Metabolic processes account for the majority of the intracellular water in log-phase Escherichia coli cells as revealed by hydrogen isotopes

It is generally believed that water transport across biological membranes is essentially a near-instantaneous process, with water molecules diffusing directly across the membrane as well as through pores such as aquaporins. As a result of these processes by which water can equilibrate across a membrane, a common assumption is that intracellular water is isotopically indistinguishable from extracellular water. To test this assumption directly, we measured the hydrogen isotope ratio of intracellular water in Escherichia coli cells. Our results demonstrate that more than 50% of the intracellular water hydrogen atoms in log-phase E. coli cells are isotopically distinct from the growth medium water and that these isotopically distinct hydrogen atoms are derived from metabolic processes. As expected, the (2)H/(1)H isotope ratio of intracellular water from log-phase cells showed an appreciably larger contribution from metabolic water than did intracellular water from stationary-phase cells (53 +/- 12 and 23 +/- 5%, respectively). The (2)H/(1)H isotope ratio of intracellular water was also monitored indirectly by measuring the isotope ratio of fatty acids, metabolites that are known to incorporate hydrogen atoms from water during biosynthesis. Significantly, the difference in the isotopic composition of intracellular water from log- to stationary-phase E. coli cells was reflected in the hydrogen isotope ratio of individual fatty acids harvested at the two different times, indicating that the isotope ratio of metabolites can be used as an indirect probe of metabolic activity. Together, these results demonstrate that contrary to the common assumption that intracellular water is isotopically identical to extracellular water, these two pools of water can actually be quite distinct.     

Investigation of metabolic objectives in cultured hepatocytes

Using optimization based methods to predict fluxes in metabolic flux balance models has been a successful approach for some microorganisms, enabling construction of in silico models and even inference of some regulatory motifs. However, this success has not been translated to mammalian cells. The lack of knowledge about metabolic objectives in mammalian cells is a major obstacle that prevents utilization of various metabolic engineering tools and methods for tissue engineering and biomedical purposes. In this work, we investigate and identify possible metabolic objectives for hepatocytes cultured in vitro. To achieve this goal, we present a special data-mining procedure for identifying metabolic objective functions in mammalian cells. This multi-level optimization based algorithm enables identifying the major fluxes in the metabolic objective from MFA data in the absence of information about critical active constraints of the system. Further, once the objective is determined, active flux constraints can also be identified and analyzed. This information can be potentially used in a predictive manner to improve cell culture results or clinical metabolic outcomes. As a result of the application of this method, it was found that in vitro cultured hepatocytes maximize oxygen uptake, coupling of urea and TCA cycles, and synthesis of serine and urea. Selection of these fluxes as the metabolic objective enables accurate prediction of the flux distribution in the system given a limited amount of flux data; thus presenting a workable in silico model for cultured hepatocytes. It is observed that an overall homeostasis picture is also emergent in the findings.     

Scavenging of extracellular H2O2 by catalase inhibits the proliferation of HER-2/Neu-transformed rat-1 fibroblasts through the induction of a stress response

High levels of reactive oxygen species (ROS) are associated with cytotoxicity. Alternatively, nontoxic levels of ROS like hydrogen peroxide (H(2)O(2)) can mediate the transmission of many intracellular signals, including those involved in growth and transformation. To identify pathways downstream of endogenous cellular H(2)O(2) production, the response of Rat-1 fibroblasts exhibiting differential HER-2/Neu receptor tyrosine kinase activity to removal of physiological H(2)O(2) concentrations was investigated. The proliferation of all cells was abolished by addition of the H(2)O(2) scavenger catalase to the culture medium. HER-2/Neu activity was not significantly affected by catalase treatment, suggesting that the target(s) of the H(2)O(2) signal lie downstream of the receptor in our model. ERK1/2 phosphorylation was blocked by catalase in fibroblasts expressing wild type Neu, however such a response did not occur in cells possessing activated mutant Neu. This indicates that the ERK1/2 response contributes little to the growth inhibition observed. By contrast, JNK1 activity increased following the addition of catalase or H(2)O(2), regardless of Neu activity or level of cell transformation. Phosphorylation of p38 MAPK was induced by H(2)O(2) but not by catalase. These observations suggest that scavenging of H(2)O(2) from the cellular environment blocks Rat-1 proliferation primarily through the activation of stress pathways.     

