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Indirect calorimetry measures oxygen consumption and carbon dioxide production to calculate resting energy expenditure and respiratory quotient. The respiratory quotient can be determined from indirect calorimetry to determine substrate utilization and used to alter the patient's nutrition support regimen. All but one indirect calorimeter manufactured in the United States are open-circuit rather than closed-circuit systems.
The interaction of the surfactant octyl glucoside (OG) with dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and soy bean phosphatidylcholine (soy bean PC) was studied using high-sensitivity titration calorimetry. We determined the partition coefficient of OG between water and lipid bilayers and the transfer enthalpy of the surfactant by addition of lipid vesicles to OG monomers or vice versa. Comparison with the micellization enthalpy of the surfactant gives information on differences in the hydrophobic environment of OG in a liquid-crystalline bilayer or a micelle. The average partition coefficient P in mole fraction units for x(e) approximately 0.12-0.2 decreases slightly from 4152 at 27 degrees C to 3479 at 70 degrees C for DMPC and from 4260 to 3879 for soy bean PC, respectively. The transfer enthalpy deltaH(T) of OG into lipid vesicles is positive at 27 degrees C and negative at 70 degrees C. Its temperature dependence is larger for the incorporation of OG into DMPC than into soy bean PC vesicles. It is concluded that OG in DMPC vesicles is better shielded from water than in soy bean PC vesicles or in micelles. Titration calorimetry was also used to determine the phase boundaries of the coexistence region of mixed vesicles and mixed micelles in the systems OG/DMPC, OG/DPPC, OG/DSPC, and OG/soy bean PC vesicles at 70 degrees C in the liquid-crystalline phase. DMPC and soy bean PC solubilization was also studied at 27 degrees C to investigate the effect of temperature. The effective surfactant to lipid ratios at saturation, R(e)(sat), for all PCs studied are in the range between 1.33-1.72 and the ratios at complete solubilization, R(e)(sol), are between 1.79-3.06. At 70 degrees C, the R(e)(sat) values decrease with increasing chain length of the saturated PC. The ratios depend also slightly on temperature and the degree of unsaturation of the fatty acyl chains. For the OG/soy bean PC system, the coexistence range for mixed vesicles and mixed micelles is larger than for the corresponding PCs with saturated chains.
Using differential scanning calorimetry and complementary ultrastructural observations, we have characterized the status of chromatin during the transformation of rat hepatocytes in the resistant hepatocyte model of Solt and Farber (1976. Nature (Lond.). 263:701-703). Differential scanning calorimetry affords a measure of the degree of condensation of chromatin in situ and has therefore been used in this work for the purpose of establishing the nature of the structural changes associated with the emergence of successive cellular populations. Since the resistant hepatocyte model generates a series of synchronous phenotypic changes, it was possible to determine unambiguously the content of heterochromatin at each step of the process. The higher-order structure undergoes a partial relaxation in early developing nodules, isolated 16 weeks after initiation; the thermal transition at 90 degrees C, which is characteristic of noninteracting core particles, increases with respect to control hepatocytes. Dramatic changes occur in persistent (46-week) nodules. The 90 degrees C endotherm dominates the thermogram, while the transition at 107 degrees C, corresponding to the denaturation of the core particle packaged within the heterochromatic domains, disappears. The complete loss of the higher-order structure at this stage of transformation has been further verified by ultrastructural observations on thin nuclear sections. Ten-nm filaments, having a beaded appearance, are scattered throughout the nucleoplasm and clearly result from the decondensation of 30-nm-thick fibers. This catastrophic relaxation process cannot be related to an effective increase in gene activity. Rather, our observations suggest that during transformation chromatin is in a state of high transcriptional competence associated with the alert of general cellular programs. This view is consistent with the finding that in persistent nodules the DNA is extensively hypomethylated with respect to normal liver.
