Organic solvents liver

Chirazymes. These are commercially available enzymes e.g. lipases, esterases, that can be used for the preparation of a variety of optically active carboxylic acids, alcohols and amines. They can cause regio and stereospecific hydrolysis and do not require cofactors. Some can be used also for esterification or transesterification in neat organic solvents. The proteases, amidases and oxidases are obtained from bacteria or fungi, whereas esterases are from pig liver and thermophilic bacteria. For preparative work the enzymes are covalently bound to a carrier and do not therefore contaminate the reaction products. Chirazymes are available form Roche Molecular Biochemicals and are used without further purification. 

Chauret, N., Gauthier, A. and Nicoll-Griffith, D.A. (1998) Effect of common organic solvents on in vitro cytochrome P450-mediated metabolic activities in human liver microsomes. Drug Metabolism and Disposition, 26 (1), 1-4. 

Thalhammer T, Kaschnitz R, Mittermayer K, et al. 1993. Organic solvents increase membrane fluidity and affect bile flow and K+ transport in rat liver. Biochem Pharmacol 46(7) 1207-1215. 

All the enzymes used in the work described above are quite stable at room temperature and can be used in a free form. They can also be used in an immobilized form to improve the stability and to facilitate the recovery. Many immobilization techniques are available today (25). The recent procedure developed by Whitesides et al using water-insoluble, cross-linked poly(aerylamide-acryloxysuccinimide) appears to be very useful and applicable to many enzymes (37). We have found that the non-crosslinked polymer can be used directly for immobilization in the absence of the diamine cross-linking reagent. Reaction of an enzyme with the reactive polymer produces an immobilized enzyme which is soluble in aqueous solutions but insoluble in organic solvents. Many enzymes have been immobilized by this way and the stability of each enzyme is enhanced by a factor of greater than 100. Horse liver alcohol dehydrogenase and FDP aldolase, for example, have been successfully immobilized and showed a marked increase in stability. 

Horse liver alcohol dehydrogenase is able to oxidise primary alcohols—except methanol—and to reduce a large number of aldehydes. Aqueous solution or organic solvents can be used [62]. As there are no new developments concerning this enzyme, the reader is referred to the review of Schreier [1]. 

The chemistry of the quinoline heterocycle has already been discussed in Chapter 4. Any alkaloid that possesses a quinoline, i.e. 1-azanaphthalene, 1-benzazine, or benzo[b]pyridine, skeleton is known as a quinoline alkaloid, e.g. quinine. Quinoline itself is a colourless hygroscopic liquid with strong odour, and slightly soluble in water, but readily miscible with organic solvents. Quinoline is toxic. Short term exposure to the vapour of quinoline causes irritation of the nose, eyes, and throat, dizziness and nausea. It may also cause liver damage. 

Retinoic acid is insoluble in water but soluble in many organic solvents. Topically applied retinoic acid remains chiefly in the epidermis, with less than 10% absorption into the circulation. The small quantities of retinoic acid absorbed following topical application are metabolized by the liver and excreted in bile and urine. 

Residue depletion studies with radiolabeled furazolidone have shown that the almost complete degradation of the drug in the body resulted in formation of a variety of protein-bound metabolites that were not solvent-extractable. Thus, when pigs were given radiolabeled furazolidone orally at 16.5 mg/kg bw/day for 14 days (123), total residual radioactivity in liver, kidney, muscle, and fat accounted for 41.1 ppm, 34.4 ppm, 13.2 ppm, and 6.2 ppm furazolidone equivalents, respectively, at zero withdrawal (132). Total residues were substantially lower by 21 days withdrawal, but were still in the ppm range at 45 days withdrawal. Extraction of the incurred muscle tissue at 0 and 45 days withdrawal with organic solvents led to removal of 21.8 and 13.7% of the total radioactivity, respectively. In contrast, 44 and 8.3% of the total radioactivity was extracted from liver on days 0 and 45, respectively. 

Following implantation of 200 mg of radiolabeled trenbolone acetate in calves and heifers, maximum levels of residues in tissues occurred at about 30 days postimplantation (31). The highest total drug-related residues expressed as trenbolone equivalents were approximately 50 and 3 ppb in liver and muscle, respectively. Only 25% and 10% of those residues could be extracted by ether or ethyl acetate from glucuronidase-treated liver and muscle samples, respectively. Tire majority of trenbolone residues were not extractable by organic solvents, a finding suggesting that they were covalently bound to tissues (32). 

In many applications, relatively large quantities of anhydrous sodium sulfate can be added to the sample, prior to extraction by organic solvents, in order to enhance the partitioning process (333-336, 341, 342, 354, 365, 370). In some instances, such as in the analysis of clorsulon in bovine liver and milk, addition of hydroxylamine hydrochloride is often recommended prior to extraction (329, 360). The use of this agent is to prevent interactions between the analyte and endogenous aldehydes that lead to loss of recovery. 

