Kinetic analysis of P-glycoprotein-mediated doxorubicin efflux.

P-glycoprotein lowers the intracellular concentration of a variety of unrelated compounds including doxorubicin by actively removing them from the cell. Mathematical modeling techniques have been applied to the transmembrane transport of doxorubicin in a nonresistant HL-60 cell line and in a P-glycoprotein-containing resistant subclone, HL-60R, to obtain clearances for inward and outward transport. Distribution of [14C]doxorubicin from extracellular fluid into the cell pellet was analyzed with a closed two-compartment system that fitted experimental measurements of drug concentrations in the extracellular fluid and in the cell pellet, represented by a third compartment that includes trapped extracellular doxorubicin along with the intact cells. Transmembrane diffusion clearance was similar for the two cell lines (1.00 vs. 1.06 microliters sec-1), yielding an apparent doxorubicin permeability coefficient of 7.4 x 10(-5) cm sec-1. However, the total efflux clearance averaged 0.99 +/- 0.05 microliters sec-1 in HL-60 and 2.29 +/- 0.62 microliters sec-1 in HL-60R. The estimated P-glycoprotein-mediated efflux clearance in HL-60R averaged 1.23 +/- 0.51 microliters sec-1. This experimental approach provides direct estimates of transport parameters in intact cells and should be useful in studying the mechanism of action and interactions of inhibitors of P-glycoprotein.     

Kinetic analysis of lead metabolism in healthy humans.

The steady state kinetics of lead metabolism were studied in five healthy men with stable isotope tracers. Subjects lived in a metabolic unit and ate constant low lead diets. Their intake was supplemented each day with 79--204 mug of enriched lead-204 as nitrate which was ingested with meals for 1--124 days. The concentration and isotopic composition of lead was determined serially in blood, urine, feces, and diet and less commonly in hair, nails, sweat, bone, and alimentary tract secretions by isotopic dilution, mass spectrometric analysis. The data suggest a three compartmental model for lead metabolism. The first compartment encompasses blood and is 1.5--2.2 times larger than the blood mass. It contains approximately 1.7--2.0 mg of lead and has a mean life of 35 days. This pool is in direct communication with ingested lead, urinary lead, and pools two and three. The second compartment is largely composed of soft tissue, contains about 0.3--0.9 mg of lead, and has a mean life of approximately 40 days. This pool gives rise to lead in hair, nails, sweat, and salivary, gastric, pancreatic, and biliary secretions. Pool three resides primarily in the skeleton, contains the vast quantity of body lead, and has a very slow mean life. Bones appear to differ in their rates of lead turnover. Within the relatively small changes in blood lead observed in the present study, the transfer coefficients between the pools remained constant.     

Norepinephrine metabolism in humans. Kinetic analysis and model.

The present study was undertaken to quantify more precisely and to begin to address the problem of heterogeneity of the kinetics of distribution and metabolism of norepinephrine (NE) in humans, by using compartmental analysis. Steady-state NE specific activity in arterialized plasma during [3H]NE infusion and postinfusion plasma disappearance of [3H]NE were measured in eight healthy subjects in the supine and upright positions. Two exponentials were clearly identified in the plasma [3H]NE disappearance curves of each subject studied in the supine (r = 0.94-1.00, all P less than 0.01) and upright (r = 0.90-0.98, all P less than 0.01) positions. A two-compartment model was the minimal model necessary to simultaneously describe the kinetics of NE in the supine and upright positions. The NE input rate into the extravascular compartment 2, estimated with the minimal model, increased with upright posture (1.87 +/- 0.08 vs. 3.25 +/- 0.2 micrograms/min per m2, P less than 0.001). Upright posture was associated with a fall in the volume of distribution of NE in compartment 1 (7.5 +/- 0.6 vs. 4.7 +/- 0.3 liters, P less than 0.001), and as a result of that, there was a fall in the metabolic clearance rate of NE from compartment 1 (1.80 +/- 0.11 vs. 1.21 +/- 0.08 liters/min per m2, P less than 0.001). We conclude that a two-compartment model is the minimal model that can accurately describe the kinetics of distribution and metabolism of NE in humans.     

