Critical study of preanalytical and analytical phases of adenine and pyridine nucleotide assay in human whole blood

Intracellular redox and energetic status play a crucial role in cardiovascular diseases and metabolic disorders. The physiological status of reducing agents, such as NADPH and NADH, and of high-energy molecules, such as ATP, is required for antioxidant system activity. For these reasons, an accurate measurement of adenine and pyridine nucleotides is fundamental. In this study we examined the preanalytical phase of reduced pyridine (RPN) and adenine and oxidized pyridine (AOPN) nucleotide assay in human whole blood. Different experimental conditions were applied to RPN alkaline and AOPN acid extracts to find the best analytical performance. Our results show that a good RPN and AOPN linearity (r from 0.994 to 0.999), recovery (near to 100%), and precision (coefficient of variation < 5%) were obtained when supernatant from acid and ultrafiltrate from alkaline extracts were neutralized, frozen, and thawed just before HPLC injection. Since NADH decays rapidly at -80 degrees C, RPN levels must be assayed within 72 h while AOPN can be stored for 1 month at the same temperature. An accurate and quantitative method for nucleotide determination can be obtained by applying the preanalytical conditions proposed in this study.     

Extracellular Adenosine, Inosine, Hypoxanthine, and Xanthine in Relation to Tissue Nucleotides and Purines in Rat Striatum During Transient Ischemia

Extracellular (EC) adenosine, hypoxanthine, xanthine, and inosine concentrations were monitored in vivo in the striatum during steady state, 15 min of complete brain ischemia, and 4 h of reflow and compared with purine and nucleotide levels in the tissue. Ischemia was induced by three-vessel occlusion combined with hypotension (50 mm Hg) in male Sprague-Dawley rats. EC purines were sampled by microdialysis, and tissue adenine nucleotides and purine catabolites were extracted from the in situ frozen brain at the end of the experiment. ATP, ADP, and AMP were analyzed with enzymatic fluorometric techniques, and adenosine, hypoxanthine, xanthine, and inosine with a modified HPLC system. Ischemia depleted tissue ATP, whereas AMP, adenosine, hypoxanthine, and inosine accumulated. In parallel, adenosine, hypoxanthine, and inosine levels increased in the EC compartment. Adenosine reached an EC concentration of 40 microM after 15 min of ischemia. Levels of tissue nucleotides and purines normalized on reflow. However, xanthine levels increased transiently (sevenfold). In the EC compartment, adenosine, inosine, and hypoxanthine contents normalized slowly on reflow, whereas the xanthine content increased. The high EC levels of adenosine during ischemia may turn off spontaneous neuronal firing, counteract excitotoxicity, and inhibit ischemic calcium uptake, thereby exerting neuroprotective effects.     

Determination of adenine and pyridine nucleotides in glucose-limited chemostat cultures of Penicillium simplicissimum by one-step ethanol extraction and ion-pairing liquid chromatography

Under specific conditions Penicillium simplicissimum excretes large amounts of organic acids, mainly citrate. As the energetic status of the hyphae might play a role in that respect, we developed a method for the determination of adenine (adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate) and pyridine (nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide (NADH)) nucleotides in hyphae of P. simplicissimum. An optimum separation of the five compounds in less than 15 min was possible on a C-8 column, utilizing 50 mM aqueous triethylamine-buffer (pH 6.5) and acetonitrile as mobile phase; detection was performed at 254 nm. With the exception of NADH, which could not be determined accurately due to stability problems, the method was sensitive (LOD < or = 0.7 ng on-column), repeatable (sigma(rel) < or = 4.4%), accurate (recovery rates between 97.9 and 104.9%), and precise (intraday variation < or = 9.4%, interday variation < or = 6.2 %). For an optimum extraction of the nucleotides the chemostat samples were directly placed into hot (90 degrees C) 50% ethanol, and shaken for 10 min, followed by evaporation of the solvent and a solid phase extraction cleanup of the redissolved aqueous samples. With this method the nucleotide concentrations in hyphae from a glucose-limited chemostat culture and the respective energy charge were determined. Additionally, the effect of the time lag between sampling and extraction and the effect of a glucose pulse on nucleotide concentrations were determined.     

