Free dG Research Articles

Spectrophotometric study of the interaction of methylene blue with poly(styrene-co-sodium styrene sulfonate)

The interaction of the cationic phenothiazine dye, the Methylene Blue (MB) with poly-(sodium styrene sulfonate) f –co-(styrene) 1-f , (PSSNa f ), has been investigated by spectrophotometric method. The polyelectrolyte induced metachromasy resulting in a blue shift of the absorption maxima of the dye, in agreement with the formation of dye H-aggregates. The stability of the PSSNa-MB complexes was studied as a function of polyelectrolyte chain length, polyelectrolyte electrostatic charge density f , polyelectrolyte concentration, NaCl salt addition, tetrahydrofurane (THF) addition and THF treatment. The stoichiometry of PSSNa-MB complex evaluated by the molar ratio method was found 4:1 for the fully charged PSSNa f = 1. Reversal of metachromasy was observed upon salt and THF addition, while THF treatment does not affect the complex and allows recovering the initial complex. Finally, thermodynamic parameters of the interaction between the polyelectrolyte and the dye at different temperatures, namely free energy DG, the enthalpy DH and the entropy DS have been evaluated to determine the binding constant and as a consequence the stability of the complex. The metachromasy effect was found to be more high as the chemical charge f increases and reaches its maximum value f = 1, when operating at optimal conditions. So, the PSSNa f = 1-MB complex is the most stable in comparison to the others based on lower charge density PSSNa f .

Lipase-Catalyzed Preparation of Diacylglycerol-Enriched Oil from High-Acid Rice Bran Oil in Solvent-Free System

The ability of immobilized lipase from Rhizomucor miehei (Lipozyme RM IM) to catalyze the reaction of high-acid rice bran oil (RBO) and monoglyceride (MG) for diacylglycerol-enriched rice bran oil (RBO-DG) preparation was investigated. The effects of substrate ratio, reaction temperature, time, and enzyme load on the respective content of free fatty acid (FFA) and DG in the final RBO-DG products was investigated. Enzyme screening on the reaction was also investigated. Response surface methodology (RSM) was used to optimize the effects of the reaction temperature (50-70 °C), the enzyme load (2-6 %; relative to the weight of total substrates), and the reaction time (4-8 h) on the respective content of FFA and DG. Validation of the RSM model was verified by the good agreement between the experimental and the predicted values. The optimum preparation conditions were as follows: MG/RBO, 0.25; temperature, 56 °C; enzyme load, 4.77 %; and reaction time, 5.75 h. Under the suggested conditions, the respective content of FFA and DG was 0.28 and 27.98 %, respectively. Repeated reaction tests indicated that Lipozyme RM IM could be used nine times under the optimum conditions with 90 % of its original catalytic activity still retained.

Preparation of TiO2 nanoparticles by sonochemical method, isotherm, thermodynamic and kinetic studies on the sorption of strontium

The objective of this study is to evaluate the use of titanium dioxide nanoparticles which were prepared by novel sonochemical method as an ion exchange material for the removal of Sr from aqueous solution. The pH effect on the Sr 2? sorption was investigated. The data obtained have been correlated with Freundlich, Temkin and Dubi- nin-Radushkevich (D-R) isotherm models. Thermody- namic parameters fort he sorption system have been determined at four temperatures. Simple kinetic models have been applied to the rate and isotherm sorption data and the relevant kinetic parameters were determined from the graphical presentation of these models at 298K. Results explained that the pseudo second-order sorption mechanism is predominant and the overall rate constant of sorption process appears to be controlled by chemical sorption process. The value of sorption energy E = 13 kJ/ mol at 298K and the value of Gibbs free energy DG = 3,222 kJ/mol at 298K prove that the sorption of strontium on titanium dioxide nanoparticles is an endo- thermic and non-spontaneous process.