We have investigated the thermotropic phase behavior of aqueous dispersions of the 1,2- and 2,3-di-O-tetradecyl-1(3)-O-(beta-D-galactopyranosyl)-sn- glycerols and their diastereomeric mixture using differential scanning calorimetry and low-angle and wide-angle x-ray diffraction. Upon heating, unannealed aqueous dispersions of these compounds all exhibit a lower temperature, moderately energetic phase transition at approximately 52 degrees C and a higher temperature, weakly energetic phase transition at approximately 63 degrees C, both of which are reversible on cooling. X-ray diffraction measurements identify these events as the L beta (or L' beta)/L alpha and L alpha/HII phase transitions, respectively. The structures of the L beta, L alpha, and HII phases of these lipids, as determined by x-ray diffraction measurements, are identical within the error bars for all of these lipids. On annealing below the L beta/L alpha phase transition temperature, the L beta phase converts to an Lc phase at a rate which is strongly dependent on the chirality of the glycerol backbone (1,2-sn > 1,2-rac > 2,3-sn). The temperature of the phase transition from the Lc phase seen on reheating is also dependent on the glycerol chirality. In addition, the nature of the Lc phase changes on subsequent heating in the 1,2-sn and 1,2-rac lipids, but we have not been able to detect this Lc1/Lc2 phase transition by calorimetry. However, wide-angle x-ray diffraction measurements indicate that these Lc phases differ mostly in their hydrocarbon chain packing modes. The Lc2 phase does not appear to be present in the 2,3-sn compound, suggesting that its formation is not favored in this diastereomeric isomer. These observations are discussed in relation to the effect of glycerol chirality on the molecular packing of these glycolipids, particularly on hydrogen bonding and hydration in the interfacial region of the bilayer.
Isothermal titration calorimetry (ITC) follows the heat change when a test compound binds to a target protein. It allows precise measurement of affinity. The method is direct, making interpretation facile, because there is no requirement for competing molecules. Titration in the presence of other ligands rapidly provides information on the mechanism of action of the test compound, identifying the intermolecular complexes that are relevant for structure-based design. Calorimetry allows measurement of stoichiometry and so evaluation of the proportion of the sample that is functional. ITC can characterize protein fragments and catalytically inactive mutant enzymes. It is the only technique which directly measures the enthalpy of binding (delta H degree). Interpretation of delta H degree and its temperature dependence (delta Cp) is usually qualitative, not quantitative. This is because of complicated contributions from linked equilibria and a single change in structure giving modification of several physicochemical properties. Measured delta H degree values allow characterization of proton movement linked to the association of protein and ligand, giving information on the ionization of groups involved in binding. Biochemical systems characteristically exhibit enthalpy-entropy compensation where increased bonding is offset by an entropic penalty, reducing the magnitude of change in affinity. This also causes a lack of correlation between the free energy of binding (delta G degree) and delta H degree. When characterizing structure-activity relationships (SAR), most groups involved in binding can be detected as contributing to delta H degree, but not to affinity. Large enthalpy changes may reflect a modified binding mode, or protein conformation changes. Thus, delta H degree values may highlight a potential discontinuity in SAR, so that experimental structural data are likely to be particularly valuable in molecular design.
The thermal denaturation of bovine fibrinogen has been investigated using differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. Differential scanning calorimetry measurements were carried out while changing the scan-rate. The transition at 57 degrees C was found to be irreversible and highly scan-rate dependent, suggesting that the denaturation is, at least in part, under kinetic control. The secondary structural changes at various temperatures were monitored by far-ultraviolet CD spectroscopy. These results show that the DSC transition for the thermal denaturation of bovine fibrinogen can be interpreted in terms of a kinetic process, N --> F, where k is a first-order kinetic constant that changes with temperature according to the Arrhenius equation. An important transition peak was observed at 78.8 degrees C which is attributed to the C-terminal parts of the Aalpha chains of fibrinogen.
A comparative study on the drug release capacity of four water swellable polymeric systems was carried out by differential scanning calorimetry (DSC). The polymeric systems chosen were alpha,beta-polyaspartahydrazide (PAHy) crosslinked by glutaraldehyde (GLU) (PAHy-GLU) or by ethyleneglycoldiglycidylether (EGDGE), (PAHy-EGDGE), polyvinylalcohol (PVA) crosslinked by glutaraldehyde (PVA-GLU) and alpha,beta-poly(N-hydroxyethyl)-DL-aspartamide (PHEA) by gamma irradiation (PHEA-gamma matrices). The degree of crosslinking for PAHy-GLU, PAHy-EGDGE and PVA-GLU samples was about 0.4 and 0.8. These hydrogels were characterized as free of drugs and were loaded with diflunisal (DFN) (approximately 2.5% w/w). Diflunisal, a non-steroidal anti-inflammatory drug, has been chosen as a model drug to be incorporated into polymeric matrices to follow the release processes of a drug from these hydrogels to a model membrane made by unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC). Differential scanning calorimetry appears to be a suitable technique to follow the transfer kinetics of the drug from the controlled release system to the biomembrane model. The drug releases from all the considered polymeric hydrogels, were compared with the release observed from the drug solid form by examining the effects on the thermotropic behaviour of DPPC unilamellar vesicles. The release kinetics of the drug from hydrogels were followed at 25, 37 and 50 degrees C to evidence the influence of temperature on the drug release and on the successive transfer to biological membrane model. Particularly, it appears evident that the total amount of drug transferred and the release rate are affected by the polymer crosslinking degree (it increases with crosslinking decrease) as well as by the nature of crosslinking agent. In fact, the drug release profiles from PAHy-GLU samples are more differentiated than those from PAHy-EGDGE. The effect of parameters correlating with the properties of starting polymer, such as water-affinity, crystallinity, glass-to-rubber transition temperature and affinity towards drug molecules, has been also evaluated.
