Gorig Brunner

Formation of S-nitroso compounds from sodium nitroprusside, nitric oxide or nitrite and reduced thiols: analysis by capillary isotachophoresis

Abstract Sodium nitroprusside (SNP) is a potent, rapidly acting intravenous hypotensive agent. The direct action on blood vessels is thought to be due to its nitroso (NO) group. This suggestion is supported by the discovery of an endothelial relaxing factor, most probably being the radical nitric oxide (NO·) or a S-nitroso compound. In this paper we describe for the first time an analytical capillary isotachophoretic (ITP) method for the determination of SNP, of some potent vasodilating and platelet anti-aggregatory S-nitroso compounds from biological and pharmacological precursors, and of their final metabolites nitrite and nitrate. ITP was applied to study SNP stability in aqueous solutions and chemical formation of S-nitroso compounds by the reaction of reduced thiols such as N-acetyl- l -cysteine, glutathione and N-acetyl- d,l -penicillamine with SNP at pH 7.4. We found that these thiols react spontaneously with SNP to give the corresponding S-nitroso compounds. This result suggests that the vasodilating and anti-aggregatory properties of SNP may be in part due to the formation of S-nitroso compounds from SNP.

New method for the quantitative assay of the enzymatic hydrolysis of udpglucuronic acid using analytical capillary isotachophoresis

Abstract The microsomal fraction of mamamalian liver homogenate contains a pyrophosphatase activity whicy catalyses the hydrolysis of UDPglucuronic acid to UMP and glucuronic acid 1-phosphate. A further phosphatase activity cleaves these products to uridine and phosphate, and glucuronic acid and phosphate, respectively. A new method for the assay of these reactions is described using analytical capillary isotachophoresis. This technique permits the simultaneous quantitative analysis of mixtures containing UDPglucuronic acid, the primary products of hydrolysis, UMP and glucuronic acid 1-phosphate, and the secondary products, phosphate and glucuronic acid. The sensitivity of the method is sufficient for the accurate determination of 0.1 nmole of these substances applied to the isotachophoresis instrument in a volume of 1–10 μl, i , e , for solutions of concentration as low as 10 μmole/l. The sample-to-sample analysis time, including washing of the capillary and resetting of the instrument is of the order of 40 min. The major advantages of this method are that a complete analysis is possible without the need for radioactively labelled compounds and the analysis time is considerably less than for a conventional chromatographic separation.

Ion-pair extraction of histamine from biological fluids and tissues for its determination by high-performance liquid chromatography with fluorescence detection

A procedure for the selective ion-pair extraction of histamine from various biological fluids and tissues by using the ion-pairing agent bis(2-ethylhexyl)hydrogen-phosphoric acid has been developed. This procedure enabled routine determination of histamine in plasma, urine and tissue samples of humans and rats by reversed-phase high-performance liquid chromatography with fluorescence detection using o-phthaldialdehyde.

Gas chromatography–mass spectrometry of cis-9,10-epoxyoctadecanoic acid (cis-EODA): I. Direct evidence for cis-EODA formation from oleic acid oxidation by liver microsomes and isolated hepatocytes

Oleic acid, cis-9-octadecenoic acid, is the major fatty acid in mammals. Its oxide, cis-9,10-epoxyoctadecanoic acid (cis-EODA), has been identified in blood and urine of humans, its origin is, however, still unknown. Lipid peroxidation and enzyme-catalyzed epoxidation of oleic acid are two possible sources. In the present article, we investigated by HPLC and GC-MS whether cis-EODA is formed enzymatically from oleic acid by the cytochrome P450 (CYP) system. Oleic acid, cis-EODA and its hydratation product threo-9,10-dihydroxyoctadecanoic acid (threo-DiHODA) were quantitated by HPLC as their p-bromophenacyl esters. For structure elucidation by GC-MS, the pentafluorobenzyl (PFB) esters of these compounds were isolated by HPLC and converted to their trimethylsilyl ether derivatives. Liver microsomes of rats, rabbits and humans oxidized oleic acid into cis-EODA. This is the first direct evidence for the enzymatic formation of cis-EODA from oleic acid. The epoxidation of oleic acid was found to depend on CYP, NADPH+H(+), and O(2). cis-EODA was measurable in incubates of liver microsomes for up to 30 min of incubation. Maximum cis-EODA concentrations were reached after 5-7 min of incubation and found to depend upon oleic acid concentration. Isolated rat hepatocytes hydratated cis-EODA into threo-DiHODA which was further converted to unknown metabolites. However, from incubation of oleic acid with these cells we could not detect threo-DiHODA or cis-EODA. Our study suggests that circulating and excretory cis-EODA may originate, at least in part, from CYP-catalyzed epoxidation of oleic acid. GC-MS of intact cis-EODA as its PFB ester in the negative-ion chemical ionization mode should be useful in investigating the physiological role of cis-EODA in man.

