Effect of acute hypoglycaemia on cerebral metabolic rate in fetal sheep

Studies were carried out in 11 fetal sheep four days after surgery for insertion of catheters and electrocortical leads. After a 3 h control period an insulin infusion was given to the ewe and maintained for the next 4 h. Fetal arterial glucose fell from 0.85 +/- 0.10 to 0.57 +/- 0.06 mM (SEM) while oxygen content was unchanged (3.80 +/- 0.24 to 3.75 +/- 0.21 mM). Cerebral uptake of oxygen and glucose were determined from samples drawn simultaneously from the axillary artery and sagittal vein and cerebral blood flow (microsphere technique). There was no significant change in uptake of either oxygen or glucose by the fetal brain. We conclude that a rapid fall in fetal glucose levels with no change in oxygen content does not result in decrease in cerebral metabolism measured over a short term.     

On‐Line Control of Glucose Concentration in High‐Yielding Mammalian Cell Cultures Enabled Through Oxygen Transfer Rate Measurements

Glucose control is vital to ensure consistent growth and protein production in mammalian cell cultures. The typical fed‐batch glucose control strategy involving bolus glucose additions based on infrequent off‐line daily samples results in cells experiencing significant glucose concentration fluctuations that can influence product quality and growth. This study proposes an on‐line method to control and manipulate glucose utilizing readily available process measurements. The method generates a correlation between the cumulative oxygen transfer rate and the cumulative glucose consumed. This correlation generates an on‐line prediction of glucose that has been successfully incorporated into a control algorithm manipulating the glucose feed‐rate. This advanced process control (APC) strategy enables the glucose concentration to be maintained at an adjustable set‐point and has been found to significantly reduce the deviation in glucose concentration in comparison to conventional operation. This method has been validated to produce various therapeutic proteins across cell lines with different glucose consumption demands and is successfully demonstrated on micro (15 mL), laboratory (7 L), and pilot (50 L) scale systems. This novel APC strategy is simple to implement and offers the potential to significantly enhance the glucose control strategy for scales spanning micro‐scale systems through to full scale industrial bioreactors.     

Enzymatic method for measuring the absolute value of oxygen concentration

An enzymatic method for measuring the absolute concentration of oxygen in aqueous solutions, using 4-hydroxybenzoate 3-monooxygenase and glucose oxidase, is described. The monooxygenase is used for quantitative oxidation of 4-hydroxybenzoate and NADPH with oxygen into 3,4-dihydroxybenzoate and NADP+; the amount of oxygen can be measured as the amount of NADPH decreased by the reaction. The monooxygenase reaction is performed in a syringe, a closed system. After the completion of the monooxygenase reaction, glucose oxidase is added to the assay solution to consume the oxygen from the atmosphere; this enables us to measure the NADPH concentration in the solution spectrophotometrically in an open system and to check the anaerobicity of closed systems. The oxygen concentrations at 25 degrees C of oxygen-saturated and air-saturated water were 1.10 and 0.23 mM, respectively. The value for argon-bubbled water was zero within the experimental error; this justifies the assay system. Thus, it is shown that a sample containing 8 microM-1.1 mM oxygen can be measured by this method.     

Identification of furoyl-containing advanced glycation products in collagen samples from diabetic and healthy rats

The compounds resulting from the reaction of glucose with proteins (advanced glycation products) can be important markers of chronic diabetic complications. To test the possible diagnostic value of advanced glycation products containing the furoyl moiety, collagen samples from diabetic and healthy rats were analyzed by parent ion spectroscopy. In our study, we compared normal collagen, diabetic collagen and normal collagen incubated with different glucose concentrations and we employed different hydrolysis procedures (HCl and proteinase). Mass spectroscopic measurements performed on hydrolyzed samples showed that either different samples or different hydrolysis procedures produce a similar set of furoyl-containing compounds. 2-(2-Furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI) which has been reported to be one of the advanced glycation products, was never found in any of the samples examined. Hence neither FFI nor furoyl-containing molecules can be considered markers of advanced glycation processes.     

