ABSTRACT
Emtricitabine (FTC) and tenofovir (TFV) are widely used antiviral agents that require intracellular phosphorylation to become active. This article describes the development and validation of an assay for the simultaneous quantification of FTC mono-, di- and triphosphate (FTC-MP, -DP and -TP), TFV and TFV mono- and diphosphate (TFV-MP and -DP) in peripheral blood mononuclear cells. Reference compounds and internal standards were obtained by thermal degradation of FTC-TP, TFV-DP, stable isotope-labeled TFV-DP and stable isotope-labeled cytosine triphosphate. Cells were lysed in methanol:water (70:30, v/v) and the extracted nucleotides were analyzed using weak anion-exchange chromatography coupled with tandem mass spectrometry. Calibration ranges in PBMC lysate from 0.727 to 36.4, 1.33 to 66.4 and 1.29 to 64.6 nM for FTC-MP, FTC-DP and FTC-TP and from 1.51 to 75.6, 1.54 to 77.2 and 2.54 to 127 nM for TFV, TFV-MP and TFV-DP, respectively, were validated. Accuracies were within -10.3 and 16.7% deviation at the lower limit of quantification at which the coefficients of variation were less than 18.2%. At the other tested levels accuracies were within -14.3 and 9.81% deviation and the coefficients of variation lower than 14.7%. The stability of the compounds was assessed under various analytically relevant conditions. The method was successfully applied to clinical samples.
Subject(s)
Adenine/analogs & derivatives , Chromatography, Ion Exchange/methods , Deoxycytidine/analogs & derivatives , Leukocytes, Mononuclear/chemistry , Nucleotides/blood , Organophosphonates/blood , Tandem Mass Spectrometry/methods , Adenine/blood , Adenine Nucleotides/blood , Anions/chemistry , Deoxycytidine/blood , Drug Stability , Emtricitabine , Humans , Linear Models , Phosphorylation , Pyrimidine Nucleotides/blood , Reproducibility of Results , TenofovirABSTRACT
MMF (mycophenolate mofetil) has been proven to provide an effective immunosuppression by non-competitive selective reversible inhibition of IMPDH (inosine monophosphate dehydrogenase), the enzyme playing a crucial role in GTP biosynthesis. However, the exact metabolic changes induced by inhibition of IMPDH in target cells of the immune system have been the subject of recent debate. The aim of the present study was to evaluate whether MMF treatment produced sustained changes in the guanosine nucleotide pool of MNLs (mononuclear leucocytes) in vivo. Sixty-two renal failure patients were divided into three groups: chronic renal failure patients undergoing haemodialysis (CRF-HD; n=20) and two groups of patients after renal transplantation, the first on AZA (azathioprine; TN-AZA; n=23) and the second treated with MMF (TN-MMF; n=19). In addition, MNLs from 25 healthy subjects were analysed as controls. Anion-exchange HPLC was used to quantify purine and pyrimidine nucleotides in MNLs. We report a significant decrease in GTP and the total MNL guanine nucleotide pool in the TN-MMF group (P<0.05) compared with control, CRF-HD and TN-AZA groups, although no significant differences were found between any of the other groups. Adenine nucleotide concentrations in MNLs were decreased in the TN-AZA group, but not in the TN-MMF group compared with the CRF-HD group and controls. There were no differences in CTP concentrations, but UTP concentrations were decreased in the CRF-HD, TN-AZA and TN-MMF groups compared with controls. MMF caused a significant and sustained decrease in the guanine nucleotide pool in MNLs from renal transplant recipients. This decrease contrasts with the elevation in GTP reported in erythrocytes of MMF-treated patients.
