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PubMed2002

Analysis of coupled bimolecular reaction kinetics and diffusion by two-color fluorescence correlation spectroscopy: enhanced resolution of kinetics by resonance energy transfer.

In two-color fluorescence correlation spectroscopy (TCFCS), the fluorescence intensities of two fluorescently-labeled species are cross-correlated over time and can be used to identify static and dynamic interactions. Generally, fluorophore labels are chosen that do not undergo Förster resonance energy transfer (FRET). Here, a general TCFCS theory is presented that accounts for the possibility of FRET between reactants in the reversible bimolecular reaction, [reaction: see text] where k(f) and k(b) are forward and reverse rate constants, respectively (dissociation constant K(d) = k(b)/k(f)). Using this theory, we systematically investigated the influence on the correlation function of FRET, reaction rates, reactant concentrations, diffusion, and component visibility. For reactants of comparable size and an energy-transfer efficiency of approximately 90%, experimentally measurable cross-correlation functions should be sensitive to reaction kinetics for K(d) > 10(-8) M and k(f) >or= approximately 10(7) M(-1)s(-1). Measured auto-correlation functions corresponding to donor and acceptor labels are generally less sensitive to reaction kinetics, although for the acceptor, this sensitivity increases as the visibility of the donor increases relative to the acceptor. In the absence of FRET or a significant hydrodynamic difference between reactant species, there is little effect of reaction kinetics on the shape of auto- and cross-correlation functions. Our results suggest that a subset of biologically relevant association-dissociation kinetics can be measured by TCFCS and that FRET can be advantageous in enhancing these effects.

Biophysical Phenomena↗

PubMed2003

Kinetic modelling of Amadori N-(1-deoxy-D-fructos-1-yl)-glycine degradation pathways. Part II--kinetic analysis.

A kinetic model for N-(1-deoxy-D-fructos-1-yl)-glycine (DFG) thermal decomposition was proposed. Two temperatures (100 and 120 degrees C) and two pHs (5.5 and 6.8) were studied. The measured responses were DFG, 3-deoxyosone, 1-deoxyosone, methylglyoxal, acetic acid, formic acid, glucose, fructose, mannose and melanoidins. For each system the model parameters, the rate constants, were estimated by non-linear regression, via multiresponse modelling. The determinant criterion was used as the statistical fit criterion. Model discrimination was performed by both chemical insight and statistical tests (Posterior Probability and Akaike criterion). Kinetic analysis showed that at lower pH DFG 1,2-enolization is favoured whereas with increasing pH 2,3-enolization becomes a more relevant degradation pathway. The lower amount observed of 1-DG is related with its high reactivity. It was shown that acetic acid, a main degradation product from DFG, was mainly formed through 1-DG degradation. Also from the estimated parameters 3-DG was found to be the main precursor in carbohydrate fragments formation, responsible for colour formation. Some indication was given that as the reaction proceeded other compounds besides DFG become reactants themselves with the formation among others of methylglyoxal. The multiresponse kinetic analysis was shown to be both helpful in deriving relevant kinetic parameters as well as in obtaining insight into the reaction mechanism.

Acetic Acid↗

PubMed1983

Kinetics of ouabain outflow from isolated frog heart evaluated by the dose-effect relationship and compared to the outflow kinetics of the tritiated drug.

In isolated frog hearts the kinetics of ouabain disappearance from the receptor biophase(s) related to the effect has been calculated from the relationship between ouabain concentration and its inotropic effect. When 10(-4) - 10(-5) M ouabain is added to the heart, the kinetics is biexponential with a fast component with a time constant of 1.7 min-1 and a slower component with a time constant of 0.28 min-1. The kinetics of the washout curve of 3H ouabain (added together with unlabelled drug, shows, in addition to the slow exponential component of about the same time constant (0.2 min-1) previously seen, another component with a 10 times longer time constant, related to ineffective drug concentrations and corresponding to a washout from aspecific sites. The kinetics of ouabain outflow from compartments which are related to the effect elicited by 10(-4) - 10(-5) M concentrations is formed mainly by two steps with time constants of 1.7 min-1 and 0.28 min-1.

Animals↗

PubMed1991

Kinetic and thermodynamic properties of wild-type and engineered mutants of tyrosyl-tRNA synthetase analyzed by pyrophosphate-exchange kinetics.