Identification of furoyl-containing advanced glycation products in collagen samples from diabetic and healthy rats

The compounds resulting from the reaction of glucose with proteins (advanced glycation products) can be important markers of chronic diabetic complications. To test the possible diagnostic value of advanced glycation products containing the furoyl moiety, collagen samples from diabetic and healthy rats were analyzed by parent ion spectroscopy. In our study, we compared normal collagen, diabetic collagen and normal collagen incubated with different glucose concentrations and we employed different hydrolysis procedures (HCl and proteinase). Mass spectroscopic measurements performed on hydrolyzed samples showed that either different samples or different hydrolysis procedures produce a similar set of furoyl-containing compounds. 2-(2-Furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI) which has been reported to be one of the advanced glycation products, was never found in any of the samples examined. Hence neither FFI nor furoyl-containing molecules can be considered markers of advanced glycation processes.     

Simultaneous analysis of physiological and electrical output changes in an operating microbial fuel cell with Shewanella oneidensis

Changes in metabolism and cellular physiology of facultative anaerobes during oxygen exposure can be substantial, but little is known about how these changes connect with electrical current output from an operating microbial fuel cell (MFC). A high‐throughput voltage based screening assay (VBSA) was used to correlate current output from a MFC containing Shewanella oneidensis MR‐1 to carbon source (glucose or lactate) utilization, culture conditions, and biofilm coverage over 250 h. Lactate induced an immediate current response from S. oneidensis MR‐1, with both air‐exposed and anaerobic anodes throughout the duration of the experiments. Glucose was initially utilized for current output by MR‐1 when cultured and maintained in the presence of air. However, after repeated additions of glucose, the current output from the MFC decreased substantially while viable planktonic cell counts and biofilm coverage remained constant suggesting that extracellular electron transfer pathways were being inhibited. Shewanella maintained under an anaerobic atmosphere did not utilize glucose consistent with literature precedents. Operation of the VBSA permitted data collection from nine simultaneous S. oneidensis MR‐1 MFC experiments in which each experiment was able to demonstrate organic carbon source utilization and oxygen dependent biofilm formation on a carbon electrode. These data provide the first direct evidence of complex cellular responses to electron donor and oxygen tension by Shewanella in an operating MFC at select time points. Biotechnol. Bioeng. 2009;103: 524–531. Published 2009 Wiley Periodicals, Inc.     

Nutrient utilization by bovine articular chondrocytes: a combined experimental and theoretical approach

A combined experimental-numerical approach was adopted to characterize glucose and oxygen uptake and lactate production by bovine articular chondrocytes in a model system. For a wide range of cell concentrations, cells in agarose were supplemented with either low or high glucose medium. During an initial culture phase of 48 h, oxygen was monitored noninvasively using a biosensor system. Glucose and lactate were determined by medium sampling. In order to quantify glucose and oxygen uptake, a finite element approach was adopted to describe diffusion and uptake in the experimental model. Numerical predictions of lactate, based on simple relations for cell metabolism, were found to agree well for low glucose, but not for high glucose medium. Oxygen did not play a role in either case. Given the close association between chondrocyte energy metabolism and matrix synthesis, a quantifiable prediction of utilization can present a valuable contribution in the optimization of tissue engineering conditions.     

Enhancement of oxygen absorption by magnetite-containing beads of immobilized glucose oxidase

Rates of oxygen absorption into glucose solutions were measured using an immobilized-enzyme reactor, in which magnetite-containing beads of immobilized glucose oxidase were moved by a revolving magnetic field to reduce the mass transfer resistances at the gas–liquid interface and around the bead. Data were also obtained for oxygen absorption into glucose solutions containing soluble or immobilized glucose oxidase (without magnetite), as well as for physical absorption of oxygen. The rates of physical absorption for the runs with the magnetite-containing beads increased because of mechanical stirring caused by spinning of the beads at the gas-liquid interface. In this case the experimental enhancement factors were found to be larger than those predicted on the basis of the film theory for gas absorption with a pseudo-first order reaction.     

Respiratory pathways and oxygen toxicity in Phanerochaete chrysosporium

Phanerochaete chrysosporium maintained on glucose as the carbon source contained severely impaired mitochondria that were characterised by the loss of both succinate dehydrogenase and cytochrome oxidase activities. These cells maintained a constant value for energy charge using anaerobic metabolism. Cells with these properties express lignin peroxidase when supplied with a pure oxygen atmosphere, which may reflect a response to accumulating reactive oxygen species. Cells maintained on cellulose retained fully functional mitochondria, but expressed lignin peroxidase without being exposed to a pure oxygen atmosphere. In the cells maintained on cellulose, mitochondrial function may be limited by the supply of glucose, leading to the accumulation of reactive oxygen species.     

