A simple method for determination of red blood cell mechanical fragility in the rat

A method for measuring the mechanical fragility of red blood cells suitable for use in small laboratory animals, such as rats, is reported because of lack of such data in the literature. Whole blood is mixed with phosphate buffered saline in a tube containing glass beads. The tubes are rocked for 90 minutes, centrifuged and the percent hemolysis determined. Varying the osmolality of the saline suspending medium had little effect on the mechanical fragility of rat red cells prior to the NaCl concentrations at which a significant change in osmotic hemolysis occurred. The duration of rocking increased the mechanical fragility. Varying the pH (6.4-8.0) had no effect. The size of the glass beads changed the mechanical fragility as did varying temperature. The mean mechanical fragility of rat red blood cells was 46% hemolysis (80 adult male animals). Because of the small volume of blood required with this method, mechanical fragility of red cells of other small laboratory animals also may be determined.     

Outward sodium and potassium cotransport in human red cells

Summary This paper reports some kinetic properties of Na–K cotransport in human red cells. All fluxes were measured in the presence of 10^–4 M ouabain. We measured Na and K efflux from cells loaded by the PCMBS method to contain different concentrations of these ions into a medium that contained neither Na nor K (MgCl₂-sucrose substitution) in the absence and presence of furosemide. Furosemide inhibited 30–60% of the total efflux depending on the internal ion concentration and the individual subject. We took the furosemide-sensitive fluxes to be a measure of Na–K cotransport. The ratio of Na to K cotransport was 1 over the entire range of internal Na and K concentrations studied. When Na was substituted for K as the only internal cation, cotransport was maximally activated when the Na and K concentrations were between 20 and 90 mmol/liter cells. The concentration of internal Na required to produce half-maximal cotransport was about 13±4 mmol/liter cells (n=4), while the comparable concentration of K was somewhat lower. The activation curve was definitely sigmoid in character, suggesting that at least two Na ions are involved in the transport process. The maximum of Na–K cotransport was about 0.5±0.15 mmol/liter cells × hr (n=5); it had a flat maximum in the medium at about pH 7.0, decreasing in both the acid and alkaline sides. furosemide-resistant effluxes were found to be linear functions of internal Na and K concentrations and to yield rate coefficients of 0.019±0.002 hr^–1 and 0.014±0.002 hr^–1 (n=7), respectively. These values are of the same order of magnitude expected of ions moving across phospholipid bilayers.Charge de Recherches CNRS.     

Active sodium and potassium transport in high potassium and low potassium sheep red cells

The kinetic characteristics of the ouabain-sensitive (Na + K) transport system (pump) of high potassium (HK) and low potassium (LK) sheep red cells have been investigated. In sodium medium, the curve relating pump rate to external K is sigmoid with half maximal stimulation (K_1/2) occurring at 3 mM for both cell types, the maximum pump rate in HK cells being about four times that in LK cells. In sodium-free media, both HK and LK pumps are adequately described by the Michaelis-Menten equation, but the K_1/2 for HK cells is 0.6 ± 0.1 mM K, while that for LK is 0.2 ± 0.05 mM K. When the internal Na and K content of the cells was varied by the PCMBS method, it was found that the pump rate of HK cells showed a gradual increase from zero at very low internal Na to a maximum when internal K was reduced to nearly zero (100% Na). In LK cells, on the other hand, no pump activity was detected if Na constituted less than 70% of the total (Na + K) in the cell. Increasing Na from 70 to nearly 100% of the internal cation composition, however, resulted in an exponential increase in pump rate in these cells to about ⅙ the maximum rate observed in HK cells. While changes in internal composition altered the pump rate at saturating concentrations of external K, it had no effect on the apparent affinity of the pumps for external K. These results lead us to conclude that the individual pump sites in the HK and LK sheep red cell membranes must be different. Moreover, we believe that these data contribute significantly to defining the types of mechanism which can account for the kinetic characteristics of (Na + K) transport in sheep red cells and perhaps in other systems.     

