1H NMR Ganglioside Ceramide Resonance Region on the Differential Diagnosis of Low and High Malignancy of Brain Gliomas

1. The high-resolution ¹H NMR (MRS) spectra of human brain tumor homogenates revealed a broad resonance at 5.3–5.4 ppm in glioblastoma multiforme (N = 16) and brain metastases (N = 2). The broad resonance was identified as ceramide, a sphingosine–fatty acid combination portion of ganglioside, indicating an elevated abundance of monounsaturated fatty acids. GLC analysis of gangliosides in the highly malignant glioblastoma multiforme revealed that the elevated monounsaturated fatty acid is oleic acid (C18:1). The resonance at 5.3–5.4 ppm region was not detectable in normal human brain (N = 2), in meningiomas (N = 2), or in low-grade astrocytomas (N = 12). In normal human brain the abundance of monounsaturated fatty acid is minimal.2. This investigation was made possible because the method of producing homogenate resulted in (i) no loss of lipids during the process and (ii) a well-homogenised sample, with (iii) no loss in chemical integrity.3. The properties of tumor gangliosides include antigenic specificity and immunosuppresive activity and the ceramide, a sphingosine–fatty acid combination, noticeably influences the ganglioside immunosuppressive activity.4. The observation of ¹H NMR ceramide resonance in high-malignant brain tumors emphasizes the dramatic role of aberrant gangliosides and ceramide precursors on the grade of malignancy and invasiveness.5. Further insight into the specific nature of the ceramide portion of gangliosides in grading the malignancy of brain tumors should be investigated further.     

Long-lived spin state of a tripeptide in stretched hydrogel

The longitudinal (T ₁), transverse (T ₂), and singlet state (T _s) relaxation times of the geminal backbone protons (CH₂) of l-Leu-Gly-Gly were studied by NMR spectroscopy at 9.4 T in a bovine hide gelatin gel composed in D₂O at 25 °C. Gelatin granules were dissolved in a hot solution of the tripeptide and then the solution was allowed to gel inside a flexible silicone tubing. With increases in gelatin content, the T ₂ and T _s of the CH₂ protons correspondingly decreased (T _s/T ₂ ~ constant), while the change in T ₁ was relatively small. The largest observed T _s/T ₁ value was 3.3 at 46 % w/v gelatin that was the lowest gelatin content examined. Stretching the tubing, and hence the gel, brought about anisotropic alignment of the constituents resulting in residual quadrupolar splitting of the resonance from D₂O in ²H NMR spectra, and residual dipolar splitting of the CH₂ resonance in ¹H NMR spectra. WALTZ-16 decoupling during the relaxation intervals extended the singlet state relaxation time, but the efficacy diminished as the gels were stretched. Theoretically predicted T ₁, T ₂, and T _s values, assuming intramolecular dipolar coupling as the only source of relaxation, were within the same order of magnitude as the experimentally observed values. Overall we showed that it is possible to observe a long-lived spin state in an anisotropic medium when T ₂ is shorter than T ₁ in the presence of non-zero residual dipolar couplings.     

Discriminating Hepatocellular Carcinoma in Rats Using a High-Tc SQUID Detected Nuclear Resonance Spectrometer in a Magnetic Shielding Box

In this study, we report the spin-lattice relaxation rate of hepatocellular carcinoma (HCC) and normal liver tissue in rats using a high-T_c superconducting quantum interference device (SQUID) based nuclear magnetic resonance (NMR) spectrometer. The resonance spectrometer used for discriminating liver tumors in rats via the difference in longitudinal relaxation time in low magnetic fields was set up in a compact and portable magnetic shielding box. The frequency-domain NMR signals of HCC tissues and normal liver tissues were analyzed to study their respective longitudinal relaxation rate T₁ ⁻¹. The T₁ ⁻¹ of liver tissues for ten normal rats and ten cancerous rats were investigated respectively. The averaged T₁ ⁻¹ value of normal liver tissue was (6.41±0.66) s⁻¹, and the averaged T₁ ⁻¹ value of cancerous tissue was (3.38±0.15) s⁻¹. The ratio of T₁ ⁻¹ for normal liver tissues and cancerous liver tissues of the rats investigated is estimated to be 1.9. Since this significant statistical difference, the T₁ ⁻¹ value can be used to distinguish the HCC tissues from normal liver tissues. This method of examining liver and tumor tissues has the advantages of being convenient, easy to operate, and stable.     

