Anoxia followed by hyperoxia: In vivo 31-P NMR of cat brain

In vivo ³¹P NMR spectroscopy was performed on a cat brain subjected to an extended period of anoxia followed by restoration of oxygen. High energy phosphate spectra were continuously obtained and pH measured. Following the onset of anoxia, phosphocreatine and ATP peaks decreased with a concomitant increase in inorganic phosphate. Following 34 min ventilation on 100% N₂, the animal was ventilated on 100% O₂. The spectral content progressively changed, inorganic phosphate decreased and ATP increased with the spectrum closely resembling that of control. Our results suggest that the absence of NMR detectable ATP signal cannot be interpreted as an irreversable change in cellular metabolic function.     

人淋巴瘤白血病细胞系Molt-4的31P核磁共振研究

用³¹P核磁共振(NMR)观察了人淋巴瘤白血病细胞系Molt-4的³¹P谱.NMR测试温度为37℃、20℃和4℃时,随着测试时间增长,细胞³¹P谱中ATP峰降低,无机磷峰升高,细胞³¹P谱的变化随测试温度降低依次减缓.实验温度为4℃,从收集细胞至开始NMR累加在30min内,NMR累加在60min内,获得活细胞的稳定的³¹P谱.根据细胞内无机磷的化学位移数值,对细胞内pH值进行了测定.     

Relayed magnetization transfer from nuclear Overhauser effect and chemical exchange observed by in vivo ³¹P MRS in rat brain

The ³¹P magnetization transfer effects among nuclear magnetic resonances (NMRs) of phosphocreatine (PCr), γ-adenosine-5'-triphosphate (γ-ATP) and inorganic phosphate (Pi) have been attributed to the chemical exchange reactions among PCr, ATP and Pi catalyzed by creatine kinase (CK) and ATPase enzymes and, therefore, are commonly applied in situ to measure chemical exchange fluxes involving two chemically coupled CK and ATPase reactions (i.e., PCr↔ATP↔Pi) by selectively saturating γ-ATP resonance. Besides the expected reductions in the Pi and PCr NMR signals upon saturating γ-ATP resonance, one particularly interesting phenomenon showing decreases in α-ATP and β-ATP signals was also observed. The underlying mechanism was investigated and identified via saturating NMR of β-ATP in the present study. The unique relayed magnetization transfer effects through spin diffusion were observed in the rat brain using in vivo ³¹P magnetic resonance spectroscopy.     

Bio-energetic impairment in human calf muscle in thyroid disorders: a P MRS study

Mitochondrial metabolism particularly oxidative phosphorylation is greatly influenced by thyroid hormones. Earlier studies have described neuromuscular symptoms as well as impaired muscle metabolism in hypothyroid and hyperthyroid patients. In this study, we intend to look in to the muscle bioenergetics including phosphocreatine recovery kinetics based oxidative metabolism in thyroid dysfunction using in vivo ³¹P nuclear magnetic resonance spectroscopy (MRS). ³¹P MRS was carried out at resting state on 32 hypothyroid, 10 hyperthyroid patients and 25 control subjects. Nine out of 32 hypothyroid patients and 17 out of 25 control subjects under went exercise protocol for oxidative metabolism study and performed plantar flexion exercise while lying supine in 1.5 T magnetic resonance scanner using custom built exercise device. MRS measurements of inorganic phosphate (Pi), phosphocreatine (PCr), phosphodiesters (PDE) and adenosine triphosphate (ATP) of the calf muscle were acquired during rest, exercise and recovery phase. PCr recovery rate constant (k_PCr) and oxidative capacity were calculated by monoexponential fit of PCr versus time (t) at the beginning of recovery. During resting condition in hypothyroid patients, PCr/Pi ratio was reduced whereas PDE/ATP and Pi/ATP were increased. However, in case of hyperthyroidism, an increased PCr/Pi ratio and reduced PDE/ATP and Pi/ATP were observed. The results confirmed differential energy status of the muscle due to increased or decreased levels of thyroid hormone. Our results also demonstrate reduced oxidative metabolism in hypothyroid patients based on PCr recovery kinetics. PCr recovery kinetics study after exercise revealed decreased PCr recovery rate constant (k_PCr) in hypothyroid patients compared to controls that resulted in decrease in oxidative capacity of muscle by 50% in hypothyroids. These findings are consistent with a defect of high energy phosphate mitochondrial metabolism in thyroid dysfunction.     

