Phospholipid synthesis in the lymphomatous mouse liver studied by 31P nuclear magnetic resistance spectroscopy in vitro and by administration of 14C-radiolabelled compounds in vivo

High phosphomonoester to ATP ratios have been found in ³¹P magnetic resonance spectra from livers of patients with hepatic lymphoma (Dixon et al. (1990) Br. J. Cancer 63, 953–958). The present study of a murine lymphoma showed that the phosphomonoester in the lymphomatous liver was largely phosphoethanolamine, which is an intermediate of phospholipid metabolism. A significant positive correlation was found between the concentration of phosphoethanolamine, measured by high resolution ³¹P nuclear magnetic resonance spectroscopy of extracts, and the degree of infiltration, assessed by quantitative histology. The phosphoethanolamine concentration reached about 10 times its normal level, but the phosphocholine concentration remained the same as in the normal liver. Radiolabelling studies showed that while the rate of phosphoethanolamine synthesis from exogenous [¹⁴]ethanolamine was higher in the lymphomatous mouse liver than in control livers, the rate of phosphatidylethanolamine synthesis was lower in the lymphomatous liver. The rate of phosphatidylcholine synthesis in lymphoma-bearing livers was not significantly different from that in control mouse livers.     

P-NMR Spectroscopy of Roots of Intact Corn Seedlings

The application of ³¹P nuclear magnetic resonance spectroscopy to the study of metabolism in roots of intact corn seedlings is described. ³¹P-NMR spectra of developmentally distinct parts of primary roots of whole seedlings are presented. The spectra are of quality comparable to those of excised pieces of plant tissue.     

Application of 31P-NMR saturation transfer techniques to investigate phospholipid motion and organization in model and biological membranes

The potential of ³¹P-NMR saturation transfer experiments for determining motional characteristics (in the millisecond to second time scale) of phospholipids in model and biological membranes is demonstrated. A technique to separate membrane phospholipid ³¹P-NMR signals from those of small water-soluble phosphates in intact cells in liver tissue is also illustrated.     

A new experimental approach and signal processing scheme for the detection and quantitation of P brain neurochemicals from in vivo MRS studies using dual tuned (H/P) head coil

Brain ³¹P-neurometabolites play an important role in energy and membrane metabolism. Unambiguous identification and quantification of these neurochemicals in different brain regions would be a great aid in advancing the understanding of metabolic processes in the nervous system. Phosphomonoester (PME), consisting of phosphoethanolamine (PE) and phosphocholine (PC), is the “building block” for membranes, while phosphodiesters (PDE), consisting of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) metabolites are involved in the membrane breakdown process. In the clinical setting, generating well-resolved spectra for PC, PE, GPC, and GPE could be crucial phospholipids in providing information regarding membrane metabolism. We present here a new experimental approach for generating well-resolved ³¹P spectra for PC and PE as well as for GPC, GPE, and other ³¹P metabolites. Our results (based on uni-dimensional (1D) and multi-voxel ³¹P studies) indicate that an intermediate excitation pulse angle (35°) is best suited to obtain well-resolved PC/PE and GPC/GPE resonance peaks. Our novel signal processing scheme allows generating metabolite maps of different phospholipids include PC/PE and GPC/GPE using the ‘time-domain–frequency-domain’ method as referred to in the MATLAB programming language.     

31P-NMR determination of phosphomonoesters in relation to phospholipid biosynthesis in testis of the rat at different ages

Changes in the phosphomonoester (PM) peak, as observed in in vivo 31P-NMR spectra, are often attributed to changes in phospholipid synthesis and therefore to changes in cell proliferation. However, this technique provides information about the absolute size of the phosphomonoester pool rather than its turnover rate. To investigate whether there is a good correlation between changes in PM concentration and its turnover rate, we studied the turnover rate of the two major PM compounds, phosphocholine and phosphoethanolamine, in rat testes at different stages of testis development. [3H]Choline and [3H]ethanolamine were injected intraperitoneally into rats at the age of 3, 6 and 13 weeks, respectively. Phosphorylation of these compounds and their incorporation into phospholipids, were followed up to 6 h after injection of the phospholipid precursors. When these data were compared with the changes observed in the in vivo 31P-NMR PM peak, the concentration of the PM compounds appeared to correlate linearly, both with the conversion of choline into phosphocholine, as well with the rate of phospholipid synthesis, and therefore with the rate of cell proliferation. Hence, it is suggested that cell proliferation can be monitored by determining the changes in the PM peak that is observed in in vivo 31P-NMR spectra.     

