A new type of migrating zone boundary in electrophoresis: 3. The hybrid boundary and stacking criteria.

This article investigates the principles of stacking of analytes by a zone of a bulk sample component that possesses one hybrid boundary. The model system investigated comprises a transient stacking zone in a BGE where the rear boundary of this zone is a hybrid one. A theoretical description of such a system is given, and general rules and mathematical criteria for quantitative stacking are presented. These criteria are based on comparison of migration velocities of analytes to be stacked and velocities of the boundaries of the stacking zone. It is shown that the presence of a hybrid boundary brings about additional constraints for stacking of analytes due to the presence of the sharp part of the hybrid boundary that migrates faster than regular sharp boundary of this zone. An experimental example related to the described theory is also shown.

Concentration and purification of orotic acid directly from whey with an expanded bed adsorption system.

The development of an expanded bed process for the concentration and purification of orotic acid directly from whey is described. Different commercially available adsorbents were tested in series of pilot batch adsorption experiments to determine the most suitable separation system. Best results were achieved using a weak anion-exchange matrix. An elution protocol was established using MgCl2 as eluting agent to recover the adsorbed orotic acid with approximately 85% yield and 10-fold concentration. Purified orotic acid was precipitated under acid conditions with a yield of 95%.

Diagnostic value of urinary orotic acid levels: applicable separation methods.

Urinary orotic acid determination is a useful tool for screening hereditary orotic aciduria and for differentiating the hyperammonemia disorders which cannot be readily diagnosed by amino acid chromatography, thus reducing the need for enzyme determination in tissue biopsies. This review provides an overview of metabolic aberrations that may be related to increased orotic acid levels in urine, and summarises published methods for separation, identification and quantitative determination of orotic acid in urine samples. Applications of high-performance liquid chromatography, gas chromatography, and capillary electrophoresis to the analysis of urinary specimens are described. The advantages and limitations of these separation and identification methodologies as well as other less frequently employed techniques are assessed and discussed.

Simultaneous separation by high-performance liquid chromatography of carbamoyl aspartate, carbamoyl phosphate and dihydroorotic acid.

Leflunomide is an immunomodulatory drug which acts by inhibiting dihydroorotic acid dehydrogenase, the fourth enzyme of pyrimidine biosynthesis. We modified our high-performance liquid chromatography method to demonstrate that the principal metabolite in mitogen-stimulated human T-lymphocytes incubated with leflunomide was not dihydroorotic acid, but carbamoyl aspartate. Identification involved preparation of [14C]carbamoyl aspartate from [14C]aspartic acid and mammalian aspartate transcarbamoylase. Accumulation of carbamoyl aspartate indicates that under these conditions the equilibrium constant for dihydroorotase favours the reverse reaction. This HPLC method, enabling simultaneous separation of the first four intermediates in the de novo pyrimidine pathway may be of use in a variety of experimental situations.

Synthesis and antiproliferative activity of threo-5-fluoro-L-dihydroorotate.

Fluorinated compounds play an important role in enzymology as well as clinical medicine. Based on the stereochemical preferences of dihydroorotate oxidase and enzymes that use fluoroaspartate, it was anticipated that threo-5-fluoro-L-dihydroorotate (t-FDHO) would have the properties of an antimetabolite. Thus, t-FDHO was synthesized via the reduction of 5-fluoroorotate using NADH and dihydroorotate dehydrogenase that was free of dihydroorotase. When the product was purified and studied by high field proton and carbon 13 NMR, the fluorine, the five carbons, and all the nonexchangeable protons were readily observed. Confirmation of threo configuration was obtained by examining the vicinal coupling constants between the substituents on carbon 5 and carbon 6 of the newly synthesized compound. Additionally, t-FDHO could be reoxidized to 5-fluoroorotate in the presence of dihydroorotate dehydrogenase and NAD+. Treatment of t-FDHO with dihydroorotase generated N-carbamyl-threo-3-fluoro-L-aspartate (CTF-ASP) which was also purified and characterized by NMR. The antiproliferative activity of t-FDHO was determined against a diploid human fibrosarcoma cell line (HT-1080). Fifty microM t-FDHO caused 50% inhibition of HT-1080 cell proliferation. During the 48-h toxicity study, extracellular t-FDHO underwent significant hydrolysis to CTF-ASP. Further extracellular degradation to fluoroaspartate was not seen. The antiproliferative activity of t-FDHO was not due to extracellular degradation since CTF-ASP itself was essentially nontoxic.

Determination of urinary orotate excretion by high-performance liquid chromatography.

A new reversed-phase high-performance liquid chromatographic procedure for the determination of urinary orotate excretion is described. It is a selective, sensitive and rapid method, suitable for the differentiation of inherited metabolic diseases with abnormal orotate metabolism.

[Isolation of orotic acid from the liver and its metabolism in the rat]. / Vydelenie orotovoi kisloty iz pecheni i izuchenie ee obmena u krys.

A method is described for isolation of orotic acid from rat liver tissue for study of the kinetics of its radioisotopic labelling. Within 24 hrs after injection of 400 mg hydroxylamine per kg of body mass, the concentration of hepatic orotate decreased, while its transport into liver tissue and the kinetics of labelling of its endogenous pool remained unaffected.