Equilibrium and Thermodynamic Studies of Cesium Adsorption on Natural Vermiculite and Optimization of Operation Conditions

Removal of cesium from synthetic aqueous solution through adsorption on vermiculite, under batch equilibrium experimental condition at six initial values of pH (3, 4, 6, 9, 11 and 12) and five temperatures (25, 50, 75, 85 and 95 °C) has been investigated. It is necessary to propose a suitable model for a better understanding of the mechanism of cesium adsorption on vermiculite. For this propose the suitability of the Langmiur, Freundlich and Redlich-Peterson (R-P) adsorption models for equilibrium data were investigated. The parameters in the adopted adsorption isotherm models were determined by Eviews software. The study of equilibrium isotherm shows that the best model for analysis of experimental data is Redlich-Peterson model with correlation coefficient higher than 0.99(both for temperature and pH). The results showed that increasing of pH and temperature increased the adsorption ability of vermiculite. Optimum conditions for adsorption were determined as T=75 °C, pH=9, vermiculite dose=1.5 g and contact time of 24 hr. Finally the thermodynamic constants of adsorption phenomena, DH° and DS° were found to be 2.672 kJ/mol and 0.563 kJ/mol K in the range of 25-95 °C respectively. The negative value of the Gibbs free energy DG demonstrates the spontaneous nature of cesium adsorption onto vermiculite.

Effect of Base Sequence and Deprotonation of Guanine Cation Radical in DNA

The deprotonation of guanine cation radical (G+*) in oligonucleotides (ODNs) was measured spectroscopically by nanosecond pulse radiolysis. The G+* in ODN, produced by oxidation with SO4-*, deprotonates to form the neutral G radical (G(-H)*). In experiments using 5-substituted cytosine-modified ODN, substitution of the cytosine C5 hydrogen by a methyl group increased the rate constant of deprotonation, whereas replacement by bromine decreased the rate constant. Kinetic solvent isotope effects on the kinetics of deoxyguanosine (dG) and ODN duplexes were examined in H2O and D2O. The rate constant of formation of G(-H)* in dG was 1.7-fold larger in H2O than D2O, whereas the rate constant in the ODN duplex was 3.8-fold larger in H2O than D2O. These results suggest that the formation of G(-H)* from G+* in the ODN corresponds to the deprotonation of the oxidized hydrogen-bridged (G+*-C) base pair by a water molecule. The characteristic absorption maxima of G+* around 400 nm were shifted to a longer wavelength in the order of G<GG<GGG-containing ODNs. In contrast, the spectra of G(-H)* were not affected by the sequence and were essentially similar to that of free dG. These results suggest that the positive charge in G+* in ODN is delocalized over the extended pi orbitals of DNA base. The rate constant of the deprotonation was altered by the sequence of ODNs, where bases adjacent to guanine are important factors for deprotonation.

Direct observation of guanine radical cation deprotonation in duplex DNA using pulse radiolysis.

The dynamics of one-electron oxidation of guanine (G) base mononucleotide and that in DNA have been investigated by pulse radiolysis. The radical cation (G+*) of deoxyguanosine (dG), produced by oxidation with SO(4)-*, rapidly deprotonates to form the neutral G radical (G(-H)*) with a rate constant of 1.8 x 10(7) s(-1) at pH 7.0, as judged from transient spectroscopy. With experiments using different double-stranded oligonucleotides containing G, GG, and GGG sequences, the absorbance increases at 625 nm, characteristic of formation of the G(-H)*, were found to consist of two phases. The rate constants of the faster ( approximately 1.3 x 10(7) s(-1)) and slower phases ( approximately 3.0 x 10(6) s(-1)) were similar for the different oligonucleotides. On the other hand, in the oligonucleotide containing G located at the 5'- and 3'-terminal positions, only the faster phase was seen. These results suggest that the lifetime of the radical cation of the G:C base pair (GC+*), depending on its location in the DNA chain, is longer than that of free dG. In addition, the absorption spectral intermediates showed that hole transport to a specific G site within a 12-13mer double-stranded oligonucleotide is complete within 50 ns; that is, the rate of hole transport over 20 A is >10(7) s(-1).

The reaction of 2′-deoxyribonucleoside with the α-pyrrolidonate-bridged Pt(III) dinuclear complex

Of the four 2′-deoxyribonucleosides (dA, dG, dC, and dT), only dG coordinated to the α-pyrrolidonate-bridged platinum(III) dinuclear complex [Pt2(NH3)4(C4H6NO)2(NO3)(H2O)](NO3)3 (1) with N7 of the guanine base by replacing the NO3− ligand. The 2′-deoxyguanosine platinum(III) dinuclear complex [Pt2(NH3)4(C4H6NO)2(C10H13N5O4)(SO4)](SO4)·4H2O (2) was prepared by oxidation of the platinum (+2,25) blue complex [Pt4(NH3)8(C4H6NO)4](NO3)5·2H2O (3) with Na2S2O8 in the presence of dG. The X-ray diffraction analysis confirmed the structure of complex 2. The 1H NMR spectroscopy showed that the complex is stable at pH<4, but free dG is liberated at pH 7.

