ATP synthesis kinetics and mitochondrial function in the postischemic myocardium as studied by 31P NMR

The effects of ischemia on mitochondrial function and the unidirectional rate of ATP synthesis (Pi----ATP rate) were studied using a Langendorff-perfused heart preparation and 31P NMR spectroscopy. There was significant postischemic depression of mechanical function assessed as the heart rate pressure product, and the myocardial oxygen consumption rate at a given rate pressure product was elevated. Experiments performed on glucose- and pyruvate-perfused hearts demonstrated the presence of a large contribution to the unidirectional Pi----ATP rate catalyzed by glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. This rate was much greater than the maximal glucose utilization rate in the myocardium, demonstrating that the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase reactions are near equilibrium both before and after ischemia. In the pyruvate-perfused postischemic hearts, the glycolytic contribution was eliminated and the net rate of ATP synthesis by oxidative phosphorylation was measurable. Despite the reduced mechanical function and increased myocardial oxygen consumption rate, the ratio of the net rate of ATP synthesis by oxidative phosphorylation to oxygen consumption rate (the P:O ratio) was not altered subsequent to ischemia (2.34 +/- 0.12 and 2.36 +/- 0.09 in normal and postischemic hearts, respectively). Therefore, mitochondrial uncoupling cannot be the cause of postischemic depression in mechanical function; instead, the data suggest the existence of ischemia-induced inefficiency in ATP utilization.     

Endothelial cell and platelet bioenergetics: effect of glucose and nutrient composition

It has been suggested that cells that are independent of insulin for glucose uptake, when exposed to high glucose or other nutrient concentrations, manifest enhanced mitochondrial substrate oxidation with consequent enhanced potential and generation of reactive oxygen species (ROS); a paradigm that could predispose to vascular complications of diabetes. Here we exposed bovine aortic endothelial (BAE) cells and human platelets to variable glucose and fatty acid concentrations. We then examined oxygen consumption and acidification rates using recently available technology in the form of an extracellular oxygen and proton flux analyzer. Acute or overnight exposure of confluent BAE cells to glucose concentrations from 5.5 to 25 mM did not enhance or change the rate of oxygen consumption (OCR) under basal conditions, during ATP synthesis, or under uncoupled conditions. Glucose also did not alter OCR in sub-confluent cells, in cells exposed to low serum, or in cells treated with added pyruvate. Likewise, overnight exposure to fatty acids of varying saturation had no such effects. Overnight exposure of BAE cells to low glucose concentration decreased maximal uncoupled respiration, but not basal or ATP related oxygen consumption. Labeled glucose oxidation to CO(2) increased, but only marginally after high glucose exposure while oleate oxidation to CO(2) decreased. Overnight exposure to linolenic acid, but not oleic or linoleic acid increased extracellular acidification consistent with enhanced glycolytic metabolism. We were unable to detect an increase in production of reactive oxygen species (ROS) from BAE cells exposed to high medium glucose. Like BAE cells, exposure of human platelets to glucose did not increase oxygen consumption. As opposed to BAE cells, platelet mitochondria demonstrate less respiratory reserve capacity (beyond that needed for basal metabolism). Our data do not support the concept that exposure to high glucose or fatty acids accelerates mitochondrial oxidative metabolism in endothelial cells or platelets.     

A biochemically structured model for Saccharomyces cerevisiae.

A biochemically structured model for the aerobic growth of Saccharomyces cerevisiae on glucose and ethanol is presented. The model focuses on the pyruvate and acetaldehyde branch points where overflow metabolism occurs when the growth changes from oxidative to oxido-reductive. The model is designed to describe the onset of aerobic alcoholic fermentation during steady-state as well as under dynamical conditions, by triggering an increase in the glycolytic flux using a key signalling component which is assumed to be closely related to acetaldehyde. An investigation of the modelled process dynamics in a continuous cultivation revealed multiple steady states in a region of dilution rates around the transition between oxidative and oxido-reductive growth. A bifurcation analysis using the two external variables, the dilution rate, D, and the inlet concentration of glucose, S(f), as parameters, showed that a fold bifurcation occurs close to the critical dilution rate resulting in multiple steady-states. The region of dilution rates within which multiple steady states may occur depends strongly on the substrate feed concentration. Consequently a single steady state may prevail at low feed concentrations, whereas multiple steady states may occur over a relatively wide range of dilution rates at higher feed concentrations.     

