Phosphodiester Peak Research Articles

Older adults with sarcopenia have distinct skeletal muscle phosphodiester, phosphocreatine, and phospholipid profiles

The loss of skeletal muscle mass and function with age (sarcopenia) is a critical healthcare challenge for older adults. 31‐phosphorus magnetic resonance spectroscopy (31P‐MRS) is a powerful tool used to evaluate phosphorus metabolite levels in muscle. Here, we sought to determine which phosphorus metabolites were linked with reduced muscle mass and function in older adults. This investigation was conducted across two separate studies. Resting phosphorus metabolites in skeletal muscle were examined by 31P‐MRS. In the first study, fifty‐five older adults with obesity were enrolled and we found that resting phosphocreatine (PCr) was positively associated with muscle volume and knee extensor peak power, while a phosphodiester peak (PDE2) was negatively related to these variables. In the second study, we examined well‐phenotyped older adults that were classified as nonsarcopenic or sarcopenic based on sex‐specific criteria described by the European Working Group on Sarcopenia in Older People. PCr content was lower in muscle from older adults with sarcopenia compared to controls, while PDE2 was elevated. Percutaneous biopsy specimens of the vastus lateralis were obtained for metabolomic and lipidomic analyses. Lower PCr was related to higher muscle creatine. PDE2 was associated with glycerol‐phosphoethanolamine levels, a putative marker of phospholipid membrane damage. Lipidomic analyses revealed that the major phospholipids, (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol) were elevated in sarcopenic muscle and were inversely related to muscle volume and peak power. These data suggest phosphorus metabolites and phospholipids are associated with the loss of skeletal muscle mass and function in older adults.

Influence of surfactants and humic acids on Artemia Franciscana's embryonic phospho-metabolite profile as measured by 31P NMR

Surfactants, such as triton X-100 (Tx-100), cetylpyridinium chloride (CPC), and sodium dodecyl sulfate (SDS) are known to be toxic to Artemia Franciscana (Artemia) – an organism, frequently used to monitor the health of the aquatic environment. The phospho-metabolite profile of a living organism is often indicative of imbalances that may have been caused by environmental stressors, such as surfactants. This study utilizes in vivo31P NMR to monitor temporal changes in the phospho-metabolite profile of Artemia caused by Tx-100, CPC, and SDS and the ability of humic acid (HA) to mitigate the toxicity of these surfactants. It was found that, while Tx-100 does not have any effect on the phospho-metabolite profile, both CPC and SDS cause a complete retardation in growth of the phosphodiester (PDE) peak in the 31P NMR spectrum, which is indicative of the inhibited cell replication. This growth inhibition was independently verified by the decreased guanosine triphosphate (GTP) concentration in the CPC and SDS-exposed Artemia. In addition, upon introduction of HA to the CPC and SDS-exposed Artemia, an increase of PDE peak over time is indicative of HA mitigating toxicity.

Ca2+-independent Binding of Anionic Phospholipids by Phospholipase C δ1 EF-hand Domain

Recombinant EF-hand domain of phospholipase C δ1 has a moderate affinity for anionic phospholipids in the absence of Ca(2+) that is driven by interactions of cationic and hydrophobic residues in the first EF-hand sequence. This region of PLC δ1 is missing in the crystal structure. The relative orientation of recombinant EF with respect to the bilayer, established with NMR methods, shows that the N-terminal helix of EF-1 is close to the membrane interface. Specific mutations of EF-1 residues in full-length PLC δ1 reduce enzyme activity but not because of disturbing partitioning of the protein onto vesicles. The reduction in enzymatic activity coupled with vesicle binding studies are consistent with a role for this domain in aiding substrate binding in the active site once the protein is transiently anchored at its target membrane.

Indexation of cerebral cell membrane phospholipid catabolism by the non-invasively determined cerebral 31-phosphorus neurospectroscopic phosphodiester peak

