Chemical assessment of phospholipid and phosphoenergetic metabolites in regenerating rat liver measured by in vivo and in vitro 31P-NMR.

For the assessment of 31P-NMR spectroscopic data, phospholipid precursors (phosphorylethanolamine (PE) and phosphocholine) and catabolites (glycerophosphorylethanolamine (GPE) and glycerophosphorylcholine (GPC)), as well as adenosine phosphates were chemically determined in regenerating rat liver. The data were compared with those obtained by in vivo and in vitro 31P-NMR spectroscopies. Chemical assay revealed a significant increase of PE and a decrease of GPE, GPC and ATP in hepatectomy group compared to sham operation group. The values obtained by in vitro NMR were in good agreements with those of chemical assay, but significant differences between the two groups were observed only in PE and inorganic phosphate (Pi). Noticeable increase in PME was not detected by in vivo 31P-NMR spectroscopy, although the increase of PE was about 2.5-times that of the control and its constitution ratio to the whole phosphomonoester (PME) was less than 15%. On the other hand, in vivo NMR showed a large phosphodiester (PDE) peak occupying approx. 40% of the total phosphorus signal, while the contribution of its constituents, GPE and GPC was about 5% found by both chemical assay and in vitro NMR. The PDE peak in in vivo NMR seemed to reflect the membrane phospholipid itself rather than its catabolites. A slight decrease of phosphoenergetic level in regenerating rat-liver was commonly suggested by all three analytical methods.     

31P-NMR study of brain phospholipid structures in vivo.

31P-NMR has been used extensively for the study of cytosolic small molecule phosphates in vivo and phospholipid structures in vitro. We present in this paper a series of studies of the brain by 31P-NMR, both in vivo and in extracts, showing the information that can be derived about phospholipids. 31P-NMR spectra of mouse brain at 73 mHz are characterised by almost a complete absence of the large phosphodiester peak in comparison to equivalent spectra at 32 mHz. Proton decoupled spectra in vivo, and spectra of extracts, show that the phosphodiester peak observed in 32 mHz spectra in vivo is mainly due to phospholipid bilayers. Homogenates of quaking and control mouse brains, and of bovine grey matter, show another narrower phosphodiester peak possibly from small phospholipid vesicles. This peak is increased in intensity in the affected mice. These experiments demonstrate the presence of three major components contributing to the phosphodiester resonance: bilayer phospholipids, more mobile phospholipids, and the freely soluble cytosolic molecules glycerophosphocholine and glycerophosphoethanolamine. These NMR methods for non-invasive investigation of phospholipid structures in the brain might be extended to studies of patients with membrane involved diseases such as multiple sclerosis.     

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.     

~（31）P－NMR谱在人急性早幼粒白血病HL－60细胞分化代谢研究中的应用

ＨＬ－６０细胞是目前研究诱导分化药物的常用细胞株,应用核磁共振~（３１）Ｐ谱,观察ＨＬ－６０细胞经分化诱导剂全反式维甲酸和新维甲类化合物ＳＬＭ９１２３作用前后的代谢改变,发现分化后的细胞对ＡＴＰ能量的需求明显增高,膜磷脂的合成前体──磷酸单酯也有明显增加,另外还发现分化后的细胞内ｐＨ值有从偏碱性转为偏酸性的趋势,文中对这些改变的可能机制作了探讨。     

A human in vivo study of the extent to which 31-phosphorus neurospectroscopy phosphomonoesters index cerebral cell membrane phospholipid anabolism

The phosphomonoester narrow resonance of human in vivo 31-phosphorus neurospectroscopy studies is believed to index the anabolism of cell membrane phospholipids and has therefore been used to study phospholipid anabolism in the brain non-invasively. However, it is an indirect measure of phospholipid metabolism and although it does contain major contributions from phosphocholine, phosphoethanolamine and L-phosphoserine, which are important precursors of membrane phospholipids, many other metabolites, including sugar phosphates, can contribute to this region of the spectrum, and separation of these different peaks is not achieved with the present in vivo methodology. Recently, it has become possible to analyze signal directly from the cell membrane motion-restricted phospholipids by analysis of a broad resonance signal. We therefore hypothesized that there should be a positive correlation between the phosphomonoester narrow resonance and the broad resonance signal if the former does indeed index cell membrane phospholipid anabolism. Cerebral 31-phosphorus magnetic resonance spectroscopy was carried out in 54 human subjects, including normal volunteers and patients with schizophrenia in order to widen the range of phosphomonoester and broad resonance values. Spectra were obtained from 70×70×70 mm³ voxels using an image-selected in vivo spectroscopy pulse sequence. There was a highly significant positive correlation between the phosphomonoester resonances and the broad resonance signals (r=0.404, P<0.005). These results are consistent with the hypothesis that the phosphomonoester narrow resonance does indeed index cell membrane phospholipid anabolism in brain studies.     

