Wayne M. Hentschel

A reproducible sodium-lithium countertransport assay: The outcome of changing key laboratory parameters

This paper describes experimental conditions for reproducible measurement of sodium-lithium countertransport in red blood cells. The assay is sensitive to temperature (10% per degree C) and the condition of the red cells; it is affected little by changes in intracellular lithium over the range 2-7 mmol/1 or by red cell concentrations with laboratory hematocrits of 0.03 to 0.07. Repeated measurements of the same subjects give day-to-day coefficients of variation of 10% or less. The mean difference for interlaboratory comparisons is 11%.

Erythrocyte cation transport activities as a function of cell age

Erythrocyte cation transport systems were evaluated on cell fractions from 17 subjects. Density centrifugation was used to separate washed red cells into fractions enriched with younger and older cells; the cell age differences in these fractions were verified by reticulocyte counts (means are 3.5% for younger cell fractions and 0.7% for older cell fractions). Red cell age has a pronounced effect on several cation transport activities. The older cell fractions had increases in lithium-potassium cotransport (p less than 0.001), the rate constant for the lithium-potassium cotransport (p less than 0.001) and cellular cation permeability. The older cells had decreases in the number of ouabain binding sites (p less than 0.001), the rate constant for sodium efflux via the sodium-potassium adenosine triphosphatase pumps (p less than 0.001) and the sodium-lithium countertransport (p less than 0.025). In subjects with markedly different cell ages, these effects should be considered when evaluating red cell cation transport activities.

A simplified method for simultaneously determining countertransport and cotransport in human erythrocytes

Both sodium countertransport and sodium-potassium cotransport are altered in erythrocytes from some hypertensive subjects and their relatives. Lithium can substitute for sodium in both of these transport mechanisms; they can then be monitored as sodium-lithium countertransport and lithium-potassium cotransport. Using erythrocytes loaded with lithium, we can determine both transport systems simultaneously by monitoring the rate of lithium efflux into three media: (1) NaCl, (2) MgCl2 and (3) MgCl2 with furosemide. The difference between the effluxes into NaCl and MgCl2 is the sodium-lithium countertransport; the difference between the effluxes into MgCl2 with and without the cotransport inhibitor furosemide is the lithium-potassium cotransport. At the intracellular Li concentrations used in these experiments, lithium-potassium cotransport is a linear function of the Li+ concentration and can be expressed by the equation for a first order reaction. The rate constant can be calculated by dividing the lithium-potassium cotransport by the intracellular lithium concentration and correlates well (r = 0.80, n = 30) with sodium-potassium cotransport measured by Dagher and Garays method. The simultaneous measurement of countertransport and cotransport requires much less time, effort and material than measuring the two transports separately.

An improved non-radioisotopic method for measuring ouabain-sensitive Na+ efflux from erythrocytes

Ouabain-sensitive Nat efflux (Na+, KC ATPase activity) from erythrocytes is most commonly measured by monitoring the efflux of radioactive sodium from previously loaded cells [l-3), a technique which requires considerable in vitro manipulation of the erythrocytes and the handling of radioisotopes. Cumberbatch and Morgan [4] have proposed a method based on the assumption that the sodium fluxes of red cells in whole blood are in equilibrium. In their method, which does not require the use of labeled sodium, ouabain is added to the whole blood to block the ouabain-sensitive Na+ efflux. Na+ influx is not blocked; therefore, the intracellular Na+ continues to increase. Cumberbatch and Morgan reason that the rate of increase in intra~llular Na+ is equal. to the ouabain-sensitive Na+ efflux, an assumption that is valid only if other Na+ efflux pathways are insignificant. We describe a simple method for measuring ouabain-sensitive Na+ efflux which does not use radioactivity and which does not rely on assumptions about other Na+ transport mechanisms. Sodium efflux via the Naf pump is determined from the difference in efflux of intracellular Na+ into two media, one containing Kf to maximize pump activity, the other containing ouabain to inhibit pump activity.

Isolation of heparan sulfates with antithrombin III affinity and anticoagulant potency from BALBc 3T3, B16.F10 melanoma, and cutaneous fibrosarcoma cell lines

The heparan sulfates synthesized in vitro by three cell lines were isolated by proteolysis and preparative anion exchange chromatography and purified free of other glycosaminoglycans by selective enzymatic degradation. The isolates from the medium of BALB/c 3T3 fibroblasts, B16.F10 melanoma cells, and a cutaneous fibrosarcoma line, along with that from the detergent-extracted cell layer of the fibroblasts, were affinity-fractionated on columns of matrix-immobilized human antithrombin III. Each heparan sulfate contained subfractions with high affinity for the proteinase inhibitor, ranging from 3-34% of the starting material. The high affinity species possessed measurable anticoagulant activities by a clotting assay (6 to 30 units/mg). Since none of the lines were derived from cell types having any known biological role in vascular homeostasis, we suggest that anticoagulant activity of the glycosaminoglycan is a random property of its primary structure.

Reserve bilirubin binding capacity assessed by difference spectroscopy: Assay statistics and results on newborn sera

Bilirubin binding properties of newborn sera and assay parameters have been investigated using a difference spectroscopy procedure [9]. Reserve bilirubin binding capacity, serum bilirubin and the total bilirubin binding capacity can be determined using only 40 microliters of serum. The measured total binding capacities agreed with the theoretical binding capacities calculated from serum albumin concentrations assuming a 1 : 1 molar binding ratio of bilirubin to albumin; in 102 assays on newborn sera, the ratio of experimental to theoretical total binding capacity was 1.04. Bilirubin binding capacity measurements were linear over the range 0--600 mg/l. Day to day precision of binding capacity determinations on 6 albumin controls yielded coefficients of variation between 4.1 and 7.2%. Recovery for the reserve bilirubin binding capacity determinations was 99.6%. In a study of 22 newborns, reserve bilirubin binding capacities showed an inverse relationship with the changes in serum bilirubin concentrations. None of the newborns included in our study appeared to be in dange of bilirubin encephalopathy.

A kinetic expression for sodium-lithium countertransport in human red cells

Sodium-lithium counter-transport in human red blood cells may be a potentially useful measurement in studies of hypertension. A kinetic expression describing this counter-transport was derived and evaluated using red cells from nine subjects at various concentrations of intracellular and extracellular Li+ and Na+. The countertransport is dependent upon all four concentrations, intracellular Li+ and Na+ as well as extracellular Li+ and Na+. We confirm that the maximum Na+-Li+ counter-transport (Vmax) is a property of the individual cells while the half-maximal saturating concentrations (K 1/2) for Li+ and Na+ are the same for all subjects. This expression permits a more thorough understanding of conditions affecting Na+-Li+ countertransport measurement.