Kinetic analysis of impaired work-cost performance in jaundiced rabbit liver

Impairment of energy metabolism was studied in jaundiced rabbit liver by kinetic analysis of energy transfer function. Free cytosolic ADP (ADP_f), as calculated from the measured components of the glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase/lactate dehydrogenase reactions, decreased from the control value of 48.1 to 37.0 μM at 24 h after bile duct ligation. The maximal velocity (V_max) of ATP synthesis, as measured by state 3 respiration of isolated mitochondria, decreased from the control value of 62.1 to 38.3 nmol ATP synthesized per min per mg mitochondrial protein, while the Michaelis constant for ADP (K _m) decreased from the control value of 19.2 to 12.8 μM. ATP synthesis velocity in vivo {v:V_max/[1+(K _m/[ADP_f])]}, as calculated by V_max,K _m and ADP_f, decreased from the control value of 44.4 to 28.5 nmol ATP synthesized per min per mg mitochondrial protein. Δv/ΔADP_f (Δv/ΔADP_f: V_max·K _m/(K _m+[ADP_f])²), which indicates work-cost performance of the liver, decreased from the control value of 0.263 to 0.198. Biochemical output of the liver, as measured by hippurate synthesis from benzoate, decreased from the control value of 98.4 to 32.7 mg/h. These results indicate that synergistic decreases in ADP_f, V_max, v and Δv/ΔADP_f take place in the course of deterioration of mitochondrial ATP synthesis and work output in jaundiced liver     

Kinetic analysis of cloning patterns by human marrow cells in leukemia

In leukemia and preleukemic disorders the progeny of a single cell proliferate and ultimately come to occupy the hemopoietic system. In the process normal stem cells are suppressed and in time may become extinct. This implies that neoplastic clones have a biological advantage. In this paper evidence is presented that the cloning of granulocytic colony forming cells in the clonal hemopathies is influenced by cell products that regulate cloning of normal colony forming cells. We have attempted to develop an approach to the study of clone-clone interactions in order to determine at what level(s) the battle between clones is fought. Future studies on relative responsiveness might help in understanding the mechanisms by which normal hemopoiesis is suppressed during the evolution of leukemia and re-established during remission induction.     

Kinetic analysis of biliary lipid excretion in man and dog.

To understand better the mechanisms involved in biliary lipid excretion and to evaluate their role in cholesterol gallstone formation, the rates of biliary excretion of bile salts, cholesterol, and phospholipids were measured in two species, man and dog. Seven cholecystectomized patients with balloon-occludable reinfusion T-tubes were studied during intact and interrupted enterohepatic circulation and four cholecystectomized dogs were studied during interrupted enterohepatic circulation. In man and dog both cholesterol and phospholipid outputs were hyperbolically related to bile salt output by the equation y = x/(a + bx). The output curves intersected the origin and showed an initial rapid rise, followed by a slower increase to a maximum, suggesting a rate-limited mechanism. The shape of the curves permitted calculation of the theoretical maximal outputs and the rates of rise to those outputs. Comparison of these values showed that in both man and dog phospholipid output was greater than cholesterol output and that cholesterol and phospholipid were excreted at different rates. These studies (a) indicate that cholesterol, phospholipids, and bile salts are not excreted in a fixed relationship and (b) demonstrate the usefulness of the derived theoretical maximal lipid output, and the rate of rise of lipid excretion to a maximum, in evaluating the kinetics of biliary lipid excretion.     

