In vivo image-guided 1H-magnetic resonance spectroscopy of the serial development of hepatocarcinogenesis in an experimental animal model

Histology on a core or open biopsy is considered the gold standard for the diagnosis of tumours. While the non-invasive technique of magnetic resonance imaging can direct some of the decision diagnostic making, it has limitations and disadvantages, that can be partly overcome with the use of in vivo magnetic resonance spectroscopy (MRS). In vivo MRS is able to provide a specific biochemical profile on tumour tissue, compared with normal tissue. The capability of this technique is demonstrated here by the long-term development of hepatocellular carcinoma in an animal model. It allows the observation of the biochemical changes that occur in tumour tissue during its progression from preneoplastic nodules to hepatocellular carcinoma. Specifically the changes in the lipid profiles of tumour tissue at various stages of development are observed with proton (¹H) MRS. Significant increases occurred in the lipid acyl chain methylene and methyl hydrogens during the early developmental stages of hepatocarcinogenesis, whereas during later stages associated with tumour development there was a significant increase in the levels of olefinic acyl chain hydrogens from unsaturated lipids. It is anticipated that this model will precede the application of the same technology to the non-invasive diagnosis and grading of human hepatocellular carcinoma.     

^H磁共振波谱在脑胶质瘤的应用进展

磁共振波谱（magnetic resonance spectroscopy,MRS）技术的出现使活体检测组织的代谢和生化信息成为可能,随着其技术的不断成熟,其在临床的应用范围日益扩大。脑胶质瘤具有与正常脑组织不同的代谢特征,借助MRS技术一方面可以反映其代谢特征,另外可将其与正常脑组织区分,因此MRS技术特别是^1H-MRS在脑胶质瘤的诊断、鉴别诊断、分级及预后评估中应用日益广泛。本文就相关进展进行综述。     

Magnetic resonance spectroscopy in vivo

Nuclear magnetic resonance (NMR) has become an important non-invasive investigative technique in medicine and biology. The most recent development has been the ability to perform magnetic resonance spectroscopy (MRS) in selected regions within the human body. Such volumes can be selected by techniques which fall into the following broad catagories: surface coil methods, surface coils with depth selection, volume selection and chemical shift mapping. The latter two methods use magnetic field gradients, present on magnetic resonance imaging systems, to select the volume. MRS can be used to measure phosphorus and proton metabolites and hence study tissue biochemistry in-vivo.     

Identification of cholesteryl esters in human carotid atherosclerosis by ex vivo image-guided proton MRS

Vulnerable atherosclerotic plaques may be identified by their large lipid component, particularly liquid cholesteryl ester (CE), covered by a fibrous cap. We hypothesized that image-guided 1H proton magnetic resonance spectroscopy (MRS) would identify mobile CE in discrete, preselected regions of atherosclerotic plaque. Human carotid endarterectomy specimens (n = 10) were imaged ex vivo by magnetic resonance imaging (MRI) at high field (11.7 T) utilizing standard T1- and T2-weighted spin echo protocols. MRS spectra were acquired from 1 mm3 voxels, localized to plaque regions that we judged by MRI to be lipid rich or lipid poor. The spectra revealed methyl and methylene resonances of fatty acyl chains with relative intensities and linewidths characteristic of pure CE, by comparison with lipid standards. Regions judged to be lipid rich by MRI showed much more intense CE resonances than did lipid-poor regions. The integrated intensities of lipid peaks were 5.5 +/- 2.0% (lipid-rich regions) versus 0.9 +/- 0.6% (lipid-poor regions) of the unsuppressed water peak (P < 0.0001). Lipid distribution by histology, MRS, and MRI showed strong correlation. Image-guided proton MRS accurately identified CE in selected regions of atherosclerotic plaque as small as 1 mm3 in an ex vivo setting. This procedure may permit the noninvasive detection and quantification of CE in atherosclerotic plaque in vivo.     

