Differential scanning calorimetry of alpha 2-macroglobulin.
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1. Energy expenditure was determined in rats fed a standard laboratory diet or a varied and palatable 'cafeteria' diet, from metabolizable energy intake and body energy gain over a 15 d experiment. This was compared with measurements of oxygen consumption (Vo2) made over 24 h during the first and second weeks of the experiment. 2. Metabolizable energy intake was elevated by 50% in 'cafeteria'-fed rats and these animals gained more weight and had a higher body fat content than stock-fed controls. Energy expenditure derived from these measurements was 45% higher in the 'cafeteria' group, and the net efficiency for energy gain was significantly reduced. 3. Vo2 was increased by 45% in the 'cafeteria' group and the daily energy expenditure, estimated from Vo2, was identical to that derived from the energy balance for controls, and differed by only 3% for the 'cafeteria' group. 4. These results confirm the high levels of diet-induced thermogenesis previously seen in hyperphagic, 'cafeteria'-fed rats and reaffirm the validity of the carcass balance method for estimating energy expenditure.
1. The rate of energy dissipation associated with Na(+)-H+ exchange in isolated, superfused soleus muscles from young rats was measured with an isothermal microcalorimeter during quasi-stationary states of oxidative metabolism. 2. Under normal physiological conditions, amiloride, an inhibitor of the Na(+)-H+ exchange across plasma membranes, had no measurable effect on the specific rate of muscle heat production (E); the ouabain-suppressible part of E was identical whether amiloride was absent or present. 3. E was increased under hyperosmotic conditions and the difference with respect to control (excess E) was proportional to the degree of hyperosmolarity of the superfusate. It was 48% of basal E during a +100 mosM stress (with no change of extracellular Na+ concentration, Na+o). Inhibition of Ca2+ release into the sarcoplasm with sodium dantrolene (10(-5) M) or tetracaine (5 x 10(-5) M) suppressed a substantial part (65 and 53%, respectively) of the steady-state excess E (1.2 mW (g wet weight)-1) induced by the +100 mosM stress. Practically 100% of excess E was suppressed in the nominal absence of extracellular sodium (Na+o = 0, Li+ substitution) or under 15 mM-Na+o, and excess E was enhanced when Na+o was increased (hyperosomolarity by addition of Na2SO4 instead of sucrose). 4. Under hyperosmotic conditions, amiloride at the 5 x 10(-7) M concentration had no effect on excess E whereas at 10(-4) M it induced a significant decrease of excess E. The absolute effect of 10(-4) M-amiloride was -0.34 mW (g wet weight)-1 (equal to 28% of the excess E due to a +100 mosM-sucrose stress and to 14% of the excess E due to a +100 mosM-Na2SO4 stress). It was left unaltered in the presence of dantrolene and was independent of the way the +100 mosM stress was obtained (i.e. 100 mM-sucrose or 50 mM-Na2SO4). It was suppressed at Na+o = 0-15 mM and could be mimicked by guanochlor, another potent inhibitor of Na(+)-H+ exchange. In the presence of 10(-4) M-amiloride, the ouabain-suppressible E was significantly reduced. In the presence of ouabain, amiloride had no effect. 5. Muscle tissue space available to [3H]inulin was measured in parallel experiments. It was 23.3% under control conditions and 30.6% after a 2 h exposure of the muscle to a +100 mosM-Na2SO4 stress.(ABSTRACT TRUNCATED AT 400 WORDS)
There is currently a need for experimental techniques to assay the biophysical response (water transport or intracellular ice formation, IIF) during freezing in the cells of whole tissue slices. These data are important in understanding and optimizing biomedical applications of freezing, particularly in cryosurgery. This study presents a new technique using a Differential Scanning Calorimeter (DSC) to obtain dynamic and quantitative water transport data in whole tissue slices during freezing. Sprague-Dawley rat liver tissue was chosen as our model system. The DSC was used to monitor quantitatively the heat released by water transported from the unfrozen cell cytoplasm to the partially frozen vascular/extracellular space at 5 degrees C/min. This technique was previously described for use in a single cell suspension system (Devireddy, et al. 1998). A model of water transport was fit to the DSC data using a nonlinear regression curve-fitting technique, which assumes that the rat liver tissue behaves as a two-compartment Krogh cylinder model. The biophysical parameters of water transport for rat liver tissue at 5 degrees C/min were obtained as Lpg = 3.16 x 10(-13) m3/Ns (1.9 microns/min-atm), ELp = 265 kJ/mole (63.4 kcal/mole), respectively. These results compare favorably to water transport parameters in whole liver tissue reported in the first part of this study obtained using a freeze substitution (FS) microscopy technique (Pazhayannur and Bischof, 1997). The DSC technique is shown to be a fast, quantitative, and reproducible technique to measure dynamic water transport in tissue systems. However, there are several limitations to the DSC technique: (a) a priori knowledge that the biophysical response is in fact water transport, (b) the technique cannot be used due to machine limitations at cooling rates greater than 40 degrees C/min, and (c) the tissue geometric dimensions (the Krogh model dimensions) and the osmotically inactive cell volumes Vb, must be determined by low-temperature microscopy techniques.