Optimization of the workup procedure for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine with electrochemical detection.

The artifactual generation of the biomarker for oxidative stress, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), during the workup procedure for its analysis is a difficult problem to solve, and the responsible factors are unclear. Here, peroxide removal and other antioxidant procedures during workup were compared using a limited amount of rat liver (50 mg) as starting material, with subsequent hydrolysis of 50 microg of DNA. A cold (0 degrees C) high salt GTC (4 M guanidine thiocyanate) nonphenol DNA extraction method was developed where DNA is quickly isolated. GSH (reduced glutathione) generated artifactual formation of 8-oxodG during the workup procedure, whereas H(2)O(2) removal using catalase, Fe(3+) removal and passivation using desferal, peroxide removal using glutathione peroxidase, ebselen and a peroxidase mimic lowered the 8-oxodG levels, all identifying peroxides as the responsible oxidants. Desferal was more protective when excluding Mg(2+) and Ca(2+) from buffers but was found to disturb the electrochemical detector when repeatedly injected five to six times, even at 100 microM. Addition of the OH(*) scavenger ethanol in all steps at 2% v/v had no protective effect. Zn(2+) was found necessary for efficient DNA hydrolysis using nuclease P(1), which was poor below 37 degrees C. Use of water substitutes was tested but inhibited DNA hydrolysis completely. H(2)(18)O could, however, work for mass spectrometry methods. Long-term (38 days) storage of 0.5% v/v Triton X-100 generated more 8-oxodG than Tween 20 when incubated with free dG. The cold GTC DNA extraction method was used for analysis of freshly isolated human lymphocytes/monocytes from 60 healthy men using catalase and TEMPO as antioxidants, giving a background level of 0.074 +/- 0.027 8-oxodG/10(5) dG (or 16 8-oxodG/10(8) nucleotides or 1943 8-oxodG/nuclei) which is probably the lowest value obtained yet. No increase with age was seen. Oxidation of dG to 8-oxodG during workup was found to fit a mathematically defined curve, and a calculated background level of 0.047 8-oxodG/10(5) dG was obtained. To obtain more reliable results it is recommended that control samples are included during the workup procedure, having an equal amount of cells (or DNA) as the exposed samples.

Kinetics and mechanisms of oxidation of 1-octene and heptanal by crown ether-solubilized potassium permanganate in non-aqueous solvents

The kinetics of oxidation of 1-octene and heptanal by 18-crown-6-ether-solubilized KMnO4 in benzene and CH2Cl2 have been investigated. In benzene, the oxidation of 1-octene is first order with respect to the oxidant and zero order with respect to the substrate, whereas in CH2Cl2 the reaction is first order with respect to both substrate and oxidant. The reaction of heptanal followed different kinetics being first order with respect to both substrate and oxidant, regardless of whether benzene or CH2Cl2 was employed as the solvent. The values of activation energy Ea, standard enthalpy DH � , standard entropy change DS � , and standard free energy DG � , for the reaction, are reported. Mechanistic pathways for the studied reactions are also proposed.

Oxidation of 2′-deoxyguanosine by H2O2–ascorbate: evidence against free OH˙ and thermodynamic support for two-electron reduction of H2O2

Of the four major nucleosides in DNA, 2′-deoxyguanosine (dG) is the most easily oxidized. The formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) by oxidation of free dG, as well as of dG in DNA, by hydrogen peroxide (H2O2) and the reducing agent ascorbate (HAsc−) was measured using HPLC with high sensitivity electrochemical detection. High concentrations of the hydroxyl radical (OH˙) scavengers PBN, DMPO, pentoxifylline and imidazole had a very limited protective effect on 8-oxodG formation, which provides evidence against free OH˙ production as the major mechanism. A possible mechanism involves a “two-electron reduction of H2O2” with oxidation of HAsc− and dG, producing the guanine radical cation (dG˙+) leading to 8-oxodG. This mechanism is, based on changes in Gibbs free energy (ΔG0′) at pH 7, much more favourable than OH˙ production. Catalase from Aspergillus niger and the aminoxyl TEMPO almost completely inhibited 8-oxodG formation, whereas bovine catalase cleaved the free dG molecule. Substantial 8-oxodG formation from free dG occurred even at 0 °C.

