A noninvasive technique for the measurement of the energetic state of free‐suspension mammalian cells

A perfusion small‐scale bioreactor allowing on‐line monitoring of the cell energetic state was developed for free‐suspension mammalian cells. The bioreactor was designed to perform in vivo nuclear magnetic resonance (NMR) spectroscopy, which is a noninvasive and nondestructive method that permits the monitoring of intracellular nutrient concentrations, metabolic precursors and intermediates, as well as metabolites and energy shuttles, such as ATP, ADP, and NADPH. The bioreactor was made of a 10‐mm NMR tube following a fluidized bed design. Perfusion flow rate allowing for adequate oxygen supply was found to be above 0.79 mL min^?1 for high‐density cell suspensions (10⁸ cells). Chinese hamster ovary (CHO) cells were studied here as model system. Hydrodynamic studies using coloration/decoloration and residence time distribution measurements were realized to perfect bioreactor design as well as to determine operating conditions bestowing adequate homogeneous mixing and cell retention in the NMR reading zone. In vivo ³¹P NMR was performed and demonstrated the small‐scale bioreactor platform ability to monitor the cell physiological behavior for 30‐min experiments. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Kinetic metabolic modelling for the control of plant cells cytoplasmic phosphate

A previously developed kinetic metabolic model for plant metabolism was used in a context of identification and control of intracellular phosphate (Pi) dynamics. Experimental data from batch flask cultures of Eschscholtiza californica cells was used to calibrate the model parameters for the slow dynamics (growth, nutrition, anabolic pathways, etc.). Perturbation experiments were performed using a perfusion small-scale bioreactor monitored by in vivo³¹P NMR. Parameter identification for Pi metabolism was done by measuring the cells dynamic response to different inputs for extracellular Pi (two pulse-response experiments and a step-response experiment). The calibrated model can describe Pi translocation between the cellular pools (vacuole and cytoplasm). The effect of intracellular Pi management on ATP/ADP and phosphomonoesters concentrations is also described by the model. The calibrated model is then used to develop a control strategy on the cytoplasmic Pi pool. From the identification of the systems dynamics, a proportional-integral controller was designed and tuned. The closed-loop control was implemented in the small-scale NMR bioreactor and experimental results were in accordance with model predictions. Thus, the calibrated model is able to predict cellular behaviour for phosphate metabolism and it was demonstrated that it is possible to control the intracellular level of cytoplasmic Pi in plant cells.     

31P NMR spectroscopy, chemical analysis, and free Mg2+ of rabbit bladder and uterine smooth muscle

31P NMR spectra of isolated rabbit bladder and uterus were obtained under steady-state arterial perfusion in vitro at rest and while stimulated. The spectra contained seven major peaks: phosphoethanolamine, sn-glycero(3)phosphocholine, inorganic phosphate (Pi), phosphocreatine, and the gamma, alpha, and beta peaks of ATP. Chemical analyses, high-pressure liquid chromatography, and NMR spectroscopy of aqueous extracts of bladders identified a number of other components that also made contributions to, but were not resolved in, the spectra of the intact tissues: UTP, GTP, UDP-Glc, NAD+, phosphocholine, and sn-glycero(3)phosphoethanolamine. Intracellular pH of unstimulated bladders and uteri, measured from the chemical shift of the Pi peak, was 7.10 +/- 0.09 S.D. and 7.01 +/- 0.12 S.D., respectively. The chemical shift of the beta-ATP peak in the smooth muscles was significantly upfield (-0.3 ppm) compared to the chemical shift observed in striated muscles (cat biceps and rat myocardium). An ADP peak was identified in stimulated and ischemic bladders. The chemical shifts of the nucleotides observed in perfused bladders were calibrated as a function of free Mg2+ concentration in solutions containing phosphocreatine, Pi, ADP, and ATP at an ionic strength of 180 mM. We derived the following estimates for the intracellular free Mg2+ concentration: uterus, 0.40 mM; unstimulated bladder, 0.46 mM; stimulated and ischemic bladder, 0.50 mM (from the ATP chemical shift) and 0.45 (from the ADP chemical shift); cat biceps, 1.5 mM; and rat myocardium, 1.4 mM.     