Modelling Xanthomonas campestris batch fermentations in a bubble column

Rate and yield expressions relating to biomass and xanthan formation and to nitrogen, glucose, and oxygen consumption were established for Xanthomonas campestris batch fermentations in a bubble column. Microbial growth was described by the logistic rate equation, characterized by a maximum specific growth rate mu(M) = 0.5 h(-1) and a maximum attainable cell concentration provided by nitrogenous compounds. With regard to carbon metabolism, the decrease with time in experimental yields and in the experimental specific rates of xanthan production and glucose assimilation demonstrated the inadequacy of the Luedeking-Piret model. These decreases were connected to the simultaneous drop in dissolved-oxygen tension observed during xanthan synthesis. The knowledge of metabolic pathways and energetic balance were used to establish the relationships between substrate utilization, ATP generation, and xanthan production. The model was structured by assuming the oxygen limitation of both the respiration rate and the efficiency of the oxidative phosphorylation mechanism (P/O ratio). Consequently, the specific rates and yield expressions became dependent on the dissolved-oxygen tension, i.e., of the volumetric oxygen transfer in the fermentor.     

A mathematical model of tumor oxygen and glucose mass transport and metabolism with complex reaction kinetics

Hypoxia imparts radioresistance to tumors, and various approaches have been developed to enhance oxygenation, thereby improving radiosensitivity. This study explores the influence of kinetic and physical factors on substrate metabolism in a tumor model, based on a Krogh cylinder. In tissue, aerobic metabolism is assumed to depend on glucose and oxygen, represented by the product of Michaelis-Menten expressions. For the base case, an inlet pO(2) of 40 mmHg, a hypoxic limit of 5 mmHg, and a tissue/capillary radius ratio of 10 are used. For purely aerobic metabolism, a hypoxic fraction of 0.16 and volume-average pO(2) of 8 mmHg are calculated. Reducing the maximum oxygen rate constant by 9%, decreasing the tissue cylinder radius by 5%, or increasing the capillary radius by 8% abolishes the hypoxic fraction. When a glycolytic term is added, concentration profiles are similar to the base case. Using a distribution of tissue/capillary radius ratios increases the hypoxic fraction and reduces sensitivity to the oxygen consumption rate, compared to the case with a single tissue/capillary radius ratio. This model demonstrates that hypoxia is quite sensitive to metabolic rate and geometric factors. It also predicts quantitatively the effects of inhibited oxygen metabolism and enhanced mass transfer on tumor oxygenation.     

Identification and control of oxidative metabolism in Ssaccharomyces cerevisiae during transient growth using calorimetric measurements

The objective of this study was to characterize the dynamic adaptation of the oxidative capacity of Saccharomyces cerevisiae to an increase in the glucose supply rate and its implications for the control of a continuous culture designed to produce biomass without allowing glucose to be diverted into the reductive metabolism. Continuous cultures subjected to a sudden shift-up in the dilution rate showed that the glucose uptake rate increased immediately to the new feeding rate but that the oxygen consumption could not follow fast enough to ensure a completely oxidative metabolism. Thus, part of the glucose assimilated was degraded by the reductive metabolism, resulting in a temporary decrease of biomass concentration, even if the final dilution rate was below Dcrit. The dynamic increase of the specific oxygen consumption rate, qO2, was characterized by an initial immediate jump followed by a first-order increase to the maximum value. It could be modeled using three parameters denoted qjumpO2, qmaxO2, and a time constant tau. The values for the first two of the parameters varied considerably from one shift to another, even when they were performed under identical conditions. On the basis of this model, a time-dependent feed flow rate function was derived that should permit an increase in the dilution rate from one value to another without provoking the appearance of reductive metabolism. The idea was to increase the glucose supply in parallel with the dynamic increase of the oxidative capacity of the culture, so that all of the assimilated glucose could always be oxidized. Nevertheless, corresponding feed-profile experiments showed that deviations in the reductive metabolism could not be completely suppressed due to variability in the model parameters. Therefore, a proportional feedback controller using heat evolution rate measurements was implemented. Calorimetry provides an excellent and rapid estimate of the metabolic activity. Satisfactory control was achieved and led to constant biomass yields. Ethanol accumulated only up to 0.49 g L-1 as compared to an accumulation of 1.82 g L-1 without on-line control in the shift-up experiment to the same final dilution rate.     