Evaluation of Nitrone Spin-Trapping Agents as Radioprotectors

The focus of this investigation was to determine whether the nitrone spin-trapping compounds alpha-phenyl-N-tert-butylnitrone (PBN), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) are radioprotectors. Two methods were used to assess for radioprotection: measurement of oxidative damage to DNA bases and mammalian cell survival assays. Oxidative damage to DNA was quantified by measuring the relative amounts of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) produced by the reaction of hydroxyl radicals (OH.) with 2-deoxyguanosine (dG) after irradiation. PBN, DMPO and POBN, when dissolved in aqueous solutions of either dG or naked salmon sperm DNA, reduced the formation of 8-OH-dG by 137Cs gamma irradiation significantly. The spin-trapping agents, especially PBN at lower concentrations, were more effective in preventing radiation-induced formation of 8-OH-dG in naked DNA than in free dG. These data suggest that PBN, DMPO and POBN act as free radical scavengers which may associate with DNA and afford protection against gamma rays. However, no enhancement of survival was observed when Chinese hamster ovary (CHO) cells were exposed to high non-toxic concentrations of PBN or POBN prior to and during irradiation with 60Co gamma rays and scored for clonogenic survival. DMPO provided only minimal protection from radiation-induced cell killing.

2-Deoxyglucose transport and metabolism in Caco-2 cells.

We investigated the kinetics of 2-deoxy-D-glucose (DG) uptake and metabolism in Caco-2 cells, because this human cell line may represent a valid enterocyte model to assess the dynamics between sugar transport and metabolism and hence to obtain insights into the factors involved during the intracellular phase of glucose absorption. When studied in 14-day-old monolayers, DG uptake is characterized by a lag phase with a time course matching the decrease in intracellular glucose concentrations, and no intracellular glucose 6-phosphate (G-6-P) can be detected at any time during incubation. After 1 h of preincubation of Caco-2 cells in substrate-free transport medium, however, steady-state DG uptake matches 2-deoxy-D-glucose 6-phosphate (DG-6-P) accumulation with undetectable levels of free DG. This complex behavior in DG uptake is linked to high hexokinase activity in Caco-2 cells, and the enzyme has a Michaelis-Menten constant K(m) for glucose that is typical of hexokinase type II (0.120 +/- 0.003 mM). Caco-2 cells also contain low-level glucose-6-phosphatase (G-6-Pase) activity, which may account for the leveling off in DG uptake, and the kinetics of DG transport may be attributed to the existence of a predominant pathway with a K(m) of 1.7 +/- 0.2 mM. Finally, analysis of the growth-related expression of DG transport and hexokinase activity clearly shows that DG uptake is lowest in postconfluent cells when hexokinase is at its highest levels. We thus conclude that 1) transport is the rate-limiting step during DG accumulation, 2) G-6-P is a potent inhibitor of hexokinase activity compared with DG-6-P, so that enzyme inhibition may have physiological relevance in diverting glucose from metabolism during its active reabsorption in the small intestine, and 3) low levels of G-6-Pase activity seem to exclude this enzyme, and hence the endoplasmic reticulum, as important factors during the intracellular phase of glucose transport.

Heterochromatic proteins specifically enhance nickel-induced 8-oxo-dG formation.

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) was measured as an indicator of nickel-induced oxidative base damage in the presence of H2O2. Heterochromatic proteins isolated from Chinese hamster liver cells enhanced the formation of 8-oxo-dG induced by NiCl2 and H2O2 in vitro, whereas euchromatic proteins inhibited this reaction. The inhibitory effect of euchromatic proteins on dG oxidation may be due to the oxygen radical scavenging effects of low molecular weight protein-rich fractions. Gel electrophoresis confirmed that histone H1 was present at a higher concentration in heterochromatin than in euchromatin. It is believed that the presence of nickel-protein complexes in cells is crucial for the formation of reactive oxygen species (ROS). We found that Ni2+ binds to histone H1 and core histones as determined by 63Ni autoradiography of proteins on nitrocellulose membranes. In vitro studies showed that commercially purified histone H1, and to a considerably lesser extent core histones, enhanced the NiCl2 and H2O2 catalyzed formation of 8-oxo-dG in a reaction containing free dG base. Since histone H1 is a lysine- and alanine-rich protein, the levels of 8-oxo-dG induced by NiCl2 and H2O2 were studied in the presence of these amino acids and found to be enhanced by them. These results suggest that nickel may specifically produce oxidative DNA damage in heterochromatin because of the nature of its binding to histone H1 and core histones. This selective oxidation of genetically inactive heterochromatin may explain why nickel compounds which generate oxygen radicals and oxidize DNA bases are inactive in most gene mutation assays.

