The 370-Da Inhibitor of the Sodium Pump in the Plasma of Haemodialysis Patients.

BACKGROUND: Previous studies have shown that a molecule of mass 370 Da that inhibits the sodium pump can be extracted from human placentas and from the concentrated plasma or ultrafiltrate of volume-expanded patients. AIM: This study aimed to study the abundance of the 370-Da molecule and its changes across dialysis in a population of patients with renal failure treated by haemodialysis. METHODS: Four millilitres of pre- and post-dialysis blood samples (2 mL plasma) were taken from patients receiving intermittent haemodialysis and analysed by high-performance liquid chromatography coupled to high sensitivity mass spectrometry. RESULTS: In over half of the study population, the 370-Da molecule was present in abundance that exceeded the limit of quantitation. Most patients experienced a marked fall in the abundance of the molecule over a haemodiafiltration session, though exceptions were seen in 2 individuals, both of whom showed clear evidence for the presence of 2 structural isomers of the 370-Da molecule. CONCLUSIONS: Advanced renal failure is frequently accompanied by an increased abundance of a 370-Da inhibitor of the sodium pump and that abundance is strongly impacted by haemodialysis. The technique described here could readily be applied to other clinical situations where sodium pump inhibition might be anticipated, such as hypertension, pregnancy, and foetal medicine, and thereby lead to a better understanding of the physiology and pathophysiology of these conditions.

Selective inhibition of the cellular sodium pump by emicymarin and 14ß anhydroxy bufadienolides.

Partial inhibition of the sodium pump (Na/K-ATP-ase) by a circulating inhibitor is known to occur in humans. The objectives of this study were to determine the effects of novel bufadienolides lacking an oxygen at C14 on sodium pumps in human erythrocytes and leucocytes, dog kidney and pig brain and to document the importance of the stereochemistry at C17 on the ability to inhibit these sodium pumps. 14α bufadienolides were weak inhibitors of all preparations studied. 3ß-OH,5ß,14ß bufadienolide produced near-total inhibition of dog kidney and pig brain Na/K-ATP-ase. Over the same concentration range, it maximally inhibited the sodium pump of erythrocytes by 70% and leucocytes by 47%. The inhibition profile induced in the leucocyte sodium pump deviated significantly from the simple sigmoidal relationship present in the other preparations over the 3×10(-5) to 1×10(-7) mol/l concentration range. Allo-emicymarin (17α) was confirmed to be a weak inhibitor of the sodium pump/ATP-ase compared with emicymarin (17ß) but both were weaker inhibitors of the leucocyte sodium pump than that of the other preparations. Molecules with the C14 in the ß configuration are more efficacious than in the α configuration. In the case of emicymarin, the attachment of the furone at C17 in the α configuration results in substantially weaker inhibitory activity than in the beta configuration, seen in most cardenolides and bufadienolides. Unlike ouabain and bufalin that show no specificity of action in these preparations, 3ß- OH,5ß,14ß bufadienolide selectively inhibits the activity of at least one low-prevalence subset of the leucocyte Na/K-ATP-ase.

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.

Elevation of anions in exercise-induced acidosis: a study by ion-exchange chromatography/mass spectrometry.

Acidosis is a major factor that determines the upper limit of exercise endurance. We have previously shown that anions usually associated with intermediary metabolism are elevated in critically ill patients with metabolic acidosis and contribute significantly to acidosis generation. This study was to determine whether volunteers with normal metabolism would exhibit similar elevations in anions associated with intermediate metabolism when exposed to a short-term physiological stress leading to a brief lactic acidosis. Physiological stress was induced on five healthy male subjects by means of a ramped exercise protocol. Blood was obtained immediately prior to and post-exercise, plasma ultrafiltrate was prepared and analysed immediately both by enzyme assay and liquid chromatography coupled to electrospray-mass spectrometry (LC/ESI-MS). Metabolic acidosis concomitant with a significant increase in blood lactate occurred in each subject, but in addition, anions normally associated with intermediate metabolism were significantly elevated after exercise. The contribution of these anions to generating an acidosis, and thus potentially limiting the extent of exercise, has never been acknowledged.

Unmeasured anions in metabolic acidosis: unravelling the mystery.

In the critically ill, metabolic acidosis is a common observation and, in clinical practice, the cause of this derangement is often multi-factorial. Various measures are often employed to try and characterise the aetiology of metabolic acidosis, the most popular of which is the anion gap. The purpose of the anion gap can be perceived as a means by which the physician is alerted to the presence of unmeasured anions in plasma that contribute to the observed acidosis. In many cases, the causative ion may be easily identified, such as lactate, but often the causative ion(s) remain unidentified, even after exclusion of the 'classic' causes. We describe here the various attempts in the literature that have been made to address this observation and highlight recent studies that reveal potential sources of such hitherto unmeasured anions.