Nuclear magnetic resonance relaxation times and plasmalemma water exchange in ivy bark

Measurement of nuclear magnetic resonance (NMR) relaxation times (transverse [T₂] and longitudinal [T₁]) for Hedera helix L. cv. Thorndale (ivy) bark water indicates the presence of at least two populations of water with different relaxation characteristics. One population of water with short T₂ and T₁ was found to be composed of both hydration water and extracellular free water. The second population of water with long T₂ and T₁ was identified as intracellular bulk water.     

Magnetic resonance imaging (MRI) of water during cold acclimation and freezing in winter wheat

Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) were used to analyse changes in the physical state of water in wheat crowns during cold acclimation and during the freezing/thawing cycle. Spectroscopically measured average spin-spin relaxation times (T₂) decreased during cold acclimation and increased when plants were grown at normal temperature. Spin-spin relaxation images whose contrast is proportional to T₂, times were calculated allowing association of water relaxation with regions of tissue in spin-echo images during acclimation and freezing. Images taken during freezing revealed nonuniform freezing of tissue in crowns and roots. Acclimated and non-acclimated wheat crowns were imaged during freezing and after thawing. Spin-echo image signal intensity and T₂ times decreased dramatically between -4°C and -8°C as a result of a decrease in water mobility during freezing. Images collected during thawing were diffuse with less structure and relaxation times were longer, consistent with water redistribution in tissue after membrane damage.     

Design and characterization of a chimeric ferritin with enhanced iron loading and transverse NMR relaxation rate

This paper describes the design and characterization of a novel ferritin chimera. The iron storage protein ferritin forms a paramagnetic ferrihydrite core. This biomineral, when placed in a magnetic field, can decrease the transverse NMR relaxation times (T ₂ and T ₂*) of nearby mobile water protons. Ferritin nucleic acid constructs have recently been studied as “probeless” magnetic resonance imaging (MRI) reporters. Following reporter expression, ferritin sequesters endogenous iron and imparts hypointensity to T ₂- and T ₂*-weighted images in an amount proportional to the ferritin iron load. Wild-type ferritin consists of various ratios of heavy H and light L subunits, and their ratio affects ferritin’s stability and iron storage capacity. We report a novel chimeric ferritin with a fixed subunit stoichiometry obtained by fusion of the L and the H subunits (L*H and H*L) using a flexible linker. We characterize these supramolecular ferritins expressed in human cells, including their iron loading characteristics, hydrodynamic size, subcellular localization, and effect on solvent water T ₂ relaxation rate. Interestingly, we found that the L*H chimera exhibits a significantly enhanced iron loading ability and T ₂ relaxation compared to wild-type ferritin. We suggest that the L*H chimera may be useful as a sensitive MRI reporter molecule.     

Conformation and dynamic structure of poly(inosinic acid) in neutral aqueous solution by 1H-, 2H-, 13C-, and 31P-NMR spectroscopy

