Glycerophosphorylethanolamine Research Articles

A promising 31P NMR-multivariate analysis approach for the identification of milk phosphorylated metabolites and for rapid authentication of milk samples

A fast and reliable method for the identification of milk from different mammalians was developed by using 31P NMR metabolite profile of milk serum coupled to multivariate analysis (PCA and classification models UNEQ, SIMCA and K-NN). Ten milk samples from six different mammalians, relevant to human nutrition (human, cow, donkey, mare, goat, sheep), were analyzed and eight monophosphorylated components were identified and quantified: phosphocreatine (PCr), glycerophosphorylcholine (GPC), glycerophosphorylethanolamine (GPE), N-acetylglucosamine-1-phosphate (NAcGlu-1P), lactose-1-phosphate (Lac-1P), galactose-1-phosphate (Gal-1P), phosphorylcholine (PC), glucose-6-phosphate (Glu-6P). PCA showed interesting clustering based on the animal genus. K-NN can be successfully used to discriminate between donkey and cow samples while UNEQ class-modeling resulted more suitable for compliance verification. Results confirm the natural variability of milk samples among different species. These data highlight the great potentials of NMR/multivariate analysis combined method in the rapid analysis of phosphorylated milk serum metabolites for milk origin assessment and milk adulteration detection.

Integrated associations of nasopharyngeal and serum metabolome with bronchiolitis severity and asthma: A multicenter prospective cohort study.

While infant bronchiolitis contributes to substantial acute (eg, severity) and chronic (eg, asthma development) morbidities, its pathobiology remains uncertain. We examined the integrated relationships of local (nasopharyngeal) and systemic (serum) responses with bronchiolitis morbidities. In a multicenter prospective cohort study of infants hospitalized for bronchiolitis, we applied a network analysis approach to identify distinct networks (modules)-clusters of densely interconnected metabolites-of the nasopharyngeal and serum metabolome. We examined their individual and integrated relationships with acute severity (defined by positive pressure ventilation [PPV] use) and asthma development by age 5years. In 140 infants, we identified 285 nasopharyngeal and 639 serum metabolites. Network analysis revealed 7 nasopharyngeal and 8 serum modules. At the individual module level, nasopharyngeal-amino acid, tricarboxylic acid (TCA) cycle, and carnitine modules were associated with higher risk of PPV use (r>.20; P<.001), while serum-carnitine, amino acid, and glycerophosphorylcholine (GPC)/glycerophosphorylethanolamine (GPE) modules were associated with lower risk (all r<-.20; P<.05). The integrated analysis for PPV use revealed consistent findings-for example, nasopharyngeal-TCA (adjOR: 2.87, 95% CI: 1.68-12.2) and serum-GPC/GPE (adjOR: 0.54, 95% CI: 0.38-0.80) modules-and an additional module-serum-glucose-alanine cycle module (adjOR: 0.69, 95% CI: 0.56-0.86). With asthma risk, there were no individual associations, but there were integrated associations (eg, nasopharyngeal-carnitine module; adjOR: 1.48, 95% CI: 1.11-1.99). In infants with bronchiolitis, we found integrated relationships of local and systemic metabolome networks with acute and chronic morbidity. Our findings advance research into the complex interplay among respiratory viruses, local and systemic response, and disease pathobiology in infants with bronchiolitis.

Serum metabolomics study of Traditional Chinese medicine formula intervention to polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a most common, heterogeneous, complex endocrinopathy disease. Traditional Chinese medicine (TCM) has been used in the treatment of PCOS for many years. However, the mechanism underlying TCM remains obscure and challenging. In this study, 30 PCOS subjects were separated into normoinsulinemic group (NI=13) and hyperinsulinemic group (HI=17), and treated for three menstrual cycles with TCM Formula, Bushen Huatan Formula (BHF). A metabolomics approach based on ultra-high-performance liquid chromatography (UPLC) coupled with linear ion trap Orbi-trap mass spectrometer (LTQ Orbi-trap MS) is used to investigate serum metabolic changes of TCM intervention to PCOS. After BHF intervention for three menstrual cycles, the serum levels of glycerophosphorylethanolamine (GPEA), creatine, creatinine decreased in both NI and HI groups. Furthermore, in NI group, the main manifestation was the changes of phospholipid metabolism. While in HI group, lysine, phenol sulfate, phe–phe etc. decreased, and ornithine, proline, betaine, acetylcholine etc. increased. Combined with clinical biochemical data, BHF was proved effective to PCOS by reducing the inflammatory reaction and oxidative stress. This study also illustrates that the LC–MS based metabolomic approach is a helpful tool to evaluate curative effect and to understand the mechanisms of TCM.

