Keto acids, detection

The major advantage of DMB is the superior sensitivity for most a-keto acids detection limits of 6-29 fmol per 5 pi sample volume are reported [428]. Similarly, 1,2-diamino-4,5-dimethoxybenzene and l,2-diamino-4,5-ethylenedioxybenzene can be used for the determination of a-keto acids. 

The compound (III) can however lose ethanol by an internal Claisen ester condensation (p. 264) to give the cyclohexane derivative (IV), which, being the ester of a (3-keto acid, in turn readily undergoes hydrolysis and decarboxylation to give 5,5Hiimethyl cyclohexan-i,3Hiione (V) or Dimedone, a valuable reagent for the detection and estimation of formaldehyde. 

In intact cell systems or vivo, the primary products of a-hydroxylation, 22. have not been detected. The principal urinary metabolites of NNN resulting from a-hydroxylation are keto acid 21 from 2 -hydroxyl at ion and hydroxy acid 21 from 5 -hydroxylation. Trace amounts of 7 y 21> H ve also been detected as urinary metabolites (34). The interrelationships of these metabolites as shown in Figure 2 have been confirmed by administration of each metabolite to F-344 rats (37). The other metabolites which are routinely observed in the urine are NNN-1-N-oxide U1 and 5-(3-pyridyl)-2-pyrrolidinone [norcotinine, ]. The p-hydroxy derivatives 2. 1 were also detected in the urine of NNN treated rats, but at less than 0.1% of the dose (36). An HPLC trace of the urinary metabolites of NNN is shown in Figure 3. Urine is the major route of excretion (80-90% of the dose) of NNN and its metabolites in the F-344 rat in contrast to NPYR which appears primarily as CO2 (70%) after a dose of 16 mg/kg (17). This is because the major urinary metabolite of NNN, hydroxy acid 21> fs not metabolized further, in contrast to 4-hy-droxybutyric acid [2, Figure 1] which is converted to CO2. In addition, a significant portion of NNN is excreted as NNN-l-N-oxide U ], a pathway not open to NPYR. 

Evidence of the formation of mono-M-octylphthalatc and phthalate ester metabolites has been shown in in vitro studies. The appearance of mono-w-octylphthalatc was observed with preparations of human small intestine, rat liver and intestine, ferret liver and intestine, and baboon liver and intestine (Lake et al. 1977). However, the amount of phthalic acid and other metabolites in these preparations was either minimal or not detected. The study authors concluded that di-ra-octylphthalate is probably absorbed primarily as mono- -octylphthalate (Lake et al. 1977). An in vitro study reported the formation of five keto acids and two diols when metabolic oxidation of the alkyl groups of di-ra-octylphthalate was simulated abiotically (Brodsky et al. 1986). Therefore, the in vivo and in vitro data indicate that major oxidation may occur in the remaining alkyl chain after di-ra-octylphthalate has been hydrolyzed to the... 

An analogous sequence to that described for ellipticine was used to convert 5-methoxyindole (1171) to 9-methoxyellipticine (229). Noteworthy is the fact that none of the other regioisomeric keto acid could be detected by analysis of the crude product 1177. Using this sequence, 9-methoxyellipticine (229) was obtained in 47% overall yield based on 1171. O-Demethylation of 9-methoxyellipticine (229)... 

When male F-344 rats were injected with NNN-2 -14c, 75-95% of the dose was excreted in the 48 hr urine. In one experiment, the urine was collected in vessels containing DNP reagent. However, the DNPs of 4-hydroxy-l-(3-pyridyl)-l-butanone and 4-hy-droxy-4-C3-pyridy1)butanal were not detected. Since this was likely due to further oxidation in vivo, methods were developed for isolation of their probable oxidation products. This resulted in identification of the lactone, 5- C3-pyridyl)—tetrahydrofuran-2-one (1-2%), the keto acid, 4-(3-pyridyl).-4-oxobutyric acid (1-2%) and the hydroxy acid, 4-(3-pyridyl)-4-hydroxybutyric acid (26-40%) as urinary metabolites. These metabolites resulted. 

The dinitrophenylhydrazine (DNPH) assay detects a-keto acids in the urine [1, 4]. 

Aldonic and ketoaldonic acids may be difficult to separate. Norris and Campbell separated ketogluconic acid from gluconic acid by preparing the phenylhydrazone of the keto acid, which is then easily separable from gluconic acid. A methanol-ethanol-water solvent mixture was used.64 Another useful solvent developer is 1-butanol-formic acid-water.133 Macek and Tadra separated a series of ketoaldonic acids in a number of acidic solvent developers.134 Color reagents which may be used include ammoniacal silver nitrate,94 o-phenylenediamine dihydrochloride,84 and aniline acid oxalate.138 Lactones are detectable with hydroxylamine followed by ferric chloride.128... 

The principal low-level impurity was later identified to be the keto acid produced by oxidation of the ketoaldehyde. A very small amount of the benzoic acid resulting from hydrolysis of the methyl ester could also be detected. 

The a-keto acids are not present as such in the alkaloids as no free keto group can be detected. It was thus assumed that an a-hydroxy-a-amino acid group was present in the alkaloids, which would decompose to the corresponding a-keto acid and 1 equivalent of NH3 (82, 86). 

Higher alcohols Higher alcohols are produced as a deviation of the metabolism of amino acids. Higher alcohols are produced when keto acids corresponding to the carbon skeleton from the different amino acids are decarboxylated and reduced. Higher alcohols are normally below their limit of detection but they are the precursors of some esters, which have a large sensory impact. 

The distribution of metabolites obtained after incubation of pineapple slices with keto acids and keto esters, potential precursors of the corresponding hydroxy compounds, is summarized in Table II. The metabolization steps comprise esterification, reduction to hydroxy compounds, formation of acetoxy esters, and cyclization to the corresponding lactones. Metabolization rate and distribution of formed products strongly depend on the structures of the precursors. The detection of these metabolites proves the enzymatic capability of pineapple tissue to catalyze these conversions, an aspect which might be interesting for future use of pineapple tissue cultures in the production of chiral compounds. 

Lipoic acid acts as one of the coenzymes in the oxidative decarboxylation of pyruvate and other a-keto acids. It can be separated in an alkaline environment on a strongly basic anion exchanger in the hydroxide form, and can be detected like carbohydrates via pulsed amperometry at a Au working electrode. The corresponding chromatogram of a lipoic acid standard is shown in Fig. 8-88. This method allows to accurately detect 0.1 nmol lipoic acid. 

The profiles of compounds (30) and (34) are shown in Table 1. As can be seen in the above table, the potency (biochemical and cellular) of keto acid 30 is superior to the sulfonamide 34. However, due to difficulties with solubility and detection of the compound in LC/MS, the full ADMET profile could not be gathered for compound 30. Compound 34 although more readily profiled in the ADMET assays was shown to have several liabilities, including microsome instability, inhibition of... 

De Schepper et al. (D15) found also a-keto-P-methylvaleric acid in normal whole blood in contradiction to the previous results (H19) they detected also a-ketoisovaleric acid, the keto acid corresponding to valine. The concentration of this keto acid in normal whole blood of children is 0.13 0.02 mg% (Kl). Traces occur also in urine (Kl, VI). 

T6. Taylor, K. W., and Smith, M. J. H., l,2-diamino-4-nitrobenzene as a reagent for the detection and determination of alpha-keto acids in blood and urine. Analyst 80, 607 (1955). 

No activity was detected with the a-keto acid analogs of glycine, glutamine, asparagine, phenylalanine, tyrosine, tryptophan, arginine, citrulline, and aspartate. 

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