Beneficial effects of dietary restriction on cerebral cortical synaptic terminals: preservation of glucose and glutamate transport and mitochondrial function after exposure to amyloid beta-peptide, iron, and 3-nitropropionic acid

Recent studies have shown that rats and mice maintained on a dietary restriction (DR) regimen exhibit increased resistance of neurons to excitotoxic, oxidative, and metabolic insults in experimental models of Alzheimer's, Parkinson's, and Huntington's diseases and stroke. Because synaptic terminals are sites where the neurodegenerative process may begin in such neurodegenerative disorders, we determined the effects of DR on synaptic homeostasis and vulnerability to oxidative and metabolic insults. Basal levels of glucose uptake were similar in cerebral cortical synaptosomes from rats maintained on DR for 3 months compared with synaptosomes from rats fed ad libitum. Exposure of synaptosomes to oxidative insults (amyloid beta-peptide and Fe(2+)) and a metabolic insult (the mitochondrial toxin 3-nitropropionic acid) resulted in decreased levels of glucose uptake. Impairment of glucose uptake following oxidative and metabolic insults was significantly attenuated in synaptosomes from rats maintained on DR. DR was also effective in protecting synaptosomes against oxidative and metabolic impairment of glutamate uptake. Loss of mitochondrial function caused by oxidative and metabolic insults, as indicated by increased levels of reactive oxygen species and decreased transmembrane potential, was significantly attenuated in synaptosomes from rats maintained on DR. Levels of the stress proteins HSP-70 and GRP-78 were increased in synaptosomes from DR rats, consistent with previous data suggesting that the neuroprotective mechanism of DR involves a "preconditioning" effect. Collectively, our data provide the first evidence that DR can alter synaptic homeostasis in a manner that enhances the ability of synapses to withstand adversity.     

On-line determination of glucose and lactate concentrations in animal cell culture based on fibre optic detection of oxygen in flow-injection analysis.

A flow-injection analysis (FIA) system based on fibre optic detection of oxygen consumption using immobilized glucose oxidase (GOD) and lactate oxidase (LOD) is described for the on-line monitoring of glucose and lactate concentrations in animal cell cultures. The consumption of oxygen was determined via dynamic quenching by molecular oxygen of the fluorescence of an indicator. GOD and LOD were immobilized on controlled pore glass (CPG) in enzyme reactors which were directly linked to a specially designed fibre optic flow-through cell covering the oxygen optrode. The system is linear for 0-30 mM glucose, with an r.s.d. of 5% at 30 mM (five measurements) and for 0-30 mM lactate, with an r.s.d. of 5% at 30 mM (five measurements). The enzyme reactors used were stable for more than 4 weeks in continuous operation, and it was possible to analyse up to 20 samples per hour. The system has been successfully applied to the on-line monitoring of glucose and lactate concentrations of an animal cell culture designed for the production of recombinant human antithrombine III (AT-III). Results of the on-line measurement obtained by the FIA system were compared with the off-line results obtained by a glucose and lactate analyser from Yellow Springs Instrument Company (YSI).     

What are the relevant parameters for the geometrical optimization of an implantable bioartificial pancreas?

A sphere within a cylinder representing the islet encapsulated in a hollow fiber can model an implantable bioartificial pancreas. Based on a finite element model for insulin response to a glucose load in the presence of various oxygen supplies, the present study aimed at pointing out the major parameters influencing this secretion. The computational results treated with the Taguchi method clearly demonstrated that geometrical parameters (fiber length and islet density) should be precisely optimized for an enhanced insulin response. This requires the collection of more relevant experimental data concerning the islet oxygen consumption. Moreover, the relative errors on glucose consumption or insulin secretion by the islets do not seem to affect the whole optimization process, which should focus on the oxygen supply to islets.     

Fast dynamic response of the fermentative metabolism of Escherichia coli to aerobic and anaerobic glucose pulses

