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Energy expenditure measurements in critically ill patients using indirect calorimetry is complex and often very inaccurate. Analysis of the data should first question their validity. The components of energy expenditure should be carefully separated to assess the effects of illness, drugs and supportive treatments.
The calorimetry studies of temperature dependence of bovine serum albumin heat capacity for the temperature interval 20-150 degrees C and pH value varying from 4 to 9 were carried out. It is shown that in the pH range considered four types of denaturation curves differing in quantity and temperatures of endothermic pikes exist. The different types of the melting curves correspond to different types of protein domain structure. At 110-120 degrees C and pH greater than or equal to 5.6 the high temperature maximum is shown to exist. The later is supposed to be due not to BSA denaturation process but to cooperative destruction of postdenaturation remnants of the secondary protein structure.
Differential scanning microcalorimetry of nuclei from cultured cells revealed differences between antitumor drugs in potency and mechanism. Scanning calorimetry of nuclei showed four structural transitions as the temperature was raised from 25 to 120 degrees C. Transitions II (76 degrees C), III (88 degrees C), and IV (105 degrees C), respectively, characterize the denaturation of the nucleosome, the unstacking of bases in nicked DNA after release from the nucleosome and unstacking in the released intact, supercoiled DNA. Nuclei from human epithelial cells treated with the DNA strand breakers bleomycin and streptonigrin showed an increase in transition III at the expense of transition IV. The effect was dose dependent. At intermediate times of treatment a substantial portion of the chromatin melted between the temperatures of transitions III and IV and this was taken to represent intact supercoiled DNA in which base-pairing had been weakened by loss of some bases. Treatment of cells with the alkylating agents N-nitroso-N-methylurea and mitomycin C gave results similar to those of the strand-breaker antitumor drugs, except that they were less potent. Irradiation by UV gave similar effects. The effects of intercalating drugs were quite distinct from those produced by strand breakers or alkylators. Nuclei from cells incubated with increasing doses of actinomycin D or ethidium bromide gave calorimetric scans that indicated progressive denaturation of the nucleosome and a concomitant stabilization of supercoiled DNA. The loss of transition IV during drug treatment was correlated with the loss of cellular capacity to divide, regardless of which drugs were used.
By the method of differential scanning calorimetry it was shown that the addition of arachidonic acid to photoreceptor membranes is accompanied by concentration-dependent shift of thermograms curve of rhodopsin value. Addition of tocopherol to photoreceptor membranes prevents the turbulent effect of the fatty acid on opsin and rhodopsin. The obtained data are discussed from the point of view of membrane protective properties of tocopherol.
Glutaraldehyde stabilizes pericardial tissue in prosthetic heart valves by forming covalent cross-links between collagen molecules. If the cross-linking is nonuniform owing to variable penetration of the glutaraldehyde, areas of the tissue may become sites for enzymatic attack or become potentially antigenic. Cross-linking can be easily assessed by measuring the shrinkage (thermal denaturation) temperature of the tissue. We used differential scanning calorimetry to perform a stratigraphic analysis of the shrinkage temperature of glutaraldehyde-treated pericardium. In the fixation conditions used (0.25 per cent glutaraldehyde for 28, seven and two days and for 2 hours), no variation was found in the shrinkage temperature measurement throughout the thickness of the tissue. This indicates uniform penetration of fixative and cross-linking throughout the tissue.
Differential scanning calorimetry (DSC) can detect stepwise melting of plasmid DNA along the molecular chain with high resolution. This method was applied to study interaction of some antitumor antibiotics with the plasmid pJL3-TB5 DNA (5277 base-pairs in length). Analysis of DSC curves of the plasmid DNA in the presence of, for example, adriamycin, an antitumor antibiotics of anthracycline group, together with theoretical analysis of the DNA melting curves obtained by calculation from the entire base sequence, led to the conclusion that adriamycin bound preferentially to the four particular regions with high G + C content. The DSC method would thus be useful for the study of properties of drugs which bind to DNA.
High sensitivity adiabatic differential scanning calorimetry was performed on lipids, membrane vesicles, and whole cells of Escherichia coli enriched in particular unsaturated fatty acids by genetic means. Information concerning the shape of the transition is discussed. Transitions with an asymmetric shape reminiscient of a second order transition were observed. Comparison between the lipid transition observed in whole cells, membrane vesicles, and extracted lipids enriched in elaidate reveal some basic similarities. Studies of synthetic lipids were undertaken in an attempt to interpret the shapes of these transitions as a function of the lipid components of the membrane.
