Lipid metabolism in large T47D human breast cancer spheroids: 31P- and 13C-NMR studies of choline and ethanolamine uptake

³¹P- and ¹³C-NMR were used to determine the kinetics of choline and ethanolamine incorporation in T47D clone 11 human breast cancer cells grown as large (300 μm) spheroids. Spheroids were perfused inside the spectrometer with 1,2-¹³C-labelled choline or ethanolamine (0.028 mM) and the buildup of labeled phosphorylcholine (PC) or phosphorylethanolamine (PE) was monitored. To analyze the NMR kinetic data, it was assumed that each signal represents a weighted average of signal from the proliferating and non-proliferating compartments of the large spheroid. The average ATP pool size was 4±1 fmol/cell compared to 8±1 fmol/cell in small (150 μm) proliferating spheroids (P < 0.0002). The average PC pool size at steady state was reduced to 11±6 fmol/cell compared to 22±8 (P < 0.007). This could be correlated with an overall reduction of choline uptake in the non-proliferating spheroid fraction. The rate of the enzyme choline kinase was 0.3 fmol/(cell h) compared to 1.0 fmol/(cell h) (P < 0.0001) for proliferating cells. The rate constant of CTP: phosphocholine cytidyltransferase (0.05 h^?1) was not significantly altered, but the rate of the enzyme was reduced from 1.3 to 0.2–0.5 fmol/(cell h). The pool size of PE in medium containing serum ethanolamine (1.7 μM) was approximately the same (15 fmol/cell) in small and large spheroids. In the presence of high ethanolamine (0.028 mM) the average PE level decreased slightly (11 fmol/cell) and the rate of the enzyme ethanolamine kinase in the non-proliferating fraction was 0.7 fmol/(cell h) versus 1.0 fmol/(cell h) in the proliferating cells (P < 0.07). The rate constant of CTP: phosphoethanolamine cytidyltransferase (0.07 h^?) was not significantly altered but the corresponding reaction rate was reduced from 1.4 to 0.2–0.8 fmol/(cell h). The kinetics of choline incorporation did not alter in the presence of 0.028 mM ethanolamine.     

The effect of headgroup class on the conformation of membrane lipids in Acholeplasma Laidlawii: A 2H-NMR study

[2-²H₂]Oleic, [2-²H₂]palmitic, [2-²H₂]dihydrosterculic and [3-²H₂]oleic acids were biosynthetically incorporated into the membrane lipids of Acholeplasma laidlawii B. ²H-NMR spectroscopy and spectral ‘de-Parking” (M. Bloom, J.H. Davis and M.I. Valic, Can. J. Phys., 58 (1980) 1510) were used to study the effect of lipid headgroup class on the conformational order in the vicinity of the C-2 position of the acyl chains of lipids in the liquid crystalline phase. The results indicate that although the orientation and conformations of the membrane lipids in the region of the C-2 position of the chains are qualitatively very similar among the various lipid classes, quantitatively there are some differences, particularly between the glycolipids and the phospholipids. These differences do not exted to the C-3 position. Unlike the headgroup class, the membrane proteins appear to have little if any effect on the molecular ordering of the lipids.     

The interaction between water and the polar head in inverted phosphatidylcholine micelles A 2H and 31P relaxation study

²H and ³¹P spin-lattice relaxation times (T₁) were studied for invented egg phosphatidylcholine micelles in CCl₄ as functions of ²H₂O concentration. When the ²H₂O/phosphatidylcholine mole ratio changed from 1.0 to 18.0, T₁ of ³¹P increased by about 2.6 fold, whereas T₁ of ²H increased by about 50 fold. A quantitative analysis of the deuterium T₁ data showed that there is only one water molecule tightly bound to the polar head, and it is in rapid exchange with the rest of the water molecules. The activation energy for the deuterium T₁ was 7.1 ± 0.8 kcal/mol (30 ± 3 kJ/mol), and was independent of the ²H₂O concentration.     

