Krebs cycle acids

Derivatives Krebs cycle acids have been analyzed using only the TMS derivatives, even though some are keto acids. 

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-... 

An analogous procedure was suggested for the methylation of the Krebs cycle acids [133], The derivatives of some acids (pyruvate, fumarate) show thermal instability, however. 

This is the case, e.g., with TMS derivatives of Krebs cycle acids, containing keto groups. These derivatives do not provide a simple peak, probably owing to on-column decomposition and, therefore, if keto acids are present in the sample conversion into an oxime [159] is recommended. A 10-mg amount of acids and 10 mg of hydroxylammo-nium chloride are placed in a 5-ml test-tube fitted with a ground-glass stopper and are dissolved in 1 ml of dry pyridine. After standing for 10 min at room temperature 0.1 ml of TMCS and 0.1 ml of HMDS are added. The reaction is completed within a few minutes... 

For the quantitative determination of Krebs cycle acids, Horning et al. [160] recommended methoxime-TMS esters. A 10-mg amount of methoxylammonium chloride is added to a solution of 1 mg of a keto acid (or a mixture of acids) in 0.5 ml of pyridine. After standing for 2-3 h, pyridine is removed with a stream of nitrogen and 0.2 ml of BSTFA is added to the residue containing methoxime and unreacted methoxylammonium chloride. After standing for 1—2 h at room temperature, 0.5—1.0 /al of the solution is taken directly for GC analysis. 

Ion-exclusion chromatography finds numerous applications for identification and determination of acidic species in complex matrix materials, such as dairy products, coffee, wine, beer, fruit juice, and other commercial products which can be quickly analyzed with minimal sample preparation before injection (usually only filtration, dilution, or centrifugation). Organic acid determination is also of great importance in biomedical research (e.g., physiological samples, in which most of the Krebs cycle acids (tricarboxylic acid cycle) are present). 

The 110-hydroxylase in adrenal homogenates of the guinea pig O is maximally stimulated by Krebs-cycle acids in the absence of NADPH while that of the rat l is stimulated by both. The response in the rat varies with the Ca concentration suggesting a partial dependence on the degree of mitochondrial swelling. Minced rat adrenals convert P to DOC and then to B and 18-OH D0C 2 Hi concentrations of NADPH Inhibit 21- but stim-... 

Stimulation of lip-hydroxylase activity by Krebs-cycle acids is inhibited by CN and AmytalS. With rat adrenal homogenates, Amytal is not active in presence of isocitrate or Ca and the CN effect is reversed by isocitrate + NADPH. Oligomycin is inactive but Antimycin A, ferricy-anide and dinitrophenol act like Amytal. This supports the concept of a link between the classical electron transfer chain and the P-if50 containing hydroxylating system. 

Some of the small molecular weight dicarboxylic acids belong to the Krebs cycle acid family. There are many dicarboxylic acids in addition to the Krebs cycle acids. Examples of this family are azelaic acid (H00C(CH2)7C00H), brassylic acid (systematic... 

Progress Report on the Quantitative Analysis by Means of Gas Chromatography of Krebs Cycle Acids in Urine... 

ION-300 Interaction Chemicals 300 X 7.8 8 Sulfonate groups Separator for Krebs cycle acids... 

Interaction Chemicals also offers two ion-exclusion columns for the separation of organic acids under the trade names ORH-801 and ION-300. Both stationary phases contain a 8-pm substrate which was optimized for its respective purpose by slight modifications of the chemical structure. While the separation properties of the ORH-801 column are very similar to those of the Dionex lonPac ICE-ASl, the ION-300 column was especially developed for the analysis of the Krebs cycle acids. Citric and pyruvic acid, succinic and lactic acid, as well as oxalacetic and a-ketoglutaric acid may also be successfully separated on this stationary phase. Such separations are of importance for the analysis of fruit juices, foods, and physiological samples. Figure 5-5 shows a respective separation of a standard mixture of organic acids, carbohydrates and alcohols, which resembles the matrix of a variety of beverages. 

Where biochemicals such as Krebs cycle acids and amino acids occur at... 

With the introduction of trimethylsilylation reactions, Horii et al. (1965b) described the gas chromatography of Krebs cycle acids as trimethylsilyl derivatives on a 3 per cent SE 52 column, while shortly after this Dalgliesh etal. (1966) also reported on their separation in addition to many other organic acids. Hydroxyl functions were converted to trimethylsilyl ethers and carboxyl groups to either methyl or trimethylsilyl esters. Polysiloxane phases were found to be the most suitable column coatings, with F-60 favoured over SE-30... 

Differences between the different methyl silicones and substituted methyl silicones for carrying out various separations should be considered in the light of the compounds of interest. For example, OV-17 separates all the Krebs cycle acids as TMS-esters, with the exception of succinic and fumaric, whilst these two acids are partially separated on OV-22 and completely separated on OV-101. Similarly the TMS-ether-TMS-esters of citric acid and isocitric acid are co-eluted on packed SE-30, OV-1 and OV-101 columns, but are resolved using a selective-phase (OV-17, OV-22, OV-25) column (Homing etal., 1967 Chalmers, 1974). 

The best polyester phase for separating methyl ester derivatives of Krebs cycle acids depends on several factors, but poly(ethylene glycol) adipate (PEGA) has been shown in one study (de Silva, 1971) to give the best separation and most stable baseline of the several phases tested. In this instance aim column of 5 per cent (w/w) PEGA on silanized Chromosorb P (100-120 mesh) support with column temperature programming from 80 to 180°C at 10°C min" separated all tricarboxylic acid cycle acids in addition to lactic and pyruvic acids within about 14 min. 

The 0X0 acids have been discussed in part in the section on the Krebs cycle acids, however, the importance of branched-chain aliphatic 2-oxo acids in valine, leucine and isoleucine metabolism and of oxo acids in other disorders prompts further comment of their chromatographic analysis. 

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