Microvascular changes of the liver preserved in UW solution. Pathological and immunohistochemical examination.

Rat livers preserved in University of Wisconsin (UW) solution for 24 h were compared with those preserved in Euro-Collins (EC) solution before and after liver transplantation using an immunohistochemical method. Tissue ATP and total tissue adenine nucleotide (TAN) were measured using HPLC. The levels of TAN in the UW group or the EC group were significantly low compared with the control group (no preservation) after 24-h storage. In the EC group, the levels of tissue adenine nucleotides (TAN) decreased 1 h after reperfusion and never reached control levels. In the UW group, the levels of TAN increased a little 1 h after reperfusion and increased more 3 h after reperfusion. After 24-h preservation, the expression of factor VIII-related antigen (FRA) in endothelial cells of central veins was weak in the EC group; in the UW group, FRA was clearly detected in these cells. After reperfusion, although severe endothelial cell damage to the central veins and numerous FRA-positive substances were observed in EC group, endothelial cells of central veins retained their normal structure and FRA-positive substances were rarely noted in the UW group. In both groups, no endothelial changes were detected in portal veins. From these results, it is concluded that UW solution prevents endothelial cell damage and microcirculatory injury in zone III during the preservation period resulting in prevention of initial graft nonfunction. Also, measurement of the TAN level after reperfusion is useful to predict the function of the graft.     

Analysis of the nucleotide pool during growth of suspension cultured cells of Nicotiana tabacum by high performance liquid chromatography

In suspension cultured cells of Nicotiana tabacum L. cv. Wisconsin 38 the .concentration of 18 nucleotides and 3 nucleosides have been determined using a new procedure developed for the extraction, purification and HPLC separation of these compounds from plant tissue. The different nucleotide pools increase in size immediately at the onset of the batch culture and reach distinct maxima at the very beginning of the proliferative phase. The main component are the uracil nucleotides with UDP-sugars as the predominant fraction followed by the adenine nucleotides; the energy charge is maintained at a high and constant value throughout the whole culture time. During the growth interval the increases in the nucleotide pools reveal that the cell proliferation phase is followed by an extensive phase of cell elongation. Whereas the concentration of the total nucleotides varies by a factor of about 3 along the growth curve, the ratio of uracil to adenine nucleotides is kept fairly constant indicating regulatory mechanisms for correlation of the individual nucleotide pools.     

Adenine nucleotide metabolism in relation to purine enzymes in liver, erythrocytes and cultured fibroblasts.

To evaluate the regulation of adenine nucleotide metabolism in relation to purine enzyme activities in rat liver, human erythrocytes and cultured human skin fibroblasts, rapid and sensitive assays for the purine enzymes, adenosine deaminase (EC 2.5.4.4), adenosine kinase (EC 2.7.1.20), hyposanthine phosphoribosyltransferase (EC 2.4.28), adenine phosphoribosyltransferase (EC 2.4.2.7) and 5'-nucleotidase (EC 3.1.3.5) were standardized for these tissues. Adenosine deaminase was assayed by measuring the formation of product, inosine (plus traces of hypoxanthine), isolated chromatographically with 95% recovery of inosine. The other enzymes were assayed by isolating the labelled product or substrate nucleotides as lanthanum salts. Fibroblast enzymes were assayed using thin-layer chromatographic procedures because the high levels of 5'-nucleotidase present in this tissue interferred with the formation of LaCl3 salts. The lanthanum and the thin-layer chromatographic methods agreed within 10%. Liver cell sap had the highest activities of all purine enzymes except for 5'-nucleotidase and adenosine deaminase which were highest in fibroblasts. Erythrocytes had lowest activities of all except for hypoxanthine phosphoribosyltransferase which was intermediate between the liver and fibroblasts. Erhthrocytes were devoid of 5'-nucleotidase activity. Hepatic adenosine kinase activity was thought to control the rate of loss of adenine nucleotides in the tissue. Erythrocytes had excellent purine salvage capacity, but due to the relatively low activity of adenosine deaminase, deamination might be rate limiting in the formation of guanine nucleotides. Fibroblasts, with high levels of 5'-nucleotidase, have the potential to catabolize adenine nucleotides beyond the control od adenosine kinase. The purine salvage capacity in the three tissues was erythrocyte greater than liver greater than fibroblasts. Based on purine enzyme activities, erythrocytes offer a unique system to study adenine salvage; fibroblasts to study adenine degradation; and liver to study both salvage and degradation.     

