Kinetics carrier-mediated transport

Carrier-mediated transport is linear with mucosal solute concentration until this concentration exceeds the number of available carriers. At this point the maximal solute flux (7max) is independent of further increases in mucosal solute concentration. In the linear range of solute flux versus mucosal concentration (C), the proportionality constant is the ratio of / to the solute-carrier affinity constant (Km). This description of Michaelis-Menten kinetics is directly analogous to time changes in mass per unit volume (velocity of concentration change) found in enzyme kinetics, while here the appropriate description is the time change in solute mass per unit surface area of membrane supporting the carrier. 

Figure 13 Mediated transport kinetic scheme. C = carrier, S = solute 1 and 2 represent sides of the membrane g are rate constants for changes in conformation of solute-loaded carrier k are rate constants for conformational changes of unloaded carrier f and bt are rate constants for formation and separation of carrier-solute complex. (From Ref. 73.)...

In addition to the mechanistic simulation of absorptive and secretive saturable carrier-mediated transport, we have developed a model of saturable metabolism for the gut and liver that simulates nonlinear responses in drug bioavailability and pharmacokinetics [19]. Hepatic extraction is modeled using a modified venous equilibrium model that is applicable under transient and nonlinear conditions. For drugs undergoing gut metabolism by the same enzymes responsible for liver metabolism (e.g., CYPs 3A4 and 2D6), gut metabolism kinetic parameters are scaled from liver metabolism parameters by scaling Vmax by the ratios of the amounts of metabolizing enzymes in each of the intestinal enterocyte compart-... 

Depending upon the mechanism that is employed by the organism to accumulate the solute, internalisation fluxes can vary both in direction and order of magnitude. The kinetics of passive transport will be examined in Section 6.1.1. Trace element internalisation via ion channels or carrier-mediated transport, subsequent to the specific binding of a solute to a transport site, will be addressed in Section 6.1.2. Finally, since several substances (e.g. Na+, Ca2+, Zn2+, some sugars and amino acids) can be concentrated in the cell against their electrochemical gradient (active transport systems), the kinetic implications of an active transport mechanism will be examined in Section 6.1.3. Further explanations of the mechanisms themselves can be obtained in Chapters 6 and 7 of this volume [24,245]. 

Yuasa H, Miyamoto Y, Iga T, Hanano M (1986) Determination of kinetic parameters of a carrier-mediated transport in the perfused intestine by two-dimensional laminar flow model Effects of the unstirred water layer. Biochim Biophys Acta 856 219-230... 

Tomei S, Torimoto M, Hayashi Y, Inoue K, Yuasa H, Watanabe J (2003) Kinetic characterization of carrier-mediated transport systems for D-glucose and taurocholate in the everted sacs of the rat colon. Biol Pharm Bull 26 899-901... 

Figure 3.3 Comparison of the kinetics of carrier-mediated transport and passive diffusion.

For carrier-mediated transport, the rate of movement across a membrane will now be constant, since flux is dependent on the capacity of the membrane carriers and not the mass of the chemical to be transported. These processes are described by zero-order kinetic rate equations of the form ... 

K0 is now the zero-order rate constant and is expressed in terms of mass/time. In an active carrier-mediated transport process following zero-order kinetics, the rate of drug transport is always equal to K once the system is fully loaded or saturated. At subsaturation levels, the rate is initially first order as the carriers become loaded with the toxicant, but at concentrations normally encountered in pharmacokinetics, the rate becomes constant. Thus, as dose increases, the rate of transport does not increase in proportion to dose as it does with the fractional rate constant seen in first-order process. This is illustrated in the Table 6.1 where it is assumed that the first-order rate constant is 0.1 (10% per minute) and the zero-order rate is 10 mg/min. 

Both processes exhibit classical saturation kinetics, since there are only a finite number of carrier molecules. Thus unlike passive absorption (paracellular or transcellular), where the rate of transport is directly proportional to the drug concentration (Figure 1.5, A), carrier-mediated transport is only proportional to the drag concentration at low concentrations of drug. At higher concentrations, the carrier mechanism becomes saturated and the rate of absorption remains constant (Figure 1.5, B). 

