We have refined the method for measuring the equilibrium exchange of the neutral phosphoryl compound dimethyl methylphosphonate across the human red cell membrane using 31P NMR spectroscopy. Using this improved technique we measured a permeability coefficient of dimethyl methylphosphonate of 9.47 +/- 1.01 X 10(-5) cm s-1 at a concentration of 164 mM, at 25 degrees C. The transport at 25 degrees C was not saturable up to a concentration of 600 mM and was not inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid, p-chloromercuriphenyl sulfonic acid, copper ions or phloretin, or by thiourea or urea at 16 degrees C. The permeability was enhanced by butanol and phloretin.

Biological Transport↗

PubMed1988

Characterization of transmembrane chemical shift differences in the 31P NMR spectra of various phosphoryl compounds added to erythrocyte suspensions.

Trimethyl phosphate, dimethyl methylphosphonate, diethyl methylphosphonate, trimethylphosphine oxide, and the hypophosphite, phenylphosphinate, and diphenylphosphinate ions all contain the phosphoryl functional group. When added to an intact erythrocyte suspension at 20 degrees C, each of the compounds gave rise to separate intra- and extracellular 31P NMR resonances, and the separation between the two resonances of each compound varied with the mean cell volume. The differences between the intra- and extracellular chemical shifts were shown to be primarily attributable to the effects of hemoglobin. The presence of hemoglobin inside the cell gave rise to a significant difference in the magnetic susceptibilities of the two compartments. In addition, it exerted a large susceptibility-independent chemical shift effect, the magnitude of which was dependent upon the chemical structure of the phosphoryl compound involved. A number of other intra- and extracellular components were also shown to cause chemical shift variations, smaller than those arising from hemoglobin but nonetheless significant. The cell volume dependence of the transmembrane chemical shift differences therefore reflected not only the cell volume dependence of the intracellular hemoglobin concentration but also the changing concentration of the other solutes in the two compartments. In addition to their cell volume dependence, the transmembrane chemical shift differences varied with temperature. In the case of the nonelectrolytes this reflected not only the temperature dependence of the mechanism(s) responsible for the susceptibility-independent shift effects but also the temperature dependence of the rates at which the compounds traversed the cell membrane.

Erythrocyte Membrane↗

PubMed1988

Physical basis of the effect of hemoglobin on the 31P NMR chemical shifts of various phosphoryl compounds.

The marked difference between the intra- and extracellular 31P NMR chemical shifts of various phosphoryl compounds when added to a red cell suspension may be largely understood in terms of the effects of hemoglobin on the 31P NMR chemical shifts. The presence of [oxy- or (carbonmonoxy)-] hemoglobin inside the red cell causes the bulk magnetic susceptibility of the cell cytoplasm to be significantly less than that of the external solution. This difference is sufficient to account for the difference in the intra- and extracellular chemical shifts of the two phosphate esters trimethyl phosphate and triethyl phosphate. However, in the case of the compounds dimethyl methylphosphonate, diethyl methylphosphonate, and trimethyl-phosphine oxide as well as the hypophosphite, phenylphosphinate, and diphenylphosphinate ions, hemoglobin exerts an additional, much larger, effect, causing the 31P NMR resonances to shift to lower frequency in a manner that cannot be accounted for in terms of magnetic susceptibility. Lysozyme is a protein structurally unrelated to hemoglobin and was shown to cause similar shifts to lower frequency of the resonances of these six compounds; this suggests that the mechanism may involve a property of proteins in general and not a specific property of hemoglobin. The effect of different solvents on the chemical shifts of the eight phosphoryl compounds provided an insight into the possible physical basis of the effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemoglobins↗

PubMed1988

Further investigation of the use of dimethyl methylphosphonate as a 31P-NMR probe of red cell volume.

We have refined a method for measuring erythrocyte volume using the 31P-NMR spectrum of a probe molecule, dimethyl methylphosphonate. This compound, when added to an erythrocyte suspension, gives rise to two 31P-NMR resonances, and the frequency separation between them is linearly dependent on the intracellular haemoglobin concentration. If, for a given cell sample (under standard conditions), the separation of the two dimethyl methylphosphonate peaks has been measured and an independent estimation of the mean cell haemoglobin content and concentration has been obtained, then changes in the mean cell volume due to altered experimental conditions may be estimated from the peak separation measured under the new conditions. Although the peak separation was independent of extracellular pH, it did vary with (i) a range of extracellular suspension media, (ii) temperature, (iii) dimethyl methylphosphonate concentration, (iv) haemoglobin ligand state and (v) different blood donors.

Carboxyhemoglobin↗

PubMed1988

The contribution of magnetic susceptibility effects to transmembrane chemical shift differences in the 31P NMR spectra of oxygenated erythrocyte suspensions.