The first step of the reaction catalyzed by the aminoacyl-tRNA synthetases is the formation of enzyme-bound aminoacyl adenylate. The steady-state kinetics of this step has conventionally been studied by measuring the rate of isotopic exchange between pyrophosphate and ATP. A simple kinetic analysis of the pyrophosphate-exchange reaction catalyzed by the tyrosyl-tRNA synthetase from Bacillus stearothermophilus is given in which all the observed rate and binding constants can be assigned to identifiable physical processes under a variety of limiting conditions. The free energies of binding to the enzyme of tyrosine, ATP, and the transition state for tyrosyl adenylate formation can be measured in relatively straightforward experiments. The excellent agreement between parameters measured in these experiments and those from earlier pre-steady-state kinetics confirms that the intermediates isolated in the presteady state are kinetically competent. The dissociation constant of ATP from the unligated enzyme, a constant that has previously been experimentally inaccessible, has been measured for wild-type and several mutant enzymes. The changes in enthalpy and entropy of activation on mutation have been measured by a rapid procedure for mutants that have altered contacts with tyrosine and ATP. Those mutants that have large changes of enthalpy and entropy of binding are likely to have structural changes and so warrant further examination by protein crystallography.

Adenosine Triphosphate↗

PubMed1988

Alternative substrate and inhibition kinetics of aminoglycoside nucleotidyltransferase 2''-I in support of a Theorell-Chance kinetic mechanism.

Aminoglycoside nucleotidyltransferase 2''-I conveys multiple antibiotic resistance to Gram-negative bacteria because the enzyme adenylylates a broad range of aminoglycoside antibiotics as substrates [Gates, C. A., & Northrop, D. B. (1988) Biochemistry (preceding paper in this issue)]. The enzyme also catalyzes the transfer of a variety of nucleotides [Van Pelt, J. E., & Northrop, D. B. (1984) Arch. Biochem. Biophys. 230, 250-263]. This doubly broad substrate specificity makes it an excellent candidate for application of the alternative substrate diagnostic [Radika, K., & Northrop, D. B. (1984) Anal. Biochem. 141, 413-417] as a means to determine its kinetic mechanism. The kinetic patterns presented here are composed of one set of intersecting lines and one coincident line and are consistent with a Theorell-Chance kinetic mechanism in which nucleotide binding precedes aminoglycosides, pyrophosphate is released prior to the nucleotidylated aminoglycoside (Q), and turnover is controlled by the rate-limiting release of the final product. Substrate inhibition by tobramycin (B) is partial and uncompetitive versus Mg-ATP, indicating that B binds to the EQ complex, but not in the usual dead-end fashion common to an ordered sequential release of products; instead, Q may escape from the abortive EQB complex at a finite rate. Dead-end inhibition by neomycin C (I) is also partial and uncompetitive versus Mg-ATP but is slope-linear, intercept-hyperbolic, partial noncompetitive versus gentamicin A; both kinetic patterns signify the formation of a partial abortive EQI complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoglycosides↗

PubMed1999

EPR kinetic studies of oxygen release in thylakoids and PSII membranes: a kinetic intermediate in the S3 to S0 transition.

Time-resolved EPR oximetry has been used to determine the oxygen release kinetics in spinach thylakoids and PSII membranes. We observe release kinetics with half-times of approximately 0.85 and approximately 1.45 ms for thylakoids and PSII membranes, respectively, which are in close agreement with the EPR determined Yz decay kinetics for the S3 --> --> S0 transition in these systems. The results show conclusively that water-oxygen chemistry is not a rate-limiting step in the donor side of PSII under normal turnover conditions. By analyzing the oxygen release kinetics in thylakoids under nonphysiological, but still functionally competent conditions (low pH or high salt), we observed an initial delay in the O2 release of up to 200 microseconds following flash turnover from the S3 state. This is the first direct indication of a probable quasi-stable intermediate in the S3 --> --> S0 turnover of PSII, possibly representing the putative S4 state. Under conditions more closely approaching physiological, no such delay was resolved, indicating that the S4 --> O2 transition occurs within 50 microseconds under such circumstances. Two possible reaction sequences for O2 formation consistent with these and other data are discussed. It is suggested that the more probable form of "S4" is in fact the S3 + Yz* combination, which must undergo some molecular rearrangement on the tens to hundreds of microseconds time scale before O2 formation chemistry occurs.

Electron Spin Resonance Spectroscopy↗

PubMed2005

Intramolecular N-H...S hydrogen bonding in the zinc thiolate complex [Tm(Ph)]ZnSCH2C(O)NHPh: a mechanistic investigation of thiolate alkylation as probed by kinetics studies and by kinetic isotope effects.

