[Simultaneous determination of sivelestat and its metabolite XW-IMP-A in human plasma using HPLC-MS/MS].

A simple and rapid method was developed based on high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to determine sivelestat and its metabolite XW-IMP-A in human plasma. After a simple protein precipitation, the samples and internal standards were analyzed on a C18 column by a gradient elution program. The mobile phase consisted of 30% acetonitrile in methanol and 5 mmol · L(-1) ammonium acetate at a flow rate of 0.7 mL · min(-1). The mass spectrometric data was collected in multiple reaction monitoring mode (MRM) in the negative electrospray ionization. The standard curves were linear in the range of 10.0-15,000 ng · mL(-1) for sivelestat, and 2.50-1000 ng · mL(-1) for XW-IMP-A. The low limits of quantitation were identified at 10.0 and 2.50 ng · mL for sivelestat and XW-IMP-A, respectively. The intra- and inter-day precision were within 11.3% and 13.1% for sivelestat and XW-IMP-A, and accuracy was 0.3% and 0.6% for sivelestat and XW-IMP-A, within the acceptable limits across all concentrations. The method was successfully validated in the pharmacokinetic study of sivelestat in healthy Chinese volunteers.

Analysis of ITPA phenotype-genotype correlation in the Bulgarian population revealed a novel gene variant in exon 6.

Mutations in the inosine triphosphate pyrophosphohydrolase (ITPA) gene causing enzyme deficiency were shown to have pharmacogenetic implications in azathioprine-induced adverse drug reactions. The distribution of ITPA activity as well as the types and the frequencies of gene variants associated with a lower enzyme activity were determined in healthy volunteers from a Bulgarian population. The ITPA activity was measured in 185 erythrocyte samples by an established high-performance liquid chromatography procedure. All samples were genotyped for 94C > A, IVS2 + 21A > C, and IVS2 + 68T > C/G by real-time polymerase chain reaction with hybridization probes. The ITPA activity ranged from 7.5 to 587.8 micromoL IMP/(g Hb x h) with a median value of 162.9 micromoL IMP/(g Hb x h). The enzyme activity showed significant differences between females and males (P = 0.006) with 17% higher values in men than women. Mutant allele frequencies were 0.038 (94C > A) and 0.130 (IVS2 + 21A > C). Mutations at IVS2 + 68 were not identified. Using a cutoff at 75 micromoL IMP/(g Hb x h) phenotyping detected all heterozygous carriers of 94C > A, two compound heterozygotes, all IVS2 + 21A > C homozygotes and 12.5% of IVS2 + 21A > C heterozygous cases. A novel frameshift mutation 359_366dupTCAGCACC in exon 6 was found in a subject with reduced enzyme activity of 61.2 micromoL IMP/(g Hb x h). The interindividual variability in ITPA activity among the Bulgarian population resembles the distribution of enzyme activity in other whites, although the observed median activity was approximately 25% lower in the Bulgarians [163 vs 219 micromoL IMP/(g Hb x h)]. The most common mutant allele IVS2 + 21A > C showed a similar frequency like in other white populations, whereas the 94C > A mutation was less frequently observed compared with other whites. Heterozygosity for the novel gene variant 359_366dupTCAGCACC was associated with 30% enzyme activity of the wild-type median value. The role of this rare variant for the thiopurine intolerance is not explored. These data further extend the knowledge for ITPA heterogeneity in whites.

The contribution of Ca+ calmodulin activation of human erythrocyte AMP deaminase (isoform E) to the erythrocyte metabolic dysregulation of familial phosphofructokinase deficiency.

Erythrocyte membrane leakage of Ca2+ in familial phosphofructokinase deficiency results in a compensatory increase of Ca2+-ATPase activity that depletes ATP and leads to diminished erythrocyte deformability and a higher rate of hemolysis. Lowered ATP levels in circulating erythrocytes are accompanied by increased IMP, indicating that activated AMP deaminase plays a role in this metabolic dysregulation. Exposure to a calmodulin antagonist significantly slows IMP accumulation during experimental energy imbalance in patients' cells to levels that are similar to those in untreated controls, implying that Ca2+-calmodulin is involved in erythrocyte AMP deaminase activation in familial phosphofructokinase deficiency. Therapies directed against activated isoform E may be beneficial in this compensated anemia.

Purine nucleotides and their metabolites in erythrocytes of streptozotocin diabetic rats.

