Accumulation assays

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]. 

The accumulated assay results obtained with the 4-alkyl-2-methylpyrrolo[2,3-d]pyrimidines are of special interest. Can-pounds 25 and 26 exhibited significant cytokinin activity in all assays utilized. On the other hand, compound 27 was a potent antagonist in the tobacco bioassay but had little activity in the other two assay systems, while 29 acted as an anticytokinin in the tobacco bioassay and a cytokinin in the lettuce seed germination assay, but was without activity in the Amaranthus test system. 

Alternatively, the transporter function can be analyzed by accumulation or efflux assay. Accumulation assay is generally used to determine substrate accumulation rate into the cells mediated by uptake transporters, whereas efflux assay is generally used to determine substrate efflux rate of the cells mediated by efflux transporters. For efflux assay, cells are preincubated with the substrate. 

Elkins, C.A. and Mullis, LB. (2007) Substrate competition studies using whole-cell accumulation assays with the major tripartite multidrug efflux pumps of Escherichia coli. Antimicrobial Agents and Chemotherapy, 51 (3), 923-929. 

Figure 6. Various designs used to study sperm accumulation in an ascending gradient of a chemoattraotant. [A] An aocumuiation assay in an apparatus consisting of two wells separated by a thin polyoarbonate membrane [59], (B) An accumulation assay in an apparatus consisting of two wells connected via a tube [28]. (C) Sperm accumulation in a capillary assay [128], (Adapted with permission from Eisenbach [48].)...

Parasorbic acid (Figure 2) was isolated from fruits of Sorbus aucuparia. Germination of mustard seed Sinapis alba) was affected adversely by parasorbic acid at 3.5 X 10-3 M and growth of excised tomato roots was inhibited at approximately 8.5 X 10 4 M (25). The acid also antagonized indoleacetic acid (IAA) in the Avena assay. Cornman 29,30) reported that parasorbic acid slowed down mitosis. Metaphase stages were observed to accumulate, but abnormalities were not detected. 

The antiinflammatory effects of statins likely result from their ability to inhibit the formation of mevalonic acid. Downstream products of this molecule include not only the end product, cholesterol, but also several isoprenoid intermediates that covalently modify ( pre-nylate ) certain key intracellular signaling molecules. Statin treatment reduces leukocyte adhesion, accumulation of macrophages, MMPs, tissue factor, and other proinflammatory mediators. By acting on the MHC class II transactivator (CIITA), statins also interfere with antigen presentation and subsequent T-cell activation. Statin treatment can also limit platelet activation in some assays as well. All these results support the concept that in addition to their favorable effect on the lipid profile, statins can also exert an array of antiinflammatory and immunomodulatory actions. 

Metabolic disorders of urea biosynthesis, while extremely rare, illustrate four important principles (1) Defects in any of several enzymes of a metabolic pathway enzyme can result in similar clinical signs and symptoms. (2) The identification of intermediates and of ancillary products that accumulate prior to a metabolic block provides insight into the reaction that is impaired. (3) Precise diagnosis requires quantitative assay of the activity of the enzyme thought to be defective. (4) Rational therapy must be based on an understanding of the underlying biochemical reactions in normal and impaired individuals. 

The results of these investigations suggest that caution should be exercised in interpreting not only the results of toxicity assays in which such organisms are employed but also data accumulated in monitoring studies that may not have taken into account the existence of metabolites. 

The metabolic and pharmacokinetic profile of sucralose (this is a novel intense sweetener with a potency about 600 times that of sucrose) in human volunteers was studied by Roberts and coworkers [82]. Part of this study was realized using PLC in the following chromatographic system in which the stationary phase was silica gel and the mobile phase was ethyl acetate-methanol-water-concentrated ammonia (60 20 10 2, v/v). Separated substances were scraped off separately, suspended in methanol, and analyzed by filtration, scintillation counting, or enzymatic assay. It was shown that the characteristics of sucralose include poor absorption, rapid elimination, limited conjugative metabolism of the fraction absorbed, and lack of bio-accumulative potential. 

The effects of glyphosate on phenolic compound production are two-fold 1) accumulation of phenolic compounds that are derivatives of aromatic amino acids is reduced and 2) pools of phenolic compounds derived from constituents of the shikimate pathway prior to 5-enolpyruvylshikimate-3-phosphate become larger. Assays that do not distinguish between effects on these two groups, such as that for hydroxyphenolics of Singleton and Rossi (18), can lead to equivocal and difficult to interpret results (e.g. 3-5). 

Anthocyanins are Che most easily assayed and commonly studied derivatives of aromatic amino acids (Figure 1). Glyphosate drastically reduces accumulation of anthocyanin flavonoids in treated tissues (6, 19) (Figure 3). Levels of rutin and procyanidin, both flavonoids, are reduced in glyphosate-treated buckwheat hypocotyls (6). Glyphosate would presumably similarly affect levels of flavonoids and flavonoid derivatives that are known to be allelochemicals. 

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