The conformation and dynamic structure of single-stranded poly(inosinic acid), poly(I), in aqueous solution at neutral pH have been investigated by nmr of four nuclei at different frequencies: ¹H (90 and 250 MHz), ²H (13.8 MHz), ¹³C (75.4 MHz), and ³¹P (36.4 and 111.6 MHz). Measurements of the proton-proton coupling constants and of the ¹H and ¹³C chemical shifts versus temperature show that the ribose is flexible and that base-base stacking is not very significant for concentrations varying from 0.04 to 0.10M in the monomer unit. On the other hand, the proton T₁ ratios between the sugar protons, T₁ (H₁′)/T₁ (H₃′), indicate a predominance of the anti orientation of the base around the glycosidic bond. The local motions of the ribose and the base were studied at different temperatures by measurements of nuclear Overhauser enhancement (NOE) of protonated carbons, the ratio of the proton relaxation times measured at two frequencies (90 and 250 MHz), and the deuterium quadrupolar transverse relaxation time T₂. For a given temperature between 22 and 62°C, the ¹³C-{¹H} NOE value is practically the same for seven protonated carbons (C₂, C₈, C_1′, C_2′, C_3′, C_4′, C_5′). This is also true for the T₁ ratio of the corresponding protons. Thus, the motion of the ribose–base unit can be considered as isotropic and characterized by a single correlation time, τ_c, for all protons and carbons. The τ_c values determined from either the ¹³C-{¹H} NOE or proton T₁ ratios, T₁(90 MHz)/T₁(250 MHz), and/or deuterium transverse relaxation time T₂ agree well. The molecular motion of the sugar-phosphate backbone (O-P-O) and the chemical-shift anisotropy (CSA) were deduced from T₁ (³¹P) and ³¹P-{¹H} NOE measurements at two frequencies. The CSA contribution to the phosphorus relaxation is about 12% at 36.4 MHz and 72% at 111.6 MHz, corresponding to a value of 118 ppm for the CSA (σ = σ∥ ? σ?). Activation energies of 2–6 kcal/mol for the motion of the ribose–base unit and the sugarphosphate backbone were evaluated from the proton and phosphorus relaxation data.     

Nuclear magnetic resonanceimaging of membrane permeability changes in plants during osmoticstress

The cell water balance of maize (Zea mays L., cv LG 11) andpearl millet (Pennisetum americanum L., cv MH 179) duringosmotic stress was studied non‐invasively using ¹H nuclearmagnetic resonance (NMR) microscopy. Single NMR parameter imagesof (i) the water content (ii) the transverse relaxation time (T₂)and (iii) the apparent diffusion coefficient (D_app)were used to follow the water status of the stem apical region duringosmotic stress. During stress there are hardly any changes in watercontent or T₂ of the stem region of maize. Incontrast, the apical tissue of pearl millet showed a ～ 30% decreaseof T₂ within 48 h of stress, whereasthe water content and D_app did not change. Thesechanges can be explained by an increase of the membrane permeabilityfor water. This conclusion is supported by results from scanningelectron microscopy, relaxation measurements of sugar solutionsand numerical simulations of the relaxation and (apparent) diffusionbehaviour of water in a plant cell.     

Pulsed NMR studies on water in striated muscle III. The effects of water content

Pulsed NMR is used to study the kinetics of dehydration of frog gastrocnemius muscle. In addition, measurements are reported of the variation of the spin-lattice (T₁) and transverse (T₂) nuclear magnetic relaxation times of the water protons as a function of water content. The proton transverse relaxation and freezing properties of the water in muscles which had been dehydrated and then rehydrated are also investigated. Correlation of the double-exponential dehydration kinetics with the transverse relaxation at various water contents provides strong evidence for the evidence of a fraction of muscle water (10–20%) which is sufficiently strongly held to the solid substance of the muscle to make it relatively slowly removed under conditions of zero relative humidity but which is still dynamically very mobile on average. This is supported by the dependence of T₁ on water content. The relaxation times are interpreted qualitatively in terms of a number of possible effects which are at present not distinguishable. The properties of the dehydrated-rehydrated muscles indicated changes in the muscle proteins which affect the transverse relaxation of the water protons and the freezing properties of the muscle water.     