Control of oxidative phosphorylation by the extramitochondrial ATP/ADP ratio

The control of mitochondrial ATP synthesis by the extramitochondrial adenine nucleotide pattern was investigated with rat liver mitochondria. It is demonstrated that any stationary state between the two limit states of maximum activity (state 3) and of resting activity (state 4) can be obtained by a hexokinase-glucose trap as an ADP-regenerating system. These intermediate states are characterized by stationary respiratory rates, stationary redox levels of the cytochromes b and c and stationary levels of extramitochondrial ATP and ADP between the rates and levels of the limit states. At a constant concentration of inorganic phosphate the activity of mitochondria between the limit states is controlled by the extramitochondrial ATP/ADP ratio independent of the total concentration of adenine nucleotides present. The control range was found to be between ratios of about 5 and 100 at 10 mM phosphate. At lower ratios the mitochondria are in their maximum phosphorylating state. With succinate + rotenone and glutamate + malate the same control range was observed, indicating that it is independent of the nature of substrate oxidized.The results suggest that in the control range the mitochondrial activity is limited by the competition of ADP and ATP for the adenine nucleotide translocator.     

Purine salvage in Entamoeba histolytica

Pulse-labeling of the nucleotide pool in Entamoeba histolytica with radioactive precursors, and subsequent high performance liquid chromatographic (HPLC) analysis of the radiolabeled nucleotides, indicate that E. histolytica is incapable of de novo synthesis of purine nucleotides. Hypoxanthine, inosine and xanthine could not be converted to nucleotides in E. histolytica, which suggests the absence of interconversion between adenine nucleotides and guanine nucleotides through formation of IMP. Adenosine was actively incorporated into nucleotides at an initial rate of 130 pmoles per minute per 10(6) trophozoites. Adenine, guanosine and guanine were also incorporated at much lower rates. The rate of adenine incorporation was enhanced by the presence of guanosine; the rate of guanine incorporation was significantly increased by adenosine. These stimulatory effects suggest that the ribose moiety of adenosine or guanosine can be transferred to another purine base to form a new nucleoside, and that the purine nucleosides are the immediate precursors of E. histolytica nucleotides. HPLC results showed that the radiolabel in adenine was exclusively incorporated into adenine nucleotides and that guanine was found only among guanine nucleotides, whereas the radioactivity associated with the ribose moiety of adenosine or guanosine was distributed among both adenine and guanine nucleotides.     

Intracellular nucleotide and nucleotide sugar contents of cultured CHO cells determined by a fast, sensitive, and high-resolution ion-pair RP-HPLC