19F-nuclear magnetic resonance study of glycerolipid fatty acyl chain order in Acholeplasma laidlawii B membranes

The technique of 19F-nuclear magnetic resonance (19F-NMR) spectroscopy offers a number of advantages for studies of lipid fatty acyl chain orientation and dynamics in biomembranes. However, the geminal difluoromethylene fatty acid probes usually employed in such studies appreciably perturb the organization of lipid bilayers. We have thus synthesized a series of specifically monofluorinated palmitic acids and carried out biophysical, biochemical, and physiological studies establishing their suitability as relatively non-perturbing probes of lipid hydrocarbon chain organization. These 19F-NMR probes were then used to determine the fatty acyl chain order profiles of Acholeplasma laidlawii B membranes highly enriched in a variety of different exogenous fatty acids, particularly those containing a methyl branch or a trans-double bond.     

Biochemical characterization of breast tumors by in vivo and in vitro magnetic resonance spectroscopy (MRS)

Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) have evolved as sensitive tools for anatomic and metabolic evaluation of breast cancer. In vivo MRS studies have documented the presence of choline containing compounds (tCho) as a reliable biochemical marker of malignancy and also useful for monitoring the tumor response to therapy. Recent studies on the absolute quantification of tCho are expected to provide cut-off values for discrimination of various breast pathologies. Addition of MRS investigation was also reported to increase the specificity of MRI. Further, ex vivo and in vitro MRS studies of intact tissues and tissue extracts provided several metabolites that were not be detected in vivo and provided insight into underlying biochemistry of the disease processes. In this review, we present briefly the role of various ¹H MRS methods used in breast cancer research and their potential in relation to diagnosis, monitoring of therapeutic response and metabolism.     

Identification of mobile cholesterol compounds in experimental gliomas by (1)H MRS in vivo: effects of ganciclovir-induced apoptosis on lipids

This letter presents a novel identification and analysis of mobile cholesterol compounds in an experimental glioma model by (1)H MRS in vivo. The cholesterol compounds turned out to comprise as much as 17 mol% of MRS visible total lipids. The results also imply partly associated accumulation of (1)H MRS detectable cholesterol compounds and unsaturated lipids during gene therapy-induced apoptosis, and indicate that the contribution of cholesterol compounds cannot be bypassed in spectral lipid analysis. The introduced (1)H MRS approach facilitates a non-invasive follow-up of mobile cholesterol compounds, paving way for studies of tumour cholesterol metabolism in vivo.     

A metabolomics perspective of human brain tumours

During the past decade or so, a wealth of information about metabolites in various human brain tumour preparations (cultured cells, tissue specimens, tumours in vivo) has been accumulated by global profiling tools. Such holistic approaches to cellular biochemistry have been termed metabolomics. Inherent and specific metabolic profiles of major brain tumour cell types, as determined by proton nuclear magnetic resonance spectroscopy ((1)H MRS), have also been used to define metabolite phenotypes in tumours in vivo. This minireview examines the recent advances in the field of human brain tumour metabolomics research, including advances in MRS and mass spectrometry technologies, and data analysis.     

Investigation of adipose tissues in Zucker rats using in vivo and ex vivo magnetic resonance spectroscopy

In vivo single-voxel magnetic resonance spectroscopy (MRS) at 4.7T and ex vivo high-resolution proton magnetic resonance spectroscopy (HR-NMR) at 500 MHz were used to study the composition of adipose tissues in Zucker obese and Zucker lean rats. Lipid composition was characterized by unsaturation and polyunsaturation indexes and mean chain lengths. In vitro experiments were conducted in known mixtures of triglycerides and oils in order to validate the method. To avoid inaccuracies due to partial peak overlapping in MRS, peak quantification was performed after fitting of spectral peaks by using the QUEST algorithm. The intensity of different spectral lines was also corrected for T2 relaxation. Albeit with different sensitivity and accuracy, both techniques revealed that white adipose tissue is characterized by lower unsaturation and polyunsaturation indexes in obese rats compared with controls. HR-NMR revealed similar differences in brown adipose tissue. The present findings confirm the hypothesis that obese and lean Zucker rats have different adipose tissue composition.     