Phosphocholine and phosphoethanolamine during chick embryo myogenesis: a (31)P-NMR study

Elevated contents of phosphoethanolamine (Etn-P) and/or phosphocholine (Cho-P), a common feature of most tumours with respect to normal counterparts, may also occur in non-cancerous proliferating tissues. The significance of these alterations in relation to cell proliferation, differentiation and maturation is scarcely understood. In this work, the Cho-P and Etn-P pools were measured by (31)P-NMR in extracts of chick embryo pectoral muscle at different days of development. The average concentration of these metabolites exhibited the highest values (respectively, 1.5 and 3.0 micromol/mg DNA) on days 9-11 and decreased at later stages of myogenesis. While, however, Cho-P maintained substantial levels (above 1.0 micromol/mg DNA) also during myotube formation (days 11-18) and stepwise decreased (to about 0.5 micromol/mg DNA) upon fibres' maturation, Etn-P gradually decreased between day 11 and hatching time (down to about 0.2 micromol/mg DNA). These results demonstrate that significant changes may occur in the steady-state pools of these metabolites during normal in vivo cellular development and differentiation, and are consistent with: (a) high rates of phospholipid biosynthesis reported in the literature for proliferating myoblasts; (b) sustained phosphatidylcholine synthesis maintained also during myoblast fusion; and (c) decreased requirement of phospholipid synthesis in the last phase of in ovo myofibre maturation.     

A 31P and 1H-NMR investigation in vitro of normal and abnormal human liver

Spectral changes in human hepatic tumours and possible systemic effects of tumour on host liver were assessed by ³¹P amnd ¹H in vitro NMR spectroscopy. The ¹H and ³¹P spectra from liver tumour biopsies showed significant elevation in phosphoethanolamine, phosphocholine, taurine, citrate, alanine, lactate and glycine, and significant reduction in GPE (glycerophosphoethanolamine), GPC (glycerophosphocholine), creatine and threonine compared to histologically normal tissue. ³¹P-NMR spectra obtained from histologically normal tissue within tumour-bearing livers showed significant elevation in phosphoethanolamine and phosphocholine compared to data from liver biopsies from nontumour-bearing patients (pancreatitis). These results suggest that alterations in membrane metabolism in host liver can be detected by ³¹P-NMR.     

Identification of phosphorylethanolamine in 31P-NMR spectra of human peripheral blood lymphocytes

The 31P-NMR spectrum of intact human peripheral blood lymphocytes contains a large unidentified peak in the phosphomonoester region. The pH dependency of the 31P-NMR chemical shift of this peak in perchloric acid extracts of peripheral blood lymphocytes was recorded. It was compared to the pH dependency of the chemical shift of phosphorylethanolamine, phosphorylcholine, and ribose 5-phosphate in model solutions. An excellent agreement was found between the behavior of phosphorylethanolamine and the unidentified peak. To further substantiate this assignment phosphorylethanolamine was added to extracts and the pH titrations were repeated. The added phosphorylethanolamine gave exactly the same chemical shift as the unidentified peak and no difference was observed with pH titrations. The concentration of phosphorylethanolamine in human peripheral blood lymphocytes was estimated by 31P NMR to be 2.4 mumol/10(9) cells (range 0.9-4.3/10(9) cells, n = 4).     

A 31P-NMR study of Propionibacterium acnes, including effects caused by near-ultraviolet irradiation

161.8 MHz 31P-NMR spectra were recorded from the light sensitive skin bacterium Propionibacterium acnes. The cells were grown anaerobically on synthetic phosphate-buffered Eagle's medium or on a complex yeast extract medium. The spectra showed a large accumulation of polyphosphates when grown on Eagles medium. A splitting of the inorganic phosphate peak indicated a difference between internal and external pH of the cells. Addition of glucose to the cell suspension gave rise to a change in the pH gradient across the cell membrane, as reported for other Gram-positive bacteria. A decrease in the polyphosphate peak was observed after addition of glucose. A lethal dose of broad-band near-ultraviolet light (corresponding to a 10% survival in a survival test), increased the amount of polyphosphates visible in the NMR-spectra. The addition of glucose to irradiated cells decreased the pH in the external solution, but no splitting of the inorganic phosphate peak could however be observed. 31P-NMR can, therefore, be used to study immediate near-ultraviolet-induced effects at the cellular level, at least in the case of P. acnes.     