1H-NMR study of the location and motion of ubiquinones in perdeuterated phosphatidylcholine bilayers

Ubiquinones (n = 1,2,3,4,7,9,10) and ubiquinols (n = 1,2,3,4,10) were incorporated into ordinary (protonated) or perdeuterated dimyristoyl phosphatidylcholine vesicles and were found to have significant local molecular motion. The motion of the quinone ring, as judged from the linewidth of the OCH₃ proton resonances, decreased in longer-chain ubiquinones. Minimum values for the transverse mobility (flip-flop rates) of ubiquinones-1,2,3,4,10, measured with the aid of lanthanide shift reagents, suggest that they are all able to function in a protonmotive ‘Q cycle’ during electron transport. As the length of the side chain increases beyond 1 isoprenoid unit, the quinone/quinol ring tends to be deeper in the outer monolayer of small sonicated vesicles and in both monolayers of larger freeze-thaw vesicles, but little or no change in depth is observed in the inner monolayer of small vesicles. The ubiquinol rings are closer to the membrane surface than are the ubiquinone rings. For side chain n = 9 or 10, a second resonance from the OCH₃ protons of ubiquinones and ubiquinols in vesicles appears in the ¹H-NMR spectrum. This is due to the presence of two types of vesicles with different ubiquinone/phospholipid ratios.     

A 31p and 15N NMR study of the extraction of uranyl nitrate by Di-2-ethylhexyl phosphoric acid

Low temperature ³¹P and ¹⁵N NMR spectroscopy was used to investigate the species forming in the organic layer following the extraction of uranium from nitric acid solutions with di-2-ethylhexyl phosphoric acid. It was found that uranium is extracted from neutral solutions as the 1:2 complex UO₂A₂ regardless of what anion is present. For dilute nitric acid solutions, the uranium is extracted both as associated and mixed nitrato species. As the nitric acid concentration of the aqueous layer increases, the mixed nitrato complex, UO2(NO₃)A·HA, becomes predominant.     

A comparison of spin probe ESR, 2H- and 31P-nuclear magnetic resonance for the study of hexagonal phase lipids

We have investigated the feasibility of the various possible magnetic resonance probes of lipids which form non-bilayer phases. As a model system we have used equimolar mixtures of phosphatidylethanolamine (PE) and cholesterol, which exhibit a thermotropic transition from a bilayer to a hexagonal phase. Variable temperature electron spin resonance (ESR) spin probe spectra were obtained using random dispersion and oriented lipid systems. Simultations of the ESR spectra were performed in order to aid in the interpretation of the experimental results for the oriented system. ³¹P- and ²H-nuclear magnetic resonance (NMR) studies were carried out using a deuterated PE. The ESR spin probes in the random dispersions show essentially no effect attributable to the phase transition. However, there are large, reversible effects in the temperature-dependent behaviour for the oriented system. The orientation dependence of the spectra above the transition temperature indicate that the hexagonal phase lipids may spontaneously assume a macroscopic organization on a flat surface. We find, however, that such an organization cannot be unambiguously assigned from the ESR spin probe spectra, and point out a potential difficulty in the interpretation of spin probe spectra in oriented systems. In contrast, the ²H-NMR method provides a reliable monitor of the phase transformation. Taken together, the ²H and ³¹P data indicate that the structure of the headgroup in PE is quite similar in both the bilayer and hexagonal phase. ²H-NMR should be very useful in probing the structural and dynamic characteristics of lipids in non-bilayer phases.     

31P nuclear magnetic resonance study of enzymatically phosphorylated glycophorin A

Glycophorin A was phosphorylated using protein kinases and the new protein was investigated using³¹P NMR spectroscopy. Most of these 30 moles of phosphate were found to be attached to Ser and Thr. Some of these phosphate residues appear to be affected by the carbohydrate residues present. The phosphorylated protein appears to be in a severe state of aggregation, with the degree of aggregationpH-dependent.     

Determination of the complete order parameter tensor for a lipid methylene group from 1H- and 2H-NMR spin labels

Proton-proton dipolar splittings are obtained as a function of temperature for the α-methylene in a potassium palmitate - (β-ω) - d₂₉ (70 wt.%) / D₂O (30 wt.%) sample above and below the gel to liquid crystal phase transition. These splittings and corresponding deuteron quadrupole splittings are used to specify the complete order parameter tensor for the α-methylene group. Deduction of lipid structural information from the complete-order parameter tensor is discussed.     

A 1H-NMR longitudinal relaxation study of the interaction between cytochrome c and cytochrome c oxidase

The interaction between the oxidized forms of cytochrome c and cytochrome c oxidase (EC 1.9.3.1) has been investigated by ¹H-NMR longitudinal relaxation measurements. It is found that relaxation of methyl groups on the heme ring of cytochrome c markedly deviates from a simple exponential behavior in the presence of small amounts of cytochrome oxidase. A comparison with the relaxation behavior of cytochrome c modified by 4-carboxy-3,5-dinitrophenyl at Lys-13 shows that the oxidase induces a conformation in native cytochrome c that is closely related to that of the derivative. It is suggested that this change in conformation consists of a rupture of the salt bridge between Lys-13 and Glu-90 and a concomitant perturbation of the methionine ligand.     