Metabolites of 2-deoxy-[14C]glucose in plasma and brain: influence on rate of glucose utilization determined with deoxyglucose method in rat brain.

The [14C]deoxyglucose ([14C]DG) method depends upon quantitative trapping of metabolites in brain at the site of phosphorylation, and in the usual procedure it is assumed that all the label in plasma is in free DG. Our previous finding of labeled nonacidic derivatives of DG in plasma raised the possibility that some metabolites of DG might not be fully retained in body tissues and therefore cause overestimation of the integrated specific activity of the precursor pool determined from assay of label in plasma and/or underestimation of the true size of the metabolite fraction in brain. In the present study, metabolism of DG in rat tissues by secondary pathways was examined and found to be more extensive than previously recognized. When 14C-labeled compounds in ethanol extracts of either plasma or brain were separated by anion exchange HPLC, eight fractions were obtained. 14C-labeled metabolites in plasma were detected after a 35-min lag and gradually increased in amount with time after an intravenous pulse. In brain, deoxyglucose-6-phosphate was further metabolized, mainly to deoxyglucose-1-phosphate and deoxyglucose-1,6-phosphate. These are acid-labile compounds and accounted for approximately 20% of the 14C in the metabolite pool in brain. The rate constants for net loss of 14C from the metabolite pool between 45 and 180 min after a pulse were similar (0.4-0.5%/min) in vivo and in intact postmortem brain. The rate constant for loss of deoxyglucose-6-phosphate (DG-6-P) in vivo (approximately 0.7%/min) was, however, about twice that for postmortem brain, suggesting that a significant fraction of the DG-6-P lost in vivo is due to its further metabolism by energy-dependent reactions. 14C-labeled metabolites of [14C]DG in plasma and brain do not interfere with determination of local rates of glucose utilization in brain in normal, conscious rats by the autoradiographic method if the prescribed procedures and a 45-min experimental period are used.

Effect of carnosine homocarnosine and anserine on hydroxylation of the guanine moiety in 2'-deoxyguanosine, DNA and nucleohistone with hydrogen peroxide in the presence of nickel(II).

The oxidation of 2'-deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in free dG and the dG residues of DNA and nucleohistone with H2O2 in the presence of Ni(II) and histidyl oligopeptides, carnosine, homocarnosine and anserine was studied at physiological pH. The oxidation of free dG with H2O2 was enhanced by the oligopeptides, but not by Ni(II) alone. Much greater enhancement was produced by equimolar mixtures of Ni(II) with any of the oligopeptides or with L-histidine. In contrast, the oxidation of dG residues in DNA and nucleohistone with H2O2 was not affected by the oligopeptides, but was enhanced by Ni(II). The latter enhancement remained practically unchanged when Ni(II) was accompanied by equimolar amounts of homocarnosine or anserine, whereas carnosine tended to attenuate that enhancement. The extent of formation of 8-OH-dG in free dG depended on time and concentration of H2O2 and was highest at pH 7.4. An electron spin resonance study of the reaction mixture containing H2O2, Ni(II), carnosine, homocarnosine and/or anserine provided evidence for the generation of .OH radical in the reaction media. Although it has previously been concluded that carnosine, anserine and homocarnosine might serve as anti-oxidants, the present study does not support that conclusion and shows that these compounds may even act as pro-oxidants, especially when they are complexed with Ni(II). The results suggest that carnosine, homocarnosine and anserine, by enhancing oxidation of the free dG pool, may potentiate the carcinogenic effects of Ni(II) since the resulting 8-OH-dG can be misincorporated into DNA and thus produce a mutagenic lesion.

Refinement of the Kinetic Model of the 2-[14C]Deoxyglucose Method to Incorporate Effects of Intracellular Compartmentation in Brain

