein contains a nucleotide-binding site(s) through which ATP modifies the catalytic properties of the transporter.     

Viability, ATP and 2,3-DPG content of resuspended erythrocytes during several-week storage in cold

Leukocyte-and thrombocyte-poor packed red cells obtained from ACD or. ACD-AG blood were resuspended to a hematocrit of about 55% and stored at 4 degrees C. The resuspension solution consisted of xylitol, inorganic phosphate, bicarbonate, adenine (A) and guanosine (G) solved in water. In one case glucose, citrate and sucrose were also added, in another one, sorbitol. The 2,3-DPG and the ATP level remained for a longer period in the sorbitol-xylitol-medium than in the glucose-xylitol-medium. The ATP content in the red cell suspension was higher than in packed cells. Higher ATP values were obtained in red blood cells from whole blood with adenine and guanosine. The survival rate of resuspended red blood cells in glucose-AG-citrate-sucrose medium was about 80--85% after 3 weeks of storage and 77% after 6 weeks with a higher range.     

Erythrocyte concentrates, low in buffy coat: production, preservation and evaluation of its quality

Buffy coat-poor packed red cells were prepared from fresh ACD-, ACD-AG- and EDTA-blood, than resuspended with a preservation solution, containing glucose, adenine, guanosine, sucrose, citric acid and sodium citrate and stored at 4 degrees C for 6 weeks. The survival rate of resuspended red cells from ACD-AG-blood amounted to 77% after 6 weeks of storage. The ATP content of resuspended red cells was approximately 25% lower than in ACD-AG whole blood during storage caused probably by increased ATP consuming reactions at the red cell membrane. The P2G-content of resuspended red cells from ACD- and ACD-AG-blood decreased above 50% of the normal level during the first week, as fast as in ACD- and ACD-AG whole blood. The P2G-breakdown in red cells from EDTA-blood was delayed for a week due to the higher pH as in CPD blood. Additions of xylitol, inorganic phosphate, and bicarbonate in 6, 5 and 20 mM final concentrations in the red cell suspensions and an increased pH at the same time delayed the breakdown of ATP and P2G. Packed red cells can be administered fast enough at hematocrits to 0.60 that will be achieved by adding 50 to 100 ml preservation solution. Leukocytes and thrombocytes were reduceds to 70 to 80%. With increasing rate of reduction a higher loss of red cells occured. Buffy coat-poor red cell concentrate contains only few microaggregates. It diminishes the risc of febrile transfusion reactions and delays the appearance of alloimmunisation. The circulatory overload of patients is less frequent than after transfusions of red cell resuspensions containing a large resuspension volume.     

Regulation of NAD and NADP synthesis in human red cell.

NAD is synthesized in red cell from nicotinic acid and PRPP through the formation of nicotinate mononucleotide and desamido-NAD. Synthesis of one mole of NAD requires two moles of ATP. NADP comes from NAD phosphorylation by NAD-kinase (EC.2.7.1.23). NAD and NADP analysis on a population with ATP level ranging from 800 to 2500 nmoles/ml red cells showed a close correlation between ATP and pyridine cofactors. Moreover, NADP level appeared to be dependent of the redox-state of NADP/NADPH couple. Subjects with low NADPH (G-6-PD) deficient red cells, Hb K?ln) showed lower NADtot/NADPtot ratio, suggesting a NAD-kinase equilibrium shift toward NADP related to lower levels of the negative effector NADPH, as already described in rat liver.     

Anomalous asymmetric kinetics of human red cell hexose transfer: role of cytosolic adenosine 5'-triphosphate

Cytosolic adenosine 5'-triphosphate (ATP) modifies the properties of human red cell sugar transport. This interaction has been examined by analysis of substrate-induced sugar transporter intrinsic fluorescence quenching and by determination of Michaelis and velocity constants for D-glucose transport in red cell ghosts and inside-out vesicles lacking and containing ATP. When excited at 295 nm, human erythrocyte ghosts stripped of peripheral proteins display an emission spectrum characterized by a scattering peak and a single emission peak centered at about 333 nm. Addition of sugar transport substrate or cytochalasin B and phloretin (sugar transport inhibitors) reduces emission peak height by 10% and 5%, respectively. Cytochalasin B induced quenching is a simple saturable phenomenon with an apparent Kd (app Kd) of 60 nM and a capacity of 1.4 nmol of sites/mg of membrane protein. Quenching by D-glucose (and other transported sugars) is characterized by at least two (high and low) app Kd parameters. Inhibitor studies indicate that these sites correspond to sugar efflux and influx sites, respectively, and that both sites can exist simultaneously. ATP induces quenching of stripped ghost fluorescence with half-maximal effects at 20-30 microM ATP. ATP reduces the low app Kd and increases the high app Kd for sugar-induced fluorescence quenching. D-Glucose transport in intact red cells is asymmetric (Km and Vmax for influx less than Km and Vmax for efflux). In addition, two operational Km parameters for efflux are detected in zero- and infinite-trans efflux conditions. Protein-mediated sugar transport in ghosts and inside-out vesicles (IOVs) is symmetric with respect to Km and Vmax for entry and exit, and only one Km for exit is detected. Addition of millimolar levels of ATP to the interior of ghosts or to the exterior of IOVs restores both transport asymmetry and two operational Km parameters for native efflux. A model for red cell hexose transport is proposed in which ATP modifies the catalytic properties of the transport system. This model mimics the behavior of the sugar transport systems of intact cells, ghosts, and inside-out vesicles.     