A phosphorus-31 nuclear magnetic resonance study of phosphate uptake and storage in cultured Catharanthus roseus and Daucus carota plant cells

High resolution 31P NMR spectra (103.2 MHz) of oxygenated Catharanthus roseus and Daucus carota cells grown in suspension cultures were obtained using a solenoidal perfusion probe. The spectra showed resonances for various phosphorylated metabolites such as ATP, ADP, NAD(P)(H), nucleoside diphosphoglucose, and sugar phosphates. The relative levels of the phosphorylated metabolites remained constant throughout the growth curve. No resonances for storage compounds such as polyphosphates, pyrophosphate, or phytates were observed. Two resolved resonances for Pi indicated an intracellular pH of 7.3 and 5.7 (or below) for the cytoplasm and vacuoles, respectively. The time course of Pi uptake and storage during growth in fresh culture medium was followed by studying the level of vacuolar Pi with 31P NMR (145.7 MHz). Simultaneously, the level of Pi in the culture medium was followed with radioactive 32P. C. roseus quickly takes up all the Pi from the culture medium (maximum rate 1.7 mumol min-1 g-1 (dry weight of cells]. The Pi is first stored in the vacuoles; subsequently, one part of this pool is used to keep a constant cytoplasmic Pi level while another part is apparently accumulated as an NMR invisible Pi store, probably in another cell organelle. In contrast, D. carota does not accumulate Pi in the vacuoles and consequently it takes up Pi from the medium at a much slower rate (0.05 mumol min-1 g-1 (dry weight of cells].     

Oxidative phosphorylation system during steady-state hypoxia in the dog brain

The relationship between biochemical and physiological responses and tissue O2 during hypoxia was investigated in vivo in the dog brain by 31P nuclear magnetic resonance (NMR) spectroscopy. Our findings demonstrate how ATP synthesis in the brain can be maintained during hypoxia because of compensatory changes in NADH, ADP, and Pi. Eleven beagle dogs were anesthetized and mechanically ventilated, and a steady-state graded hypoxia was induced by decreasing the fraction of inspired O2 (FIO2) stepwise at 20-min intervals. Biochemical metabolites were measured using 31P-NMR and fluorescence spectroscopy. When sagittal sinus O2 partial pressure (PVO2) had decreased to 15 Torr, NADH increased by 30%, Pi increased by 50%, and phosphocreatine (PCr) decreased by 20%. In contrast, ATP remained constant. There was a 10% increase in ADP in dogs that maintained a steady temperature, but ADP decreased by as much as 30% in dogs in which body temperature decreased with the falling PVO2. PCr/Pi was logarithmically related to the phosphorylation potential during steady-state hypoxia. Compensation for the O2 lack is attributed to increases in ADP, Pi, and NADH as a result of the reciprocal relationship of the Michaelis-Menten equation. If the Michaelis-Menten constants (Km) of ADP, Pi, and O2 are the same as determined in vitro in mitochondria, the minimum brain cytosolic O2 capable of maintaining a steady-state ATP is near its Km (0.1 Torr) at a PVO2 of 7.5 Torr. At this critical O2 level, PCr/Pi is 0.9, intracellular pH is 6.75, phosphorylation potential is 38.5 mM-1, and the calculated maximum velocity of ATP formation by oxidative phosphorylation is 55% of normal.     

Use of gated perfusion to study early effects of anoxia on cardiac energy metabolism: a new 31P NMR method