The effects of calcium deficiency on Cucurbita pepo L. hypocotyl cells: A 31P nuclear-magnetic-resonance study

Calcium deficiency in zucchini (Cucurbita pepo L.) is associated with reduced growth and a reduced ability to transport auxin (Allan and Rubery, 1991, Planta 183, 604–612). An investigation of the effects of calcium-deficiency on zucchini hypocotyl cells was made using weak-acid uptake and ³¹P-nuclear-magneticresonance (³¹P-NMR) spectroscopy in vivo and in tissue extracts. Calcium-deficient tissue had the same cytoplasmic and vacuolar pHs as normal tissue when extracellular pH was near neutral. At acidic external pH the vacuolar pH was lower in deficient tissue. Adenine nucleotides were present predominantly as ATP in both control and calcium-deficient tissues. Addition of calcium to calcium-deficient tissue, under conditions which cause recovery of auxin transport induced no changes in the ³¹P-NMR spectra of deficient tissue. The content of mobile, phosphorylated metabolites was reduced in calcium-deficient tissue in comparison to control tissue. However, a substantial increase in the content of phosphorylcholine occurs in calcium-deficient tissues compared with controls; this may reflect changes in lipid turnover in calcium-stressed cells. We wish to thank Drs. Terry Moore and Jamie Vandenberg for technical assistance and Professor Peter Morris for providing the gated oxygen device. A.C.A. thanks the Cambridge Commonwealth Trust for a Prince of Wales Scholarship and the O.R.S. Awards Scheme for an award.     

Platelet procoagulant activity and microvesicle formation. Its putative role in hemostasis and thrombosis.

Spontaneously hypertensive rats recieved 1 mg/kg of Adriamycin intravenously once a week for up to 12 weeks; their hearts were excised and perfused with buffer containing 4 mM [1-¹³C]glucose. Histological evidence of Adriamycin cardiotoxicity was evident after 8 and 12 weeks of treatment and was accompanied by a significant decrease in cardiac function. There were only minor changes in the ³¹P-NMR spectra in hearts following treatment; however, ¹³C-NMR spectra revealed decreased incorporation of label into the lactate, alanine and glutamate pools in hearts with severe tissue damage to hearts from untreated animals.     

Phosphocholine synthesis in spinach: Characterization of phosphoethanolamine N-methyltransferase

Phosphocholine is a precursor for phosphatidylcholine or it may be hydrolysed to choline. Choline can be oxidized to form the compatible osmolyte glycine betaine which is accumulated by many plants under conditions of osmotic stress. In Spinacia oleracea phosphocholine is synthesized by 3 sequential N‐methylations of phosphoethanolamine with the first step catalysed by the enzyme phosphoethanolamine N‐methyltransferase (EC 2.1.1.103). This enzyme has been partially purified 5400‐fold from spinach leaves using a combination of ammonium sulphate fractionation, followed by chromatographic separations on DEAE‐Sepharose, phenyl‐Sepharose, Ω‐aminohexyl‐agarose, Mono Q and adenosine‐agarose. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) separation and silver‐staining of the final preparation revealed several polypeptides present, only one of which with an estimated molecular mass of 54 kDa could be photoaffinity cross‐linked to the substrate [³H] S‐adenosyl‐l ‐methionine. HPLC gel permeation chromatography was used to obtain an estimate for the native molecular mass of 77 kDa. Enzyme activity was optimal at pH 7.8 in HEPES‐KOH buffer, it was inhibited by S‐adenosyl‐l ‐homocysteine, phosphocholine, phosphate, Mn²⁺ and Co²⁺ but not by ethanolamine, methylethanolamine, dimethylethanolamine, choline, glycine betaine or Mg²⁺. Using phosphoethanolamine as substrate, the final preparation had a specific activity of 189 nmol mg^?1 protein min^?1. The reaction products were identified and their relative abundance estimated following separation by TLC as phosphomethylethanolamine (87%), phosphodimethylethanolamine (10%) and phosphocholine (2%). Thus, a highly purified preparation of phosphoethanolamine N‐methyltransferase was shown to catalyse 3 successive N‐methylations of phosphoethanolamine. Photoaffinity cross‐linking of proteins extracted from leaves of spinach followed by SDS‐PAGE and autoradiography shows that a 54‐kDa radiolabelled polypeptide was more prominent in extracts from salinized plants and barely visible in extracts from plants exposed to prolonged dark periods, a pattern which corresponds to the salt and light‐responsive changes in phosphoethanolamine N‐methylating activity. Thus, the production of phosphocholine for glycine betaine accumulation in spinach can be mediated by a single phosphobase N‐methyltransferase which is more abundant in salt‐stressed plants.     