Controlled ventilation during NMR spectroscopic studies: hemodynamic and biochemical consequences

The effects of different ventilation methods on cardiac output measured by the indicator-dilution method, liver blood flow measured by a deuterium washout technique using 2H nuclear magnetic resonance (NMR) and liver concentrations of ATP and intracellular pH determined with 31P NMR were compared in anesthetized rats. No differences in mean arterial blood pressure were demonstrable with the different modes of ventilation. However, significant drops in cardiac output were observed between freely breathing and animals ventilated with positive pressure but not the high frequency oscillatory method (407 +/- 46 and 520 +/- 88 vs. 633 +/- 86 ml/min/kg, p less than 0.05 and p = NS, respectively). Moreover, liver blood flow was significantly reduced during positive pressure but not high frequency oscillatory ventilation compared with free breathing rats (32 +/- 4 and 43 +/- 10 vs. 46 +/- 8 ml/100 g, p less than 0.05 and p = NS, respectively). 31P NMR spectroscopy revealed no effects of either ventilation method on tissue ATP or intracellular pH as estimated by the chemical shift of inorganic phosphate. These data suggest that controlled ventilation in normal rats accomplished with standard positive pressure methods is associated with major decreases in cardiac output and liver blood flow despite maintenance of normal blood pressure. High frequency oscillatory ventilation appears to effect less compromise of cardiac output and hepatic perfusion than positive pressure ventilation and may, therefore, be preferable for some biological studies.     

Role of inorganic phosphate during electron transfer in the redox system NADH+flavin

We have used ³¹P NMR and EPR to study the effect of inorganic phosphate (P_i) on the process of electron transfer in oxidation of reduced nicotinamide adenine dinucleotide (NADH) by flavin in aqueous solutions. Observation of a significant (up to 40%) drop in the integrated intensity of the ³¹P NMR signal for the inorganic phosphate over the course of the reaction is interpreted as a manifestation of the effect of chemically induced polarization of the phosphorus nuclei during singlet-triplet conversion in charge-transfer complexes [NADH…P_i], and is evidence for a possible catalytic role for P_i in processes involving transfer of an electron (hydrogen atom) from NADH to appropriate acceptors. The results presented support the possibility of direct involvement of P_i in a chain of one-electron conversions in chemical systems. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 73, No. 3, pp. 392–396, May–June, 2006.     

Considerations for brain pH assessment by 31P NMR

Recently in vivo NMR spectroscopy has been used to measure brain pH non-invasively. Both the inorganic orthophosphate (Pi) chemical shift (delta) and the difference between the chemical shifts of phosphocreatine (PCr) and Pi(delta delta PCr-Pi) have been proposed as indicators of brain pH. However, the precise delta of Pi may be difficult to determine under normoxic conditions as is the delta of PCr under hypoxic/ischemic conditions. Ideally one needs a NMR delta parameter that: (1) linearly changes between pH 6.0-8.0, (2) is either relatively unaffected or predictably affected by cations (e.g., Mg2+) other than H+, and that (3) comes from readily observable 31P NMR resonances whose delta's can be accurately assessed under all physiological conditions. Therefore, we undertook a systematic 31P NMR study of the pH and Mg2+ titration curves for 16 phosphorus-containing metabolites observed in brain by 31P NMR. On the basis of the titration curves, the delta delta's for PCr-Pi, phosphoethanolamine (PE)-Pi, and PCr-PE fulfill criteria (1) and (2), but not criterion (3). However, the delta delta of ATP gamma-alpha fulfills all three criteria and potentially provides information on the intracellular Mg2+ concentration.     

