R.B. Kemp

ANAEROBIC METABOLISM IN AEROBIC MAMMALIAN CELLS : INFORMATION FROM THE RATIO OF CALORIMETRIC HEAT FLUX AND RESPIROMETRIC OXYGEN FLUX

Calorimetric and respirometric studies of cultured cells show that both neoplastic and non-neoplastic cell types maintain an anaerobic contribution to their total heat flux. In many mammalian cells this can be explained quantitatively by lactate production observed under fully aerobic conditions. Uncoupling and enhanced futile substrate cycling increase the ratio of heat flux to oxygen flux, the calorimetric-respirometric (CR) ratio. The interpretation of calorimetric and respirometric measurements requires an energy balance approach in which experimentally measured CR ratios are compared with thermochemically derived oxycaloric equivalents. The oxycaloric equivalent is the enthalpy change per mole of oxygen consumed, and equals -470 kJ/mol O2 in the aerobic catabolism of glucose, assuming that catabolism is 100% dissipative (the net efficiency of metabolic heat transformation is zero). CR ratios more negative than -470 kJ/mol O2 have been reported in well-oxygenated cell cultures and are discussed in terms of integrated aerobic and anaerobic metabolism.

Specific heat flow rate: an on-line monitor and potential control variable of specific metabolic rate in animal cell culture that combines microcalorimetry with dielectric spectroscopy.

One of the requirements for enhanced productivity by the animal culture systems used in biotechnology is the direct assessment of the metabolic rate by on-line biosensors. Based on the fact that cell growth is associated with an enthalpy change, it is shown that the specific heat flow rate is stoichiometrically related to the net specific rates of substrates, products, and indeed to specific growth rate, and therefore a direct reflection of metabolic rate. Heat flow rate measured by conduction calorimetry has a technical advantage over estimates for many material flows which require assays at a minimum of two discrete times to give the rate. In order to make heat flow rate specific to the amount of the living cellular system, it would be advantageous to divide it by viable biomass. This requirement has been fulfilled by combining a continuous flow microcalorimeter ex situ with a dielectric spectroscope in situ, the latter measuring the viable cell mass volume fraction. The quality of the resulting biosensor for specific heat flow rate was illustrated using batch cultures of Chinese hamster ovary cells (CHO 320) producing recombinant human interferon-gamma (IFN-gamma) during growth in a stirred tank bioreactor under fully aerobic conditions. The measuring scatter of the probe was decreased significantly by applying the moving average technique to the two participant signals. It was demonstrated that the total metabolic rate of the cells, as indicated by the specific heat flow rate sensor, decreased with increasing time in batch culture, coincident with the decline in the two major substrates, glucose and glutamine, and the accumulation of the by-products, ammonia and lactate. Furthermore, the specific heat flow rate was an earlier indicator of substrate depletion than the flow rate alone. The calorimetric-respirometric ratio showed the intensive participation of anaerobic processes during growth and the related IFN-gamma production. Specific heat flow rate was monotonically related to specific cell growth rate and associated with specific IFN-gamma production. Specific heat flow rate is potentially a valid control variable for the growth of genetically engineered cell lines producing target proteins.

On the audio- and radio-frequency dielectric behaviour of anchorage-independent, mouse L929-derived LS fibroblasts☆

Abstract The utility of dielectric methods as a means for estimating the biomass of animal cells in suspension culture was assessed, using mouse L929-derived LS fibroblasts. The dielectric increment of the β-dispersion was found to be a linear function of both cell number (30–70 permittivity units per 10 6 cells/ml, depending on the batch of cells) and volume fraction in the range measured (up to 1.28 × 10 8 cells/ml, volume fraction = 0.14). The notional distribution of relaxation times as encompassed in the Cole/Cole α (0.13 ± 0.03 SD) was rather modest. If the cells were treated as spherical shell capacitors of their observable diameter and number, the apparent capacitance of the plasma membrane was some 1.9–4.0 μF/cm 2 . This value significantly exceeded those (0.5–1 μF/cm 2 ) usually encountered or claimed, due predominantly to the possession by these cells of numerous plasma membrane protrusions. As the osmolarity of the suspending medium was increased using the non-permeant solute sorbitol, the apparent specific capacitance of the plasma membrane and the Cole/Cole αa were increased, whilst the dielectric increment per 10 6 cells/ml was unchanged. In addition, a secondary β-dispersion, with a characteristic frequency greater than that of the main β-dispersion, became increasingly prominent as the medium osmolarity was increased. It is proposed that this β 2 -dispersion is dominated by a Maxwell-Wagner mechanism taking place in the region of the plasma membrane protrusions of these cells.