Subject(s)
Guanosine Triphosphate/blood , Immunosuppressive Agents/pharmacology , Kidney Transplantation , Leukocytes, Mononuclear/metabolism , Mycophenolic Acid/analogs & derivatives , Mycophenolic Acid/pharmacology , Adolescent , Adult , Aged , Azathioprine/pharmacology , Child , Drug Administration Schedule , Enzyme Inhibitors/pharmacology , Female , Humans , IMP Dehydrogenase/antagonists & inhibitors , Kidney Failure, Chronic/blood , Leukocytes, Mononuclear/drug effects , Male , Middle Aged , Postoperative Period , Purine Nucleotides/blood , Pyrimidine Nucleotides/bloodABSTRACT
Pyrimidine 5' nucleotidase (P5'N-1) deficiency is an autosomal recessive condition causing hemolytic anemia characterized by marked basophilic stippling and the accumulation of high concentrations of pyrimidine nucleotides within the erythrocyte. It is implicated in the anemia of lead poisoning and is possibly associated with learning difficulties. Recently, a protein with P5'N-1 activity was analyzed and a provisional complementary DNA (cDNA) sequence published. This sequence was used to study 3 families with P5'N-1 deficiency. This approach generated a genomic DNA sequence that was used to search GenBank and identify the gene for P5'N-1. It is found on chromosome 7, consists of 10 exons with alternative splicing of exon 2, and produces proteins 286 and 297 amino acids long. Three homozygous mutations were identified in this gene in 4 subjects with P5'N-1 deficiency: codon 98 GAT-->GTT, Asp-->Val (linked to a silent polymorphism codon 92, TAC-->TAT), codon 177, CAA-->TAA, Gln-->termination, and IVS9-1, G-->T. The latter mutation results in the loss of exon 9 (201 bp) from the cDNA. None of these mutations was found in 100 normal controls. The DNA analysis was complicated by P5'N-1 pseudogenes found on chromosomes 4 and 7. This study is the first description of the structure and location of the P5'N-1 gene, and 3 mutations have been identified in affected patients from separate kindreds. (Blood. 2001;97:3327-3332)
Subject(s)
5'-Nucleotidase/deficiency , 5'-Nucleotidase/genetics , Anemia, Hemolytic/genetics , 5'-Nucleotidase/chemistry , Alternative Splicing , Amino Acid Sequence , Anemia, Hemolytic/enzymology , Base Sequence , Child , Child, Preschool , Chromosomes, Human, Pair 7 , DNA, Complementary/chemistry , Deoxyribonucleases, Type II Site-Specific/metabolism , Erythrocytes/enzymology , Erythrocytes/metabolism , Exons , Female , Humans , Infant, Newborn , Male , Middle Aged , Molecular Sequence Data , Mutation , Norway , Pedigree , Pyrimidine Nucleotides/blood , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , South AfricaABSTRACT
Purine and pyridine metabolism were studied in ten Lesch-Nyhan patients, with virtually no hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity in erythrocytes. Increased NAD erythrocyte concentrations were found in all patients. Raised activities of two enzymes catalysing NAD synthesis from nicotinic acid (nicotinic acid phosphoribosyltransferase: NAPRT, and NAD synthetase: NADs) was found in erythrocyte lysates from all patients. The two enzymes had normal apparent Km for their substrates and increased Vmax. The rate of synthesis of pyridine nucleotides from nicotinic acid by intact erythrocytes in vitro was also increased in most patients. These findings suggest that raised NAD concentrations in HPRT- erythrocytes are due to enhanced synthesis as a result of increased enzyme activities.
Subject(s)
Erythrocytes/enzymology , Hypoxanthine Phosphoribosyltransferase/deficiency , Lesch-Nyhan Syndrome/blood , NAD/biosynthesis , Pyridines/blood , Adolescent , Adult , Amide Synthases/blood , Child , Child, Preschool , Erythrocytes/metabolism , Female , Humans , Infant , Kinetics , Lesch-Nyhan Syndrome/enzymology , Male , Middle Aged , NAD/blood , Nicotinic Acids/blood , Pentosyltransferases/blood , Purine Nucleotides/blood , Purines/blood , Pyrimidine Nucleotides/blood , Tryptophan/bloodABSTRACT
BACKGROUND: Leflunomide is an experimental drug with demonstrated ability to prevent and reverse acute allograft and xenograft rejection. The two biochemical activities reported for the active metabolite of leflunomide, A77 1726, are inhibition of tyrosine phosphorylation and inhibition of dihydroorotate dehydrogenase, an enzyme necessary for de novo pyrimidine synthesis. These activities can be distinctly separated in vitro by the use of uridine, which reverses the anti-proliferative effects of A77 1726 caused by inhibition of de novo pyrimidine synthesis. We report the effect of uridine on the in vivo immunosuppressive activities of leflunomide. METHODS: We first quantified the serum levels of A77 1726, the active metabolite of leflunomide, after a single treatment of leflunomide (5, 15, and 35 mg/kg). Additionally, we quantified the levels of serum uridine and of nucleotide triphosphates in the liver, spleen, and lymph nodes of Lewis rats after the administration