The response of Escherichia coli cells to transient exposure (step increase) in substrate concentration and anaerobiosis leading to mixed‐acid fermentation metabolism was studied in a two‐compartment bioreactor system consisting of a stirred tank reactor (STR) connected to a mini‐plug‐flow reactor (PFR: BioScope, 3.5 mL volume). Such a system can mimic the situation often encountered in large‐scale, fed‐batch bioreactors. The STR represented the zones of a large‐scale bioreactor that are far from the point of substrate addition and that can be considered as glucose limited, whereas the PFR simulated the region close to the point of substrate addition, where glucose concentration is much higher than in the rest of the bioreactor. In addition, oxygen‐poor and glucose‐rich regions can occur in large‐scale bioreactors. The response of E. coli to these large‐scale conditions was simulated by continuously pumping E. coli cells from a well stirred, glucose limited, aerated chemostat (D = 0.1 h^?1) into the mini‐PFR. A glucose pulse was added at the entrance of the PFR. In the PFR, a total of 11 samples were taken in a time frame of 92 s. In one case aerobicity in the PFR was maintained in order to evaluate the effects of glucose overflow independently of oxygen limitation. Accumulation of acetate and formate was detected after E. coli cells had been exposed for only 2 s to the glucose‐rich (aerobic) region in the PFR. In the other case, the glucose pulse was also combined with anaerobiosis in the PFR. Glucose overflow combined with anaerobiosis caused the accumulation of formate, acetate, lactate, ethanol, and succinate, which were also detected as soon as 2 s after of exposure of E. coli cells to the glucose and O₂ gradients. This approach (STR‐mini‐PFR) is useful for a better understanding of the fast dynamic phenomena occurring in large‐scale bioreactors and for the design of modified strains with an improved behavior under large‐scale conditions. Biotechnol. Bioeng. 2009; 104: 1153–1161. © 2009 Wiley Periodicals, Inc.     

Effects of Glucose, pH, and Dissolved-Oxygen Tension on Bacillus cereus Growth and Permeability Factor Production in Batch Culture

The production of a Bacillus cereus enterotoxin, measured as rabbit skin permeability factor (PF), in response to differences in glucose availability, pH, and dissolved oxygen tension was studied in a 1-liter batch fermentor system. Glucose had to be present for toxigenesis to occur. In uncontrolled fermentation an increasing inhibition of PF production and growth occurred as pH dropped occurred below 6.5. Optimum pH for toxigenesis was 7.0 to 7.5, and fermentations maintained at this level yielded 10- to 20-fold more PF than comparable uncontrolled fermentations. PF production was appreciably diminished at or below pH 6.0 and at or above pH 8.5. Peak PF titer was associated with a drop in acid output, and the titrant utilization profile could be used as an indication of this point. Productivity was greatest in the early exponential phase of growth and decreased to zero at the transition phase. Differences in dissolved oxygen tension affected both the maximum productivity early in the fermentation and the rate of its decrease as growth progressed. The optimum dissolved oxygen tension for toxigenesis was 0.002 atm, and the most rapid growth occurred at 0.10 atm. Productivity and growth were reduced under anerobic conditions, whereas a hyperoxic environment severely reduced productivity, but not growth. Postexponential-phase loss of toxic activity coincided with a rapid increase in cellular oxygen demand. Neither was inhibited by the presence of glucose. However, PF loss was completely prevented by stringent oxygen limitation. Extracellular proteolytic activity did not appear to be responsible for the loss of toxic activity.     

Effect of dissolved oxygen and Eh and Bacteroides fragilis during continuous culture.

Bacteroides fragilis subsp. fragilis was maintained in a chemostat modified for anaerobic conditions to test the effects of dissolved oxygen and Eh on growth. Using a defined medium containing glucose and a dilution rate of 0.16 h -1, a stable population of 3 X 10(9) colony-forming units/ml was present. At this steady state, the pH was 5.6, the Eh was -50 mV, and the dissolved oxygen concentration was 0% atmospheric saturation. The Eh was then adjusted to +300 mV by adding potassium ferricyanide while oxygen was excluded; in this system there were no demonstrable changes from the steady state in viable cells, pH, glucose concentration, or volatile fatty acid production. In other experiments oxygen was introduced into the original steady state at a dissolved oxygen concentration of 10% atmospheric saturation for a period of 6 to 8 h. During O2 exposure, the viable cell count decreased at a rate comparable to the theoretical washout rate for a static bacterial culture. Similar results were obtained with a dissolved oxygen concentration of 25 and 100%. Other effects of O2 exposure included an increase in Eh from -50 to +250 mV, a decrease in glucose consumption, and a decrease in volatile fatty acid production. These results suggest that dissolved oxygen has a bacteriostatic effect on B. fragilis in continuous culture, which may be independent of changes in Eh alone.     

Effects of glucose, pH, and dissolved-oxygen tension on Bacillus cereus growth and permeability factor production in batch culture.

