Absence of glycochenodeoxycholic acid (GCDCA) in human bile is an indication of cholestasis: A 1H MRS study

The utility of ¹H MR spectroscopy in detecting chronic cholestasis has been investigated. The amide proton region of the ¹H MR spectrum of human bile plays a major role in differentiating cholestatic (Ch) patterns from the normal ones. Bile obtained from normal bile ducts contains both taurine and glycine conjugates of bile acids – cholic acid (CA), chenodeoxycholic acid (CDCA), and deoxycholic acid (DCA). Absence of a glycine‐conjugated bile acid glycochenodeoxycholic acid (GCDCA) has been observed in bile samples obtained from primary sclerosing cholangitis (PSC) patients. A total of 32 patients with various hepatobiliary diseases were included in the study. Twenty‐one patients had PSC and 11 had normal cholangiograms. One PSC patient was excluded from the study because of a bad spectrum. Seventeen out of the 20 PSC patients showed an absence of GCDCA in their ¹H MR spectrum of bile. Six of the 11 reference patients with normal cholangiogram also showed spectra similar to those of PSC, indicating the possibility of cholestasis. DQF‐COSY and TOCSY experiments performed on bile samples from PSC patients also revealed absence of phosphatidylcholine (PC) in some of the bile samples, suggesting possible damage to the cholangiocytes by the toxic bile. These observations suggest that analysis of human bile by ¹H MRS could be of value in the diagnosis of chronic Ch liver disorders. Copyright © 2008 John Wiley & Sons, Ltd.     

Characterization of the peak at 2.06 ppm in 31P magnetic resonance spectroscopy of human liver: phosphoenolpyruvate or phosphatidylcholine?

High field MR scanners can resolve a metabolite resonating at 2.06 ppm in the in vivo proton‐decoupled liver ³¹P MR spectrum. Traditionally this peak has been assigned to phosphoenolpyruvate (PEP), the key metabolite for gluconeogenesis. However, recent evidence supported the assignment to biliary phosphatidylcholine (PtdCh), which is produced in the liver and stored in the gall bladder. To elucidate the respective contributions of PtdCh and PEP to the in vivo resonance at 2.06 ppm (PEP–PtdCh), we made phantom measurements that confirmed that both biliary PtdCh and PEP resonate approximately at 2 ppm. The absolute quantification of PEP–PtdCh yielded concentrations ranging from 0.6 to 2.0 mmol/l, with mean coefficients of variation of 4.8% for intraday and 7.2% for interday reproducibility in healthy volunteers. The T₁ relaxation time of PEP–PtdCh was 0.97 ± 0.30 s in the liver and 0.44 ± 0.11 s in the gallbladder. Ingestion of a mixed meal decreased the concentration of PtdCh‐PEP by approximately 12%. In the retrospective analysis, PEP–PtdCh was 68% higher in the liver of subjects with gallbladder infiltration of the volume of interest (VOI) compared with those without gallbladder infiltration. PEP–PtdCh was also significantly higher in the liver of cholecystectomy patients compared with volunteers without gallbladder infiltration, which suggests increased intrahepatic bile fluid as a compensation for gall bladder removal. These results show that liver PtdCh is the major component of the resonance at 2.06 ppm and that careful VOI positioning is mandatory to avoid interference from the gallbladder. Copyright © 2015 John Wiley & Sons, Ltd.     

Explanation of colon cancer pathophysiology through analyzing the disrupted homeostasis of bile acids

Background

The colon plays a key role in regulating the homeostasis of bile acids.

Aim

The present study aims to evaluate the influence of colon cancer towards the homeostasis of bile acids.

Methods

The free and conjugated bile acids were determined using ultraperformance LC (UPLC) coupled with ABI 4000 QTRAP triple quadrupole instruments.

Results

The results showed that the free bile acids in serum of patients with colon cancers tend to increase, and the conjugated bile acids tended to decrease, especially for taurolithocholate (TLCA) (p<0.001).

Conclusion

The alteration of bile acids balance in colon cancers indicated the possibility of complicated diseases due to the disrupted balance of bile acids.     