Characteristics of tumour vessels in cytological squash smears of astrocytic tumours

S. Sato, Y. Sato, K. Marutsuka, H. Takeshima and Y. Asada Characteristics of tumour vessels in cytological squash smears of astrocytic tumours Objective: Smear preparations are useful tools from which to diagnose brain tumours intraoperatively. Although vascular proliferation is histologically a key feature of high‐grade astrocytoma, the characteristics of tumour vessels in smear preparations have not been determined. Methods: We examined the density and morphological parameters (area, width, nuclear layer and branches of vessel wall) of tumour vessels in squash smears of 43 primary astrocytomas (grade II diffuse astrocytomas, n = 9; grade III anaplastic astrocytomas, n = 13; grade IV glioblastomas, n = 21) and normal brain tissues (n = 11). Results: Vessel density and all morphological parameters were significantly higher in grade IV than in the other grades of tumours and in normal brain tissue. Vessel area, width and nuclear layer were greater in grade III than in normal brain tissue. The sensitivity and specificity of these vessel parameters for astrocytomas were 75–100% and 82–100%, respectively. Conclusions: Tumour vessel evaluations from squash smears provide useful information for the intraoperative diagnosis and grading of astrocytic tumours.     

高分辨魔角旋转核磁共振和主成分分析研究人类低级星形细胞瘤和脑膜瘤的代谢组特征

采用高分辨魔角旋转核磁共振（HRMAS ^1H NMR）技术结合主成分分析（PCA）方法研究了39例人体脑肿瘤组织的代谢组特征．39例肿瘤样本分别来自39个脑肿瘤患者,包括15例低级星形细胞瘤,13例纤维型脑膜瘤和11例过渡型脑膜瘤．核磁共振波谱分析结果表明,脑肿瘤组织的代谢组中丰要含有脂肪酸、乳酸、胆碱代谢物（如胆碱、磷酸胆碱和甘油磷酸胆碱）、氯基酸（如丙氨酸、谷氨酸、谷氮酰胺、牛磺酸）、N-乙酰天门冬氨酸（NAA）和谷胱甘肽等代谢物．通过对核磁共振谱进行主成分分析（PCA）,发现低级星形细胞瘤和脑膜瘤的代谢组之间具有明显的差异,而在过渡型和纤维型两个亚类脑膜瘤之间该差别相对较小．与脑膜瘤相比,低级星形细胞瘤中甘油磷酸胆碱、磷酸胆碱、肌醇与肌酸的含量较高,而丙氨酸、谷氨酸、谷氨酰胺、谷胱甘肽和牛磺酸的含量较低．NAA的含量在低级星形细胞瘤中尽管较低但能观察到,而脑膜瘤中却未发现NAA的信号．结果衷明,HRMAS ^1H NMR和多变量统计分析相结合的组织代谢组学方法,不仅能有效区分不同类型的脑肿瘤,而且还可以为脑肿瘤提供丰富的代谢组信息,这些信息对研究肿瘤发生发展的机制具有潜在的意义．     

High resolution <Superscript>13</Superscript>C-detected solid-state NMR spectroscopy of a deuterated protein

High resolution ¹³C-detected solid-state NMR spectra of the deuterated beta-1 immunoglobulin binding domain of the protein G (GB1) have been collected to show that all ¹⁵N, ¹³C′, ¹³Cα and ¹³Cβ sites are resolved in ¹³C–¹³C and ¹⁵N–¹³C spectra, with significant improvement in T ₂ relaxation times and resolution at high magnetic field (750 MHz). The comparison of echo T ₂ values between deuterated and protonated GB1 at various spinning rates and under different decoupling schemes indicates that ¹³Cα T ₂′ times increase by almost a factor of two upon deuteration at all spinning rates and under moderate decoupling strength, and thus the deuteration enables application of scalar-based correlation experiments that are challenging from the standpoint of transverse relaxation, with moderate proton decoupling. Additionally, deuteration in large proteins is a useful strategy to selectively detect polar residues that are often important for protein function and protein–protein interactions.     

