Philip J. Hilton

ABNORMAL LEUCOCYTE COMPOSITION AND SODIUM TRANSPORT IN ESSENTIAL HYPERTENSION

Cation transport and electrolyte composition were studied in leucocytes from 17 patients with uncomplicated essential hypertension. Significant increases in cell sodium and water contents, associated with a depression of the rate-constant for active sodium efflux, were found in the hypertensive patients. These abnormalities in cell sodium transport may possibly be related to mechanisms of hypertension.

Circulating anions usually associated with the Krebs cycle in patients with metabolic acidosis

IntroductionAcute metabolic acidosis of non-renal origin is usually a result of either lactic or ketoacidosis, both of which are associated with a high anion gap. There is increasing recognition, however, of a group of acidotic patients who have a large anion gap that is not explained by either keto- or lactic acidosis nor, in most cases, is inappropriate fluid resuscitation or ingestion of exogenous agents the cause.MethodsPlasma ultrafiltrate from patients with diabetic ketoacidosis, lactic acidosis, acidosis of unknown cause, normal anion gap metabolic acidosis, or acidosis as a result of base loss were examined enzymatically for the presence of low molecular weight anions including citrate, isocitrate, α-ketoglutarate, succinate, malate and d-lactate. The results obtained from the study groups were compared with those obtained from control plasma from normal volunteers.ResultsIn five patients with lactic acidosis, a significant increase in isocitrate (0.71 ± 0.35 mEq l-1), α-ketoglutarate (0.55 ± 0.35 mEq l-1), malate (0.59 ± 0.27 mEq l-1), and d-lactate (0.40 ± 0.51 mEq l-1) was observed. In 13 patients with diabetic ketoacidosis, significant increases in isocitrate (0.42 ± 0.35 mEq l-1), α-ketoglutarate (0.41 ± 0.16 mEq l-1), malate (0.23 ± 0.18 mEq l-1) and d-lactate (0.16 ± 0.07 mEq l-1) were seen. Neither citrate nor succinate levels were increased. Similar findings were also observed in a further five patients with high anion gap acidosis of unknown origin with increases in isocitrate (0.95 ± 0.88 mEq l-1), α-ketoglutarate (0.65 ± 0.20 mEq l-1), succinate (0.34 ± 0.13 mEq l-1), malate (0.49 ± 0.19 mEq l-1) and d-lactate (0.18 ± 0.14 mEq l-1) being observed but not in citrate concentration. In five patients with a normal anion gap acidosis, no increases were observed except a modest rise in d-lactate (0.17 ± 0.14 mEq l-1).ConclusionThe levels of certain low molecular weight anions usually associated with intermediary metabolism were found to be significantly elevated in the plasma ultrafiltrate obtained from patients with metabolic acidosis. Our results suggest that these hitherto unmeasured anions may significantly contribute to the generation of the anion gap in patients with lactic acidosis and acidosis of unknown aetiology and may be underestimated in diabetic ketoacidosis. These anions are not significantly elevated in patients with normal anion gap acidosis.

An inhibitor of the sodium pump obtained from human placenta

BACKGROUND Much effort has been expended in the search for an endogenous inhibitor of the cellular sodium/potassium pump, a compound of major physiological importance, which has been implicated in the mechanism of essential hypertension. Others have suggested that ouabain or an isomer of ouabain may be the endogenous pump inhibitor. Neonatal cord serum contains an inhibitor of the sodium pump; we attempted to isolate and characterise this substance from human placentas. METHODS Homogenised placentas were dialysed and the resulting solutes were trapped on octadecylsilyl silica and then separated by high-performance liquid chromatography. Measurement of the activity of the sodium pump of human leucocytes was used to test each fraction for the presence of the inhibitor. FINDINGS An inhibitor of the sodium pump was obtained by this technique in a mass spectrometrically pure form with a mass of 370 Da, an empirical formula of C24H34O3 and only one hydroxyl group. The characteristic fragmentation pattern observed in negative-ion mass spectrometry was compared with those of various model compounds; this comparison suggested that the active material was a dihydropyrone-substituted steroid. INTERPRETATION These results suggest that a dihydropyrone-substituted steroid is an endogenous regulator of the sodium pump in humans and, presumably, other mammals. Proof of the endogenous origin will require the demonstration of a previously unrecognised biosynthetic pathway.

