Assay uptake

Accumulation/efflux studies can be performed on different cell systems or membrane vesicle preparations. In the accumulation assays, uptake of a probe over time, typically either fluorescent (e.g. calcein-AM (CAM) [25-27]) or radiolabeled, into the cell or membrane vesicles is measured in the presence or absence of a known P-gp inhibitor. As P-gp transports substrates out of the cells, the inhibition of the protein would result in an increase in the amount of the probe in the cell. Accumulation studies in cells that overexpress P-gp can be compared to those obtained in the parental cell line that does not have as high a level of P-gp expression. The probe in the absence of inhibitors shows lower accumulation in P-gp expressing cells than in P-gp deficient cells. Similarly, probe accumulation is increased under conditions where P-gp is inhibited such that the difference in accumulation in P-gp deficient and overexpressing cells, respectively, becomes smaller. Accumulation assays poorly distinguish substrates and inhibitors of P-gp and, as far as transport assays are concerned, are also influenced by a passive diffusion property of molecules [20]. In contrast to transport assays, both accumulation (i.e. calcein-AM assay) and ATPase assays tend to fail in the identification ofrelatively low permeable compounds as P-gp active compounds [20]. 

Thyroid Uptake Systems. Studies involving absolute thyroid uptake can be performed without imaging using small amounts of or and a simple scintillation probe. This is caUbrated using a phantom, ie, a model of a portion of the human body, loaded with the isotope being used. This instmment is also useful for assaying thyroid exposure to radioiodine among personnel. 

Figure 6.10b shows a pattern of antagonism often observed in isolated tissue studies but not so often in cell-based assays. Saturation of uptake systems for the agonist or saturation of an adsorption site for the agonist can account for this effect. The linear portion of the regression can be used to estimate the pKB or the pA2. If there is a loss of concentration dependence of antagonism, as seen in... 

Fatty Acid Transporters. Figure 2 Quencher-based real-time fatty acid uptake assay with a fluorescently labeled FFA analogue (C1-Bodipy-C12). Predominantly protein-mediated fatty acid uptake by 3T3-L1 adipocytes (diamonds) was compared with diffusion-driven uptake by fibroblasts (squares) using the QBT Fatty Acid Uptake reagent (Molecular Devices Corp., CA, USA), which contains C1-Bodipy-C12 as substrate in conjunction with a cell impermeable quencher. Uptake kinetics was recorded using a Gemini fluorescence plate reader. Error bars indicate the standard deviations from 12 independent wells. RFU relative fluorescence units. 

Phloretin inhibits FATP-mediated traversing of fatty acids across lipid bilayers. Phloretin is the aglycon of phlorizin and has been used to terminate the uptake of LCFAs and VLCFAs in timed in vitro uptake assays with cultured cells or in ex vivo uptake assays with isolated primary cells. 

NAGEL s c, VOM SAAL F s, WELSHONS w V (1999) Developmental effects of oestrogenic chemicals are predicted by an in vitro assay incorporating modification of cell uptake by serum. J Steroid Biochem Mol Biol. 69 343-57. 

Texturization is not measured directly but is inferred from the degree of denaturation or decrease of solubility of proteins. The quantities are determined by the difference in rates of moisture uptake between the native protein and the texturized protein (Kilara, 1984), or by a dyebinding assay (Bradford, 1976). Protein denaturation may be measured by determining changes in heat capacity, but it is more practical to measure the amount of insoluble fractions and differences in solubility after physical treatment (Kilara, 1984). The different rates of water absorption are presumed to relate to the degree of texturization as texturized proteins absorb water at different rates. The insolubility test for denaturation is therefore sometimes used as substitute for direct measurement of texturization. Protein solubility is affected by surface hydrophobicity, which is directly related to the extent of protein-protein interactions, an intrinsic property of the denatured state of the proteins (Damodaran, 1989 Vojdani, 1996). 

PAMPA is typically used to make a prediction of the passive, transcellular absorption of a compound. Compounds which may be absorbed by a paracellular mechanism or may be substrates for active transport (uptake or efflux) are usually better assessed in a cell based system. A combination of assays can be applied to gain a greater understanding of the permeability and transport properties of a compound. 

An elegant study of lycopene uptake in LNCaP, PC-3, and DU-145 cells using beadlet-delivered 1.48 J.M all-trans lycopene (a maximal level in human plasma) found that all three cell lines rapidly took up lycopene during the first 10 h of incubation. Cells continued to accrue lycopene, but more slowly, over the next 48h. The uptake by the LNCaP cells was 2.5-fold higher than PC-3 cells and 4.5-fold higher than DU-145 cells at 24h of incubation but lycopene showed no affinity for the AR receptor, which is expressed in the LNCaP cells (Liu et al. 2006). LNCaP uptake followed Michaelis-Menten kinetics with a V m i, of 66.3pmol/106 cells/h and a Km of 7.72 pM lycopene. Because of the sensitivity of their LC-MS-MS lycopene assay, Liu et al. were also able to investigate the subcellular lycopene distribution. The nuclear membrane contained 55%, the nuclear matrix 26%, and the microsomal fraction 19% of the intracellular fraction. The cytosol contained no lycopene(Liu et al. 2006). 

Figure 7.3 Dose-response effects of MDMA on the release of preloaded [3H]5-HT (left panel) and [3H]DA (right panel) from synaptosomes in vitro. [3H]Transmitter release is expressed as percent of tritium retained in tissue. Various concentrations of MDMA were incubated with or without the 5-HT uptake blocker fluoxetine (10 n/W) in [3H]5-HT assays, whereas various concentrations of MDMA were incubated with or without the DA uptake blocker GBR12909 (10 n/W) in [3H]DA assays. Data are mean SD for three separate experiments, each performed in triplicate. See Baumann et al.39 for methods.

The two patents described above were particularly important in the initiation of the developments of biodesulfurization catalysts. The bioreactor arrays required for operation and growth method constituted key elements in the following developments of the area, which would condition viability and successful path to industrialization. A sulfur bioavailability assay was incorporated into the screen for monitoring the sulfur uptake by the microorganisms, and the concept formed a claim in the patents [67,91], The objective... 

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