N‐acetyl resonances in in vivo and in vitro NMR spectroscopy of cystic ovarian tumors

An unassigned and prominent resonance in the region from δ 2.0–2.1 ppm has frequently been found in the in vivo MR spectra of cancer patients. We demonstrated the presence of this resonance with in vivo MRS in the cyst fluid of a patient with an ovarian tumor. ¹H‐NMRS on the aspirated cyst fluid of this patient confirmed the observation. A complex of resonances was observed between 2.0 and 2.1 ppm. It was also present in 11 additional ovarian cyst fluid samples randomly chosen from our biobank. The resonance complex was significantly more prominent in samples from mucinous tumors than in samples from other histological subtypes. A macromolecule (>10 kDa) was found responsible for this complex of resonances. A correlation spectroscopy (COSY) experiment revealed cross peaks of two different types of bound sialic acid suggesting that N‐glycans from glycoproteins and/or glycolipids cause this resonance complex. In the literature, plasma α‐1 acid glycoprotein (AGP), known for its high content of N‐linked glycans, has been suggested to contribute to the δ 2.0–2.1 spectral region. The AGP cyst fluid concentration did not correlate significantly with the peak height of the δ 2.0–2.1 resonance complex in our study. AGP may be partly responsible for the resonance complex but other N‐acetylated glycoproteins and/or glycolipids also contribute. After deproteinization of the cyst fluid, N‐acetyl‐L ‐aspartic acid (NAA) was found to contribute significantly to the signal in this spectral region in three of the 12 samples. GC‐MS independently confirmed the presence of NAA in high concentration in the three samples, which all derived from benign serous tumors. We conclude that both NAA and N‐acetyl groups from glycoproteins and/or glycolipids may contribute to the δ 2.0–2.1 ppm resonance complex in ovarian cyst fluid. This spectral region seems to contain resonances from biomarkers that provide relevant clinical information on the type of ovarian tumor. Copyright © 2009 John Wiley & Sons, Ltd.     

Investigation of breast cancer using two‐dimensional MRS

Proton (¹H) MRS enables non‐invasive biochemical assay with the potential to characterize malignant, benign and healthy breast tissues. In vitro studies using perchloric acid extracts and ex vivo magic angle spinning spectroscopy of intact biopsy tissues have been used to identify detectable metabolic alterations in breast cancer. The challenges of ¹H MRS in vivo include low sensitivity and significant overlap of resonances due to limited chemical shift dispersion and significant inhomogeneous broadening at most clinical magnetic field strengths. Improvement in spectral resolution can be achieved in vivo and in vitro by recording the MR spectra spread over more than one dimension, thus facilitating unambiguous assignment of metabolite and lipid resonances in breast cancer. This article reviews the recent progress with two‐dimensional MRS of breast cancer in vitro, ex vivo and in vivo. The discussion includes unambiguous detection of saturated and unsaturated fatty acids, as well as choline‐containing groups such as free choline, phosphocholine, glycerophosphocholine and ethanolamines using two‐dimensional MRS. In addition, characterization of invasive ductal carcinomas and healthy fatty/glandular breast tissues non‐invasively using the classification and regression tree (CART) analysis of two‐dimensional MRS data is reviewed. Copyright © 2008 John Wiley & Sons, Ltd.     

Enterohepatic bile-acid circulation in the pregnant cat

Pregnancy increases the risk of gallstones. The physiological changes responsible for this are not clearly demonstrated. Adjustments in the enterohepatic circulation of bile acids have earlier been studied in pregnancy by methods involving dilution of labelled bile acids. In the present study the bile-acid circulation was measured with direct drainage methods in pregnant animals and controls. It was found that the total bile-acid-pool size was reduced to 65% in the pregnant cat (P < 0.01) and there was a reduced accumulation of bile acids in the gallbladder after fasting 24 h (P < 0.01). Bile-acid synthesis by the liver was not reduced and the relation between water and bile-acid secretion by the liver was unchanged. It is concluded that, in the pregnant cat, the bile-acid-pool size is reduced due to a decreased accumulation of bile acids in the gallbladder and an increased interdigestive recycling rate of the bile-acid pool (P < 0.05). One possible explanation for the reduced accumulation of bile acids in the gallbladder is delayed emptying of the stomach, inducing a late refilling of the gallbladder after a meal.     

