Study of the interaction of Saccharomyces cerevisiae with glucose by particle microelectrophoresis.

Saccharomyces cerevisiae NCYC 239 in the presence of glucose at temperatures under 303 K shows a time-dependent lowering of electrophoreric mobility v. At temperatures above 303 K, this time-dependent change in v is in the direction of increased mobilities. Cells suspended in buffer indicate a surface pKa of less than 4, whereas for cells suspended in buffered glucose it is impossible to derive a surface pKa. A kinetic study of the interaction of S. cerevisiae with glucose as a function of temperature allows calculation of an activation energy of 140 kJ X mol-1 for the combined processes of (i) uptake of glucose onto the cell wall, (ii) transfer through the cell wall and membrane, and (iii) the establishment of a steady glucose flux through the wall and membrane.

The effect of chemical modification of Saccharomyces cerevisiae on electrophoretic mobility, cell-wall structure and amphotericin B uptake.

Saccharomyces cerevisiae NCYC 239 suspended in solutions of NaCl showed two distinct plateaus in plots of electrophoretic mobility vs. pH, corresponding to pKa values of approx. 2 and 5. This is in contrast to cells suspended in buffer where only a single pKa (4) can be determined. Modification of cells with KI/I2 or nitrous acid led to altered electrophoretic mobility, indicating the presence of sulphydryl and amino groups, respectively, in the yeast cell surface, whereas uranyl nitrate modification had little effect, suggesting phosphate groups to be absent. Electron micrographs showed visible effects of KI/I2 and nitrous acid modification on cell membrane structure, and in these modified cells amphotericin B uptake was rapid. It is suggested that diffusion through the cell wall is the rate-limiting step for amphotericin B uptake. An activation energy of 20 kJ X mol-1 was determined for uptake of amphotericin B by unmodified cells.

Inter- and intralaboratory variation of in vitro diffusion cell measurements: an international multicenter study using quasi-standardized methods and materials.

In vitro measurements of skin absorption are an increasingly important aspect of regulatory studies, product support claims, and formulation screening. However, such measurements are significantly affected by skin variability. The purpose of this study was to determine inter- and intralaboratory variation in diffusion cell measurements caused by factors other than skin. This was attained through the use of an artificial (silicone rubber) rate-limiting membrane and the provision of materials including a standard penetrant, methyl paraben (MP), and a minimally prescriptive protocol to each of the 18 participating laboratories. "Standardized" calculations of MP flux were determined from the data submitted by each laboratory by applying a predefined mathematical model. This was deemed necessary to eliminate any interlaboratory variation caused by different methods of flux calculations. Average fluxes of MP calculated and reported by each laboratory (60 +/- 27 microg cm(-2) h(-1), n = 25, range 27-101) were in agreement with the standardized calculations of MP flux (60 +/- 21 microg cm(-2) h(-1), range 19-120). The coefficient of variation between laboratories was approximately 35% and was manifest as a fourfold difference between the lowest and highest average flux values and a sixfold difference between the lowest and highest individual flux values. Intralaboratory variation was lower, averaging 10% for five individuals using the same equipment within a single laboratory. Further studies should be performed to clarify the exact components responsible for nonskin-related variability in diffusion cell measurements. It is clear that further developments of in vitro methodologies for measuring skin absorption are required.

Microcalorimetry as an immunological tool: preliminary studies with beef heart mitochondrial adenosine triphosphatase and its antiserum.

The calorimetric technique was applied to a complex immunological system based upon purified beef heart mitochondrial adenosine triphosphatase (ATPase). Whole serum containing antibodies to the ATPase was obtained from a rabbit and used to study the antigen-antiserum interaction in comparison with control serum pooled from 50 non-immune rabbits. The results suggest that the immunological interaction may be accompanied by complex processes which were not detectable by other immunological methods. It was shown that kinetic analysis of the calorimetric data may resolve an apparently complex process of interaction into different component stages of reaction. The advantages of this type of application of calorimetry are discussed.

Oscillations in some linear free energy relationships derived from partition coefficients of phenols between octanol and water.

In the partition of some resorcinol alkyl ethers between water and 1-octanol, the values of delta Gtrs do not increase in a regular way. Odd and even chain alkyl compounds show different, regular increases in delta Gtrs for addition of each methylene group. The unrecognized occurrence of this phenomenon in earlier data is pointed out, and its possible significance in medicinal chemistry is discussed.

QSAR based on biological microcalorimetry.