A novel 31P NMR method is described that is capable of determining rapid changes in the intracellular levels of various phosphorus-containing compounds in an isolated, perfused working rat heart. This technique involves the gating of 31P NMR measurements to a heart that is alternately perfused with a modified Krebs-Henseleit medium containing 10 mM pyruvate and equilibrated with either 95% O2/5% CO2 or 95% N2/5% CO2. The experimental design allows up to three NMR measurements to be made during a single O2/N2 perfusion cycle. When these measurements are repeated at different intervals during the cycle, rapid changes in metabolite levels can be determined. Preliminary studies have shown that hearts remain hemodynamically stable to the aerobic/anoxic perfusion cycle as judged by heart rate, peak systolic pressure, aortic output, and coronary flow for at least 80 min in the magnet when subjected to cycle times of 4.5-s O2 and 1.5-s N2 perfusions. NMR measurements made under these conditions showed that a transition from full aerobic perfusion to this cycle revealed a new steady state, with an increased inorganic phosphate level from 6% total observable phosphorus to 10% and a possibly significant decreased measurement of creatine phosphate level (from 35 to 31%). Comparison of individual NMR measurements made during this perfusion cycle shows apparent rapid cyclical variations in intracellular pH and the levels of Pi, ATP, and NAD(H). These changes, expressed as variations above and below mean values measured during the cycle, showed that (a) intracellular pH, as measured by the chemical shift of Pi, reversibly decreases by more than 0.1 pH unit within 0.5-1 s following maximal anoxic perfusion and (b) coincident with a decrease in intracellular pH, Pi levels increased by a maximum of 30-40% whereas ATP levels decreased by a maximum of 15-20%. The amount of total observable phosphorous detected during the cycle is essentially constant. Unexpectedly, creatine phosphate levels are most stable, indicating that their levels are being maintained at the expense of ATP. Also unexpected is the finding that NAD(H) levels varied from maximal to undetectable levels during the perfusion cycle. The current method of aerobic/anoxic perfusion is capable of resolving metabolic events much faster than previous NMR methods and yielding information that is unobtainable by any other technique.     

Phosphorus-31 nuclear-magnetic-resonance study of phosphorylated metabolites compartmentation, intracellular pH and phosphorylation state during normoxia, hypoxia and ethanol perfusion, in the perfused rat liver

A quantitative analysis of the phosphorus-31 NMR spectra of excised perfused rat liver has been carried out at 80.9 MHz using a 30-mm sample cell. The results indicate that in liver from fed rats, all intracellular ATP is detected by NMR. In contrast, only the cytosolic fractions of Pi and ADP can be observed as indicated by careful analysis of spectra obtained from perchloric acid liver extracts and intact liver under valinomycin perfusion. In well-oxygenated perfused liver the ATP concentration is 7.4 mM. Values of 5.3 mM and 0.9 mM are found respectively for Pi and ADP concentrations in the cytosolic compartment. Cytosolic pH value (pHi) is 7.25 +/- 0.05 and free magnesium concentration 0.5 mM. Addition of 70 mM (0.4%) ethanol to the perfusate of a fed rat liver induces 25% and 38% reduction of ATP and Pi levels, respectively. A large amount of sn-glycerol 3-phosphate is synthesized (up to 11 mM) in the cytosol. After ethanol withdrawal, a large overshoot in cytosolic Pi is observed, which is indicative of a net uptake of Pi across the plasma membrane that occurred during ethanol oxidation. No significant pH variation is observed during ethanol infusion. In perfused liver of rats subjected to 48-h fasts, the concentrations of cytosolic phosphorylated metabolites are 5.3 mM, 0.8 mM and 11.5 mM for ATP, ADP and Pi, respectively. The perfusion of the liver with 70 mM ethanol does not change the adenine nucleotide levels, while the Pi content is decreased by 10%. During a 4-min hypoxia, induced by reducing the perfusion flow rate from 12 ml to 3 ml min-1 (100 g body weight)-1, ATP concentration decreases to 5.8 mM in the fed rat liver. Cytosolic Pi and ADP increase to 8.7 mM and 1.6 mM, respectively. The cytosolic pH evolves to more acidic values and reaches 7.02 +/- 0.05 at the end of the 4-min hypoxic period.     

