PubMed Health⌕ Search

Biomedical subjects

A Bevington

Publications and source records attributed to A Bevington.

At least 19 recordsLinked to original sources
PubMed1992

Creatine and creatinine metabolism in uraemia.

Measurements of serum creatinine to monitor the progression of chronic renal failure may be misleading and influenced by factors other than GFR. Creatinine is produced by the degradation of creatine which is stored in muscle. The effect of biochemical changes observed in uraemia on the transport of [1-14C]creatine were examined in a cultured muscle model. In a Na-free medium flux of [1-14C]creatine was reduced (p < 0.001), however the addition of various uraemic toxins, insulin, parathyroid hormone and the alteration of pH had no effect on creatine influx. The variance of red cell creatine levels in uraemic patients was significantly different from controls (p < 0.02) and the plasma to red cell creatine gradient was low in some patients.

Animals↗
PubMed1990

Calcium and orthophosphate deposits in vitro do not imply osteoblast-mediated mineralization: mineralization by betaglycerophosphate in the absence of osteoblasts.

It has been shown in several laboratories that addition of beta-glycerophosphate (beta GP), a substrate for alkaline phosphatase (AP), to cultured osteoblast-like cells induces deposition of orthophosphate (Pi) and Ca within seven days. Even though this effect is regarded as an in vitro model of bone mineralization, it is not known whether it is specific for osteoblasts. We have, therefore, studied the amounts of Pi and Ca deposited after seven days with 10 mM beta GP in culture wells containing confluent cultures of osteoblast-like cells (OB) derived from human trabecular bone explants, human skin fibroblasts (SF), or culture medium alone (MED). Ox liver AP at an activity considerably greater than the endogenous AP activity of the cells, but comparable with that of other osteoblast models, was added to ensure a similar rate of Pi generation from beta GP in all wells. beta GP was converted quantitatively to Pi within seven days, leading to a nonphysiological 10-fold increase in the Pi concentration in the culture medium. After thorough rinsing on day seven, the OB and SF wells contained deposits of Pi and Ca, but the amounts were comparable for the two cell types. Smaller, but significant, amounts of Pi and Ca were also detectable even in rinsed MED wells. This suggests that the detection of such deposits in beta GP experiments cannot necessarily be interpreted as a specific property of osteoblast cultures in vitro, and may simply reflect the presence of AP.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1989

32P-labelling anomalies in human erythrocytes. Is there more than one pool of cellular Pi?

1. Human erythrocytes were incubated in autologous plasma containing [32P]Pi, and sampled by a method which avoids washing the cells. 2. In experiments of up to 3 h duration, the specific radioactivity of cellular Pi stabilized at a value below that of extracellular Pi. This can be explained on the basis of a single cellular Pi pool exchanging with a large unlabelled pool of cellular organic phosphates. 3. However, a rapid initial phase of labelling, occurring within 30 s, was inconsistent with the situation described in point 2. A possible explanation is that about 1/4 of cellular Pi occurs in a separate, fast-labelling pool. 4. When the extracellular Pi concentration was doubled, most of the corresponding increase in the steady-state cellular Pi concentration was accounted for by the apparent fast-labelling Pi pool, which also doubled. 5. The observed initial rate of labelling of cellular organic phosphates [which probably occurs through the reaction catalysed by glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12)] was considerably lower than that predicted from the flux through the Embden-Meyerhof pathway. This implies that the enzyme is exposed to Pi whose specific radioactivity is lower than the mean specific radioactivity of cellular Pi, and fails to support earlier suggestions that this enzyme uses extracellular Pi. 6. In 3 h incubations, the rate of organic phosphate labelling was roughly constant throughout, even though the specific radioactivity of cellular Pi had risen slowly to a plateau. Viewed in conjunction with point 5, this again suggests some inhomogeneity in cellular Pi. 7. Cellular Pi and extracellular Pi only reached isotopic steady state after 2 days. At this stage some organic phosphates were probably still incompletely labelled. 8. We conclude that, whatever their physical or technical reasons, such labelling inhomogeneities and slow attainment of isotopic steady state may cause serious misinterpretation of results if ignored during 32P-labelling of intact cells.

Erythrocytes↗
PubMed1989

Selective extraction, concentration, and assay of orthophosphate from microliter quantities of cultured mammalian cells.

A colorimetric procedure is described for determination of orthophosphate (0.2-2.5 nmol) in sample volumes up to 400 microliters. Orthophosphate is selectively extracted (in the form of phosphomolybdate) into an organic solvent mixture (2-methylpropan-1-ol and petroleum spirit) leaving interfering substances, such as labile organic phosphates, in the aqueous phase. Orthophosphate is then back-extracted into a small volume of aqueous sodium hydroxide. By keeping this volume small, orthophosphate from large dilute samples can be concentrated into small volumes and assayed colorimetrically in microcuvettes using the dye malachite green. The procedure is highly reproducible and insensitive to interfering substances, as shown by comparison with a conventional malachite green assay without the solvent extraction.

Animals↗
PubMed1988

Theoretical interpretation of isotope labelling experiments in cells in which the label is chemically incorporated: the example of orthophosphate.

