Simultaneous separation of intracellular and extracellular lactate NMR signals of human erythrocytes.

Intracellular/extracellular lactate (Lac) distribution has been determined before in human and animal erythrocytes (red blood cells [RBCs]) with various methods. However, all previous methods determine intra- and extracellular Lac separately or indirectly. Now, (13)C-NMR spectroscopy has been used to monitor intra- and extracellular Lac simultaneously in intact RBCs. Isolated human RBCs were incubated with [3-(13)C]-Lac, [3-(13)C]-pyruvate (Pyr), and [1-(13)C]-glucose (Gluc). A distortionless enhancement by polarization transfer (DEPT) sequence was used (TR = 3.3 s, N = 128) to monitor the (13)C-NMR resonances in both compartments. The intra- and extracellular methyl group resonances of Lac and Pyr were clearly separated by 9.6 Hz and 7.0 Hz, respectively, under normoxic conditions due to the RBC chemical-shift effect. The results show that the chemical-shift effect of RBCs is convenient to monitor intra- and extracellular Lac simultaneously in intact RBCs under normoxic conditions.     

Cholesterol effects on nonelectrolyte membrane transport in human erythrocytes: NMR magnetization transfer studies

The cholesterol content of human erythrocytes was altered by incubating them with sonicated dispersions of cholesterol/phosphatidylcholine at 37°C. ³¹P NMR saturation transfer experiments were used to measure the rate constant for efflux of dimethyl methylphosphonate (DMMP) from the cells, and thereby gain an estimate of the permeability coefficient. It was shown that up to 39% depletion of membrane cholesterol (cholesterol/phospholipid molar ratio of 0.46) increased the efflux rate constant and permeability coefficient of DMMP 1.55-and 1.86-fold, respectively. Enrichment of the membranes with cholesterol by 45% (cholesterol/phospholipid molar ratio of 1.57) on the other hand, decreased the efflux rate constant and permeability coefficient 1.63-and 1.79-fold, respectively. It was concluded that DMMP may be used as a probe molecule to study the functional consequences of changes in the lipid composition of erythrocytes in diseases that are associated with disorders of lipid metabolism.     

核磁共振测定蛋白质构象

核磁共振(NMR)已成为与X射线晶体衍射相补充的研究蛋白质构象的方法,而且其应用日益深入。本文综述了NMR测定蛋白质构象的基本原理、主要步骤、现状及前景,并对NMR的应用和成就与晶体衍射法进行了简要的比较讨论。     

A 7Li NMR study of visibility, spin relaxation, and transport in normal human erythrocytes.

The behavior of the lithium (Li) ion in normal human erythrocytes has been studied by 7Li NMR. The uptake of Li into the cells was followed as a function of solution conditions, temperature, hematocrit, and blood age using dysprosium tripolyphosphate shift reagent. Under our conditions the uptake of Li increases with increasing hematocrit and blood age. For packed cells the extracellular 7Li spin-lattice relaxation time was only slightly longer than the intracellular relaxation time. Thus, T1 may not be useful for separate observation of intra- and extracellular Li in vivo. The intra- and extracellular T2s were substantially shorter than the corresponding T1s. Also, the intracellular T2 was considerably shorter than that for the extracellular compartment, suggesting that T2 may provide a noninvasive handle for observation of intracellular Li. Nuclear Overhauser enhancements could be observed for both extra- and intracellular 7Li, confirming that dipolar coupling to 1H is a contributing relaxation mechanism. The 7Li NMR visibility was essentially 100% at high Li concentrations, decreasing to about 84% at 1 mM Li. Based on time course studies of the invisibility, and a comparison of NMR and inductively coupled plasma results, it appears that the invisibility of the intra- and extracellular compartments for packed cells is the same. Although a 23Na double-quantum signal could be observed for red blood cells, no double-quantum signal was observed for 7Li.     

13C NMR investigation of synovial fluids

¹³C NMR spectra of synovial fluids from 20 patients suffering from rheumatic diseases have been recorded. Structural changes in hyaluronic acid, the main carrier of the viscoelastic properties of synovial fluid, could be observed in the NMR spectra of the native biological fluid. By comparing these spectra with those of purified hyaluronic acid, a rough estimation of the degree of depolymerization of synovial hyaluronlic acid was possible. The patients with rheumatoid arthritis appeared to have a lower degree of polymerization compared to patients with osteoarthrosis. Thus, ¹³C NMR spectroscopy prolvides useful information about biophysical properties of sartorial fluid.     