31P-NMR studies of phosphate transfer rates in T47D human breast cancer cells

The concentration of phosphates and the kinetics of phosphate transfer reactions were measured in the human breast cancer cell line, T47D, using 31P-NMR spectroscopy. The cells were embedded in agarose filaments and perifused with oxygenated medium during the NMR measurements. The following phosphates were identified in spectra of perifused cells and of cell extracts: phosphorylcholine (PC), phosphorylethanolamine (PE), the glycerol derivatives of PC and PE, inorganic phosphate (Pi), phosphocreatine (PCr), nucleoside triphosphate (primarily ATP) and uridine diphosphate glucose. The rates of the transfers: PC----gamma ATP (0.2 mM/s), Pi----gamma ATP (0.2 mM/s) and the conversion beta ATP----beta ADP (1.3 mM/s) were determined from analysis of data obtained in steady-state saturation transfer and inversion recovery experiments. Data from spectrophotometric assays of the specific activity of creatine kinase (approx. 0.1 mumol/min per mg protein) and adenylate kinase (approx. 0.4 mumol/min per mg protein) suggest that the beta ATP----beta ADP rate is dominated by the latter reaction. The ratio between the rate of ATP synthesis from Pi and the rate of consumption of oxygen atoms (4 X 10(-3) mM/s) was approx. 50. This high value and preliminary measurements of the rate of lactate production from glucose, indicated that aerobic glycolysis is the main pathway of ATP synthesis.     

31P nuclear magnetic resonance of metabolic changes associated with cyanide intoxication in the perfused rat liver.

Metabolic changes associated with cyanide intoxication were observed for the first time in perfused rat liver using ³¹P nuclear magnetic resonance (NMR) at 60.7 MHz. Well-oxygenated control livers showed strong ATP peaks and little discernable internal orthophosphate (Pi). Perfusion with 2 mM cyanide eliminated the observable ATP peaks and caused internal Pi to increase. Despite clear evidence for ATP hydrolysis, resonances from cytoplasmic ADP were conspicuously absent. Resumption of perfusion with cyanide-free buffer caused a dramatic return of the ATP peaks with a concomitant fall in internal Pi. These metabolic changes are consistent with reversible binding of cyanide to mitochondrial cytochromes and their observation by ³¹P NMR indicates the potential of this method for studying metabolism in whole, perfused rat liver under physiologic conditions.     

The alpha beta-methylene analogues of ADP and ATP act as substrates for creatine kinase. delta G0 for this reaction and for the hydrolysis of the alpha beta-methylene analogue of ATP

The alpha beta-methylene analogues of ATP and ADP, [alpha beta CH2]ATP and [alpha beta CH2]ADP, are substrates for creatine kinase. However, the rate of the phosphoryl transfer reaction catalysed is about 10(-5)-times lower than that with normal ATP. The affinities of the analogues (especially [alpha beta CH2]ADP) for the enzyme are lower than those of the normal substrates. The equilibrium constant at 25 degrees C, measured using 31P NMR, for the reaction Mg[alpha beta CH2]ATP + creatine in equilibrium Mg[alpha beta CH2]ADP + phosphocreatine + H+ is 2.2 X 10(-12) M compared with a value of 2.5 X 10(-10) M for the same reaction with the normal substrates, corresponding to a difference in delta G0 values of 11.7 kJ X mol-1. It follows that delta G0 for the hydrolysis of the terminal phosphate group of Mg[alpha beta CH2]ATP is less favourable by 11.7 kJ X mol-1 than that for MgATP.     

Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart

The bioenergetic basis by which the Krebs cycle substrate pyruvate increased cardiac contractile function over that observed with the Embden-Meyerhof substrate glucose was investigated in the isovolumic guinea pig heart. Alterations in the content of the high energy phosphate metabolites and the rate of high energy phosphate turnover were measured by 31P NMR. These were correlated to the changes in contractile function and rates of myocardial oxygen consumption. Maximum left ventricular developed pressure (LVDP) and high energy phosphates were observed with 16 mM glucose or 10 mM pyruvate. In hearts perfused with 16 mM glucose, the intracellular phosphocreatine (PCr) concentration was 15.2 +/- 0.6 mM with a PCr/Pi ratio of 10.3 +/- 0.9. The O2 consumption was 5.35 mumol/g wet weight/min, and these hearts exhibited a LVDP of 97 +/- 3.7 mm Hg at a constant paced rate of 200 beats/min. In contrast, when hearts were switched to 10 mM pyruvate, the PCr concentration was 18.3 +/- 0.4 mM, the PCr/Pi ratio was 30.4 +/- 2.2, the O2 consumption was 6.67 mumol/g wet weight/min, and the LDVP increased to 125 +/- 3.3 mm Hg. From NMR saturation transfer experiments, the steady-state flux of ATP synthesis from PCr was 4.9 mumol/s/g of cell water during glucose perfusion and 6.67 mumol/s/g of cell water during pyruvate perfusion. The flux of ATP synthesis from ADP was measured to be 0.99 mumol/s/g of cell water with glucose and calculated to be 1.33 mumol/s/g of cell water with pyruvate. These results suggest that pyruvate quite favorably alters myocardial metabolism in concert with the increased contractile performance. Thus, as a mechanism to augment myocardial performance, pyruvate appears to be unique.     