Coupling of microbial kinetics and oxygen transfer for analysis and optimization of gluconic acid production with Aspergillus niger

Gluconic acid fermentation has been widely used for the analysis of various aspects of kinetics and gas liquid transfer of oxygen. Most of these studies are, however, restricted to processes with bacteria. Mathematical models for industrially important productions with fungi have not been elaborated. In the experimental part of this work computer coupled fermentations of gluconic acid production with Aspergillus niger NRRL 3 have been performed. Knowledge of the stoichiometric relationship in the key reaction (glucose oxidase) provides an excellent opportunity for on-line estimation of glucose, biomass and product gluconate from oxygen uptake and carbon dioxide evolution rates. Starting then from experimental observations on the pH-depending oxygen kinetics of gluconic acid formation and influences of product concentrations on the growth of Aspergillus niger a mathematical framework is developed in which the kinetics of growth and production are coupled with gas liquid oxygen transfer. The model can be successfully applied to simulations of the experimental results of gluconic acid fermentations with cyclic addition of glucose. An important aspect in the coupling of transport and microbial reaction in this model is the incorporation of the influence of sugar and gluconate on the solubility of oxygen and k _La via changes of viscosities and molecular diffusivities. With the development of such a comprehensive model, it appears feasible to investigate the influence of various process conditions (sugar feeding, pressure, optimal pH profiles) and to study their possible impacts on the productivity of the overall process.     

Metabolic gradients as key regulators in zonation of tumor energy metabolism: A tissue-scale model-based study

Characteristics of many tumor types are the reprogramming of metabolism and the occurrence of regional hypoxia. In this work, we investigated the hypothesis that metabolic reprogramming in combination with metabolic zonation of cellular energy metabolism are important factors in promotion of the growth capacity of solid tumors. A tissue-scale model of the two main ATP delivering pathways, glycolysis (GLY) and oxidative phosphorylation (OXP), was used to simulate the energy metabolism within solid tumors under various metabolic strategies. Remarkably, despite the high diversity in the usage of glucose, lactate and oxygen in various spatial regions, the tumor as a whole clearly displays the hallmark of the so-called Warburg effect, i.e. a high rate of glucose consumption and lactate production in the presence of sufficiently high levels of oxygen. Our simulations suggest that an increase in GLY capacity and concomitant decrease in OXP capacity from the periphery towards the center of the tumor improves the availability of oxygen to pericentral tumor cells. The found relationship between the regional oxygen level and the relative share of GLY and OXP capacities supports the view that metabolite availability functions as key regulator of tumor energy metabolism.     

A method for the determination of confidence limits for the P/2e − ratio for chosen values of Y ATP max from the results of continuous culture experiments

A model is described, which allows the determination of 95% confidence limits for the maintenance coefficient and the efficiency of oxidative phosphorylation for chosen values of the growth yield for ATP corrected for energy maintenance (Y _ATP ^max ). As experimental data the specific rates of substrate consumption, product formation and oxygen uptake in chemostat cultures at various growth rates are used.     

Impact of altered substrate utilization on cardiac function in isolated hearts from Zucker diabetic fatty rats

The goal of this study was to determine whether changes in cardiac metabolism in Type 2 diabetes are associated with contractile dysfunction or impaired response to ischemia. Hearts from Zucker diabetic fatty (ZDF) and lean control rats were isolated and perfused with glucose, lactate, pyruvate, and palmitate. The rates of glucose, lactate, pyruvate, and palmitate oxidation rates and glycolysis were determined during baseline perfusion and low-flow ischemia (LFI; 0.3 ml/min for 30 min) and after LFI and reperfusion. Under all conditions, ATP synthesis from palmitate was increased and synthesis from lactate was decreased in the ZDF group, whereas the contribution from glucose was unchanged. During baseline perfusion, the rate of glycolysis was lower in the ZDF group; however, during LFI and reperfusion, there were no differences between groups. Despite these metabolic shifts, there were no differences in oxygen consumption or ATP production rates between the groups under any perfusion conditions. Cardiac function was slightly depressed before LFI in the ZDF group, but during reperfusion, function was improved relative to the control group despite the increased dependence on fatty acids for energy production. These data suggest that in this model of diabetes, the shift from carbohydrates to fatty acids for oxidative energy production did not increase myocardial oxygen consumption and was not associated with impaired response to ischemia and reperfusion.     