Breakdown of mammalian cerebral cell membrane phospholipids releases phosphorylated polar head groups from the sn-3 phospholipid position, including phosphorylcholine and phosphorylethanolamine. Glycerophosphorylcholine and glycerophosphorylethanolamine are on their catabolic pathways and have been assigned to the phosphodiester narrow resonance obtained from 31-phosphorus neurospectroscopy, accounting for approximately 38% of the overall signal; therefore in human in vivo 31-phosphorus neurospectroscopy neuropsychiatric studies this narrow resonance has been used to index the catabolism of cerebral cell membrane phospholipids non-invasively. However, for ethical reasons direct assessment of this assumption has not hitherto been possible in humans. Recently, it has become possible to analyze signal directly from the cell membrane motion-restricted phospholipids by analysis of a broad resonance signal. It was therefore hypothesized that there should be a negative correlation between the phosphodiester narrow resonance and the broad resonance signal if the former does indeed index cell membrane phospholipid catabolism. Cerebral 31-phosphorus magnetic resonance spectroscopy was carried out in 54 human subjects (mean age 38 years; 41 male), including normal volunteers and patients with schizophrenia, in order potentially to widen the range of phosphodiester and broad resonance values. Spectra were obtained from 70 × 70 × 70 mm 3 voxels using an image-selected in vivo spectroscopy pulse sequence. There was a highly significant negative correlation between the phosphodiester resonances and the broad resonance signals ( r = −0.509, P < 0.0001). This result is consistent with the hypothesis that the phosphodiester narrow resonance does index cell membrane phospholipid catabolism in non-invasive human neuropsychiatric studies.

Skeletal muscle metabolism in individuals with spinal cord injury

With increasing survival rates in people with spinal cord injuries (SCI), detection and prevention of metabolic and cardiovascular disease have become increasingly important. Few studies have evaluated in vivo mitochondrial function in paralyzed skeletal muscle. The purpose of this study was to compare oxidative muscle metabolism using the rate of phosphocreatine (PCr) resynthesis measured by magnetic resonance spectroscopy (MRS) in people with SCI and able-bodied (AB) controls. Eight subjects with complete SCI (American Spinal Injury Association Impairment Scale A, levels T3-T12, injury duration 2-13 years) were compared with 12 AB controls. T1-weighted (1)H MR images of the thigh were taken to identify skeletal muscle. Phosphorous MRS was performed with a 13 × 13-cm(2) surface coil placed on the right vastus lateralis in a 3 Tesla clinical MRI scanner. PCr resynthesis was measured after electrical stimulation for 60 s at 4 Hz in SCI and AB and in AB subjects after 39 s of voluntary isometric contractions. Resting metabolites were not different between SCI and AB, except for an elevated phosphodiester peak. PCr recovery was slower in AB subjects using electrical stimulation compared with voluntary exercise (28.4 ± 6.1 vs. 41.5 ± 4.3 s; P < 0.05). PCr recovery rates and calculated muscle maximum oxidative capacity in SCI were both 52% of electrically stimulated AB (P < 0.001). In vivo oxidative metabolism was reduced in paralyzed muscle to a similar extent as seen in people with mitochondrial myopathies and heart failure.

Evidence from in vivo 31-phosphorus magnetic resonance spectroscopy phosphodiesters that exhaled ethane is a biomarker of cerebral n-3 polyunsaturated fatty acid peroxidation in humans

BackgroundThis study tested the hypothesis that exhaled ethane is a biomarker of cerebral n-3 polyunsaturated fatty acid peroxidation in humans. Ethane is released specifically following peroxidation of n-3 polyunsaturated fatty acids. We reasoned that the cerebral source of ethane would be the docosahexaenoic acid component of membrane phospholipids. Breakdown of the latter also releases phosphorylated polar head groups, giving rise to glycerophosphorylcholine and glycerophosphorylethanolamine, which can be measured from the 31-phosphorus neurospectroscopy phosphodiester peak. Schizophrenia patients were chosen because of evidence of increased free radical-mediated damage and cerebral lipid peroxidation in this disorder.MethodsSamples of alveolar air were obtained from eight patients and ethane was analyzed and quantified by gas chromatography and mass spectrometry (m/z = 30). Cerebral 31-phosphorus spectra were obtained from the same patients at a magnetic field strength of 1.5 T using an image-selected in vivo spectroscopy sequence (TR = 10 s; 64 signal averages localized on a 70 × 70 × 70 mm3 voxel). The quantification of the 31-phosphorus signals using prior knowledge was carried out in the temporal domain after truncating the first 1.92 ms of the signal to remove the broad component present in the 31-phosphorus spectra.ResultsThe ethane and phosphodiester levels, expressed as a percentage of the total 31-phosphorus signal, were positively and significantly correlated (rs = 0.714, p < 0.05).ConclusionOur results support the hypothesis that the measurement of exhaled ethane levels indexes cerebral n-3 lipid peroxidation. From a practical viewpoint, if human cerebral n-3 polyunsaturated fatty acid catabolism can be measured by ethane in expired breath, this would be more convenient than determining the area of the 31-phosphorus neurospectroscopy phosphodiester peak.