OBJECTIVES: In the present study it was tried to obtain a complete overview of purine nucleotide metabolism in erythrocytes of streptozotocin (STZ) induced diabetes mellitus rats. METHODS: Erythrocyte levels of the main nucleotides (ATP, ADP, AMP, GTP, GDP, GMP, IMP, NAD+, NADP+), nucleosides (Ado, Guo, Ino) and the base Hyp were measured using the HPLC method. The parameters that can be deduced from their concentrations: TAN, TGN and AEC, GEC expressed by the ratio of high/low energy nucleoside phosphates were calculated. The effects of streptozotocin-induced diabetes on the concentration and metabolism of rat erythrocyte purine and pyridine nucleotides and the activity of Na+, K+-ATPase as well as Ca2+-ATPase were investigated. RESULTS: Increased dephosphorylation of adenine nucleotides (found as the increased concentration of Ado and Hyp and the decrease in AEC value) and the decrease in ATP and TAN and the changes in the concentrations of NAD+ and NADP+ suggest serious purine and pyridine metabolism disruptions in diabetic erythrocytes and decrease in ATPases activity. CONCLUSION: The observations suggest that purine nucleotide degradation is markedly accelerated in erythrocytes of STZ diabetic rats.

Effect of methotrexate polyglutamates on thioguanine nucleotide concentrations during continuation therapy of acute lymphoblastic leukemia with mercaptopurine.

Methotrexate is widely administered with mercaptopurine, a prodrug requiring activation into thioguanine nucleotides (TGN) to exert antileukemic effects. In vitro, methotrexate enhances TGN formation, but in vivo, such enhancement has yet to be demonstrated. We investigated whether TGN concentrations were related to methotrexate concentrations in children with acute lymphoblastic leukemia who received a weekly intravenous methotrexate (40 mg/m(2)) dose combined with daily mercaptopurine (75 mg/m(2)). A total of 141 erythrocyte TGN concentrations were measured with erythrocyte methotrexate polyglutamates (MTX-PG) concentrations in 87 patients. Average TGN concentrations ranged from 137 to 958 pmol/8 x 10(8) cells (median 389), average total MTX-PG concentrations (MTX- PG(1-7)) from 0.60 to 97.7 pmol/10(9)cells (median 29), and average long chain polyglutamate concentrations (MTX-PG(5-7)) from 0 to 8.35 pmol/10(9) cells (median 2.43). Higher TGN concentrations correlated with higher MTX-PG(5-7) concentrations (P = 0.002). These data support the practice of administering methotrexate with mercaptopurine during continuation therapy of acute lymphoblastic leukemia.

Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stress.

To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5-10 mM) of hydrogen peroxide for 1 h at 37 degrees C and the main substances of energy metabolism (ATP, AMP, GTP and IMP) and one index of lipid peroxidation (malondialdehyde) were determined by HPLC on cell extracts. Using the same incubation conditions, the activity of AMP-deaminase was also determined. Under nonhaemolysing conditions (at up to 4 mM H2O2), oxidative stress produced, starting from 1 mM H2O2, progressive ATP depletion and a net decrease in the intracellular sum of adenine nucleotides (ATP + ADP + AMP), which were not paralleled by AMP formation. Concomitantly, the IMP level increased by up to 20-fold with respect to the value determined in control erythrocytes, when cells were challenged with the highest nonhaemolysing H2O2 concentration (4 mM). Efflux of inosine, hypoxanthine, xanthine and uric acid towards the extracellular medium was observed. The metabolic imbalance of erythrocytes following oxidative stress was due to a dramatic and unexpected activation of AMP-deaminase (a twofold increase of activity with respect to controls) that was already evident at the lowest dose of H2O2 used; this enzymatic activity increased with increasing H2O2 in the medium, and reached its maximum at 4 mM H2O2-treated erythrocytes (10-fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H2O2, being detectable at the lowest H2O2 concentration and increasing without appreciable haemolysis up to 4 mM H2O2. Besides demonstrating a close relationship between lipid peroxidation and haemolysis, these data suggest that glycolytic enzymes are moderately affected by oxygen radical action and strongly indicate, in the change of AMP-deaminase activity, a highly sensitive enzymatic site responsible for a profound modification of erythrocyte energy metabolism during oxidative stress.

Changes in purine metabolism and production of oxygen free radicals by intermittent partial umbilical cord occlusion in chronically instrumented fetal lambs.