The zinc thiolate complex [Tm(Ph)]ZnSCH2C(O)N(H)Ph, which features a tetrahedral [ZnS4] motif analogous to that of the Ada DNA repair protein, may be obtained by the reaction of Zn(NO3)2 with [Tm(Ph)]Li and Li[SCH2C(O)N(H)Ph] ([Tm(Ph)] = tris(2-mercapto-1-phenylimidazolyl)hydroborato ligand). Structural characterization of [Tm(Ph)]ZnSCH2C(O)N(H)Ph by X-ray diffraction demonstrates that the molecule exhibits an intramolecular N-H...S hydrogen bond between the amide N-H group and thiolate sulfur atom, a structure that is reproduced by density functional theory (DFT) calculations. The thiolate ligand of [Tm(Ph)]ZnSCH2C(O)N(H)Ph is subject to alkylation, a reaction that is analogous to the function of the Ada DNA repair protein. Specifically, [Tm(Ph)]ZnSCH2C(O)N(H)Ph reacts with MeI to yield PhN(H)C(O)CH2SMe and [Tm(Ph)]ZnI, a reaction which is characterized by second-order kinetics that is consistent with either (i) an associative mechanism or (ii) a stepwise dissociative mechanism in which the alkylation step is rate determining. Although the kinetics studies are incapable of distinguishing between these possibilities, a small normal kinetic isotope effect of kH/kD = 1.16(1) at 0 degrees C for the reaction of [Tm(Ph)]ZnSCH2C(O)N(H*)Ph (H* = H, D) with MeI is suggestive of a dissociative mechanism on the basis of DFT calculations. In particular, DFT calculations demonstrate that a normal kinetic isotope effect requires thiolate dissociation because it results in the formation of [PhN(H)C(O)CH2S]- which, as an anion, exhibits a stronger N-H...S hydrogen bonding interaction than that in [Tm(Ph)]ZnSCH2C(O)N(H)Ph. Correspondingly, mechanisms that involve direct alkylation of coordinated thiolate are predicted to be characterized by kH/kD < or = 1 because the reaction involves a reduction of the negative charge on sulfur and hence a weakening of the N-H...S hydrogen bonding interaction.

Alkylation↗

PubMed2005

Kinetic capillary electrophoresis (KCE): a conceptual platform for kinetic homogeneous affinity methods.

We propose kinetic capillary electrophoresis (KCE) as a conceptual platform for the development of kinetic homogeneous affinity methods. KCE is defined as the CE separation of species that interact during electrophoresis. Depending on how the interaction is arranged, different KCE methods can be designed. All KCE methods are described by the same mathematics: the same system of partial differential equations with only initial and boundary conditions being different. Every qualitatively unique set of initial and boundary conditions defines a unique KCE method. Here, we (i) present the theoretical bases of KCE, (ii) define four new KCE methods, and (iii) propose a multimethod KCE toolbox as an integrated kinetic technique. Using the KCE toolbox, we were able to, for the first time, observe high-affinity (specific) and low-affinity (nonspecific) interactions within the same protein-ligand pair. The concept of KCE allows for the creation of an expanding toolset of powerful kinetic homogeneous affinity methods, which will find their applications in studies of biomolecular interactions, quantitative analyses, and selecting affinity probes and drug candidates from complex mixtures.

DNA, Single-Stranded↗

PubMed2005

Kinetic justification of the solubility product: application of a general kinetic dissolution model.

Using a simple, feldspar-like model and the crystal-based reaction mechanism for water-rock kinetics being developed before, we show directly how the dissolution of euhedral faces of crystals are governed by the nonlinear quantity represented by the solubility product. The kinetic approach requires recognition of the essential role played by the correlation of the dynamics of neighboring sites in a crystal, the statistical dynamics of steps, the coupling of the various kink sites on the surface by the crystal structure, and the inclusion of bond formation as well as bond rupture into the kinetic reaction mechanism. The same kinetic approach, which accounts for the role of the solubility product (or DeltaG) in the overall rate, is then shown also to explain the observed inhibition behavior in feldspars as well as the often-written phenomenological rate law, involving a product of a pH term, an activation energy term, and a DeltaG term.

Aluminum Silicates↗

PubMed1989

Kinetic and chemometric studies of the determination of creatinine using the Jaffé reaction. Part I. Kinetics of the reaction: analytical conclusions.