Analysis of intracellular nucleotide and nucleotide sugar contents is essential in studying protein glycosylation of mammalian cells. Nucleotides and nucleotide sugars are the donor substrates of glycosyltransferases, and nucleotides are involved in cellular energy metabolism and its regulation. A sensitive and reproducible ion-pair reverse-phase high-performance liquid chromatography (RP-HPLC) method has been developed, allowing the direct and simultaneous detection and quantification of some essential nucleotides and nucleotide sugars. After a perchloric acid extraction, 13 molecules (8 nucleotides and 5 nucleotide sugars) were separated, including activated sugars such as UDP-glucose, UDP-galactose, GDP-mannose, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine. To validate the analytical parameters, the reproducibility, linearity of calibration curves, detection limits, and recovery were evaluated for standard mixtures and cell extracts. The developed method is capable of resolving picomolar quantities of nucleotides and nucleotide sugars in a single chromatographic run. The HPLC method was then applied to quantify intracellular levels of nucleotides and nucleotide sugars of Chinese hamster ovary (CHO) cells cultivated in a bioreactor batch process. Evolutions of the titers of nucleotides and nucleotide sugars during the batch process are discussed.     

In vitro alteration of the size of the liver mitochondrial adenine nucleotide pool: Correlation with respiratory functions

Mitochondrial respiration was studied as a function of the total adenine nucleotide content of rat liver mitochondria. The adenine nucleotide content was varied by treating isolated mitochondria with pyrophosphate or by incubating pyrophosphate-treated mitochondria with ATP. Mitochondria with at least 4 nmol adenine nucleotides/mg protein maintained at least 80% of the State 3 activity of control mitochondria, which had approximately 10 nmol/mg protein. However, State 3 decreased rapidly once the adenine nucleotide content fell below 4 nmol/mg protein. Between 2 and 4 nmol adenine nucleotides/mg, State 3 was not limited by the maximal capacity of electron flow as measured by the uncoupled respiration. However, at very low adenine nucleotide levels (<2 nmol/mg), the uncoupled rates of respiration were markedly depressed. State 4 was not affected by changes in the mitochondrial adenine nucleotide content. Adenine translocase activity varied in almost direct correlation with changes in the adenine nucleotide content. Therefore, adenine translocase activity was more sensitive than State 3 to changes in total adenine nucleotides over the range of 4 to 10 nmol/mg protein. The results suggest that (i) State 3 is dependent on the level of intramitochondrial adenine nucleotides, particularly in the range below 4 nmol/mg protein, (ii) adenine translocase activity is not rate-limiting for oxidative phosphorylation in mitochondria with the normal complement of adenine nucleotides, however, at low adenine nucleotide levels, depressed State 3 rates may be explained in part by the low rate of ADP translocation, and (iii) a mechanism of net ATP uptake exists in mitochondria with low internal adenine nucleotides.     

Adenine nucleotide metabolism in relation to purine enzymes in liver,erythrocytes and cultured fibroblasts

To evaluate the regulation of adenine nucleotide metabolism in relation to purine enzyme activities in rat liver, human erythrocytes and cultured human skin fibroblasts, rapid and sensitive assays for the purine enzymes, adenosine deaminase (EC 2.5.4.4), adenosine kinase (EC 2.7.1.20), hypoxanthine phosphoribosyltransferase (EC 2.4.28), adenine phosphoribosyltransferase (EC 2.4.2.7) and 5′-nucleotidase (EC 3.1.3.5) were standardized for these tissues. Adenosine deaminase was assayed by measuring the formation of product, inosine (plus traces of hypoxanthine), isolated chromatographically with 95% recovery of inosine. The other enzymes were assayed by isolating the labelled product or substrate nucleotides as lanthanum salts. Fibroblast enzymes were assayed using thin-layer chromatographic procedures because the high levels of 5′-nucleotidase present in this tissue interferred with the formation of LaCl₃ salts. The lanthanum and the thin-layer chromatographic methods agreed with-in 10%.Liver cell sap had the highest activities of all purine enzymes except for 5′-nucleotidase and adenosine deaminase which were highest in fibroblasts. Erythrocytes had lowest activities of all except for hypoxanthine phosphoribosyltransferase which was intermediate between the liver and fibroblasts. Erythrocytes were devoid of 5′-nucleotidase activity. Hepatic adenosine kinase activity was thought to control the rate of loss of adenine nucleotides in the tissue.Erythrocytes had excellent purine salvage capacity, but due to the relatively low activity of adenosine deaminase, deamination might be rate limiting in the formation of guanine nucleotides. Fibroblasts, with high levels of 5′-nucleotidase, have the potential to catabolize adenine nucleotides beyond the control of adenosine kinase. The purine salvage capacity in the three tissues was erythrocyte > liver > fibroblasts. Based on purine enzyme activities, erythrocytes offer a unique system to study adenine salvage; fibroblasts to study adenine degradation; and liver to study both salvage and degradation.     

