Liver human, enzyme concentration

The level of enzyme needed can influence the choice of preparation used for the study. Microsomal preparations from cell cultures allow the use of higher concentrations of active enzyme per unit volume than use of whole cells or cell lysates. The use of whole, viable cells allows the use of longer incubation times but at a lower enzyme concentration per unit volume. In addition, adequate oxygen transfer and nutrient concentrations are needed to maintain culture viability. These requirements impose limitations on cell concentration. In addition, microsomes cannot be efficiently prepared from all cultured cell types. We have found that standard microsome preparation procedures as used for human or rodent liver were unsuitable for isolating active enzymes from human lymphoblasts, and this appears to be a general property of cultured cell lines. Specific catalytic activities in microsomes were lower than for whole cell lysates. This loss of activity appears to happen in other mammalian cell systems which has led to the common use of whole cell lysates.With human lymphoblasts, shortening the length of... 

The oxidation of acetaldehyde to acetic acid has been studied with NAD-linked ALDH purified from human, rat and Syrian hamster liver (Klyosov et al., 1996). The mitochondrial enzymes from these species have very similar kinetic properties, whereas human cytosolic ALDHl has a value of about 180 pM, compared with 15 pM and 12 pM for rats and hamsters, respectively. Apparently, in human liver, only mitochondrial ALDH oxidizes acetaldehyde at physiological concentrations, whereas both mitochondrial and cytosolic ALDHs of rodents can participate in acetaldehyde metabolism. The rodent cytosolic ALDHs are at least 10 times more sensitive that the human enzyme to inhibition by disulfiram. 

TABLE I - Enzyme concentration in developing human liver... 

The major enzyme involved in the formation of ammonia in the liver, brain, muscle, and kidney is glutamate dehydrogenase, which catalyzes the reaction in which ammonia is condensed with 2-oxoglutarate to form glutamate (Sec. 15.1). Small amounts of ammonia are produced from important amine metabolites such as epinephrine, norepinephrine, and histamine via amine oxidase reactions. It is also produced in the degradation of purines and pyrimidines (Sec. 15.6) and in the small intestine from the hydrolysis of glutamine. The concentration of ammonia is regulated within narrow limits the upper limit of normal in the blood in humans is 70/tmol L-1. It is toxic to most cells at quite low concentrations hence there are specific chemical mechanisms for its removal. The reasons for ammonia toxicity are still not understood. The activity of the urea cycle in the liver maintains the concentration of ammonia in peripheral blood at 20/ molL. ... 

Alveoli highly permeable to many biologies most small molecules and many macromolecules capable of absorption throughout the respiratory tract Relatively rapid, first-order absorption Much less first-pass metabolism and degradation in the gastrointestinal tract and liver Cytochrome P450 enzyme concentration in human lungs <0.7% that of liver... 

Microsomal Incubation Conditions Incubations in animal or human liver microsomes are the most common way to determine activity in the presence of added substrate, UDPGA, Mg, and a buffer. As there is no method available to directly determine enzyme concentration, the incubations are standardized by addition of the same amount of protein (typically 0.25-1.0 mg protein/ImL) after determination of linearity of product formation with respect to protein concentration and time. In general, the enzyme is stable up to 45 min to 1 h. Because of the location of the enzyme, a portion of the microsomal vesicle will be obtained in the normal configuration with the enzyme active site entrapped within the vesicle. Since UDPGA must have access to the active site, and the UDPGA influx transporter is not operative without ATP, it may be necessary to activate or remove latency of the enzyme. In the past this has been achieved by a variety of methods, but most commonly by addition of detergents such as Brij 58, Lubrol, or Triton X... 

Miwa M, Ura M, Nishida M, Sawada N, Ishikawa T, Mori K, Shimma N, Umeda I, Ishitsuka H. Design of a novel oral fluoro-pyrimidine carbamate, capecitabine, which generates 5-fluorouracil selectively in tumours by enzymes concentrated in human liver and cancer tissue. Eur J Cancer 1998 34(8) 1274-81. 

Selden, C., Seymour, C.A. and Peters, T.J. (1980). Activities of some free radical scavenging enzymes and glutathione concentrations in human and rat liver and their relationship to the pathogenesis of tissue damage in iron overload. Clin. Sci. 58, 211-219. 

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