13C MRS of occipital and frontal lobes at 3 T using a volume coil for stochastic proton decoupling

Previously, we devised a novel strategy for in vivo ¹³C MRS using [2‐¹³C]glucose infusion and low‐power proton decoupling, and proposed that this strategy could be used to acquire ¹³C MR spectra from the frontal lobe of the human brain. Here, we demonstrate, for the first time, in vivo ¹³C MRS of human frontal lobe acquired at 3 T. Because the primary metabolites of [2‐¹³C]glucose can be decoupled using very‐low‐radiofrequency power, we used a volume coil for proton decoupling in this study. The homogeneous B₁ field of the volume coil was found to significantly enhance the decoupling efficiency of the stochastic decoupling sequence. Detailed specific absorption rates inside the human head were analyzed using the finite difference time domain method to ensure experimental safety. In vivo ¹³C spectra from the occipital and frontal lobes of the human brain were obtained. At a decoupling power of 30 W (time‐averaged power, 2.45 W), the spectra from the occipital lobe showed well‐resolved spectral resolution and excellent signal‐to‐noise ratio. Although frontal lobe ¹³C spectra were affected by local B₀ field inhomogeneity, we demonstrated that the spectral quality could be improved using post‐acquisition data processing. In particular, we showed that the frontal lobe glutamine C5 at 178.5 ppm and aspartate C4 at 178.3 ppm could be spectrally resolved with effective proton decoupling and B₀ field correction. Because of its large spatial coverage, volume coil decoupling provides the potential to acquire ¹³C MRS from more than one brain region simultaneously. Copyright © 2010 John Wiley & Sons, Ltd.     

Assessment of early docetaxel response in an experimental model of human breast cancer using DCE‐MRI,ex vivo HR MAS,and in vivo 1H MRS

The purpose of this study was to evaluate the use of dynamic contrast‐enhanced (DCE) MRI, in vivo ¹H MRS and ex vivo high resolution magic angle spinning (HR MAS) MRS of tissue samples as methods to detect early treatment effects of docetaxel in a breast cancer xenograft model (MCF‐7) in mice. MCF‐7 cells were implanted subcutaneously in athymic mice and treated with docetaxel (20, 30, and 40 mg/kg) or saline six weeks later. DCE‐MRI and in vivo ¹H MRS were performed on a 7 T MR system three days after treatment. The dynamic images were used as input for a two‐compartment model, yielding the vascular parameters K^trans and v_e. HR MAS MRS, histology, and immunohistochemical staining for proliferation (Ki‐67), apoptosis (M30 cytodeath), and vascular/endothelial cells (CD31) were performed on excised tumor tissue. Both in vivo spectra and HR MAS spectra were used as input for multivariate analysis (principal component analysis (PCA) and partial least squares regression analysis (PLS)) to compare controls to treated tumors. Tumor growth was suppressed in docetaxel‐treated mice compared to the controls. The anti‐tumor effect led to an increase in K^trans and v_e values in all the treated groups. Furthermore, in vivo MRS and HR MAS MRS revealed a significant decrease in choline metabolite levels for the treated groups, in accordance with reduced proliferative index as seen on Ki‐67 stained sections. In this study DCE‐MRI, in vivo MRS and ex vivo HR MAS MRS have been used to demonstrate that docetaxel treatment of a human breast cancer xenograft model results in changes in the vascular dynamics and metabolic profile of the tumors. This indicates that these MR methods could be used to monitor intra‐tumoral treatment effects. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of hepatic fatty acids in mice with reduced liver fat by ultra‐short echo time 1H‐MRS at 14.1 T in vivo