Network identification and flux quantification of glucose metabolism in Rhodobacter sphaeroides under photoheterotrophic H(2)-producing conditions

The nonsulfur purple bacteria that exhibit unusual metabolic versatility can produce hydrogen gas (H(2)) using the electrons derived from metabolism of organic compounds during photoheterotrophic growth. Here, based on (13)C tracer experiments, we identified the network of glucose metabolism and quantified intracellular carbon fluxes in Rhodobacter sphaeroides KD131 grown under H(2)-producing conditions. Moreover, we investigated how the intracellular fluxes in R. sphaeroides responded to knockout mutations in hydrogenase and poly-β-hydroxybutyrate synthase genes, which led to increased H(2) yield. The relative contribution of the Entner-Doudoroff pathway and Calvin-Benson-Bassham cycle to glucose metabolism differed significantly in hydrogenase-deficient mutants, and this flux change contributed to the increased formation of the redox equivalent NADH. Disruption of hydrogenase and poly-β-hydroxybutyrate synthase resulted in a significantly increased flux through the phosphoenolpyruvate carboxykinase and a reduced flux through the malic enzyme. A remarkable increase in the flux through the tricarboxylic acid cycle, a major NADH producer, was observed for the mutant strains. The in vivo regulation of the tricarboxylic acid cycle flux in photoheterotrophic R. sphaeroides was discussed based on the measurements of in vitro enzyme activities and intracellular concentrations of NADH and NAD(+). Overall, our results provide quantitative insights into how photoheterotrophic cells manipulate the metabolic network and redistribute intracellular fluxes to generate more electrons for increased H(2) production.     

Phenotypic conversion of TK-deficient cells following electroporation of functional TK enzyme.

The ability to phenotypically rescue a mutant (Rat-3, thymidine kinase-deficient) cell line by electroporation of functional TK enzyme has been investigated. Extracts of electroporated cells showed a 35-fold increase in TK enzyme levels under conditions where greater than 90% of the cells remained viable. The electroporated enzyme was intracellular, as demonstrated by the fact that cells were able to utilize exogenous [3H]thymidine for DNA synthesis. By in situ autoradiography, 82% of electroporated cells contained functional enzyme and incorporated [3H]thymidine into DNA. Thus, this technique can efficiently provide a missing metabolic function to cultured mammalian cells.     

Lysophosphatidic acid increases intracellular H2O2 by phospholipase D and RhoA in rat-2 fibroblasts.

We have investigated the possible roles of phospholipase D (PLD) and RhoA in the production of intracellular H2O2 and actin polymerization in response to lysophosphatidic acid (LPA) in Rat-2 fibroblasts. LPA increased intracellular H2O2, with a maximal increase at 30 min, which was blocked by the catalase from Aspergillus niger. The LPA-stimulated production of H2O2 was inhibited by 1-butanol or PKC-downregulation, but not by 2-butanol. Purified phosphatidic acid (PA) also increased intracellular H2O2 and the increase was inhibited by the catalase. The role of RhoA was studied by the scrape-loading of C3 transferase into the cells. The C3 toxin, which inhibited stress fiber formation stimulated by LPA, blocked the H2O2 production in response to LPA or PA, but had no inhibitory effect on the activation of PLD by LPA. Exogenous H2O2 increased F-actin content by stress fiber formation. In addition, catalase inhibited actin polymerization activated by LPA, PA, or H2O2, indicated the role of H2O2 in actin polymerization. These results suggest that LPA increased intracellular H2O2 by the activation of PLD and RhoA, and that intracellular H2O2 was required for the LPA-stimulated stress fiber formation.     

Metabolic flux analysis of hybridoma cells in different culture media using mass balances

