Metabolic product, label distribution

Intracellular fluxes can be estimated more precisely through 13C tracer experiments. Following 13C feeding to a cell it is possible to analyze metabolic products, such as amino acids, and measure 13C enriched patterns, so to be able to reconstruct the flux distribution from the measured data [91]. To obtain flux data from the labeling patterns, two techniques can be applied NMR [92, 93] and MS [94, 95]. Due to the low intracellular concentration of metabolites, these are often difficult to measure therefore the analysis of the labeling pattern of amino acids in proteins is used as input for flux quantification. Here proteins are hydrolyzed to release labeled amino acids and further analyzed by NMR of GC-MS. Once NMR or MS spectra are recorded, the next step is the quantitative interpretation of the isotopomer data by using mathematical models that describe the relationship between fluxes and the observed isotopomer abundance [96, 97], Some of the mathematical approaches used include cumulative isotopomer (cumomers) [98], bondomers [99], and fractional labeling [100], For a more comprehensive review on the methods we refer to Sauer [91]. 

The use of C -labeled a-tocopherol offers obvious advantages over chemical methods in determining the distribution of a-tocopherol in the tissues. By measuring the total radioactivity, metabolic products of -tocopherol are included whereas they may be exeluded if the a-tocopherol is determined by chemical methods. On the other hand, if chemical determinations are used, Emmerie-Engel positive compounds not related to o-tocopherol may falsify the picture. Several investigators (Martius and Costeli, 1957 Alaupovic et al., 1961) have used this technique to follow the isolation and separation of metabolites from the liver. Other workers (Niedner, 1957 Sternberg and Pascoe-Dawson, 1959) have reported on the distribution of radioactivity in the tissues after administration of C -labeled a-tocopherol. 

Experiments with C -tryptophan have established that this amino acid is fairly readily oxidized to completion in the intact animal (883), about as readily as is phenylalanine, according to Dalgleish and Tabechian (884). I 18 true for the carbon atoms of the aromatic ring as well as the side chain. The distribution of the in the metabolic products of tryptophan is given in Table III. The large fraction of C in the tissues was present largely as tryptophan, according to Henderson et al. (883, 886). A considerable fraction of the label is always found in the urine, once a variety of incompletely oxidized products are invariably produced in the metabolism of tiyptophan. These will be discussed below. 

Hydroxylated metabolites are conjugated as glucuronides and sulfates. The balance of products in this last step and their distribution between urine and feces distinguishes the metabolism between humans, rats, and rabbits (Baldwin and Hutson 1980 Bedford et al. 1975b Hutson 1981 Hutson et al. 1975), as discussed in Section 2.3.4. Similarly, studies in lactating cows ingesting radio-labeled endrin in the diet for 21 days suggest metabolic pathways similar to those in rats and rabbits with apparent differences between the 3 species attributed more to differences in biliary versus renal excretion (Baldwin et al. 1976). 

There is a substantial literature on the transformation of simple phenolic acids by microorganisms.2,7,11,16,18,20,22,25,29,44 For example, ferulic acid is transformed by fungi to either caffeic acid or vanillic acid, and these are transformed to protocatechuic acid. Next the ring structure of protocatechuic acid is broken to produce 3-carboxy-c/s,c/s-muconic acid, which is then converted to (3-oxoadipic acid (Fig. 3.1), which in turn is broken down to acetic acid and succinic acid, and these ultimately are broken down to C02 and water.11,18,29 Flowever, distribution of residual 14C-activity after growth of Hendersonula toruloidea, a fungus, in the presence of specifically 14C-labeled ferulic acid ranged from 32 to 45% in C02, 34 to 45% in cells, 9 to 20% in humic acid and 4 to 10% in fulvic acid.29 Thus, a considerable portion of the ferulic-acid carbon was bound/fixed over a 12-week period, and the initial ferulic acid transformation products (e.g., caffeic acid, vanillic acid and protocatechuic acid) were clearly of a transitory nature. Similar observations have also been made for other simple phenolic acids 22,23 however, the proportions metabolized to C02 and fixed into cells and the soil... 

We labeled the lactose permease with a yellow fluorescent protein. The two specific phenotypes under discussion are first, above a certain threshold number of the permease, a cell has a fluorescent membrane and is capable of lactose metabolism second below this threshold, a cell is non-fluorescent and is incapable of lactose metabolism. The two phenotypes coexist in a population of cells (Fig. 22.7B) and show a bimodal distribution (Fig. 22.7C). This threshold is determined to be 300, corresponding to a big burst of permease production. 

Only metabolites of carbon disulfide were found 3 hours after a dose of 14C- or 35S-labeled carbon disulfide was intraperitoneally administered (Snyderwine and Hunter 1987). Distribution varied with the age of the rat and the radiolabel injected. Following intraperitoneal administration of 14C-carbon disulfide, 4-9% of the dose was metabolized to carbon dioxide depending on age. Significantly more carbon disulfide was metabolized to carbon dioxide by 30- and 40-day-old rats than by 1-20-day-old rats. The biotransformation products of carbon disulfide which were covalently bound remained in tissues from rats of all ages. Twenty-four hours after dosing with 35S-labeled carbon disulfide, up to 13 times more labeled... 

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