Alterations in the hepatic lipid content (HLC) and fatty acid composition are associated with disruptions in whole body metabolism, both in humans and in rodent models, and can be non‐invasively assessed by ¹H‐MRS in vivo. We used ¹H‐MRS to characterize the hepatic fatty‐acyl chains of healthy mice and to follow changes caused by streptozotocin (STZ) injection. Using STEAM at 14.1 T with an ultra‐short T_E of 2.8 ms, confounding effects from T₂ relaxation and J‐coupling were avoided, allowing for accurate estimations of the contribution of unsaturated (UFA), saturated (SFA), mono‐unsaturated (MUFA) and poly‐unsaturated (PUFA) fatty‐acyl chains, number of double bonds, PU bonds and mean chain length. Compared with in vivo ¹H‐MRS, high resolution NMR performed in vitro in hepatic lipid extracts reported longer fatty‐acyl chains (18 versus 15 carbons) with a lower contribution from UFA (61 ± 1% versus 80 ± 5%) but a higher number of PU bonds per UFA (1.39 ± 0.03 versus 0.58 ± 0.08), driven by the presence of membrane species in the extracts. STZ injection caused a decrease of HLC (from 1.7 ± 0.3% to 0.7 ± 0.1%), an increase in the contribution of SFA (from 21 ± 2% to 45 ± 6%) and a reduction of the mean length (from 15 to 13 carbons) of cytosolic fatty‐acyl chains. In addition, SFAs were also likely to have increased in membrane lipids of STZ‐induced diabetic mice, along with a decrease of the mean chain length. These studies show the applicability of ¹H‐MRS in vivo to monitor changes in the composition of the hepatic fatty‐acyl chains in mice even when they exhibit reduced HLC, pointing to the value of this methodology to evaluate lipid‐lowering interventions in the scope of metabolic disorders. Copyright © 2015 John Wiley & Sons, Ltd.     

Differences in gallstone structure in primary common bile duct lithiasis and gallbladder lithiasis

Summary Some differences between gallbladder lithiasis and primary common bile duct lithiasis are described. Microbiological cultures and biochemical analyses were carried out on the bile of two groups of patients: 27 suffering from gallbladder and 5 from primary common duct lithiasis. The microstructure and composition of gallstones were also examined by polarized light microscopy and X-ray diffraction. Women predominated in gallbladder lithiasis but not in primary common duct lithiasis group (P<0.05) and body weight was higher in the former group (P<0.02). Primary common duct lithiasis patients had a higher, although not significant, incidence of duodenal diverticulosis (P=0.15), and a higher incidence ofE. coli-positive cultures in bile (P<0.001). No significant difference in the biochemical composition of the bile was found between the groups. Brown pigment stones predominated in primary common duct lithiasis, while cholesterol stones did in gallbladder and secondary common duct lithiasis (P<0.0001). Stones formed in the gallbladder generally show linear, radial growths of cholesterol crystals, while those from the common duct present a polystratified, concentric deposition of microgranules composed mainly of pigmentary salts.These differences should be taken into account as additional criteria in the differential diagnosis between primary and secondary common duct lithiasis, as the classical criteria for diagnosing of the former greatly underestimate its actual incidence. The distinction between primary and secondary common duct lithiasis is of practical significance, since each entity requires different treatment.Abbreviations CBD common bile duct - CBDL common bile duct lithiasis - ERCP endoscopic retrograde cholangiopancreatography - GBL gallbladder lithiasis - HDL high density lipoproteins - PCBDL primary common bile duct lithiasis - SCBDL secondary common bile duct lithiasis - SGOT serum glutamic-oxalacetic transaminase - SGPT serum glutamic-pyruvic transaminase     

A quantitative comparison of metabolite signals as detected by in vivo MRS with ex vivo 1H HR‐MAS for childhood brain tumours