Evidence from in vivo 31-phosphorus magnetic resonance spectroscopy phosphodiesters that exhaled ethane is a biomarker of cerebral n-3 polyunsaturated fatty acid peroxidation in humans

This study tested the hypothesis that exhaled ethane is a biomarker of cerebral n-3 polyunsaturated fatty acid peroxidation in humans. Ethane is released specifically following peroxidation of n-3 polyunsaturated fatty acids. We reasoned that the cerebral source of ethane would be the docosahexaenoic acid component of membrane phospholipids. Breakdown of the latter also releases phosphorylated polar head groups, giving rise to glycerophosphorylcholine and glycerophosphorylethanolamine, which can be measured from the 31-phosphorus neurospectroscopy phosphodiester peak. Schizophrenia patients were chosen because of evidence of increased free radical-mediated damage and cerebral lipid peroxidation in this disorder. Samples of alveolar air were obtained from eight patients and ethane was analyzed and quantified by gas chromatography and mass spectrometry (m/z = 30). Cerebral 31-phosphorus spectra were obtained from the same patients at a magnetic field strength of 1.5 T using an image-selected in vivo spectroscopy sequence (TR = 10 s; 64 signal averages localized on a 70 × 70 × 70 mm3 voxel). The quantification of the 31-phosphorus signals using prior knowledge was carried out in the temporal domain after truncating the first 1.92 ms of the signal to remove the broad component present in the 31-phosphorus spectra. The ethane and phosphodiester levels, expressed as a percentage of the total 31-phosphorus signal, were positively and significantly correlated (r s = 0.714, p < 0.05). Our results support the hypothesis that the measurement of exhaled ethane levels indexes cerebral n-3 lipid peroxidation. From a practical viewpoint, if human cerebral n-3 polyunsaturated fatty acid catabolism can be measured by ethane in expired breath, this would be more convenient than determining the area of the 31-phosphorus neurospectroscopy phosphodiester peak.

Long-term observation of in vivo 31P NMR spectra in carbon tetrachloride-intoxicated rabbit liver using implanted wireless surface coil.

Phosphorus-31 NMR spectra in rabbit liver were successively observed for more than 5 months in a chronic CCl4 intoxication model using implanted wireless surface coils. The quality of the obtained NMR spectra was satisfactory and there was sufficient time resolution to follow the dynamic changes that occurred during a fructose tolerance test. The implanted coil did not lead to any serious adverse effects such as infection or liver dysfunction. During an 18-week administration of CCl4, the area of the phosphodiester peak significantly decreased. The ratio of P(i)/beta-ATP was transiently elevated and gradually returned to the control level. In the fructose tolerance test, the increase in phosphomonoester and the decrease in P(i) and beta-ATP after i.v. fructose loading were apparent, but the degrees in these changes became smaller during the CCl4 intoxication. This wireless surface coil was useful for consecutive and non-invasive 31P NMR observations of the liver to follow chronically treated animal models over a period of several months.

Phosphorus metabolism during growth of lymphoma in mouse liver: a comparison of 31P magnetic resonance spectroscopy in vivo and in vitro

Large phosphomonoester (PME) signals are detected in the phosphorus magnetic resonance spectra (31P MRS) of many neoplastic and rapidly dividing tissues. In addition, alterations in phosphodiester (PDE) signals are sometimes seen. The present study of a murine lymphoma growing in liver showed a positive correlation between the hepatic PME/PDE ratio measured in vivo by 31P MRS at 4.7 T and the degree of lymphomatous infiltration in the liver, quantified by histology. High-resolution 31P MRS of liver extracts at 9.7 T showed that the PME peak consists largely of phosphoethanolamine (PE) and to a lesser extent of phosphocholine (PC). The concentration of both PE and PC increased positively with lymphomatous infiltration of the liver. In vivo, the PDE peak contains signals from phospholipids (mostly phosphatidylethanolamine and phosphatidylcholine) and the phospholipid breakdown products glycerophosphoethanolamine (GPE) and glycerophosphocholine (GPC). Low levels of GPE and GPC were detected in the aqueous extracts of the control and infiltrated livers; their concentrations remained unchanged as the infiltration increased. The total concentration of phospholipids measured by 31P MRS of organic extracts decreased about 3-fold as the infiltration increased to 70%. Thus, our data showed that the increased PME/PDE ratio in vivo is due to both an increase in the PME metabolites and a decrease in the PDE metabolites. We propose that this ratio can be used as a non-invasive measure of the degree of lymphomatous infiltration in vivo.

