Kinetic analysis of microcalorimetric data derived from microbial growth: Basic theoretical, practical and industrial considerations.

We report here a mathematical framework for the quantitative interpretation of exponential bacterial growth measured with isothermal microcalorimetry. The method allows determination of many parameters that define the exponential growth phase. To automate the analysis, we also wrote a coding program, so that the approach could be embedded in a commercial setting. As an exemplar, we apply the method to a commercial probiotic product. The outcome was that we could identify characteristic parameters of growth (including rate constant and doubling time), and hence authenticate product quality, within 15 h. This compares favourably with the current 7-10 days required for conventional microbiological assessment (to allow release of product for bottling and marketing) via plating methods. The method would lend itself to growth analysis of single and mixed bacterial cultures.

In vitro inhibition of Clostridium difficile by commercial probiotics: A microcalorimetric study.

The aim of the study was to investigate the influence of some commercial probiotics on the growth of Clostridium difficile using isothermal microcalorimetry, a technique which can monitor the real time growth of bacteria. Commercial probiotic strains and products, Lactobacillus acidophilus LA-5®, Bifidobacterium lactis BB-12®, Probio 7® and Symprove™ were co-cultured with C. difficile in Brain Heart Infusion (BHI) broth supplemented with 0.1% (w/v) l-cysteine hydrochloride and 0.1% (w/v) sodium taurocholate and monitored in the microcalorimeter. Pseudomonas aeruginosa NCIMB 8628 was also co-cultured with C. difficile and studied. The results indicated inhibition of C. difficile by the probiotics. The inhibition of C. difficile was shown to be pH-dependent using neutralized and unmodified cell free supernatant (CFS) produced by the probiotic strains. However, concentrated CFS of the probiotics also inhibited the intestinal pathogen in a non pH-dependent manner, likely due to specific antimicrobial substances produced. The results also indicated that C. difficile growth was greatly influenced by the presence of sodium taurocholate and by the pH of the medium. A medium pH of between 6.45 and 6.9 demonstrated maximum growth of the organism in the microcalorimeter.

Development of a flow system for studying biofilm formation on medical devices with microcalorimetry.

Isothermal microcalorimetry (IMC) is particularly suited to the study of microbiological samples in complex or heterogeneous environments because it does not require optical clarity of the sample and can detect metabolic activity from as few as 10(4) CFU/mL cells. While the use of IMC for studying planktonic cultures is well established, in the clinical environment bacteria are most likely to be present as biofilms. Biofilm prevention and eradication present a number of challenges to designers and users of medical devices and implants, since bacteria in biofilm colonies are usually more resistant to antimicrobial agents. Analytical tools that facilitate investigation of biofilm formation are therefore extremely useful. While it is possible to study pre-prepared biofilms in closed ampoules, better correlation with in vivo behaviour can be achieved using a system in which the bacterial suspension is flowing. Here, we discuss the potential of flow microcalorimetry for studying biofilms and report the development of a simple flow system that can be housed in a microcalorimeter. The use of the flow system is demonstrated with biofilms of Staphylococcus aureus.

An in vitro test of the efficacy of an anti-biofilm wound dressing.

Broad-spectrum antimicrobial agents, such as silver, are increasingly being formulated into medicated wound dressings in order to control colonization of wounds by opportunistic pathogens. Medicated wound dressings have been shown in-vitro to be effective against planktonic cultures, but in-vivo bacteria are likely to be present in biofilms, which makes their control and eradication more challenging. Recently, a functional wound dressing (AQUACEL(®) Ag+ Extra™ (AAg + E)) has been developed that in addition to silver contains two agents (ethylenediaminetetraacetic acid (EDTA) and benzethonium chloride (BC)) designed to disrupt biofilms. Here, the efficacy of AAg + E is demonstrated using a biofilm model developed in an isothermal microcalorimeter. The biofilm was seen to remain viable in the presence of unmedicated dressing, silver-containing dressing or silver nitrate solution. In the presence of AAg + E, however, the biofilm was eradicated. Control experiments showed that neither EDTA nor BC alone had a bactericidal effect, which means it is the synergistic action of EDTA and BC disrupting the biofilm with silver being bactericidal that leads to the product's efficacy.

An in vitro test of the efficacy of silver-containing wound dressings against Staphylococcus aureus and Pseudomonas aeruginosa in simulated wound fluid.