Fructose 3-phosphate and 5-phosphoribosyl-1-pyrophosphate formation in perhsed human erythrocytes: 31P NMR studies

³¹P NMR was used to study the formation of fructose 3-phos-phate (F3P) and 5-phosphoribosyl-1-pyrophosphate (PRPP) in perfused human erythrocytes, in the presence of 10 different combinations and concentrations of glucose, inosine, pyru-vate, fructose, and inorganic phosphate (P_i). (1) The cells were immobilized in alginate-coated agarose threads and perfused with a medium containing fructose, and the level of F3P increased continuously over more than 10 h. The net rate of F3P formation was independent of the concentration of 2,3-bis-phosphoglycerate (2,3-DPG) present in the cells. (2) PRPP was formed in high concentrations, relative to normal, in immobilized cells when they were perfused with a medium containing P_i at a low pH (6.6). (3) The 2,3-DPG level decreased simultaneously when the sample was perfused with a medium containing fructose, but without inosine or pyruvate. The measured intracellular pH and free Mg²⁺ concentration were constant in these experiments. (4) The experiments confirmed the presence of fructose-3-phosphokinase (E.C. 2.7.1.-) and ribose-phosphate pyrophosphokinase (E.C. 2.7.6.1) activity in the human erythrocytes and that the biosynthetic pathways are active in immobilized cells at 37°C. (5) The rates of accumulation of 2,3-DPG and phosphomonoesters (PME) appeared to be strongly correlated.     

NMR studies of erythrocytes immobilized in agarose and alginate gels.

31P and 13C NMR were used to study the energy metabolism in perfused, human erythrocytes. The erythrocytes were immobilized in agarose threads, Ca- or Ba-alginate beads, and Ba-alginate-coated agarose threads. Erythrocytes were easily washed out from the agarose threads, but not from alginate-containing gels. Various small molecules, such as hypophosphite, dimethyl methylphosphonate, and methylphosphonate, were taken up from the perfusion medium in a normal manner. In addition, the 2,3-bisphosphoglycerate (2,3-DPG) chemical shifts were sensitive to the oxygen partial pressure suggesting that O2 molecules were diffusing through the gel and modifying the binding of 2,3-DPG to hemoglobin. A combination of inosine and pyruvate stimulated the synthesis of 2,3-DPG, but only if inorganic phosphate was present in the perfusion medium. Inosine only resulted in a dramatic rise in the intracellular sugarphosphate concentrations. Furthermore, [2-13C]glucose was converted to [2-13C]lactate by immobilized cells at a rate which was comparable to that in a control suspension. In summary, immobilization in Ba-alginate-coated agarose threads was an efficient way of trapping human erythrocytes for whole cell NMR investigations.     

NMR visibility of 39K and 35Cl in erythrocytes and kidney tubules

The NMR visibility of 39K and 35Cl has been investigated in erythrocytes and in dog renal tubules. In erythrocytes, the 39K NMR visibility was determined by comparing the signal intensities before and after hemolysis with water and by comparing the NMR and flame photometry results. Both procedures showed a NMR visibility of 100% for intracellular potassium. The visibility of intracellular chloride in erythrocytes was estimated at 40% by monitoring the intensity of the 35Cl signal as a function of the hematocrit value. In the case of kidney proximal tubules, the 39K visibility appeared to be very low but could not be accurately determined due to the low sensitivity of the nucleus. The 35Cl signals for intracellular chloride in renal tubules were too broad to be detected.     

Ascorbate-assisted, phthalocyanine-sensitized photohaemolysis of human erythrocytes.

The rate of photohaemolysis of human red blood cells sensitized by chloroaluminium phthalocyanine sulphonate is increased by ascorbate, with or without added FeCl3. Stimulation of haemolysis by ascorbate without addition of metal salt, and in the presence of a strong chelator such as desferrioxamine, is an unexpected phenomenon. Lysis rate and ascorbate concentration were directly related, suggesting that ascorbate acts as a reactant and not as a catalyst. The process also requires oxygen; azide and D2O tests indicate some participation of singlet oxygen, although to a lesser extent than in the photosensitized haemolysis in the absence of ascorbate. Kinetic considerations suggest a reaction path initiated by excited sensitizer and ascorbate, parallel to the singlet oxygen-mediated process. Because of the ubiquitous presence of ascorbate in human tissues in concentrations comparable to those of dissolved oxygen, it is quite possible that in photodynamic therapy a fraction of the photodynamic damage proceeds via a Type I, ascorbate-assisted, mechanism.     