High resolution in vivo 31P-MRS of the liver: potential advantages in the assessment of non-alcoholic fatty liver disease.

Biopsy remains the current gold-standard for assessing non-alcoholic fatty liver disease (NAFLD). To develop a non-invasive means of assessing the disease, 31P magnetic resonance spectroscopy (31P-MRS) has been explored, but the severe spectral overlaps and low signal-to-noise-ratio in 31P-MRS spectra at clinical field strength are clearly limiting factors. To investigate potential advantages of high resolution invivo 31P-MRS in assessing NAFLD. The study was conducted at 9.4T in control and carbon tetrachloride (CCl4)-treated rats. Rats were divided according to histopathologic findings into a control group (n = 15), a non-alcoholic steatohepatitis group (n = 17), and a cirrhosis group (n = 12). Data were presented with different reference peaks that are commonly used for peak normalization such as total phosphorous signal, phosphomonoester + phosphodiester (PME + PDE), and nucleotide triphosphate (NTP). Then, multivariate analyses were performed. In all spectra PME and PDE were well resolved into phosphoethanolamine (PE) and phosphocholine (PC), and into glycerophosphorylethanolamine (GPE) and glycerophosphorylcholine (GPC), respectively. Those MRS measures quantifiable only in highly resolved spectra had higher correlations with histology than those conventional MRS measures such as PME, PDE, and NTP. The optimized partial least-squares discriminant analysis (PLS-DA) model correctly classified 79% (22/28) of the rats in the training set and correctly predicted 69% (11/16) of the rats in the test set. PE, PC, GPE, GPC, and nicotinamide adenine dinucleotide phosphate (NADP) that can be separately quantifiable in highly resolved spectra may further improve the potential efficacy of 31P-MRS in the diagnosis of NAFLD.

Nonalcoholic Fatty Liver Disease: Detection of Elevated Nicotinamide Adenine Dinucleotide Phosphate with in Vivo 3.0-T31P MR Spectroscopy with Proton Decoupling

To determine if 3.0-T proton-decoupled phosphorus 31 ((31)P) magnetic resonance (MR) spectroscopy can be used to differentiate between stages of nonalcoholic fatty liver disease (NAFLD) by resolving the components of phosphomonoester (PME) and phosphodiester (PDE) and enabling detection of a greater number of other phosphorus-containing compounds. This study was approved by the ethics committee of Helsinki University Central Hospital, and written informed consent was obtained from all study subjects. A 3.0-T clinical imager was used to obtain proton-decoupled (31)P MR spectra in the liver of control subjects (n = 12), patients with biopsy-proved simple steatosis due to nonalcoholic causes (nonalcoholic fatty liver, n = 13; nonalcoholic steatohepatitis [NASH], n = 9), and patients with cirrhosis (n = 9) to determine PME, phosphoethanolamine (PE), phosphocholine, PDE, glycerophosphocholine (GPC), glycerophosphoryl ethanolamine, uridine diphosphoglucose, nicotinamide adenine dinucleotide phosphate (NADPH), inorganic phosphate, phosphoenolpyruvate, and alpha-, beta- and gamma-nucleotide triphosphate levels. Liver fat was determined with hydrogen 1 MR spectroscopy. Differences between the disease groups were analyzed with one-way analysis of variance. The PME/(PME + PDE), PME/PDE, and PE/(PME + PDE) ratios were higher and the GPC/(PME + PDE) ratio was lower in patients with cirrhosis than in the other study groups (P < or = .001, one-way analysis of variance). The NADPH/(PME + PDE) ratio was higher in patients with NASH and those with cirrhosis than in control subjects (P < .05, post hoc analyses) and correlated with disease severity (P = .007). NADPH, a marker of inflammation and fibrinogenic activity in the liver, is increased in patients with NASH and those with cirrhosis. Proton-decoupled (31)P 3.0-T MR spectroscopy shows promise in the differentiation of NAFLD stages.