The thermal denaturation of aspartate transcarbamoylas of Escherichia coli was investigated by differential scanning calorimetry. Isolated regulatory and catalytic subunits were heat denatured at 55 and 80 degrees C, respectively. In contrast, the intact enzyme was denatured in two steps. A small endotherm near 73 degrees C was assoicated with denaturation of the regulatory subunits and the major endotherm at 82 degrees C with denaturation of the catalytic subunits. Thus regulatory subunits are stabilized against heat denaturation by more than 17 degrees C when incorporated in the enzyme. Similar conclusions were obtained from measurements of the enthalpy of heat denaturation. Regulatory subunits yielded a much lower value of the enthalpy of denaturation, 1.91 cal/g, than that found for the catalytic subunit, 3.94 cal/g, or typical globular proteins (4 to 6 cal/g). When the regulatory subunits were incorporated into aspartate transcarbamoylase their enthalpy of denaturation was increased 125% (to 4.3 cal/g). The enthalpy of the catalytic subunits in the intact enzyme was increased 38% (enthalpy of denaturation of 5.43 cal/g). Stabilization of the isolated catalytic subunit as well as the intact enzyme was achieved by the addition of the bisubstrate analog N-(phosphonacetyl)-L-aspartate. Similarly the allosteric effectors, CTP and ATP, stabilized the isolated regulatory subunits or those subunits within the intact enzyme. However, the addition of the bisubstrate analog caused a decrease in the enthalpy of denaturation of the regulatory subunits within the enzyme. These results are consistent with other studies of the ligand-promoted conformational changes in the native enzyme.
The phase transition characteristics of cardiolipin and phosphatidylglycerol suspensions were investigated by differential scanning calorimetry. The phase transition temperatures for dimyristoylphosphatidylglycerol, tetramyristoylcardiolipin, dipalmitoylphosphatidylglycerol and tetrapalmitoylcardiolipin were 25.0, 47.0, 40.5 and 62.2 degrees C, respectively. The phase transition temperature for a mixture of two analogous phospholipids was higher than that for phosphatidylglycerol alone, but lower than that for cardiolipin alone. It increased along with cardiolipin content. The phase transition temperature for cardiolipin was increased in the presence of divalent cations, particularly Ca2+. The results indicate that the head group of cardiolipin by itself can increase the phase transition temperature.
The possible structure of lipophorin in insect blood (hemolymph) was investigated by differential scanning calorimetry (DSC) and 13C nuclear magnetic relaxation studies. The DSC heating curves of intact lipophorins showed endothermic peaks between -3 and 40 degrees C for lipophorins which contain hydrocarbons, whereas no such peaks were observed for lipophorins which do not contain this lipid. Hydrocarbon fractions isolated from the lipophorins showed endothermic peaks similar to those obtained from intact lipophorin in terms of the transition temperatures, the shapes, and the enthalpy changes. 13C spin lattice relaxation times of the (CH2)n resonance of hydrocarbons of intact lipophorin were measured as a function of temperature and revealed that the motions of hydrocarbon chains changed coincidentally with the onset and offset of phase transition. These data suggest the presence of a hydrocarbon-rich region within the lipophorin particles.
Differential scanning calorimetry has been used in combination with thermal gel analysis and electron microscopy to identify and study the structural domains of the membrane-enclosed vesicular stomatitis virus and its isolated internal components. Three major endothermic transitions centered at approximately 52, 76, and 80 degrees C and at least two minor transitions are observed at pH 7.0 for the intact virion. Thermal gel analysis suggests the possibility that specific proteins of vesicular stomatitis virus are involved in two or more of the calorimetric transitions. The effect of heating to defined temperatures on the morphology of the virion was studied by negative stain electron microscopy. The results of these "thermal EM" studies show discrete irreversible morphological changes in the virion which seem to coincide with the three major calorimetric transitions.
Differential scanning calorimetry has been employed to monitor structural alterations induced in the dimeric enzyme alkaline phosphatase on binding of Cd(II) (to the metal-free apoenzyme) and phosphate (Pi) (to the Cd(II) enzyme). Cd(II) addition to the apoenzyme at pH 6.5 results in an increased transition temperature, suggesting a stabilizing effect of the bound metal ion. Two distinct structural forms of the protein are detected as discrete calorimetric transitions (Tm = 69-84 degrees C; 87-94 degrees C, respectively). Distribution of the enzyme between these forms is found to depend on the exogenous Cd(II) concentration and the protocol of Cd(II) addition. These results indicate that conversion between the conformational forms is a slow process which appears to require specific levels of metal ion site occupancy. These studies, in which the exogenous Cd(II) concentration was varied from 10(-5) M to 10(-3) M suggest a structural basis for previously observed hysteretic phenomena observed on Cd(II) binding to the enzyme. Even at a minimum stoichiometry of Cd(II) (2 eq/mol of dimer) a single equivalent of Pi is sufficient to accelerate assumption of a stabilized form of the protein (Tm = 90 degrees C). This is followed by a slow structural change paralleling the time course of formation of the functional 2 Cd(II) phosphoryl enzyme which displays two calorimetric transitions (Tm = 65 degrees C, 88 degrees C). The low temperature transition does not appear if Pi is initially present at millimolar concentrations and is abolished on addition of Pi at concentrations in excess of 0.1 mM. These observations suggest the presence of a second, distinct Pi binding site on the 2 Cd(II) phosphoryl enzyme. This is supported by the changes observed in the 31P NMR chemical shift of Pi added to comparable enzyme samples. These data, including assessment of the effect of the presence of Mg(II), are discussed in terms of the mechanism of metal ion association to the enzyme and rearrangement of bound metal ions induced by Pi binding.