Stability constants of Mg2+ and Cd2+ complexes of adenine nucleotides and thionucleotides and rate constants for formation and dissociation of MgATP and MgADP

Stability constants for the Mg2+ and Cd2+ complexes of ATP, ADP, ATP alpha S, ATP beta S, and ADP alpha S have been determined at 30 degrees C and mu = 0.1 M by 31P NMR. Besides being of the utmost importance for determining species distributions for enzymatic studies, these constants allow an estimation of the preference of Cd2+ for sulfur vs. oxygen coordination in phosphorothioate complexes. Stability constants for Mg2+ complexes decreases when sulfur replaces oxygen (log K: ADP, 4.11; ADP alpha S, 3.66; ATP, 4.70; ATP alpha S, 4.47; ATP beta S, 4.04) because of (a) a statistical factor resulting from the loss of one potential phosphate oxygen ligand and (b) either an alteration in the charge distribution between oxygen and sulfur or destabilization of the chelate ring structure by loss of an internal hydrogen bond between an oxygen of coordinated phosphate and metal-bound water. Cd2+ complexes with sulfur-substituted nucleotides are more stable than those without sulfur (log K: ADP, 3.58; ADP alpha S, 4.95; ATP, 4.36; ATP alpha S, 4.42; ATP beta S, 5.44) because of the preferential binding of Cd2+ to sulfur rather than oxygen, which we estimate to be approximately 60 in CdADP alpha S and CdATP beta S. The proportion of tridentate coordination is estimated to be 50-60% in MgATP and MgATP beta S, approximately 27% in MgATP alpha S, approximately 16% in CdATP or CdATP beta S, but approximately 75% in CdATP alpha S. By analysis of the data of Jaffe and Cohn [Jaffe, E. K., & Cohn, M. (1979) J. Biol. Chem. 254, 10839], we conclude that the preference for oxygen over sulfur coordination to ATP beta S is 31 000 for Mg2+, 3100-3900 for Ca2+, and 158-193 for Mn2+. Proton NMR demonstrates that bidentate Cd2+ complexes form intramolecular chelates with the N-7 of adenine while Mg2+ nucleotides and the tridenate CdATP alpha S do not. An analysis of the 31P NMR line widths shows that the rate constants for dissociation of MgADP and MgATP are both 7000 s-1 while the association rate constants are 7 X 10(7) and 4 X 10(8) M-1 s-1, respectively. The observed dependence of the line width on nucleotide concentration is best explained by a base-stacking model at nucleotide concentrations above 5 mM.     

Studies on photophosphorylation utilizing methylene diphosphonate analogs of ADP and ATP.

Spinach chloroplasts were able to photophosphorylate the ADP analog alpha,beta-methylene adenosine 5'-diphosphate (AOPCP). Phosphorylation of AOPCP was catalyzed by chloroplasts that were washed or dialyzed to remove free endogenous nucleotides. In the presence of glucose, hexokinase, AOPCP and 32Pi, the 32P label was incorporated into alpha,beta-methylene adenosine 5'-triphosphate (AOPCPOP). In contrast to photophosphorylation of AOPCP, the ATP analog AOPCPOP was a poor substrate for the ATP-Pi exchange reaction and its hydrolysis was neither stimulated by light and dithiothreitol nor inhibited by Dio-9. Photophosphorylation of AOPCP was inhibited by the alpha,beta- and beta,gamma-substituted methylene analogs of ATP, while phosphorylation of ADP was unaffected by them. The ATP-Pi exchange was also unaffected by both ATP analogs, while the weak AOPCPOP-Pi exchange was inhibited by the beta,gamma-methylene analog of ATP. Direct interaction of methylene analogs with the chloroplast coupling factor ATPase was indicated by the enzymatic hydrolysis of AOPCPOP on polyacrylamide gels.     