Kinetic and physiological analysis of the GAME(Wheels) system

For individuals with a spinal cord injury or dysfunction (SCI/D), opportunities to exercise are limited and are usually not highly motivating experiences. Exercise programs or extracurricular activities may help increase or maintain the cardiovascular fitness level of individuals with SCI/D. The GAME(Wheels) system, an interface between a portable roller system and a computer, enables an individual to control a video game by propelling his or her wheelchair. The purpose of this study was to investigate whether the propulsive forces used during video play, both with and without the GAME(Wheels) system, were different. A secondary purpose was to examine differences in metabolic parameters during exercise under these two conditions. Ten manual wheelchair users exercised on the GAME(Wheels) system with and without controlling a video game. Physiological and kinetic data were collected six times during two exercise trials. Kinetic data were recorded with the SMART(Wheel) and used to investigate propulsion forces. No significant differences were found in the resultant force, rate of rise, or number of hand contacts with the pushrims. This study showed that propulsion pattern did not change significantly when wheelchair users exercised while playing a computer video game. Oxygen consumption, ventilation, and heart rate were significantly different (p < 0.05) between the two groups during the last three exercise intervals and cooldown. Playing a video game while exercising may help to motivate manual wheelchair users to exercise longer and regularly, something that was reported by this study's subjects; likewise, exercising while playing a video game may not be associated with higher pushrim forces and stroke frequencies.     

Kinetic analysis of hyaluronidase activity using a bioactive MRI contrast agent

One of the attractions of molecular imaging using ‘smart’ bioactive contrast agents is the ability to provide non‐invasive data on the spatial and temporal changes in the distribution and expression patterns of specific enzymes. The tools developed for that aim could potentially also be developed for functional imaging of enzyme activity itself, through quantitative analysis of the rapid dynamics of enzymatic conversion of these contrast agents. High molecular weight hyaluronan, the natural substrate of hyaluronidase, is a major antiangiogenic constituent of the extracellular matrix. Degradation by hyaluronidase yields low molecular weight fragments, which are proangiogenic. A novel contrast material, HA‐GdDTPA‐beads, was designed to provide a substrate analog of hyaluronidase in which relaxivity changes are induced by enzymatic degradation. We show here a first‐order kinetic analysis of the time‐dependent increase in R₂ as a result of hyaluronidase activity. The changes in R₂ and the measured relaxivity of intact HA‐GdDTPA‐beads (r_2B) and HA‐GdDTPA fragments (r_2D) were utilized for derivation of the temporal drop in concentration of GdDTPA in HA‐GdDTPA‐beads as the consequence of the release of HA‐GdDTPA fragments. The rate of dissociation of HA‐GdDTPA from the beads showed typical bell‐shaped temperature dependence between 7 and 36 °C with peak activity at 25 °C. The tools developed here for quantitative dynamic analysis of hyaluronidase activity by MRI would allow the use of activation of HA‐GdDTPA‐beads for the determination of the role of hyaluronidase in altering the angiogenic microenvironment of tumor micro metastases. Copyright © 2006 John Wiley & Sons, Ltd.     

Kinetic analysis of mechanisms regulating granulopoiesis in cultured human bone marrow

In the past, in vitro perturbations of granulocytic progenitor cells have been measured by comparing the number of colonies that have grown to a threshold size in a given time. In our hands this method has proven to be both insensitive and unreliable. In this paper kinetic evidence is presented that the number of colonies, however defined, is subject to the influence of unpredictable independent variables which include the age distribution of the clonogenic cells, the proportion of them which are dormant, and the factors which promote their recruitment, their expansion into colonies and the rate at which colonies die. Results are improved by an early analysis of total clonal number (TC) as a measure of recruitment and mean clonal size (MCS) as a measure of clonal expansion rate.     

Kinetic analysis of Enterococcus faecium L,D-transpeptidase inactivation by carbapenems

Bypass of classical penicillin-binding proteins by the L,D-transpeptidase of Enterococcus faecium (Ldt(fm)) leads to high-level ampicillin resistance in E. faecium mutants, whereas carbapenems remain the lone highly active β-lactams. Kinetics of Ldt(fm) inactivation was determined for four commercial carbapenems and a derivative obtained by introducing a minimal ethyl group at position 2. We show that the bulky side chains of commercial carbapenems have both positive and negative effects in preventing hydrolysis of the acyl enzyme and impairing drug binding.     