An isothermal microcalorimetric assay was used to quantify the efficacy of a silver-containing wound dressing against two common wound pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. The growth patterns of the two species were unique and varied depending on the environment in which the organisms were grown. Addition of non-silver-containing dressing altered the growth kinetics while addition of silver (contained either in a dressing or as AgNO3 solution) was seen to elicit inhibition and/or kill depending on concentration. Tests were conducted in nutrient broth and simulated wound fluid. It was found that minimum inhibitory and minimum bactericidal concentration values were higher in simulated wound fluid and under anaerobic conditions. Bioavailability of silver from the wound dressing was 35% against S. aureus in nutrient broth and 68% against both species in simulated wound fluid. The data highlight the importance of developing and conducting in vitro assays in biorelevant media.

Calorimetric determination of rate constants and enthalpy changes for zero-order reactions.

Calorimetry is a general method for determination of the rates of zero-order processes, but analysis of the data for the rate constant and reaction enthalpy is difficult because these occur as a product in the rate equation so evaluation of one requires knowledge of the other. Three methods for evaluation of both parameters, without prior knowledge, are illustrated with examples and compared with literature data. Method 1 requires the reaction to be studied in two buffers with different enthalpies of ionization. Method 2 is based on calculation of reaction enthalpy from group additivity functions. Method 3 applies when reaction progresses to completion. The methods are applied to the enzymatic hydrolysis of urea, the hydrolysis of acetylsalicylic acid, and the photodegradation of nifedipine, respectively.

Alcohol enhanced permeation in model membranes. Part II. Thermodynamic analysis of membrane partitioning.

The role of solvents in drug transport has not been properly addressed in the literature, despite its well known influence on drug permeation. Previously we have conducted thermodynamic and kinetic analyses to probe the molecular mechanisms of alcohol enhanced permeation. In the present study, the influence of temperature on the partitioning of methyl paraben into silicone membranes is investigated. In line with previous membrane transport studies of methyl paraben in silicone membranes, butanol and heptanol are used as representative alcohols. The results show higher amounts of methyl paraben extracted from the silicone membrane following equilibration with butanol, at all experimental temperatures. This was in line with alcohol uptake data. In fact, a linear correlation (r(2) ∼0.97) was found between the amount of methyl paraben in the silicone membrane and the corresponding alcohol uptake. Calculated "specific" vehicle-membrane partition coefficients for both alcohols were approximately one, suggesting that the effective concentrations of methyl paraben inside and outside the membrane were the same. Thermodynamic analysis of the alcohol-membrane partition coefficients as a function of temperature showed no apparent trend for butanol, with an associated enthalpy change of approximately zero. Conversely, there was a positive trend in the van't Hoff plot for methyl paraben in heptanol, indicative of an exothermic process. Moreover, the partitioning trends of methyl paraben in silicone membranes obtained from membrane transport and equilibrium experiments were not the same. This reflects the fundamental differences between the calculated vehicle-membrane partition coefficients in the two studies. Finally, the findings from membrane transport and equilibrium experiments support a model of alcohol enhanced permeation where high solvent sorption promotes high solute concentrations in the overall volume of the membrane (i.e. K), thus leading to modified solute transport (i.e. increased flux). The same model also accounts for changes in membrane diffusivity (i.e. D) related to the properties of the imbibed alcohol.

Quantitative analysis of solid-state processes studied with isothermal microcalorimetry.

Quantitative analysis of solid-state processes from isothermal microcalorimetric data is straightforward if data for the total process have been recorded and problematic (in the more likely case) when they have not. Data are usually plotted as a function of fraction reacted (α); for calorimetric data, this requires knowledge of the total heat change (Q) upon completion of the process. Determination of Q is difficult in cases where the process is fast (initial data missing) or slow (final data missing). Here we introduce several mathematical methods that allow the direct calculation of Q by selection of data points when only partial data are present, based on analysis with the Pérez-Maqueda model. All methods in addition allow direct determination of the reaction mechanism descriptors m and n and from this the rate constant, k. The validity of the methods is tested with the use of simulated calorimetric data, and we introduce a graphical method for generating solid-state power-time data. The methods are then applied to the crystallization of indomethacin from a glass. All methods correctly recovered the total reaction enthalpy (16.6 J) and suggested that the crystallization followed an Avrami model. The rate constants for crystallization were determined to be 3.98 × 10(-6), 4.13 × 10(-6), and 3.98 × 10(-6) s(-1) with methods 1, 2, and 3, respectively.

Quantifying the rates of relaxation of binary mixtures of amorphous pharmaceuticals with isothermal calorimetry.