The estimation of the intracellular fluxes of mammalian cells using only the mass balances of the relevant metabolites is not possible because the set of linear equations defined by these mass balances is underdetermined. Either additional experimental flux data or additional theoretical constraints are required to find one unique flux distribution out of the solution space that is bound by the mass balances. Here, a method is developed using the latter approach. The uptake and production rates of amino acids, glucose, lactate, O(2), CO(2), NH(4), MAB, and the intracellular amino acid pools have been determined for two different steady-states. The cellular composition {total protein and protein composition, total lipids and fatty acid distribution, total carbohydrates, DNA and RNA} has been measured to calculate the requirements for biosynthesis. It is shown to be essential to determine the uptake/production rates of ammonia and either carbon dioxide or oxygen. In mammalian cells these are cometabolites of cyclic metabolic pathways. The flux distribution that is found using the Euclidean minimum norm as the additional theoretical constraint and taking either the CO(2) or the NAD(P)H mass balance into account is shown to be in agreement with the measured O(2) and CO(2) metabolic rates.The metabolic fluxes in hybridoma cells in continuous culture at a specific growth rate of 0.83 day(-1) are estimated for a medium with (optimal medium) and without (suboptimal medium) Primatone RL, an enzymatic hydrolysate of animal tissue that causes a more than twofold increase in cell density. It is concluded that (i)The majority of the consumed glucose (>90%) is channeled through the pentose-phosphate pathway in rapidly proliferating cells.(ii)Pyruvate oxidation and tricarboxylic acid (TCA) cycle activity are relatively low, i.e., 8% of the glucose uptake in suboptimal and 14% in optimal medium, respectively. Under both conditions, only a small fraction of pyruvate is further oxidized to CO(2).(iii)The flux from glutamate to alpha-ketoglutarate (catalyzed by glutamate dehydrogenase) is almost zero in medium with and even slightly reversed in medium without Primatone RL. Almost all glutamate enters the TCA cycle due to the action of transaminases.(iv)Transhydrogenation plays a significant role in hybridoma cells under our experimental conditions. NADPH is produced at relatively high rates (11 x 10(-12) to 13 x 10(-12) mol . cell(-1) . day(-1)) compared to other fluxes in both culture media. (c) 1996 John Wiley & Sons, Inc.     

Metabolic-flux analysis of continuously cultured hybridoma cells using (13)CO(2) mass spectrometry in combination with (13)C-lactate nuclear magnetic resonance spectroscopy and metabolite balancing

Protein production of mammalian-cell culture is limited due to accumulation of waste products such as lactate, CO(2), and ammonia. In this study, the intracellular fluxes of hybridoma cells are measured to determine the amount by which various metabolic pathways contribute to the secretion of waste products derived from glucose. Continuously cultured hybridoma cells are grown in medium containing either 1-(13)C-, 2-(13)C-, or 6-(13)C-glucose. The uptake and production rates of amino acids, glucose, ammonia, O(2), and CO(2) as well as the cellular composition are measured. In addition, the (13)C distribution of the lactate produced and alanine produced by the hybridomas is determined by (1)H-NMR spectroscopy, and the (13)CO(2)/(12)CO(2) ratio is measured by on-line mass spectrometry. These data are used to calculate the intracellular fluxes of the glycolysis, the pentose phosphate pathway, the TCA cycle, and fluxes involved in amino acid metabolism. It is shown that: (i) approximately 20% of the glucose consumed is channeled through the pentose shunt; (ii) the glycolysis pathway contributes the most to lactate production, and most of the CO(2) is produced by the TCA cycle; (iii) the pyruvate-carboxylase flux is negligibly small; and (iv) the malic-enzyme flux is estimated to be 10% of the glucose uptake rate. Based on these flux data suggestions are made to engineer a more efficient glucose metabolism in mammalian cells.     

Kinetic flux profiling for quantitation of cellular metabolic fluxes

This protocol enables quantitation of metabolic fluxes in cultured cells. Measurements are based on the kinetics of cellular incorporation of stable isotope from nutrient into downstream metabolites. At multiple time points, after cells are rapidly switched from unlabeled to isotope-labeled nutrient, metabolism is quenched, metabolites are extracted and the extract is analyzed by chromatography-mass spectrometry. Resulting plots of unlabeled compound versus time follow variants of exponential decay, with the flux equal to the decay rate multiplied by the intracellular metabolite concentration. Because labeling is typically fast (t(1/2)相似文献   

Remodeling of the myoblast membrane accompanies development

We have prepared a library of cloned hybridomas that produce monoclonal antibodies reactive with the surface of E63 rat myoblasts. Using immunofluorescence analysis of antigens on single cells we have studied the expression of determinants at distinct stages of development. Conditions were established for quantitative photometry and were used to confirm the diversity in stage-specific expression that accompanies development. The remodeling of the myoblast membrane also involves stage-specific and transient changes in topography and aggregation of many antigens, and the period surrounding fusion is one of particular activity. The localization of antigens on the upper and attached surfaces of myogenic cells was often distinct, and quantitative and spatial stage-specific reorganizations of antigens differed with respect to these two surfaces. This polarity represents an additional level of complexity in the continuous remodeling of the muscle cell membrane. Comparisons of quantitative and topographic analyses of antigens on E63 cells with Rat-1 fibroblasts and developmentally defective (fu-) myoblasts indicate that the outer membranes of these nonmyogenic cells are distinct from differentiating myoblasts. One determinant, H36, is absent on Rat-1 cells and on all fu- lines tested, and undergoes interesting stage-specific changes in expression and topography.     

