A 1H-NMR study of the permeation of glycolic acid through phospholipid membranes.

The transmembrane permeability coefficient of the alpha-hydroxyacid, glycolic acid, has been measured for egg phosphatidylcholine large unilamellar vesicles. The determination of the vesicle concentration independent first-order rate constant for membrane transport and the permeability coefficient were made using an NMR technique employing shift agents. Both the temperature dependence and the dependence on cholesterol content were investigated. The activation energy and the Arrhenius pre-exponential factor were found to be dependent on the cholesterol content. A marked increase in both parameters was observed up to 20 mol% cholesterol, with a further, small increase up to 50%. The pH dependence of permeability was also investigated. An increase in permeability is observed with a decrease in pH, providing a possible explanation for the effectiveness of glycolic acid in skin treatment.

Analysis of Dictyostelium discoideum plasma membrane fluidity by electron spin resonance.

Dictyostelium discoideum grown axenically in media containing polyunsaturated fatty acids exhibited normal growth rates but impaired differentiation (Weeks, G. (1976) Biochim. Biophys. Acta 450, 21--32). Since cell-cell contact is vital for differentiation but unnecessary for growth we have examined the isolated plasma membranes of these cells. The lipids of the plasma membranes of cells grown in the presence of polyunsaturated fatty acids contain considerable quantities of these acids, but the total phospholipid and sterol contents of the plasma membrane are close to normal. Electron spin resonance studies using 5-doxyl-stearic acid as the spin probe reveal two things. Firstly, there are no detectable characteristic transition temperatures in the plasma membranes of D. discoideum. Secondly, the plasma membranes of cell grown in the presence of polyunsaturated fatty acids have essentially the same fluidity as that of the control cells. The possible significance of this result to impaired cell-cell interaction is discussed.

The fluidity of plasma membranes of Dictyostelium discoideum. The effects of polyunsaturated fatty acid incorporation assessed by fluorescence depolarization and electron paramagnetic resonance.

Two probe techniques, fluorescence depolarization (using diphenylhexatriene) and electron paramagnetic resonance (using 5-doxyl stearic acid), have been used to assess the fluidity of the purified plasma membranes of Dictyostelium discoideum. Both techniques indicate that a large incorporation of polyunsaturated fatty acids into the plasma membranes does not significantly change membrane fluidity In addition, phosphatidylcholines isolated from cells grown on both polyunsaturated fatty acid-supplemented and unsupplemented media exhibit similar mobilities of an incorporated spin probe when dispersed in aqueous solution. This result suggests that the enrichment of a membrane already high in content of fatty acyl chains containing two double bonds with those containing three or more double bonds does not markedly change fluidity.

Electron spin resonance studies of lipid fluidity changes in membranes of an uncoupler-resistant mutant of Escherichia coli.

The fluidity of the lipids in membrane preparations from a mutant of Escherichia coli resistant to the uncoupler CCCP, grown at different temperatures with and without CCCP, was examined by electron spin resonance using the spin probe 5-doxyl stearic acid. The fluidity of the membrane lipids at the growth temperature, as estimated using electron spin resonance, was less in cells grown at lower temperatures. Precise homeoviscous adaptation was not observed. Growth in the presence of CCCP resulted in a decrease in membrane lipid fluidity, particularly in the inner (cytoplasmic) membrane. There was no change in the proportion of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin in the cell envelope. However, there was an increase in the proportion of unsaturated fatty acids in membranes from cells grown with uncoupler. This was reflected in the increased fluidity of the lipids extracted from these membranes. This result is contrary to that expected from measurements of the fluidity of the lipid in these membranes. The decreased fluidity of the lipid in these membranes may be a consequence of the observed increase in the ratio of protein to phospholipid.

Equations describing passive transport through vesicular membranes.