Heterogeneity in dog red blood cells: sodium and potassium transport

After incubation in isotonic KCl, dog red blood cells can be separated by centrifugation into subgroups which assume different cell volumes and possess different transport characteristics. Those red cells which swell in isotonic KCl exhibit a higher permeability to K and possess a greater volume dependence for transport of K than those red cells which shrink. A high Na permeability characterizes cells which shrink in isotonic KCl and these cells exhibit a larger volume-dependent Na flux than those red cells which swell. These two subgroups of red cells do not seem to represent two cell populations of different age. The results indicate that the population of normal cells is evidently heterogeneous in that the volume-dependent changes in Na and K permeability are distributed between differnt cell types rather than representing a single cell type which reciprocally changes its selectivity to Na and K.     

A simple method for quantitative determination of polysaccharides in fungal cell walls

A simple and reliable method for quantitative determination of cell wall polymers in fungal cell with an s.e.m. of 5% is described. This protocol is based on the hydrolysis by sulfuric acid of beta-glucan, mannan, galactomannan and chitin present at different levels in the wall of yeasts and filamentous fungi into their corresponding monomers glucose, mannose, galactose and glucosamine. The released monosaccharides are subsequently separated and quantified by high-performance ionic chromatography coupled to pulse amperometry detection, with a detection limit of 1.0 mug ml(-1). This procedure is well suited to screening a large collection of yeast mutants or to evaluating effects of environmental conditions on cell wall polysaccharide content. This procedure is also applicable to other fungal species, including Schizosaccharomyces pombe, Candida albicans and Aspergillus fumigatus. Results can be obtained in 3 d.     

p-Chloromercuribenzenesulfonic acid stimulation of chloride-dependent sodium and potassium transport in human red blood cells

The organic mercurial p-chloromercuribenzensulfonic acid (PCMBS) reversibly increases fluxes of sodium and potassium across the human red blood cell membrane. We examined the effect of different monovalent anions on cation fluxes stimulated by PCMBS. A substantial portion of the fluxes of both cations was found to have a specific anion requirement for chloride or bromide, and was not observed when chloride was replaced by nitrate, acetate or methylsulfate. The chloride-dependent component of the cation fluxes was only observed when the cells were exposed to PCMBS concentrations of 0.5 mM or greater. Furosemide (1 mM) did not inhibit the PCMBS-stimulated cation fluxes. The observed anion specificity is directly associated with the transport process rather than PCMBS binding to the membrane. A portion of the potassium transport stimulated by PCMBS appears to involve K+-K+ exchange; however, Na+ + K+ cotransport is not stimulated by this sulfhydryl reagent.     

The interphase technique: a simple method of cell immobilization in gel-beads

A variation of the emulsion techniques for microbial cell immobilization by encapsulation in gel beads is described. The utilized gels are silica gel or natural polymers like agar, agarose, phytagel or carrageenan. This technique utilizes the interfacial tension that is formed between two liquids of different polarity. The gel-beads are easily obtained and homogeneous in size.     

The ATP dependence of a ouabain-sensitive sodium efflux activated by external sodium, potassium and lithium in human red cells.

The stimulation of ouabain-sensitive Na+ efflux by external Na+, K+ and Li+ was studied in control and ATP-depleted human red cells. In the presence of 5 mM Na+, with control and depleted cells, Li+ stimulated with a lower apparent affinity than K+, and gave a smaller maximal activation than K+. The ability of Na+, K+ and Li+ to activate Na+ efflux was a function of the ATP content of the cells. Relative to K+ both Na+ and Li+ became more effective activators when the ATP was reduced to about one tenth of the control values. At this low ATP concentration Na+ was absolutely more effective than K+.     

Further studies of hormone-sensitive sodium and potassium transport in red cells from developing chick embryos

Sodium and potassium transport in the definitive series of chick embryo red cells changes significantly, both qualitatively and quantitatively, during maturation. Sodium efflux and potassium influx consist of three parts: a ouabain-sensitive, a furosemide-sensitive, and a ouabain-furosemide-insensitive component. In chick red cells of most ages, the ouabain-sensitive and furosemide-sensitive parts of the cation fluxes do not overlap. Cation transport in the more mature red cells is increased significantly by epinephrine, whereas cation transport in red cells from younger embryos is stimulated much less. This is a beta-adrenergic effect of epinephrine and is mediated by cyclic AMP. The relative lack of response in younger embryos is not due to the absence of beta-adrenergic receptor or the lack of production of cyclic AMP. Ouabain has no effect on the hormone-sensitive sodium or potassium transport. On the other hand, furosemide nearly completely abolishes the effect of epinephrine. In addition, there is a good correlation between furosemide-sensitive components of both sodium and potassium transport and the epinephrine-sensitive component. Furosemide has no effect on cyclic AMP levels in the presence or absence of epinephrine. This suggests that furosemide may act directly on the cation transport system. In the red cells from younger embryos, furosemide-sensitive units are present but cannot be fully activated by epinephrine. Therefore, the lack of the hormone effect on cation movements in these cells is consistent with the view that the appropriate units are present, but do not respond fully to intracellular cyclic AMP levels.     

