Metabolomics/Analytical Methods/NMR/P NMR

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Examining the Metabolic Processes Underpinning Critical Swimming in Atlantic cod (Gadus morhua L.) using in vivo 31P NMR Spectroscopy

In a recent study, in vivo 31P NMR spectroscopy was used in the examination of the metabolic processes underpinning critical swimming in Atlantic cod Critical swimming speed (Ucrit) is identified as the speed at which a fish can no longer propel itself forward and becomes exhausted. 31P-NMR spectroscopy was used in combination with a Brett-type swim tunnel in order to perform a detailed analysis of the processes occurring during a Ucrit swim test.

The Ucrit swim test was originally intended for analyzing the dependence of salmon fitness on temperature. Brett defined this critical speed as the swimming velocity of a fish that could no longer propel itself forward as a result of exhaustion. Previous analyses of different swimming modes and metabolic processes that fuel muscle contractions in fish have established that red muscle is oxidative and produces slow contractions during subcarangiform swimming, while white muscle is glycolytic and responsible for fast twitch contractions, which produce tail kicks during burst swimming. Increases in inorganic phosphate and acidification of the intracellular milieu were observed during the muscular fatigue that occurs during the critical swimming speed, but the exact processes involved have not been established. However, the products of anaerobic kick-and-glide – fast twitch – bursts leave products to indicate the metabolic state of the fish. These products have allowed researches to examine the relationship between swimming gait and metabolic processes. Although the Ucrit swim test provides information about the swimming performance of Atlantic cod and metabolic products from Atlantic cod muscles during the Ucrit, they do not elaborate on the underlying processes such as the causes of fatigue during this speed.

In vivo 31P NMR spectroscopy has been previously established as an efficient method for non-invasively monitoring intracellular pH changes in muscle and the metabolic statuses of phosphorous based compounds such as ATP, phosphocreatine (PCr) and intracellular phosphate (Pi). Although previous studies were limited to analyzing resting or restrained animals, there have been increasingly developed techniques for non-invasively examining unrestrained fish under resting conditions. In this study, in vivo 31P NMR proved to be a practical and effective method for analyzing the metabolic processes of Atlantic cod during Ucrit tests.

First generation cultured North Eastern Arctic cod Gadus morhua L. were held at 4ºC for a minimum of two weeks prior to being moved to a 10ºC aquarium and were fed to satiation twice a week. Feeding was halted for five to seven days prior to experimentation.

The swim tunnel apparatus comprised three major constituents: A Perspex™ pipe was attached to a circulatory system and was fed through a 40 cm inner diameter Bruker Biospec 47/40 that operated at 4.7 T. When the pipe was closed, seawater was hermetically sealed to measure oxygen consumption. When open, a reservoir of constantly aerated seawater was used to flush the system. Three large taps had been used to alternate between the two circulations. The hermetic seal of the closed system was periodically checked by purging the seawater with nitrogen gas overnight and then monitoring the oxygen content of seawater over a twelve hour period. There were no detected increases in oxygen content, during the course of the experiment. A digital motion camera system was connected to a computer and used to monitor the fish. Circulations were temperature controlled and all swimming speeds were corrected for solid blocking effects. A substantial amount of pioneer studies in 31P-NMR involved freshwater fish studies. Early studies were conducted exclusively on resting or restrained animals, but recent innovations have allowed for the non-invasive analysis of unrestrained fish under resting conditions, such as the experimental procedures followed in this study.

In vivo 31P-NMR spectra were prominently obtained from white muscle with miniscule contribution from red muscle. Spectra were collected using a 200 μs bp32 pulse with a flip angle of 45º, sweep width of 5000 Hz at 4 k and a repetition time of 0.8 s. A total of 256 scans were collected, which resulted in an overall acquisition time of approximately three minutes. NMR spectra were recorded over a 30 min. time period, for each swimming speed.

Experimental findings indicated that a metabolic transition point occurred as the cod approached Ucrit. This transition was from steady state aerobic metabolism to anaerobic metabolism, which occurred simultaneously as the fish changed gait from subcarangiform to kick-and-glide swimming. Metabolic processes that underpin the swimming transition from slow swimming to kick-and-glide and then to complete fatigue were measured online and established the Ucrit as the point of complete fatigue; as Ucrit was approached, oxygen consumption and tail-beat frequency increased. It was determined that the observed muscular fatigue was a result of ΔG/dξATP dropping to –49.8±0.7•kJ•mol–1. This value appeared to fall below a specific threshold value that was required by transporters to maintain ion gradients and fuel muscles.

