Metabolic assays

Fialho E.T., Vassalo M., Lima J.A.F. and Bertechine A.G. (1998). Probiotics utilization for piglets from 10 to 30 kg (performance and metabolism assay) , in Proceedings Contributed Papers - Vol. 1. The 8th World Conference on Animal Production. June 28-July 4, 1998, Seoul National University, Seoul, Korea, 622-623. 

Trubetskoy, O.V., Gibson, J.R., and Marks, B.D. 2005. Highly miniaturized formats for in vitro drug metabolism assays using vivid fluorescent substrates and recombinant human cytochrome P450 enzymes. J. Biomol. Screen. 10 56. 

Several alternative methods for the determination of sialic acid in body fluids and tissues have been described. Most of these methods make use of the classic periodate-TBA assay in combination with purification using HPLC [13]. Another method makes use of fluorometric HPLC of sialic acids after derivatization with a fluorogenic compound [9]. The most promising new method for the determination of free sialic acid in urine (and probably also other body fluids and tissues) is the HPLC-tandem mass spectrometry method [19]. This method is rapid, accurate, and sensitive, and is more robust than earlier methods. The only disadvantage is the expensive equipment that is required, which makes it only economical for specialized metabolic laboratories. Since this equipment is used for many different metabolic assays, the investment is certainly warranted, and nowadays almost essential for any metabolic laboratory. 

Perrot S, DutertreCatella H, Martin C et aL (2003) Rezasurin metabolism assay is a new sensitive alternative test in isolated pig cornea. Toxicol Sci 72 122-129 Prinsen MK, Koeter HB (1993) Justification of the enucleated eye test with eyes of slaughterhouse animals as an alternative to the Draize eye irritation test with rabbits. Food Chem Toxicol 31 69-76... 

High-throughput in vitro drug metabolism assays... 

Creatinine in urine. The assay of creatinine in body fluids is one of the core assays in clinical chemistry since its level in blood and urine reflects the functional status of the kidney. There are many methods for its assay ranging from the simple colorimetric Jafle reaction to dedicated creatinine analysers using discrete sampling technologies. For many metabolic assays, the so-called creatinine correction can be applied since creatinine excretion is considered to be constant throughout the day. The clinic therefore only needs to collect a random specimen of urine rather than a full 24 h specimen. 

Because poor bioavailability represents one of the principal causes of compound failure, absorption and metabolism assays feature prominently in lead optimization screens. As oral administration represents the ideal route of drug delivery, models that predict low absorption across the gastrointestinal (GI) tract are commonly employed. Caco-2 cell assays have been... 

FIGURE 1.2 Four types of mass spectrometers that are used for various drug metabolism assays. (From Pappas, J., Thermo Fisher Scientific Inc., Waltham, MA. With permission.)... 

Varoglu, M. et al., Simultaneous quantitative and qualitative measurements of in vitro microsomal metabolism assays by Orbitrap LC-MS methods, Proceeding of 56th ASMS Conference on Mass Spectrometry and Allied Topics. Denver, CO, June 1-5, 2008. 

Catechol Amine Biosynthesis and Metabolism, Assay of Enzymes of Catecholamines and Catecholamine Metabolites, Estimation of Total... 

Figure 1 Structure of INS37217 and INS542. INS37217 is a deoxcytidine-uridine dinucleotide [P1-(Uridine 5/)-P4-(2/-deoxycytidine 5 )tetraphosphate, tetrasodium salt] and INS542 is a cytidine-uridine dinucleotide [P1-(Uridine 5 )-P4-(cytidine 5 )tetraphosphate, tetrasodium salt]. The only difference between the two molecules is the absence of the OH-group in the 2 position of the cytidine ribose in INS37217. Both molecules have nearly identical activity in a variety of pharmacological and metabolism assays.

Bioassay is the quantitative method in which the endpoint is an observable effect on a biological system or an organism. The classical approach to microbial detection involves the use of differential metabolic assays (monitored colormetrically) to determine species type in the case of most bacteria, or the use of cell culture and electron microscopy to diagnose viruses and some bacteria that are intracellular parasites. Samples taken from the environment, such as soil and water, and most clinical samples must be cultured in order to obtain sufficient numbers of various cell types for reliable identification. The time required for microbial outgrowth is typically 4-48 hours (or two weeks for certain cases, such as Mycobacterium tuberculosis). Furthermore, bacterial culture suffers from an inherent drawback cells that are viable may not be culturable, because they possess unanticipated nutritional requirements as a result of genetic mutation. 

The cell cultures were routinely maintained and the metabolism assays were incubated at 27°C on an orbital shaker set at 120 rpm. A time course with 4 time points was generally utilized. Aliquots of 0.5 mL were taken from the samples containing d e cell cultures and also from those widi media. The aliquots were taken at time 0 (within a few minutes of when the compound is added), at 6 hours, 24 hours, and 48 hours. Each sample taken was transferred to an individual well of a 96-deep-well plate with 2 mL capacity per well. Two 5mm... 

The Commercial Standard Used to Evaluate Metabolism Assay... 

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