A kinetic-spectrophotometric study of the Jaffé reaction was carried out and the kinetic behaviour, calibration step and interfering effect of albumin on creatinine standard solutions were studied. It was concluded that there is a variation in the kinetic behaviour of the system when higher concentrations of creatinine, picrate or sodium hydroxide are tested. The experimental conditions for quantifying creatinine must be chosen so that the kinetic behaviour is the same in the dynamic concentration range. Changes in the absorbance (delta A) versus concentration equations were chosen as the most suitable for calibration graphs. It was also shown that creatinine results will have a proportional bias error if the interfering effect of albumin is not taken into consideration.

Creatinine↗

PubMed1973

Normalization in the fitting of data by iterative methods. Application to tracer kinetics and enzyme kinetics.

1. The normalization of biochemical data to weight them appropriately for parameter estimation is considered, with reference particularly to data from tracer kinetics and enzyme kinetics. If the data are in replicate, it is recommended that the sum of squared deviations for each experimental variable at each time or concentration point is divided by the local variance at that point. 2. If there is only one observation for each variable at each sampling point, normalization may still be required if the observations cover more than one order of magnitude, but there is no absolute criterion for judging the effect of the weighting that is produced. The goodness of fit that is produced by minimizing the weighted sum of squares of deviations must be judged subjectively. It is suggested that the goodness of fit may be regarded as satisfactory if the data points are distributed uniformly on either side of the fitted curve. A chi-square test may be used to decide whether the distribution is abnormal. The proportion of the residual variance associated with points on one or other side of the fitted curve may also be taken into account, because this gives an indication of the sensitivity of the residual variance to movement of the curve away from particular data points. These criteria for judging the effect of weighting are only valid if the model equation may reasonably be expected to apply to all the data points. 3. On this basis, normalizing by dividing the deviation for each data point by the experimental observation or by the equivalent value calculated by the model equation may both be shown to produce a consistent bias for numerically small observations, the former biasing the curve towards the smallest observations, the latter tending to produce a curve that is above the numerically smaller data points. It was found that dividing each deviation by the mean of observed and calculated variable appropriate to it produces a weighting that is fairly free from bias as judged by the criteria mentioned above. This normalization factor was tested on published data from both tracer kinetics and enzyme kinetics.

Data Display↗

PubMed1994

Experimental approach to the kinetic study of unstable site-directed irreversible inhibitors: kinetic origin of the apparent positive co-operativity arising from inactivation of trypsin by p-amidinophenylmethanesulphonyl fluoride.

Experimental characterization of enzyme inactivation by unstable irreversible inhibitors has only previously been carried out by using discontinuous methods involving preincubation, removal of samples and further residual activity assays. A continuous method for the kinetic study of these inhibitors in the presence of an auxiliary substrate was recently proposed in a theoretical study. This method was based on approximate expressions for the evolution of the product concentration, which contained series expansions with five or more exponential terms, seriously complicating their use in practice. In the present paper, a new experimental method has been developed for the kinetic study of unstable and site-directed irreversible inhibitors, considering two different ranges of inhibitor concentration. Thus at low inhibitor concentrations, the system evolves from an initial to a final steady state, the rates of which are described by exact analytical equations. At high inhibitor concentrations, however, the product accumulation can be described by an exact uniexponential equation. This simple and efficient method has been applied to the kinetic study of trypsin inactivation by p-amidinophenylmethanesulphonyl fluoride, near the optimum pH of the enzyme. The dependence of the final steady-state rate on the substrate concentration shows apparent positive co-operativity which has not previously been reported. The kinetic origin of this type of co-operativity is predicted by one of the exact analytical equations derived here. The instability of new protein and non-protein irreversible inhibitors has to be carefully characterized to prevent true unstable irreversible inhibitors being wrongly described as allosteric reversible inhibitors.

Animals↗

PubMed1978

The steady-state kinetics of yeast phosphoglycerate kinase. Anomalous kinetic plots and the effects of salts on activity.