Localization and identification of the compound causing peak ‘X’ in the 31P-NMR spectrum of Saccharomyces cerevisiae

The present study is aimed at identifying the unidentified compound which gives rise to the so-called resonance ‘X’ in the ³¹P-NMR spectra of yeast cells. In addition, it is attempted to determine the localization of X (inside or outside the cell). Enzymic removal of the cell wall causes resonance ‘X’ to disappear in the spectra of the cells. This observation indicates an extracellular localization of X. The ³¹P-NMR spectrum of the phosphomannan extracted from the yeast shows a single resonance at exactly the same position as that of resonance ‘X’. Extraction of the phosphomannan from delipidized cells causes resonance ‘X’ to disappear from the ³¹P-NMR spectrum of the cells. The intensity of resonance ‘X’ in the spectrum of the intact cells can be almost quantitatively attributed to the amount of phosphomannan present in the yeast. The present results demonstrate that the resonance ‘X’ in the ³¹P-NMR spectrum of yeast cells is caused by phosphomannan in the cell wall.     

Synthesis of methylated ethanolamine moieties: regulation by choline in soybean and carrot

The results of experiments in which intact plants of Lemna paucicostata were labeled with either l-[³H₃C]methionine, l-[¹⁴CH₃]methionine, or [1,2-¹⁴C]ethanolamine support the conclusion that growth in concentrations of choline of 3.0 micromolar or above brings about marked decreases in the rate of biosynthesis of methylated forms of ethanolamine (normally present chiefly as phosphatidylcholine, with lesser amounts of choline and phosphocholine). The in vivo locus of the block is at the committing step in the biosynthetic sequence at which phosphoethanolamine is methylated by S-adenosylmethionine to form phosphomethylethanolamine. The block is highly specific: flow of methyl groups originating in methionine continues into S-adenosylmethionine, S-methylmethionine, the methyl moieties of pectin methyl ester, and other methylated metabolites. When choline uptake is less than the total that would be synthesized by control plants, phosphoethanolamine methylation is down-regulated to balance the uptake; total plant content of choline and its derivatives remains essentially constant. At maximum down-regulation, phosphoethanolamine methylation continues at 5 to 10% of normal. A specific decrease in the total available activity of AdoMet: phosphoethanolamine N-methyltransferase, as well as feedback inhibition of this enzyme by phosphocholine, and prevention of accumulation of phosphoethanolamine by down-regulation of ethanolamine synthesis may each contribute to effective control of phosphoethanolamine methylation. This down-regulation may necessitate major changes in S-adenosylmethionine metabolism. Such changes are discussed.     

Structural properties of phospholipids in the rat liver inner mitochondrial membrane. A P-NMR study

1. 1. The ³¹P-NMR characteristics of intact rat liver mitochondria, mitoplasts and isolated inner mitochondrial membranes, as well as mitochondrial phosphatidylethanolamine and phosphatidylcholine, have been examined.