Kinetic study of a change in intracellular ATP level associated with aerobic catabolism of ethanol by Streptococcus mutans.

Streptococcus mutans, a group of lactic acid bacteria and a normal inhabitant of the human oral cavity, generates ATP by substrate-level phosphorylation coupled to oxidation of ethanol (an end product of fermentation of sugars) into acetate in the presence of oxygen (K. Fukui, K. Kato, Kodama, H. Ohta, T. Shima moto, and T. Shimono, Proc. Jpn. Acad. 64B:13-16, 1988). Kinetic measurements were made of the cellular responses of S. mutans FA-1 to ethanol in comparison with those to glucose. In contrast to oxygen-independent acid production from glucose, oxygen was absolutely required for acid production from ethanol. Ethanol elicited a marked increase in the intracellular ATP concentration (ATPi) from a starved level to a steady level which was held constant as long as oxygen was present in the medium. Once oxygen was exhausted, ATPi returned to the starved level without delay. On the contrary, ATPi changes induced by glucose, which were independent of oxygen, followed a rather complicated time course before a steady level was established. Both the steady ATPi and the rate of accompanying oxygen consumption were functions of the ethanol concentration. These two parameters were linearly correlated, indicating that the unimolecular ATP turnover rate, which is independent of the rate of ATP generation in the steady state, can be calculated for cells energized by ethanol. The estimated turnover rate was 1.5 s-1 at 37 degrees C, which is comparable to that for other bacteria energized by glucose under nongrowing conditions.     

Effect of oxygen supply and dilution rate on the production of 2,3-butanediol in continuous bioreactor by Klebsiella pneumoniae

Kinetics of 2,3-butanediol production by Klebsiella pneumoniae (NRRL B199) from glucose have been studied in a continuous bioreactor. The effect of oxygen supply rate and dilution rate on the product output rate and yield of 2,3-butanediol were investigated. For a feed glucose concentration of 100 g l⁻¹, the optimum oxygen transfer rate is between 25.0–35.0 mmol l⁻¹ h⁻¹. Under these conditions, maximum product concentration obtained was 35 g l⁻¹ at a dilution rate of 0.1 h⁻¹ and the maximum product output rate obtained was 4.25 g l⁻¹ h⁻¹. The product yield based on the substrate utilized approached the theoretical value (50%) at low values of oxygen transfer rate but decreased with increasing oxygen transfer rate.     

Oxygen transfer rate,respiration and yields in batch and chemostat cultures ofKlebsiella aerogenes

The effect was studied of oxygen supply on the changes in total and specific rate of oxygen consumption by the cells, oxygen transfer rate, saturation concentrations of dissolved oxygen and the yields of batch and continuous cultivations. Experiments were done on the microorganismKlebsiella aerogenes CCM 2318 growing on synthetic glucose medium. Continuous cultivations were carried out at dilution rates of 0.96 and 0.178 h⁻¹. The rate of oxygen transfer was determined by the sulphite method and the coefficient K_La was assessed using the dynamic method with a correction for changes in the saturations of dissolved oxygen. A lowered oxygen supply in batch cultivations caused deformations in the course of cell respiration. Comparison of results of batch and continuous cultivations showed that the highest yields Y_x/s and Y_x/o are attained at low dilution rates without oxygen limitation. Batch cultivations, on the other hand, exhibit the lowest yields and the highest cell respiration levels. In both types of cultivations, a respiration peak was ascertained under the conditions of growth limitation by oxygen.     

Both superficial and deep zone articular chondrocyte subpopulations exhibit the crabtree effect but have different basal oxygen consumption rates

