Metabolite extraction procedure liver

Zile and DeLuca reported the existence of a metabolite of retinoic acid that was found in rat liver and that was able to support the growth of retinoid-deficient rats. This metabolite was later identified as 13-a.r-retinoic acid (E6) and most, if not all, of it appeared to be generated from all-rrans-retinoic acid by the extraction procedure (Emerick et al., 1967). Sundaresan (1966) has likewise reported diat an acidic metabolite of retinoic acid extracted from normal rat liver was able to restore to near normal the activity of a sulfur-activating enzyme (ATP-sulliiiylase) whose activity was depressed in retinoid-deficient rat liver supernatants. Except for the 13-c/s-retinoic acid, none of these metabolites have been characterized further. 

Liquid-liquid partitioning cleanup on a hydrophilic matrix has also been employed for purification of the primary sample extract. This procedure was only applied in the determination of fenbendazole and four metabolites in plasma and liver using a Chem Elut disposable column to partition an alkalinized aqueous sample extract into dichloromethane (371). In a few instances, further cleanup can also be achieved by submitting an organic extract to freezing at 20 C, a procedure that can precipitate dissolved matrix components (359, 366, 367). 

The MAS does not represent a rapid throughput procedure, but because there is no sample extraction or other manipulation prior to analysis, it represents an unbiased method in which changes in tissues can be studied, and as appropriate, compared to alterations observed in biofluids. In this manner, MAS is synergistic with and complimentary to analysis of biofluids by metabonomic applications. Figure 2 shows the typical NMR-MAS spectra from the liver of a control and galactosamine-treated rat. This method readily detects glucose and glycogen, choline and related metabolites and a variety of lipids, and fatty acids. An example of the utility of NMR-MAS data in concert with biofluid analyses is described later in this review. 

In the present study, three groups of six cattle (a total of eighteen) were administered the Captec device intra-ruminally via a fistula. Drug release rates were determined by HPLC assay of ABZ and its major metabolites in plasma as well as by physical measurement of the plunger movement. ABZ marker residue levels were determined using the established regulatory procedure in total liver, an ethyl acetate extractable fraction as well as the remaining intractable (bound) residue on each of three cattle at 0- and 5-Day withdrawal. 

Liver and Kidney. Liver and kidney tissues collected after the sacrifice of cow 59 (0-day withdrawal) were utilized for isolation of metabolites. The isolation procedure involved acetonitrile extraction followed by solid-phase (C-18 Bond Elut cartridge) extraction of the extracts. Sample cleanup after solid-phase extraction was similar to that of urine. The procedure is described in flow diagram 2. 

Comparative Metabolism. Extracts of dog and rat urine, liver, and kidneys, obtained by the same procedures as the swine extracts, were analyzed by reverse phase Ci8 HPLC. The data showed that urine and both tissues of dogs and rats contained the same four major metabolites A, B, C, and D as swine urine and tissues. Table V contains comparative quantitative data on ractopamine HCl and its metabolites in liver and kidneys of swine, dogs, and rats. The amounts as a percent of total residues in all fractions of ractopamine HCl and the four metabolites were almost the same in the three species in both liver and kidney tissue. The notable differences were that dog liver contained less ractopamine and more Metabolite C, and rat kidney contained more Metabolites A and B and less Metabolite C than the other species. The nonextractable C residues were low in the two tissues of all species. 

The first quantitative determinations of HPLC of CoA compounds have been presented by King and Reiss (57). The authors developed a reversed-phase HPLC procedure to measure CoA and short-chain CoA compounds in rat liver tissue. Seventeen CoA-related standards in model experiments were separated and quantified in a 37-min run employing a 75 X 4.6 mm ODS Ultrasphere octadecylsilica column (Fig. 19). In rat liver CoA, acetyl-CoA and six minor CoA-related metabolites could be quantitated (Fig. 20). The separation was achieved using an Na2HP04-acetonitrile gradient. The eluate was monitored at 254 nm. Recovery of CoA standards added in tissue extracts ranged from 83% to 107%. The procedure allowed measurement of as little as 12 pmol of CoA compounds injected. 

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