2. 2. Rat liver mitochondrial phosphatidylethanolamine hydrated in excess aqueous buffer undergoes a bilayer-to-hexagonal (H_II) polymorphic phase transition as the temperature is increased through 10°C, and thus prefers the H_II) arrangement at 37°C. Rat liver mitochondrial phosphatidylcholine, on the other hand, adopts the bilayer phase at 37°C.

3. 3. Total inner mitochondrial membrane lipids, dispersed in an excess of aqueous buffer, exhibit ³¹P-NMR spectra consistent with a bilayer arrangement for the majority of the endogeneous phospholipids; the remainder exhibit spectra consistent with structure allowing isotropic motional averaging. Addition of Ca²⁺ results in hexagonal (H_II) phase formation for a portion of the phospholipids, as well as formation of ‘lipidic particles’ as detected by freeze-fracture techniques.

4. 4. Preparations of inner mitochondrial membrane at 4 and 37°C exhibit ³¹P-NMR spectra consistent with a bilayer arrangement of the large majority of the endogenous phospholipids which are detected. Approx. 10% of the signal intensity has characteristics indicating isotropic motional averaging processes. Addition of Ca²⁺ results in an increase in the size of this component, which can become the dominant spectral feature.

5. 5. Intact mitochondria, at 4°C, exhibit ³¹P-NMR spectra arising from both phospholipid and small water-soluble molecules (ADP, P_i, etc.). The phospholipid spectrum is characteristic of a bilayer arrangement. At 37°C the phospholipids again give spectra consistent with a bilayer; however, the labile nature of these systems is reflected by increased isotropic motion at longer (at least 30 min) incubation times.

6. 6. It is suggested that the uncoupling action of high Ca²⁺ concentrations on intact mitochondria may be related to a Ca²⁺-induced disruption of the integrity of the inner mitochondrial phospholipid bilayer. Further, the possibility that non-bilayer lipid structures such as inverted micelles occur in the inner mitochondrial membrane cannot be excluded.

NMR study of in vivo RIF-1 tumors: Analysis of perchloric acid extracts and identification of 1H, 31P and 13C resonances

Perchloric acid extracts of radiation-induced fibrosarcoma (RIF-1) tumors grown in mice have been analyzed by multinuclear NMR spectroscopy and by various chromatographic methods. This analysis has permitted the unambiguous assignment of the ³¹P resonances observed in vivo to specific phosphorus-containing metabolites. The region of the in vivo spectra generally assigned to sugar phosphates has been found in RIF-1 tumors to contain primarily phosphorylethanolamine and phosphorylcholine rather than glycolytic intermediates. Phosphocreatine was observed in extracts of these tumor cells grown in culture as well as in the in vivo spectra, indicating that at least some of the phosphocreatine observed in vivo arises from the tumor itself and not from normal tissues. In the ³¹P-NMR spectra of the perchloric acid extract, resonances originating from purine and pyrimidine nucleoside di- and triphosphate were resolved. HPLC analyses of the nucleotide pool indicate that adenine derivatives were the most abundant components, but other nucleotides were present in significant amounts. The ¹H and ¹³C resonance assignments of the majority of metabolites present in RIF-1 extracts have also been made. Of particular importance is the ability to observe lactate, the levels of which may provide a noninvasive measure of glycolysis in these cells in both the in vivo and in vitro states. In addition, the aminosulfonic acid, taurine, was found in high levels in the tumor extracts.     

Lymphocyte Activation-31P Magnetic Resonance Studies of Energy Metabolism and Phospholipid Pathways

³¹P NMR spectra of perfused lymphocytes embedded in alginate capsules and activated by interleukin-2 (IL-2) are remarkably different from those of control lymphocytes. The main differences are the appearance and gradual increase of phosphodiester signals, glycerophosphocholine and glycerophosphoethanolamine. These metabolic changes also occur following perfusion with phorbol ester and after incubation with phytohemagglutinin (PHA) and are not dependent on a special growth medium. Nifedipine, a calcium channel blocking drug, inhibits the effects of PHA, but not of IL-2. There are no NMR spectral differences between peripheral lymphocytes, stimulated for 3 weeks, and tumor-infiltrating lymphocytes. Thus, sustained accelerated turnover of phosphatidylcholine (PC) and phosphatidylethanolamine is an inherent feature of the activation process. ³¹P NMR spectra of lymphocytes are characterized by a low phosphocholine signal. Perfusion studies with high concentrations of choline and the use of dapsone, an inhibitor of phosphocholine cytidyltransferase, indicate that choline kinase plays a key role in regulating PC synthesis in human lymphocytes.     