Studies of transport across the plasma membrane in intact cells frequently involve measuring the incorporation of a labelled extracellular species into the cells. Unfortunately, if the labelled species is metabolized in the cell, the kinetics of labelling are made more complicated. Using the example of the incorporation of 32P-labelled orthophosphate into cells, we describe a mathematical model which allows for this complication, and show how this may alter the interpretation of experiments. The analysis is widely applicable to cellular labelling studies with any species that undergoes chemical exchange with a large cellular pool.

Animals↗
PubMed1988

Regulation of phosphate metabolism in human red cells following prolonged incubation to steady state in vitro.

Human red cells were incubated aseptically in vitro for 24 or 48 h to allow the cellular concentrations of orthophosphate (Pi) and organic phosphates to attain steady state. In plasma at pH 7.0-8.0, the transmembrane Pi concentration ratio R (cellular Pi/plasma Pi) decreased with increasing pH, with a slope which was 2.7-times greater than that predicted if Pi simply distributed passively across the cell membrane. The concentration of 2,3-bisphosphoglycerate (2,3-BPG), the most abundant cytosolic organic phosphate, decreased at acidic pH and increased at alkaline pH, but stabilised at these values after 24 h. Therefore, while net generation or consumption of Pi by 2,3-BPG may initially have contributed to the steep dependence of R on pH, some other factor must have maintained this anomaly after 24 h. In plasma in which the Pi concentration was increased from 1 to 2.5 mM, the cellular Pi concentration increased from 0.6 to only 1.0 mmol/l cells, and 2,3-BPG increased by less than 20%. Thus, cellular Pi and 2,3-BPG concentrations seemed to be buffered or regulated in the face of changes in extracellular Pi. However, this regulation failed in a Pi-free balanced salt solution, as the 2,3-BPG concentration declined to half that observed in freshly drawn blood, although cell Pi remained at about 0.3 mM. Incubation in Pi-free solution with ouabain for 24 h to decrease the transmembrane sodium gradient, or incubation for 2 h in the absence of sodium, decreased this residual cellular Pi by about 20%, but did not abolish it. In Pi-free solution, but not with 1 mM Pi, cellular Pi increased when passive transmembrane Pi leakage was inhibited with 4-acetamido-4'-iso-thiocyanatostilbene-2,2'-disulphonate (SITS). We conclude that red cell Pi concentration cannot be explained fully by passive transmembrane distribution of Pi, nor by changes in 2,3-BPG, and that part of the anomaly may arise from sodium-linked active Pi transport.

2,3-Diphosphoglycerate↗
PubMed1988

Net fluxes of orthophosphate across the plasma membrane in human red cells following alteration of pH and extracellular Pi concentration.

Even though net fluxes of Pi (orthophosphate) across the cell membrane may be important in clinical disorders involving the abnormal extracellular Pi concentration, in acid-base disturbances, and in the responses of some cells to hormones, relatively few studies have been made of these fluxes, owing to the complexities of interpretation. Here we have studied net fluxes in response to changes in extracellular pH and Pi concentration in the simple case of the human red cell. The permeability of the cell membrane to net Pi fluxes was described in terms of a first-order rate constant, epsilon. By means of a mathematical model, it was possible to discriminate between transmembrane Pi movement, net intracellular generation or consumption of Pi by organic phosphates, and extracellular generation of Pi from the cells lysing during the experiment. We show that net Pi influx into the cell during experimental alkalosis was probably driven by net consumption of Pi by organic phosphates, and that this was reversed during acidosis. Inhibition of net Pi influx by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonate (SITS) suggests that, like Pi self-exchange, net influx is at least partly mediated by the band 3 transport protein. Unexpectedly, epsilon increased from 2 h-1 at extracellular pH 7.4 to approx. 7 h-1 at pH 7.8. From the value of epsilon at pH 7.4, we conclude that the apparent buffering or regulation of steady-state Pi concentrations, previously reported in red cells in vitro, was not an artifact of intracellular generation of Pi from organic phosphates.

Erythrocyte Membrane↗
PubMed1988

Erythrocyte phosphate metabolism and pH in vitro: a model for clinical phosphate disorders in acidosis and alkalosis.

When whole blood from 9 normal donors was incubated at 37 degrees C for 90 min under 95%, O2/5% CO2, over a range of pH from 7.1 to 7.9, the concentrations of orthophosphate (Pi) in plasma, in whole blood and in the cells decreased with increasing pH. At all pHs the ratio of cell to plasma concentrations of Pi was higher than that predicted from the cell to plasma distribution ratio for chloride, on the assumption that the mono- and di-anionic forms of Pi distributed passively in response to the membrane potential. Both the observed and predicted distribution ratios for Pi, and the difference between them, decreased with increasing pH. It is concluded that the observed distribution of Pi between erythrocytes and plasma is not consistent with a steady-state passive distribution, and that small changes of pH in vitro can lead to marked alterations in cellular Pi concentration. These measurements provide a direct example of the redistribution of Pi between cells and plasma, which has been postulated to occur in glycolyzing cells, leading to Pi depletion, during pH disturbances in vivo.