Metabolic changes in the rat brain after acute and chronic ethanol intoxication: A 31P NMR spectroscopy study

In this work, 31P phosphorus NMR (31P NMR) studies of the brain have been conducted in rats acutely and chronically intoxicated with ethanol. In both groups, changes in levels of high-energy phosphates were observed: increase of phosphocreatinine (PCr)/beta AaTP and PCr/inorganic phosphate (Pi) in acute and long-term ethanol exposure, and decrease of Pi/beta ATP after acute ethanol administration. These changes in high-energy phosphates, indicative of a reduction of adenosine triphosphate (ATP) and PCr consumption (PCr+ ADP+ H+ ATP+ Cr; ATP ADP+ Pi), suggest a reduction of cerebral metabolism both in acute and chronic ethanol exposure. In addition, in the group of rats chronically intoxicated with ethanol, there were variations in phosphodiester peak intensities (decrease of phosphomonoester (PME)/phosphodiester (PDE), increase of PDE/beta ATP), suggesting increased breakdown of membrane phospholipids. These changes could provide a metabolic explanation for the development of cerebral atrophy in chronic alcoholism.

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.

Endoplasmic reticulum: the major contributor to the PDE peak in hepatic 31P-NMR spectra at low magnetic field strengths

31P-NMR spectra of liver in vivo, subcellular fractions and model systems were acquired in order to characterise further the hepatic phosphodiester peak seen at low magnetic field strengths previously shown to be predominantly due to phospholipid bilayers. The data obtained in this study in vitro suggested that the phospholipid membranes of the endoplasmic reticulum provide the dominant contribution to this phosphodiester peak. Support for this hypothesis was provided by experiments on rats. Phenobarbitone, which is known to induce proliferation of the endoplasmic reticulum produced a considerable increase in intensity of the phosphodiester peak in liver spectra in vivo.

Nuclear magnetic resonance spectroscopy of excised human hearts.

Phosphorus nuclear magnetic resonance spectroscopy has been proposed as a method of studying the metabolism of the myocardium in patients. Little is known about 31P nuclear magnetic resonance spectroscopy of diseased human hearts. Two donor hearts meeting the requirements for heart transplantation and 11 diseased hearts were removed during a transplantation procedure and were studied in a horizontal 2.35 T superconducting magnet. Spectra were obtained at 0 degrees C about 30 minutes after the excision. The areas of the inorganic phosphate peak (Pi) and of the phosphocreatine peak (PCr) were summed and expressed as a ratio with respect to the area of the beta ATP peak. The ratio (Pi + PCr)/beta ATP was found to be significantly lower in five hearts with a myocardial infarct (0.77 (0.18)) than in hearts with dilated cardiomyopathy (1.25 (0.29)) and in normal hearts (1.69 (0.11)). The area of the phosphodiester peak was expressed as a ratio with respect to the area of the beta ATP peak: no differences were found between the three groups. These results suggest that the phosphocreatine concentration is lower in ischaemic heart disease than in dilated cardiomyopathy and that the phosphodiester peak is probably not useful in distinguishing between these two types of heart disease.

Visibility of phospholipids in 31P NMR spectra of human breast tumours in vivo.

The aims of this study were two-fold: to characterize the in vivo 31P NMR spectrum of human breast tumours by identifying the metabolites contributing to each peak; and to demonstrate changes in the detectability of the phosphodiester (PDE) peak at varying field strengths. This was achieved by obtaining 31P spectra from 23 patients in vivo at 1.5 T and also of 11 perchloric acid (PCA) extracts and 3 chloroform-methanol (CM) extracts of tumour biopsy samples at 9.4 T. In spectra acquired in vivo the percentage areas for each peak were: phosphomenoester (PME), 15%; Pi, 11%; PDE 35%; phosphocreatine, 5%; gamma-NTP, 11%; alpha-NTP, 11% and beta-NTP, 12% of the total spectral area. PCA extracts showed the PME peak to be greater than 70% phosphorylethanolamine whilst the PDE peak included almost equal proportions of glycerophosphorylethanolamine and glycerophosphorylcholine. CM extracts of tumours revealed additional metabolites in the PDE region suggesting that the large PDE peak observed in vivo could arise mainly from phospholipids. Spectra of human breast tumour xenografts examined in vivo at both 1.9 T and 9.4 T confirmed that the presence of a large PDE peak in vivo was a function of field strength. Further experiments with microsomal membranes from rat mammary tumours at 1.5 and 9.4 T demonstrated that phospholipids are more clearly visible at the lower field strength due to a substantial decrease in linewidth.

Infrared spectroscopy of exfoliated human cervical cells: evidence of extensive structural changes during carcinogenesis.