Changes in the functions of the intestinal brush border membrane during the development of the ruminant habit in lambs

1. Brush border membrane vesicles were prepared from lamb enterocytes. These were used to study the changes in the enzyme contents and the transport capacities which occur during the change from a milk to a roughage diet. 2. Na+-dependent transport of D-glucose was present in all regions of the small intestine of pre-ruminant lambs and absent in ruminants. 3. Na+-dependent transport of L-proline was present in all regions of the small intestine irrespective of the age of the animal. 4. Phosphate transport was seen only in the presence of a transmembrane pH gradient (acid outside). The transport was not stimulated by either Na+ or K+. The transport capacity increases 2-fold as the animal becomes ruminant. 5. The activities of lactase and maltase diminished with age. Alkaline phosphatase and aminopeptidase N activities remain constant. Sucrase activity cannot be detected in lambs of any age.

Calorimetric studies of heat flux in animal cells

Abstract This overview concerning calorimetric studies of animal cells in vitro firstly highlights some of the thermodynamic principles underlying measurement of heat flux. The resurgence of such measurements is based on refined instrumentation and a growing awareness of the undoubted contribution heat flux measurements can make to the accumulating knowledge of cellular physiology. Direct calorimetry has been employed to investigate medical, pharmacological and lexicological problems, and can be a valuable diagnostic tool. It is advocated, however, that it exercises a greater scientific contribution when it is harnessed to indirect calorimetry, especially measurement of oxygen flux. The calorimetric-respirometric (CR) ratio is explained in terms of its value in thermochemical analysis of cellular physiology based on rigorous thermodynamics.

“Fire burn and cauldron bubble” (W. Shakespeare): what the calorimetric–respirometric (CR) ratio does for our understanding of cells?

Abstract In this paper, it has been shown that the existence of anaerobic pathways under aerobic conditions is not always due to poor environmental conditions. There are two sources of lactate in cultured cells, oxidative glutaminolysis and reductive glycolysis. Only the latter is measured in the CR ratio as a value more negative than the oxycaloric equivalent for the relevant catabolic substrate. The validity of the value for the CR ratio can be determined by Mayer’s enthalpy balance method. Highly exothermic ratios are no reflection of thermodynamic efficiency but in many cases indicate the need for ATP not supplied by, or insufficiently available from, oxidative phosphorylation. For other types of cell grown in culture, a highly exothermic CR ratio is due to the fact that there are not sufficient quantities of the appropriate anabolic precursors in the culture medium. Then, biosynthetic precursors must be constructed from substrates using the catabolic pathways and this leads to the reduction of pyruvate in order to conserve NAD+. An on-line measurement of the CR ratio would monitor cell growth and could be used to control fed-batch cultures.

Direct and indirect calorimetry of lactate oxidation: implications for whole-body energy expenditure

Whole-body energy expenditure for heavy/severe exercise is currently accounted for by either: (1) anaerobic and oxygen uptake measures during exercise where recovery energy expenditure is omitted; or (2) oxygen uptake during, and an EPOC (excess post-exercise oxygen consumption), measure following exercise where substrate level phosphorylation during exercise is considered part of EPOC. Simultaneous direct/indirect calorimetry enabled us to determine if a thermodynamic reversal (i.e. heat consumption) takes place as the highly exothermic pyruvate to lactate reaction proceeds in the opposite direction. Reversibility implies that oxygen uptake (e.g. EPOC) can indeed account for rapid glycolytic ATP production regardless if lactate is formed or not (e.g. 1.2 g glucose catabolism = 20.9 kJ · l O2  - 1). Cultured hybrid cells and mouse cardiac muscle fibres were utilized in simultaneous calorimetry and respirometry experiments where pyruvate or lactate was predominantly oxidized. The calorimetric to respiratory ratio was determined using heat flux (pW · cell - 1) and oxygen flux (pmol · s - 1 · cell - 1) measures. Ten cell experiments gave calorimetric to respiratory ratios that showed no statistical difference (P = 0.97) whether cells respired predominantly on lactate (−516 ± 53 kJ · mol O2  - 1) or pyruvate (−517 ± 89 kJ · mol O2  - 1). In three cardiac preparations, the calorimetric to respiratory ratio was - 502 ± 15 kJ · mol O2  - 1 for lactate and - 506 ± 47 kJ · mol O2  - 1 for pyruvate, again a non-significant difference (P = 0.91). Heat consumption did not occur during lactate oxidation. These results suggest that rapid glycolytic ATP and lactate production, and lactate oxidation, are both independently associated with heat production and thus represent separate and additive components to the measurement of total energy expenditure for exercise and recovery.