Volume regulation by Amphiuma red blood cells. The membrane potential and its implications regarding the nature of the ion-flux pathways

After osmotic perturbation, the red blood cells of Amphiuma exhibited a volume-regulatory response that returned cell volume back to or toward control values. After osmotic swelling, cell-volume regulation (regulatory volume decrease; RVD) resulted from net cellular loss of K, Cl, and osmotically obliged H2O. In contrast, the volume-regulatory response to osmotic shrinkage (regulatory volume increase; RVI) was characterized by net cellular uptake of Na, Cl, and H2O. The net K and Na fluxes characteristic of RVD and RVI are increased by 1-2 orders of magnitude above those observed in studies of volume-static control cells. The cell membrane potential of volume-regulating and volume-static cells was measured by impalement with glass microelectrodes. The information gained from the electrical and ion-flux studies led to the conclusion that the ion fluxes responsible for cell-volume regulation proceed via electrically silent pathways. Furthermore, it was observed that Na fluxes during RVI were profoundly sensitive to medium [HCO3] and that during RVI the medium becomes more acid, whereas alkaline shifts in the suspension medium accompany RVD. The experimental observations are explained by a model featuring obligatorily coupled alkali metal-H and Cl-HCO3 exchangers. The anion- and cation-exchange pathways are separate and distinct yet functionally coupled via the net flux of H. As a result of the operation of such pathways, net alkali metal, Cl, and H2O fluxes proceed in the same direction, whereas H and HCO3 fluxes are cyclic. Data also are presented that suggest that the ion-flux pathways responsible for cell-volume regulation are not activated by changes in cell volume per se but by some event associated with osmotic perturbation, such as changes in intracellular pH.     

Metabolite-balancing techniques vs. 13C tracer experiments to determine metabolic fluxes in hybridoma cells

The estimation of intracellular fluxes of mammalian cells using only mass balances of the relevant metabolites is not possible because the set of linear equations defined by these mass balances is underdetermined. In order to quantify fluxes in cyclic pathways the mass balance equations can be complemented with several constraints: (1) the mass balances of co-metabolites, such as ATP or NAD(P)H, (2) linear objective functions, (3) flux data obtained by isotopic-tracer experiments. Here, these three methods are compared for the analysis of fluxes in the primary metabolism of continuously cultured hybridoma cells. The significance of different theoretical constraints and different objective functions is discussed after comparing their resulting flux distributions to the fluxes determined using 13CO2 and 13C-lactate measurements of 1 - 13C-glucose-fed hybridoma cells. Metabolic fluxes estimated using the objective functions "maximize ATP" and "maximize NADH" are relatively similar to the experimentally determined fluxes. This is consistent with the observation that cancer cells, such as hybridomas, are metabolically hyperactive, and produce ATP and NADH regardless of the need for these cofactors.     

A dual-color luciferase assay system reveals circadian resetting of cultured fibroblasts by co-cultured adrenal glands