A theoretical description of the kinetics of the passive transport of both lipophobic and lipophilic nonelectrolytes, weak acids, and weak bases through membranes of large unilamellar vesicles is discussed. Equations are derived which may be used to obtain permeability coefficients and predict the extent of LUV entrapment of permeant molecules. Theoretical curves are generated to illustrate the difference between lipophobic and lipophilic permeation. By applying a diffusional approach rather than a simple first order kinetic approach to the problem of passive transport, some of the inconsistencies observed in other works are corrected.

Vesicular membrane permeability of monomethylarsonic and dimethylarsinic acids.

The transmembrane permeability coefficients of the environmentally sensitive arsenicals, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) have been measured for egg phosphatidylcholine large unilamellar vesicles. The determination of the vesicle concentration-independent first-order rate constants for membrane transport and the permeability coefficients were made using an NMR technique employing shift agents. The permeability coefficient of DMA was found to be two orders of magnitude greater than that of MMA. This is attributed to the presence of MMAs extra hydroxyl group. Both species are shown to permeate membranes in the neutral form. Both the temperature dependence and the dependence on cholesterol content were investigated for DMA. The activation energy and the Arrhenius preexponential factor were found to be dependent on the cholesterol content. A marked increase in both parameters was observed up to 20 mol.% cholesterol, with a further, small increase up to 60%. The significance of these results to our understanding of membrane transport is discussed. The importance of using permeability coefficients rather than n-octanol/water partition coefficients in determining bioavailability and bioaccumulation of environmentally sensitive compounds is also discussed.

Spin label studies of erythrocytes with abnormal lipid composition: comparison of red cells in a hereditary hemolytic syndrome and lecithin: cholesterol acyltransferase deficiency.

Erythrocytes from patients with familial lecithin : cholesterol acyltransferase (LCAT) deficiency have been shown to exhibit an increase in membrane fluidity which is surprisingly small in view of the extensive alterations both in membrane lipid composition (namely, an elevation in cholesterol and phosphatidylcholine contents as well as a decrease in phosphatidylethanolamine) and in the functional properties of these cells. In the hope of deriving some information concerning the interrelationship between the structural and functional abnormalities, we have used the spin probe 5-doxyl stearic acid to investigate the temperature-dependent fluidity properties of red cells from two patients with a hereditary hemolytic syndrome (HHS) whose red cells are also characterized by qualitatively similar alterations in phosphatidylcholine and phosphatidylethanolamine but, unlike those in LCAT deficiency, have relatively normal levels of membrane cholesterol. A small increase in membrane fluidity of HHS erythrocytes equivalent to that previously observed in LCAT deficiency was found, indicating that membrane cholesterol level does not exert an important modulatory influence on membrane fluidity in these cells. It is concluded that while the distinct patterns of structural and functional erythrocyte alterations in these two disorders cannot be explained on the basis of differences in bulk membrane fluidity, the marginally increased fluidity may underlie the abnormalities in osmotic fragility and membrane p-nitrophenylphosphatase activity which are shared in common by both types of modified red cells.

The lipid composition and membrane fluidity of Dictyostelium discoideum plasma membranes at various stages during differentiation.

There are only minor changes in the amounts of the major lipid constituents of Dictyostelium discoideum plasma membranes during the early stages in the differentiation of this organism. By the time cells reach the pseudoplasmodial stage of development there are small increases in the amounts of phosphatidylinositol, phosphatidylglycerol, and lysophosphatidylethanolamine, and small decreases in the amounts of phosphatidylethanolamine and its plasmalogen form. There is also a slight decrease in the total amount of sterol in the plasma membrane during the transition from aggregation to pseudoplasmodium formation. However, no significant change in membrane fluidity as determined by electron paramagnetic resonance (EPR) accompanies these minor changes in lipid composition. It can be concluded that the establishment of cell-cell interaction in D. discoideum does not involve gross changes in plasma membrane fluidity or lipid composition. It was found that the plasmalogen form of phosphatidylethanolamine is a major phospholipid constituent in D. discoideum, and that this species is somewhat enriched in the plasma membrane.-Weeks, G., and F. G. Herring. The lipid composition and membrane fluidity of Dictyostelium discoideum plasma membranes at various stages during differentiation.