While the use of isothermal calorimetry to quantify the rate of relaxation of one-phase amorphous pharmaceuticals, through application of models, is well documented, the resolution of the models to detect and quantify relaxation in systems containing two independent amorphous phases is not known. Addressing this knowledge gap is the focus of this work. Two fitting models were tested; the Kohlrausch-Williams-Watts model (KWW) and the modified-stretch exponential (MSE). The ability of each model to resolve relaxation processes in binary systems was determined with simulated calorimetric data. It was found that as long as the relaxation time constants of the relaxation processes were with 10(3) of each other, the models could determine that two events were occurring and could quantify the correct reaction parameters of each. With greater differences in the time constants, the faster process always dominates the data and the resolving power of the models is lost. Real calorimetric data were then obtained for two binary amorphous systems (sucrose-lactose and sucrose-indomethacin mixtures). The relaxation behaviour of all the single components was characterised as they relaxed individually to provide reference data. The ability of the KWW model to recover the expected relaxation parameters for two component data was impaired because of their inherently noisy nature. The MSE model reasonably recovered the expected parameters for each component for the sucrose-indomethacin system but not for the sucrose-lactose system, which may indicate a possible interaction in that case.

Alcohol enhanced permeation in model membranes. Part I. Thermodynamic and kinetic analyses of membrane permeation.

While it is well recognised that formulation components influence drug permeation, few studies have addressed the influence of vehicles on drug transport in artificial or biological membranes Previously we have investigated the effects of temperature on the uptake of model vehicles (i.e. alcohols) into silicone membrane. The present study evaluates the permeation of the model drug methyl paraben in the presence of butanol or heptanol. Drug permeation through silicone membranes was studied at different temperatures for each vehicle. Thermodynamic and kinetic analyses of the permeation data were conducted to elucidate the possible mechanisms of drug transport. Independent examination of the partition and diffusion coefficients estimated for the permeation studies at different temperatures showed a break point occurring near 20 degrees C for butanol, but not heptanol. This transition temperature separated two different mechanisms of solute diffusion and partitioning, which may be associated with a change in the properties of the solvent. This was not observed from an analysis of flux data, owing to compensatory influences on the diffusion and partition behaviour of the drug. The study underlines the importance of appropriate temperature control when studying drug permeation.

An in vitro method for the quantitative determination of the antimicrobial efficacy of silver-containing wound dressings.

Treatment with silver-containing wound dressings is becoming an increasingly popular strategy to eliminate growth of opportunistic wound pathogens during the healing process. However, there are concerns over the possible side-effects of silver to the patient; coupled to the cost of silver as an ingredient there is a desire to ensure that wound dressings contain the least quantity of active ingredient to ensure the minimum bactericidal concentration (MBC) of silver is maintained in the wound environment. This requires the ability to determine the efficacy of silver directly within the wound environment; an extremely complicated task that is difficult using classical (plate counting) microbiological assays because these cannot be conducted in situ. Here, we report a quantitative method for determining the efficacy of silver in wound dressings using an isothermal calorimetric method. The growth curves of P. aeruginosa (NCIMB 8628) were recorded in growth medium and in growth medium containing AQUACEL Ag Hydrofiber dressing. It was found that 10 mg of dressing was sufficient to ensure no detectable growth of organism in 2.5 mL of medium inoculated to 10(6) cfu/mL. This corresponded to a silver load of 1.1x10(-6) moles (equivalent to 4.4x10(-4) M, in the volume of medium used in the experiment). Experiments conducted with silver nitrate rather than dressing indicated the MBC of silver against P. aeruginosa was 1x10(-4) M. The results suggested that not all of the silver in the dressing was bioavailable, at least over the lifetime of the experiment. One advantage of this effect would be the lack of excess availability of the silver, which allays fears of potential toxicity to the patient and may provide an extended period of time over which the dressing is bactericidal.

LED-array photocalorimetry: instrument design and application to photostability of nifedipine.

A photocalorimeter was designed to analyse quantitatively the photostability of pharmaceuticals. Its application is demonstrated with reference to two solution phase test systems; the photodegradations of 2-nitrobenzaldehyde (2-NB) and nifedipine. Light emitting diode (LED) arrays were used to illuminate the sample and reference channels of the calorimeter. Five LEDs were used to create an array from 360 to 700nm. A power supply system was constructed that zeroed the instrument by supplying a preset voltage to the sample side LEDs and varying that supplied to the reference LEDs until a zero calorimetric signal was obtained. The photodegradation of 2-NB was zero-order and varied in proportion to the input voltage supplied to the LED array. Analysis of the data (the rate of reaction was determined to be equal 1.04x10(-6)moldm(-3)s(-1) by pH titration) determined a reaction enthalpy of 5.0+/-0.6kJmol(-1). In the case of nifedipine, the LEDs in the array were operated individually in order to determine the causative wavelength of degradation. This was found to be 360nm, in agreement with the literature.