Investigation of the dependence of the intramitochondrial [ATP]/[ADP] ratio on the respiration rate

The relationship between the respiration rate and the intra- and extramito-chondrial adenine nucleotides was investigated in isolated rat liver mitochondria.

For the determination of adenine nucleotide patterns in both compartments a new procedure was developed, based on the evaluation of these metabolites from incubation of various amounts of mitochondria under identical stationary states of oxidative phosphorylation. These identical states were adjusted by addition of appropriate amounts of hexokinase to a glucose-containing incubation mixture.

Adenine nucleotides were measured in aliquots of the total extract of the incubation mixture without any separation. The concentrations of the adenine nucleotides in both compartments were obtained from a plot of the total concentration of these species versus mitochondrial protein. Disturbances of this method by unspecific efflux of adenine nucleotides could be excluded.

The results obtained for the total adenine nucleotide content (12 nmol · mg⁻¹ protein) and the intramitochondrial [ATP]/[ADP] ratio (about 4 in the resting state) are in good agreement with data obtained by other methods.

Strong evidence is provided for a decrease of the intramitochondrial [ATP]/[ADP] ratio with increasing rate of oxygen consumption. Therefore it is not necessary to assume a microcompartmentation of the intramitochondrial adenine nucleotide pool in respect to the ATPase reaction and the adenine nucleotide translocation.     

Immunofluorescent localization of phosphoenolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase/oxygenase proteins in leaves of C3, C4 and C3−C4 intermediate Flaveria species

The concentrations of 17 nucleotides and three nucleosides have been determined in a batch suspension culture of Datura innoxia using a new procedure for extraction, purification and high-performance liquid chromatography separation of these compounds. The nucleotide pools change appreciably in the different phases of growth. These changes indicate the preparation for and initiation of cell proliferation, and reflect metabolic events during cell division, cell elongation and starvation. The main components of the nucleotide pool are uracil nucleotides, with uridine 5-diphosphate sugars as the predominant fraction, and the adenine nucleotides. Although their concentrations vary by a factor of more than 6 the ratio of the uracil to adenine nucleotides is kept fairly constant during growth. The energy charge is maintained at a rather high value. The correlation of these events with nutrient uptake and macromolecular synthesis by the batch culture is presented in the following paper.Abbreviations Glc glucose - GlcNAc 2-acetamido-2-deoxy-d-glucose - HPLC high performance liquid chromatography - UDP uridine 5-diphosphate     

Degradation and resynthesis of adenine nucleotides in adult rat heart myocytes

The degradation and short-term resynthesis of adenine nucleotides have been examined in a preparation of isolated rat heart myocytes. These myocyte preparations are essentially free of vascular and endothelial cells, contain levels of adenine nucleotides quite comparable to those of intact heart tissue, and retain these components remarkably well for up to 2 h of aerobic incubation in the presence of 1 mM Ca2+. When the cells are rapidly and synchronously de-energized by addition of uncoupler, an inhibitor of respiration and iodoacetate, cellular ATP is degraded almost quantitatively to AMP. The AMP is then converted to either intracellular adenosine, which accumulates to high concentrations before release to the cell exterior, or to IMP. The relative contribution of these two pathways depends on the metabolic state of the cells just prior to de-energization, with IMP production favored when respiring cells are de-energized and adenosine formation predominant when glycolyzing myocytes are subjected to this treatment. Cells de-energized by anaerobiosis in the absence of glucose lose ATP and adenine nucleotides with the production of IMP and adenosine. Upon reoxygenation, these cells restore a high adenylate energy charge and about 60% of control levels of GTP. There is a net resynthesis of 5-7 nmol of adenine nucleotides.mg-1 protein with a corresponding decline in IMP. Added [14C]adenosine labels the adenine nucleotide pool, but little net resynthesis of adenine nucleotides via adenosine kinase can be detected. It therefore appears that a rapid regeneration of adenine nucleotides can occur via the enzymes of the purine nucleotide cycle in heart myocytes and is limited by the size of the IMP pool retained.     