Various acetylcholine receptor-rich membrane preparations from Torpedo californica electroplax tissue were examined using the techniques of differential scanning calorimetry coupled with gel electrophoretic analysis of heat-denaturing material and functional assays following passage through discrete transitions. In unfractionated membranes, four irreversible calorimetric transitions were observed, one of which (Td = 59 degrees C) could be assigned to a complete loss of acetylcholine receptor function. A second lower temperature transition apparently corresponds to loss of certain peripheral membrane proteins including the Mr = 43,000 polypeptide and the acetylcholinesterase activity. Membrane preparations highly enriched in acetylcholine receptor polypeptides contained a major transition at 59 degrees C which could be shown to be sensitive to the presence of added ligands of the acetylcholine receptor, supporting its assignment to structural alterations of the receptor protein or its arrangement in the membrane.
Isolated rat livers were perfused at 37 degrees C with blood-free, defined medium containing delipidized bovine serum albumin (BSA), BSA-oleate, or BSA-palmitate. Very low density lipoproteins (VLDL) were isolated from the perfusate at 12 degrees C and lipid components were extracted and purified. Differential scanning calorimetry indicated multiple phase alterations in intact VLDL. The extracted triglycerides exhibited phase alterations at similar temperatures as the intact VLDL. The small quantities of cholesteryl esters present in the VLDL generally did not greatly affect the VLDL triglyceride transitions. The extracted phospholipids showed detectable transitions that were abolished by cholesterol at mole ratios found in the respective VLDL. The phase behavior of VLDL and its component triglycerides was associated with the degree of unsaturation of the infusate fatty acid, and by variation of fatty acid chain length. The structural differences between VLDL lipid fractions noted by DSC were also monitored by fluorescence probes. The data indicated that in the intact VLDL the physical properties of the 'interior core' lipids can affect the properties of the 'surface monolayer'. In addition, Arrhenius plots of corrected fluorescence indicated that trans-parinarate and diphenyl-hexatriene detected different characteristic breakpoint temperatures in the phospholipids. The breakpoints of triglycerides were highly dependent on the type of fatty acid in the infusate, but were similar to those noted in intact VLDL. Finally, the breakpoints in Arrhenius plots of fluorescence probe parameters did not necessarily coincide with onset or end temperatures of DSC transitions.
Data derived from indirect calorimetry in patients with stable chronic pneumopathies were evaluated together with their pulmonary function data (spirometry, diffusing capacity, blood gases). Their basal metabolic rate was significantly higher (116.1% of the predicted value), this increase being more expressed in the patients with hypoxemia and with more severe obstructive disorder. In chronic obstructive pulmonary disease, the increased energy expenditure in hypoxemia was accompanied with increased lipid oxidation. There also was a correlation between the patients' nutritional status (evaluated by their body mass index) and their pulmonary function. These results confirm the impairment of pulmonary function in malnourished patients.
The application of scanning calorimetry to investigate laccase I from the lignin-degrading basidomycete PM1 (CECT 2971) showed three thermal transitions beneath the overall endotherm following the previous heating of the sample up to 60 degrees C. The thermodynamic parameters of these three transitions satisfy a model of two-state independent unfolding, supporting a three-domain organization of the enzyme. It is shown that the catalytic site of laccase I is located in the domain with the thermally-induced transition at 76 degrees C.
Twenty-one obese patients with non-insulin dependent diabetes mellitus, 16 female and 5 male ones, were fed similar isocaloric rations, differing only by the share of substitution of the traditional protein products (0%, 60%, 30%, 45%) in the Danpro-S, Danpro-Fibre soybean protein concentrate. The parameters tested were daily glycemia, C-peptides, blood hydrocortisone, and urinary excretion of nitrous metabolites. Indirect calorimetry was used to assess the protein, fat, and carbohydrate oxidation rates at rest. Addition of proteins of a plant origin to the diets of such patients was associated with significant changes of the energy metabolism at rest at the expense of increased oxidation of carbohydrates and reduced protein catabolism, that may be regarded as a favorable effect. In this patient population a 30% soybean diet brings about an almost maximal positive effect in patients with the first degree of obesity, whereas in those with the second degree of obesity such effect is attained by the 45% soybean diet.
We evaluated the influence of epidural and spinal anesthesia on VO2 and VCO2 measured by the indirect calorimetry. According to the spreading of anesthetic level, VO2, VCO2 and EE increased in the epidural group (EP group), but they decreased in spinal group (SP group). RQ was unchanged in both groups. Rate pressure product (RPP), in proportion to the MVO2 and the REE, did not correlate with the EE. The VO2, VCO2 and EE in EP group increased probably because; (1) epidural anesthesia does not affect the VO2 change, as it does not markedly suppress respiration; (2) cardiac stroke work is retained during epidural anesthesia, as it does markedly suppress circulation. In conclusion, epidural anesthesia exerts less influence on the respiratory and circulatory systems of patients compared with spinal anesthesia.