A translocase to transport hexose phosphate formed in the cytosol into the cisterns of the endoplasmic reticulum, where the phosphatase resides, is absent in brain (Fishman and Karnovsky, 1986). 2-Deoxyglucose-6-phosphate (DG-6-P) may therefore have limited access to glucose-6-phosphatase (G-6-Pase), and transport of the DG-6-P across the endoplasmic reticular membrane may be rate limiting to its dephosphorylation. To take this compartmentation into account, a five-rate constant (5K) model was developed to describe the kinetic behavior of 2-deoxyglucose (DG) and its phosphorylated product in brain. Loss of DG-6-P was modeled as a two-step process: (a) transfer of DG-6-P from the cytosol into the cisterns of the endoplasmic reticulum; (b) hydrolysis of DG-6-P by G-6-Pase and subsequent return of the free DG to the precursor pool. Local CMRglc (LCMRglc) was calculated in the rat on the basis of this model and compared with values calculated on the basis of the three-rate constant (3K) and the four-rate constant (4K) models of the DG method. The results show that under normal physiological conditions all three models yield values of LCMRglc that are essentially equivalent for experimental periods between 25 and 45 min. Therefore, the simplest model, the 3K model, is sufficient. For experimental periods from 60 to 120 min, the 4K and 5K models do not correct completely for loss of product, but the 5K model does yield estimates of LCMRglc that are closer to the values at 45 min than those obtained with the 3K and 4K models.

The use of the creatine kinase reaction to determine free energy change of ATP hydrolysis in anoxic cardiomyocytes

In isolated cardiomyocytes from adult rat heart the free energy change of ATP hydrolysis (dG) was determined under conditions of substrate-free anoxia. Changes of free cytosolic ADP concentrations, needed for the calculation of dG, were determined by two indirect methods since a direct measurement is not feasible: (i) via the mass action ratio of the creatine kinase reaction (CK) assuming near equilibrium conditions, and (ii) via quantification of the net hydrolysis of ATP to ADP by a detailed balancing of possible contribution to Pi production. Both approaches gave virtually identical results, showing that in anoxia only 6% of the ATP hydrolysed are hydrolysed to ADP and 94% completely to adenosine and further degradation products. The convergence of both methods also indicates that in this model the CK reaction is indeed catalysed near its equilibrium. Therefore estimations of free ADP and dG using its mass action ratio are valid. In anoxic cardiomyocytes dG values fell from 57 kJ/mol in normoxia to 42 kJ/mol after 120 min anoxia, corresponding to a decrease of ATP contents from 24 to 4 nmol/mg protein.

Quantification of glucose utilization in an experimental brain tumor model by the deoxyglucose method.

Reevaluation of lumped and rate constants is necessary when Sokoloff's 2-deoxyglucose (DG) method is used to measure glucose utilization in pathological tissue. We describe here a modification of Sokoloff's lumped constant measurement that permits simultaneous estimation of both lumped and rate constants from a single animal experiment. A subcutaneous tumor model (AA ascites tumor) was used for measurement of these constants with a procedure similar to Sokoloff's that kept the plasma tracer concentration constant. Measured constants were as follows: lumped constant, 0.654 +/- 0.081; k1, 0.196 +/- 0.038 min-1; k2, 0.262 +/- 0.067 min-1; k3, 0.117 +/- 0.044 min-1. These constants were used to quantify glucose utilization in the implanted brain tumor. To test the validity of this method, we compared a fraction of the free DG pool calculated using the tumor constants with a fraction measured directly by chromatographic analysis of tissue samples from both subcutaneous tumor and implanted brain tumor. The values derived by chemical analysis agreed well with those predicted by the calculations. The value of k4 varied from 0.0031 +/- 0.0018 min-1 for the tumor tissue to 0.0214 +/- 0.0024 min-1 for tumors with a large necrotic center. This method would be especially useful when applied to xenograft human gliomas in nude mice for quantification of glucose utilization in human gliomas by means of positron emission tomography.

2-Deoxy-D-glucose uptake by rat granular pneumocytes in primary culture.

Uptake of 2-deoxy-D-glucose (DG) was investigated with rat granular pneumocytes isolated in primary culture. Cells attached to flasks were incubated in Minimal Essential Medium usually containing 5 mM DG in place of glucose. Uptake of DG increased progressively with time of incubation and approached a plateau value of 35-40 mumol/10(6) cells at 60 min. Uptake increased as a function of external DG concentration with half-maximal uptake at approximately 2.0 mM DG. DG uptake was inhibited by the presence of glucose, alpha-methylglucoside, phlorizin, ouabain, or sodium-free medium. After 60 min incubation, approximately 20% of total intracellular DG was in the free form, and the calculated mean intracellular concentration of free DG (n = 4) was approximately twice the external concentration. Phosphatase activity was indicated by increase in free DG and efflux from cells after removal of external DG. In comparison with pneumocytes, uptake of DG by alveolar macrophages showed different kinetics, and intracellular free DG did not exceed the extracellular concentration. These findings indicate that type II cells take up DG by a sodium-dependent, carrier-mediated transport process that results in accumulation of free sugar against a concentration gradient.