The production of a Bacillus cereus enterotoxin, measured as rabbit skin permeability factor (PF), in response to differences in glucose availability, pH, and dissolved oxygen tension was studied in a 1-liter batch fermentor system. Glucose had to be present for toxigenesis to occur. In uncontrolled fermentation an increasing inhibition of PF production and growth occurred as pH dropped occurred below 6.5. Optimum pH for toxigenesis was 7.0 to 7.5, and fermentations maintained at this level yielded 10- to 20-fold more PF than comparable uncontrolled fermentations. PF production was appreciably diminished at or below pH 6.0 and at or above pH 8.5. Peak PF titer was associated with a drop in acid output, and the titrant utilization profile could be used as an indication of this point. Productivity was greatest in the early exponential phase of growth and decreased to zero at the transition phase. Differences in dissolved oxygen tension affected both the maximum productivity early in the fermentation and the rate of its decrease as growth progressed. The optimum dissolved oxygen tension for toxigenesis was 0.002 atm, and the most rapid growth occurred at 0.10 atm. Productivity and growth were reduced under anerobic conditions, whereas a hyperoxic environment severely reduced productivity, but not growth. Postexponential-phase loss of toxic activity coincided with a rapid increase in cellular oxygen demand. Neither was inhibited by the presence of glucose. However, PF loss was completely prevented by stringent oxygen limitation. Extracellular proteolytic activity did not appear to be responsible for the loss of toxic activity.     

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.     

The effect of elevation of maternal plasma catecholamines on the fetus and placenta of the pregnant sheep

To study the effects of reduced uterine blood flow on fetal and placental metabolism, adrenaline has been infused at physiological doses (0.5 microgram/min per kg) into the circulation of the pregnant sheep. This gives a reduction of about one third of uterine blood flow at days 120-143 of pregnancy, but causes no significant change in umbilical blood flow. In contrast to the effects of constricting the uterine artery to reduce blood flow to a similar degree, placental oxygen consumption was reduced and that, together with a large increase in lactate production, indicated the placenta became hypoxic. The fetal blood gas status and hence oxygen consumption was not affected significantly. A consistent arterio-venous difference for glucose across the umbilical or uterine circulations was not detected unless the uterine blood flow was comparatively high. Glucose balance across the uterus showed a close linear relationship with uterine blood flow and more particularly with the supply of glucose to the uterus. There was clear evidence for glucose uptake by the placenta and fetus and also glucose output by both. The latter was more common when uterine blood flow was comparatively low or reduced by adrenaline infusion. The results are consistent with the concept that glucose supply has to be maintained to the placenta even at the expense of fetal stores, although lactate can substitute if there is enhanced output because of fetal hypoxia. They indicate that placental mobilisation of glycogen can lead to a net output of glucose to the mother. The manner of communicating to the fetus changes in placental state that occur during maternal adrenaline infusion is not clear. However towards the end of the 60 min infusion, elevation of fetal plasma adrenaline, probably resulting from a breakdown of the placental permeability barrier, may be an important signal.     

Altered multiaxial mechanical properties of the porcine anterior lens capsule cultured in high glucose

Hyperglycemia can alter the mechanical properties of tissues through the formation of advanced glycation endproducts in matrix proteins that have long half-lives. We used a custom experimental system and subdomain finite element method to quantify alterations in the regional multiaxial mechanical properties of porcine lens capsules that were cultured for 8 or 14 weeks in high glucose versus control media. Findings revealed that high glucose significantly stiffened the capsules in both the circumferential and the meridional directions, but it did not affect the known regional variations in anisotropy. Such information could be important in the design of both improved clinical procedures and intraocular implants for diabetic patients.     

A fluorescent glucose biosensor based on immobilized glucose oxidase on bamboo inner shell membrane

A fluorescent glucose biosensor was constructed by immobilizing glucose oxidase on a bamboo inner shell membrane with glutaraldehyde as a cross-linker. The detection scheme was based on the depletion of dissolved oxygen content upon exposure to glucose solution with a concomitant increase in the fluorescence intensity of an oxygen transducer, tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(Pi) ditetrakis(4-chlorophenyl)borate. The enzyme immobilization, effect of pH, temperature and ionic strength have been studied in detail. The biosensor exhibited repeatable response to a 2.0 mM glucose solution with a relative standard deviation of 3.0% (n = 10). It showed good storage stability and maintained 95% of its initial response after it had been kept at 4 degrees C for 8 months. The biosensor has a linear response range of 0.0-0.6 mM glucose with a detection limit of 58 microM (S/N = 3). Common potential interferants in samples do not pose any significant interference on the response of the glucose biosensor. It was successfully applied to the determination of glucose content in some commercial wines and medical glucose injections.