A simple and sensitive fluorescence method for the determination of trace ozone in air using acridine red as a probe

The ozone in an air sample was trapped by H₃BO₃‐LK solution to produce iodine (I₂) that interacted with excess I^– to form I₃^–. In pH 4.0 acetate buffer solutions, the I₃^– reacted with acridine red to form acridine red–I₃ ion association particles that resulted in the fluorescence peak decreased at 553 nm. The decreased value ΔF_{553 nm} is linear to the O₃ concentration in the range 0.08–53.3 × 10^–6 mol/L, with a detection limit of 4 × 10^–8 mol/L. This fluorescence method was used to determine ozone in air samples, and the results were in agreement with that of indigo carmine spectrophotometry. Copyright © 2014 John Wiley & Sons, Ltd.     

Sidedness of (sodium, potassium)-adenosine triphosphate of inside-out red cell membrane vesicles. Interactions with potassium.

Inside-out membrane vesicles of human red cells, prepared according to the method of Steck et al. (1970) Science 168, 255-257) have sufficiently low cation permeability to allow the examination of the side-specific interactions of ligands with the asymmetric sodium pump complex. In accordance with the known properties of the pump in intact cells the following results were observed: (a) ATP-dependent sodium influx and (b) maximal (sodium, potassium)-ATPase with K+ present inside the vesicles with larger than or equal to 20 micronM ATP. With much lower [ATP], K+ inhibited sodium-activated ATPase. K+ was inhibitory at either surface. Inhibition was different on the two sides since cytoplasmic (extravesicular) Na+ counteracted inhibition by cytoplasmic (extravesicular) K+ but not inhibition by K+ at the plasma or external membrane surface, i.e. intravesicular K+. A decrease in the steady state level of the phosphenzyme intermediate of sodium-activated ATPase was caused also by K+ at either surface. The effect of cytoplasmic K+ is compatible with its competitive inhibition of activation of phosphorylation of the enzyme by cytoplasmic Na+. At 37 degrees, the inhibitory effect of external K+ is due to interaction with the phosphoenzyme to form a stable complex of K+ with the dephosphenzyme resulting in a decreased overall reaction rate but increased turnover of the phosphoenzyme (E-P + K leads to EK + Pi). At 0 degree, external K+ inhibits by interacting with the unphosphorylated enzyme to form an occluded enzyme-K complex. This results in a decreased overall rate but relatively small change in apparent turnover of the phosphoenzyme. At 0 degree, but not at 37 degrees, external Na+ counteracted the inhibitory effects of external K+.     

Spectrophotometric method for determination of tocopherol in red blood cells

A relatively rapid procedure is described for the spectrophotometric determination of total tocopherol in red blood cells (RBC) based on a modification of the original Emmerie-Engel reaction. The critical feature in this method is the presence of a large amount of an added antioxidant, pyrogallol or ascorbic acid, during the saponification and extraction stages and the use of thin-layer chromatography for tocopherol purification. The total tocopherol levels of plasma and erythrocytes were determined for a number of human subjects, for patients with abetalipoproteinemia, and for rats. It was found that these levels had a wide range in normal human subjects but that the ratio of RBC to plasma tocopherol was relatively constant and equal to 0.18, uncorrected, and 0.21 when both RBC and plasma values were corrected to 100% recovery. The RBC-to-plasma ratio for rats was 0.39. The accuracy of this ratio determined by the spectrophotometric procedure was verified by measuring the distribution of [(14)C]tocopherol in RBC and plasma when radioactive vitamin E was introduced into the blood by both in vitro and in vivo techniques. The addition of radioactive tocopherol to RBC or plasma at the initial stage of the analysis permits an accurate determination of the total tocopherol in RBC or plasma by calculations based on the recovery of the added isotope. This procedure for erythrocyte tocopherol analysis is compared with a gas-liquid chromatographic method in current use.