Developments in cellular microcalorimetry with particular emphasis on the valuable role of the energy (enthalpy) balance method

Abstract This review of microcalorimetric studies of animal cells includes muscle and nerve as well as blood tissue, non-erythroid cells and transformed cells. It highlights the wide range of heat flow rate (φ = d Q /d t ) data obtained for cells but points to their limited inherent value unless expressed per unit volume or biomass—scalar heat flux. Measurements of heat flow have been used in detecting clinical, pharmacological, toxicological and immunological changes—calorimetry as an analytical tool to assay metabolic activity in the face of pathological and xenobiosis-induced alterations. Microcalorimetry is a powerful and non-destructive technique of itself but its true strength is revealed when combined with other analytical procedures to allow access to the energy (enthalpy) balance method, which has its basis in the First Law of Thermodynamics. Its early use in dissecting all the chemical sources of heat in the thermogenesis of muscle is summarized, together with more recent studies of the metabolic burst in phagocytosis and catalytic pathways in cultured, anchorage-independent T-lymphoma and LS-L929 mouse fibroblasts. The theoretical basis of the calorimetric-respirometric (CR) ratio is explained and its value in detecting anaerobic pathways in respiring cells is emphasized with reference to several cell types. Two ways of calculating ATP turnover (μ mol ∝ ATPh −1 ) are described, namely from heat flow and catabolic coupling flow; and there is opuscular reference to thermodynamic efficiency.

(Ca2++Mg2+)-activated ATPases in the plasma membrane of mouse liver cells

1. Purified plasma membranes from dissociated adult mouse liver cells posses a (Ca-2+ + Mg-2+)-stimulated ATPase (EC 3.6.1.3) activity. 2. Enzyme activity is at a maximum with the addition of 0.3 mM Ca-2+ and 3 mM Mg-2+. 3. Using medium devoid of alkali metal ions (Ca-2+ + Mg-2+)-ATPase enzyme activity was observed with Km1 = 0.35 - 10-3 M at a substrate concentration of 1 mM or less and an apparent Km2 = 0.88 - 10-3 M at higher substrate concentrations. 4. In the presence of Na+ and 4 mM ATP, an increase in activity was seen, suggesting the presence of a (Ca-2+ + Mg-2+ + Na+)-activated ATPase. 5. In the presence of both Na+ and K+ the (Ca-2+ + Mg-2+)-dependent enzyme activity was further increased, indicating that a (Ca-2+ +Mg-2+ + K+)-stimulated ATPase may also be present.

The application of heat conduction microcalorimetry to study the metabolism and pharmaceutical modulation of cultured mammalian cells

Abstract The heat produced by animal cells in culture can be used as the primary indicator of the kinetics of their metabolism because the scalar flux of it is a function of the metabolic flux. The validity of the relationship between heat and metabolism was demonstrated theoretically through the concept of thermal advancement and in experiments by the use of continuous cultures. This validation permitted the application of heat flux as a probe of the metabolic state of cells in culture. It consisted of an on-line heat conduction microcalorimeter that measures the instantaneous heat flow and dividing the smoothed signal with one obtained simultaneously using a dielectric spectrometer that records the change in capacitance as an estimate of the amount of viable biomass. In this mini-review, it is shown with Chinese hamster ovary cells (CHO320) genetically engineered to produce interferon-γ (IFN-γ) that heat flux is an early signal of deteriorating metabolism in cultures that produce considerable amounts of toxic lactate under fully aerobic conditions. The early detection favours the use of heat flux as the control variable in fed-batch cultures. This is a particularly useful finding in the context of the pharmaceutical industry because it will help to ensure the high fidelity of the cytokines, antibodies and vaccines produced in large-scale cultures. The monotonic relationship between the fluxes for heat and metabolism means that the enthalpy balance method can be employed to test the validity of the growth reaction for cells in culture. This showed that the crucial ratio between the substrates, glucose and glutamine, in the culture medium was incorrect at 5.5:1 instead of about 3:1, depending on the phase of the culture. Together with other changes to the medium composition, an improved formulation was made that ensured faster cell growth and greater specific rate (flux) of IFN-γ constitutive secretion while decreasing glucose utilisation and, most importantly, halving the excretion of lactate, that is toxic to the cells and harmful to the fidelity of their secondary products. Indirect calorimetry (oxygen uptake rate, OUR) is often favoured over the direct technique, but the former only measures aerobic metabolism. The environmental conditions in cultures favours lactate production even under fully aerobic conditions. Developments in measuring OUR mean that the stationary liquid phase balance can be used successfully to make the calorimetric:respirometric (CR) ratio a valuable tool in optimising cell culture to grow cells that synthesise the maximum amounts of the high fidelity secondary products. Besides the value of heat flux in improving the cultures of animal cells producing heterologous products, three different techniques are examined that should be valuable in the testing the many compounds that are produced on a speculative basis as potential drugs. They are: (i) a thin-film thermopile transducer as an immunosensor; (ii) infra-red imaging of cells cultured in multi-well microtitre plates and (iii) integrated circuit (IC) calorimetry for small samples and low detection limit. One or more of these methods could well find favour with industry in the near future.