Determination of outer layer and bulk dehydration kinetics of trehalose dihydrate using atomic force microscopy, gravimetric vapour sorption and near infrared spectroscopy.

Knowledge of the kinetics of solid state reactions is important when considering the stability of many medicines. Potentially, such reactions could follow different kinetics on the surface of particles when compared with their interior, yet solid state processes are routinely followed using only bulk characterisation techniques. Atomic force microscopy (AFM) has previously been shown to be a suitable technique for the investigation of surface processes, but has not been combined with bulk techniques in order to analyse surface and bulk kinetics separately. This report therefore describes the investigation of the outer layer and bulk kinetics of the dehydration of trehalose dihydrate at ambient temperature and low humidity, using AFM, dynamic vapour sorption (DVS) and near infrared spectroscopy (NIR). The use of AFM enabled the dehydration kinetics of the outer layers to be determined both directly and from bulk data. There were no significant differences between the outer layer dehydration kinetics determined using these methods. AFM also enabled the bulk-only kinetics to be analysed from the DVS and NIR data. These results suggest that the combination of AFM and bulk characterisation techniques should enable a more complete understanding of the kinetics of certain solid state reactions to be achieved.

Application of chemometric analysis to complexity in isothermal calorimetric data.

The interpretation of complexity in isothermal calorimetric data is demanding. The observed power signal is a composite of the powers arising from each of the individual events occurring (which can involve physical, as well as chemical, change). The challenge, therefore, lies in deconvoluting the observed data into their component parts. Here, we discuss the potential use of chemometric analysis, because it offers the significant advantage of being model-free, using principal component analysis to deconvolute data. Using model data, we discovered that the software required a minimum trivariate data matrix to be constructed. Two variables, power and time, were available from the raw data. Selection of a third variable was more problematic, but it was found that by running multiple experiments the small variation in the number of moles of compound in each experiment was sufficient to allow a successful analysis. In general we noted that it required a minimum 2n + 2 repeat experiments to allow analysis (where n is the number of reaction processes). The data outputted from the chemometric software were of the form intensity (arbitrary units) versus time, reflecting the fact that the software was written for analysis of spectroscopic data. We provide a mathematical treatment of the data that allows recovery of both reaction enthalpy and rate constants. The study demonstrates that chemometric analysis is a promising approach for the interpretation of complex calorimetric data.

Development of an isothermal heat-conduction photocalorimeter.

Assessing photostability (particularly of pharmaceuticals) is of growing importance, but hampered by a lack of reliable, rapid experimental testing protocols and instrumentation. In particular, most approaches require irradiation of the sample separately from the analytical measurement, which increases both experimental complexity and the number of assumptions that must be made when calculating stability. One technique that may obviate this is photocalorimetry, principally because the reporter of change (heat) is measured directly as a sample is irradiated. Although not a new idea, the design challenges of photocalorimeters are complex, primarily because light power is being introduced to the calorimeter which can thus both saturate the amplifiers and swamp the response of the sample. Careful instrument design is thus paramount. The aim of this work was to develop a robust, compact, and easy to use photocalorimeter with the immediate focus of developing photostability assays for pharmaceuticals. The final instrument design, arrived at through a series of iterative design modifications, is based on a twin differential heat-conduction principle and achieves an average base line deflection of -0.04+/-0.11 microW with light irradiating the sample cell. The performance capabilities of the instrument were demonstrated using a model system; the photodegradation of 2-nitrobenzaldehyde in solution.

Stability assessment of pharmaceuticals by isothermal calorimetry: two component systems.

Isothermal calorimetry offers the potential to determine rapidly the stability of formulated pharmaceuticals because it is indifferent to physical form and sensitive enough to detect extremely small powers; ca. 50 nW at 25 degrees C. However, its use in this area is not widespread, principally because the power-time data obtained often comprise contributions from more than one process and are thus difficult to analyse quantitatively. In this work, we demonstrate how power-time data recorded for systems in which two components are degrading in parallel (in this case, binary mixtures of selected parabens) can be analysed using a kinetic-based model; the methodology allows the determination of the first-order rate constant and reaction enthalpy for each process, so long as one rate constant is at least twice the magnitude of the other. It was found that the reactions did not need to run to completion in order for the analysis to be successful; a minimum of 15 min of data were required for samples with one degrading component and a minimum of 4 h of data were required for samples with two degrading components. It was observed that the rate constants for paraben degradation in binary systems were significantly lower than expected. This was ascribed to the fact that the parabens degrade to a common product and is an important factor that should be accounted for when the two or more parabens are formulated together.