Is the detection of fucose cancer markers by 2D 1H NMR unequivocal?

Fucose is believed to be a marker for cancer that may be detectable by the 2D proton NMR (COSY) of human plasma. However, the COSY technique is time consuming and prone to error. These problems may be alleviated by using the SUPERCOSY pulse sequence; this technique shows that there are many other cross peaks in the region (1.3, 4.2 ppm) which are not readily distinguishable from fucose.

Malignant hyperthermia: characterization of erythrocyte membranes from individuals at risk.

Structural and functional characteristics of erythrocytes and isolated erythrocyte membranes from known malignant hyperthermia (MH) carriers have been examined in the hope of deriving some information concerning the underlying molecular basis of this genetic abnormality, which may represent a state of generalized membrane involvement. The increase in erythrocyte osmotic fragility which has previously been noted in porcine MH was found not to apply to the human disorder and there was evidence that in some individuals at risk osmotic fragility was in fact reduced. Although no alteration in erythrocyte membrane phospholipid profiles was detected, membrane cholesterol levels were reduced in all three definite carriers examined as well as in approximately half of the possible MH carriers investigated. No evidence for associated changes in membrane protein sulfhydryl group latency or in temperature-dependent perturbations of membrane fluidity using a stearic acid spin probe could be detected. Finally, since alterations at the level of skeletal muscle membrane -Ca++ interaction have been implicated in the pathogenesis of MH, we have examined in detail the influence of temperature on the Ca++-stimulated components of the Mg++-dependent ATPase of erythrocyte membranes from known MH carriers but no evidence of any abnormality could be found. Since MH carriers detection based solely on measurements of plasma creatine phosphokinase elevations may yield equivocal results, a decrease in erythrocyte membrane cholesterol content may provide a convenient means of identifying such individuals at risk.

A spin label study of the membranolytic effects of crystalline monosodium urate monohydrate.

The nature of the membranolytic interaction between monosodium urate monohydrate (MSUM) crystals and phospholipid membranes was studied using electron spin resonance. Two spin probe molecules were incorporated into intact human erythrocytes and incubated with MSUM crystals. The apparent increased fluidity of 5-doxyl stearic acid incorporated erythrocytes after a 2 h incubation with MSUM was probably due to an electrostatically induced redistribution of probe from the outer more rigid layer to the fluid inner leaflet via a flip-flop mechanism. It was suggested that the MSUM induced redistribution of cationic amphiphilic probe population in the whole erythrocyte was also due to an electrostatic interaction between negatively charged MSUM crystals and positively charged probe. Possible mechanisms of MSUM induced membranolysis are discussed.

Flexibility of end-labeled polymers from electron spin resonance line-shape analysis: 3' terminus of transfer ribonucleic acid and 5S ribonucleic acid.

Saccharomyces cerevisiae tRNA and 5S RNA, Escherichia coli 5S RNA, and wheat germ 5S RNA have each been specifically spin-labeled at the 3'-terminal ribose to give morpholino-spin-labeled (MSL) RNAs. Enzymatic hydrolysis with pancreatic RNase, followed by anion-exchange chromatography, confirms the site of attachment of the spin-label. Effective rotational correlation times, TB and TC, have been determined from electron spin resonance (ESR) peak heights and widths as a function of temperature for each MSL RNA, and Arrhenius plots of -log T vs. 1/T have been constructed. TC is a measure of internal flexibility at the link between the label and the RNA, while TB is a measure of rotational flexibility of the RNA near the labeled site. Validity of the TB and TC determination has been confirmed from simulation of the experimental EPR spectra by theoretical spectra computed for various attachment geometries and motional rates. Discontinuities in the slope of Arrhenius plots for TB were seen at 34 and 66 degrees C (yeast MSL tRNA), 37 and 60 degrees C (E. coli MSL 5S RNA), 37 and 57 degrees C (yeast MSL 5S RNA), and 36 and 54 degrees C (wheat germ MSL 5S RNA). Temperature-induced hydrolysis of each MSL RNA was less than 5% as determined by gel-filtration chromatography. The melting curves are consistent with a recently proposed universal secondary structural model for prokaryotic and eukaryotic 5S RNA.