The thermodynamic parameters of interactions between six variants of the anti-idiotypic monoclonal antibody (mAb) CGP 60686 produced by the hybridoma MK2-23 with an idiotypic mAb and five different anti-anti-idiotypic mAb were studied with high sensitivity titration calorimetry. CGP 60686 recognizes an epitope in the antigen-combining region of the human high-molecular-weight-melanoma-associated antigen (HMW-MAA)-specific mouse mAb CGP 76873 produced by the hybridoma 763.74. The five HMW-MAA-specific anti-anti-idiotypic mAbs GH 464, GH 518, GH 149, GH 386 and GH 586 were generated from mice immunization with mAb CGP 60686. All interactions between the anti-idiotypic mAb and the idiotypic mAb or the anti-anti-idiotypic mAb showed large exothermic binding enthalpies between -15 and -23 kcal/mol and binding affinities larger than 6 x 10(9) M-1. Four of the five anti-anti-idiotypic mAbs tested exhibited significantly higher binding enthalpies for the interaction with the anti-idiotypic than the idiotypic mAbs. Replacement of either the heavy or the light chain variable region of the anti-idiotypic mAbs with an unrelated variable region abolished the idiotype to anti-idiotype interaction. Thus, both the heavy and the light chain variable region of the anti-idiotypic mAbs are required for binding to the idiotype. The values of the binding enthalpy showed only small variations when binding of the idiotypic mAb CGP 76873 to four variants of the anti-idiotypic mAb CGP 60686 with different immunoglobulin constant regions, but identical variable regions were compared. Furthermore, Fab fragments of the idiotypic mAbs showed almost the same binding enthalpy per binding site as the whole IgG molecules. Immunoglobulin constant regions thus had little influence on the idiotype to anti-idiotype interactions. Taken together, the observed thermodynamic parameters suggest that the idiotype to anti-idiotype interactions studied here are enthalpy-driven processes with only minor entropic contributions. High sensitivity titration calorimetry was used to monitor protein-protein interactions within an anti-idiotypic antibody cascade. It was found that the direct measurement of the interaction enthalpy allowed a quantitative characterization of the binding processes studied.
The characteristics of polymer-DNA complexes formed by positively-negatively charged interaction have a great influence on their transfection potential. Since the limit changes in thermal transitions which were hardly measured in conventional calorimetry, now in this study they have been successfully carried out by highly-sensitive differential scanning calorimetry for better understanding the pDMAEMA-plasmid DNA complexing process. Thermal behaviors of plasmid DNA, polymer and their formed complexes were recorded to give insights into their conformational changes when temperature was raised. In results, the supercoiled or open-circular plasmid DNA is not thermal reversible indicated by the decrease of denaturation peak and disappearance of DNA conformational transition related to its twist status at 50-70 degrees C. The cationic polymer is thermally stable by showing reversible transition peaks after two heating processes. For the cationic polymer-plasmid DNA complexes, electrostatic forces lead to a higher denaturation temperature of plasmid DNA and transition temperature of polymer. Also, heat can cause a topological change in plasmid DNA and then change their mutual complexation capacity.
The presence of multiple polymorphic forms in seven batches of raw material of a model compound having poor wettability properties (cimetidine) was studied by solution calorimetry. Due to the large number of polymorphic forms described in the literature ('Gazz. Chim. Ital., 109 (1979) 535'; 'J. Pharm. Sci., 73 (1983) 1436'; 'J. Pharm. Biomed. Anal., 3 (1985) 303') and its poor wettability characteristics, cimetidine was chosen as a model compound to illustrate the possible use of solution calorimetry in the detection of polymorphism using surfactant systems as solvents for dissolution. Due to the closeness of the melting points of the different polymorphic forms of cimetidine, DSC was not the best investigational tool. As initially suspected, the measurement of enthalpy of solution values in water of the cimetidine batches was not possible. However, the use of sodium dodecyl sulfate (SDS) and polysorbate 20 (Tween 20(R)) at concentrations above their respective cmc values permitted the detection of significant differences in enthalpy of solution among several batches. The presence of different polymorphic forms was confirmed by microscopy, X-ray powder diffractometry, and Fourier transform infrared spectroscopy.
