Tricarboxylic acids, separation

Plant metabolism can be separated into primary pathways that are found in all cells and deal with manipulating a uniform group of basic compounds, and secondary pathways that occur in specialized cells and produce a wide variety of unique compounds. The primary pathways deal with the metabolism of carbohydrates, lipids, proteins, and nucleic acids and act through the many-step reactions of glycolysis, the tricarboxylic acid cycle, the pentose phosphate shunt, and lipid, protein, and nucleic acid biosynthesis. In contrast, the secondary metabolites (e.g., terpenes, alkaloids, phenylpropanoids, lignin, flavonoids, coumarins, and related compounds) are produced by the shikimic, malonic, and mevalonic acid pathways, and the methylerythritol phosphate pathway (Fig. 3.1). This chapter concentrates on the synthesis and metabolism of phenolic compounds and on how the activities of these pathways and the compounds produced affect product quality. 

Aconitase, an unstable enzyme,4 is concerned with the reversible conversion of cis-aconitate to either citric acid or isocitric acid. It may be noted that the entire system of tricarboxylic cycle enzymes are present in the mitochondria separated from cells, and, furthermore, it has been found that the mitochondrial enzymes differ from the isolated enzymes in that the former require no addition of D.P.N. (co-enzyme I) or T.P.N. (co-enzyme II) for activity. Peters suggests that the citrate accumulation is caused by the competitive reaction of the fluorocitrate with aconitase required for the conversion of citrate to isocitrate. This interference with the tricarboxylic acid... 

One example is the separation of tricarboxylic acid cycle organic acids. These organic acids were originally separated on sulfonic acid-treated silica gel as the stationary phase material, and a chloroform and isopentyl alcohol mixture was used as the eluent. However, this eluent is not suitable for UV detection. A mixture of -hexane, THF, and ter/-butanol was therefore selected for the same separation.74... 

Free fatty acids can be separated on a Ci8 column based on carbon number using 50% MeOH/water pH 2.5 at UV, 280 nm a fatty acid column (actually a phenyl column) will also separate them based on the number of double-bonds. Fatty acids have also been analyzed at UV, 210 nm, or by refractive index. For high-sensitivity work, they are derivatized with bromophenacylbromide and separated on Ci8 in a 15-80% AN/water gradient at 254 nm. Increase in early running C2 and C4 fatty acids measured by HPLC is used as an indicator of bacterial action. Krebs cycle acids are di- and tricarboxylic acids involved in metab-... 

Knappe, E. and Rohdewald, I., Thin-layer chromatography of dicarboxylic acids. V. Separation and identification of hydroxy dicarboxylic acids, of di- and tricarboxylic acids of the citrate cycle, and some other dicarboxylic acids of plant origin, Z. Anal. Chem., 211, 49, 1965 Chem. Abs., 63, 7333c, 1965. 

These organelles are the sites of energy production of aerobic cells and contain the enzymes of the tricarboxylic acid cycle, the respiratory chain, and the fatty acid oxidation system. The mitochondrion is bounded by a pair of specialized membranes that define the separate mitochondrial compartments, the internal matrix space and an intermembrane space. Molecules of 10,000 daltons or less can penetrate the outer membrane, but most of these molecules cannot pass the selectively permeable inner membrane. By a series of infoldings, the internal membrane forms cristae in the matrix space. The components of the respiratory chain and the enzyme complex that makes ATP are embedded in the inner membrane as well as a number of transport proteins that make it selectively permeable to small molecules that are metabolized by the enzymes in the matrix space. Matrix enzymes include those of the tricarboxylic acid cycle, the fatty acid oxidation system, and others. 

Aliphatic tricarboxylic acids such as citric acid exhibit a remarkably high affinity toward the stationary phase of an anion exchanger. Hence, low ionic strength bicarbon-ate/carbonate buffer solutions are not particularly suited as eluents. However, when a sodium hydroxide solution at a comparatively high concentration (c 0.08 mol/L) is used, citric acid may be eluted, and may even be separated from its structural isomer isocitric acid. When the detection of these compounds is carried out via electrical con-... 

Di- and tricarboxylic acids such as oxalic and citric acid elute between the excluded and the total permeated volume. Apart from Donnan exclusion, the predominating separation mechanism is, in this case, mainly steric exclusion. The retention is determined by the size of the sample molecule. Since the pore volume of the resin is established by its degree of crosslinking, the resolution can only be improved by applying another or by coupling with another separator column, respectively. 

Gas chromatography, which has been applied to the separation of the tricarboxylic acid cycle acids in the liver (F8), might prove useful in the future in the field of organic acids of blood and urine. 

Tbe next mejor comasereial success was the family of composite membranes. They feature a very thin RG membrane on a suitable substrate, usually a UF membrane. Most of the RO composite membranes are polyamide cnatings in which the separating layer is produced by interfacial polymerization of a diamine and a multibasic acid chloride. The most snecessfol recent memhrane is based on an inteifacial polymer of 1,3-diamino benzene and 1,3,5-benzene tricarboxylic acid chloride coated on a polysulfone membrane substrate. 