Kinetic analysis of the interactions of agonists and antagonists with beta adrenergic receptors

The affinity of the beta adrenergic receptor for antagonists is frequently higher than that for agonists. It has been assumed that the binding of agonists and antagonists is diffusion limited and that the high affinity of the receptor for typical antagonists is due to slow rates of dissociation. To test this hypothesis, the kinetics of binding of unlabeled agonists and antagonists were determined using the method described by Motulsky and Mahan (Mol. Pharmacol. 25: 1-9, 1984). The time course of the binding of a radioligand in the presence of a competing unlabeled ligand was analyzed in terms of rate constants of association (kon) and dissociation (koff) for binding of the radioligand and the competitor. This approach was validated by showing that the rate constants for binding of [3H]dihydroalprenolol and [3H]CGP-12177 [[3H]-4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2-on ] determined directly were similar to values determined when the binding of [125I]iodopindolol was measured in the presence of [3H]dihydroalprenolol or [3H]CGP-12177. Computer simulations suggested that this method was experimentally limited to competing ligands with rate constants of dissociation below approximately 0.50 min-1. The apparent rate constants for binding of four unlabeled agonists and eight antagonists were determined experimentally at 10 degrees C. Although the values of koff for agonists and antagonists were similar, the values for kon for binding of agonists were consistently lower than the values for binding of antagonists. The relatively slow rate constant for association of agonists may be explained by a two-step mechanism or may involve agonist-induced isomerization of the receptor.     

Kinetic analysis of Ia expression on T cells in patients with systemic lupus erythematosus

An increased proportion of Ia positive circulating T cells was observed in patients with systemic lupus erythematosus (SLE) (17.2 +/- 5.8%, n = 20, p less than 0.005), compared with normal subjects (3.2 +/- 0.9%, n = 10) by the analysis with flow cytometry using OKIa1. There was a significant difference (p less than 0.05) in the proportion of Ia positive T cells (Ia +T cells) between inactive patients (19.6 +/- 5.8%, n = 12) and active patients (13.6 +/- 3.7%, n = 8). The kinetic analysis in normal subjects revealed that Ia expression on T cells stimulated with Concanavalin A (ConA) was significantly increased on day 4 (10.2 +/- 6.2%, n = 8). However, when stimulated with native DNA from calf thymus (nDNA), Ia expression was not increased (1.4 +/- 1.6%, n = 8). In SLE patients, initial Ia expression faded out within 4 days without stimulus (3.9 +/- 4.0%, n = 10). In spite of the decline of Ia expression with stimulation of both Con A and nDNA, the initial proportion of Ia positive T cells was relatively well maintained with the stimulation of nDNA (16.2 +/- 4.7%). Further analysis of T-cell subpopulations in SLE patients revealed that both OKT8- (T4) and OKT4- (T8) cells expressed Ia antigens. Interestingly, in 4 out of 5 patients with active disease, the incidence of Ia expression in OKT4- cells was increased with nDNA stimulation. Thus, in patients with SLE, circulating Ia positive T cells might be activated in vivo, and be implicated in the development of autoimmunity to DNA.     

Kinetic analysis of Mycobacterium tuberculosis-specific cytokine production by PBMC in adults after BCG vaccination

