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.

Differential adsorption of analyte and deuterated analogue onto the rotor seal of the injector in high-performance liquid chromatography/electrospray ionisation mass spectrometry.

Quantitation of analytes by liquid chromatography/mass spectrometry (LC/MS) is now very widely used, usually with an appropriate internal standard, but this does not guarantee problem-free analyses. Investigation of a problem that arose during method development is described, where it was surmised that differential adsorption onto the high-performance liquid chromatography (HPLC) injector of the analyte (Rac GTPase inhibitor NSC23766) and its deuterated analogue, as internal standard, was taking place. METHODS: Samples were injected onto the HPLC system via either a Valco C14W.1 internal sample injector or a Rheodyne 7125 injector, both with dissimilar Vespel rotor seals, and linked to a quadrupole time-of-flight (QTOF) mass spectrometer. Alternatively, samples were infused directly into the mass spectrometer via a syringe-driver. Electrospray ionisation in positive mode was used in both cases. RESULTS: Experiments demonstrated significant differential adsorption of the analyte (RAC inhibitor, NSC23766) and its tri-deuterated internal standard onto the Vespel rotor seal, with the latter seeming to be preferentially adsorbed. The deuterated analogue also showed lower electrospray sensitivity, as demonstrated by syringe infusion of a mixture of the compounds, compared with their separate infusion at the same concentration. CONCLUSIONS: This study has demonstrated the problem of differential adsorption onto HPLC Vespel rotor seals, and that the use of a stable-isotope-labelled analogue as internal standard does not guarantee the constancy of the analyte/internal response ratio in quantitative methods. The partial solution was to work at much higher concentration where adsorption, whilst still apparent, was relatively insignificant.

PBTK model for assessment of operator exposure to haloxyfop using human biomonitoring and toxicokinetic data.

Physiologically-based toxicokinetic (PBTK) models are mathematical representations of chemical absorption, distribution, metabolism and excretion (ADME) in animals. Each parameter in a PBTK model describes a physiological, physicochemical or biochemical process that affects ADME. Distributions can be assigned to the model parameters to describe population variability and uncertainty. In this study to assess potential crop sprayer operator exposure to the herbicide haloxyfop, a permeability-limited PBTK model was constructed with parameter uncertainty and variability, and calibrated using Bayesian analysis via Markov chain Monte Carlo methods. A hierarchical statistical model was developed to reconstruct operator exposure using available measurement data: experimentally determined octanol/water partition coefficient, mouse and human toxicokinetic data as well as human biomonitoring data from seven operators who participated in a field study. A chemical risk assessment was performed by comparing the estimated systemic exposure to the acceptable operator exposure level (AOEL). The analysis suggested that in one of the seven operators, the model estimates systemic exposure to haloxyfop of 49.04 ±â€¯10.19 SD µg/kg bw in relation to an AOEL of 5.0 µg/kg bw/day. This does not represent a safety concern as this predicted exposure is well within the 100-fold uncertainty factor applied to the No Observed Adverse Effect Level (NOAEL) in animals. In addition, given the availability of human toxicokinetic data, the 10x uncertainty factor for interspecies differences in ADME could be reduced (EFSA, 2006). Thus the AOEL could potentially be raised tenfold from 5.0 to 50.0 µg/kg bw/day.

Cavitation of liquid flow in electrospray mass spectrometry especially for addition of internal calibrant in 'accurate mass' measurements.

'Accurate mass' measurements in electrospray mass spectrometry are becoming more prevalent with the increasing availability of mass spectrometers with sufficient resolution. A reference compound is ideally admitted separately but almost simultaneously with the analyte and this is achieved by use of a 'dual sprayer' or voltage switching between reference and sample sprayers. This paper describes a novel third method, relying on cavitation (segmentation) of the liquid stream containing the reference compound, allowing the sample to ionise independently from the sample, thus preventing interference. The technique may also find application in kinetic experiments, such as protein folding studies. A high-performance liquid chromatography (HPLC) injector was also used to provide a reference compound, producing Gaussian-shaped profiles of varying ion intensity, thus allowing easier selection of a desirable measurement point where the intensities of the reference and analyte were similar.

Desferrioxamine dehydrogenates bilirubin in two stages, leading to a 1:1 red-coloured adduct. Characterization of the products by high-performance liquid chromatography/electrospray ionization mass spectrometry.