Calcium citrate is produced as an intermediate in the purification of citric acid (CeHgO , or 2-hydroxypropane-l,2,3-tricarboxylic acid). A broth of citric acid is produced by the fermentation of sugar using a strain of Aspergillusniger mould. Suspended solids are removed by filtration. The solution is reacted with slaked lime to precipitate calcium citrate, which is separated by filtration. 

Acetyl CoA feeds into a remarkable biochemical sequence, variously known as the tricarboxylic acid (TCA) cycle, the citric acid cycle or, after its discoverer, the Krebs cycle (Fig. 14.1). It is called a cycle because it does indeed end up where it started, at a compound called oxaloacetate. Oxaloacetate combines with acetyl CoA, releasing the CoA and forming citric acid (so named because plentiful in citrus fruit juices). A sequence of seven further reactions takes citrate back to oxaloacetate. What has happened is that the two carbons in the acetyl group of acetyl CoA have been taken up (into citrate) and two carbons have been separately released as CO2 in the reactions of the cycle. If we tracked individual carbon atoms like ringed birds (as we can with radioactive isotopes - see Chemistry X), we would find that the cycle does not release precisely the same two carbon atoms that entered, but that need not concern us. In net chemical terms, it does not matter where the atoms came from. At the end of the cycle, the amounts of aU the TCA cycle intermediates are unchanged, and in effect an acetyl group has been oxidised to 2CO2 (see Appendix 8). 

Another long-known example of 2D inclined interpenetration is the structure of trimesic acid (1,3,5-benzene-tricarboxylic acid). It contains hydrogen-bonded (6.3) sheets that interpenetrate such that each window of each sheet is penetrated by three other inclined sheets. It is also possible for more than two stacks of parallel sheets to show inclined interpenetration—the remarkable structure of Co2(azpy)3(N03)4-Me2CO-3H20 [a /73 =4,4 -azo/ zT-(pyridine)] contains four separate stacks of parallel (6,3) sheets that all interpenetrate at mutually inclined angles.Combinations of network and interpenetration topologies are also possible. Inclined interpenetration between (4,4) and (6.3) sheets has been reported,and... 

The following series of tests facilitate separation of the two genera of AAB based on their relative ability to oxidize acetic acid. Overoxidizers Acetobacter sp.) convert the acid via the Tricarboxylic Acid (TGA) cycle to COg and HgO, whereas acetate is a end product in the case of Cluconobacter. 

In considering the effect of insulin or diabetes on carbohydrate metabolism of muscle preparation in vitro, the effect of insulin on the conversion of glucose to CO2 and the fate of the various possible pathways for glucose—glycolysis, the tricarboxylic acid cycle, the hexose monophosphate shunt, and glycogen synthesis—will be examined separately. 

Goebell andKuNGENBEBG [34] have separated several acids of the tricarboxylic acid cycle, using two-dimensional TLd on cellulose layers and the solvents, ethanol-25 % ammonium hydroxide-water (72.6 + 18.2 + 9.2) and isobutanol-5M formic acid (40 + 60). They concluded with quantitative evaluation by autoradiography. The combination of TLC with enzyme test methods permitted accurate quantitative determination of these acids in the nanomole region. 

Thermo Fisher Scientific offers two ion-exclusion columns Dionex lonPac ICE-ASl and AS6. The former is a moderately hydrophilic, microporous poly (styrene-co-divinylbenzene)-based cation exchanger with a particle diameter of 7.5 pm and ion-exchange capacity of 27 mequiv/column, functionalized with sulfonate groups. The lonPac ICE-ASl is primariliy used for the separation of weak inorganic acids, short-chain fatty acids, and alcohols. Difficulties are encountered, however, in the separation of aliphatic mono-, di-, and tricarboxylic acids. These acids elute from such stationary phases within the totally permeated volume. The selectivity of the separation in this retention range is usually very poor. Since the totally permeated volume is determined... 

Two of the enzymes of the tricarboxylic acid cycle, aconitase and fumarase, catalyze reactions in which water is added reversibly to an unsaturated polycarboxylic acid. Both enzymes exhibit rigid stereospecificity fumarase forms only L-malate from fumarate and forms only fumarate (trans) and not maleate (czs-ethylenedicarboxylic acid), and aconitase reacts with only cis-, not imns-aconitate, and with D-, not L-isocitrate. Citrate is a symmetrical molecule, with no optical isomers, but it will be shown that steric factors also enter into the reaction of this substrate with aconitase. The enzymes of the tricarboxylic acid cycle, in contrast to the glycolytic enzymes, are associated with intracellular granules known as mitochondria. Studies of the individual enzymes have depended to a large extent on the separation of soluble activities from these particles. Aconitase and fumarase are released from the particles very rapidly under mild conditions often in the preparation of cell-free homogenates these activities are largely solubilized, and special care must be taken to demonstrate their origin in mitochondria. 

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