Wolfram Schneider

Ion-pair reversed phase HPLC determination of nucleotides, nucleosides and nucleobases — Application to nucleotide metabolism in hepatocytes

SummaryThree groups of metabolites were analyzed in extracts of rat hepatocytes by an HPLC method: (i) nucleotides (ATP, ADP, AMP, GTP, GDP, UTP, UDP, IMP, UMP), (ii) nucleosides and nucleobases (adenosine, adenine, guanosine, inosine, xanthine, hypoxanthine, uridine) and (iii) inhibitors of xanthine oxidoreductase (oxypurinol, allopurinol). Perchloric acid extracts were neutralized with K2CO3/triethanolamine and analyzed at 254 nm by reversed-phase ion-pair high-performance liquid chromatography. The nucleotides and their derivatives were separated with a gradient elution using 10 mM NH4H2PO4/2 mM t-butylammonium-phosphate and acetonitrile. Recovery values were estimated for the extraction procedure used. The method was applied to the investigation of nucleotide metabolism of hepatocytes of starved rats at anoxia and reoxygenation.

Nucleotide and aldehyde analysis by HPLC for determination of radical induced damage

SummaryThe analysis of two metabolite groups, nucleotides and aldehydes, is necessary for assessment of oxygen radical metabolism during hypoxia and reoxygenation.Nucleotides and their derivatives were determined by HPLC using gradient elution with 10 mM NH4H2PO4 buffer containing 2 mM t-butylammoniumphosphate and acetonitrile.Aldehydes occuring after lipid peroxidation were analyzed by derivatisation to dinitrophenylhydrazones followed by TLC and HPLC separation with methanol/water on an ODS column.

Regulation of purine nucleotide metabolism in hypoxic liver and intestine of rats: radical scavenging effects of allopurinol and oxypurinol.

The relationship between nucleotide catabolism and generation of activated oxygen species was investigated in liver, hepatocytes and small intestine of rats. In severe hypoxia nucleotide degradation via xanthine oxidase and urate oxidase requires about half of the oxygen consumed. Data on the changes of nucleobase compounds in rat hepatocytes and small intestine during ischemia and reoxygenation and the effects of allopurinol and oxypurinol thereon are presented. From EPR measurements it is concluded that OH. radicals induce reactions of allopurinol yielding long-living products which are able to react with DMPO-OH with loss of its radical properties.

Mechanisms of Protection of Hepatocytes at Anoxia and Reoxygenation by the Xanthine Oxidase Inhibitor Oxypurinol

Oxygen-free radicals generated during postischemic reperfusion are thougt to be responsible for the damage which occurs on reoxygenation. However, the participation of free oxygen radicals on reperfusion injury in liver is still controversial,1,2 including the effects of xanthine oxidase inhibitors allopurinol and oxipurinol. The enzyme release which occurs upon reperfusion in the ischemic perfused rat liver was decreased by allopurinol.3,4 In contrast, allopurinol had no appreciable effect on recovery of the ATP level in ischemic perfused rat liver during reoxygenation.5 The discussion was stimulated since a radical scavinging activity of allopurinol and oxipurinol was prescribed.6,7

Dynamics in the Purine Nucleotides of Liver During Various Periods of Hypoxia/Ischaemia and Reoxygenation

The constituents forming the purine pool play a pivotal role in virtually all cellular functions.1,2 That implicates thermodynamic, kinetic as well as regulatory aspects. ATP, for instance, is best described as a kinetically versatile agent integrated in cell functions as an energy transducer, not as a source or store of energy.3 There exists a mutual dependence: ATP and the related nucleotides need other constituents of the purine pool as precursors the synthesis de novo or by salvage reactions of constituents of the purine pool requires energy transduced by ATP.

Influence of oxypurinol on colitis induced in rat colon by trinitrobenzene sulfonic acid treatment.

Chronically inflammatory intestinal diseases such as Morbus Crohn and Colitis ulcerosa are severe diseases highly impairing the condition of affected patients, not seldom leading to inability to work, and often require radical surgical measures with corresponding consequences.