In the absence of in vivo measurements, the oxygen concentration within articular cartilage is calculated from the balance between cellular oxygen consumption and mass transfer. Current estimates of the oxygen tension within articular cartilage are based on oxygen consumption data from full‐depth tissue samples. However, superficial and deep cell subpopulations of articular cartilage express intrinsic metabolic differences. We test the hypothesis that the subpopulations differ with respect to their intrinsic oxygen consumption rate. Chondrocytes from the full cartilage thickness demonstrate enhanced oxygen consumption when deprived of glucose, consistent with the Crabtree phenomena. Chondrocyte subpopulations differ in the prevailing availability of oxygen and glucose, which decrease with distance from the cartilage–synovial fluid interface. Thus, we tested the hypothesis that the oxygen consumption of each subpopulation is modulated by nutrient availability, by examining the expression of the Crabtree effect. The deep cells had a greater oxygen consumption than the superficial cells (V_max of 6.6 compared to 3.2 fmol/cell/h), consistent with our observations of mitochondrial volume (mean values 52.0 vs. 36.4 µm³/cell). Both populations expressed the Crabtree phenomena, with oxygen consumption increasing ～2.5‐fold in response to glycolytic inhibition by glucose deprivation or 2‐deoxyglucose. Over 90% of this increase was oligomycin‐sensitive and thus accounted for by oxidative phosphorylation. The data contributes towards our understanding of chondrocyte energy metabolism and provides information valuable for the accurate calculation of the oxygen concentration that the cells experience in vivo. The work has further application to the optimisation of bioreactor design and engineered tissues. J. Cell. Physiol. 223:630–639, 2010. © 2010 Wiley‐Liss, Inc.     

Effect of pH and dilution rate on specific production rate of extra cellular metabolites by <Emphasis Type="Italic">Lactobacillus salivarius</Emphasis> UCO_979C in continuous culture

The effect of pH and dilution rate on the production of extracellular metabolites of Lactobacillus salivarius UCO_979 was studied. The experiments were carried out in continuous mode, with chemically defined culture medium at a temperature of 37 °C, 200 rpm agitation and synthetic air flow of 100 ml/min. Ethanol, acetic acid, formic acid, lactic acid and glucose were quantified through HPLC, while exopolysaccharide (EPS) was extracted with ethanol and quantified through the Dubois method. The results showed no linear trends for the specific production of lactic acid, EPS, acetic acid and ethanol, while the specific glucose consumption and ATP production rates showed linear trends. There was a metabolic change of the strain for dilution rates below 0.3 h^?1. The pH had a significant effect on the metabolism of the strain, which was evidenced by a higher specific glucose consumption and increased production of ATP at pH 6 compared with that obtained at pH 7. This work shows not only the metabolic capabilities of L. salivarius UCO_979C, but also shows that it is possible to quantify some molecules associated with its current use as gastrointestinal probiotic, especially regarding the production of organic acids and EPS.     

Effects of dissolved oxygen on hybridoma cell growth, metabolism, and antibody production kinetics in continuous culture.

The effects of dissolved oxygen concentration (DO) on hybridoma cell physiology were examined in a continuous stirred tank bioreactor with a murine hybridoma cell line (167.4G5.3). Dissolved oxygen concentration was varied between 0% and 100% air saturation. Cell growth and viability, carbohydrate, amino acid, and energy metabolism, oxygen uptake, and antibody production rates were investigated. Cell growth was inhibited at both high and low DO. Cells could grow at 0% DO and maintain viability under a nitrogen atmosphere. Cell viability was higher at low DO. Glucose, glutamine, and oxygen consumption rates changed little at DO above 1% air saturation. However, the metabolic uptake rates changed below 1% DO, where growth became oxygen limited, and a Km value of 0.6% DO was obtained for the specific oxygen uptake rate. The metabolic rates of glucose, glutamine, lactate, and ammonia increased 2-3-fold as the DO dropped from 1% to 0%. Amino acid metabolism followed the same general pattern as that of glutamine and glucose. Alanine was the only amino acid produced. The consumption rates of amino acids changed little above 1% DO, but under anaerobic conditions the consumption rates of all amino acids increased severalfold. Cells obtained most of their metabolic energy from glutamine oxidation except under oxygen limitation, when glucose provided most of the energy. The calculated ATP production rate was only slightly influenced by DO and rose at 0% DO. Antibody concentration was highest at 35% DO, while the specific antibody production rate was insensitive to DO.     

Reaction rates of creatine kinase and ATP synthesis in the isolated rat heart. A 31P NMR magnetization transfer study

The NMR technique of magnetization transfer can be used to define intracellular reaction kinetics. In order to determine the relationship between ATP synthesis and flux through the creatine kinase reaction in the intact heart, we used this technique to measure flux through the creatine kinase reaction in the isolated, isovolumic rat heart at five levels of cardiac performance and oxygen consumption. The unidirectional reaction rate constants (s-1) calculated from a two-site exchange model for both the forward and reverse creatine kinase reactions increased with cardiac performance and oxygen consumption. As the rate-pressure product varied from 0 to 44.7 X 10(3) mm Hg/min and oxygen consumption rose from 5.9 to 45.8 mumol of O2/g dry weight/min, kforward increased from 0.27 to 1.30 and kreverse increased from 0.31 to 1.14. The relationship between creatine kinase flux and oxygen consumption, and thus ATP synthesis, took the form of the Michaelis-Menten equation. Rates of ATP synthesis estimated from magnetization transfer were similar to values calculated from oxygen consumption. The longitudinal relaxation time of creatine phosphate (2.06 s), the gamma-phosphorus atom of ATP (0.75 s), and inorganic phosphate (0.81 s) did not change with cardiac performance. These results show that myocardial energy transfer via the creatine kinase reaction is closely coupled to energy production.     