Hepatic phospholipids in alcoholic liver disease assessed by proton-decoupled 31P magnetic resonance spectroscopy

Alteration of the phospholipid composition of hepatic biomembranes may be one mechanism of alcoholic liver disease (ALD). We applied proton-decoupled (31)P magnetic resonance spectroscopic imaging ({(1)H}-(31)P MRSI) to 40 patients with ALD and to 13 healthy controls to confirm that metabolic alterations in hepatic phospholipid intermediates could be detected non-invasively. All patients underwent liver biopsy. Specimens were scored in non-cirrhosis [fatty liver (n=3), alcoholic hepatitis (n=2), fibrosis (n=4), alcoholic hepatitis plus fibrosis (n=16)], and cirrhosis (n=15). {(1)H}-(31)P spectra were collected on a clinical 1.5-Tesla MR system and were evaluated by calculating signal intensity ratios of hepatic phosphomonoester (PME), phosphodiester (PDE), phosphoethanolamine (PE), phosphocholine (PC), glycerophosphorylethanolamine (GPE), and glycerophosphorylcholine (GPC) resonances. The signal intensity ratio GPE/GPC was significantly elevated in cirrhotic (1.19+/-0.22; P=0.002) and non-cirrhotic ALD patients (1.01+/-0.13; P=0.006) compared to healthy controls (0.68+/-0.04), while PE/PC and PME/PDE were significantly elevated in cirrhotic ALD patients compared to controls (1.68+/-0.60 vs. 0.97+/-0.31; P=0.02, and 0.38+/-0.02 vs. 0.25+/-0.01; P=0.002, respectively) and non-cirrhotic patients. The data support that {(1)H}-(31)P MRSI appears to distinguish cirrhotic from non-cirrhotic ALD patients and confirms changes in hepatic phospholipid metabolism observed in an animal model.

Metabolite mapping of human filarial parasite, Brugia Malayi with nuclear magnetic resonance

Metabolite mapping of human filarial parasite, Brugia malayi was carried out in vitro as well as in situ in host Mastomys coucha by 31P nuclear magnetic resonance (NMR) spectroscopy. Detection of parasites by visualizing contrast spots due to pathologic changes was observed by 1H magnetic resonance imaging (MRI). Major metabolites of adult B. malayi observed by 31P-NMR spectroscopy were of sugar phosphates (SP), phosphomonoesters (PME), glycerophosphoryl-ethanolamine (GPE), -choline (GPC), phosphoenolpyruvate (PEP), inorganic phosphate (Pi), nucleoside diphosphosugar and nucleotides-mono, -di and -tri phosphates. PEP and GPC were present in high concentration; PEP being the major energy reservoir and GPC the major phospholipid in this species of filaria. The 31P NMR spectra of testis of mastomys, showed seven major peaks of SP, PME, phosphocreatine (PCr), phosphodiesters (PDE), Pi, and nucleotides di- and tri-phosphates. The 31P-NMR spectra of testis of B. malayi infected animal also consisted of seven major peaks with significant decrease in the SP and PME peak showing changes in the carbohydrate and lipid metabolism of filaria infected testis. Thus, in vivo 31P MRS provided a non-invasive assessment of tissue bioenergetics and phospholipid metabolism.

Ab initio study of phospholipid headgroups: GPE and GPC

A systematic conformational study of the stable headgroup structures for phospholipid single molecule using five model compounds: CH 3PO 4 −CH 3, CH 3CH 2PO 4 −CH 2CH 3, CH 3CH 2PO 4 −CH 2CH 2NH 3 +, glycerophosphorylethanolamine (GPE) and glycerophosphorylcholine (GPC) is presented. The conformational potential energy surface was searched for local minima using the rigid-rotor approximation. Fully geometry optimized structures were then obtained using the molecular orbital program Gaussian 92. The standard 3-21G* basis set was employed in most calculations. The conformational energy differences for the rotations around the CO and PO bonds were determined to be in the range of 3–6 kJ/mol. It was found that structural parameters for isolated compounds agree reasonably well with the crystallographic data. The variation of geometrical parameters with the increasing size of the model compound is also discussed. Phospholipid structural parameters such as molecular area, thickness of the headgroup and dipole moments are estimated.

