Isolation of heparan sulfates with antithrombin III affinity and anticoagulant potency from BALB/c 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.

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.

Hemodialysis does not affect erythrocyte sodium-lithium countertransport.

Sodium-lithium countertransport measurements on erythrocytes are currently of interest because some hypertensive subjects and their relatives have abnormally high values. Woods et al [1] reported that red cells taken from dialysis patients after hemodialysis had significantly lower sodium-lithium countertransport than before dialysis. They suggested that sodium-lithium countertransport is influenced by 'a dialyzable plasma factor'. We conducted experiments to further evaluate their observations relating to the 'dialyzable plasma factor'. However, we have been unable to confirm their findings. Neither an effect of hemodialysis on sodium-lithium countertransport in erythrocytes from maintenance dialysis patients nor any effect of dialysis on normal erythrocytes in vitro was evident. Our results do not support the existence of a dialyzable plasma factor affecting sodium-lithium countertransport.

Three red cell sodium transport systems in hypertensive and normotensive Utah adults.

Sodium-lithium countertransport (SLC), sodium-potassium cotransport (CoT), and ouabain binding to sodium-potassium adenosine triphosphatase (Na, K-ATPase) sites were measured on fresh erythrocytes from hypertensive and normotensive Utah subjects with and without a first-degree relative with hypertension. SLC was measured as Li+ efflux into NaCl and MgCl2 media from Li+-loaded cells (5-7 mM). CoT was measured by monitoring Na+ and K+ efflux from cells loaded to 20-30 mM Na+ and 20-30 mMK+. Ouabain binding was determined for fresh cells using 3H-ouabain. Subjects were selected from pedigrees that showed a prevalence of hypertension. SLC was significantly elevated in 26.5% of the hypertensive subjects (p less than 0.001) as well as in 12.8% of the normotensives with a hypertensive first-degree relative (p less than 0.05). Although elevated SLC and decreased CoT have previously been associated with hypertension, no hypertensive subject in this study exhibited both abnormalities. All subjects with elevated SLC had normal CoT. A positive correlation between SLC and CoT was observed. Few hypertensive subjects (11.8%) had decreased CoT. In the majority of subjects studied, both SLC and CoT were normal: hypertensives 61.8%, normotensives with a hypertensive first-degree relative 61.7%, and other normotensives 58.7%. The number of ouabain-binding sites was not significantly altered among hypertensives, or their relatives, even though there was a positive correlation between SLC and the number of ouabain-binding sites.

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 Garay's method. The simultaneous measurement of countertransport and cotransport requires much less time, effort and material than measuring the two transports separately.

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%.

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.

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.

High-density lipoproteins estimated by an enzymatic cholesterol procedure, with a centrifugal analyzer.

We describe an assay for high-density lipoprotein cholesterol, adapted to a centrifugal analyzer, the GEMSAEC System 3, which includes use of an increased Mn2+concentration (91 mmol/liter) [J. Lipid Res. 19, 65 (1978)] and ethylenediaminetetraacetate [Clin. Chem. 22, 98 (1976)]. Modifications to the GEMSAEC system include reducing the mixing burst and preconditioning the sample tip. Accuracy of this procedure, as assessed by analysis of a control pool from the Center for Disease Control, was 99.2%. Day-to-day precision for two control pools was 320 +/- 13 and 506 +/- 17 mg/liter. Serum sample volume was decreased to 0.5 ml. In blanks with heparin/Mn2+ present, the pseudocholesterol concentrations resulting from a reaction of the enzymatic cholesterol reagent and the heparin/Mn2+ precipitating reagent depend on the source of the enzymatic reagent and appear to be enhanced slightly by the use of ethylenediaminetetraacetate. Pseudocholesterol concentrations reach a maximum at heparin/Mn2+ concentrations well below those needed to completely precipitate the low-density and very-low-density lipoprotein fractions. Population reference values were obtained from analyses done on 224 local physicians (mean: male, 500 mg/liter; female, 620 mg/liter) and 156 ambulatory patients (mean: male, 463 mg/liter; female, 553 mg/liter).

Ethionine-resistant mutants of the filamentous blue-green alga Plectonema boryanum.

Plectonema boryanum mutants that are resistant to ethionine are unable to incorporate ethionine into acid-precipitable material. Ethionine causes bleaching of chlorophyll in sensitive cells.