维拉帕米对离体大鼠心脏缺血-再灌注损伤保护作用的31P核磁共振研究

为评价维拉帕米(Ver)防治心脏缺血-再灌注损伤的作用,采用³¹P核磁共振(^3lP NMR)技术对大鼠心肌缺血前,缺血过程中及缺血后高能磷化物的含量及细胞内pH (pH_i)的变化过程进行了动态跟踪测定,离体心脏于37℃下平衡灌流30min,停止灌流30min,再灌注30min.灌流液中始终含有Ver (0.2μmol·L^-1).Ver可使再灌注后心脏的冠脉流量有较高程度的恢复,^3lP NMR测定显示Ver可使心脏产生代谢上的改善作用.缺血10min后对照组心脏即检测不到磷酸肌酸(PCr),而Ver组心脏PCr仍保持在缺血前的20%.缺血过程中治疗组比未治疗组心脏ATP下降减缓,至缺血结束时心肌ATP分别为缺血前的53%和34%.再灌注后两组心肌的ATP均未回升,但Ver使PCr的恢复显著提高(P<0.05),给药心脏PCr/P_i(无机磷酸盐)无论在缺血前或再灌注阶段,都非常显著(P<0.01)地高于对照组心脏.Ver还可显著减轻缺血过程中的酸中毒并防止再灌注后心肌出现严重酸化的区域.     

<Superscript>31</Superscript>P NMR Manifestation of Metabolic Changes in Blood of Spinal Cord Injured Rats

The ³¹P nuclear magnetic resonance spectroscopy technique was applied to study the blood of rats with a 3-day spinal cord injury and control rats. Phosphorus-containing blood metabolites: 2,3-diphosphoglycerates, inorganic phosphates, phospholipids, ATP and adenosine monophosphates were detected and quantitatively evaluated. It was found that the amount of 2,3-diphosphoglycerates, inorganic phosphates, phospholipids and adenosine monophosphates increase, and pH of the blood decreases after spinal cord injury. The results demonstrate increased hypoxia in injured rats.     

Quantitation of phosphorus metabolites in newborn human brain using internal water as reference standard

A new method for noninvasive, in vivo quantitation of cerebral phosphorus (³¹P) metabolites is described. The technique employs point-resolved spectroscopy (PRESS) to obtain both ³¹P-metabolite and proton (¹H) water spectra: brain water is used as an internal concentration reference. Spin-spin relaxation times (T₂s) of cerebral ³¹P metabolites are much longer than the minimum echo time (TE) usable on a spectrometer equipped with actively shielded gradient coils. With short-TE (≈10 ms) ³¹P PRESS, T₂ relaxation is minimal and phase modulation of the nucleotide triphosphate (NTP) multiplets can be accounted for. ¹H water spectra were acquired using several TEs so that extra- and intracellular water signals could be separated from that due to cerebrospinal fluid. Prior calibration of the ³¹P and ¹H spectrometer channels and an assumed brain-water concentration enabled estimations of metabolite concentrations. Using this method, mean ³¹P metabolite concentrations in the brains of eight normal infants of gestational plus postnatal age 34 to 39 wk were: phosphomonoester (PME) 5.6 (SD 0.9); inorganic phosphate 1.4 (0.4); mobile phosphodiester 2.3 (0.6); phosphocreatine 2.9 (0.3); nucleotide triphosphate 2.8 (0.6); and total mobile phosphate 21.4 (2.8) mmol/kg wet.     

离体心脏的31P NMR谱

³¹P NMR谱自70年代后期开始被用于离体心脏中含磷化合物代谢的动态研究.^3lP NMR谱不仅可以鉴定心肌组织中浓度较高的含磷化合物,即ATP、磷酸肌酸和无机磷酸盐,而且可提供有关这些化合物化学环境(包括pH和Mg²⁺)和细胞内分布的信息.利用这一非损伤性的定量分析方法,可连续观测心肌的代谢并可同时测定离体心脏的机械功能.利用饱和转移技术还能对完整心脏中肌酸激酶和腺苷酸激酶反应的速率加一测定.本文综述介绍了近年来进行离体灌流心脏^3lP NMR谱测定的实验方法以及这一技术所提供的各种信息.     

Pre-transplantation 31P-magnetic resonance spectroscopy for quality assessment of human pancreatic grafts – A feasibility study