The thermal properties of the drug sulfapyridine were studied using a combination of differential scanning calorimetry (DSC), modulated temperature differential scanning calorimetry (MTDSC) and thermo optical analysis with a view to examining the combined use of these methods as a characterisation strategy. Conventional DSC indicated that quenched sulfapyridine exhibited a series of transitions on reheating at 10 degrees C min(-1) which were ascribed to a glass transition (56.9 degrees C), cold crystallisation (103.7 degrees C), a solid-solid transition (131.4 degrees C) and metastable and stable polymorphic melting (177.3 and 186.3 degrees C). MTDSC studies were able to show the glass transition with much greater clarity in the reversing signal than was possible using the conventional technique, while it was also possible to observe the phase angle which again allowed clearer visualisation of the Tg. Thermooptical analysis confirmed the interpretation of the DSC and MTDSC data, showing the formation of spherulitic crystals which converted to a needle-shaped morphology on heating, these being ascribed to metastable and stable polymorphs respectively. The study has therefore demonstrated that using the three techniques in combination allows unique insights into the glass transitional and polymorphic behaviour of a drug substance.
Few analytical techniques allow to evaluate the inclusion yield of cyclodextrin-drug complexes, because most manufacturing processes give amorphous products. In this study, we have developed an alternative method to differential scanning calorimetry, to accurately determine the free/complexed piroxicam ratio by UV spectroscopy. This method is based on the differential solubility of the piroxicam-beta-cyclodextrin 1:2.5 mol/mol complex in water-acetonitrile (1:1, v/v) (Solvent A) or in anhydrous acetonitrile (Solvent B), both containing 0.05 M HCl. In anhydrous acetonitrile, beta-cyclodextrin is insoluble and the included drug remains entrapped, allowing the free piroxicam determination, while with 50% of water, the complex is totally dissolved, allowing the determination of the total guest content. This method was validated for linearity, precision and accuracy. The presence of cyclodextrin does not influence the assays, but more than 0.5% of water in Solvent B significantly affects the determination of the free piroxicam content. In comparison with differential scanning calorimetry, both detectability and precision were improved. It is now possible to analyse complexes with an inclusion purity greater than 99%.
Of all the techniques that are currently available to measure binding, isothermal titration calorimetry is the only one capable of measuring not only the magnitude of the binding affinity but also the magnitude of the two thermodynamic terms that define the binding affinity: the enthalpy (AH) and entropy (AS) changes. Recent advances in instrumentation have facilitated the development of experimental designs that permit the direct measurement of arbitrarily high binding affinities, the coupling of binding to protonation/deprotonation processes and the analysis of binding thermodynamics in terms of structural parameters. Because isothermal titration calorimetry has the capability to measure different energetic contributions to the binding affinity, it provides a unique bridge between computational and experimental analysis. As such, it is increasingly becoming an essential tool in molecular design.
The sulfonylureas are a class of oral hypoglycemic agents used to treat type II diabetes mellitus, and tolbutamide is a "first generation" member of this family. It is a nonpolar, weakly acidic drug that binds to serum albumin in the circulation. In the present study, we have examined the interactions of tolbutamide with human serum albumin by isothermal titration calorimetry and heteronuclear multiple-quantum coherence NMR spectroscopy. Calorimetric titrations revealed that tolbutamide binds to albumin at three independent sites with the same or comparable affinity. This result was independently confirmed by NMR experiments which resolved three resonances at 1H chemical shifts of 2.07, 2.11 and 2.14 ppm, corresponding to [methyl-13C]tolbutamide bound to three discrete binding sites. The binding affinity quantitated by calorimetry (Kd = 21 +/- 9 microM at pH 7.4, 37 degrees C) was approximately 5 times lower than the most frequently reported value. Tolbutamide titrations of albumin complexed with three other drugs whose binding sites have been localized by X-ray crystallography (salicylate, clofibric acid, and triiodobenzoic acid) demonstrated direct competition for common binding sites. NMR experiments with samples containing [methyl-13C]tolbutamide and these competing drugs permitted assignment of the resonances at 2.07 and 2.14 ppm to tolbutamide bound to the aspirin sites in albumin subdomains IIIA and IIA, respectively. These findings permit the first assignment of tolbutamide binding sites to specific locations on the albumin molecule within the context of the recently published crystal structure of human serum albumin. In addition, this information provides a molecular basis for predicting unfavorable drug interactions involving tolbutamide in patients with type II diabetes.
