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The metabolism of the aldehydic lipid peroxidation product, 4-hydroxynonenal (HNE),was studied in suspensions of mouse thymocytes. Thymocytes are characterized by low lipid peroxidation in comparison with other cell types notwithstanding their high content of arachidonic acid. In our study a very high capacity of HNE metabolism in thymocytes was observed: 27.7 nmol/mg w.w./min. That is about the same HNE degradation rate as determined in liver cells or small intestinal enterocytes, which are the cells with the by far highest capacity for the degradation of HNE and other aldehydic lipid peroxidation products in comparison with other cell types. The primary and secondary HNE metabolites in thymocytes were identified and quantified after the addition of 100 microM HNE to thymocyte suspensions: the glutathione-HNE conjugate, the hydroxynonenoic acid, the 1,4-dihydroxynonene, water, and the glutathione-dihydroxynonene conjugate. Furthermore, the HNE binding to proteins was measured. The very rapid HNE degradation in thymocytes besides the high amounts of lipophilic chain-breaking antioxidants is postulated to be an important secondary antioxidative mechanism and the main factor for the low accumulation of lipid peroxidation products in these cells.1668

Radioprotective action of natural carotenoid Lycopene in mice during exposure to lethal dose of 6.5 Gy has been shown. Lycopene was found to be two-fold more effective radioprotector than beta-carotene at the same conditions. This correlates with the difference in their antioxidant activity. Lycopene also exhibits antimutagenic effect resulting in increased survival of Drosophila melanogaster chrysalis under exposure to 40 Gy gamma-radiation.

The pool sizes of purine nucleotides, nucleosides, and nucleobases were measured in the host tissues liver, skeletal muscle, and blood of Ehrlich ascites tumor-bearing mice during the different periods of tumor growth. There were large differences of tissue concentrations of these metabolites between control animals, animals in the logarithmic growth period of the Ehrlich ascites tumor, and animals in the resting phase of tumor growth. The ATP concentrations in liver, muscle, and erythrocytes were higher during the proliferating phase of the tumor compared with the ATP levels of these organs in healthy animals. In liver and skeletal muscle the ATP concentration decreased during the transition from proliferating into resting phase of tumor growth. The concentrations of nucleosides and nucleobases within the RBC and blood plasma deceased during the logarithmic growth phase but restored during the plateau period. As well as in the organs/cells investigated and in the body fluids (plasma, ascites fluid) a tremendous increase of adenosine concentration during the resting phase of tumor growth was observed. From changes of total purine ribonucleotide pattern an activation period in the nucleotide metabolic pathways of liver and skeletal muscle during the proliferating phase of tumor growth is postulated.

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.

Isocratic methods are described for the separation of purine compounds in specimens of jejunal mucosa available by peroral biopsy from patients with coeliac disease. The first mode provided separation of both the ionic nucleoside monophosphates and the non-ionic nitrogen bases and nucleosides. The second mode was applicable to analyses of the nucleoside mono-, di- and triphosphates only. These chromatographic procedures were carried out with tetrabutylammonium phosphate as an ion-pair modifier and different concentrations of acetonitrile in the mobile phase. The modes are flexible in that they permit the optimization of the separation of these compounds according to the purpose of the investigation. The results of studies on purine metabolite patterns in coeliac mucosa are discussed.

The pattern of purine derivatives was studied in the erythrocytes of C3HA and ICR mice during Hepatoma 22 and Ehrlich ascites tumor cells growth. Host erythrocytes purine nucleotides, nucleosides and bases were affected by the implanted tumors. The results indicated that the host erythrocytes markedly concentrated adenine and guanine nucleotides on the 5th and 11th-12th day of tumor growth. By contrast, content of nucleosides and bases were sharply decreased during the log growth phase (5th day) with the restoring of these precursors within the 11th-12th day (plateau phase). These observations indicate that aspects of the purine compounds metabolism in host erythrocytes are linked with tumor development.

The degradation of intramitochondrial adenine nucleotides to nucleosides and bases was investigated by incubating isolated rat liver mitochondria at 37 degrees 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.

The pattern of purine and pyrimidine derivatives was studied in the erythrocytes of C3HA and ICR mice during Hepatoma 22 and Ehrlich ascites tumor cell growth. Concentrations of purine nucleotides, nucleosides and nucleobases of host erythrocytes were changed after the implantation of the tumors. The host erythrocytes markedly concentrated adenine and guanine nucleotides both during logarithmic and plateau phase of tumor growth (5th and 11-12th days after inoculation of the tumor). The contents of nucleosides and nucleobases in the red blood cells were decreased during the logarithmic growth phase but restored within the plateau phase.