It was observed that as Ucrit began, Pi started to increase while the pHi began to acidify. Researchers concluded that determining when the pHi decreased (at or before Ucrit) would be crucial information for evaluating the transition from steady state aerobic metabolism to non-steady state anaerobic metabolism. It had been previously hypothesized that physiological accumulations of Pi are responsible for decreases in power generated in muscles as Pi moves into the sarcoplasmic reticulum to precipitate calcium ions and reduce steady state tension. Mean Pi concentration at Ucrit was elevated, which led to increased force re-development during rapid contraction-relaxation cycles. These results were indicative of kick-and-glide bursts while swimming. In addition, detected drops in pHi resulted in increased excitability of working skinned muscle fibres. It was established that as Pi increased and PCr decreased, cellular energy stores were depleted at Ucrit in order to buffer cellular ATP concentrations. Consequential drops in pHi indicated that anaerobic metabolism was used to maintain ATP levels.

Altogether, researchers concluded that in vivo 31P-NMR spectroscopy combined with a Brett-type swim tunnel was an efficient method for analyzing metabolic traits of Atlantic cod during the critical swimming velocity. With this method, it was determined that as swimming speed increased, and gait was changed from subcarangiform to kick-and-glide swimming prior to the critical swimming speed, intracellular pHi decreased during an exponential increase of oxygen consumption. Simultaneously, PCr levels had dropped while Pi levels increased. These changes had maximized at Ucrit and were restored during recovery. The Gibbs free energy change of ATP hydrolysis was minimal at Ucrit, and was determined to be the most significant factor leading to exhaustion in the cod. This implied that a transition from steady state aerobic metabolism to non-steady state anaerobic metabolism ultimately led to the complete exhaustion of aerobic and anaerobic fuel reserves, at the critical swimming velocity.

Bock, C.H.; Lurman, G.J.; Portner, H.O. An examination of the metabolic processes underpinning critical swimming in Atlantic cod (Gadus morhua L.) using in vivo 31P-NMR spectroscopy. J Exp Biol. 2007, 210(Pt 21), 3749-56.

31P MR Spectroscopy in Assessment of Response to Antiviral Therapy for Hepatitis C Virus-Related Liver Disease

31P MR spectroscopy has been recruited to assess responses to antiviral therapy for hepatitis C virus-related liver disease. The purpose of this study was to assess the implementation of 31P-NMR as a biomarker of response to interferon and ribavirin treatment. Interferon and ribavirin have been proven to eradicate the virus in at least half of recorded cases, preventing progression of HCV infection to cirrhosis. Researchers used 31P-NMR spectroscopy to observe increases in the ratio of phosphomonoester (PME) to phosphodiester (PDE) of the liver with increasing severity of hepatitis C-related disease.

An estimated 3% of the global population has chronic infection with the hepatitis C virus (HCV), while approximately four million people are newly infected, each year. In approximately 20% of cases, fibrosis develops into cirrhosis, which leads to hepatocellular cancer in 5% of cases each year. Liver biopsies serve as the reference standards for staging and grading of chronic liver disease. However, this is an invasive procedure with a low mortality rate but high error rate, which is prominently caused by undersampling. This has compelled researchers to develop more effective noninvasive methods for evaluating liver damage. One such technique for characterizing chronic liver disease is 31P-NMR spectroscopy. In the past, this method proved to be an efficient means for determining disease severity.

Forty-seven patients (29 men and 18 women with a mean age of 47.5 years) with biopsy-proven HCV-related liver disease underwent viral eradication treatment with interferon and ribavirin, and were enrolled in routine clinical protocols in the United Kingdom. All patients experienced baseline liver biopsies in order to assess the severity of hepatic inflammation before any decision was made to follow through with antiviral treatment. All patients also underwent baseline pretreatment with 31P MR spectroscopy within three months of commencing viral eradication treatment. After treatment began, all patients underwent repeated imaging every six months for up to eighteen months. All patients were also administered with interferon α and ribavirin or pegylated interferon α and ribavirin treatment for at least twelve weeks.

Hepatic 31P MR spectra were obtained with a 1.5 T MRI unit with an enveloping transmitter coil and separate surface receiver coil. Both coils were double-turned for protons at 64 MHz and phosphorus at 26 MHz. A T1-weighted image confirmed patient positioning and was obtained with the proton signals. Spectra were localized to a centrally placed voxel within the liver with an image-selected in vivo spectroscopy sequence. A voxel location within the right liver and away from major vessels had been used for each patient and was consistent for all baseline and follow-up images. Total examination time lasted forty minutes with ten-minute acquisition periods, for each 31P MR sequence. All imaging was conducted overnight.

Quantitation of 31P signals were recorded with respect to time with the Advanced Method for Accurate, Robust, and Efficient Spectral fitting (AMARES) algorithm. This algorithm was in the Magnetic Resonance User Interface (MRUI) software program. Blinded observers analyzed and rechecked MR spectra in order to assure anonymity. PME, PDE and inorganic phosphate (Pi) peak areas were obtained with respect to total phosphorous signal intensity.