MAGNETIC RESONANCE RENOGRAPHY: OPTIMISATION OF PULSE SEQUENCE PARAMETERS AND Gd-DTPA DOSE, AND COMPARISON WITH RADIONUCLIDE RENOGRAPHY

The aim of this study was to assess the feasibility of magnetic resonance renography (MRR) using gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) in comparison with conventional radionuclide renography (RR) using technetium-99m-DTPA (99mTc-DTPA). MRR has many advantages over RR, including lack of ionising radiation, increased spatial resolution, and visible background anatomy. By optimising the pulse sequence, we developed an MRR protocol in which signal intensity is linear with Gd-DTPA concentration over a clinically relevant range. Twenty-nine patients and a volunteer were studied using this protocol. Magnetic resonance renography was performed using three different doses of Gd-DTPA: 0.1 mmol kg-1 (n = 13), 0.05 mmol kg-1 (n = 7), and 0.025 mmol kg-1 (n = 9). Each patient was also assessed using radionuclide renography. The resulting renograms were assessed in terms of time to peak signal intensity, signal decrease after peak, and kidney function ratios calculated from both the areas underneath and the slopes of the uptake curves. We have shown that the MR renograms obtained using low dose Gd-DTPA correlate best with the radionuclide renograms. Remaining discrepancies may be explained by variations in the injection procedures (hence in arterial input functions) and the limited coverage of the three MRR slices compared to the whole body projection of RR. Furthermore, at high local concentrations, signal becomes independent of T1 and is dominated by T2.

Ouabain, a circulating hormone secreted by the adrenals, is pivotal in cardiovascular disease. Fact or fantasy?

Substantial evidence points to the presence in human plasma of an inhibitor of the sodium/potassium pump which plays a central role in the pathophysiology of circulatory disorders, including essential hypertension. Studies from the 1980/90s claimed that this inhibitor was identical or very similar in structure to plant-derived ouabain and was synthesized by the adrenal cortex. However, the physical evidence in studies reporting isolation and identification of ouabain in human plasma appears insecure on closer examination. Additionally, reported circulating levels of immunoreactive ouabain in humans vary greatly, the ability of the human adrenal glands to secrete ouabain is questionable and the original commercial assay for measuring immunoreactive ouabain is no longer available. We submit that the position of ouabain as an endogenous, adrenally produced regulator of the sodium pump is of such importance that the current evidence needs either to put on a more secure footing or to lose its current status.

Effects of ouabain and low sodium on contractility of human resistance arteries.

Earlier work with rat arteries has resulted in a widely held assumption that resistance artery smooth muscle will not contract on exposure to a reduced transplasmalemmal sodium gradient. In view of the well-recognized low sensitivity of rat tissue to cardiac glycosides, we have investigated the effects of altering the transplasmalemmal sodium gradient on vascular smooth muscle tone by using human resistance arteries. Incubation of arteries in low sodium or in ouabain to inhibit active sodium efflux for 1 hour increased the contractile response to caffeine stimulation; this finding indicated enhanced calcium buffering by the sarcoplasmic reticulum. Prolonged incubation in ouabain in the presence of phentolamine or diltiazem resulted in a concentration-dependent increase in the tone of resting human resistance arteries. Reduction of the transplasmalemmal sodium gradient by incubation in low sodium buffer effected an increase in tone similar to that obtained in the presence of ouabain. These results suggest that alteration of the transplasmalemmal sodium gradient may increase the vascular smooth muscle tone of human resistance arteries by altering intracellular calcium handling. This is a new finding in human resistance arteries and may involve inhibition and, indeed, reversal of sodium-dependent calcium efflux. A concentration-dependent potentiation of tone was found after the addition of ouabain to submaximally activated arteries. Sodium-calcium exchange may also play a pivotal role in this mechanism.

Cyclosporin A inhibits endothelium-dependent, prostanoid-induced relaxation in human subcutaneous resistance vessels

The effect of cyclosporin on endothelium-dependent relaxation of human subcutaneous resistance vessels was investigated. Resistance vessels were obtained from normal subjects undergoing routine surgery. Acetylcholine-induced relaxation was shown to consist of at least two components, one mediated through vasodilatory prostanoids, the other through endothelium-derived relaxing factor (EDRF). Incubation with cyclosporin inhibited endothelium-dependent relaxation, as assessed by the response to acetylcholine. The inhibitory effect of cyclosporin on the acetylcholine response was most marked in the presence of methylene blue, which is an inhibitor of EDRF. This suggests that the inhibition of endothelium-dependent relaxation by cyclosporin is mediated by an effect on vasodilatory prostanoids.