Multicomponent spectra from 31P-NMR studies of the phase equilibria in the system dioleogylphosphatidylcholine-dioleoylphosphatidylthanolamine-water

The phase equilibria in mixtures of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and water were studied by ³¹P-NMR and ²H-NMR. The chemical shift anisotropy is greater for DOPC than for DOPE (6–9 ppm in the lamellar phase). This difference can most probably be ascribed to different order parameters for the two lipid head groups. ³¹P-NMR spectra recorded from a lamellar phase formed by DOPC-DOPE-water below maximum hydration exhibit two resolved, superimposed powder spectra. The chemical shift anisotropy for both phospholipids has greater values at excess water contents than below maximum hydration, and the spectral resolution between DOPC and DOPE in the lamellar phase is strikingly diminished at excess water contents. From ³¹P-NMR spectra it is possible to observe relative differences in composition between different lipid phase existing in equilibrium. The proportion of DOPE is decreased in the lamellar phase, and is increased in the reversed hexagonal phase, when these phases exist in equilibrium.     

Relation between lipid polymorphism and transbilayer movement of lipid in rat liver microsomes

We have studied the effects of trinitrophenylation on the transbilayer movement of phosphatidylcholine and the macroscopic lipid structure in rat liver microsomal membranes. The transbilayer movement of phosphatidylcholine was investigated using the PC-specific transfer protein. ³¹P-NMR was employed to monitor the phospholipid organization in intact microsomal vesicles. The results indicate that modification of microsomes with trinitrobenzenesulfonic acid enhances the transbilayer movement of phosphatidylcholine at 4°C. Furthermore, phosphatidylethanolamine headgroup trinitrophenylation in microsomes increases the isotropic component in the ³¹P-NMR spectra even at 4°C, possibly representing the appearance of intermediate non-bilayer lipid structures. The observed parallel between these data suggests that phosphatidylethanolamine molecules in the microsomal membrane, probably in combination with a protein component, are able to destabilize the bilayer organization, thereby facilitating the transmembrane movement of phospholipids.     

Metabolism of peripheral lymphocytes, interleukin-2-activated lymphocytes and tumor-infiltrating lymphocytes from 31P NMR studies

31P NMR spectra of tumor-infiltrating lymphocytes (TILs) were found to be significantly different from those of normal peripheral lymphocytes. The greatest difference was in the phosphodiester (PDE) region, mainly in the glycerophosphocholine (GPC) signal. Short-term activation of peripheral lymphocytes with interleukin-2 induced a small increase in ATP levels. In all lymphocytes the phosphomonoester (PME) region is dominated by phosphoethanolamine (PE), while there is an unusual absence of phosphocholine (PC). Perfusion of these cells with high concentrations of choline caused only a minimal increase in PC, indicating that choline kinase is not the rate limiting step of lecithin synthesis in lymphocytes.     