Patients were classified as responders to viral eradication treatment on the basis of sustained viral clearance that was obtained from quantitative HCV RNA polymerase chain reaction studies. Researchers defined a sustained virologic response as repeatedly undetectable HCV RNA measured at least six months after finishing antiviral treatment.

Evaluation and treatment of increasing numbers of patients with HCV infection has developed into a major clinical concern. Although biopsies remain the reference standard for the staging and grading of most cases of HCV-related liver disease, there are still considerable risks for this procedure. It has also become increasingly evident that liver biopsies produce results that are less accurate than those obtained with 31P-NMR methods. One of the more significant advantages of 31P MR spectroscopy over liver biopsies is its noninvasive application and substantially larger sampling of the liver – many times larger than liver biopsy samples. Treatment with interferon-α or pegylated interferon-α and ribavirin was proven to be an efficient form of viral eradication therapy for patients with HCV-related liver disease via 31P MR. Viral clearance with this method occurred in at least half of all patients, but was dependent upon the underlying genotype. PME/PDE ratio determinations via 31P MR spectroscopy have proven to be significant indicators of cirrhosis. In this study, a significant decrease in the PME/PDE ratio toward normal was observed in the sustained virologic responder group. However, this ratio remained static or increased in patients who were virologic nonresponders. PME resonance had also contained contributions from cell membrane precursors, while PDE resonance contained contributions from cell membrane degradation complexes. These features allowed the PME/PDE ratio to provide information on liver cell turnover. With this knowledge, researchers were surprised to observe in a reduction of this ratio after effective viral eradication treatment and within three of four responders in the cirrhosis group.

31P MR spectroscopic assessment of PME/PDE ratios was not without faults. Specifically, three patients from this study were discovered to have a PME/PDE ratio reduction, which suggested attenuation of liver disease. This was contrary to findings that revealed these patients to not have any sustained response, based upon results of longer-term follow-up virologic studies. Two patients were also discovered to have worsening PME/PDE ratios, although abatement of the virus was established with longer-term virologic follow-up studies. It seems that the most practical approach to assessing PME/PDE ratios in HCV-related liver disease patients would include long-term virologic follow-up studies. Researchers had also correlated histological findings at least twenty-four months after antiviral treatment, in order to secure the accuracy and reliability of the 31P MR spectroscopic method that was used. Another potential fault in this study could have resulted from excluding correlative serologic biomarkers of fibrosis. This led to difficulty during the follow-up procedures of the patients. To limit experimental error from excluding these biomarkers, a larger group of patients could have been used.

Altogether, it seems that PME/PDE ratio determinations via 31P MR spectroscopy likely serve as significant indicators of responses to antiviral treatment. In addition, 31P MR spectroscopy has proven to be an efficient substitution for liver biopsy in this assessment. Researchers stated that the MR spectroscopic sequence used for this study may be freely added to any standard MR liver imaging protocol. Although the sequence does lengthen the imaging protocol by approximately ten minutes, there is a notable increase in significant data pertinent to the metabolites in patients with hepatitis C.

Kuo, Y.T.; et al. 31P MR Spectroscopy in Assessment of Response to Antiviral Therapy for Hepatitis C Virus–Related Liver Disease. J Exp Biol. 2007, 189(4), 819-23.

Real-Time Noninvasive Assessment of Pancreatic ATP Levels During Cold Preservation

31P-NMR spectroscopy has been used to non-invasively monitor rat and porcine pancreatic ATP:Pi ratios. This method allowed investigators to simultaneously assess the efficacy of existing protocols for cold preservation (CP) in maintaining organ quality. In the rat pancreas model, it was observed that CP with histidine-tryptophan-ketoglutarate solution (HTK)/perfluorodecalin two-layer method (TLM) substantially improved ATP:Pi ratio levels when compared to CP in (HTK) exposed to air. When compared to TLM, preservation of rat pancreata in an HTK solution saturated with molecular oxygen resulted in a slightly greater ATP:Pi ratio improvement. As a result, TLM was determined to be effective for improving oxygenation and elevating ATP levels in the rat pancreas model. Although, when the TLM stored porcine pancreata was analyzed, researchers found no recognizable ATP peaks in the NMR spectra. This indicated that TLM was less effective in this model. These results support previous studies that indicate TLM cannot fully oxygenate larger porcine and human pancreata.