ObjectiveTo investigate the feasibility of using ³¹P-MRS for objective non-invasive quality assessment of human pancreas grafts prior to transplantation or islet isolation.Materials and methodsPancreata from 5 human donors, 3 males and 2 females, aged 49–78 years, with body mass index (BMI) 22–31 kg/m², were included. Pancreata were perfused with histidine-tryptophan-ketoglutarate solution during procurement and stored in hypothermic condition (4 °C) for 21–44 h. During the period of hypothermic storage repeated spectra were obtained for each graft by ³¹P-MRS (1.5 Tesla) to measure the cold ischemia time (CIT) dependent changes of the phosphorous metabolites adenosine triphosphate (ATP), phosphomonoesters (PME), phosphodiesters (PDE) and inorganic phosphate (Pi), in the grafts. Graft temperature was measured immediately before and after MR-examination. Reference spectrum for non-viable tissue was obtained after graft exposure to room temperature.ResultsPME/Pi, PDE/Pi and ATP/Pi spectral intensities ratios decreased with increasing CIT, reflecting the decreased viability of the grafts. PME/Pi ratio was the most discriminatory variable at prolonged CIT. ³¹P-MRS could be performed without significantly increasing graft temperature.Conclusions³¹P-MRS may provide quantitative parameters for evaluating graft viability ex vivo, and is a promising tool for objective non-invasive assessment of the quality of human pancreas grafts prior to transplantation or islet isolation.     

用3lP NMR研究小鼠脑在缺氧状态下能量代谢的动态变化

用³¹P体内核磁共振方法无损伤地观察了C57小鼠脑在缺氧时与能量代谢有关的含磷化合物Pi,PCr,ATP的动态变化,当气体中氧气含量降到5%时,小鼠大脑的PCr,ATP逐渐减少,Pi不断增加,脑组织内pH值降低至6.83~6.93.当Pi与PCr的峰高比等于1时给予复氧,发现复氧时Pi立即降低且pH立即升高的小鼠可以恢复,反之则不可恢复,表明Pi和pH反映了大脑缺氧损伤程度.     

ESR and31P NMR study of metabolic changes in mouse tumor and liver after combined misonidazole and irradiation treatment

A combination of ESR and³¹P NMR techniques was used to study the mechanism of the radiosensitizing influence of misonidazole (MISO) on mouse tumors and liver. The MISO injection (1 g/kg) was shown to cause significant metabolic changes in tumors, such as an increase in the content of nitrosyl complexes, increases in adenosine diphosphate and inorganic phosphate levels, and decreases in phosphomonoesters, phosphodiesters, adenosine triphosphate and phosphocreatine in the initial period of observation. These alterations were accompanied by slight cytosol alkalinization (pH shift ca 0.15). A decrease in the ESR signal intensity of Mo⁵⁺-containing enzymes, an increase in the cytochrome P-450 signal and a significant increase in the free radical signal were observed in liver after MISO treatment. Local irradiation of tumors (30 Gy) 30 min after MISO injection led to the enhancement and prolongation of nearly all the effects induced by MISO treatment. The combined effect of MISO and irradiation was not simply the sum of their separate effects. It is suggested that radiation enhances cytotoxic effects of nitrocompounds on hypoxic tumor cells, inhibiting glycolysis and inducing greater production of toxic and highly reactive intermediates resulting from incomplete nitro group reduction. The latter appears to be considered as the major factor involved in the radiosensitizing action of nitrocompounds.     

Observations of energy metabolism in neuroectodermal tumors using in vivo 31P-NMR

The energy metabolism of living tumors in rats and hamsters were investigated by obtaining in vivo 31P-NMR spectra, and the effects of chemotherapy on tumors were evaluated by observing the changes of these spectra. Tumor cells of rat glioma, human glioblastoma and human neuroblastoma were inoculated subcutaneously in the lumbar region of the animals. After the tumor grew to over 1.5 cm in diameter, in vivo 31P-NMR spectrum data was obtained selectively from the tumor with a TMR-32 spectrometer (Oxford Research Systems, U.K.). Several peaks (ATP, inorganic phosphate (Pi), phosphodiesters and phosphomonoesters (PME) were observed in the tumors. The heights of these peaks varied widely corresponding to the tumor growth. However, the spectrum pattern of each tumor in an active stage was found to be essentially the same regardless of histological type or tumor origin. The phosphocreatine (PCr) peak was small, ATP and PME peaks were large and tissue pH calculated from the chemical shift of Pi was low in each tumor group. After intravenous injection of a large dose of a chemotherapeutic agent, ATP peaks decreased and the Pi peak increased gradually, resulting in a dominant Pi peak pattern after several hours in all groups. With lower drug doses, spectrum changes were temporarily seen in the tumors. These findings indicated that drugs with a high dose have a selective and a direct action on the energy metabolism of tumor tissues. In vivo 31P-NMR spectra measurement is very valuable not only to investigate the energy metabolism in tumor tissue but also to evaluate the effects of chemotherapy on the tumor.     