Infrared spectra were obtained from exfoliated cervical cells from 156 females, of whom 136 were normal, 12 had cervical cancer, and 8 had dysplasia. The spectra of the normal women, essentially identical, differed from those obtained from patients with either cancer or dysplasia. In malignant samples we noted (i) significant changes in the intensity of the glycogen bands at 1025 cm-1 and 1047 cm-1, the bands at 1082 cm-1 and 1244 cm-1, the C--O stretching band at 1155 cm-1, and the band at 1303 cm-1, (ii) significant shifts of the peaks normally appearing at 1082 cm-1, 1155 cm-1, and 1244 cm-1, and (iii) an additional band at 970 cm-1. Further study of several of these bands, including the pressure dependence of their frequencies, revealed that in the malignant cervical tissue there were extensive changes in the degree of hydrogen bonding of phosphodiester groups of nucleic acids and C--OH groups of proteins, as well as changes in the degree of disorder of methylene chains of lipids. The IR spectra of samples with dysplasia demonstrated the same changes with cancer samples, except that the changes were of lesser magnitude and the phosphodiester peak normally appearing at 1082 cm-1 did not shift. These spectroscopic changes appear to progress in tandem with the morphological changes that lead normal cervical epithelium to cancer through the premalignant stage of dysplasia. The diagnostic potential of IR spectroscopy is discussed.

Biochemical changes within a multiple sclerosis plaque in vivo.

Magnetic resonance spectroscopy (MRS) was performed on a superficial part of the brain containing a large multiple sclerosis (MS) lesion. Reduced levels of phosphocreatine (PCr) relative to adenosine triphosphate (ATP) were found suggesting an abnormality in energy metabolism, with an increase in the phosphodiester (PDE) peak. A follow up study 16 months later revealed reduction in size of the lesion on MRI and normal intracellular biochemistry by MRS. Four MS patients without visible superficial cerebral lesions showed no significant changes in phosphorus metabolism.

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.

A comparison of in vivo and in vitro 31P NMR spectra from human breast tumours: variations in phospholipid metabolism

An in vivo 31P NMR spectrum was obtained from each of four human breast tumours. The phosphomonoester and phosphodiester region of each spectrum consisted of a broad peak. Chemical extracts from samples of each of the tumours obtained at resection were examined on a high field strength NMR system. The phosphomonoester region in the spectrum from each extract resolved into three peaks consisting of phosphocholine, phosphoethanolamine and a nucleoside monophosphate. The phosphodiester region resolved into two components, glycerophosphorylcholine and glycerophosphorylethanolamine. Comparing the in vivo and in vitro data from each tumour showed that the contribution of phosphodiester was much lower in the in vitro spectra. We believe this to be a consequence of phospholipid, which would not appear in the aqueous extract, contributing to the phosphodiester peak in vivo.

Characterization of astrocytomas, meningiomas, and pituitary adenomas by phosphorus magnetic resonance spectroscopy

Phosphorus magnetic resonance (MR) spectroscopy allows noninvasive measurement of phosphate-containing compounds and pH within brain cells. The authors obtained localized phosphorus MR spectra from 10 normal brains, four low-grade astrocytomas, six glioblastomas, four meningiomas, and three pituitary adenomas and found differences in the spectra of each tumor type. Compared to normal brain, the spectra from low-grade astrocytomas showed a significant reduction of the phosphodiester (PDE) peak. Glioblastomas were characterized by a significant reduction of the PDE peak, elevation of the phosphomonoester (PME) peak, and a relatively alkaline intracellular pH. The spectra from meningiomas and pituitary adenomas were markedly different from the glial tumors. Meningiomas showed significant reductions in phosphocreatine, PDE, and inorganic phosphate, as well as a relatively alkaline pH. Pituitary adenomas resembled meningiomas, but had a much higher PME peak. Although the number of tumors studied was small, there appears to be a characteristic spectrum associated with these different tumor types. The present findings can be useful in the preoperative identification of these tumors and in furthering understanding of their growth and metabolism in vivo.

Spin-spin relaxation of the phosphodiester resonance in the 31P NMR spectrum of human brain : The determination of the concentrations of phosphodiester components

The phosphodiester peak in 31P nuclear magnetic resonance spectra of human brain in vivo is often the most prominent feature of the spectrum. We have demonstrated that this resonance exhibits bi-exponential spin-spin relaxation, giving relaxation times of 2 and 10 ms. We interpret this in terms of the two components which make up the peak, bilayer lipids and the small cytosolic phosphates glycerophosphoethanolamine and glycerophosphocholine. Using the relaxation times and the relative peak heights of the two components we have also been able to quantitate the concentration of the bilayer lipids as 20–40 times that of ATP.