Venous blood acid-soluble fraction was investigated by means of high-efficiency liquid chromatography in patients with myocardial infarction and angina pectoris. Myocardial ischemia is shown to result in marked changes of purine and pyrimidine metabolism. A rise in intermediate and end products of purine metabolism (inosine, hypoxanthine, xanthine, uric acid), and the emergence of thymin and thymidine were demonstrated in venous blood of patients with myocardial infarction and angina pectoris.

Alterations in the levels of hypoxanthine and some other purine derivatives occurring in the erythrocytes from umbilical cord blood of newborn infants with perinatal hypoxia were examined. Newborn babies were divided into two groups which included infants with favourable (Group 1) and complicated (Group 2) periods of early adaptation. Purine metabolites of erythrocytes were analyzed by isocratic reversed-phase HPLC. It was shown a reciprocal relation between concentrations of hypoxanthine and IMP in erythrocytes of infants from two groups that was expressed in elevation (Group 1) and a fall (Group 2) of hypoxanthine level while in Group 1 the IMP content had a tendency to decrease and Group 2 was characterized by an elevated level of this compound. The alterations in the concentrations of these interrelated metabolites may be due to the effects of hypoxia upon purine metabolism in erythrocytes.

The purine metabolism was studied in the thymus and spleen lymphocytes of C3HA mice during hepatoma 22 growth in comparison with the transport characteristics of hypoxanthine and inosine in lymphocytes. An increase in the transport of these metabolites from erythrocytes was observed in the early hepatoma growth period. It may be associated with a certain pattern in the dynamics of purine metabolic pool in the lymphocytes. The inverse relationship is found between the rates of hypoxanthine transport into thymocytes and its intracellular concentration.

A method is described for the simultaneous analysis of twelve major nucleotides, certain biochemically essential nucleosides and nitrogen bases. The separation was achieved using an ion-pair reversed-phase high-performance liquid chromatographic (HPLC) system with a radially compressed NovaPak C18 cartridge and tetrabutylammonium phosphate (TBA) as a mobile phase modifier under acetonitrile gradient conditions. The effects of TBA and acetonitrile on the retention of major nucleotides were studied. The retention of nucleotides increased significantly with an increase in the TBA concentration from 0 to 2 mM and remained constant up to 5 mM TBA. The logarithm of the capacity factor decreased in proportion to the concentration of acetonitrile in the mobile phase. The separation of nitrogen bases, nucleosides and some of their deoxy forms was performed on an isocratic ion-pair reversed-phase HPLC system with pentanesulphonic and heptanesulphonic acid using a mu Bondapak C18 column. The described chromatographic procedures were applied to the separation of various biological extracts.

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.

The catabolism of intramitochondrial guanine nucleotides was examined. During 30 min incubation of rat liver mitochondria at 37 degrees C in the presence of oligomycin and carboxyatractyloside, guanine and xanthine were formed and appeared in the medium. Under these conditions, the direct conversion of GMP to guanine by hypoxanthine-guanine phosphoribosyltransferase is suggested to be the main catabolic route within the organelles. Only very small amounts of guanosine were produced and detected both inside and outside the organelles. [14C]Guanosine and [14C]inosine were taken up by the mitochondria. Therefore, guanosine is suggested to be a precursor of intramitochondrial guanine nucleotides.

Simultaneous separation of the main ribonucleotides, nucleosides and bases was carried out using tetrabutyl ammonium phosphate (TBA) hetaerons with a reversed-phase (C18) packing material. Chromatographic properties of purine and pyrimidine metabolites in radially compressed NovaPak C18 system was studied at various concentrations of acetonitrile and TBA in the mobile phase. The optimal conditions for gradient separation of nucleotides, nucleosides and bases were evaluated. The procedure was used for analysis of blood from healthy persons and patients with acute myocardial infarction.

The distribution of purine compounds in the skeletal muscles of the anterior and posterior limbs of 129/Re mice with hereditary muscular dystrophy (HMD) was investigated in a comparative study. The results revealed unidirectional metabolic disorders in both groups of muscles which was manifested in a quantitative redistribution of phosphorus-containing purine components and a decrease in the pool of adenylates in muscle tissue. Elevated concentrations of purine metabolites (inosine, hypoxanthine, xanthine, and uric acid) indicated an augmented metabolism of purines in this abnormality. Impairment of metabolic transformations of purines in the muscle tissue in HMD may be one of the factors responsible for the development of the pathological process in the muscle in the given disease.

The degradation of endogenous adenine nucleotides was compared in mitochondria isolated from mouse and rat liver, rat renal cortex and solid hepatoma. Mitochondria were incubated for 30 min at 37 degrees C in the presence of carboxyatractyloside and oligomycin. In rat liver mitochondria ATP, ADP and AMP were degraded by about 25% each, whereas in kidney and hepatoma mitochondria a rapid decline of ATP and ADP but no change in the AMP contents were observed. Main products formed were adenosine, inosine and hypoxanthine in all mitochondria examined. Compartment studies revealed that the main route of intramitochondrial adenine nucleotide catabolism seems to proceed via AMP dephosphorylation and subsequent adenosine deamination.