Relaxation Along a Fictitious Field (RAFF) and Z-spectroscopy using Alternating-Phase Irradiation (ZAPI) in Permanent Focal Cerebral Ischemia in Rat

Cerebral ischemia alters the molecular dynamics and content of water in brain tissue, which is reflected in NMR relaxation, diffusion and magnetization transfer (MT) parameters. In this study, the behavior of two new MRI contrasts, Relaxation Along a Fictitious Field (RAFF) and Z-spectroscopy using Alternating-Phase Irradiation (ZAPI), were quantified together with conventional relaxation parameters (T₁, T₂ and T_1ρ) and MT ratios in acute cerebral ischemia in rat. The right middle cerebral artery was permanently occluded and quantitative MRI data was acquired sequentially for the above parameters for up to 6 hours. The following conclusions were drawn: 1) Time-dependent changes in RAFF and T_1ρ relaxation are not coupled to those in MT. 2) RAFF relaxation evolves more like transverse, rather than longitudinal relaxation. 3) MT measured with ZAPI is less sensitive to ischemia than conventional MT. 4) ZAPI data suggest alterations in the T₂ distribution of macromolecules in acute cerebral ischemia. It was shown that both RAFF and ZAPI provide complementary MRI information from acute ischemic brain tissue. The presented multiparametric MRI data may aid in the assessment of brain tissue status early in ischemic stroke.     

A proton magnetic resonance study of water in human stratum corneum

Proton magnetic resonance spectroscopy has been used to study the nature of water in human stratum corneum. For a single planar sheet of stratum corneum mounted at a specific orientation to the applied magnetic field, three distinct absorptions may be seen having different chemical shifts and spin-lattice relaxation times (T₁). All T₁ values for these resonances are smaller than that for normal liquid water. One absorption is unusual in that the resonance position is dependent upon the orientation of the sample within the field.     

Cell surface involvement in cancer metastasis: an NMR study

NMR spectroscopy is one of the few techniques which has the sensitivity to detect subtle changes to the surface chemistry of cells. It has previously been demonstrated that high resolution ¹H NMR methods can distinguish tumour cells with the capacity to metastasise and this information appears to arise from a type of proteolipid in or attached to the plasma membrane. Here we report that the ¹H NMR signal, which we have used to identify metastatic cells in rat tumours, is significantly reduced in intensity after cultured cells are treated with trypsin/EDTA. The long T₂ relaxation value (⪢ 350 ms) observed in metastatic cells is absent after enzyme treatment. 2D scalar correlated NMR (COSY) spectra of these treated cells show that a cross peak normally associated with malignancy and metastatic disease is markedly reduced. These findings indicate that the plasma membrane lipid particle which generates the high resolution spectrum is directly affected by trypsin/EDTA. Alterations to the cell surface properties were also demonstrated in vivo since reduced numbers of metastases were observed in animals injected with enzyme-treated cells. The correlation between the absence of a long T₂ relaxation value and the diminished numbers of metastases in animals suggests that the plasma membrane particle is involved in the metastatic process.     

Faster Metabolite 1H Transverse Relaxation in the Elder Human Brain

¹H magnetic resonance spectroscopy (MRS) is unique among imaging modalities because signals from several metabolites are measured during a single examination period. Each metabolite reflects a distinct intracellular process. Furthermore transverse (T₂) relaxation times probe the viability of the cell microenvironment, e.g., the viscosity of the cellular fluids, the microscopic susceptibility distribution within the cells, and the iron content. In this study, T₂s of brain metabolites were measured in the occipital lobe of eighteen young and fourteen elderly subjects at a field strength of 4 tesla. The T₂s of N-acetylaspartate, total creatine, and total choline were 23%, 16% and 10% shorter in elderly than in young subjects. The findings of this study suggest that noninvasive detection of T₂ provides useful biological information on changes in the cellular microenvironment that take place during aging.     