Quinoxaline-1,4-dioxides are synthetic antimicrobial agents the best-known members are carbadox and olaquindox. Both compounds are rapidly metabolized to monoxy- and desoxy- compounds. The final product from carbadox and the one most often determined, mainly in liver (target tissue), is quinoxaline-2-carboxylic acid. Carbadox and olaquindox are light-sensitive compounds and sample manipulations should be performed only under the minimum of indirect incandescent illumination. Carbadox and desoxycarbadox are insoluble in water but are soluble in chloroform and methanol, while olaquindox is slightly soluble in water and some organic solvents. The solubility, however, of quinoxaline-2-carboxylic acid can be easily monitored by adjusting the pH because it is a strong carboxylic acid (pK 2.88). 

Harden, L., Bengtsson, N.O., Jonsson, U., Erikssonn, S. Larsson, L.G. (1984) Aetiological aspects of primary liver cancer with special regard to alcohol, organic solvents and acute intermittent porphyria—an epidemiological investigation. Br. J. Cancer. 50, 389-397... 

Determinative and confirmatory methods of analysis for PIR residue in bovine milk and liver have been developed, based on HPLC-TS-MS (209). Milk sample preparation consisted of precipitating the milk proteins with acidified MeCN followed by partitioning with a mixture of -butylchloride and hexane, LLE of PIR from aqueous phase into methylene chloride, and SPE cleanup. The dry residue after methylene chloride extraction was dissolved in ammonium hydroxide, and this basic solution was transferred to the top of Cl8 SPE column. The PIR elution was accomplished with TEA in MeOH. For liver, the samples were extracted with trifluoroacetic acid (TFA) in MeCN. The aqueous component was released from the organic solvent with n-butyl chloride. The aqueous solution was reduced in volume by evaporation, basified with ammonium hydroxide, and then extracted with methylene chloride. The organic solvent was evaporated to dryness, and the residue was dissolved in ammonium acetate. The overall recovery of PIR in milk was 94.5%, RSD of 8.7%, for liver 97.6%, RSD of 5.1 %. A chromatographically resolved stereoisomer of PIR with TS-MS response characteristics identical to PIR was used as an internal standard for the quantitative analysis of the ratio of peak areas of PIR and internal standard in the pro-tonated molecular-ion chromatogram at m/z 411.2. The mass spectrometer was set for an 8 min SIM-MS acquisition. Six samples can be processed and analyzed in approximately 3 hours. 

Alcohol dehydrogenase-catalyzed reduction of ketones is a convenient method for the production of chiral alcohols. HLAD, the most thoroughly studied enzyme, has a broad substrate specificity and accommodates a variety of substrates (Table 11). It efficiently reduces all simple four- to nine-membered cyclic ketones and also symmetrical and racemic cis- and trans-decalindiones (167). Asymmetric reduction of aliphatic acyclic ketones (C-4-C-10) (103,104) can be efficiently achieved by alcohol dehydrogenase isolated from Thermoanaerobium brockii (TBADH) (168). The enzyme is remarkably stable at temperatures up to 85°C and exhibits high tolerance toward organic solvents. Alcohol dehydrogenases from horse liver and T. brockii... 

Chloroform is a common industrial organic solvent that can be a hepatotoxicant or a nephrotoxicant in both humans and animals. As a nephrotoxicant it is both species and gender dependent. For example, following chloroform administration male mice develop primarily kidney necrosis whereas female develop liver necrosis. 

Note Moderately polar solvent soluble in water and most organic solvents flammable highly toxic by ingestion and inhalation absorbed through the skin may cause central nervous system depression, necrosis of the liver and kidneys incompatible with strong oxidizers. Synonyms diethylene ether, 1,4-diethylene dioxide, diethylene dioxide, dioxyethylene ether. 

Another gap of almost 10 years occurred before work in the area of CLCs picked up again. In 1985, a group at the Louis Pasteur University in Strasbourg, France, prepared cross-linked crystals of horse liver alcohol dehydrogenase [4], The activity of the enzyme in CLC form was maintained and the coenzyme was found to be firmly bound to the crystals. The cross-linked crystals could be used as redox catalysts with no addition of coenzyme. The authors also reported the increased stability of CLCs toward organic solvents. 

KM Lee, M Blaghen, J-P Samama, J-F Biellmann. Crosslinked crystalline horse liver alcohol dehydrogenase as a redox catalyst activity and stability toward organic solvent. Bioorg Chem 14 202-210, 1986. 

The FDA-approved and acceptable chemical inhibitors for reaction phenotyping are included in Table 2. Many of the inhibitors listed in Table 2 are metabolism-dependent inhibitors that, in order to inhibit CYP, require preincubation with NADPH-fortified human liver microsomes for 15 minutes or more. In the absence of the metabolism-dependent inhibitor, this preincubation of microsomes with NADPH can result in the partial, spontaneous loss of several CYP enzyme activities (see sec. II.C.7.c). Furthermore, the organic solvents commonly used to dissolve chemical inhibitors can themselves inhibit (or possibly activate) certain CYP enzymes, as discussed in section II.C.4. Therefore, appropriate solvent and preincubation controls should be included in all chemical inhibition experiments. 

Paris BL, Marcum AE, Clarin JR, et al. Effects of common organic solvents on CYP2E1 activity in human liver microsomes effects of order of addition and preincubation with NADPH. Drug Metab Rev 2003 35(suppl 2) 180. 

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