Identification of some major cytokinins in Phaseolus vulgaris and their distribution

The determination of the various endogenous cylokinins and their distribution among organs is important in understanding their role in growth and development in the intact plant. Cytokinins in young plants of Phaseolus vulgaris were purified by immunoaffinity chromatography and HPLC, and characterised by UV spectra. Zeatin nucleotide (zeatin riboside-5'-monophosphate) and isopentenyladenine nucleotide (isopentenyladenosne-5'-monopnosphate) were the most abundant cytokinins in all organs. Their identities were confirmed by GC-MS. The levels of zeatin, zeatin riboside, isopentenyladenine and isopentenyladenosine never exceeded 5% of the nucleotides, as assessed by a methodology that preserves cytokinin nucleotides. Three extraction methods were compared with qualitatively similar results, though differing in their suppression of nucleotidase activity. Cytokinin nucleotide levels were greater in the stems and petioles than in the roots and leaves on a per gram fresh weight basis, and were greater in the stems than in the other organs on a per plant basis. Levels of the zeatin and isopentenyladenine nucleotides were about equal in the stems and leaves, but in the petioles the zeatin nucleotide levels were about twice the level of isopentenyladenine nucleotide, while in the roots they were about half the isopentenyladenine nucleotide level. The importance of considering the cytokinin form is emphasised.     

Changes in the energy state of tissues in spontaneously hypertensive rats]

The contents of adenine nucleotides (ATP, ADP, AMP), phosphocreatine (PCr) and creatine (Cr) in the heart, skeletal muscle, liver and spleen in spontaneously hypertensive (SHR) and normotensive (WKY) rats. The ATP/ADP ratio in cardiac tissue was lower in SHR compared with WKY, while myocardial contents of adenine nucleotides, PCr and Cr did not differ significantly between the groups. A lower ATP/ADP ratio in the skeletal muscle SHR of was accompanied by a reduction of PCr content comparing with these indices in WKY rats. The liver and spleen of SHR exhibited lower ATP contents and higher ADP and AMP levels compared with those ones in WKY rats, despite of the close values of adenine nucleotide pools (sigma AN = ATP + ADP + AMP). This redistribution of tissue adenine nucleotides was corresponded to lower energy charges (EC = (ATP + 0.5 ADP)/sigma AN) and ATP/ADP ratios in SHR group. The reduction of the energy state of tissues in SHR rats increased in the following rank: heart > skeletal muscle > liver > spleen, thus, reflecting progressive decrease of intensity of oxidative metabolism. The results suggest changes in the balance of rates of ATP formation and hydrolysis occur at the system level in primary hypertension. Probably, consequences of such rearrangement in energy metabolism are functional disturbances of plasma membrane and sacroplasmic reticulum well-documented in a number of experimental and clinical studies.     

A sensitive and robust method for quantification of intracellular short-chain coenzyme A esters

A procedure for the analysis of short-chain intracellular coenzyme A (CoA) esters and adenine nucleotide pools in microbial cells is described. The simultaneous isolation of bacterial cells from media, quenching of their metabolism, and extraction of metabolites was accomplished by centrifugation of cells through a layer of silicone oil into a denser solution of trichloroacetic acid. The acid was neutralized by extraction into Freon containing tri-n-octylamine to provide a salt-free solution of cell metabolites. After high-performance liquid chromatography separation, CoA, CoA esters, and adenine-containing nucleotides were derivatized by postcolumn reaction with bromoacetaldehyde to form the fluorescent 1,N6-ethenoadenine adducts which were analyzed by a fluorescence detector at picomolar levels.     

