Extraction methods hepatic

The methods for P450 measurements require the extraction of hepatic tissue at necropsy to obtain microsomal preparations for the measurement of some of the relevant cytochromes (Batt et al. 1992,1994 Halpert et al. 1994 loannides 1996 Wang et al. 1999). The relationships of microsomal enzyme induction with liver pathology in dogs and rats have been described by Amacher and colleagues (1998, 2001). 

Choudhury RB, Poddar MK. (1984) Andrographolide and Kahnegh (Andrographis paniculata) extract In vivo and in vitro effect on hepatic lipid peroxidation. Methods Find Exp Clin Pharmacol 6 481. 

A reference for this method is P. S. Venkateswaran, 1. Millman, and B. S. Blumberg, Effect of an extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses in vitro and in vivo studies, Proc. Natl. Acad. Sci., USA, 1987, 84, 274-278, which is incorporated herein by reference. 

Available control materials include patient specimens that have been well characterized by a similar method, synthetic controls such as Armored RNA (Ambion Inc., Austin, Texas), or purified nucleic acid, although purified nucleic acid would not be appropriate for use as an extraction control. Armored RNA uses a bacteriophage coat around an RNA target to produce pseudoviral particles. Control material is currently available for a variety of pathogens, including HIV-1, HCV, hepatitis A virus, enterovirus, Norwalk virus, and West Nile virus. These armored RNA preparations are stable for at least 11 months and are compatible with a large number of different platforms commonly used for quantitative molecular assays. Table 42-3 lists other manufacturers of control materials. 

DNA and RNA viruses. For example, 152 methanol and water extracts of different parts of 71 plants commonly used in Sudanese traditional medicine were screened for their inhibitory effects on hepatitis C vims protease using in vitro assay methods. Thirty-four extracts showed significant inhibitory activity. From the Embelia schimperi D. C. extract, two benzoquinones, embelin and 5-O-methylembelin, were isolated and found to be potent inhibitors [148,149],... 

A series of mouse bioassay procedures (biological methods), differing in the test portion (hepat-opancreas or whole body) and in the solvents used for the extraction and purification steps, can be used for detection of the toxins DSR Sensitivity and selectivity depend on the choice of the solvents used for the extraction and purification steps, and this should be taken into account when making a decision on the method to be used, in order to cover the full range of toxins. 

A second in vivo model system that is very useful in sorting through problem of low oral bioavailability is portal vein carmulated animals. There are two ways this experiment can be conducted to determine hepatic extraction (1) measure systemie plasma concentration after oral, portal vein and systemic administration and (2) measure portal vein and hepatic vein concentrations after an oral dose. Both methods yield information on hepatic extraction and the percentage of dose reaching the portal circulation (the product of the fraction absorbed and the fraction metabolized by the gut wall). 

The in vivo tissue distribution, excretion, and hepatic metabolism of microcystins have been primarily investigated using variously radiolabeled ones. " The amounts of the injected microcystins were too small and the amounts of the contaminants in the tissues were too large to investigate the metabolites by instrumental analysis, such as HPLC with UV detection and LC/MS. Fig. 4 shows the HPLC profiles of a cytosolic extract from mouse liver spiked with 5 p.g each of microcystins-LR and -RR. When the cytosolic extract is prepared by the method described by Robinson et al., which consists of heat-denaturation, pronase digestion, and ODS silica gel treatment (Fig. 4a), the two spiked microcystins cannot be precisely analyzed because of a substantial amount of coexisting substances. When the cytosolic extract is further purified with the immunoaffinity column, the coexisting substances are effectively eliminated... 

An LC-MS-MS method measures directly the concentration of amantadine (1-adamantylamine, used for treatment of influenza, hepatitis C, parkinsonism, and multiple sclerosis) without protein precipitation, centrifugation, extraction, and derivatization steps. Only 50 p,l sample is needed. Internal standard is l-(l-adamantyl)pyridinium bromide. The serum sample is diluted by water in a 96-well plate. The chromatographic separation is performed using an eluent of isocratic water/acetonitrile (60/40, v/v) with 5 g/1 formic acid on a C8 column. Run time is 3 min. Electrospray atmospheric pressure ionization, positive ion, and selective reaction monitoring mode were used. Detection hmit 20 mg/1, linearity 20-5000 mg/1, intraassay/interassay coefficient of variation <6%/<8% recovery 99-101%. 

Serum cholesterol and TG levels were determined by enzymatic methods (28,29). Serum free fatty acid (FFA) level was determined by a colorimetric method (30). Human LDL was obtained by ultracentrifugation. The density range between 1.006-1.063 g/mL was collected as LDL. To determine the oxidative susceptibility in Cu -induced oxidation, LDL was dialyzed in phosphate-buffered saline (PBS) (10 mM, pH 7.4) and subjected to oxidation without prolonged storage (25,31). Hepatic cholesterol contents of hamsters were determined by a modified colorimetric assay after saponification and extraction (32). To avoid interference from plant sterols in the colorimetric assay, fecal cholesterol content was determined by reversed-phase HPLC (mobile phase methanol acetonitrile = 56 44, v/v). 

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