Precision scanning calorimetry has been used to examine the thermal behavior of mixed micelles formed between bile salts and dipalmitoylphosphatidylcholine (DPPC). Complex thermal transitions are observed which change dramatically with the mole ratio of bile salt to DPPC, dilution, and ionic strength. Comparison of the behavior of sodium taurocholate (TC) mixed micelles with sodium taurodeoxy-cholate (TDC) mixed micelles indicates similarity in the thermal transitions at high dilution or when the actual micellar composition is similar. It was found through equilibrium dialysis that considerably less TC than TDC is incorporated into mixed micelles with DPPC at a given bile salt concentration. Accounting for these concentration differences provides a means for more direct analysis of changes in the thermal transitions with mole ratio and dilution for the two bile salt components. Resolution of the thermal transitions into several component contributions is employed as an aid to interpretation of the differential scanning calorimetry curves. The curve resolutions lead to estimates of van't Hoff and calorimetric enthalpies of the individual contributions. The results of the curve resolutions, along with the behavior of the total enthalpies of the transitions, are consistent with a transformation in micellar structure occurring when the actual micellar composition is a mole ratio of bile salt to DPPC of about 1 to 1. The transformation region is near that found from X-ray evidence and is thought to correspond to a change from disk-shaped to spherical micelles.
The effects of a Naja mossambica mossambica cardiotoxin on the thermotropic properties of charged phospholipids have been studied by fluorescence polarization, differential scanning calorimetry, and Raman spectroscopy. The binding of the toxin is only governed by the net charge at the interface and is not affected by the polar head group structure of the phospholipids or by the acyl chains physical state, degree of insaturation, or length. The effect of the toxin on the phospholipid structure is drastic. In all cases, the gel to liquid-crystalline phase transition monitored by fluorescence and Raman spectroscopies is progressively abolished without notable shift in temperature as the proportion of toxin is increased. The endothermic peaks detected by differential scanning calorimetry decrease in intensity as the toxin content is increased but always remain sharp. All the techniques used give complementary results, and none of them reveals the presence of secondary transitions at higher or lower temperatures. We thus believe that the lipid molecules that are perturbed by the toxin, approximately 10 +/- 2 molecules, do not undergo a phase transition. Raman results demonstrate that these "boundary" lipids display a population of gauche rotamers that is as high as the one found in the liquid-crystalline phase of the pure phospholipid and this even well below the phase transition temperature. On the other hand, fluorescence results are interpreted as due to a partial immobilization of the lipids in contact with the toxin above the transition temperature. Thus, even though the interaction is governed by electrostatic forces, the toxin penetrates at least partially into the bilayers, inducing a disorganization of the aliphatic chains and changes in their mobility; this could explain their lytic activity.
The interaction between dipalmitoylphosphatidylcholine (DPPC) and the aqueous form of the myelin proteolipid apoprotein (PLA) has been investigated. Lyophilization was found to be an efficient and nonperturbing method for membrane reconstitution. Mixtures of different lipid/protein ratios were analyzed by means of differential calorimetry, fluorescence polarization, and sucrose gradient centrifugation. The presence of two coexisting lipid populations, termed "bulk" and "interacting" lipids, was demonstrated by these three techniques. By differential calorimetry, 23 DPPC molecules per molecule of protein (30 kDa) were shown to be excluded from the lipid phase transition. By fluorescence polarization, we detected above the phase-transition temperature a large perturbation of the lipid acyl chain dynamics induced by the aqueous form of PLA. Increasing the protein content above 35% by weight within the recombinants caused drastic changes in both delta H values and the fluorescence anisotropy parameter, which could stem from protein aggregation.
A differential scanning calorimetry study of the thermal denaturation of Bacillus thermoproteolyticus rokko thermolysin was carried out. The calorimetric traces were found to be irreversible and highly scan-rate dependent. The shape of the thermograms, as well as their scan-rate dependence, can be explained by assuming that the thermal denaturation takes place according to the kinetic scheme N k----D, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation, N the native state, and D the unfolded state or, more probably, a final state, irreversibly arrived at from the unfolded one. On the basis of this model, the value of the rate constant as a function of temperature and the activation energy have been calculated. It is shown that the proposed model may be considered as being one particular case of that proposed by Lumry and Eyring [Lumry, R., & Eyring, H. (1954) J. Phys. Chem. 58, 110] N in equilibrium D----I, where N is the native state, D the unfolded one, and I a final state, irreversibly arrived at from D. Lastly, some comments are made on the use of the scan-rate effect on the calorimetric traces as an equilibrium criterion in differential scanning calorimetry.