细胞灌注-核磁共振联用方法的研究

该文报道了研制的一套细胞灌注装置,并运用该细胞灌注装置与核磁共振连用的方法在线监测药物作用下肿瘤细胞的能量代谢过程. 结果表明核磁共振细胞灌注方法可有效观察到细胞能量代谢的变化,可作为追踪筛选药物的活性成分及探索药物治疗肿瘤细胞的作用机理的一种有力手段.     

Langendorff灌流心脏的31P NMR谱测定

测定了Langendorff灌流大鼠和兔心脏的³¹P NMR谱.观测到PCr、ATP、SP、P_i以及PME和GPC等含磷代谢物的共振峰,各谱峰之间实现了很好的分辨.在3~5min的累加时间内测得的图谱具有较好的S/N,可用于以上含磷代谢物的定量测定,以高能含磷化合物ATP和PCr相对峰强度以及冠脉流量的变化对灌流大鼠心脏代谢的稳定性进行了考察,结果表明,在本实验条件下大鼠心脏的能量储存至少可稳定2h,用所建方法以3min的时间间隔对同一大鼠心脏缺血21min及再灌注12min过程中心脏的³¹P NMR谱进行了连续跟踪测定,并初步观测了缺血及再灌注过程中心肌细胞内ATP、PCr与P_i之间的消长关系.     

31P nmr study of I–IV group polycrystalline phosphates

Polycrystalline phosphates of group I–IV elements have been studied using solid state ³¹P NMR. The relationship between the spectral parameters (the principal components of the chemical shielding tensor and obtained from MAS values of the isotropic shifts of ³¹P) and the environment of the phosphorus atoms in these compounds has been considered. The shape of the lines in stationary ³¹P NMR spectra reflects the near neighbour environment of a phosphorus atom and the degree of distortion of the PO₄ tetrahedron. The type of cation, as well as the presence of the water of crystallization in the structure affect considerably an isotropic shift of ³¹P in phosphates. The high sensitivity of the solid state ³¹P NMR to even slight variations in the structure of the phosphate anions is shown.     

Soluble Complexes of Pyridoxamine Phosphate-Pr(EDTA)−

Abstract

The equilibrium involved in the complexation of Pr(EDTA)^? ions by pyridoxamine phosphate have been studied by potentiometric and ¹H NMR titrations. Two main sites - phosphate and phenolate groups - are implied in the coordination of lanthanide ions. Vitamin B6, in it's various forms, has been demonstrated to interact with metal cationslY2. As the coordination site may vary at any state of protonation of the molecule, several forms of labile complexes may be simultaneously present when pyridoxamine phosphate (Fig. 1) is titrated with lanthanide cations. According to the pH range, phosphate, phenolic, amine or pyridine groups are potentiel candidate^3,4. These will readily coordinate to the open half sphere of the soluble Pr(EDTA)^? ions. This complex had to be used as free metal cations will precipitate in the investigated pH range^5,6. The paramagnetic shifts induced in the NMR spectra of the pyridoxamine phosphate molecule allow a monitoring of the complexation process. A correct treatement of the experimental values is described in previously report^7,8. The present note gives the ¹H chemical shifts and their ratio values for these series of complex in the range 3.2< pH< 10.3.     

美兰光化学法对红细胞损伤的NMR研究

运用³¹P NMR研究红细胞中高能磷酸化合物的代谢变化,探讨美兰光化学法对红细胞的损伤. 采用MSL-300 MHz型超导核磁共振仪分别测量健康成人红细胞对照组、经美兰（亚甲基蓝）光化学处理组及预先加入L-组氨酸组的高能磷酸化合物,通过分析比较红细胞中2,3-二磷酸甘油酸（2,3-DPG）、三磷酸腺苷（ATP）及磷酸化糖（SP）相对含量,了解美兰光化学法对红细胞代谢的影响. 经亚美兰光化学法处理的红细胞,葡萄糖进入戊糖磷酸途径的分量增加,使红细胞中2,3-DPG和ATP含量显著减少,而SP含量大幅上升,在某种程度上影响红细胞的理化性质和功能指标;L-组氨酸对此有一定的保护作用.