Magnetic resonance studies on membrane and model membrane systems: II. Phosphorus spectra and relaxation rates in dispersions of lecithin

We present phosphorus magnetic resonance (PhMR) spectra, relaxation rates, and chemical shifts for unsonicated and sonicated lecithins in aqueous dispersions and for egg lecithin in chloroform and methanol. Aqueous lecithin dispersions are characterized by long values for T₁ and considerably shorter values for T₂. Both of these values as well as the value of the linewidth change with sonication. Lecithin dispersions in methanol and chloroform have relaxation rates shorter than those seen for sonicated lecithin. We do not, at this time, present a detailed interpretation of these results. On an empirical level, however, since the relaxation rates are sensitive to the type of dispersion and possibly to the solvent, we are optimistic that they will be sensitive to structural changes involving the headgroup region.     

Enolase Activity and Isoenzyme Distribution in Human Brain Regions and Tumors

Abstract: The distribution of enolase (EC 4.2.1.11) activity and isoenzymes in various regions of human brain at different ages (from 23 weeks of gestation to 95 years) and in brain tumors has been determined. Total enolase activity increased in all regions with age. No significant differences were found in the relative proportions of αα-, αγ-, and γγ-enolase isoenzymes in the various brain regions, determined by agarose gel electrophoresis. Type αα-enolase was the predominant isoenzyme, and αγ-enolase represented a substantial proportion of the total enolase activity. Astrocytomas, anaplastic astrocytomas, glioblastomas, and meningiomas possessed lower enolase activity than normal brain. Among astrocytic tumors, total enolase activity correlated with malignancy. Astrocytomas possessed the lowest and glioblastomas the highest enolase activity. All tumors possessed a higher proportion of αα-enolase and a lower proportion of γγ-enolase than the normal human brain. Among astrocytic tumors, glioblastomas were the tumors with the highest proportion of αα-enolase and lowest proportion of γγ-enolase.     

A proton nuclear magnetic resonance study of the physical changes in growing plant cell walls

Changes in broadline proton nuclear magnetic resonance parameters of cell walls during growth of etiolated hypocotyls of bean (Phaseolus vulgaris L.) indicate that cell wall structure becomes more rigid during development. Most of the changes are completed in the first 6 cm below cotyledon insertion and are correlated with increased restriction of proton movements in regions of dense polymer packing.Abbreviations FID free induction decay - M₂ second moment - M_2interpair interpair second moment - NMR nuclear magnetic resonance - T_1D dipolar relaxation time - T₂ spin-spin relaxation time This work was supported by grants from Natural Sciences and Engineering Research Council of Canada to A.L.M., I.E.P.T. and M. Bloom.     

High-frequency1H NMR studies of the effects of growth factors and phorbol esters on normal syrian hamster diploid fibroblast cells

The nuclear magnetic resonance (NMR) parameters, spin-lattice (T₁), and spin-spin (T₂) relaxation time, are usually longer for neoplastic cells than for normal cells of the same cell type. This has generally been true at low NMR frequencies (≤100 MHz) when comparisons have been made between normal and neoplastic cells that have both spent a short time in culture. We have previously demonstrated that although the T₁ values of paired normal and neoplastic Syrian hamster (SH) fibroblastic cells in culture are not significantly different when measured at 300 MHz, the 300 MHz T₂ values for the neoplastic cells are smaller than those of the normal cells. (Xin et al. (1986),Cell Biophysics 8, 213.) Since treatment of normal diploid cells with polypeptide growth factors or tumor promoters frequently results in reversible expression of neoplasia-associated phenotypes, T₁ and T₂ were obtained at 300 MHz for treated and untreated SH cells to see if these compounds could also produce smaller 300 MHz T₂ values. Secondary culture SH fetal fibroblast cells were treated with epidermal growth factor (EGF), fibroblast growth factor (FGF), phorbol-12,13-didecanoate (PDD) and 4-α-phorbol-12,13-didecanoate (4αPDD). Treatment with either growth factor resulted in smaller T₂ values, but a statistically significant decrease was not observed for PDD or 4αPDD. The observed reductions in T₂ values were correlated with the morphological and growth-stimulatory effects of these compounds on the cells.