026 Determination of histamine in food by combined ion-pair extraction and reversed-phase HPLC with fluorescence detection

The limit of detection for the described method using UV detection is 5 ng. An increased sensitivity is obtained with fluorescence detection with a detection limit of 1 ng. The linearity and sample recovery were tested by using spiked serum samples. The linearity of the method was confirmed in the range of 0 4 5 gg/ ml for standards and 0 30 gg/ml for serum. The recovery was between 90 and 100%. To determine the within-day and dayto-day precision of this method we used serum pools within and out of the reference area ( 5 20 gg/ml, 11 .6 46.4 gmol/l). For UV detection the coefficient of variation within-day (n = I 0) was between 1.I and 1.9% for the standards and 1.2 and 2.2% for the serum pools. The day-to-day (n = 10) coefficient of variation was 1.4 to 2.4% for the standards and 4.1 to 4.8% for the samples. For fluorescence detection the coefficient of variation within-day (n = 10) was between 0.3 and 0.7% for the standards and 0.3 and 0.9% for the serum samples. The day-to-day coefficient of variation ( n = 10) was 0.5 to 0.9% for the standards and 1.1 to 1.5% for the serum pools. Typical chromatograms are shown in Fig. 1. The presented method allows a routine determination of alpha-tocopherol in serum with UV detection. Using fluorescence detection it may be proposed as a candidate reference method.

Capillary isotachophoretic determination of cysteinyl leukotrienes

The cysteinyl leukotrienes (LTs) C4, D4 and E4 are among the most potent lipid mediators of anaphylaxis and inflammation. A capillary isotachophoretic method is described for the determination of these cysteinyl LTs. The method is based on anionic separation and detection using UV (254 nm) and conductivity detectors. The total analysis time is of the order of 30 min. The limit of detection of the method was determined to be 0.5 nmol of LTE4. Despite of similar chemical structures, all three cysteinyl LTs can be determined simultaneously.

Analytical capillary isotachophoresis of bis(2-ethylhexyl) hydrogenphosphate and 2-ethylhexyl dihydrogenphosphate

Abstract Bis(2-ethylhexyl) hydrogenphosphate (H2DEHP) and 2-ethylhexyl dihydrogenphosphate (HDEHP) are widely used liquid cation exchangers. An analytical capillary anionic isotachophoretic method was developed for the determination of H2DEHP and HDEHP in methanolic and aqueous phosphate-buffered solutions. The detection limit of the method was 3 nmol for H2DEHP and 0.16 nmol for HDEHP. H2DEHP was determined in aqueous phases at concentrations down to 15 μmol/l when it was previously ion-pair extracted by use of tetrabutylammonium hydrogensulphate. The recovery and the intraassay relative standard deviation of the method including the ion-pair extraction procedure were 78% and 6.8%, respectively.

Agarose-Encapsulated Adsorbents I. Concept and General Properties

The development of a new material for use in haemoperfusion systems, agarose-encapsulated adsorbents, is reported. In contrast to conventional „coating“ methods, the required adsorbing phase, such as active charcoal or resin, is actually trapped within the matrix of the agarose gel, which is formed into smooth, soft, but resilient beads of virtually any desired diameter, conveniently in the range 3–5 mm. It is our belief, derived from the unusual properties of this system which have so far emerged, that the technique of agarose-encapsulation could provide a further step forward in the field of extracorporeal detoxification. The decisive advantage of the new material is undoubtedly the possible use of adsorbent phases in powder form encapsulated in the beads, which affords a vastly improved degree of adsorption without a negative effect on haemocompatibility. An approach to a semi-selective system is offered by the use of combinations of adsorbers in the beads.

Detoxification of Phenols by Sulphate Conjugation: An Alternative to Glucuronidation in an Enzymatic Liver Support System

The relevance of phenolic compounds in acute liver failure is well established. There are two conjugating processes in the liver which are normally responsible for the detoxification of this class of substance, namely the glucuronyl and sulphate transferases, of which the latter play a subordinate role in vivo. In an artificial liver support device, however, sulphate conjugation presents several advantages over glucuronidation. The apparent superiority of glucuronidation is simply attributable to the availability of the donor molecule, a situation which is reversible in an artificial system, where the quantity of donor molecule can be controlled. Some characteristics of sulphate transferase are discussed in this contribution, together with some suggestions for the application of this enzyme in an extracorporeal detoxification device.

Helicobacter pylori, Its Urease and Carbonic Anhydrases, and Macrophage Nitric Oxide Synthase

In their recent review article entitled “The immune battle against Helicobacter pylori infection: NO offense”, Gobert and Wilson [1] provided an excellent review on the most recent literature reporting on interactions between Helicobacter pylori infection and nitric oxide ( NO) synthesis in host cells and on potential underlying mechanisms. Emphasis was given to the involvement of urease in the detoxification of peroxynitrite (ONOO−), the reaction product of the radicals NO and superoxide anion (O2–) [1].