1. A re-investigation of the kinetics of yeast phosphoglycerate kinase in the direction of 1,3-bisphosphoglycerate formation has been carried out, covering a 1000-fold range in substrate concentrations. A variety of improved spectrophotometric and fluorimetric assay procedures have been used. 2. Kinetic plots proved to be non-linear for each variable substrate. A variety of checks have been carried out to show that this is not due to artifacts in the assay procedures or heterogeneity of the enzyme preparation. 3. The effects of a variety of salts on the activity of the enzyme have been examined. Most salts, especially those with multivalent anions, can cause activation of the enzyme, but inhibit at high concentration. 4. The salt effect is shown to be principally due to anions rather than cations, and not to ionic strength changes. Sulphate, as one of the most effective anions has been used in most comparisons. 5. Salt activation is steepest when the substrate concentrations are low; maximum activation has been about 5-fold with 0.2 mM MgATP and 0.2 mM 3-phosphoglycerate. Inhibition at the higher salt concentrations is strongest at the same substrate concentrations as when activation is steepest, indicating a link between the two effects. 6. The presence of 20 mM or more Na2SO4 converted non-linear kinetic plots to linear ones. A study of the kinetics in the presence of 40 mM Na2SO4 was interpreted in terms of a random sequential binding mechanism, with sulphate acting as a competitive inhibitor. 7. Possible explanations for these anomalous results are discussed in terms of several mechanisms which have been shown to apply in other systems.

Anions↗

PubMed1982

Polymerization/excision kinetics of Escherichia coli DNA polymerase I. Stability in kinetic behaviour and variations of the rate constants with temperature and pH.

1. The incorporation and excision kinetics of Escherichia coli DNA polymerase I in the replication of (dA)n with dUTP and (dI)n with dCTP was studied at various temperatures and pH. When the incorporation/excision ratio (dy/dx) is plotted against the concentration of deoxynucleoside triphosphate [S] two kinds of curves are obtained. With (dI)n, dy/dx increases with [S], then reaches an asymptotic value. This behaviour, consistent with a kinetic amplification or kinetic proofreading mechanism, is observed at all temperatures and pH. With (dA)n, dy/dx increases with [S] but in a convex, instead of a concave manner. In this case, we approximated the curves by straight lines at the origin, in conformity with the prediction of the frayed-unfrayed model. Again a single behaviour is consistently observed at all temperatures and pH. 2. The data were analyzed in terms of ratios or products of three kinetic constants: ki for incorporation, ke for excision and, in the (dI)n system, theta for the processing of the incoming dNTP. The dNMP production in the (dI)n system is dominated by the ke theta term which increases with temperature and pH. Temperature influences excision more than incorporation, the net result being a linear decrease of ki/ke with temperature. The effect is more pronounced with (dI)n than with (dA)n and is probably related to the stability of the template-primer complex. The ki/ke term shows a bell-shaped dependency with pH in the (dI)n system. With (dA)n it remains constant between pH 7.5 and 8.5 then decreases with a transition midpoint at pH 9.0. We suggest that the pH profiles may reflect the ionization of the template in the first case, and of the substrate in the second.

Bacterial Proteins↗

PubMed1985

Kinetic co-operativity of monomeric mnemonical enzymes. The significance of the kinetic Hill coefficient.

The expression of the kinetic Hill coefficient for a two-substrate, two-product mnemonical enzyme has been derived. Its relation with the gamma coefficient, that is the slope of the reciprocal plots for 1/[A]----O, has been established. The variation of this Hill coefficient, as a function of the second substrate and product concentrations, has been studied theoretically. Whereas the gamma coefficient does not vary as a function of the substrate and first product concentrations, the kinetic Hill coefficient does. If the enzyme is positively co-operative, the Hill coefficient increases upon increasing the second substrate concentration and decreases if the first product concentration is increased. The converse is expected to occur if the enzyme displays a negative co-operativity. The last product may either reverse a positive co-operativity into a negative one or, alternatively, strengthen an already negative co-operativity. The co-operativity generated by the mnemonical model has been compared to the kinetic behaviour of a random model. These two models have been shown to be discriminated on the basis of the departure they show with respect to the Michaelis-Menten behaviour. These theoretical considerations have been applied to previously published data, obtained with wheat germ hexokinase LI. This monomeric enzyme has a negative co-operativity with respect to the preferred substrate, glucose. The Hill coefficient decreases with MgATP concentration, increases with MgADP concentration and decreases with glucose-6-phosphate concentration. This is exactly what is to be expected on the basis of the above theory of kinetic co-operativity.

Allosteric Site↗

PubMed2000

Analysis of urea nitrogen and creatinine kinetics in hemodialysis: comparison of a variable-volume two-compartment model with a regional blood flow model and investigation of an appropriate solute kinetics model for clinical application.