Conversion of 2,5-dimethylfuran to 2-hydroxy-5-hydroperoxy-2,5-dimethyldihydrofuran,a true 1O2-derived reaction in aqueous 1O2 generating systems

It has been studied whether 2,5-dimethylfuran (DMF) is a specific ¹O₂ trapping agent in aqueous system. The exposure of DMF to aqueous ¹O₂ generating system (Rose Bengal photooxygenation system) gave 2-hydroxy-5-hydroperoxy-2,5-dimethyldihydrofuran (a hydrated form of endoperoxide, ¹O₂-derived reaction product) and cis-diacetylethylene (cis-DAE), while the bromine-catalyzed autoxidation of DMF afforded only trans-DAE. In Fenton system (·OH generating system) DMF was converted in the main to cis-DAE, but not to the hydrated form of endoperoxide. The exposure of DMF to acetaldehyde-xanthine oxidase system failed to detect the hydrated form of endoperoxide, but chiefly yielded a non-specific oxidation product, cis-DAE.     

2H- and 31P-NMR studies of bilayers composed of 1-acyllysophosphatidylcholine and fatty acids

1-Palmitoyllysophosphatidylcholine has been mixed in equimolar amounts with specifically deuterated palmitic acid and the structural properties of the lipid/water phase have been studied by ²H- and ³¹P-nuclear magnetic resonance. The order profile of the free palmitic acid is very similar to that of the parent compound $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero-3-phosphocholine}$ at temperatures above the gel-to-liquid crystal phase transition. The bending of the $\text{sn-2}$ chain which is typical for diacyl lipids is not observed for the free palmitic acid. The mixture of lysolipid and palmitic acid exhibits well-defined quadrupole splittings even at temperatures below the gel-to-liquid crystal phase transition. Hence it is possible for the first time to establish an order profile in the gel-state of the lipid bilayer phase. Between carbon atoms 5 to 12 the palmitic acid chain is found to assume the extended all-trans conformation with a very small contribution from gauche defects. Towards the methyl terminal a distinct increase in the gauche probability can be noted. The motion of the phosphocholine headgroup was also studied by ²H- and ³¹P-NMR using selectively deuterated 1-palmitoyllysophosphatidylcholine. The headgroup has a considerably larger motional freedom in the mixture of lysolipid and palmitic acid than in $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero-3-phosphocholine}$. In addition, the average headgroup conformations are also different in the two systems.     

A 31P-NMR study of the intact liver fluke Fasciola hepatica

³¹P-NMR techniques offer a useful method of studying changes in the metabolism of intact parasitic worms. The liver flukes, Fasciola hepatica, provide good quality ³¹P high resolution NMR spectra for at least 6 h under anaerobic conditions. The levels of ATP remain constant throughout this period. There is no signal for phosphocreatine or phosphoarginine. In contrast to the findings in mammalian tissues, there is a distinct peak for the terminal phosphate of ADP. A number of signals are observed in the phosphodiester region of the spectrum the largest of which is identified as l-α-glycerophosphoryl choline. Serotonin (5-hydroxytryptamine) causes an appreciable increase in the levels of sugar phosphates when the flukes are incubated in the absence of glucose. The addition of glucose also causes a marked increase in the signals for the hexose phosphate.     

The kinetics of ionophore X-537A-mediated transport of manganese through dipalmitoylphosphatidylcholine vesicles A 1H-NMR study