In this paper we describe a QSAR based on biological microcalorimetry for a set of antimicrobial hydrazides acting against Saccharomyces cerivisiae and Escherichia coli. Results show that an extrathermodynamic relationship exists based upon partitioning (log P(TA)) and microcalorimetrically measured biopotencies using the same cell systems. Moreover, the extrathermodynamic relationship between drug potencies for these two cell systems shows that both cellular systems appear to behave in the same way with respect to the importance of partitioning. This means that the same set of congeneric compounds experience a similar environment in the two systems. This represents a lateral validation of the method and discloses the validity of the QSAR model.

Pharmaceutical microcalorimetry: applications to long-term stability studies.

Calorimetry has been a mainstay of stability analyses for some time in the form of differential scanning microcalorimetry (DSC). This technique exploits high (relatively) temperature studies of pure materials and of formulations to accelerate any degradation or interactions. The behaviour of the material at storage or ambient conditions is then estimated via extrapolation from the Arrhenius equation. Recent developments in isothermal microcalorimetry allow the direct determination of both kinetic and thermodynamic parameters for long, slow reactions from studies conducted at appropriate temperatures and under designated environmental control (pH, pO2, RH etc.). This review introduces the kinetic analysis of microcalorimetric data and, through selected examples, shows applications of the method.

The stability of benzoyl peroxide formulations determined from isothermal microcalorimetric studies.

Recent developments in the analysis of microcalorimetric data output allow the possibility of determining both thermodynamic and kinetic parameters for complex reaction systems. Such experiments routinely take around 50 h, hence qualifying for the description rapid. The methods have earlier been applied to a study of the stability of benzoyl peroxide itself in aqueous suspension. This paper reports the results of isothermal microcalorimetric study of the stability of benzoyl peroxide in the presence of a wide range of excipients and in formulated materials. The results are shown to assist in formulation design, are achieved rapidly and are derived from direct experimental study of the complex systems themselves. That is, no ancillary information is required nor are the studies invasive or destructive.

The stability of benzoyl peroxide by isothermal microcalorimetry.

Isothermal microcalorimetry may be used to determine kinetic and thermodynamic parameters for chemical reactions. This paper reports rate constants, determined as a function of temperature, and the activation enthalpy for the degradation of solid benzoyl peroxide as determined by isothermal microcalorimetry. Studies were conducted on aqueous suspension phase, solid benzoyl peroxide. In addition, supporting evidence is cited from work carried out in this laboratory on the solution phase degradation of benzoyl peroxide using UV-visible spectrophotometry. The activation energy obtained by microcalorimetry was E(a)=137.8+/-6.6 kJ mol(-1) and the activation energy obtained from UV-visible spectrophotometry was E(a)=112.7+/-4.2 kJ mol(-1).

UV-spectrophotometry study of membrane transport processes with a novel diffusion cell.

A novel diffusion cell has been constructed which allows study of membrane diffusion processes without the need for sampling of the receiver compartment, that is highly sensitive and, being based around a diode array spectrophotometer also allows for continuous, real-time recording of multi-species concentration changes in the receiving compartment. The system is controlled to operate isothermally (via a Peltier control system) at temperatures between 15 and 85 degrees C. To examine the performance of this novel design, the transfer of tetracaine from a preparation in PEG 400 (20% tetracaine in PEG 400) has been studied. The results have been used to determine flux, lag time and related parameters. The performance of the novel cell is compared with results from traditional Franz cell diffusion studies.

Combined microassay and determination of bioactivity of N-acetylnystatin.

The application of flow microcalorimetric bioassay for polyene antibiotics to nystatin and its N-acetyl derivative, and its extension to the measurement of their relative bioactivities are reported. The responding organism was Saccharomyces cerevisae. The bioactivity of the N-acetyl derivative is shown to be less than that of the parent antibiotic and to be concentration-dependent. A limited diffusion bioassay study supports these conclusions.

Combined assay and determination of bioactivity of polyene antibiotics by flow nephelometry.

The application of a simple and inexpensive method, flow nephelometry, to the assay and measurement of relative bioactivity of polyene antibiotics is reported. The responding organism was Saccharomyces cerevisiae NCYC 239 in the mid-exponential phase of growth. The relative bioactivity of the polyene antibiotics is shown to be in the order lucensomycin > nystatin > candicidin > amphotericin B > pimaracin. The method is suitable f automation.

Bioassay of nystatin: measurement of Mg2+ efflux by atomic-absorption spectroscopy.

A rapid bioassay for the polyene antibiotic nystatin, based on the leakage of Mg(2+) from sensitive cells of Saccharomyces cerevisiae, is described. The assay employs atomic-absorption spectrophotometry to measure the Mg(2+) leaked. It compares favourably with the classical method of diffusion on an agar-plate, in terms of speed, reproducibility and convenience.