Cellular responses to excess phospholipid

Phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells, and its synthesis is controlled by the activity of CDP:phosphocholine cytidylyltransferase (CCT). Enforced CCT expression accelerated the rate of PtdCho synthesis. However, the amount of cellular PtdCho did not increase as a result of the turnover of both the choline and glycerol components of PtdCho. Metabolic labeling experiments demonstrated that cells compensated for elevated CCT activity by the degradation of PtdCho to glycerophosphocholine (GPC). Phospholipase D-mediated PtdCho hydrolysis and phosphocholine formation were unaffected. Most of the GPC produced in response to excess phospholipid production was secreted into the medium. Cells also degraded the excess membrane PtdCho to GPC when phospholipid formation was increased by exposure to exogenous lysophosphatidylcholine or lysophosphatidylethanolamine. The replacement of the acyl moiety at the 1-position of PtdCho with a non-hydrolyzable alkyl moiety prevented degradation to GPC. Accumulation of alkylacyl-PtdCho was associated with the inhibition of cell proliferation, demonstrating that alternative pathways of degradation will not substitute. GPC formation was blocked by bromoenol lactone, implicating the calcium-independent phospholipase A2 as a key participant in the response to excess phospholipid. Owing to the fact that PtdCho is biosynthetically converted to PtdEtn, excess PtdCho resulted in overproduction and exit of GPE as well as GPC. Thus, general membrane phospholipid homeostasis is achieved by a balance between the opposing activities of CCT and phospholipase A2.     

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.     

In vivo and in vitro 31P-NMR spectroscopy of rat liver treated with halocarbons

Both in vivo and in vitro 31P-NMR spectroscopy were used to demonstrate metabolic changes in rat liver as a function of time after exposure to either carbon tetrachloride (CCl4) or bromotrichloromethane (BrCCl3). The inorganic phosphate resonance, measured in vivo, moves upfield, which is associated with a decrease in cytosolic pH over a 12 or 20 h period (for BrCCl3 or CCl4, respectively). Intoxication by CCl4 or BrCCl3 causes an intracellular acidosis to pH 7.05 or 6.82 (+/- 0.05), respectively. Also, it has been found that halocarbon exposure increases the amounts of phosphomonoesters (PME) detected. High resolution in vitro 31P-NMR spectroscopy studies of perchloric acid extracts of CCl4-treated rat livers indicated a significant increase in the height of the phosphocholine resonance in the PME region 4-5 h after CCl4 exposure.     

Phospholipid metabolism in cancer cells monitored by 31P NMR spectroscopy

Addition of choline, ethanolamine, or hemicholinium-3 (a choline kinase inhibitor) to the perfusate of human breast cancer cells monitored by 31P NMR spectroscopy resulted in significant changes to phosphomonoester (PME) and phosphodiester (PDE) signals. These results enable us to assign the PMEs to phosphcholine (PC) and phosphoethanolamine (PE), the PDEs to glycerophosphorylcholine and glycerophosphorylethanolamine, and to define the pathways producing them. The PMEs are products of choline and ethanolamine kinases, the first steps in phospholipid synthesis; and the PDEs are substrates of glycerophosphorylcholine phosphodiesterase, the last step in phospholipid catabolism. Furthermore, PC and PE peaks are twice as intense in cells at log phase versus confluency. We also observed these signals in vivo in human colon and breast tumors grown in mice. Since PMEs are low in most nonproliferating tissues, they could form a basis for noninvasive diagnosis. Also, PE and PC are situated between the control enzymes of two major synthetic pathways and will allow noninvasive 31P NMR studies of these pathways in intact cells and in vivo.     

An in Vivo31P Magnetic Resonance Spectroscopy Study of Uridine Excess in Rats Fed Orotic Acid

Spatially localized ³¹P NMR spectroscopy was used to assay in vivo the liver of intact rats fed erotic acid (OA) in a diet which produces hepatic steatosis. Twenty-three sets of multiple volume spectra were obtained from twenty-one 265- to 315-g female rats after 0-9 days of feeding either a 1% OA/64% sucrose diet (12 rats) or a 65% sucrose control diet (9 rats). The intensity of the in vivo diphosphodiester resonance ascribed to UDP-hexos(amin)es increased and the phosphomonoester resonance decreased in intensity prior to fatty infiltration. High resolution NMR spectroscopy of extracts of these livers indicated that the UDP-hexos(amin)e peak included four different UDP-sugars including UDP-N-acetylglucosamine (UDP-glcNAc), and that lower phosphocholine (P-Cho) accounted for the lower phosphomonoester resonance in vivo. Increased UDP-glcNAc is thought to reflect impaired lipoprotein glycosylation as a mechanism for hepatic steatosis in orotic acid feeding. P-Cho deficiency has been shown to be due to an increased rate of phosphatidylcholine synthesis. Low P-Cho concentration has been shown to be associated with lipid accumulation in a choline-deficient diet, but was not previously associated with hepatic steatosis in OA feeding. Changes in phosphorus metabolites were observed 2 days prior to the development of fatty liver, HPLC assay of uridine nucleotides showed a good correlation between magnetic resonance spectroscopy and HPLC quantitation. In this study there were two biochemical correlates of impaired hepatic lipid secretion detectable by in vivo assay with ³¹P NMR spectroscopy. This method has application for noninvasive assays in ornithine transcarbamylase-deficient patients.     