Developmental changes in choline- and ethanolamine-containing compounds measured with proton-decoupled (31)P MRS in in vivo human brain.

Cerebral phosphorylated metabolites, possibly involved in membrane and myelin sheath metabolism, were measured and quantified using proton-decoupled (31)P ({(1)H}-(31)P) MRS in 32 children and 28 adults. Age-dependent changes were determined for phosphorylethanolamine (PE), phosphorylcholine (PC), glycerophosphorylethanolamine (GPE), glycerophosphorylcholine (GPC), and phosphocreatine (PCr) concentrations. In the neonate, PE dominates the spectrum and decreases with age along with PC, whereas GPE, GPC, and PCr increase in concentration with postnatal age. PE (1.23 +/- 0.13 mM) and GPE (0.57 +/- 0.08 mM) co-resonate with choline in (1)H MRS. Together with PC (0.57 +/- 0.12 mM) and GPC (0. 94 +/- 0.13 mM) these four metabolites accounted for all of the visible (1)H MRS choline in normal adult brain. Children with diseases that affect myelination were found to have abnormal ¿(1)H¿-(31)P MRS. The new quantitative assay may provide novel insights in determining and monitoring normal and abnormal brain maturation noninvasively. Magn Reson Med 42:643-654, 1999.

A 31P nuclear magnetic resonance study of the hydrothermal vent tube worm Riftia pachyptila

31P nuclear magnetic resonance (NMR) was used to study the major phosphorylated compounds visible in perchloric extracts of three body regions of the vestimentiferan worm Riftia pachyptila: winged vestimentum, trunk and segmented posterior opisthosome. Two phosphagens (PGs) were present in vestimentum and opisthosome. The major resonance corresponded to those of phosphoarginine and phosphotaurocyamine, which cannot be discriminated on 31P NMR spectra. We have identified four distinct phosphodiesters (PDEs) in these tissues: glycerophosphorylethanolamine (GPE), serine ethanolamine phosphodiester (SEP), glycero-phosphorylcholine (GPC) and threonine ethanolamine phosphodiester (TEP). Three phosphonates or derivates (PAs) were observed in the three body regions. The minor one was identified as 2-aminoethyl phosphonate (2-AEP). The phosphorus profile of the trunk was appreciably different: one additional resonance in the PDE region and only one phosphagen peak were observed.

Changes in fatty chain compositions of lipid classes during frozen storage of the adductor muscle of giant ezo scallop ( Patinopecten yessoensis)

Changes in lipid components, particularly glycerophospholipids in the adductor muscle of giant ezo scallop during storage at −20°C, were investigated. During storage, the contents of total lipid (TL) and polar lipid (PL) decreased but that of non-polar lipid (NL) increased. Glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) decreased by 50 and 15% of the each initial content, while lyso-GPC and free fatty acids increased. The percentages of polyunsaturated fatty acids such as 20:5n-3 and 22:6n-3 in the TL and PL fractions decreased during storage, but those of the polyunsaturated fatty acids in the NL increased. In the alkenylacyl-GPE and diacyl-GPC, the percentages of relatively longer acids in the sn-1 positions of glycerol moieties decreased at higher rates than did the shorter chains, whereas the results for diacyl-GPE were opposite to those of alkenylacyl-GPE and diacyl-GPC. In the prominent fatty acids in the sn-2 positions of alkenylacyl-GPE and diacyl-GPC, the percentage of 22:6n-3 decreased from compared to the high level of 20:5n-3, while that of diacyl-GPE increased.

Lack of effect of oral choline supplement on the concentrations of choline metabolites in human brain.

Recent reports suggest that oral choline supplement may alter the cerebral choline/creatine (Cho/Cr) ratio and might be used to treat neurodegenerative disorders of cholinergic transmission. Using both 1H and 31P MRS, we reexamined the Cho/Cr ratio and quantified cerebral choline and its major constituents: phosphoethanolamine (PE), phosphorylcholine (PC), glycerophosphorylethanolamine (GPE), and glycerophosphorylcholine (GPC). In the four brain locations examined, no significant increases in Cho/Cr, [Cho], or in its major constituents were found in response to an oral challenge of 50 mg/kg of choline bitartrate. Oral choline did not significantly affect human cerebral metabolism in the short term.