Although bacille Calmette–Guérin (BCG) has been used worldwide as the only vaccine for tuberculosis, its protective efficacy in human adults is controversial. To investigate human immunological responses to Mycobacterium tuberculosis after BCG vaccination, we analyzed IFN- and IL-10 production by peripheral blood mononuclear cells (PBMC) from health-care workers five times throughout the year after BCG vaccination. Of 449 health-care workers, 36 (8.0%) were negative by the tuberculin skin test, and of these, 20 were vaccinated with BCG. Because all the subjects had received BCG vaccination as infants, the present vaccination was considered to be a revaccination. The cytokine responses of the vaccinated and control tuberculin skin-test-positive subjects (n = 6) were followed at 0, 2, 4, and 8 weeks and at 12 months. The mean IFN- production by PBMC when cultured with purified protein derivative (PPD) gradually increased, reached a peak at week 8, and then declined until 12 months, with four exceptions who showed no IFN- elevation. The IFN- level of the vaccinated group at week 0 was significantly lower than that of the controls. The mean IL-10 production in response to PPD reached a peak at week 2, and then declined to its lowest point at week 8. These results indicate that the BCG vaccine can induce a type I cytokine response to M. tuberculosis in most tuberculin skin-test-negative adults at week 8, suggesting the immunological efficacy of vaccination.     

Kinetic analysis of 25-hydroxyvitamin D3 metabolism in strontium-induced rickets in the chick.

Kinetic data analysis was used to derive a six-compartment computer model which describes the in vivo [3H]25-hydroxyvitamin D3 ([3H]25-OHD3) metabolism in control and strontium rachitic chicks. Plasma concentrations of 25-OHD3 (13 pmol/ml) and 25, 25-dihydroxyvitamin D3 (0.9 pmol/ml) were 18 and 125% greater than controls, respectively, whereas the corresponding level for 1alpha,25-dihydroxyvitamin D3 (0.3 pmol/ml) was only 30% of control. Plasma disappearance of 25-HOD3 was fitted using a two-compartment model in which the metabolite extrapolated half-life was nearly twice as large for strontium rachitic chicks (71 compared to 41 h). Intestinal sequestration of 1alpha,25-dihydroxyvitamin D3 was assumed to be irreversible and was fitted by a single exponential term in which metabolite uptake rate and tissue concentration in strontium rickets was suppressed to 20 and 10% of control, respectively. In contrast, uptake of 25-OHD3 by the intestine was observed to occur by a reversible process in which metabolite concentration was 45% greater in the strontium rachitic compared to control group. The developed compartment model accepts time-dependent control or perturbed metabolite data for the plasma and (or) intestinal pools and provides quantitative values for metabolite pool size, flux rate, and turnover time.     

Kinetic analysis in cell culture of the reversal of antiherpes activity of nucleoside analogs by thymidine

The inhibition of herpes simplex virus type 1 plaque formation by acyclovir, bromovinyldeoxyuridine, 9-(3,4-dihydroxybutyl)guanine, and 9-(4-hydroxybutyl)guanine at different thymidine concentrations was analyzed in Lineweaver-Burk plots. Linear competitive patterns between thymidine and the nucleoside analogs were observed for the inhibition of herpes simplex virus type 1 plaque formation. A new constant, the reversal constant Kr, was introduced to describe the sensitivity in cell culture of an antiviral drug to the reversal of its viral activity by a metabolite (e.g., thymidine).     

Kinetic analysis of the renal handling of 2,2-bis(p-chlorophenyl) acetic acid by the rat

The kinetics of uptake and efflux of organic acids in rat renal cortical slices were used to examine the affinity of 2,2-bis(p-chlorophenyl)acetic acid (DDA) for the organic acid transport system and to assess intracellular binding of this polar 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) metabolite. As judged by its ability to inhibit p-aminohippuric acid transport, DDA was a potent competitive inhibitor, almost as strong as probenecid, the classical inhibitor of this system. Efflux of DDA from slices demonstrated that the bulk (85%) of the DDA within the slice was reversibly bound to proteins or other macromolecules. Cortical slices incubated 60 minutes with 10 micron DDA contained a total concentration of 160 micron DDA within the tubular cells, but the actual free concentration in the cells was only 20 to 30 micron. Thus, although DDA was accumulated against a concentration gradient by the kidney, the gradient was much smaller than the measured tissue/medium ratio. Potential consequences of DDA exposure through its interaction with the organic acid system and roles of DDA binding sites in the toxicity and transport of DDA are discussed.