¹H MRS provides a powerful method for investigating tumour metabolism by allowing the measurement of metabolites in vivo. Recently, the technique of ¹H high‐resolution magic angle spinning (HR‐MAS) has been shown to produce high‐quality data, allowing the accurate measurement of many metabolites present in unprocessed biopsy tissue. The purpose of this study was to evaluate the agreement between the techniques of in vivo MRS and ex vivo HR‐MAS for investigating childhood brain tumours. Short‐TE (30 ms), single‐voxel, in vivo MRS was performed on 16 paediatric patients with brain tumours at 1.5 T. A frozen biopsy sample was available for each patient. HR‐MAS was performed on the biopsy samples, and metabolite quantities were determined from the MRS and HR‐MAS data using the LCModel? and TARQUIN algorithms, respectively. Linear regression was performed on the metabolite quantities to asses the agreement between MRS and HR‐MAS. Eight of the 12 metabolite quantities were found to correlate significantly (P < 0.05). The four worst correlating metabolites were aspartate, scyllo‐inositol, glycerophosphocholine and N‐acetylaspartate, and, except for glycerophosphocholine, this error was reflected in their higher Cramer–Rao lower bounds (CRLBs), suggesting that low signal‐to‐noise was the greatest source of error for these metabolites. Glycerophosphocholine had a lower CRLB implying that interference with phosphocholine and choline was the most significant source of error. The generally good agreement observed between the two techniques suggests that both MRS and HR‐MAS can be used to reliably estimate metabolite quantities in brain tumour tissue and that tumour heterogeneity and metabolite degradation do not have an important effect on the HR‐MAS metabolite profile for the tumours investigated. HR‐MAS can be used to improve the analysis and understanding of MRS data. Copyright © 2009 John Wiley & Sons, Ltd.     

J‐refocused 1H PRESS DEPT for localized 13C MR Spectroscopy

Proton point‐resolved spectroscopy (PRESS) localization has been combined with distortionless enhanced polarization transfer (DEPT) in multinuclear MRS to overcome the signal contamination problem in image‐selected in vivo spectroscopy (ISIS)‐combined DEPT, especially for lipid detection. However, homonuclear proton scalar couplings reduce the DEPT enhancement by modifying the spin coherence distribution under J modulation during proton PRESS localization. Herein, a J‐refocused proton PRESS‐localized DEPT sequence is presented to obtain simultaneously enhanced and localized signals from a large number of metabolites by in vivo ¹³C MRS. The suppression of J modulation during PRESS and the substantial recovery of signal enhancement by J‐refocused PRESS‐localized DEPT were demonstrated theoretically by product operator formalism, numerically by the spin density matrix simulations for different scalar coupling conditions, and experimentally with a glutamate phantom at various TEs, as well as a colza oil phantom. The application of the sequence for localized detection of saturated and unsaturated fatty acids in the calf bone marrow and skeletal muscle of healthy subjects yielded high signal enhancements simultaneously obtained for all components. Copyright © 2013 John Wiley & Sons, Ltd.     

Monitoring of gliomas in vivo by diffusion MRI and 1H MRS during gene therapy‐induced apoptosis: interrelationships between water diffusion and mobile lipids

The measurement of water diffusion by diffusion‐weighted MRI (DWI) in vivo offers a non‐invasive method for assessing tissue responses to anti‐cancer therapies. The pathway of cell death after anti‐cancer treatment is often apoptosis, which leads to accumulation of mobile lipids detectable by ¹H MRS in vivo. However, it is not known how these discrete MR markers of cell death relate to each other. In a rodent tumour model [i.e. ganciclovir‐treated herpes simplex thymidine kinase (HSV‐tk) gene‐transfected BT4C gliomas], we studied the interrelationships between water diffusion (Trace{D}) and mobile lipids during apoptosis. Water diffusion and water‐referenced concentrations of mobile lipids showed clearly increasing and interconnected trends during treatment. Of the accumulating ¹H MRS‐visible lipids, the fatty acid ? CH ?CH ? groups and cholesterol compounds showed the strongest associations with water diffusion (r² = 0.30; P < 0.05 and r² = 0.48; P < 0.01, respectively). These results indicate that the tumour histopathology and apoptotic processes during tumour shrinkage can be interrelated in vivo by DWI of tissue water and ¹H MRS of mobile lipids, respectively. However, there is considerable individual variation in the associations, particularly at the end of the treatment period, and in the relative compositions of the accumulating NMR‐visible lipids. The findings suggest that the assessment of individual treatment response in vivo may benefit from combining DWI and ¹H MRS. Absolute and relative changes in mobile lipids may indicate initiation of tumour shrinkage even when changes in tissue water diffusion are still small. Conversely, greatly increased water diffusion probably indicates that substantial cell decomposition has taken place in the tumour tissue when the ¹H MRS resonances of mobile lipids alone can no longer give a reliable estimate of tissue conditions. Copyright © 2008 John Wiley & Sons, Ltd.     