The transport of some solutes across membranes does not resemble diffusion and suggests a temporary, specific interaction of the solute with some component (protein) of the membrane characterized as carrier, e.g., the small-peptide carrier of the intestinal epithelium. The rate of transport increases in proportion to concentration only when this is small, and it attains a maximal rate that cannot be exceeded even with a large further increase in concentration. The kinetics of carrier-mediated transport is theoretically treated by considering carrier-solute... 

Macheras, P., Carrier-mediated transport can obey fractal kinetics, Pharmaceutical Research, Vol. 12, No. 4, 1995, pp. 541-548. 

Fractal kinetics was successfully shown to be a useful indicator for carrier-mediated transport (CMT) of substrates than classical Michaelis-Menten kinetics... 

Ogihara, T. Tamai, I. Tsuji, A. Application of fractal kinetics for carrier-mediated transport of drugs across intestinal epithelial membrane. Pharm. Res. 1998, 15 (4), 620-625. 

Kinetics of carrier-mediated transport processes is similar to enzyme-substrate reactions and can be described by the Michaelis-Menten equation (Eq. (9.2)), assuming that each transport system has one specific binding site for its substrates. Maximum transport velocity (Vmax) is reached when all binding sites of the respective carrier proteins are occupied by substrate molecules. Substrate turnover can be delineated by the Michaelis constant Km corresponding to the substrate concentration [S], at which half-maximum transport velocity has been reached (Figure 9.5). Km also depends on pH and temperature. In cotransport systems transferring several substrates, the transport protein has a characteristic Km for each molecule transported. 

After five days, dopamine, L-tryptophan, and l-DOPA passively permeated through the membrane as indicated by fittings with a first-order kinetic process equation. After seven days of co-culture, occludin localizes at EC periphery, dopamine does not cross the barrier to any further extent, while the transfer of L-tryptophan and l-DOPA fits well with a saturable Michaelis-Menten kinetic process, thus indicating the involvement of a specific carrier-mediated transport mechanism. Permeation studies confirmed that culture of ECs in the presence of neurons induces the characteristic permeability limitations of a functional BBB. 

The PEP-fructophosphotransferase system does not exist in Spirillum itersoii, Pseudomonas aeruginosa,181,182 or several other genera of aerobic, oxidative bacteria.183 The transport system for D-glucose, D-fructose, and D-mannitol is energy- and temperature-dependent, obeys saturation kinetics, and is inducible.181,182 This indicates the presence of a carrier-mediated transport-system.184 D-Fructose is transported as the free sugar, and trapped intracellularly by phosphorylation, An inducible fructokinase (EC 2.7.1.4) converts transported D-fructose into D-fructose 6-phosphate.181... 

The second method in this group is the intestinal rings or slices. This method for studying drug absorption has been used extensively for kinetic analysis of carrier-mediated transport of glucose, amino acids and peptides (Kararli 1989 Osiecka et al. 1985 Porter et al. 1985 Kim et al. 1994 Leppert and Fix 1994). The method is easy to use the intestine of the animal is cut... 

Nearly all enzymes follow what is known as Michaelis-Menten kinetics, which was encountered in Section 10.2.2 for carrier-mediated transport processes. The Michaelis-Menten equation for the rate of metabolism f mei) c.an be written as... 

This derivation is commonly used to describe the kinetics of product formation in enzyme-catalyzed reactions (substitute enzyme for carrier protein and product formation for the conformational change of the carrier protein). Under the assumptions of this simple model, carrier conformational change is the rate-limiting step, so it is reasonable to assume further that k2 is much less than k. n this case, the constant is approximately equal X.o Ki=k i/kx, the dissociation constant for the binding of solute to carrier. For this reason, it is common to refer to as the affinity of the solute for the carrier (note the analogy to Equation 3-53). F ax is the maximum flux due to this carrier-mediated transport, which occurs when all of the carrier-binding sites are occupied (Figure 5.11). 

For each assay, incubate 100 pL of synaptosomes, equivalent to 300 pg of protein, with 300 nM [ H]AEA for different time intervals at 37°C or 4°C to discriminate carrier-mediated from non-carrier-mediated transport of AEA through cell membranes. Alternatively, to determine the kinetic constants of AMT, incubate for 15 min with different concentrations of [ H]AEA, in the range 0-1000 nM (in this case the uptake at 4°C has to be subtracted from that at 37°C). 

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