31P NMR spectroscopy, chemical analysis, and free Mg2+ of rabbit bladder and uterine smooth muscle

31P NMR spectra of isolated rabbit bladder and uterus were obtained under steady-state arterial perfusion in vitro at rest and while stimulated. The spectra contained seven major peaks: phosphoethanolamine, sn-glycero(3)phosphocholine, inorganic phosphate (Pi), phosphocreatine, and the gamma, alpha, and beta peaks of ATP. Chemical analyses, high-pressure liquid chromatography, and NMR spectroscopy of aqueous extracts of bladders identified a number of other components that also made contributions to, but were not resolved in, the spectra of the intact tissues: UTP, GTP, UDP-Glc, NAD+, phosphocholine, and sn-glycero(3)phosphoethanolamine. Intracellular pH of unstimulated bladders and uteri, measured from the chemical shift of the Pi peak, was 7.10 +/- 0.09 S.D. and 7.01 +/- 0.12 S.D., respectively. The chemical shift of the beta-ATP peak in the smooth muscles was significantly upfield (-0.3 ppm) compared to the chemical shift observed in striated muscles (cat biceps and rat myocardium). An ADP peak was identified in stimulated and ischemic bladders. The chemical shifts of the nucleotides observed in perfused bladders were calibrated as a function of free Mg2+ concentration in solutions containing phosphocreatine, Pi, ADP, and ATP at an ionic strength of 180 mM. We derived the following estimates for the intracellular free Mg2+ concentration: uterus, 0.40 mM; unstimulated bladder, 0.46 mM; stimulated and ischemic bladder, 0.50 mM (from the ATP chemical shift) and 0.45 (from the ADP chemical shift); cat biceps, 1.5 mM; and rat myocardium, 1.4 mM.     

Phase behavior of the major lipids of Tetrahymena ciliary membranes

The major lipids of Tetrahymena membranes have been purified by thin-layer and high pressure liquid chromatography and the phosphatidylethanolamine and aminoethylphosphonate lipids were examined in detail. ³¹P-NMR, X-ray diffraction and freeze-fracture electron microscopy were employed to describe the phase behavior of these lipids. The phosphatidylethanolamine was found to form a hexagonal phase above 10°C. The aminoethylphosphonate formed a lamellar phase up to 20°C, but converted to a hexagonal phase structure at 40°C. Small amounts of phosphatidylcholine stabilized the lamellar phase for the aminoethylphosphonate. ³¹P-NMR spectra of the intact ciliary membranes were consistent with a phospholipid bilayer at 30°C, suggesting that phosphatidylcholine in the membrane stabilized the lamellar form, even though most of the lipid of that membrane prefers a hexagonal phase in pure form at 30°C. ³¹P-NMR spectra also showed a distinctive difference in the chemical shift tensor of the aminoethylphosphonolipid, when compared to that of phosphatidylethanolamine, due to the difference in chemical structure of the polar headgroups of the two lipids.     

Assessment of Preparation Methods for Organic Phosphorus Analysis in Phosphorus-Polluted Fe/Al-Rich Haihe River Sediments Using Solution 31P-NMR

Fe/Al-rich river sediments that were highly polluted with phosphorus (P) were used in tests to determine the optimum preparation techniques for measuring organic P (Po) using solution ³¹P nuclear magnetic resonance spectroscopy (³¹P-NMR). The optimum pre-treatment, extraction time, sediment to solution ratio and sodium hydroxide-ethylenediaminetetraacetic acid (NaOH-EDTA) extractant solution composition were determined. The total P and Po recovery rates were higher from freeze- and air-dried samples than from fresh samples. An extraction time of 16 h was adequate for extracting Po, and a shorter or longer extraction time led to lower recoveries of total P and Po, or led to the degradation of Po. An ideal P recovery rate and good-quality NMR spectra were obtained at a sediment:solution ratio of 1∶10, showing that this ratio is ideal for extracting Po. An extractant solution of 0.25 M NaOH and 50 mM EDTA was found to be more appropriate than either NaOH on its own, or a more concentrated NaOH-EDTA mixture for ³¹P-NMR analysis, as this combination minimized interference from paramagnetic ions and was appropriate for the detected range of Po concentrations. The most appropriate preparation method for Po analysis, therefore, was to extract the freeze-dried and ground sediment sample with a 0.25 M NaOH and 50 mM EDTA solution at a sediment:solution ratio of 1∶10, for 16 h, by shaking. As lyophilization of the NaOH-EDTA extracts proved to be an optimal pre-concentration method for Po analysis in the river sediment, the extract was lyophilized as soon as possible, and analyzed by ³¹P-NMR.