Investigations of the partial reactions catalyzed by pyruvate phosphate dikinase

The kinetic mechanism of pyruvate phosphate dikinase (PPDK) from Bacteroides symbiosus was investigated with several different kinetic diagnostics. Initial velocity patterns were intersecting for AMP/PPi and ATP/Pi substrate pairs and parallel for all other substrate pairs. PPDK was shown to catalyze [14C]pyruvate in equilibrium phosphoenolpyruvate (PEP) exchange in the absence of cosubstrates, [14C]AMP in equilibrium ATP exchange in the presence of Pi/PPi but not in their absence, and [32P]Pi in equilibrium PPi exchange in the presence of ATP/AMP but not in their absence. The enzyme was also shown, by using [alpha beta-18O, beta, beta-18O2]ATP and [beta gamma-18O, gamma, gamma, gamma-18O3]ATP and 31P NMR techniques, to catalyze exchange in ATP between the alpha beta-bridge oxygen and the alpha-P nonbridge oxygen and also between the beta gamma-bridge oxygen and the beta-P nonbridge oxygen. The exchanges were catalyzed by PPDK in the presence of Pi but not in its absence. These results were interpreted to support a bi(ATP,Pi) bi(AMP,PPi) uni(pyruvate) uni(PEP) mechanism. AMP and Pi binding order was examined by carrying out dead-end inhibition studies. The dead-end inhibitor adenosine 5'-monophosphorothioate (AMPS) was found to be competitive vs AMP, noncompetitive vs PPi, and uncompetitive vs PEP. The dead-end inhibitor imidodiphosphate (PNP) was found to be competitive vs PPi, uncompetitive vs AMP, and uncompetitive vs PEP. These results showed that AMP binds before PPi. The ATP and Pi binding order was studied by carrying out inhibition, positional isotope exchange, and alternate substrate studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of inositol 1,4,5-trisphosphate and calcium in the action of adenine nucleotides on aortic endothelial cells

ADP and ATP, in the 1-100 microM range of concentrations, increased the formation of inositol phosphates in bovine aortic endothelial cells. The accumulation of inositol trisphosphate in response to adenine nucleotides was rapid (maximum at 15 s) and transient. This material was identified as the biologically active isomer inositol 1,4,5-trisphosphate on the basis of its retention time by high-performance liquid chromatography on an anion-exchange resin. AMP and adenosine have no effect on inositol phosphates. The action of ATP and ADP was mimicked with an equal potency and activity by their phosphorothioate analogs, ATP gamma S and ADP beta S, and with a lower potency by adenosine 5'-(beta,gamma-imido)triphosphate, whereas adenosine 5'-(alpha,beta-methylene)triphosphate, was inactive. In the same range of concentrations, ADP and ATP induced an efflux of 45Ca2+ from prelabeled bovine aortic endothelial cells and increased the fluorescence emission by cells loaded with quin-2. Here, too, AMP and adenosine were completely inactive. The outflow of 45Ca2+ induced by ADP was partially maintained in a calcium-free medium. These data suggest that in aortic endothelial cells, P2-purinergic receptors, of the P2Y subtype, are coupled to the hydrolysis of phosphatidylinositol bisphosphate by a phospholipase C. It is likely that the release of prostacyclin and endothelium-derived relaxing factor in response to ADP and ATP is a consequence of this initial event.     

