Liver extract, purification

In the USA, purity alone often facilitates patenting of a product of nature (Table 2.3). The PTO recognizes purity as a change in form of the natural material. For example, although vitamin B12 was a known product of nature for many years, it was only available in the form of a crude liver extract, which was of no use therapeutically development of a suitable production (fermentation) and purification protocol allowed production of pure, crystalline vitamin B12 which could be used clinically. On this basis, a product patent was granted in the USA. 

There is abundant evidence indicating that a natural hydrophobic inhibitor of acetyl-CoA carboxylase is present in crude enzyme extracts of liver and adipose tissue [128,129,182,192,236-238]. The activating effect of (+)-palmityl carnitine on fatty acid synthesis in crude liver extracts and on impure acetyl-CoA carboxylase preparations has tentatively been ascribed to the displacement of hydrophobic inhibitors such as fatty acids or fatty acyl-CoA derivatives [129,182,192,236-238]. Inhibition of rat liver acetyl-CoA carboxylase by added palmityl-CoA can be reversed in part by (+)-palmityl carnitine [236], but not by citrate. This activating effect does not appear to be specific with respect to (+)-palmityl carnitine in that cetyl trimethylammonium ion is also effective [192]. Furthermore, impure preparations of acetyl-CoA carboxylase from adipose tissue or rat liver are markedly activated by serum albumin [123,129,238] or extensive dilution of the enzyme preparation prior to assay [129,182]. On the other hand, none of these agents [(+)-palmityl carnitine, serum albumin, or dilution], which activate the impure carboxylase, have an activating effect on the homogeneous acetyl-CoA carboxylases from adipose tissue or liver [129,182, 239]. It is evident that an inhibitory substance, apparently hydrophobic in nature, is removed either by purification of the enzyme or by the agents or treatments mentioned above. 

Ion-exchange chromatography may be used for purification and group separation. As mentioned, Okishio et al. (24) purified a liver extract by applying it in 0.1 M NaOH to an Amberlyst A-26 column as described above for solid extraction. An extract can also be dissolved in chloroform-methanol, 1 1, saturated with water, and applied with a slow flow rate to an Amberlyst A-26 column in OH" form in the same solvent. A 10-ml column should be used for 250 mg of sample. Neutral lipid contaminants are removed by washing with a suitable organic solvent and bile acids are eluted with 0.2 M ammonium carbonate in 80% ethanol as described above (34). 

Early studies for the purification of the antipernicious anemia factor used either minced liver preparations or proteolyzed liver extracts in which the concentration of the factor was of the order of one part per million. Adsorption chromatography, partition chromatography, and extraction were the methods of choice for purification. In the initial fractionation steps, the factor was adsorbed on either activated carbon or fuller s earth, and eluted with either aqueous ethanol, phenol, or p3U-idine. Intermediate purification steps were accomplished by partition chromatography adsorption chromatography on either silica or alumina was also effective. At various stages of these column procedures, the activity could be removed from aqueous solution by butanol extraction or with phenol or cresol in combination with solvents such as butanol or toluene. Final purification was usually accomplished by crystallization from aqueous acetone solution. 

UMP in a reaction that greatly resembles that of adenylate kinase. The reaction requires ATP and results in the formation of ADP and UDP. The complete purification of kinases of this type has not been achieved, but the partial separations already accomplished indicate that at least three, and possibly more enzymes with varying substrate specificities exist. These enzymes, studied in calf liver extracts, all use ATP as the phosphate donor, and show at least relative specificity for adenylic acid, uridylic acid, and cytidylic acid as phosphate acceptors. 

Inasmuch as the chief difficulty in the work on the purification of active materials has been the relative scarcity of pernicious anemia patients, many worthy attempts have been made to develop an animal assay the guinea pig, dog, cat, pigeon, swine, monkey, and rabbit have all been tried without definite success if any. Creskoff and Fritz Hugh have covered this subject admirably in their review of standardization and assay of liver extracts (20). At any rate the clinical assay still is the only reliable way of following the fractionation procedures. 