Effect of ConA—receptor interaction on the structure of cell membrane

Recently,the effect of ligand receptor interaction on the membrane structure of liposomes has been studied extensively,However,little is known about how it exists on biological membranes,In this paper,the effect of Concanavalin A(ConA) receptorinteratcion on the structure of cell membranes was studied by Circular DIchrosim(CD) and 31P Nuclear Magnetic Resonance(NMR).CD results of both the purified macrophage membranes and human erythrocyte hgosts(EG) showed that the conformation of membrane proteins changed after ConA binding.For further research,31P-NMR was used to detect the orgainzation of phosp[holipid molecules on macrophage membranes.After ConA binding,the tendercy to form non bilayer structure increased with the amount of ConA.The changes of 31P-NMR spectra of living macrophages might be partly due to the above stated reason too.In addition,ConA-receptor interaction also induced similar results of 31P-NMR spectra in EG.In contrast,wheat germ agglutinin (WGA),another kind of lectin,rarely showed the same influence.     

Light-induced membrane protein phosphorylation in the bovine rod outer segment. A magic angle spinning 31P-NMR study

Magic angle spinning 31P-NMR (MAS 31P-NMR) spectra of bovine rod outer segments, unphosphorylated and phosphorylated, were obtained. In the phosphorylated samples the spectra showed new resonances not assignable to phospholipids. These signals were present only when stimulation of receptor phosphorylation occurred. These resonances were not due to exogenous, soluble phosphorus-containing compounds. Limited proteolysis to remove the carboxyl-terminal region of the photoreceptor that contains the phosphorylation sites removed these resonances. The chemical shifts were in the usual range for serine phosphate and threonine phosphate. The pKa obtained from a pH titration of the 31P chemical shift was typical of serine phosphate. Therefore, these 31P-NMR resonances were assigned to the phosphorylation sites on membrane proteins in the rod outer segment disk membranes. Static 31P-NMR measurements revealed that at least some of these sites gave rise to relatively narrow resonances, indicative of considerable motional freedom of the carboxyl-terminal segment of the photoreceptor when phosphorylated. These data indicate that it is possible to study phosphorylation sites on membrane proteins using MAS 31P-NMR, and that using in vivo 31P 'spin labelling' one can study directly and selectively regions of receptors crucial to receptor function.     

Proton nuclear magnetic resonance spectroscopy of isolated rabbit fundic glands

1H-nuclear magnetic resonance spectroscopy (NMR) was adapted to isolated rabbit fundic glands and identification made of compounds responsible for several observed spectral resonances. A minimum gland concentration of 0.5 mg dry weight or 5 mg wet weight per 0.5 ml was needed for adequate signal-to-noise ratio. At physiological temperature and pH, the glands demonstrated reproducible spectra, stability for accumulation times greater than 30 min and responsiveness to histamine stimulation, as measured by oxygen consumption and aminopyrine uptake. The relatively anaerobic conditions favored use of proton compared to phosphorus NMR, since 1H-NMR allowed significantly shorter spectral accumulation times and therefore did not compromise glandular viability to the same extent as 31P-NMR. The most conspicuous resonance in the gland spectrum was assigned to the -N+(CH3)3 protons of choline and related compounds. In membrane-free lysates, several components of the signal were resolvable and assigned to choline, phosphatidylcholine, phosphocholine and L-alpha-glycerophosphocholine. Thin-layer chromatography verified that phosphatidylcholine and phosphatidylethanolamine were the major phospholipids present in gland lipid. Presumably, they represent the source of the surface-active phospholipids present in gastric juice, which may play a role in gastric cytoprotection.     

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.     

Short chain phospholipids in membrane protein crystallization: a 31P-NMR study of colloidal properties of dihexanoyl phosphatidylcholine

The colloidal features of short chain phospholipids can be deduced from 31P-NMR analysis by comparison with available data on phospholipid aqueous dispersion. In this study with dihexanoyl phosphatidylcholine, detergent phase separation was obtained by temperature shift and by addition of the precipitating agent polyethylene glycol. The 31P-NMR spectra indicate that the detergent micelles fuse to enter the hexagonal HII and lamellar phases. Consequences for the crystallization of membrane proteins are discussed.