Very high density of Chinese hamster ovary cells in perfusion by alternating tangential flow or tangential flow filtration in WAVE bioreactor™—part II: Applications for antibody production and cryopreservation

A high cell density perfusion process of monoclonal antibody (MAb) producing Chinese hamster ovary (CHO) cells was developed in disposable WAVE Bioreactor? using external hollow fiber (HF) filter as cell separation device. Tangential flow filtration (TFF) and alternating tangential flow (ATF) systems were compared and process applications of high cell density perfusion were studied here: MAb production and cryopreservation. Operations by perfusion using microfiltration (MF) or ultrafiltration (UF) with ATF or TFF and by fed‐batch were compared. Cell densities higher than 10⁸ cells/mL were obtained using UF TFF or UF ATF. The cells produced comparable amounts of MAb in perfusion by ATF or TFF, MF or UF. MAbs were partially retained by the MF using ATF or TFF but more severely using TFF. Consequently, MAbs were lost when cell broth was discarded from the bioreactor in the daily bleeds. The MAb cell‐specific productivity was comparable at cell densities up to 1.3 × 10⁸ cells/mL in perfusion and was comparable or lower in fed‐batch. After 12 days, six times more MAbs were harvested using perfusion by ATF or TFF with MF or UF, compared to fed‐batch and 28× more in a 1‐month perfusion at 10⁸ cells/mL density. Pumping at a recirculation rate up to 2.75 L/min did not damage the cells with the present TFF settings with HF short circuited. Cell cryopreservation at 0.5 × 10⁸ and 10⁸ cells/mL was performed using cells from a perfusion run at 10⁸ cells/mL density. Cell resuscitation was very successful, showing that this system was a reliable process for cell bank manufacturing. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:768–777, 2013     

Carbon-13 and phosphorus-31 NMR study of hepatic metabolism in the perfused rat liver

Phosphorus-31 nuclear magnetic resonance (NMR) has been used to determine non-invasively absolute concentrations of phosphorylated metabolites in the perfused rat liver. It has been shown that the NMR method does detect cytoplasmic ATP and ADP (ATP:ADP ratio of 15 +/- 3) with no contribution from mitochondrial adenine nucleotides. The concentration of ATP was 7.2 +/- 0.3 mM in the cytosol of well-oxygenated liver, after two hours of perfusion with a Krebs-Ringer buffer. Other phosphorylated metabolites were detected, mainly inorganic phosphate (1.1 mumol/g liver wet weight), phosphorylcholine (1.0 mumol/g wet weight), glycerophosphorylethanolamine (0.34 mumol/g wet weight) and glycerophosphorylcholine (0.30 mumol/g wet weight). The intracellular pH measured from the position of the Pi resonance has a value of 7.2 +/- 0.1. It is likely that the detectable Pi originates from the cytosolic compartment since a pH value of 7.4-7.6 would be expected for the mitochondrial matrix. Natural abundance carbon-13 NMR has also been used to follow the glycogen breakdown in situ by measuring the intensity of the glycogen C-1 resonance in the perfused liver spectrum as a function of the perfusion time. The glycogenolytic process has been studied as a function of the glucose content of the perfusate. Rate of glycogenolysis from 2.7 to 0.16 muEq glycosyl units g wet weight-1 min-1 were found when glucose concentration in the perfusate was varied from 0 to 50 mM. The fate of 90% enriched [2-13C] acetate has been studied in the perfused rat liver by 13C-NMR in order to investigate the mitochondrial metabolism and the interrelations between cytosolic and mitochondrial pools of metabolites. Some compounds of the intermediary metabolism where found to be extensively labelled, e.g. glutamate, glutamine, acetoacetate and beta-hydroxybutyrate. Under our experimental conditions, labelling of glutamate reached a steady-state within 30 min after the onset of perfusion of 20 mM acetate. In addition, the observed incorporation of carbon-13 isotope into glutamine can be linked to the operation of the glutamate-glutamine antiporter and to the high activity of the cytosolic glutamate synthetase. The finding of both active glutaminase and glutamine synthetase activity in the same liver cells is an evidence of the existence of an active glutamine-glutamate futile cycle.     