In vivo MR spectroscopy in diagnosis and research of neuropsychiatric disorders

Magnetic resonance spectroscopy is one of the most important tools for quantitative analysis of chemical composition and structure, and this non-invasive technique is now being applied in vivo to study biochemical processes in those neuropsychiatric disorders that are part of the phospholipid spectrum. Interpretation of a clinical magnetic resonance spectrum can provide information about membrane phospholipid turnover, cellular energetics, neuronal function, selected neurotransmitter activity and intracellular pH. Cerebral proton and phosphorus magnetic resonance spectroscopy findings are summarized in relation to schizophrenia, dyslexia and chronic fatigue syndrome.     

In vivo MRS measurement of liver lipid levels in mice

A magnetic resonance spectroscopy (MRS) procedure for in vivo measurement of lipid levels in mouse liver is described and validated. The method uses respiratory-gated, localized spectroscopy to collect proton spectra from voxels within the mouse liver. Bayesian probability theory analysis of these spectra allows the relative intensities of the lipid and water resonances within the liver to be accurately measured. All spectral data were corrected for measured spin-spin relaxation. A total of 48 mice were used in this study, including wild-type mice and two different transgenic mouse strains. Different groups of these mice were fed high-fat or low-fat diets or liquid diets with and without the addition of alcohol. Proton spectra were collected at baseline and, subsequently, every 4 weeks for up to 16 weeks. Immediately after the last MRS measurement, mice were killed and their livers analyzed for triglyceride level by conventional wet-chemistry methods. The excellent correlation between in vivo MRS and ex vivo wet-chemistry determinations of liver lipids validates the MRS method. These results clearly demonstrate that in vivo MRS will be an extremely valuable technique for longitudinal studies aimed at providing important insights into the genetic, environmental, and dietary factors affecting fat deposition and accumulation within the mouse liver.     

Quantitative imaging of oil storage in developing crop seeds

In this article, we present a tool which allows the rapid and non-invasive detection and quantitative visualization of lipid in living seeds at a variety of stages using frequency-selected magnetic resonance imaging. The method provides quantitative lipid maps with a resolution close to the cellular level (in-plane 31 µm × 31 µm). The reliability of the method was demonstrated using two contrasting subjects: the barley grain (monocot, 2% oil, highly compartmentalized) and the soybean grain (dicot, 20% oil, economically important oilseed). Steep gradients in local oil storage were defined at the organ- and tissue-specific scales. These gradients were closely coordinated with tissue differentiation and seed maturation, as revealed by electron microscopy and biochemical and gene expression analysis. The method can be used to elucidate similar oil accumulation processes in different tissues/organs, as well as to follow the fate of storage lipids during deposition and subsequent mobilization.     

In vivo and in vitro intervention with L-carnitine prevents abnormal energy metabolism in isolated diabetic rat heart: chemical and phosphorus-31 NMR evidence

The beneficial effects of in vivo injections (200 mg/kg, twice daily) or in vitro perfusion (5.0 mM) of L-carnitine on an intrinsic abnormality in energy metabolism was investigated in isolated, perfused diabetic rat heart. Hearts were aerobically perfused for 60 min with elevated fatty acid substrate to simulate diabetic conditions. Phosphorus-31 nuclear magnetic resonance spectroscopy revealed a temporal decline in myocardial ATP levels (to approx 82%) during perfusion of diabetic hearts, but not in control hearts. This reduction was prevented by prior treatment in vivo with L-carnitine or by providing L-carnitine acutely in the perfusion medium. Chemical analysis of tissue extracts indicated that L-carnitine injections were effective in replenishing the decrease in total myocardial carnitine content which was present in diabetic hearts and in preventing the accumulation of long chain fatty acyl CoA. Perfusion with L-carnitine also attenuated the elevation of long chain fatty acyl CoA in diabetic hearts. This study gives additional support to the hypothesis that decreases in ATP which occur in the isolated, perfused diabetic heart are correlated with a concomitant elevation in long chain fatty acyl CoA, a known inhibitor of adenine nucleotide translocase. In the presence of elevated exogenous fatty acids, a primary deficiency in the total myocardial carnitine pool would result in elevations in tissue concentrations of long chain fatty acyl CoA since carnitine is a required carrier for transport of fatty acids into mitochondria. Replenishment of the carnitine in vivo was shown to be sufficient to prevent subsequent alteration in long chain fatty acyl CoA and ATP in isolated perfused diabetic hearts despite the burden of elevated fatty acid substrates.     