Cyclosporine A inhibits relaxation but does not induce vasoconstriction in human subcutaneous resistance vessels.

The effects of cyclosporine on the functional characteristics of human subcutaneous resistance vessels were investigated. Resistance vessels were obtained from normal subjects undergoing routine surgery. Incubation with cyclosporine did not alter the resting tone of the vessels, but decreased the maximum contractile response and the sensitivity of the vessels to stimulation with exogenous noradrenaline and potassium. Cyclosporine decreased the rate of spontaneous relaxation and inhibited endothelium-dependent relaxation as assessed by the response to acetylcholine. Paradoxically, endothelium-independent relaxation as assessed by the response to sodium nitroprusside was augmented. Hypertension induced by cyclosporine may, in part, be explained by a reduction in relaxation of peripheral resistance vessels.

Endogenous Ouabain Is Not Ouabain

The concept of a circulating digitalis-like inhibitor of the sodium pump, Na+, K+-ATPase, evolved from studies performed in the 1960s. De Wardener et al1 addressed the question of whether a small increase in the glomerular filtration rate together with changes in the concentration of the more recently discovered hormone aldosterone could explain the natriuresis that generally followed salt (sodium) loading. In their studies, dogs had their renal blood flow reduced significantly by constricting the aorta above the renal arteries and were given supramaximal doses of fludrocortisone, a synthetic analogue of aldosterone, and vasopressin before being challenged with intravenous saline. Their ability to develop a natriuresis clearly demonstrated that a third factor (beyond changes in glomerular filtration rate and aldosterone concentrations) was involved in the natriuresis of salt loading. Subsequent experiments demonstrated that the responsible agent could be transmitted by the plasma of the volume-expanded animal.2 Although we may now ask whether in these experiments the effect was mediated, at least in part, by the release of atrial and B-type natriuretic peptide from the heart, it was nearly 2 decades before those hormones were discovered during which time it was demonstrated that the plasma of volume-expanded animals had the ability to inhibit the sodium pump3—which is not a target of atrial natriuretic peptide and B-type natriuretic peptide. Essentially parallel studies were performed by Welt and colleagues4 in uremia where inhibition of the sodium pump of erythrocytes was demonstrated along with the ability of uremic plasma to induce such a defect in normal erythrocytes. In 1975, it was shown that patients with essential hypertension had, as a group, reduced activity of the sodium pump of leukocytes, with corresponding elevated values for intracellular sodium.5 This finding proved to be reproducible in various laboratories, …

Leucocyte intracellular pH and Na(+)-H+ exchange activity in essential hypertension: an in vitro study under physiological conditions

The cellular basis for essential hypertension remains obscure. Abnormal ion transport has been demonstrated in both experimental and essential hypertension, raised levels of sodium-lithium (Na(+)-Li+) and sodium-proton (Na(+)-H+) exchange in blood cells being a consistent feature. However, Na(+)-H+ exchange is not the main regulator of intracellular pH at resting pH, while the importance of the contribution of bicarbonate to cellular pH regulation is now increasingly appreciated. Serum and serum-derived growth factors are known to affect intracellular pH and the activity of the Na(+)-H+ antiporter. This study was designed to investigate the activity of Na(+)-H+ exchange in the leucocytes of patients with essential hypertension in the presence of bicarbonate in vitro and to measure the effect of autologous serum on intracellular pH and Na(+)-H+ exchange. Paired serum samples from essential hypertensives and their controls were used on leucocytes from other (unrelated, normotensive) donors to investigate the same parameters. In a study of 30 patients with untreated essential hypertension and 30 controls matched for age, sex, race and body habitus we found no difference in resting pH or buffering capacity (pH 7.28 +/- 0.01 and 32.0 +/- 1.6 mmol/l per pH, hypertensives, versus 7.27 +/- 0.02 and 34.5 +/- 1.8 mmol/l per pH, controls) but a marked difference in the maximal rate of Na(+)-H+ exchange in response to intracellular acidification (57.8 +/- 3.2 mmol/l per min versus 47.2 +/- 1.4 mmol/l per min, P = 0.004).(ABSTRACT TRUNCATED AT 250 WORDS)