Effects of buffers and pH on rabbit red blood cell hexokinase

The kinetic properties of rabbit red blood cell hexokinase in different buffer systems have been studied. At pH 8.0 the reaction velocity (v) is about 30% higher in glycylglycine compared to Tris, Tea, Hepes or ammonium acetate buffers. The enzyme stability, heat-dependence and spectral properties of the enzyme are also affected by the buffer utilized. None of the following kinetic properties of red blood cell hexokinase varies with pH in the range 6.8-8.5: Km of glucose; Km of ATP and Ki of glucose 6-phosphate.     

Two classes of site for ATP in the Ca2+-ATPase from human red cell membranes.

(1) The response of the Ca2+-ATPase activity from human red cell membranes to ATP concentrations can be represented by the sum of two Michaelis-like curves: one with a Km of 2.5 micrometer and the other with a Km of 145 micrometer. (2) The maximum Ca2+-ATPase activity elicited by occupation of the site with lower Km represents about 10% of the activity attainable at non-limiting ATP concentrations. (3) 30--50% of the Ca2+-ATPase activity with lower Km remains in the absence of Mg2+ . Mg2+ increases V and the maximum effect of Ca2+, having no effect on the apparent affinities for ATP and Ca2+. (4) The large increase in Ca2+-ATPase activity which results from the occupation of the site with higher Km only takes place when Mg2+ is present. (5) Results are compatible with the idea that the Ca2+-ATPase from human red cell membranes has two classes of site for ATP binding, both of which are occupied when the enzyme catalyzes the hydrolysis of ATP at maximum rate. (6) The properties of the high affinity site suggest that this is the catalytic site of the Ca2+-ATPase. It is proposed that binding of ATP at the low affinity site regulates the turnover of the system.     

Delivery of ion pumps from exogenous membrane-rich sources into mammalian red blood cells.

Using polyethylene glycol-mediated fusion of ATP-ase-enriched (native) microsomes with red blood cells, we have delivered sarcoplasmic reticulum (SR) Ca-ATPase and kidney Na,K-ATPase into the mammalian erythrocyte membrane. Experiments involving delivery of the SR Ca-ATPase into human red cells were first carried out to assess the feasibility of the fusion protocol. Whereas there was little detectable 45Ca2+ uptake into control cells in either the absence or presence of extracellular ATP, a marked time-dependent uptake of 45Ca2+ was observed in the presence of ATP in cells fused with SR Ca-ATPase. Comparison of the kinetics of uptake into microsome-fused cells versus native SR vesicles supports the conclusion of true delivery of pumps into the red cell membrane. Thus, the time to reach steady state was more than two orders of magnitude longer in the (large) cells versus the native SR vesicles. Na,K-ATPase from dog and rat kidney microsomes were fused with red cells of humans, sheep, and dogs. Using dog kidney microsomes fused with dog red cells which are practically devoid of Na,K-ATPase, functional incorporation of sodium pumps was evidenced in ouabain-sensitive Rb+ uptake and Na+ efflux energized by intracellular ATP, as well as in ATP-stimulated Na+ influx and Rb+ efflux from inside-out membrane vesicles prepared from the fusion-treated cells. From analysis of the biphasic kinetics of ouabain-sensitive Na+ efflux under conditions of limited intracellular Na+ concentration, it is concluded that the kidney pumps are incorporated into a relatively small fraction (approximately 15%) of the red cells. This system provides a uniquely useful system for studying the behavior of native sodium pumps in a compartment (red cell) of small surface/volume ratio. The newly incorporated native kidney pumps, while of the same isoform as the endogenous red cell pump, behave differently from the endogenous red cell sodium pump with respect to their very low "uncoupled" Na+/O flux activity.     

Effect of phosphoenolpyruvate on metabolic and morphological recovery of red cells after prolonged liquid storage and subsequent freezing in glycerol medium.

The present study was designed to determine the effects of (i) phosphoenolpyruvate (PEP) treatment of red blood cells (RBCs) previously cold stored for a prolonged period in a liquid medium and (ii) the freezing of these treated cells in glycerol. RBCs stored for 21 days at 4 degrees C were incubated for 30 min at 37 degrees C with rejuvenant solution containing 50 mM PEP, 60 mM mannitol, 30 mM sodium chloride, 25 mM glucose, and 1 mM adenine, pH 6.0, and then frozen at -80 degrees C for 4 weeks. Red cell recovery as frozen and thawed red cells (FTRCs) after deglycerolization was increased to 80 +/- 4% compared to 43 +/- 9% in units without rejuvenation; the percentage of PEP-treated FTRCs was similar to the percentage of FTRCs recovered from fresh RBCs within 5 days after donation. Incubation of RBCs with PEP solution restored ATP and 2,3-DPG to levels seen in fresh RBCs, and also facilitated transformation of crenated RBCs to discocytes. These results indicate that maximum recovery of viable RBCs can be attained when FTRCs are processed from cells stored in the frozen state after they had been rejuvenated with PEP even after prolonged liquid storage.