In vivo and in vitro hepatic phosphorus-31 magnetic resonance spectroscopy and electron microscopy in chronic ductopenic rejection of human liver allografts

Background—In vivo hepatic phosphorus-31 magnetic resonance spectroscopy (MRS) provides non-invasive information about phospholipid metabolism.Aims—To delineate MRS abnormalities in patients with chronic ductopenic rejection (CDR) and to characterise spectral changes by...

31P-NMR determinations of cytosolic phosphodiesters in turtle hearts

As part of our ongoing research on cardiac hypoxia tolerance we have conducted 31P nuclear magnetic resonance (NMR) studies of isolated, perfused, working hearts from freshwater turtles, animals that are well known for their ability to tolerate prolonged periods of anoxia. A striking feature of turtle heart spectra is an extremely high concentration of NMR visible phosphodiesters (PDEs). Cardiac spectra from mammals, on the other hand, typically exhibit only a small resonance in the PDE region. Our aim in this study was to compare myocardial PDE profiles between the highly hypoxia tolerant western painted turtle ( Chrysemys picta bellii) and the relatively hypoxia sensitive softshelled turtle ( Trionyx spinifer) in order to begin to test the hypothesis that high constitutive levels of cytosolic PDEs may play a role in conferring hypoxia and ischemia tolerance on the myocardium. We also collected 31P-NMR spectra of PCA extracts of tissue from these species and from Kemp's ridley sea turtles ( Lepidochelys kempi), as well as spectra from isolated hearts and PCA extracts of redeared sliders ( Trachemys [formerly Pseudemys] scripta]). Total NMR visible phosphodiesters make up 24 ± 8.6% of the total NMR visible phosphorus in chrysemys hearts, 20.7 ± 5.9% in Trachemys hearts, but only 12.2 ± 5.1% in Trionyx hearts ( P < 0.05). We have identified three distinct PDEs in turtle hearts: glycerophosphorylcholine (GPC); glycerophosphorylethanolamine (GPE); and serine ethanolamine phosphodiester (SEP). SEP is the dominant compound in Chrysemys and Trachemys (79.3 ± 10.2% and 84.7 ± 3.7% of total PDE, respectively), while GPC is most abundant in Trionyx (74.0 ± 4.3% of total PDE) and Lepidochelys (not quantitated). The function of this class of compounds is unclear but it has been suggested that cytosolic PDEs may function as lysophospholipase inhibitors, a role that would decrease the rate of membrane phospholipid turnover. Our comparative data suggest that cytosolic PDEs could play a role in phospholipid sparing during anoxic or ischemic stress in turtles but a direct test of this hypothesis awaits future experimentation.

A Phosphorus-31 Nuclear Magnetic Resonance Study of Phospholipid Metabolism in Mesangial Cells Treated with Arginine Vasopressin

Aqueous solutions containing extracts of cultured rat mesangial cells treated with and without arginine vasopressin (AVP) were measured by phosphorus-31 nuclear magnetic resonance (³¹P-NMR) spectroscopy. NMR signal intensities of phospholipid metabolites in the solutions were measured as the areas of those resonance lines. The signal intensity of phosphorylcholine (PC) and the ratio of phosphorylcholine/phosphorylethanolamine (PC/PE) increased significantly 5 min after AVP administration. The signal intensity of glycerophosphoryleth-anolamine (GPE) increased significantly 30 min after AVP administration, and the ratio of GPE/PE also increased significantly 30 and 60 min after administration of AVP. Our data showed that AVP stimulates phospholipid metabolism in cultured rat mesangial cells, and that the changes in signal intensities of each phospholipid metabolite in ³¹P-NMR induced by AVP might be based on phospholipid metabolism closely related to the hormonal regulation of mesangial function.