A portable indirect calorimeter adapted from adult use was validated for use in preterm infants. Oxygen consumption (VO2) and carbon dioxide production (VCO2) were subsequently measured in 16 preterm infants breathing spontaneously in room air (canopy mode) and in nine preterm infants receiving intermittent positive pressure ventilation (ventilator mode). Validation of the system was performed using a gas injection technique with nitrogen to simulate VO2 and carbon dioxide for VCO2. Mean errors in validation of the canopy mode were 1.4% and 0.2% for VO2 and VCO2 with limits of agreement of 0.6 (+2SD) ml/min and -1.3 (-2SD) ml/min, and 0.9 (+2SD) ml/min and -2.3 (-2SD) ml/min respectively. In validation of the ventilator mode mean errors were -1.8% and -5.05% for VO2 and VCO2 with limits of agreement of 1.02 (+2SD) ml/min and -0.74 (-2SD) ml/min, and 0.93 (+2SD) ml/min and -1.45 (-2SD) ml/min respectively. Values of VO2 and VCO2 in 16 preterm infants in the canopy mode were 6.2 ml/kg/min (0.5 1SD) and 6.7 ml/kg/min (0.6 1SD) and in nine preterm infants in the ventilator mode 4.98 ml/kg/min (1.09 1SD) and 4.74 ml/min/kg (1.08 1SD) respectively. Mean energy expenditure was 45.5 kcal (191 kJ)kg/day for infants measured in the canopy mode and 35.5 kcal (149 kJ)/kg/day for ventilated infants. This metabolic system can be adapted for use in the newborn but accuracy is reduced when it is used in those weighing less than 1000 g.
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A method has been developed to measure myocardial heat production simultaneously with mechanical (developed tension, rate of contraction and relaxation) and metabolic parameters in the arterially perfused interventricular septum of the rabbit. The system allows control of rest tension, frequency of contraction, temperature, and composition of the perfusate. The technique is based on the differential measurement of the heat flux from the muscle to the calorimetric bath. This technique is able to resolve changes in heat production as small as 0.06 mW. The resting heat production measured with the present calorimeter (1.62 +/- 0.1 mW/g wet tissue) agrees with that obtained with thermopiles and with that calculated from measurements of oxygen consumption. The heat per contraction (6.5 +/- 1.4 mJ/g wet tissue) also agreed with that measured with thermopiles in rabbit papillary muscles. The heat production measured at 22 degrees C under severe hypoxia can be fully explained by the addition of the expected change of enthalpy due to the reaction 0.5 glucose-lactate, calculated on the basis of the lactate measured in the perfusate, and the expected change of enthalpy of oxygen consumption [assuming that all remaining O2 in perfusate (0.09 vol%) is used for combustion of glucose]. These results clearly demonstrated the feasibility of this method for the correlation of changes in energy turnover, through the measurement of the heat production, with mechanical and metabolic processes on-line in arterially perfused septum.
Sodium flux rate and energy consumption of the Na(+)-K+ pump in vascular endothelial cells of porcine aorta grown on micro-carrier beads were studied using a combination of nuclear magnetic resonance spectroscopy of intracellular 23Na and microcalorimetry. The Na+ flux into the cells was determined in the presence of the shift reagent Dy(P3O10)2(7-), while the Na(+)-K+ pump was inhibited with ouabain. Basal Na+ influx was 17 +/- 3 nmol.min-1.mg protein-1, and intracellular Na+ concentration was 23.5 +/- 3.8 mM, resulting in a complete exchange of intracellular Na+ within 5-6 min. Spin-lattice relaxation time (T1) measurements of intracellular Na+ showed a T1 of 19 +/- 1 ms under basal conditions and a T1 of 26.2 +/- 1.6 ms after pump inhibition with 50 microM ouabain. Such an increase is typical for a system in which the total amount of Na+ increases but where the amount of bound Na+ remains constant. The Na+ ionophore nystatin maximally increased the Na(+)-K+ pump rate about twofold, whereas the amount of intracellular Na+ only increased 14%. With microcalorimetry a cellular heat flux of 183 +/- 18 microW per mg endothelial protein was determined, which relates to 7.6 microW/mg endothelial protein generated by the Na(+)-K(+)-adenosinetriphosphatase. Our data demonstrate that small intracellular changes of Na+ can stimulate the endothelial Na(+)-K+ pump activity. The contribution of the Na(+)-K+ pump to total endothelial energy expenditure is approximately 4-5%.