OBJECTIVE: Undetected umbilical-cord compression has been suggested to be implicated in unexplained fetal brain damage. We tested the hypothesis that the generation of oxygen-free radicals (OFRs) during intermittent partial umbilical cord occlusion might play a causal role in antenatal CNS injury. METHODS: Using 7 established chronically instrumented fetal lambs, intermittent partial occlusion of the umbilical circulation was produced according to the method of Clapp et al. for 1 of every 3 minutes for 2 hours. A microdialysis probe was implanted in the fetal brain white matter. Normal saline was infused, and the perfusate obtained at 2 micrograms/ min. Hypoxanthine (HX), xanthine (XA), and inosine-5-monophosphate (IMP) concentrations in the perfusate and blood samples obtained from fetal jugular veins were assayed by HPLC. Concurrently, the perfusate, which contained superoxide produced in the brain, when mixed with cypridina luciferin analogue extracorporally, caused chemiluminescence that in 4 cases was measured by a highly sensitive electronic fluorescence detector. RESULTS: (1) HX, XA, and IMP concentrations in the blood and perfusate were higher than in the control during the intermittent partial occlusion of the umbilical circulation period and returned to control levels during the recovery period. (2) The residual chemiluminescence of perfusate revealed that the intermittent partial occlusion of the umbilical circulation level was about 100% higher than the control level, and during the recovery period the level returned to the control level. (3) The chemiluminescence during the intermittent partial occlusion of the umbilical circulation period was inhibited by infusion through the fetal jugular vein of 1 micron polyethylene glycol conjugated superoxide dismutase. CONCLUSION: During the intermittent partial occlusion of the umbilical circulation period the fetal brain tissue releases a large amount of OFRs, and a portion of these might be synthesized by the increased conversion of HX to XA. This phenomenon might play an important role in the etiology of fetal brain injury.

Effect of muscular exercise by bicycle ergometer on erythrocyte purine nucleotides.

The effect of muscular exercise by bicycle ergometer on erythrocyte purine nucleotides was investigated in 6 athletes. Muscular exercise increased the concentration of inosine monophosphate from 5.9 +/- 1.1 to 7.3 +/- 1.3 nmol/ml in venous erythrocytes and from 5.7 +/- 1.0 to 6.8 +/- 1.4 nmol/ml in arterial erythrocytes, respectively, while it decreased the concentrations of adenosine diphosphate and adenosine monophosphate from 189.3 +/- 42.7 to 141.2 +/- 26.9 and from 26.0 +/- 7.8 to 15.7 +/- 4.3 nmol/ml in venous erythrocytes and also decreased their concentrations from 195.1 +/- 51.0 to 141 +/- 29.2 and from 26.5 +/- 9.6 to 14.8 +/- 3.0 nmol/ml in arterial erythrocytes, respectively. The muscular exercise also increased the concentration of inorganic phosphate in venous plasma from 1.12 +/- 0.12 to 1.46 +/- 0.22 mmol/l, that of NH3 in blood from 41.90 +/- 6.91 to 150.22 +/- 50.80 mumol/l, that of lactic acid in blood from 7.90 +/- 1.71 to 61.03 +/- 18.43 mg/dl and that of hypoxanthine in venous plasma from 1.32 +/- 0.36 to 18.14 +/- 4.87 mumol/l, respectively. Therefore, in vitro study was performed to investigate whether inorganic phosphate, NH4Cl, lactic acid or hypoxanthine affects nucleotides in erythrocytes. After 2 hour-incubation, 2 mM inorganic phosphate increased the erythrocyte concentration of inosine monophosphate 1.6 fold but decreased the erythrocyte concentrations of adenosine monophosphate and adenosine diphosphate 0.72 and 0.89 fold, respectively, in the suspension (pH 7.35), as compared with 1 mM inorganic phosphate. However NH4Cl, lactic acid or hypoxanthine did not affect erythrocyte purine nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased resting levels of adenine nucleotides in human skeletal muscle after high-intensity training.