Determination of nickel and selenium by direct injection enthalpimetry.

The construction of a simple thermometric titrator is described. The titrator has been used in a direct injection enthalpimetric procedure to determine nickel and selenium with precisions of 1.0 and 0.3% respectively at a concentration of 10(-2M). The titrant was quinoxaline-2,3-dithiol (disodium salt). The thermometric procedure is shown to be successful in the determination of selenium in contrast to an absorptiometric method using the same reagent.

Application of flow microcalorimetry to analytical problems-I. Determination of organophosphorus pesticides by inhibition of cholinesterase.

Flow microcalorimetry has been applied to the determination of organophosphorus pesticides by inhibition of cholinesterase. One direct inhibitor (TEPP) and one latent inhibitor (parathion) were investigated. The former is determinable at concentrations of about 10(-6)M and the latter at about 10(-4)M. The inhibitory power of parathion is increased if methanol is used as solvent.

Analysis of drugs by microcalorimetry Isothermal power-conduction calorimetry and thermometric titrimetry.

Microcalorimetric analysis has been the subject of a few review's in recent years, but these reviews have mainly dealt with the wide-ranging capabilities of calorimetric assay. This review, however, discusses the experimental basis and practical exploitation of the method in the particularly important area of pharmaceuticals. This field of analysis embraces both conventional chemical assays and bioassays which involve living microbial species. The review highlights the design of calorimetric instruments appropriate for study of microbial metabolism and interaction with drug substances. For comprehensiveness, both microcalorimetric and thermometric assay systems are discussed and critically assessed.

Application of flow-microcalorimetry to analytical problems-II Urea-urease system.

A discussion of the importance of the kinetic parameters, particularly the Michaelis constant K(m), in designing an analytical kinetic technique is given. The magnitude of K(m) is shown to affect only the range of substrate concentrations accessible and not the sensitivity of the method. The limitations and advantages of the microcalorimetric method in the determination of K(m) are outlined. The microcalorimetric technique and the principles discussed were tested by application to the urea-urease system.

Flow microcalorimetric assay of antibiotics--I. Polymyxin B sulphate and its combinations with neomycin sulphate and zinc bacitracin on interaction with Bordetella bronchiseptica (NCTC 8344).

A flow microcalorimetric assay for polymyxin B sulphate has been developed which has a better reproducibility (relative standard deviation less than 3%) and sensitivity (0.35 micrograms ml-1) than conventional microbiological assays, and requires an assay time of ca. 4.5 h. The combinations with zinc bacitracin, with neomycin sulphate, and with both zinc bacitracin and neomycin sulphate indicate antagonism between these antibiotics upon interaction with Bordetella bronchiseptica (NCTC 8344). The combinations of all three antibiotics assayed were: (1) equimolar proportions; and (2) those proportions present in the commercial preparation TrisepR (ICI, Macclesfield, UK).

Flow microcalorimetric assay of antibiotics--II. Neomycin sulphate and its combinations with polymyxin B sulphate and zinc bacitracin on interaction with Bacillus pumilus (NCTC 8241).

A flow microcalorimetric assay for Neomycin has been developed which is monitored through interaction of the antibiotic with Bacillus pumilus as the test organism. The assay has better reproducibility (relative standard deviation 2.3%) and is more sensitive than conventional microbiological bioassay (0.5-2 micrograms ml-1). The effects of combinations with zinc bacitracin, with polymyxin B sulphate, and with both zinc bacitracin and polymyxin B sulphate (both in equimolar proportions), and in those proportions present in the commercial preparation TrisepR (ICI, Macclesfield, UK) have also been investigated. Synergy was observed for the combinations of Neomycin with the other two antibiotics in binary mixtures at the relative proportions found in TrisepR. The addition of all three antibiotics at the levels used in TrisepR did not show synergy. However, addition of all three antibiotics at equimolar concentrations did show synergy. It is suggested that microcalorimetry may be useful in in vitro experiments for exploring the relative proportions required for maximal effect in antibiotic combinations.

Flow microcalorimetric assay of antibiotics--III. Zinc bacitracin and its combinations with polymyxin B sulphate and neomycin sulphate on interaction with Micrococcus luteus.

A flow microcalorimetric assay for zinc bacitracin has been developed which has better reproducibility (relative standard deviation less than 2%) and sensitivity (0.02 micrograms ml-1) than conventional microbiological assays, and requires an assay time of between 7.5-9 h. The assay is not suitable for zinc bacitracin determinations in the presence of equimolar concentrations of polymyxin B sulphate or neomycin sulphate, or of these antibiotics in the proportions in which they occur in the commercial preparation Trisep (ICI, Macclesfield, UK).