Various methods are described for the analysis of purine derivatives in biological samples by ion-pair high-performance liquid chromatography (HPLC) with both gradient and isocratic systems. A new approach is proposed that is suitable for the separation of nuclei acid constituents in different cells with a specific enzymatic activity pattern. The ion-pair HPLC methods were developed for the analysis of erythrocytes, lymphocytes and mitochondria acid-soluble fractions in clinical and experimental studies of normal and altered nucleotide metabolism. The results of studies of purine metabolite redistribution in mouse liver mitochondria during a 30-min incubation at 37 degrees C and data on purine metabolic alterations in mouse thymocytes during hepatoma growth are discussed.

Utilization of ion-air reagents in a reversed-phase chromatographic system allows solving a number of problems related to the separation of purine and pyrimidine derivatives. Simultaneous analysis of nucleotides, nucleosides and their bases was carried out by acetonitrile gradient elution using tetrabutyl ammonium phosphate as a counterion in the mobile phase. Besides, optimal conditions were selected for isocratic separation of adenine nucleotides and their metabolites. Furthermore, isocratic separation of certain purines and pyrimidines was achieved by modifying the stationary C18-phase with pentane- and heptane sulphonic acids.

Using reverse phase ion pair high performance liquid chromatography, the levels of free adenosine, inosine, adenine, xanthine, hypoxanthine, guanine and deoxycytidine in thymocytes and splenic T- and B-lymphocytes of C3HA mice, were studied under normal conditions and at different times (5 hrs, 1, 2, 3, 4, 5, 8 and 20 days) after transplantation of solid hepatoma 22a. The adenosine and inosine levels in thymus and spleen lymphocytes were 5 to 10 times as low as that of purine bases. Inosine was totally absent in T-and B-lymphocytes. The absolute content of adenine and guanine in thymus and spleen lymphocytes was higher compared to purine bases. It was shown that in all cases studied the decrease in hypoxanthine, xanthine and guanine levels in T- and B-lymphocytes during maximal tumour growth, i.e., on the 5th and 8th post-inoculation days as well as at the terminal period (20th day), was correlated with the decrease in the adenosine deaminase and functional activities of these cells. The level of free adenine in thymocytes and spleen T-lymphocytes during tumour growth showed a 2-4-fold increase in comparison with normal values. A dramatic decrease of intracellular concentration of deoxycytidine was observed in thymocytes and spleen T- and B-lymphocytes beginning with the 5th hour and over the whole subsequent period. The key role of the deoxycytidine decline during tumour growth as a possible cause of simultaneous impairment of DNA synthesis and purine deoxyribonucleoside phosphorylation in lymphocytes is discussed.

Applying a recently developed reverse-phase ion-pair HPLC technique we determined purine compounds in acid extracts of mitochondria isolated from rat and mouse liver, rat renal cortex, and hepatoma. Intramitochondrial purine nucleosides and bases were evaluated quantitatively for the first time. All mitochondria examined contain significant amounts of adenosine, guanosine, inosine, guanine, and hypoxanthine. Moreover, IMP was identified in all extracts. Adenine was detected in traces only. These results are in accordance with the concept of mitochondrial metabolism of purine compounds.

Tonsils from 10 patients (18-40 years old), with chronic tonsillitis were studied by means of histological methods as well as by high pressure liquid chromatography. In 5 patients with decompensated forms of the disease, accompanied by heart impairments during 1-3 years, only slight alterations in epithelial and follicular parts of tonsils were observed, while pronounced destruction of tonsil cells was detected in other 5 patients suffering from repeated anginas. The following purine and pyrimidine derivatives were identified in faucial tonsil tissue of all the patients with chronic tonsillitis studied: adenine, guanine, adenosine, guanosine, inosine and uridine. Distinct increase in content of these derivatives as well as occurrence of xanthine and uric acid (non-identified in the first group of patients with chronic tonsillitis) was noted in the patients with maximal destruction of faucial tonsil cells.

During development of transmitted solid hepatoma 22 pools of purine metabolites were studied as well as the rate of 3H-hypoxanthine and inosine incorporation was measured in acid soluble fraction of liver tissue and erythrocytes of mice C3HA as well as of the tumor tissue. Specific alterations in metabolism of adenine and guanine nucleotides and of their derivatives were detected at various steps of the hepatoma development in the tissues studied. Specific interaction of the hepatoma cells with host tissues appears to be realized via the pool of purine nucleotides.