Figure 7. Purification of Tj nuclear receptors on the SW 3000 column. Crude liver nuclear extract was prepared and incubated with as outlined in the...

Y. Shibusawa, T. Fujiwara, H. Shindo and Y. Ito, Purification of alcohol dehydrogenase from bovine liver crude extract by dye-ligand affinity counter current chromatography. J. Chromatogr.B, 799 (2004) 239-244. 

BDE 47, 99 100 Fish (Muscle tissues of salmon and conger eel and liver tissues of sea bass) green mussel Homogenization, MSPD with sodium sulfate, microwave-assisted extraction with pentane-dichloromethane (1 1) and purification with GPC Gas Chromatography (DB-5MS) Q-MS <100 ng/Kg [41]... 

Kirkbride (1987) described the estimation of diazinon in human omental tissue (fatty tissue) after a fatal poisoning. In this method, the tissue was pulverized and extracted with acetone. After extract concentration and purification by sweep co-distillation and Florisil fractionation, diazinon was measured by gas chromatography (GC) with nitrogen-phosphorus detection (NPD). After another fatal diazinon poisoning, diazinon was quantified by GC/electron capture detection (ECD) and GC/flame ionization detection (FID) by Poklis et al. (1980). The diazinon in human adipose, bile, blood, brain, stomach contents, kidney, and liver was recovered by macerating the sample with acetonitrile followed by the addition of aqueous sodium sulfate and extraction into hexane. Following an adsorption chromatography clean-up, the sample was analyzed. 

In another method for the determination of sulfamethazine in muscle and liver tissues (59), the extraction problem was successfully addressed by applying a matrix solid-phase dispersion procedure for rapid and efficient purification of the tissue extracts. Determination was made by ELISA on the basis of antibodies raised against sulfamethazine-diazo-bovine serum albumin conjugates. 

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). 

Diphasic dialysis can also be used for purification of the primary sample extract. This procedure was only applied in the determination of clenbuterol residues in liver using tert.-butylmethyl ether as the extraction solvent (483). 

The investigation of a single-step pathway usually begins with a crude cell-free extract from a source abundant in the enzyme that catalyzes the conversion. Two of the most popular sources of cells for many biochemical studies are rat liver and E. coli. To investigate a particular reaction, the precursor (substrate) is added to the extract, and the amount of product formed as a function of time is determined. Once an assay for disappearance of precursor and appearance of product has been developed, the crude extract can be processed into fractions that can be tested to see which are active in the conversion. Through further fractionation and assays it should ultimately be possible to purify the enzyme of interest. Some procedures followed in enzyme purification were discussed in chapter 6, and many procedures used to determine the mechanisms of action of the purified enzymes were considered in chapters 8 and 9. 

L-Ping, H. Nagasawa, K. Matsumoto, A. Suzuki, K. Fuwa, Extraction and purification of a new compound containing selenium and mercury accumulated in dolphin liver, Biol. Trace Elem. Res., 11 (1986), 185-199. 

Albro PW, Schroeder JS, Harvan DJ, Corbett BJ (1984), J. Chromat. 312 165-182. Characteristics of an extraction and purification procedure for chlorinated dibenzo-p-dioxins and diben-zofurans in soil and liver"... 

Beef liver (or lung) was minced and then autolyzed for twenty-four hours before extraction with an alkaline solution saturated with ammonium sulfate. Protein was precipitated by warming the extract, and the heparin-protein complex was precipitated from the supernatant liquor on acidification. Extraction of the complex with ethanol removed fatty material, and tryptic digestion removed most of the protein. The heparin was precipitated with ethanol, redissolved in warm alkaline solution to destroy trypsin, and reprecipitated with acetone. This material, crude heparin, was isolated in a yield of 15-50 g. per 100 lb. of animal tissue. In a later paper," the purification of crude heparin by fractionation successively with Lloyd s reagent, cadmium chloride, and acetone, was described. The purified heparin w-as 100 times as active as the crude material. Scott and Charles" reported the presence of nitrogen... 

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