The effect of high-intensity intermittent training on the adenine nucleotide content of skeletal muscle was studied. Eleven male subjects (group A) performed high-intensity intermittent training on a cycle ergometer three times per week for 6 wk, followed by 1 wk of the same kind of training with two sessions per day. Nine males (group B) exclusively performed 1 wk of training with two sessions per day. In group A, skeletal muscle total adenine nucleotide (TAN) levels decreased from 25.1 +/- 0.7 (SE) to 22.0 +/- 0.6 mmol/kg dry wt over the 6-wk period (P < 0.01). The subsequent intensive week did not further alter TAN levels. In group B, the intensive week of training reduced TAN levels from 25.1 +/- 0.5 to 19.4 +/- 0.6 mmol/kg dry wt (P < 0.001). The decrease was sustained 72 h after training (P < 0.001). During the intensive week, there was no change in plasma creatine kinase activity in either group A or group B. The plasma activity was, however, higher in group B than in group A on days 4 and 7 of the intensive week (P < 0.05). The results from this study indicate that high-intensity intermittent exercise causes a decrease in resting levels of skeletal muscle adenine nucleotide without a concomitant indication of muscle damage. A training-induced adaptation appears to occur with training by which a further loss of adenine nucleotides is prevented despite an increased training dose.

High-performance liquid chromatographic assay of the methyl and nucleotide metabolites of 6-mercaptopurine: quantitation of red blood cell 6-thioguanine nucleotide, 6-thioinosinic acid and 6-methylmercaptopurine metabolites in a single sample.

A reversed-phase high-performance liquid chromatographic assay was developed to quantify intracellular metabolites of the cytotoxic drug 6-mercaptopurine in the human red blood cell. The 6-thioguanine nucleotides, 6-thioinosinic acid and 6-methylmercaptopurine metabolites are measured in a single sample. A similar assay measures the parent thiopurine compounds. The limit of quantitation of the assay is 0.03, 0.03 and 0.12 nmol per 8 x 10(8) red blood cells for the 6-thioguanine nucleotides, 6-thioinosinic acid and the 6-methylmercaptopurine metabolites, respectively.

Adenine nucleotide degradation in the thoroughbred horse with increasing exercise duration.

Adenine nucleotide (AN) degradation has been shown to occur during intense exercise in the horse and in man, at or close to the point of fatigue. The aim of the study was to compare the concentrations of muscle inosine 5'-monophosphate (IMP) and plasma ammonia (NH3) during intense exercise with the concentrations of muscle and blood lactate. Seven trained thoroughbred horses were used in the study. Each exercised on a treadmill for periods of between 30 s and 150 s, at 11 and/or 12 m.s-1. Blood and muscle samples were taken and analysed for lactate and NH3 and adenosine 5'-triphosphate (ATP), phosphorylcreatine (PCr), IMP, creatine, lactate and glycerol-3-phosphate respectively. Horses showed varying degrees of AN degradation as indicated by plasma [NH3] and muscle [ATP] and [IMP]. Comparisons of [IMP] with muscle [lactate], and plasma [NH3] with that of blood [lactate] indicated a threshold to the start of AN degradation. This threshold corresponded to a lactate content of around 80 mmol.kg-1 dry muscle and 15 mmol.l-1 in blood. We discuss the mechanisms which have been proposed to account for AN degradation and suggest that IMP formation occurs as a result of a sudden rise in the concentration of adenosine 5'-diphosphate (ADP) and consequently the concentration of adenosine 5'-monophosphate. The data suggest a critical pH below which there may be a substantial reduction in the kinetics of ADP rephosphorylation provided by PCr resulting in an increase in [ADP], which is the stimulus to AN degradation during intense exercise.

Increased 5-HT-2 receptor function as measured by serotonin-stimulated phosphoinositide hydrolysis in platelets of depressed patients.

1. The present study was undertaken to examine whether or not 5-HT-induced inositol monophosphate (IP-1) accumulation in human platelets is mediated by 5-HT-2 receptors and to assess 5-HT-2 receptor function as measured by 5-HT-stimulated IP-1 accumulation in platelets from normal controls and depressed patients before drug treatment. 2. In platelets prelabeled with 3H-myo-inositol, in Ca ion free HEPES buffer containing 10 mM LiCl, 5-HT caused a dose-dependent accumulation of IP-1 during 15 min incubation. A maximal increase in IP-1 formation was observed at 30 microM of 5-HT and its EC50 value was 4 microM. 3. Ketanserin, a selective 5-HT-2 antagonist, was a potent inhibitor of 5-HT-stimulated IP-1 accumulation with a Ki value of 12 nM, but (-)propranolol (10 microM), a 5-HT-1 antagonist, failed to block the 5-HT response. 4. The potencies of various compounds tested to inhibit 5-HT-stimulated IP-1 accumulation in human platelets correlated positively with the affinities to 5-HT-2 receptor as defined by radioligand binding in rat cerebral cortex. 5. In a group of unmedicated patients with major depressive disorder matched for age with normal control group, we found a significant increase in 5-HT (100 microM)-induced accumulation of IP-1 (150 +/- 7% of basal for depressed patients, 132 +/- 3% for controls).