Glycerophosphorylethanolamine (GPEA) Identified as an Hepatocyte Growth Stimulator in Liver Extracts

Extracts from weanling pig liver were found to act synergistically with growth factors such as hepatocyte growth factor and transforming growth factor-α to stimulate hepatocyte growth in serum-free cultures. In the absence of added growth factors, the extracts had no activity. The compound responsible for this activity was isolated by passing heat-treated liver extract through anion-exchange and heparin columns followed by gel filtration at neutral and low pH, reversed-phase HPLC, and a final gel filtration column at low pH. The activity was followed throughout the purification by its ability to increase substantially the incorporation of [3H]thymidine into primary rat hepatocytes cultured serum-free in the presence of hepatocyte growth factor. The active compound was identified by NMR and mass spectrometry as glycerophosphorylethanolamine (GPEA), a breakdown product of the phospholipid phosphatidylethanolamine. The ethanolamine portion of the molecule was critical for the observed activity, whereas the glycerol phosphate portion was not necessary. In the absence of added growth factors, neither GPEA nor ethanolamine had any stimulatory effect on the cells. These results demonstrate that hepatocytes grown in culture, and especially those grown in serum-free media, require a supplement of ethanolamine and/or GPEA. In the absence of these compounds, their response to growth stimuli is greatly reduced.

Hepatic nucleotide triphosphate regeneration after hypothermic reperfusion in the pig model: an in vitro P-NMR study.

The aim of this study was to assess the possibility of regenerating nucleotide triphosphates (NTP) in the pig liver following its harvest and subsequent storage on ice. This study has used a pig model that allowed human donor liver retrieval techniques and methods of storage to be utilized. In vitro phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy was used to evaluate the changes associated with phosphorus containing metabolites such as NTP, phosphomonoesters (PME), phosphodiesters (PDE), and inorganic phosphate (Po). During 4 hr storage NTP levels were reduced to undetectable levels but its regeneration was possible over a period of 2 hr of oxygenated hypothermic reperfusion. Resynthesized NTP reached values that were only 30% reduced from pre-harvest values. There was a corresponding reduction in Pi over the same period. Glycolytic intermediates, 3-phosphoglycerate and 2,3 diphosphoglycerate, both increased significantly during the period of storage and subsequently declined following hypothermic reperfusion. Cellular damage, indicated by the concentrations of glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) was minimal during cold storage. However upon hypothermic reperfusion, concentrations of GPC and GPE reduced, indicating a degree of cellular damage caused by reperfusion. This study has shown for the first time that is possible to regenerate high energy phosphate nucleotides following a period of hypothermic reperfusion in a large, clinically related animal model. This technique warrants investigation clinically to improve the outcome of orthotopic liver transplantation. It also provides a method to study the effects of different preservation fluids and methods of storage and organ reperfusion.

Cirrhosis of the human liver: an in vitro 31P nuclear magnetic resonance study

Human livers with histologically proven cirrhosis were assessed using in vitro 31P NMR spectroscopy. Spectra were compared with those from histologically normal livers and showed significant elevations in phosphoethanolamine (PE) and phosphocholine (PC) and significant reductions in glycerophosphorylethanolamine (GPE) and glycerophosphorylcholine (GPC). There were no significant differences in spectra from livers with compensated and decompensated cirrhosis. These results help to characterise the alterations in membrane metabolism in cirrhosis of the liver.

Phosphate metabolites of Tenebrio molitor (Coleoptera: tenebrionidae) infected with metacestodes of Hymenolepis diminuta.

In vivo phosphorus-31 nuclear magnetic resonance (31P NMR) spectra and those of extracts of Tenebrio molitor L. infected with metacestodes of Hymenolepis diminuta R. showed modifications in the amounts of phosphorous-containing metabolites when compared with those of uninfected beetles. Infected females were more affected than infected males, having significantly more glucose-6-phosphate (Glu-6-P), glycerol-3-phosphate (Gly-3-P), and phosphorylethanolamine (PE), but less inorganic orthophosphate (Pi), glycerophosphorylethanolamine (GPE), gylcerolphosphorylcholine (GPC), phosphoarginine (PAr), and adenosine diphosphate (ADP). Infected males had more Glu-6-P and PE, but less Pi, GPE, and GPC. These changes directly reflected the phosphorylation potential of infected hosts, which increased approximately 100% and 50% in infected females and males, respectively.

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 (P i). 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.