Proton magnetic resonance spectroscopy of plasma from patients with dyslipoproteinemia: identification of factors governing methyl and methylene proton line widths.

The line width of the proton magnetic resonance spectrum (MRS) of the composite methylene and methyl resonances of plasma has been reported as a marker for the presence of malignancy. In this study, the contribution of very low density (VLDL), low density (LDL), and high density lipoproteins (HDL) to the MRS line width was determined. This was achieved by measuring the MRS line widths for the plasma from patients with primary disorders of lipoprotein metabolism and from normal individuals. A negative correlation between plasma trigylceride levels and the average line width was observed and this was confirmed in normal plasma to which pure VLDL was added. Also, computer simulations were employed to demonstrate how the line width varies in such complex mixtures of lipoproteins. We demonstrate that the line width is governed by the relative contribution of VLDL and HDL to the composite line shape. This is particularly important when the shoulder from the HDL line lies near the half-height of the VLDL line. As changes in VLDL/HDL ratio occur in patients with malignancy, we propose that this is the basis of the narrowed MRS lines observed in the proposed test for malignancy. However, any individual with elevated VLDL will be false positive in this test.

Detection of erythrocyte membrane structural abnormalities in lecithin: cholesterol acyltransferase deficiency using a spin label approach.

The membrane fluidity of erythrocytes from patients with Lecithin: cholesterol acyltransferase (LCAT) deficiency was studied by means of electron spin resonance. The temperature dependence of the separation of the outer extrema of the spectra of 2-(3-carboxy-propyl)-4,4-dimethyl, 2-tridecyl-3-oxazolidinyloxyl spin probe was monitored for normal, presumed carrier and clinically affected subjects. The temperature profile of controls was significantly different from that of the presumed carriers and the clinically affected individuals. The results show that the compositional abnormalities previously noted in erythrocyte membranes from patients with LCAT deficiency are associated with alterations in the physiocochemical state of the membrane. An investigation of the spectral lineshapes below 10 degrees C allowed a distinction to be made at the membrane level between clinically affected subjects and clinically normal heterozygous carriers. Alterations in the temperature dependence of elec-ron spin resonance parameters may provide a sensitive index of red cell membrane alterations in pathological states of generalized membrane involvement.

Membranolytic effects of monosodium urate monohydrate: influence of grinding.

The effect of grinding on the membranolytic interaction between monosodium urate monohydrate (MSUM) crystals and intact erythrocyte membranes was studied. Crystals were ground for between 1-72 h, and percent hemolysis and zeta potentials determined. A cationic amphiphilic probe (CAT12) was incorporated into the erythrocyte membrane and incubated with MSUM. Increasing grinding times caused a decrease in both the crystallinity and zeta potential of the samples, a decrease in percent hemolysis values and a change in the distribution of free and bound spin label populations. The probe redistribution is thought to be due to an electrostatic interaction between negatively charged MSUM and the CAT12 probe.

The proton NMR of blood plasma and the test for cancer.

The water-suppressed proton NMR spectra of plasma from healthy controls and cancer patients have been recorded along with the high-density lipoprotein (HDL) and triglyceride (TG) levels in the plasma. The dependence of the average of methylene and methyl resonance linewidths on the HDL/TG ratio is demonstrated. It is shown that the deranged lipoprotein levels caused by the presence of cancer lead to the serological test for cancer proposed by Fossel. The work provides the formation for a complete understanding of the proposed serological test for cancer. However, the wide variation in lipoprotein composition in the plasma from both healthy adults and cancer patients means that the serological test proposed by Fossel cannot be used as a screen for cancer.