Jean B. Smith

A gene for high urinary kallikrein may protect against hypertension in Utah kindreds.

The inheritance of 12-hour overnight total urinary kallikrein excretion and its association with family history of essential hypertension were studied in 405 normotensive adults and 391 youths in 57 Utah pedigrees. Total urinary kallikrein excretion was highly familial with 51% of the total variance attributable to a dominant allele for high total urinary kallikrein excretion and 27% attributable to the combined effects of polygenes and shared family environment. An estimated 28% of the population has one or two copies of the dominant allele for high total urinary kallikrein excretion (2.3 SD units higher than the low homozygotes). About 83% of the population could be assigned to one of the two genotypic populations. Individuals with the high total urinary kallikrein excretion genotype were significantly less likely to have one or two hypertensive parents (relative odds = 0.56, p = 0.042). We conclude that a dominant allele expressed as high total urinary kallikrein excretion may be associated with decreased risk of essential hypertension. Further studies should be performed to confirm this finding and to test for interactions between this apparently protective gene and other genetic and environmental determinants of essential hypertension.

Associations of three erythrocyte cation transport systems with plasma lipids in Utah subjects.

To investigate the pathophysiology of essential hypertension, detailed biochemical and clinical variables were collected and analyzed for 2091 Utah subjects aged 3 to 83 years. Three different measurements of erythrocyte cation transport were obtained: Na+-Li+ countertransport, Li+-K+ cotransport, and furosemide-insensitive Li+ efflux into MgCl2. Total plasma cholesterol, triglycerides, and high density lipoprotein cholesterol levels were obtained from fasting subjects. Levels of high density lipoprotein subfractions 2 and 3 were also obtained from 350 subjects. Standardized data collection also included blood pressure, height, weight, and presence or absence of a diagnosis or treatment of essential hypertension. In univariate analyses of all 1420 adults, each of the three transport systems showed the same significant correlations with triglyceride levels (r = 0.33-0.35, p less than 0.0001), high density lipoprotein concentration (r = -0.19 to -0.21, p less than 0.001), and weight (r = 0.22-0.28, p less than 0.0001). In multivariate regression analyses, values for each transport system were significantly higher in hypertensive subjects; values for triglycerides, high density lipoprotein, and usually, the high density lipoprotein subfractions continued to have strong significant independent associations with all three transport systems; and weight remained significantly related only to Na+-Li+ countertransport. In separate logistic regressions, plasma triglyceride levels (positively, p less than 0.001) and high density lipoprotein subfraction 3 levels (inversely, p less than 0.03) were associated with hypertension itself. In multivariate analyses among 671 children, high density lipoprotein and high density lipoprotein subfraction 3 levels showed significant (p less than 0.05) inverse correlations with Na+-Li+ countertransport and furosemide-insensitive Li+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

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 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.

A source of error in equilibrium dialysis

Equilibrium dialysis is often used to study the binding of steroids to proteins. With this technique it is customary to determine the percent bound and unbound steroid in the sample, the affinity constant for the steroid-protein binding reaction, and the concentration of binding sites on the protein. Investigators have used many different ratios of dialysis buffer to sample volumes in their experiments assuming that the equilibrium in the post-dialysis sample was the same as existed before dialysis. Chemical equilibrium expressions for the system before and after dialysis indicate that during dialysis the concentration of steroid in the sample decreases resulting in a new equilibrium in which the percent bound and unbound are different from the original sample. The magnitude of the difference between the pre- and post-dialysis systems is proportional to the ratio of dialysis buffer to sample volumes. Accurate values for the affinity constant and binding site can be obtained only if this change in the equilibrium is considered.

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.

The Relationship of Lithium-Potassium Cotransport and the Passive Lithium Leak to Hypertension in Utah Subjects

Rate constants for lithium-potassium cotransport (kLPC) and the lithium efflux into MgCl2 with furosemide (passive lithium leak) along with sodium-lithium countertransport (SLC) were measured in erythrocytes from 351 normotensive adults age 18 and over, 220 youth under age 18 and in 27 hypertensives. The kLPC was significantly higher in the hypertensives than the adult normotensives with means and standard deviations of 13.9 +/- 9.2 vs. 8.7 +/- 5.9 10(-3)/hr (p less than 0.01). Adjusting for the significant weight (p = 0.014) and sex (p = 0.066, normotensive males higher than females) associations with kLPC in an analysis of covariance, increased the significant difference between the hypertensives and normotensives (p = 0.0004). The passive lithium leak rate constant was also higher in hypertensives than normotensives (20.2 +/- 7.6 vs. 15.5 +/- 5.3 10(-3)/hr, p less than 0.01). Weight (p=0.0003), but not sex, was related to the leak but did not account for the difference between hypertensives and normotensives (p = 0.0009). Mean blood pressure was positively associated with the lithium leak but not the kLPC or SLC values in a multivariate regression.

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.