The in vivo J‐difference editing MEGA‐PRESS technique for the detection of n‐3 fatty acids

In this study, we present a method for the detection of n‐3 fatty acid (n‐3 FA) signals using MRS in adipose tissue in vivo. This method (called oMEGA‐PRESS) is based on the selective detection of the CH₃ signal of n‐3 FA using the MEGA‐PRESS (MEshcher–GArwood Point‐RESolved Spectroscopy) J‐difference editing technique. We optimized the envelope shape and frequency of spectral editing pulses to minimize the spurious co‐editing and incomplete subtraction of the CH₃ signal of other FAs, which normally obscure the n‐3 FA CH₃ signal in MR spectra acquired using standard PRESS techniques. The post‐processing of the individual data scans with the phase and frequency correction before data subtraction and averaging was implemented to further improve the quality of in vivo spectra. The technique was optimized in vitro on lipid phantoms using various concentrations of n‐3 FA and examined in vivo at 3 T on 15 healthy volunteers. The proportion of n‐3 FA estimated by the oMEGA‐PRESS method in phantoms showed a highly significant linear correlation with the n‐3 FA content determined by gas chromatography. The signal attributed to n‐3 FA was observed in all subjects. Comparisons with the standard PRESS technique revealed an enhanced identification of the n‐3 FA signal using oMEGA‐PRESS. The presented method may be useful for the non‐invasive quantification of n‐3 FA in adipose tissue, and could aid in obtaining a better understanding of various aspects of n‐3 FA metabolism. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantification of amounts and 13C content of metabolites in brain tissue using high‐ resolution magic angle spinning 13C NMR spectroscopy

Metabolic pathway mapping using ¹³C NMR spectroscopy has been used extensively to study interactions between neurons and glia in the brain. Established extraction procedures of brain tissue are time consuming and may result in degradation of labile substances. We examined the potential of mapping ¹³C‐enriched compounds in intact brain tissue using high‐resolution magic angle spinning (HR‐MAS) NMR spectroscopy. Sprague–Dawley rats received an intraperitoneal injection of [1,6‐¹³C]glucose, and 15 min later the animals were subjected to microwave fixation of the brain. Quantification of concentration and ¹³C labelling of metabolites in intact rat thalamus were carried out based on exogenous ethylene glycol concentrations measured from ¹H NMR spectra using an ERETIC (Electronic REference To access In vivo Concentrations) signal. The results from intact tissue were compared with those from perchloric acid‐extracted brain tissue. Amounts of ¹³C labelling at different positions (C2, C3 and C4) in glutamate, glutamine, γ‐aminobutyric acid and aspartate measured in either intact tissue or perchloric acid extracts were not significantly different. Proton NMR spectra were used for quantification of six different amino acids plus lactate, inositol, N‐acetylaspartate, creatine and phosphocreatine. Again, results were very similar when comparing the methods. To our knowledge, this is the first time quantitative ¹³C NMR spectroscopy measurements have been carried out on intact brain tissue ex vivo using the HR‐MAS technique. The results show that HR‐MAS ¹³C NMR spectroscopy in combination with ¹H NMR spectroscopy and the ERETIC method is useful for metabolic studies of intact brain tissue ex vivo. Copyright © 2008 John Wiley & Sons, Ltd.     

On the theoretical limits of detecting cyclic changes in cardiac high‐energy phosphates and creatine kinase reaction kinetics using in vivo 31P MRS