We have studied the kinetics of ionophore X-537A-mediated transport of manganese ions into small unilamellar vesicles formed from dipalmitoylphosphatidylcholine. To follow the transport we used the paramagnetic effect of manganese on the ¹H-NMR signal from choline trimethylammonium groups on the inner phospholipid monolayer. The transport of only one manganese ion produces an intravesicular concentration which is high enough (approx. 1 mM) to substantially broaden this signal. The observed signal thus arises predominantly from those vesicles which contain no manganese. Therefore, as manganese is transported into the vesicles the observed signal decreases in intensity, but does not broaden. The initial time-dependence of the intensity of the signal, $\text{S(t)}$, can be approximated by the simple first-order rate law: $\text{S(t) = S}\text{(O)}\text{exp}\text{(?K′t)}$, where $\text{K′}$ is the probability per unit time for the transport of a manganese ion from the external medium to the intravesicular space. From the dependence of $\text{K′}$ on the ionophore X-537A concentration we conclude that manganese is transported into the vesicles via both 1 : 1 and 2 : 1 complexes with ionophore X-537A. At low ratios of ionophore X-537A to vesicles transport via the 1 : 1 complex predominates; at high ratios transport via the 2 : 1 complex predominates. From the dependence of $\text{K′}$ on manganese concentration we determined that under our conditions the equilibration of ionophore X-537A between vesicles is much faster than the transport of manganese through the vesicles. Lastly, from the dependence of $\text{K′}$ on temperature, we conclude that the ionophore X-537A-mediated transport of manganese into the dipalmitoylphosphatidylcholine vesicles is very sensitive to the gel-liquid crystalline phase transition.     

Nonlamellar liquid crystalline nanostructured particles: advances in materials and structure determination

Analogous to the dispersion of lamellar phase-forming lipids to form liposomes, dispersion of lipids that form alternative liquid crystalline structures, such as cubic and hexagonal phase, forms particles termed cubosomes and hexosomes, respectively. Although these particles possess alternative structural forms and hence behavior, when compared to liposomes, they have received significantly less attention in the literature. While most studies have utilized glyceride lipids to prepare nonlamellar dispersions, recent advances in identifying new materials from which to prepare these particles has broadened the interest in this field. This review focuses on the materials used to form nonlamellar dispersions and the methods used to characterize their structure. Increased awareness of their structural characteristics and hence potential benefits in applications, such as drug delivery, is hoped to stimulate further studies that will ultimately see their uptake in commercial products.     

A high-resolution NMR study (1H, 13C, 31P) of the interaction of paramagnetic ions with phospholipids in aqueous dispersions

¹H-, ¹³C-and ³¹P-NMR spectra of egg-yolk phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidic acid (PA) and cosonicated mixtures of these phospholipids were obtained from ultrasonicatcd dispersions containing Pr³⁺, Eu³⁺, Gd³⁺ and Mn²⁺ ions.The differences in chemical shift values. °_n, between the “inner” and “outer” resonance signals for the different nuclei of the polar head group of egg-yolk phosphatidyl choline provide information about the average distances of the paramagnetic ion within the polar groups of the phospholipid molecules. In the Pr(²H₂O)³⁺_n/egg-yolk phosphatidylcholine system the ions are nearest to the phosphate and -CH₂CH₂ group, respectively but relatively far from the N(CH₃)₃ group of the polar head group of the lipid.The integral analysis of the¹ H-NMR spectra obtained from dispersions containing Pr³⁺ and Mn²⁺ ions enables us to calculate the number of the polar groups in both sides of the egg-yolk phosphatidylcholine bilayer, the size of the lipid vesicle and to give some features of the arrangement of the phospholipid molecules in cosonicated egg-yolk phosphatidylcliotine/ phosphatidytserine vesicles. At p²H 8.3 in PC/PS mixtures an extreme asymmetry is observed with PS preferentially in the outer side of the membrane. This side contains approximately three times more PS than PC molecules.Some comments are made concerning the quantitative integral analysis of proton-noise decoupled ³¹ P-NMR spectra as obtained from similar phospholipid mixtures by Michaelson et al. and Berden et at.     

Effects of divalent cations and pH on phosphatidylserine model membranes: A 31P NMR study

The influence of divalent cations, and pH on the behaviour of phosphatidylserine, derived from egg phosphatidylcholine, has been examined employing ³¹P-NMR techniques. The addition of Ca²⁺ results in the observation of a “rigid lattice” ³¹P-NMR spectra and more than an order of magnitude increase in the spin-lattice relaxation time T₁. This corresponds to a strong and specific headgroup immobilization by Ca²⁺, similar to that observed for anhydrous phosphatidylserine. At pH 7.4 the hydrated sodium salt of (egg) phosphatidylserine adopts the bilayer phase, whereas when the pH is decreased through 3.5 a bilayer to hexagonal (H_II) polymorphic phase transition is observed at 50°C, which is unaffected by equimolar cholesterol. The same transition is shown to occur at 37°C for phosphatidylserine isolated from human erythrocytes.     