The Metabolism of Purine and Pyrimidine Nucleotides in Rat Cortex During Insulin-Induced Hypoglycemia and Recovery

Brains of paralysed rats with insulin-induced hypoglycemia were frozen in situ after spontaneous EEG activity had been absent for 5 or 15 min (“coma”). Recovery (30 min) was achieved in a different group of rats by administering glucose after a 30-min coma period. Purine and pyrimidine nucleotides, nucleosides and free bases were determined in the cortical extracts by high pressure liquid chromatography (HPLC). The ATP values obtained with the HPLC method were in excellent agreement with those obtained using standard enzymatic/fluorometric techniques, while values for ADP and AMP obtained with the HPLC method were significantly lower. Comatose animals showed a severe (40-80%) reduction in the concentrations of all nucleoside triphosphates (ATP. GTP, UTP and CTP) and a simultaneous increase in the concentrations of all nucleoside di- and monophosphates, including that of IMP. The adenine nucleotide pool size decreased to 50% of control level. The concentrations of the nucleosides adenosine, inosine, and uridine increased 50- to 250-fold, while the concentrations of the purine bases, xanthine and hypoxanthine, rose 2- and 30-fold, respectively. There were no increases in the concentrations of adenine, guanine, or xanthosine. Following glucose administration there was a partial (ATP, UTP and CTP) or almost complete (GTP) recovery of the nucleoside triphosphate levels. During recovery, the levels of nucleosidc di- and monophosphates and of adenosine decreased to values close to control; the rise in the inosine level was only partially reversed, and the concentrations of hypoxanthine and xanthine rose further. The adenine nucleotide pool size was only partially restored (to 67% of control value). The adenine nucleotide pool size was not increased by i.p. injection of adenosine or adenine under control condition, or during the posthypoglycemic recovery period.     

Sensitive determination of tenofovir in human plasma samples using reversed-phase liquid chromatography

A new high-performance liquid chromatography assay was developed for the determination of tenofovir, a nucleotide analogue, in plasma. A solid-liquid extraction procedure was coupled with a reversed-phase HPLC system. The system requires a mobile phase containing Na(2)HPO(4) buffer, tetrabutylammonium hydrogen sulfate and acetonitrile for different elution through a C(18) column with UV detection. The method proved to be accurate, precise and linear between 10 and 4000 ng/ml. The method was applied to determine trough levels of tenofovir in 11 HIV-infected patients with virologic failure under multiple antiretroviral therapy. This method was also successfully applied to a pharmacokinetic study in an HIV infected patient with renal failure.     

Interaction of intracellular RNAase from Aspergillus clavatus with derivatives of its substrates]

Using spectrophotometric and kinetic methods and also the methods of protection of Aspergillus clavatus RNAse (EC 3.1.4.23) by adenine nucleotides and their components against inactivation by means of acylation or heating, it was found that RNAse-nucleotide complex was formed by association of one enzyme molecule with one nucleotide molecule. It was also shown that all components of nucleotides (base, ribose and phosphate) take part in the formation of such complex and the removal of one of them (base or phosphate) lead to loosening of bindings of remaining fragments (ribose-5'-monophosphate, adenine) with the active site of RNAse, and to disappearance of bends within the pH range of 3.0-4.0 on the plot of pKi (5'-MP) versus pH, within the pH range of 5.5-7.0 on the plot of oKi (Ado) versus pH. The possibility of participation of associative pair RNAse imidasole groups - nucleotide phosphate groups and RNAse carboxylic group - nucleotide base in the mechanism of formation of enzyme-nucleotide (enzyme-substrate) complexes is postulated.