Antimicrobial properties of silver-containing wound dressings: a microcalorimetric study.

The studies reported here have been undertaken to assess the potential use of isothermal microcalorimetry in studying the antimicrobial efficacy of wound dressings that contain antimicrobial agents. The microcalorimetric technique allows non-invasive and non-destructive analysis to be performed directly on a test sample, regardless of whether it is homogeneous or heterogeneous in nature. Microcalorimetry is an established procedure that offers quantitative measurements and has the distinct advantage over traditional antimicrobial test methodologies in that calorimetric measurements are made continuously over real-time, thus the dynamic response of microorganisms to an antimicrobial agent is observed in situ. The results described in this paper are for interaction of two silver-containing wound care products AQUACEL Ag Hydrofiber (ConvaTec, Deeside, UK) and Acticoat 7 with SILCRYST (Smith and Nephew Healthcare, UK) with the wound pathogenic organisms Staphylococcus aureus and Pseudomonas aeruginosa. Both dressings are shown, microcalorimetrically, to have the capacity to kill these common wound pathogens within 1-2 h of contact. A dose-response study was conducted with the AQUACEL Ag dressing. Microcalorimetry is shown to be rapid, simple and effective in the study of the antimicrobial properties of gel forming wound dressings.

The determination of equilibrium constants, DeltaG, DeltaH and DeltaS for vapour interaction with a pharmaceutical drug, using gravimetric vapour sorption.

The application of gravimetric vapour sorption (GVS) to the characterisation of pharmaceutical drugs is often restricted to the study of gross behaviour such as a measure of hygroscopicity. Although useful in early development of a drug substance, for example, in salt selection screening exercises, such types of analysis may not contribute to a fundamental understanding of the properties of the material. This paper reports a new methodology for GVS experimentation that will allow specific sorption parameters to be calculated; equilibrium constant (K), van't Hoff enthalpy change (DeltaH(v)), Gibbs free energy for sorption (DeltaG) and the entropy change for sorption (DeltaS). Unlike other reports of such type of analysis that require the application of a specific model, this method is model free. The analysis does require that over the narrow temperature range of the study DeltaH(v) is constant and there is no change in interaction mechanism.

A kinetic and thermodynamic study of seratrodast polymorphic transition by isothermal microcalorimetry.

The development of isothermal microcalorimetry to a study of the kinetic and thermodynamics of polymorphic transitions in seratrodast ((+/-)-7-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-7-phenylheptanoic acid) Form II is reported. Sieved samples of Form II were allowed to convert to Form I, in a reaction vessel of an isothermal microcalorimeter, under 13, 31, 63 and 93% relative humidity (RH) between 48 and 65 degrees C. The power (Phi, in Watts) versus time curves from the microcalorimeter were integrated into the heat output (q, in Joules) versus time curves to yield fractional extent of Form I converted versus time curves. The change in enthalpy (-5.70 kJmol(-1)) agreed very closely with that obtained by differential scanning calorimetry and solution calorimetry, which indicated that the power measured by the microcalorimeter was due only to the Form II-to-Form I transition. Application of the theoretical kinetic method [J. Am. Ceram. Soc. 55 (1972) 74] revealed that the transition took place via a two-dimensional growth of nuclei mechanism at all the studied relative humidities and temperatures. The rate constant increased with increasing RH and temperature, and with decreasing the particle size of sample. The activation energies obtained from Arrhenius plots were 292, 290, 280 and 284 kJmol(-1), and the extrapolated rate constants at 25 degrees C were also 3.01 x 10(-10), 3.11 x 10(-10), 9.65 x 10(-10) and 3.84 x 10(-9)s(-1) for 13, 31, 63 and 93% RH, respectively.

Interaçäo de compostos mesoiônicos com Saccharomyces cerevisiae medida pela microcalorimetria biológica / On the interaction of some mesoionic compounds with saccharomyces-cerevisiae by biological microcalorimetry

Resumo: Avaliou-se a sensibilidade de Saccharomyces cerevisiae frente a compostos mesoiônicos dos tipos 1, 3, 4-tiadiazólio-2-aminida e 1, 3, 4-triazólio-2-tiolato através de microcalorimetria bioógica de fluxo comparando-se os resultados com os obtidos através do método de difusäo em ágar.Apesar dos resultados fornecidos pelos dois métodos terem sido compatíveis, há vantagem no uso do métodomicrocalorimétrico por ser mais rápido e preciso, podendo, além disso, distinguir entre diferentes modos de açäo.