Gerhard Gerber

Determination of nucleotides, nucleosides and nucleobases in cells of different complexity by reversed-phase and ion-pair high-performance liquid chromatography.

Procedures are presented for the analysis of profiles of purine and pyridine compounds in human and rabbit red blood cells by reversed-phase high-performance liquid chromatography and in Ehrlich ascites tumour cells of mouse by ion-pair high-performance liquid chromatography. These compounds are present in rabbit erythrocytes in higher concentrations than in human blood cells, and in rabbit reticulocytes the concentration of purine compounds is still higher. During glucose-free incubation, human red cells accumulate adenosine and adenine in the presence of coformycin owing to the inhibition of adenosine and AMP deamination. Ehrlich ascites tumour cells lose major portions of purine mono-, di- and triphosphates between the seventh and eleventh day after inoculation into mouse peritoneal cavities.

Response of the glycolysis of human erythrocytes to the transition from the oxygenated to the deoxygenated state at constant intracellular pH

The time course of the rate of the glycolysis of human erythrocytes and of some metabolites were determined before and after rapid deoxygenation at constant intracellular pH. For this purpose stripped deoxygenated haemoglobin was used as a rapid oxygen acceptor. Deoxygenation causes an increase of the glycolytic rate by 26%. Glucose 6-phosphate is decreased while the adenine nucleotides and 2,3-bisphosphoglycerate remain constant. Fructose 1,6-bisphosphate and the triose phosphates decrease transiently before rising. The data can be explained by increased binding of phosphocompounds to deoxygenated as compared with oxygenated haemoglobin. Thereby the control enzymes hexokinase and phosphofructokinase are influenced. It is concluded that under physiological conditions changes in the oxygenation state of haemoglobin per se alter the glycolytic rate.

A mathematical model to study short-term regulation of mitochondrial energy transduction

A mathematical model is presented which includes the following elementary process of mitochondrial energy transduction: hydrogen supply, proton translocation by the respiratory chain, proton-driven ATP synthesis by the F0F1-ATPase, passive back-flow of protons (leak) and carrier-mediated exchange of adenine nucleotides and phosphate. For these processes empirical rate laws are used. The model is applied to calculate time-dependent states of energy transduction in isolated rat liver mitochondria. From the general agreement of the computational results with experimental data (Ogawa, S. and Lee, T.M. (1984) J. Biol. Chem. 259, 10004-10011) the following conclusions can be drawn. (1) The length of the time interval during which mitochondria are able to maintain a relatively high and constant delta pH in the absence of oxygen (anaerobiosis) is limited by the availability of intramitochondrial ATP. (2) The overshoot kinetics of delta pH which appear when reoxigenating mitochondria after a preceeding anaerobiosis might be due to a lag phase kinetics of the F0F1-ATPase. (3) In phosphorylating mitochondria the homeostasis of delta pH is brought about by a high sensitivity of the respiration rate and the rate of the F0F1-ATPase as to changes of delta pH. (4) Analysis of the mean transient times shows that the rate of ATP synthesis in State 3 is controlled to almost the same extent by the hydrogen supply, the respiratory chain, the adenine nucleotide translocator and the proton leak.

Radical formation in the rat small intestine during and following ischemia.

Oxidative loading during the reperfusion of the proximal jejunum of rats following a one hour-period of complete ischemia was demonstrated in in vivo-experiments by the increases of the GSSG: total glutathione ratio and the concentration of TBA-RS. The pretreatment of the animals with the xanthine oxidoreductase inhibitor allopurinol diminished the accumulation of GSSG and of TBA-RS. It was concluded that the purine nucleotide degradation is an important source of oxygen reduction products in reoxygenated small intestine. The tissue concentrations of nucleotides, nucleosides and nucleobases were measured by an ion-pair reversed-phase HPLC separation. There occurred fast declines of ATP and GTP concentrations during ischaemia leading to temporary increases of nucleoside mono- and diphosphate pools. The hypoxanthine concentration is increased about twentyfold during oxygen deficiency. The ATP and GTP restoration during the reperfusion was accelerated in presence of allopurinol. The shares of the beneficial allopurinol effects are not yet clarified.

Determination of the ultraviolet absorbance and radioactivity of purine compounds separated by high-performance liquid chromatography. Application to metabolic flux rate analysis.

A double detection system for the determination of adenine metabolism in biological tissues using isocratic ion-pair reversed-phase chromatography is presented. Two isocratic ion-pair separations were used: (i) 10 mM NH4H2PO4, 2 mM tetrabutylammonium phosphate (PIC reagent A) and 18% acetonitrile for the determination of nucleotides and (ii) 50 mM KH2PO4, 1 mM PIC reagent A and 1% acetonitrile for the determination of monophosphorylated nucleotides, nucleosides and nucleobases. The parallel detection of ultraviolet absorbance at 254 nm and the radioactivity of separated purine compounds allows the detection of pool sizes and of the specific radioactivities in tracer kinetic experiments. The high-performance liquid chromatography methods were applied to the determination of flux rates during adenine nucleotide metabolism in suspensions of Ehrlich mouse ascites tumour cells. The pathways of adenine metabolism in cells during the proliferation and plateau phases of tumour growth were compared.