31P NMR relaxation time studies of 2,3-diphosphoglycerate in solution and intact erythrocytes

The 31P T1 spin-lattice relaxation times and nuclear Overhauser effects of 3-P and 2-P in 2,3-diphosphoglycerate (2,3-DPG) have been measured in pure water solutions and in suspensions of intact erythrocytes. It was found that extremely careful purification of the water solutions from paramagnetic impurities was necessary in order to obtain reproducible results. The dominant relaxation mechanism in purified samples was shown to be the dipole-dipole interaction. Contributions from the chemical-shift anisotropy mechanism were demonstrated to be important at higher magnetic field strengths. Based upon the measurements in water solutions and intact erythrocyte suspensions it was concluded that there could be observed no significant influence of the oxygenation state of hemoglobin on the 31P T1 values of 2,3-DPG.     

长春新碱载体红细胞的体外抗肿瘤作用

目的探讨长春新碱(VCR)载体红细胞的体外抗肿瘤活性。方法采用改良低渗预膨胀技术制备VCR载体红细胞,并将VCR载体红细胞与人红白血病细胞株K562共培养,MTT法检测K562细胞增殖,PI/Rhodamin123染色,流式细胞分析K562细胞周期和细胞凋亡情况,Hoechest33258细胞核染色观察凋亡K562细胞核形态。结果VCR载体红细胞可明显抑制K562细胞增殖并促其凋亡,且该作用随剂量增高而加强。与载体红细胞混合培养24h后,S+G2/M期K562细胞明显增多,G0/G1期细胞减少,与VCR原药作用一致,与未载药红细胞的作用相比差异显著(P<0.01)。结论经红细胞载药并释放出来的VCR在体外具有与原药相同的抗肿瘤活性。     

31P NMR measurements of intracellular pH in erythrocytes: direct comparison with measurements using freeze-thaw and investigation into the influence of ionic strength and Mg2+

The pH-dependent chemical shift behavior in 31P NMR spectroscopy of inorganic phosphate (Pi) and methylphosphonic acid (MeP) in solutions of various composition at 37 degrees C are presented. An ionic strength dependent lowering of the pKa values of both Pi and MeP is observed. 3 mmol/liter Mg2+ did not affect the pH-dependent chemical shift behavior of Pi and MeP in solutions containing 135 mmol/liter KCl and 5 mmol/liter NaCl. This finding was reinforced by adding 5 mmol/liter 2,3-diphosphoglycerate (2,3-DPG) and 1.5 mmol/liter ATP to solutions with 3 mmol/liter Mg2+. The presence of these Mg2+-binding substances had no detectable effect on the chemical shift of Pi and MeP at various pH values. The pH dependence of the chemical shift of MeP, Pi, and 2,3-DPG in oxygenated erythrolysates was compared to protein free model solutions. No significant differences could be detected for MeP and Pi whereas there was a change in the chemical shift of about 0.1-0.2 ppm for both phosphates in 2,3-DPG. Finally intracellular pH estimated by 31P NMR were compared to pH measured by electrode in freeze-thawed hemolysates. At an extracellular pH of 7.341 +/- 0.045 (SD) (n = 6) the intracellular pH was 7.160 +/- 0.014 (SD) by the freeze-thaw procedure and 7.213 +/- 0.027 (SD), 7.172 +/- 0.029 (SD), and 7.152 +/- 0.033 (SD) estimated by the chemical shift of MeP, and 3-phosphorus and 2-phosphorus of 2,3-DPG, respectively.     

Multinuclear NMR investigation of probe construction materials at 9.4T.

This work investigates probe construction materials for their signal contribution to ultrashort echo time spectroscopy and imaging. (1)H, (13)C, and (31)P spectra were obtained at a field strength of 9.4 T for 16 materials considered for use in probe and holder design and construction. Four of the materials were found to be suited for the construction of NMR probes, housing of RF coils, and holders for in vivo experiments.     