Adenosine triphosphate (ATP) is absolutely required to fuel normal cyclic contractions of the heart. The creatine kinase (CK) reaction is a major energy reserve reaction that rapidly converts creatine phosphate (PCr) to ATP during the cardiac cycle and at times of stress and ischemia, but is significantly impaired in conditions such as hypertrophy and heart failure. Because the magnitudes of possible in vivo cyclic changes in cardiac high‐energy phosphates (HEPs) during the cardiac cycle are not well known from previous work, this study uses mathematical modeling to assess whether, and to what extent, cyclic variations in HEPs and in the rate of ATP synthesis through CK (CK flux) could exist in the human heart, and whether they could be measured with current in vivo ³¹P MRS methods. Multi‐site exchange models incorporating enzymatic rate equations were used to study the cyclic dynamics of the CK reaction, and Bloch equations were used to simulate ³¹P MRS saturation transfer measurements of the CK reaction. The simulations show that short‐term buffering of ATP by CK requires temporal variations over the cardiac cycle in the CK reaction velocities modeled by enzymatic rate equations. The maximum variation in HEPs in the normal human heart beating at 60 min^–1 was approximately 0.4 m m and proportional to the velocity of ATP hydrolysis. Such HEP variations are at or below the current limits of detection by in vivo ³¹P MRS methods. Bloch equation simulations show that ³¹P MRS saturation transfer estimates the time‐averaged, pseudo‐first‐order forward rate constant, k_f,ap′, of the CK reaction, and that periodic short‐term fluctuations in k_f′ and CK flux are not likely to be detectable in human studies employing current in vivo ³¹P MRS methods. Copyright © 2015 John Wiley & Sons, Ltd.     

In vivo mouse myocardial 31P MRS using three‐dimensional image‐selected in vivo spectroscopy (3D ISIS): technical considerations and biochemical validations

³¹P MRS provides a unique non‐invasive window into myocardial energy homeostasis. Mouse models of cardiac disease are widely used in preclinical studies, but the application of ³¹P MRS in the in vivo mouse heart has been limited. The small‐sized, fast‐beating mouse heart imposes challenges regarding localized signal acquisition devoid of contamination with signal originating from surrounding tissues. Here, we report the implementation and validation of three‐dimensional image‐selected in vivo spectroscopy (3D ISIS) for localized ³¹P MRS of the in vivo mouse heart at 9.4 T. Cardiac ³¹P MR spectra were acquired in vivo in healthy mice (n = 9) and in transverse aortic constricted (TAC) mice (n = 8) using respiratory‐gated, cardiac‐triggered 3D ISIS. Localization and potential signal contamination were assessed with ³¹P MRS experiments in the anterior myocardial wall, liver, skeletal muscle and blood. For healthy hearts, results were validated against ex vivo biochemical assays. Effects of isoflurane anesthesia were assessed by measuring in vivo hemodynamics and blood gases. The myocardial energy status, assessed via the phosphocreatine (PCr) to adenosine 5′‐triphosphate (ATP) ratio, was approximately 25% lower in TAC mice compared with controls (0.76 ± 0.13 versus 1.00 ± 0.15; P < 0.01). Localization with one‐dimensional (1D) ISIS resulted in two‐fold higher PCr/ATP ratios than measured with 3D ISIS, because of the high PCr levels of chest skeletal muscle that contaminate the 1D ISIS measurements. Ex vivo determinations of the myocardial PCr/ATP ratio (0.94 ± 0.24; n = 8) confirmed the in vivo observations in control mice. Heart rate (497 ± 76 beats/min), mean arterial pressure (90 ± 3.3 mmHg) and blood oxygen saturation (96.2 ± 0.6%) during the experimental conditions of in vivo ³¹P MRS were within the normal physiological range. Our results show that respiratory‐gated, cardiac‐triggered 3D ISIS allows for non‐invasive assessments of in vivo mouse myocardial energy homeostasis with ³¹P MRS under physiological conditions. Copyright © 2015 John Wiley & Sons, Ltd.     

Pitfalls and advantages of different strategies for the absolute quantification of N‐acetyl aspartate,creatine and choline in white and grey matter by 1H‐MRS