To investigate an appropriate solute kinetics model for clinical application, we analyzed urea nitrogen (UN) and creatinine (Cr) kinetics by a variable-volume two-compartmental model (2CM) and a regional blood flow model (RBF) in 44 hemodialysis patients with varying proportions of first compartmental volume and regional volume (p(1)). Solute kinetics could not be solved in some of the patients with higher p(1) values, and there were more solution failures by the RBF than by the 2CM. The solute generation rate (g) and solute distribution volume in the dry state (V(D)) increased with increases in p(1) in both models, but there were some differences between the two models. When g was normalized by V(D), it became relatively constant, irrespective of the p(1) value or model used (0.133 +/- 0.029 mg/min/l by the 2CM and 0.132 +/- 0.029 mg/min/l by the RBF for UN; 0.0200 +/- 0.0049 mg/min/l by the 2CM and 0.0198 +/- 0.0048 mg/min/l by the RBF for Cr). The intercompartmental mass transfer coefficient (K(c); liters/min) calculated by the 2CM decreased as p(1) increased (K(c) = -1.77.p(1) + 1.16, p < 0.0001, R = 0.999 for UN; K(c) = -0.847.p(1) + 0.556, p < 0.0001, R = 1.000 for Cr). The systemic blood flow (Q(sys); liters/min) calculated by the RBF also decreased as p(1) increased (Q(sys) = -11.1.p(1) + 6.21, p < 0.0005, R = 1.000 for UN; Q(sys) = -5.22.p(1) + 2.90, p < 0.001, R = 0.999 for Cr). Since the RBF more frequently failed to solve the solute kinetics and since there was a difference in its Q(sys) values for UN and Cr, the 2CM was considered to be a superior model. When p(1) was extremely low, the 2CM could be transformed into a modified variable-volume one-compartment model (1CM) which presented a similar g/V(D) (0.133 +/- 0.029 for UN; 0.0200 +/- 0.0048 for Cr). This modified 1CM was considered to satisfy appropriate conditions for clinical application, since it is simpler than the 2CM and provides useful information on the dialysis dose.

Aged↗

PubMed2006

Multivariate curve resolution-alternating least squares and kinetic modeling applied to near-infrared data from curing reactions of epoxy resins: mechanistic approach and estimation of kinetic rate constants.

This study describes the combination of multivariate curve resolution-alternating least squares with a kinetic modeling strategy for obtaining the kinetic rate constants of a curing reaction of epoxy resins. The reaction between phenyl glycidyl ether and aniline is monitored by near-infrared spectroscopy under isothermal conditions for several initial molar ratios of the reagents. The data for all experiments, arranged in a column-wise augmented data matrix, are analyzed using multivariate curve resolution-alternating least squares. The concentration profiles recovered are fitted to a chemical model proposed for the reaction. The selection of the kinetic model is assisted by the information contained in the recovered concentration profiles. The nonlinear fitting provides the kinetic rate constants. The optimized rate constants are in agreement with values reported in the literature.

Algorithms↗

PubMed1976

Computer simulation of leukemia therapy: combined pharmacokinetics, intracellular enzyme kinetics, and cell kinetics of the treatment of L1210 leukemia by cytosine arabinoside.

An integrated mathematic computer-based model of the pharmacokinetics, intracellular enzyme kinetics, and cell kinetics of the treatment of L1210 leukemia by cytosine arabinoside (ara-C) is described. The compartment model of Bischoff and Dedrick is extended to the intracellular level by inclusion of equations describing the phosphorylation, dephosphorylation, and deamination of ara-C with enzymatic feedback control. The activities of kinase, deaminase, and phosphatase are explicitly included in the models and are estimated from relevant data. Cell proliferation is described by a continuous-flow mathematic model in which cellular maturation and cell-to-cell variability in maturation rates are key variables. Cell proliferation is related to intracellular biochemistry through mathematic expressions which relate cell lethality and progression delay to the time course of intracellular ara-CTP. In vitro and in vivo experiments performed in a number of laboratories are compared by simulation. The most sensitive parameters in dose-response and cell-survival simulations are deoxycytidine kinase activity, ara-CTP half-life, renal clearance of ara-C, and cell-kinetic parameters for proliferation and cell killing. Progression delay is vital to the realistic simulation of divided-dose schedules. By comparative simulation we have identified areas of uncertainty which can be classified by a few additional measurements. The applications of simulations combining pharmacokinetic, biochemical, and cell-kinetic data in vitro and in vivo are discussed, exploring consistency among different measurements, and relating experimental protocols to clinical treatment.

Animals↗