The increase in the ATP:Pi ratio over the first nine hours of CP with the TLM might have reflected reoxygenation dynamics and ATP regeneration that followed ischemia during procurement. In addition, the ATP:Pi ratio reduction after twelve hours of CP was consistent with the decreased isolation yields after eight to nine hours of CP. This was believed to indicate the presence of crucial, additional factors that limit CP. Researchers determined that the noninvasive capability of 31P-NMR to provide real time information allowed it to serve as efficient tool when identifying factors that limit CP as well as gauging and evaluating modern methods of organ preservation.

Avqoustiniatos, E.S.; et al. Real-Time Noninvasive Assessment of Pancreatic ATP Levels During Cold Preservation. Transplant Proc. 2008, 40(2), 403-6.

Effects of Lipid Chain Length and Unsaturation on Bicelles Stability: A Phosphorus NMR Study

31P NMR has been used to determine the effects of lipid chain length and unsaturation on bicelles stability. The majority of previous studies involving the magnetically alignable system formed by a binary mixture of long- and short-chain lipids were based on the mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (D14PC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (D6PC) lipids. Researchers have recently revealed that a substantial portion of the phase diagrams of this lipid mixture may be understood by considering partial miscibility of long-chain and short-chain lipids when heated above the melting transition temperature (Tm) of the long-chain lipids. When the chain lengths of either one of the two lipids were modified, researchers were capable of controlling their miscibility. This allowed manipulation of the intervals of temperature and composition where spontaneous alignment is observed in a magnetic field. Using 31P-NMR, researchers demonstrated that the unique properties of these binary lipid mixtures are correlated with the tendency for short-chain lipids to diffuse into bilayer regions. Researchers also established that lipid mixtures with comparable properties could be formed with unsaturated lipids such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC).

It was determined that the majority of the distinctive bicellular mixture properties are preserved when the lipid chain lengths of the long- and short-chain lipids are modified by one or two carbons. It was also discovered that these same properties were maintained among a large fraction of unsaturated lipids such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Experimental results indicated that the temperature domain where spontaneous alignment was observed in a magnetic field is contingent upon the lipids used. This temperature contingency was determined to be a result of the miscibility variation between lipids. At a given temperature, it was discovered that this miscibility depends upon mismatched lipid chain lengths, the main transition temperature of the long-chain lipid and the affinity of short-chain lipids for the edge sections. Researchers grasped an understanding of the underlying forces that are responsible for forming alignable lipidic structures. As a result, it is now possible to predict which lipid mixtures will align and to design mixtures that will align within a desired temperature range. These capabilities have proven to be especially useful in biological applications that include partial orientation of soluble proteins, membrane protein incorporation or drug encapsulation.

Devaux, P.F.; Triba, M.N.; Warschawski, D.E. Effects of Lipid Chain Length and Unsaturation on Bicelles Stability: A Phosphorus NMR Study. Biophys J. 2006, 91(4), 1357-67.

31P NMR has proven to be an efficient, noninvasive technique for monitoring metabolic systems, profiling the roles of ATP and other phosphorous containing compounds in biological systems, and observing the impact of particular stimuli on biological systems that utilize phosphorous containing compounds. Disease research appears to be a practical, up-and-coming application for 31P-NMR. In particular, 31P-NMR has already been applied to detailing the responses of antiviral therapy for hepatitis C virus-related liver disease and has also been used to profile phosphorous based compound activity in Alzheimer’s and McArdle’s disease. 31P NMR has also been recently applied to food analysis and appears to be a significant future application for profiling levels of phosphorous-based compounds in foods. It is also likely that this technique will have increased usage in future studies of metabolomics, which involves mapping human metabolic processes, the metabolites involved in those processes, and assessing the impact of stimuli on these processes. Current metabolomics research recruits 31P NMR spectroscopy in recording the toxic actions of dinoseb in medaka (Oryzias latipes) embryos. 31P NMR had been used to characterize ATP and phosphocreatine metabolism within the intact embryos.

Brown, G. G.; et al. In vivo 31P NMR profiles of Alzheimer's disease and multiple subcortical infarct dementia. Neurology. 1989, 39, 1423.

Lewis, S. F.; Haller, R. G.; Cook, J. D.; Nunnally, R. L. Muscle fatigue in McArdle's disease studied by 31P-NMR: effect of glucose infusion. J Appl Physiol. 1985, 59, 1991 – 1994.

Spyros, A.; Dais, P. Application of 31P NMR Spectrsoscopy in Food Analysis. 1. Quantitative Determination of the Mono- and Diglyceride Composition of Olive Oils. J Agric Food Chem. 2000, 48(3), 802 – 805.

Articles and Web Pages for Review and Inclusion
Peer-Reviewed Article #1:

Toxic actions of dinoseb in medaka (Oryzias latipes) embryos as determined by in vivo 31P NMR, HPLC-UV and 1H NMR metabolomics

Aquatic Toxicology Volume 76, Issues 3-4, 10 March 2006, Pages 329-342 

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