31P NMR investigation of energy metabolism in perifused MMQ cells

The MMQ cell line is a unique prolactin-secreting rat pituitary cell line. MMQ cells entrapped in agarose gel threads are metabolically active, as determined by the uptake and phosphorylation of creatine and the maintenance of high energy phosphates for over 15 h. Forskolin activates the catalytic subunit of adenylyl cyclase and, in MMQ cells, elevates the level of cAMP and stimulates prolactin secretion. ³¹P NMR spectroscopy was used to investigate the energy metabolism of the MMQ cells during stimulation by forskolin. The ability to measure small changes in the energy status of these cells was enhanced by increasing the PCr levels in the cells. Administration of forskolin to the perifused MMQ cells resulted in acute, reversible, and dose-dependent changes in the ³¹P NMR spectra of the cells within 12 to 24 min of the beginning of forskolin exposure. Several lines of evidence indicate that the changes observed in the MMQ cells are the composite result of the interaction of forskolin with adenylyl cyclase and the plasma membrane glucose transporter. Also, preincubation of the MMQ cells with the dopamine agonist, bromocriptine, attenuates the forskolin-stimulated decrease in the PCr resonance by approximately 50%. This attenuation indicates that the forskolin-stimulated changes in energy metabolism are probably related to the prolactin secretion process.     

Characteristics of metabolism of human erythrocytes after long-term space flight

Before and after the 150-day Salyut-7 flight the crewmembers were examined for their red blood metabolism, viz: major metabolic pathways (glycolytic and pentosophosphate), erythrocyte resistance, membrane permeability and lipid peroxidation rate. The resulting data indicate that the metabolic and membrane changes were not pathological and can be classified as adaptive.     

In vitro NMR evaluation of human thyroid lesions

In vitro analysis of spin-lattice relaxation times (T1), water self-diffusion coefficients (DH2O), and proton NMR spectroscopy were performed in a study of 88 patients with thyroid lesions in order to determine the usefulness of these parameters in the differentiation of benign and malignant tissues. Thyroid tissue sample proton NMR spectral patterns were examined at 360 MHz. Proton NMR spectra were different for normal thyroid tissues, benign, and cancerous lesions. Significantly prolonged T1 (0.5T) and decreased DH2O were found in cancerous thyroid lesions relative to normal thyroid tissues. Considerable overlap was found, however, in comparing T1 and DH2O values for benign and malignant thyroid lesions. This study suggests that proton NMR spectroscopy may be more useful than T1 and DH2O in differentiating benign and malignant thyroid lesions.     

戊二醛对长春新碱载体红细胞生物学特性的影响

目的 探讨控制长春新碱(VCR)载体红细胞体外药物释放,增强载体红细胞稳定性的方法和条件.方法 采用改良的低渗预膨胀-等渗重封闭法制备VCR载体红细胞,分别用0.16%和0.25%戊二醛溶液进行处理,同时将载体红细胞悬液与药量PBS混匀作为对照组.通过4℃保存定时提取上清液,以高效液相色谱法测定VCR含量,比较不同处理方式对VCR载体红细胞载药释放的影响,通过观察上清液可见溶血和细胞渗透脆性检测,比较各种方式处理后VCR载体红细胞的贮存稳定性和渗透脆性,并观察处理后VCR载体红细胞的形态变化.结果 各组VCR载体红细胞的载药累积释放均随时间延长而逐渐增大.与对照组相比,.25%戊二醛溶液处理组载药累积释放下降了71.67%±4.20%,稳定贮存时间由5天延长至21天,差异有显著性(P＜0.01),渗透脆性和细胞形态无明显改变;0.16%戊二醛溶液处理组载药累积释放无明显变化,稳定贮存时间增加至7.5天,而渗透脆性有所增大.结论 0.25%戊二醛溶液对人源VCR载体红细胞具有较好的细胞膜封闭和稳定作用.     

高胆固醇血症红细胞超微结构研究

 目的 探讨高胆固醇血症红细胞超微结构及发病机制.方法 以透射电镜观察高胆固醇血症(>6 mmol/L)20例亚健康人血红细胞超微结构.结果 所选20例亚健康人血红细胞超微结构均有不同程度变异.结论 高胆固醇血症红细胞超微结构的改变是导致血液流变学异常的主要原因之一.