The Value of Proton Magnetic Resonance Spectroscopy in High-Intensity Focused Ultrasound Treatment of Experimental Liver Cancer

High-intensity focused ultrasound (HIFU) is a rapidly developing, non-invasive technique for local treatment of solid tumors that produce coagulative tumor necrosis. This study is aimed to investigate the feasibility of proton magnetic resonance spectroscopy (MRS) on early assessing treatment of HIFU ablation in rabbit with VX2 liver tumor. HIFU ablation was performed on normal liver and VX2 tumor in rabbit, and MRS was performed on normal liver and VX2 tumor before and 2 days after 100% HIFU ablation or 80% ablation in tumor volume. Choline (Cho) and choline/lipid (Cho/Lip) ratios between complete and partial HIFU ablation of tumor were compared. Tissues were harvested and sequentially sliced to confirm the necrosis. In normal liver, the Cho value liver was not obviously changed after HIFU (P > .05), but the Cho/Lip ratio was decreased (P < .05). Cho in liver VX2 tumor was much higher than that in normal liver (P < .001). Cho and Cho/Lip ratio were significantly decreased in tumor after complete HIFU ablation and partial HIFU ablation, and the Cho value in complete HIFU tumor ablation did not show any difference from that in normal liver after HIFU (P > .05); however, the Cho value in partial ablation was still higher than that in normal liver before or in tumor after complete HIFU treatment due to the residual part of tumors, and Cho/Lip ratio is lower than that in complete HIFU treatment (P < .001). The changes in MRS parameters were consistent with histopathologic changes of the tumor tissues after treatment. MRS could differentiate the complete tumor necrosis from residual tumor tissue, when combined with magnetic resonance imaging. We conclude that MRS may be applied as an important, non-invasive biomarker for monitoring the thoroughness of HIFU ablation.     

Non-invasive assessment of oxidative capacity in young Indian men and women: a 31P magnetic resonance spectroscopy study

It is generally assumed that men display greater strength and muscle capacity than women. However, previous biochemical and histological studies have shown that men have greater capacity for anaerobic metabolism and women have higher or similar oxidative metabolism. Therefore, in the present study, we estimated oxidative capacity of gastrocnemius muscle and compared in Indian men and women using non-invasive in vivo 31P magnetic resonance spectroscopy (MRS). Healthy subjects (8 young males and 9 females, age-matched) performed plantar flexion exercise within a magnet and MRS measurements of inorganic phosphate (Pi), phosphocreatine (PCr), ADP, and pH of the calf muscles were carried out using a 1.5 T whole-body MRI system. PCr values during recovery were fitted to an exponential curve, and oxidative capacity was calculated using rate constant (k(PCr)), as an index of oxidative phosphorylation. When men and women were compared for different metabolic ratios, ADP, pH, k(PCr) and oxidative capacity, all parameters turned out to be statistically insignificant. The results showed no gender effect on skeletal muscle oxidative metabolism. The study demonstrated the usefulness of such non-invasive method to indirectly measure the oxidative capacity of the muscle based on PCr recovery.     

Biochemical characterization of human brain and kidney tissues by magnetic resonance spectroscopy

In vivo and in vitro Magnetic Resonance Spectroscopy is useful for monitoring changes in intracellular metabolites of human cerebral and renal tissues. Healthy and tumoral tissues of different histologic types have been characterized from a biochemical point of view. In vitro molecular characterization is performed on both the aqueous and lipid extracts of surgically removed tissue biopsies, after in vivo MRS, yielding a full picture of tissue biochemistry. Biochemical markers of healthy brain and kidney and of their relative neoplastic lesions have been disclosed. Moreover, some biochemical features can differentiate neoplasm within the same histological type. Ex vivo MRS also gives molecular information related to necrotic phenomena in glial tumors. MRS finding paralleled histologic data and new knowledge about the molecular base of proliferative neoplastic phenomena can be obtained.     