This study extensively investigates different strategies for the absolute quantitation of N‐acetyl aspartate, creatine and choline in white and grey matter by ¹H‐MRS at 1.5 T. The main focus of this study was to reliably estimate metabolite concentrations while reducing the scan time, which remains as one of the main problems in clinical MRS. Absolute quantitation was based on the water‐unsuppressed concentration as the internal standard. We compared strategies based on various experimental protocols and post‐processing strategies. Data were obtained from 30 control subjects using a PRESS sequence at several TE to estimate the transverse relaxation time, T₂, of the metabolites. Quantitation was performed with the algorithm QUEST using two different metabolite signal basis sets: a whole‐metabolite basis set (WhoM) and a basis set in which the singlet signals were split from the coupled signals (MSM). The basis sets were simulated in vivo for each TE used. Metabolites' T₂s were then determined by fitting the estimated signal amplitudes of the metabolites obtained at different TEs. Then the absolute concentrations (mM) of the metabolites were assessed for each subject using the estimated signal amplitudes and either the mean estimated relaxation times of all subjects (mean protocol, MP) or the T₂ estimated from the spectra derived from the same subject (individual protocol, IP). Results showed that MP represents a less time‐consuming alternative to IP in the quantitation of brain metabolites by ¹H‐MRS in both grey and white matter, with a comparable accuracy when performed by MSM. It was also shown that the acquisition time might be further reduced by using a variant of MP, although with reduced accuracy. In this variant, only one water‐suppressed and one water‐unsuppressed spectra were acquired, drastically reducing the duration of the entire MRS examination. However, statistical analysis highlights the reduced accuracy of MP when performed using WhoM, particularly at longer echo times. Copyright © 2009 John Wiley & Sons, Ltd.     

In vivo and ex vivo evidence for ketamine-induced hyperglutamatergic activity in the cerebral cortex of the rat: Potential relevance to schizophrenia

Subanesthetic doses of ketamine, a noncompetitive N‐methyl‐D ‐aspartate (NMDA) receptor antagonist, impair prefrontal cortex (PFC) function in the rat and produce symptoms in humans similar to those observed in patients with schizophrenia. In the present study, in vivo ¹H‐MRS and ex vivo ¹H high‐resolution magic angle spinning (HR‐MAS) spectroscopy was used to examine the brain metabolism of rats treated with subanesthetic doses of ketamine (30 mg/kg) for 6 days. A single voxel localization sequence (PRESS, TR/TE = 4000/20 ms and NEX = 512) was used to acquire the spectra in a 30‐µl voxel positioned in the cerebral cortex (including mainly PFC) of the rats (ketamine group: n = 12; saline group: n = 12) anesthetized with isoflurane. After the in vivo ¹H‐MRS acquisition, the animals were sacrificed and the cerebral cortex tissues were extracted (ketamine group: n = 7; saline group: n = 7) for ex vivo ¹H HR‐MAS spectroscopy (CPMG sequence, 2.0‐s presaturation delay, 2.0‐s acquisition time, 128 transients and 4‐ms inter‐pulse delay) using a 500‐MHz NMR spectrometer. All proton metabolites were quantified using the LCModel. For the in vivo spectra, there was a significant increase in glutamate concentration in the cerebral cortex of the ketamine group compared with the controls (p < 0.05). For the ex vivo HR‐MAS spectra, there was a significant increase in the glutamate/total creatine ratio, and a decrease in the glutamine/total creatine and glutamine/glutamate ratios in the cerebral cortex tissue of the ketamine group compared with the controls. The results of the present study demonstrated that administration of subanesthetic doses of ketamine in the rat may exert at least part of their effect in the cerebral cortex by activation of glutamatergic neurotransmission. Copyright © 2011 John Wiley & Sons, Ltd.     

Quantification of skeletal muscle mitochondrial function by 31P magnetic resonance spectroscopy techniques: a quantitative review

Magnetic resonance spectroscopy (MRS) can give information about cellular metabolism in vivo which is difficult to obtain in other ways. In skeletal muscle, non‐invasive ³¹P MRS measurements of the post‐exercise recovery kinetics of pH, [PCr], [Pi] and [ADP] contain valuable information about muscle mitochondrial function and cellular pH homeostasis in vivo, but quantitative interpretation depends on understanding the underlying physiology. Here, by giving examples of the analysis of ³¹P MRS recovery data, by some simple computational simulation, and by extensively comparing data from published studies using both ³¹P MRS and invasive direct measurements of muscle O₂ consumption in a common analytical framework, we consider what can be learnt quantitatively about mitochondrial metabolism in skeletal muscle using MRS‐based methodology. We explore some technical and conceptual limitations of current methods, and point out some aspects of the physiology which are still incompletely understood.