In mammals, circadian rhythms of various organs and tissues are synchronized by pacemaker neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus. Glucocorticoids released from the adrenal glands can synchronize circadian rhythms in other tissues. Many hormones show circadian rhythms in their plasma concentrations; however, whether organs outside the SCN can serve as master synchronizers to entrain circadian rhythms in target tissues is not well understood. To further delineate the function of the adrenal glands and the interactions of circadian rhythms in putative master synchronizing organs and their target tissues, here we report a simple co-culture system using a dual-color luciferase assay to monitor circadian rhythms separately in various explanted tissues and fibroblasts. In this system, circadian rhythms of organs and target cells were simultaneously tracked by the green-emitting beetle luciferase from Pyrearinus termitilluminans (ELuc) and the red-emitting beetle luciferase from Phrixothrix hirtus (SLR), respectively. We obtained tissues from the adrenal glands, thyroid glands, and lungs of transgenic mice that expressed ELuc under control of the promoter from a canonical clock gene, mBmal1. The tissues were co-cultured with Rat-1 fibroblasts as representative target cells expressing SLR under control of the mBmal1 promoter. Amplitudes of the circadian rhythms of Rat-1 fibroblasts were potentiated when the fibroblasts were co-cultured with adrenal gland tissue, but not when co-cultured with thyroid gland or lung tissue. The phases of Rat-1 fibroblasts were reset by application of adrenal gland tissue, whereas the phases of adrenal gland tissue were not influenced by Rat-1 fibroblasts. Furthermore, the effect of the adrenal gland tissue on the fibroblasts was blocked by application of a glucocorticoid receptor (GR) antagonist. These results demonstrate that glucocorticoids are strong circadian synchronizers for fibroblasts and that this co-culture system is a useful tool to analyze humoral communication between different tissues or cell populations.     

Modification of endoplasmic reticulum Ca2+ stores by select oxidants produces changes reminiscent of those in cells from patients with Alzheimer disease

Abnormalities in calcium homeostasis and oxidative processes occur in fibroblasts from patients with Alzheimer disease (AD) and in fibroblasts and neurons from transgenic mice bearing a presenilin-1 (PS-1) mutation. Bombesin-releasable endoplasmic reticulum Ca2+ stores (BRCS) are exaggerated in all of these cells. Our previous studies show that H2O2 exaggerates BRCS. The goal of the present study was to determine whether select reactive species exaggerate BRCS in cultured human fibroblasts and to determine if the ability of fibroblasts to handle these specific oxidant species is altered in cells from AD patients. Two fluorescent indicators were used to distinguish different reactive oxygen species (ROS): 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (c-DCF) and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM). ROS were produced by a variety of oxidants, including tert-butyl-hydroxyperoxide (t-BHP), hypoxanthine/xanthine oxidase, S-nitroso-N-acetylpenicillamine, 3-morpholinosydnonimine, and sodium nitroprusside. Different oxidants selectively induced various ROS in distinct patterns. These oxidants also induced selective modification in [Ca2+]i and/or BRCS. Of the several oxidants tested, t-BHP was most specific for exaggerating BRCS without affecting basal [Ca2+]i and inducing only c-DCF-detectable ROS. On the other hand, the results show that NO that reacted with DAF-FM was not responsible for alterations in BRCS. Furthermore, the c-DCF-detectable ROS production induced by t-BHP was higher in fibroblasts from AD patients bearing a PS-1 mutation (n = 7) than in those from aged controls (n = 8). The higher production of c-DCF-detectable ROS may underlie the exaggeration of BRCS in fibroblasts from AD patients. Thus, these results are consistent with the hypothesis that abnormalities in selective cellular ROS cause AD-related changes in intracellular calcium regulation.     

Esterification of exogenously derived free cholesterol by cultured fibroblasts

The incorporation and esterification by cultured human fibroblasts of vesicle- or low density lipoprotein-derived free [3H]cholesterol was examined. The rate of the cellular uptake of free [3H]cholesterol from lipid vesicles was similar in both LDL-receptor positive lung fibroblasts and in LDL-receptor negative fibroblasts. When human LDL was used as the carrier of free [3H]cholesterol, however, the LDL-receptor positive lung fibroblasts incorporated significantly more [3H]cholesterol than did the LDL-receptor negative cells. The exchangeable free [3H]cholesterol was available for intracellular esterification. The formation of [3H]cholesteryl esters was markedly inhibited by lysosomotropic drugs, either indicating a partly lysosomal esterification reaction, or implying that free [3H]cholesterol moves through the lysosomal compartment on its way to intracellular esterification sites. Either way, the lysosomes appear to have a metabolic role in the metabolism of exchangeable free [3H]cholesterol.     