A rapid LC-MS method for determination of plasma anion profiles of acidotic patients.

In metabolic acidosis, the concentrations of anions associated with intermediary metabolism are increased and can make a significant contribution to the observed acidosis. Here we describe a method for the rapid determination of the plasma ultrafiltrate profile of these anions using liquid chromatography coupled to electrospray ionisation mass spectrometry (LC/ESI-MS). The ultrafiltrate from patients with acidosis resulting from various causes were examined and the results compared to control values. Using the LC/ESI-MS method described, a unique plasma ultrafiltrate anion profile was obtained for each of the groups studied that provides rapid diagnosis of the type of underlying acidosis.

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

INTRODUCTION: Acute 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. METHODS: Plasma 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, alpha-ketoglutarate, succinate, malate and d-lactate. The results obtained from the study groups were compared with those obtained from control plasma from normal volunteers. RESULTS: In five patients with lactic acidosis, a significant increase in isocitrate (0.71 +/- 0.35 mEq l-1), alpha-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), alpha-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), alpha-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). CONCLUSION: The 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.

Differentiating midazolam over-sedation from neurological damage in the intensive care unit.

INTRODUCTION: Midazolam is used routinely to sedate patients in the intensive care unit (ICU). We suspected that midazolam over-sedation was occurring in the ICU of the Guy's and St. Thomas' Trust and that it could be difficult to differentiate this from underlying neurological damage. A sensitive assay for detecting midazolam and 1-hydroxymidazolam glucuronide (1-OHMG) in serum was developed and applied in the clinical setting. METHODS: In the present study we evaluated a series of cases managed in a mixed medical, surgical and trauma ICU. Serum was collected from 26 patients who received midazolam, were 'slow to wake' and in whom there was suspicion of neurological damage. Patient outcome was followed in terms of mortality, neurological recovery and neurological damage on discharge. RESULTS: Out of 26 patients, 13 had detectable serum levels of midazolam and/or 1-OHMG after a median of 67 hours (range 36-146 hours) from midazolam cessation. Of these 13 patients in whom midazolam/1-OHMG was detectable, 10 made a full neurological recovery. Of the remaining 13 patients with no detectable midazolam/1-OHMG, three made a full neurological recovery; 10 patients were subsequently found to have suffered neurological damage (P < 0.002), eight of whom died and two were discharged from the ICU with profound neurological damage. CONCLUSION: These findings confirm that prolonged sedation after midazolam therapy should be considered in the differential diagnosis of neurological damage in the ICU. This can be reliably detected by the assay method described. The effects of midazolam/1-OHMG persist days after administration of midazolam has ceased. After prolonged sedation has been excluded in this patient group, it is highly likely that neurological damage has occurred.

Circulating sodium pump inhibitors in five volume-expanded humans.

BACKGROUND: We have previously reported the isolation from human placentas of an inhibitor of the sodium pump (Na/K ATP-ase) of molecular weight 370 Da, which is considered to have a dihydropyrone-substituted steroid (bufenolide) structure. OBJECTIVE: To examine if this inhibitor is present outside of the pregnant state. METHODS: We examined the plasma ultrafiltrate of patients who were clinically volume-expanded. During the period of this study five such patients were identified. One was receiving haemofiltration for acute renal failure and four were being treated by plasma exchange. High performance liquid chromatograph (HPLC) purified fractions obtained from each of these five patients inhibited the human leucocyte sodium pump in vitro. RESULTS: Each of the purified fractions that inhibited the leucocyte ATP-ase in vitro contained a compound of mass 370 Da, the same mass as that found previously in placental extracts. This inhibitory factor was absent from HPLC purified fractions of plasma ultrafiltrate obtained from fifty-five patients who were clinically normovolaemic. Negative ion mass spectrometry (MS)/MS of the inhibitory material produced the fragmentation pattern characteristic of the placenta-derived pump inhibitor in only one of the five samples. The other four samples, although having the same mass, exhibited a different fragmentation pattern. CONCLUSION: The results suggest that an inhibitor of the sodium pump, identical in mass to that obtained from human placentas, circulates in the plasma of volume-expanded patients. The fragmentation pattern observed in negative ion mass spectrometry in the majority of the volume expanded patients may represent the presence of an isomer of the sodium pump inhibitor previously described in placental material.