Adenine nucleotides modulate phosphatidylcholine metabolism in aortic endothelial cells

ATP and ADP, in concentrations ranging from 1-100 microM, increased the release of [3H]choline and [3H]phosphorylcholine (P-choline) from bovine aortic endothelial cells (BAEC) prelabelled with [3H]choline. This action was detectable within 5 minutes and was maintained for at least 40 minutes. ATP and ADP were equiactive, and their action was mimicked by their phosphorothioate analogs (ATP gamma S and ADP beta S) and adenosine 5'-(beta, gamma imido) triphosphate (APPNP), but not by AMP, adenosine, and adenosine 5'-(alpha, beta methylene)triphosphate (APCPP): these results are consistent with the involvement of P2Y receptors. ATP also induced an intracellular accumulation of [3H]choline: the intracellular level of [3H]choline was increased 30 seconds after ATP addition and remained elevated for a least 20 minutes. The action of ATP on the release of choline metabolites was reproduced by bradykinin (1 microM), the tumor promoter phorbol 12-myristate 13-acetate (PMA, 50 nM), and the calcium ionophore A23187 (0.5 microM). Down-regulation of protein kinase C, following a 24-hour exposure of endothelial cells to PMA, abolished the effects of PMA and ATP on the release of choline and P-choline, whereas the response to A23187 was maintained. These results suggest that in aortic endothelial cells, ATP produces a sustained activation of a phospholipase D hydrolyzing phosphatidylcholine. The resulting accumulation of phosphatidic acid might have an important role in the modulation of endothelial cell function by adenine nucleotides. Stimulation of phospholipase D appears to involve protein kinase C, activated following the release of diacylglycerol from phosphatidylinositol bisphosphate by a phospholipase C coupled to the P2Y receptors (Pirotton et al., 1987a).     

Phosphate free perfusion prevents washout of tissue creatine in Langendorff perfused rabbit heart.

Rabbit hearts were perfused with Krebs-Henseleit bicarbonate buffer supplemented with 15 mM glucose and 10 mU/ml of insulin +/- Pi. At the end of 60 min the hearts were freeze-clamped and the content of ATP, creatine phosphate, creatine, lactate, pyruvate, DHAP and 3-P glycerate were determined enzymatically in neutralized perchloric acid tissue extracts. The free cytosolic ADP and Pi and the cytosolic NAD+ redox and phosphorylation potentials were calculated from the measured metabolite concentrations. Pi free perfusion resulted in increased creatine, free cytosolic ADP and cytosolic phosphorylation potential, decreased calculated free Pi and no change in cardiac ATP and creatine phosphate content. The increase in the cytosolic phosphorylation potential was due to the lowering of cytosolic free Pi. The increase in ADP was due to the increase in creatine. The increase in creatine appeared to be due to an inhibition of creatine efflux from the heart during Pi free perfusion which was mediated by an enhanced Na+ electrochemical gradient.     

Effects of fructose on the energy metabolism and acid-base status of the perfused starved-rat liver. A 31phosphorus nuclear magnetic resonance study.

Fructose metabolism has been studied with 31P n.m.r. in perfused livers from rats starved for 48h. The time course of changes in liver ATP, Pi and sugar phosphate (fructose l-phosphate) concentrations, and intracellular pH were followed in each perfusion after infusion of fructose to give an initial concentration of either 5mM or 10mM. Rapid falls in the concentrations of ATP and Pi and intracellular pH occurred after infusion of fructose, reaching a minimum after 4-5 min, which was lower in the 10mM group than in the 5mM group. These changes were accompanied by a rapid rise in fructose 1-phosphate, reaching a plateau also after 4-5 min. At both concentrations of fructose, after the early falls, some recovery of ATP, Pi and intracellular pH occurred; this was complete for Pi and intracellular pH in the 5mM-fructose experiments (within 12-30 min). Complete restoration of ATP to the pre-fructose value was not achieved in either the 5mM of 10mM groups. Measurements of the uptake of lactate by the liver indicated that the fall in intracellular pH was caused primarily by production of protons accompanying the formation of lactate from fructose with possibly a transient contribution generated during the rise in fructose 1-phosphate.