Importance of nicotinamide as an NAD precursor in the human erythrocyte.

The effect of variation in the concentration of inorganic phosphate and of the pyridine precursors nicotinamide (NAm) and nicotinic acid (NA) on pyridine nucleotide synthesis was studied using intact human erythrocytes. A wide range of incubation times was employed. The results showed that under physiological conditions the rate of synthesis of NAD from NAm exceeded that from NA twofold, while the reverse situation pertained at higher and unphysiological substrate levels. The two pathways had different regulation points. For NAm the rate-limiting factor was the initial step, namely its conversion into the mononucleotide, while for NA it lay at the second step, conversion of NA mononucleotide (NAMN) to its adenine dinucleotide. At physiological substrate levels the uptake of NA and conversion to NAMN were rapid, while the uptake and conversion of NAm were time dependent. This process was stimulated significantly by inorganic phosphate only for NAm. These results indicate that while NA is the predominant precursor of human erythrocyte NAD at high (unphysiological) substrate and phosphate levels, NAm is more efficient as an NAD precursor under physiological conditions, suggesting an important and hitherto unrecognized role for nicotinamide in NAD synthesis in vivo.

[Effect of intracellular organic phosphates on erythrocyte deformability].

Organic phosphates in human erythrocytes were selectively varied by incubating fresh human erythrocytes in phosphate-buffered saline containing inosine, pyruvate, adenine, and/or adenosine in various concentrations. The deformability of erythrocytes was measured at 24 degrees C with a rheoscope under shear stress of 8-82 dyn/cm2. (1) With increasing 2, 3-DPG (5 approximately 15 mM/l cells), undeformable erythrocytes increased due to the increased mean corpuscular hemoglobin concentration (MCHC). However, these cells became deformable, when the MCHC was reduced by suspending in hypotonic medium. (2) At the same MCHC, the deformability of 2, 3-DPG-enriched erythrocytes was still reduced, compared with that of control erythrocytes, probably due to altered membrane viscoelastic properties. (3) 2, 3-DPG-reduced erythrocytes (2.2 mM/l cells) was not altered in their deformability. (4) Deformability of 2, 3-DPG-enriched erythrocytes was not changed by lowering oxygen tension. (5) Deformability of erythrocytes was not affected by varying intracellular ATP in the range of 0.5 approximately 2.2 mM/l cells (ATP in control cells was 1 mM/l cells). (6) Increment of IMP (approximately 0.9 mM/l cells) and ITP (approximately 0.5 mM/l cells) did not alter the deformability of erythrocytes. (7) Interaction of intracellular organic phosphates with membrane proteins was discussed.

Radioisotopic assay for erythrocyte adenosine 5'-monophosphate deaminase.

Adenosine-5'-monophosphate deaminase is a critical enzyme in the regulation of adenine nucleotide levels in the erythrocyte. The routine examination of this enzyme in crude hemolysates is difficult with the commonly used assay which monitors ammonia generated by the deamination reaction. This report details a radioisotopic assay for AMP deaminase which allows separation of the [14C]inosine 5'-monophosphate product from the [14C]adenosine 5'-monophosphate substrate by ion-exchange chromatography at pH 2.2. The radioisotopic assay is linear with respect to time and enzyme concentration over a considerable range and thereby significantly simplifies the monitoring of crude or dilute enzyme preparations.

Influence of perinatal hypoxia on purine contents in erythrocytes of newborn infants.

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.

Sensitivity of the adenine nucleotide metabolism of platelets from malignant hyperthermia patients to halothane.

Blood platelets from malignant hyperthermia patients share the same defect as skeletal muscle. This could be verified by the effect of halothane on the energy metabolism of blood platelets under optimal experimental conditions. The sensitivity of changes in adenine nucleotide metabolization to halothane was significantly higher in platelets from malignant hyperthermia patients than in controls. The enhanced sensitivity is not caused by an adenylate kinase deficiency.