Metabolic flux analysis of HEK-293 cells in perfusion cultures for the production of adenoviral vectors

To meet increasing needs of adenovirus vectors for gene therapy programs, development of efficient and reproducible production processes is required. Perfusion cultures were employed to allow infection at greater cell concentrations. In an effort to define culture conditions resulting in enhanced productivities, experiments performed at different feed rates and infected at various cell densities were compared using metabolic flux analysis. The highest specific product yields were achieved in experiments performed at high perfusion rates and/or low cell concentrations. The intracellular flux analysis revealed that these experiments exhibited greater glycolytic fluxes, slightly higher TCA fluxes, and greater ATP production rates at the time of infection. In contrast, cultures infected at high cell density and/or low medium renewal rates were characterized by a more efficient utilization of glucose at the time of infection, but the specific product yields achieved were lower. The intracellular flux analysis provided a rational basis for the implementation of a feeding strategy that allowed successful infection at a density of 5x10(6)cells/ml.     

Application of multivariate analysis to optimize function of cultured hepatocytes

Understanding the metabolic and regulatory pathways of hepatocytes is important for biotechnological applications involving liver cells, including the development of bioartificial liver (BAL) devices. To characterize intermediary metabolism in the hepatocytes, metabolic flux analysis (MFA) was applied to elucidate the changes in intracellular pathway fluxes of primary rat hepatocytes exposed to human plasma and to provide a comprehensive snapshot of the hepatic metabolic profile. In the current study, the combination of preconditioning and plasma supplementation produced distinct metabolic states. Combining the metabolic flux distribution obtained by MFA with methodologies such as Fisher discriminant analysis (FDA) and partial least squares or projection to latent structures (PLS) provided insights into the underlying structure and causal relationship within the data. With the aid of these analyses, patterns in the cellular response of the hepatocytes that contributed to the separation of the different hepatic states were identified. Of particular interest was the recognition of distal pathways that strongly correlated with a particular hepatic function. The hepatic functions investigated were intracellular triglyceride accumulation and urea production. This study illustrates a framework for optimizing hepatic function and a possibility of identifying potential targets for improving hepatic functions.     

Membrane potentials, electrolyte contents, cell pH, and some enzyme activities of fibroblasts

The resting membrane potential of the cultured fibroblasts derived from rabbit subcutaneous tissues was -10.2 +/- 0.20 mV (n = 390). This potential was affected by the potassium concentration in the culture medium, but not by other chemical or hormonal preparations, such as dibutyryladenosine 3',5'-cyclic monophosphate (0.5 to 5.0 mmol/l), sodium fluoride (10(-5) to 10(-4) M), hydrocortisone (10(-7) to 10(-6) M), parathyroid extract (0.5 to 1.0 U/ml), or thyrotrophin (5 to 10 mU/ml). The Na+, K+, and Cl- concentrations of the cultured fibroblasts were 35.4, 85.7, and 22.6 mmol/l cell water, respectively. The water and protein contents of these cells were 82.1 and 9.18 g/100-g cells, respectively. The intracellular pH of fibroblasts as determined by [14C] dimethyloxazolidine-2, 4-dione, and 3H2O ranged between 6.9 and 7.1 when the pH of the culture medium was maintained at 7.4. The activities of Na+, K+-, HCO3(-)-, and Ca++, Mg++-ATPases in these cultured cells were 19.0 +/- 2.1, 13.6 +/- 2.1, and 6.6 +/- 1.2 nmol pi/mg protein per minute, respectively, and the carbonic anhydrase activity was 0.054 U/mg protein. Calculations based on the values for the membrane potential and the electrolyte concentrations observed in this study indicate that Na+, K+, Cl-, and H+ are not distributed according to their electrochemical gradients across the cell membrane. Na+, Cl-, and H+ are actively transported out of the cells and K+ into the cells.     

Simultaneous measurement of metabolic heat rate, CO2 production, and O2 consumption by microcalorimetry

This study describes methods and equipment for measurement of metabolic heat rates of cells and tissues under conditions that provide simultaneous determinations of the flux rates of both O2 and CO2. Isothermal measurement of metabolic heats are conducted in a sealed ampule. A trapping solution is employed to absorb metabolic CO2. Absorption of CO2 produces heat at a rate proportional to the rate of CO2 production. Under these conditions, O2 consumption by the tissue results in a decrease in the partial pressure of O2 within the sealed ampule. The decrease in pressure can be monitored with a pressure sensor and related to O2 consumption rates. The combined measurements of heat rates, CO2, and O2 fluxes provide important information on bioenergetic efficiency of cell metabolism. These data can also suggest possible shifts in metabolic pathways or substrate sources as cells develop, or are exposed to effectors, inhibitors, and environmental factors.