In vivo assessment of hepatic triglycerides in murine non-alcoholic fatty liver disease using magnetic resonance spectroscopy

In vivo¹H magnetic resonance spectroscopy (MRS) was used to examine the progression of fatty liver in two murine models of progressive hepatic steatosis: leptin-deficient obese (ob/ob) mice and mice maintained on a diet deficient in methionine and choline (MCDD). Ob/ob mice displayed high levels of intracellular hepatic triglycerides as early as 9 weeks after birth, as observed with MRS and histopathology. Single voxel spectra of ob/ob liver displayed strong resonances arising from saturated (1.3 ppm) and unsaturated (2.8 and 5.3 ppm) fatty acyl chains that could be resolved in the absence of water suppression. Hepatic inflammation, induced by lipopolysaccharide administration, led to a significant increase in unsaturated and polyunsaturated fatty acyl chain resonances (P < 0.05), indicating a change in the composition of hepatic triglycerides in lipid droplets. Mice maintained on the MCDD displayed histological evidence of hepatic steatosis as early as two weeks, progressing to macrovesicular steatohepatitis at 10 weeks. The histological changes were accompanied by significant increases in saturated and unsaturated fatty acyl chain resonances and a significant decrease in the lipid/(water + lipid) ratio (P < 0.05). These results indicate that in vivo¹H MRS may be a suitable method to monitor the progression of steatohepatitis.     

Proton and carbon-13 nuclear magnetic resonance studies of rhodopsin-phospholipid interactions

Proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR) spectra of rhodopsin-phospholipid membrane vesicles and sonicated disk membranes are presented and discussed. The presence of rhodopsin in egg phosphatidylcholine vesicles results in homogeneous broadening of the methylene and methyl resonances. This effect is enhanced with increasing rhodopsin content and decreased by increasing temperature. The proton NMR data indicate the phospholipid molecules exchange rapidly (less than 10(-3) s) between the bulk membrane lipid and the lipid in the immediate proximity of the rhodopsin. These interactions result in a reduction in either or both the frequency and amplitude of the tilting motion of the acyl chains. The 13C NMR spectra identify the acyl chains and the glycerol backbone as the major sites of protein lipid interaction. In the disk membranes the saturated sn-1 acyl chain is significantly more strongly immobilized than the polyunsaturated sn-2 acyl chain. This suggest a membrane model in which the lipid molecules preferentially solvate the protein with the sn-1 chain, which we term an edge-on orientation. The NMR data on rhodopsin-asolectin membrane vesicles demonstrate that the lipid composition is not altered during reconstitution of the membranes from purified rhodopsin and lipids in detergent.     

Neuroimaging of mitochondrial disease

Mitochondrial disease represents a heterogeneous group of genetic disorders that require a variety of diagnostic tests for proper determination. Neuroimaging may play a significant role in diagnosis. The various modalities of nuclear magnetic resonance imaging (MRI) allow for multiple independent detection procedures that can give important anatomical and metabolic clues for diagnosis. The non-invasive nature of neuroimaging also allows for longitudinal studies. To date, no pathonmonic correlation between specific genetic defect and neuroimaging findings have been described. However, certain neuroimaging results can give important clues that a patient may have a mitochondrial disease. Conventional MRI may show deep gray structural abnormalities or stroke-like lesions that do not respect vascular territories. Chemical techniques such as proton magnetic resonance spectroscopy (MRS) may demonstrate high levels of lactate or succinate. When found, these results are suggestive of a mitochondrial disease. MRI and MRS studies may also show non-specific findings such as delayed myelination or non-specific leukodystrophy picture. However, in the context of other biochemical, structural, and clinical findings, even non-specific findings may support further diagnostic testing for potential mitochondrial disease. Once a diagnosis has been established, these non-invasive tools can also aid in following disease progression and evaluate the effects of therapeutic interventions.