Metabolic control over the oxygen consumption flux in intact skeletal muscle: in silico studies

It has been postulated previously that a direct activation of all oxidative phosphorylation complexes in parallel with the activation of ATP usage and substrate dehydrogenation (the so-called each-step activation) is the main mechanism responsible for adjusting the rate of ATP production by mitochondria to the current energy demand during rest-to-work transition in intact skeletal muscle in vivo. The present in silico study, using a computer model of oxidative phosphorylation developed previously, analyzes the impact of the each-step-activation mechanism on the distribution of control (defined within Metabolic Control Analysis) over the oxygen consumption flux among the components of the bioenergetic system in intact oxidative skeletal muscle at different energy demands. It is demonstrated that in the absence of each-step activation, the oxidative phosphorylation complexes take over from ATP usage most of the control over the respiration rate and oxidative ATP production at higher (but still physiological) energy demands. This leads to a saturation of oxidative phosphorylation, impossibility of a further acceleration of oxidative ATP synthesis, and dramatic drop in the phosphorylation potential. On the other hand, the each-step-activation mechanism allows maintenance of a high degree of the control exerted by ATP usage over the ATP turnover and oxygen consumption flux even at high energy demands and thus enables a potentially very large increase in ATP turnover. It is also shown that low oxygen concentration shifts the metabolic control from ATP usage to cytochrome oxidase and thus limits the oxidative ATP production. respiration rate; parallel activation; oxidative phosphorylation; metabolic control analysis; flux control coefficient; muscle contraction     

Substrate oxidation and ATP supply in AS-30D hepatoma cells

The oxidation of several metabolites in AS-30D tumor cells was determined. Glucose and glycogen consumption and lactic acid production showed high rates, indicating a high glycolytic activity. The utilization of ketone bodies, oxidation of endogenous glutamate, and oxidative phosphorylation were also very active: tumor cells showed a high respiration rate (100 ng atoms oxygen (min x 10(7) cells)(-1)), which was 90% oligomycin-sensitive. AS-30D tumor cells underwent significant intracellular volume changes, which preserved high concentrations of several metabolites. A high O(2) concentration, but a low glucose concentration were found in the cell-free ascites liquid. Glutamine was the oxidizable substrate found at the highest concentration in the ascites liquid. We estimated that cellular ATP was mainly provided by oxidative phosphorylation. These data indicated that AS-30D hepatoma cells had a predominantly oxidative and not a glycolytic type of metabolism. The NADH-ubiquinol oxido reductase and the enzyme block for ATP utilization were the sites that exerted most of the control of oxidative phosphorylation (flux control coefficient = 0.3-0.42).     

Hybridoma growth and antibody production as a function of cell density and specific growth rate in perfusion culture

Steady state metabolic parameters for hybridoma cell line H22 were determined over a wide range of cell densities and specific growth rates in a filtration based homogeneous perfusion reactor. Operating the reactor at perfusion rates of 0.75, 2.0, and 2.9 day(-1)(each at four different specific growth rates), viable cell densities as high as 2 x 10(7) cells/mL were obtained. For the cell line under investigation, the specific monoclonal antibody production rate was found to be a strong function of the viable cell density, increasing with increasing cell density. In contrast, most of the substrate consumption and product formation rates were strong functions of the specific growth rate. Substrate metabolism became more efficient at high cell densities and low specific growth rates. The Specific rates of metabolite formation and the apparent yields of lactate from glucose and ammonia from glutamine decreased at low specific growth rates and high cell densities. While the specific oxygen consumption rate was independent of the specific growth rate and cell density, ATP production was more oxidative at lower specific growth rate and higher cell density. These observed shifts are strong indications of the production potential of high-density perfusion culture. (c) 1995 John Wiley & Sons, Inc.