2,3-Diphosphoglycerate↗
PubMed1986

Cellular phosphate metabolism in patients receiving bisphosphonate therapy.

Patients with Paget's disease of bone were treated with oral disodium dihydrogen ethylidene-1-hydroxy-1,1-bisphosphonate (EHBP), a drug that is known to stimulate renal tubular reabsorption of orthophosphate (Pi). After 2 weeks of treatment, plasma Pi rose from 1.02 to 1.67 mmol/l. No increase in Pi was observed with the related drug, dichloromethylene bisphosphonate, which also reduces bone turnover in Paget's disease. Intravenous EHBP caused a more rapid increase in plasma Pi, but maximum hyperphosphatemia was not observed until 7-11 days after treatment commenced. It is therefore unlikely that this effect is due to an immediate action of EHBP on the luminal face of the renal brush border Pi transporter. After 2 weeks of oral EHBP, the increase in the Pi concentration in patients' erythrocytes was 31% compared with 64% in plasma. In blood platelets and leukocytes, negligible changes in cellular Pi occurred. The concentrations of 2,3-diphosphoglycerate, adenosine 5'-diphosphate (ADP) and adenosine 5'-triphosphate (ATP) were unaltered, indicating that these organic phosphates were not offsetting a potential change in cellular Pi. The decrease in erythrocyte/plasma distribution ratio for Pi was also observed in patients receiving intravenous EHBP. However, no change occurred in cell/plasma distribution of chloride, suggesting that this apparent regulation of cellular Pi did not arise from changes in erythrocyte membrane potential, pH, or water content.

Administration, Oral↗
PubMed1986

A study of intracellular orthophosphate concentration in human muscle and erythrocytes by 31P nuclear magnetic resonance spectroscopy and selective chemical assay.

In order to study the relationship between extracellular and intracellular concentrations of orthophosphate (Pi), phosphorus nuclear magnetic resonance spectra were recorded, at rest, from the flexor digitorum superficialis muscle of hypophosphataemic patients with vitamin D-resistant rickets, and patients with Paget's disease of bone before and after they had been made hyperphosphataemic by treatment with the drug ethylidene-1-hydroxy-1,1-bisphosphonate. Changes in intramuscular P1 were estimated from the ratio of the areas of the Pi to adenosine 5'-triphosphate peaks. Even though the plasma Pi concentration in these patients spanned a fourfold range (0.5-2.0 mmol/l) the corresponding intramuscular Pi concentration increased by only 70%. A similar effect was observed in erythrocytes, from patients with these disorders, which were incubated in autologous plasma at 37 degrees C, under an atmosphere of O2 + CO2 (95:5, v/v). However, chloride ions, which are transported passively across the cell membrane, showed no change in distribution between cells and plasma, indicating that there was no general effect on passive anion distribution. When erythrocytes from normal subjects were incubated in autologous plasma (1.0 mmol of Pi/l) and in plasma supplemented with Pi (2.3 mmol of Pi/l), the Pi concentration in the cells, at steady state, increased only from 0.57 to 0.78 mmol/l cells, suggesting that the effect was not an artifact of disease or drug therapy. It is concluded that, in human skeletal myocytes and erythrocytes, the percentage change in the concentration of cytoplasmic Pi is lower than that in plasma. This implies that these cells can buffer or regulate cytoplasmic Pi when the extracellular concentration is disturbed.

Adult↗
PubMed1985

Declining catecholamine secretion in adrenal medulla on prolonged stimulation with acetylcholine.

Perfused pig adrenal glands and cortex-free ox adrenal medullae were stimulated by continuous infusion of 10(-4) M acetylcholine (ACh). Secretion of adrenaline rose to a maximum in approximately 5 min but, after a further 15 min, declined to 36 +/- 19% (+/- S.D.) of maximum for pig (N = 5) and 27 +/- 10% of maximum for ox (N = 3), in spite of continued infusion of ACh. After 20 min, no further significant decline was detectable. Nevertheless, in ox medullae, oxygen consumption measured after stimulation showed no significant change relative to the pre-stimulation value, indicating that the decline in secretion did not arise from a failure of oxidative energy metabolism. In 4 pig adrenal glands subjected to a 1 hr infusion of ACh, adrenaline secreted in the last 20 min was only 52 +/- 10% of that secreted in the first 20 min but, after a 2 hr rest interval, recovered to 74 +/- 18% (P less than 0.05) in the first 20 min of a second 1 hr stimulation. In the same glands, no reproducible recovery was detectable for noradrenaline and, by the last 20 min of the second 1 hr stimulation, noradrenaline secretion had declined to 36 +/- 20% of the initial value, even though only 15% of the noradrenaline originally in the gland had been secreted. It is concluded that, while decline and recovery of adrenaline secretion may, in part, have arisen from desensitization and resensitization of the ACh receptor; the decline in noradrenaline secretion arose mainly from depletion of a readily secreted pool which was considerably smaller than the total in the gland.

Acetylcholine↗