31P NMR study of head group behavior in sonicated phosphatidylcholine liposomes in the gel and in the liquid state

We measured the ³¹P[¹H] Nuclear Overhauser Effect (NOE) as a function of temperature and of ¹H irradiation frequency, the linewidth $\text{Δν}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ as a function of temperature and the relaxation time T₁ above and below the thermal transition temperature, of the ³¹P-NMR signal in sonicated liposomes of 1,2-dimiristoyl-3-sn-phosphatidylcholine (DMPC), 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) and 1,2-dimiristoyl-3-sn-phosphatidylcholine (DSPC). The same measurements were repeated in the presence of high molecular weight dextrans. They strongly reduce the NOE and produce longer relaxation times T₁. According to the current models, we were able to evaluate, in the different situations, the correlation time of the internal motion τ_G and the distance r between interacting groups in the region of the polar head groups. While the first parameter changes abruptly through the phase transition and under the effect of dextrans, the latter does not appear modified in any case. These results are discussed in terms of a conformational change of the phosphocholine head groups.     

A 31P-NMR study of structural and functional aspects of phosphate and phosphonate distribution in Tetrahymena

³¹P-NMR has been used to study the chemical nature of cytoplasmic components of live Tetrahymena in a non-invasive manner. The technique has further been used to characterize the physical behaviour of lipids extracted from this organism. In particular, we have shown the presence of large quantities of pyrophosphate and of tripolyphosphate in acid extracts of the organism. These are not detectable in the live cell due to the motionally rigid nature of the storage granules. We have characterized the distribution of phosphonic acids in the organism and followed the phase behaviour of the extracted cell lipids. Aqueous dispersions of extracted lipid show both bilayer and non-bilayer behaviour in the range of the growth temperature. The phosphonolipid in Tetrahymena appears to play a role similar to that of phosphatidylethanolamine in regulating the phase behaviour of the membrane. The high degree of unsaturation in the fatty acids of Tetrahymena is most likely responsible for the polymorphic phase behaviour observed near the growth temperature.     

Induction of a relatively fast transbilayer movement of phosphatidylcholine in vesicles. A 13C NMR study

[$\text{N-}{}^{13}\text{CH}{}_3$] Phosphatidylcholines are introduced into the outer monolayer of phosphatidylcholine vesicles with the phosphatidylcholine exchange protein from bovine liver. The transbilayer distribution of the [$\text{N-}{}^{13}\text{CH}{}_3$] phosphatidylcholine is measured with ¹³C NMR. The transbilayer movements of $\text{[N-}{}^{13}\text{CH}{}_3\text{]-}\text{dioleoyl}$ phosphatidylcholine and [$\text{N-}{}^{13}\text{CH}{}_3$] dimyristoyl phosphatidylcholine at 30°C in vesicles composed of these phosphatidylcholines are extremely slow processes with estimated half-times of days. [$\text{N-}{}^{13}\text{CH}{}_3$] Dioleoyl phosphatidylcholine introduced into dimyristoyl phosphatidylcholine vesicles migrates from the outer to the inner monolayer with a half-time of less than 12 h. The data suggest that differential changes in the lateral packing of the two monolayers might be a driving force for transbilayer transport of phospholipids.     

A 31P-NMR study of the cross-membrane pH gradient induced by ATP hydrolysis in mitochondria

³¹P-NMR has been used to study the increase of ΔpH in mitochondria by externally added ATP. Freshly prepared mitochondria was treated with N-ethylmaleimide to inhibit the exchange between internal and external P_i. Upon addition of ATP, phosphocreatine (30 mM) and creatine kinase to a NMR sample of mitochondria suspension (approx. 120 mg protein/ml) at 0°C, an increase of ΔpH by approx. 0.5 pH unit was observed. However the increased ΔpH could not be maintained, but slowly decayed along with the increase of external ADP/ATP ratio. Further addition of valinomycin to the suspension induced a larger ΔpH (approx. 1) which was maintained by the increased rate of internal ATP hydrolysis as seen in the growth of the internal P_i peak intensity in NMR spectra and the concomitant decrease of the external phosphocreatine peak. The external P_i and ATP peaks stayed virtually constant. When carboxyatractyloside was added to inhibit the ATP/ADP translocase, the internal P_i increase was stopped and the ΔpH decayed. These observations in conjunction with those made earlier in respiring mitochondria clearly show the reversible nature of the ATPase function in which the internal ATP hydrolysis is associated with outward pumping of protons.