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.

Changes of nucleotide patterns in liver, muscle and blood during the growth of Ehrlich ascites cells: application of the reversed-phase and ion-pair reversed-phase high-performance liquid chromatography with radial compression column☆

The pool of purine compounds was analysed in liver, skeletal muscle and blood of mice during the growth of Ehrlich ascites tumour cells. Three fast isocratic high-performance liquid chromatographic methods were used. (1) Determination of nucleotides by an isocratic ion-pair reversed-phase chromatography with a 10 mM ammonium phosphate buffer containing acetonitrile and tetrabutylammonium phosphate. (2) Separation of nucleosides and nucleobases in cell extracts by a reversed-phase system with methanol and 50 mM potassium phosphate buffer as eluent. (3) Nucleosides and nucleobases in body fluids were analysed by a reversed-phase system with 10 mM potassium phosphate containing methanol. These methods allow the rapid determination of purine compounds in small biological samples from various cell types and body fluids, with high accuracy and sensitivity. The pool of cellular nucleotides increased during the exponential phase of tumour growth. Adenosine accumulated significantly in all tissues in the stationary phase of tumour growth.

Interrelationships between purine nucleotide degradation and radical formation during intestinal ischaemia and reperfusion: An application of ion-pair high-performance liquid chromatography of nucleotides, nucleosides and nucleobases

There are peroxidative changes during the reperfusion of the rat small intestine following a 1h period of total ischaemia. That is demonstrated by the increases of the concentrations of glutathione disulphide and of thiobarbituric acid-reactive substances. An important source of the active oxygen species leading to peroxidations is the degradation of purine nucleotides. The nucleotides and their derivatives were measured by an ion-pair reversed-phase high-performance liquid chromatographic separation in a single analysis within 40 min. Modification of the elution gradient resulted in a high resolution of nucleosides and nucleobases, including allopurinol and oxypurinol. The decrease of the nucleoside triphosphate concentration and the increase of nucleoside monophosphate concentration, followed by accumulations of nucleosides and nucleobases in the course of the ischaemia were measured. During reperfusion the nucleotide pools are filled up. Restoration of adenosine triphosphate and guanosine triphosphate can be accelerated by application of the xanthine oxidoreductase inhibitor allopurinol. Pretreatment of the animals with allopurinol also diminished the formation of glutathione disulphide and thiobarbituric acid-reactive substances.

Application of Reversed-Phase and Ion-Pair HPLC for investigation of Nucleotide Metabolism in Erythrocytes, Reticulocytes and Tumour Cells

SummaryThe determination of nucleotides, nucleosides, and nucleobases was carried out in cells of different metabolic complexity: in mature and immature red blood cells, in Ehrlich ascites tumour cells from different proliferation stages, and in other tumour cells.The maturation of reticulocytes to erythrocytes is accompanied by loss of organelles and energy-requiring processes as well as the switch from aerobic to anaerobic ATP production. The profile of purine nucleotides, nucleosides, bases, and pyridine dinucleotides, by reversed-phae HPLC, shows large concentration changes during the maturation of red blood cells. The concentrations of purine mono and triphosphates are two to four times greater in reticulocytes in comparison with erythrocytes; the difference in the concentrations of nucleosides and nucleobases between reticulocytes and erythrocytes is even greater. Application of ion-pair HPLC showed that the Ehrlich ascites cells loose major portions of purine mono-, diand triphosphates between the 7th and 11th day after inoculation. Fast growing solid sarcoma tumours of rats (MV 202 Ner) contain higher amounts of nucleotides than slowly growing tumours of identical cell type.

The catabolism of endogenous adenine nucleotides in rat liver mitochondria.

SummaryThe degradation of intramitochondrial adenine nucleotides to nucleosides and bases was investigated by incubating isolated rat liver mitochondria at 37°C under non-phosphorylating conditions in the presence of oligomycin and carboxyatractyloside. Within 30 min the adenine nucleotides were degraded by about 25 per cent. The main products formed were adenosine and inosine the contents of which increased five- to sevenfold.Compartmentation studies revealed that about 50 to 60 per cent of the adenosine formed remained inside the organelles whereas inosine was almost completely released into the surrounding medium. Outside the mitochondria only very small amounts of adenine nucleotides were detected. Similar incubations in the presence of [14C]-adenosine yielded no [14C]-inosine ruling out extramitochondrial adenosine deamination.It is concluded that endogenous adenine nucleotides can be degraded in mitochondria via AMP dephosphorylation and subsequent adenosine deamination. A purine nucleoside transport system mediating at least the efflux of inosine from the mitochondria is suggested.