Triethyl phosphate, dimethyl methylphosphonate, and the hypophosphite ion all contain the phosphoryl functional group. When added to an oxygenated erythrocyte suspension, the former compound gives rise to a single 31P NMR resonance, whereas the latter compounds give rise to separate intra- and extracellular 31P NMR resonances. On the basis of experiments with intact oxygenated cell suspensions (in which the hematocrit was varied) and with oxygenated cell lysates (in which the lysate concentration was varied), it was concluded that the chemical shifts of the intra- and extracellular populations of triethyl phosphate differ as a consequence of the diamagnetic susceptibility of intracellular oxyhemoglobin but that this difference is averaged by the rapid exchange of the compound across the cell membrane. The difference in the magnetic susceptibility of the intra- and extracellular compartments contributes to the observed separation of the intra- and extracellular resonances of dimethyl methylphosphonate and hypophosphite. The magnitude of this contribution is, however, substantially less than that calculated using a simple two-compartment model and varies with the hematocrit of the suspension. Furthermore, it is insufficient to fully account for the transmembrane chemical shift differences observed for dimethyl methylphosphonate and hypophosphite. An additional effect is operating to move the intracellular resonances of these compounds to a lower chemical shift. The effect is mediated by an intracellular component, and the magnitude of the resultant chemical shift variations depends upon the chemical structure of the phosphoryl compound involved.

Alcohol Oxidoreductases↗

PubMed1988

Hypophosphite ion as a 31P nuclear magnetic resonance probe of membrane potential in erythrocyte suspensions.

Hypophosphorus acid has a single pKa of 1.1 and at physiological pH values it is therefore present almost entirely as the univalent hypophosphite ion. When added to a red cell suspension the ion crosses the cell membrane rapidly, via the anion exchange protein, and the intra- and extracellular populations of the ion give rise to separate 31P NMR resonances. From a single 31P NMR spectrum it was possible to determine the relative amounts of hypophosphite in the intra- and extracellular compartments and thereby estimate the corresponding concentrations. The ratio of intracellular to extracellular hypophosphite concentration was independent of the total hypophosphite concentration for cells suspended in NaCl solutions and was independent of hematocrit. The hypophosphite distribution ratio increased as extracellular NaCl was replaced iso-osmotically with citrate or sucrose, through it remained very similar to the corresponding hydrogen ion distribution ratio. Incorporation of the hypophosphite distribution ratio into the Nernst equation yielded an estimate of the membrane potential. For cells suspended in NaCl solutions the estimated potential was consistently around -10 mV.

Erythrocyte Membrane↗

PubMed1988

Moving onto Mainstreet: the value of developing a chaplaincy organization affiliated with a state hospital association.

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Chaplaincy Service, Hospital↗

PubMed1988

Cancer survivors: is there a need for group support?

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Adult↗

PubMed1987

Induction of antibody and cellular immune responses in chickens against Eimeria tenella by anti-idiotype.

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Animals↗

PubMed1987

Fast transmembrane exchange in red cells studied with NMR.

13C NMR, with two fundamentally different procedures, was used to measure the H13CO3- permeability coefficient (P) of human erythrocytes. The values of P (approximately 3 X 10(-4) cm s-1) were similar to those obtained by other, non-NMR methods. A third procedure was used with 31P NMR to measure the permeability coefficient of human erythrocytes for the non-electrolyte dimethyl methylphosphonate; the effects on transmembrane exchange of varying haematocrit and butanol were studied.

Bicarbonates↗

PubMed1986

Bicarbonate exchange kinetics at equilibrium across the erythrocyte membrane by 13C NMR.

The rate of exchange of 13C-labelled bicarbonate across the membranes of human erythrocytes in suspension, at thermal and chemical equilibrium, was measured using 13C NMR spectroscopy: the permeability coefficient (3.34 X 10(-4) cm s-1) agrees well with previous values obtained with other methods. Data analysis was complicated by the need to consider the Donnan ratio of the charged species inside and outside the cells. This work appears to be the first, involving the present NMR procedure, for studying fast membrane transport of a molecule other than water.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1986

Intracellular pH in stored erythrocytes. Refinement and further characterisation of the 31P-NMR methylphosphonate procedure.

Methylphosphonate in conjunction with 31P-NMR spectroscopy was used for the measurement of transmembrane delta pH in human erythrocytes stored at 4 degrees C for up to 5 weeks in a nutrient medium. Intra- and extracellular pH was determined using calibration curves based on the pH-dependent separation between the NMR resonances of methylphosphonate and orthophosphate (Pi). A comprehensive statistical procedure is presented for the determination of the variance of NMR-based pH estimates. The entry of methylphosphonate into erythrocytes was more rapid at low pH and uptake was fully inhibited by the band 3 reagent, disodium 4,4-diisothiocyano-2,2'-disulphonic acid stilbene. The distribution ratio of methylphosphonate concentration inside and outside the cells was used to calculate the membrane potential; the analysis depends on a consideration of the Donnan equilibrium for an anion with one or two charges. Furthermore, the analysis does not depend on the pH estimates but relies solely on concentration estimates. The chemical shift of methylphosphonate was not subject to the variations associated with specific intracellular binding encountered with many other phosphorus compounds, including Pi. On the other hand, the ionic strength dependence of the chemical shift of methylphosphonate, contrary to earlier reports, is comparable in magnitude (but opposite in sign) to that of Pi.

Blood Preservation↗