We have used open-chain tetrapyrroles, such as bilirubin, as molecular probes to investigate the pro-oxidant activity of desferrioxamine (DES) and its modulation by Trolox. On exposure to Fe-EDTA/H2O2, bilirubin and mesobilirubin underwent bleaching. When DES was present, bleaching was prevented and both rubins were converted into green-coloured derivatives and then into red pigments. Trolox added with DES inhibited the colour changes induced by DES. The oxidative products were resolved from their parent compounds by high-performance liquid chromatography (HPLC) and studied by electrospray ionization mass spectrometry and by UV/visible spectroscopy. The green products were identified as biliverdin or mesobiliverdin; the red pigments as the 1:1 molar adduct of DES with biliverdin or mesobiliverdin, less two hydrogens in both cases. It is concluded that DES exercises its oxidative activity through nitroxyl oxidizing radicals capable of efficient hydrogen abstraction, dehydrogenating either rubin to the corresponding verdin. A diradical derivative of DES (bearing two nitroxyl radicals in the same molecule) may be involved in the oxidation of verdins to red pigments, through concerted dehydrogenation and adduct formation. These results shed further light on the redox properties of bilirubin, DES and Trolox, and their interactions. They provide further evidence of the pro-oxidant activity of DES and suggest a more general biological significance, as rapid removal of bilirubin by bleaching or dehydrogenation may have pharmacological/toxicological implications in severe jaundice.

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.

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.

Bilirubin degradation by uncoupled cytochrome P450. Comparison with a chemical oxidation system and characterization of the products by high-performance liquid chromatography/electrospray ionization mass spectrometry.

Bilirubin is a protective antioxidant; however, when its conjugation and excretion are impaired, as in neonatal and hereditary jaundice, bilirubin accumulates and may cause severe neurotoxicity. Degradation of bilirubin takes place (a) on interaction with oxidative free radicals and (b) when cytochrome P450 (CYP) enzymes are uncoupled by polyhalogenated substrate analogues. The products of pathways (a) and (b) above have now been characterized by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and the mechanisms of fragmentation in part clarified. Oxidation of bilirubin by uncoupled CYP1A5 and by a Fe-EDTA/H2O2 system produced both biliverdin and an identical profile of dipyrrolic fragments, as detected by positive ESI-MS. A similar profile of oxidation products was found from mesobilirubin, all showing the expected increase in mass, thus providing direct evidence for fragmentation at the central methene bridge of the tetrapyrroles. Two degradation products, also detected by negative ESI-MS, were characterized as dipyrroles retaining the central bridge carbon, with one or two oxygen atom(s) bound (probably as the aldehyde and hydroperoxide derivatives). Ions compatible with propentdyopents and bilifuscins were also detected, but here the assignment was less certain. It is concluded that the first step in the oxidation of bilirubin may be hydrogen abstraction at the central methene bridge. This is followed either by loss of another hydrogen to give biliverdin, or by oxygen binding and fragmentation. Fe-EDTA/H2O2 and uncoupled CYP(Fe=O) may both initiate the reaction, the latter in an attempt to reduce the ferryl oxygen to water. These studies shed light on the CYP uncoupling mechanism and are of potential significance for the therapy of severe jaundice.

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.

Capillary electrochromatography of caffeine and its metabolites in rat brain microdialysate.

A capillary electrochromatography (CEC) method has been developed for the separation of caffeine and its two metabolites 1-methylxanthine (1-MX) and 1,7-dimethylxanthine (1,7-DX). The stationary phase was 3-(1,8-naphthalimido) propyl-modified silyl silica gel (NAIP) and the best separations were achieved with 4.0 mM citrate buffer (pH 5.0) containing 80% methanol at an applied voltage of 25 kV. The compounds were completely separated in less than 3.5 min with good repeatability, which was approximately 3-times less than that in high-performance liquid chromatography (HPLC) with NAIP. The proposed method coupled with microdialysis was successfully applied to the monitoring of caffeine concentration in rat brain with detection limits of 1.11 microg/mL.

Capillary electrochromatographic analysis of barbiturates in serum.

A capillary electrochromatographic method was developed for the separation of barbiturates. The separation was optimized in a 75 microm ID capillary, packed with 3-(1,8-naphthalimido)propyl-modified silyl silica gel (NAIP), studying the effect of buffer pH, buffer concentration, and mobile phase composition. Using an applied voltage of 20 kV and the short-end injection method (9 cm capillary effective length), the mobile phase of 1.0 mM citrate buffer (pH 5.0) containing 40% methanol provided the baseline separation of barbital, phenobarbital, secobarbital, and thiopental (internal standard) in less than 4.5 min. The method was successfully applied to the analysis of barbiturates in human serum. Under the optimal conditions, good repeatability and linearity were obtained in the range of 2.90-43.29 microg/mL for barbital, phenobarbital, and secobarbital.

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.