of a single dose of uridine (500 mg/kg; i.p.). Lewis rats heterotopically transplanted with brown Norway or Golden Syrian hamster hearts were treated for 50 or 75 days with leflunomide (5, 15, and 35 mg/kg/day; gavage) alone or in combination with uridine (500 mg/ kg/day; i.p.). Hematocrits were determined and the levels of alloreactive or xenoreactive immunoglobulin (Ig)M and IgG were determined by flow cytometric analysis. The allograft and xenografts, small bowel, liver, kidney, and spleen were subjected to pathological examination. RESULTS: A linear relationship was observed between the serum A77 1726 concentrations in Lewis rats and the dose of leflunomide administered. Peak A77 1726 concentrations were 20.9, 71.8 and 129.3 mg/l (77.5, 266.1 and 478.8 microM) for the 5, 15, and 35 mg/kg doses of leflunomide, respectively. The concentration of uridine in the serum of normal Lewis rats is 6.5 microM; after i.p. administration of 500 mg/kg uridine, the serum uridine concentrations peaked at 384.1 microM in 15-30 min. The rapid elimination of uridine was not reflected in the lymphoid compartments, and the pharmacokinetics of pyrimidine nucleotides in the spleen resembled that of A77 1726. This dose of uridine, when administered daily (500 mg/kg/day, i.p.), weakly antagonized the immunosuppressive activities of leflunomide (5, 15, and 35 mg/kg/day) in the allotransplantation model. In contrast, in the xenotransplantation model, the same concentration of uridine completely antagonized the immunosuppressive activities of low-dose leflunomide (15 mg/kg/day) and partially antagonized the immunosuppressive activities of high-dose leflunomide (35 mg/kg/day). Toxicities associated with high-dose leflunomide (35 mg/kg/day) were anemia, diarrhea, and pathological changes in the small bowel and liver. These toxicities were significantly reduced by uridine co-administration. CONCLUSION: These studies reveal that the blood levels of A77 1726 in Lewis rats satisfy in vitro requirements for both inhibition of de novo pyrimidine synthesis and protein tyrosine kinase activity. Our data also illustrate that the in vivo mechanism of immunosuppression by leflunomide is complex and is affected by at least the following four factors: type and vigor of the immune response, availability of uridine for salvage by proliferating lymphocytes, species being investigated, and concentration of serum A77 1726.
Subject(s)
Isoxazoles/metabolism , Aniline Compounds/blood , Aniline Compounds/pharmacokinetics , Animals , Cricetinae , Crotonates , Graft Rejection/prevention & control , Heart Transplantation/immunology , Humans , Hydroxybutyrates/blood , Hydroxybutyrates/pharmacokinetics , Immunosuppressive Agents/metabolism , Immunosuppressive Agents/pharmacokinetics , Isoxazoles/pharmacokinetics , Isoxazoles/therapeutic use , Leflunomide , Liver/chemistry , Lymph Nodes/chemistry , Mesocricetus , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Nitriles , Pyrimidine Nucleotides/biosynthesis , Pyrimidine Nucleotides/blood , Rats , Rats, Inbred Lew , Spleen/chemistry , Toluidines , Transplantation, Heterologous/immunology , Transplantation, Homologous/immunology , Uridine/pharmacokineticsABSTRACT
Sensitive high performance liquid chromatography techniques, which differentiate between purine and pyrimidine ribonucleoside and deoxyribonucleoside triphosphates, were used to quantify pools in phytohemagglutinin-stimulated T-lymphocytes (98% CD4+ and CD8+) from healthy volunteers. The importance of de novo synthesis and salvage was evaluated by incubating the cells with 14C-radiolabeled precursors (40 microM), azaserine (20 microM; a glutamine antagonist), and ribavirin (50 microM; an IMP dehydrogenase inhibitor). We confirmed that resting T-lymphocytes meet their metabolic requirements by salvage. Noteworthy observations were as follows. First, nucleotide pool expansion over 72 h is disproportionate, with that for purines (ATP and GTP) being 2-fold compared with up to 8-fold for pyridine (NAD) or pyrimidine (UTP, UDP-Glc, and CTP) pools. This supports an additional role for the latter in membrane lipid biosynthesis, protein glycosylation, and strand break repair. Second, intact de novo pathways are essential for such expansion. Azaserine not only inhibited purine synthesis (confirmed by N-formylglycinamide polyphosphate accumulation), but also reduced expansion of pyrimidine and NAD pools by 70%. Ribavirin depleted GTP pools by 40% and reduced pyrimidine pool expansion by 40% at 72 h. These findings underline the importance of pyrimidine ribonucleotide availability as well as GTP synthesis de novo to proliferating T-lymphocytes. They also demonstrate an absence of coordinate regulation between de novo purine and pyrimidine biosynthesis.
Subject(s)
DNA/biosynthesis , Lymphocyte Activation , Ribonucleotides/blood , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Azaserine/pharmacology , Carbon Radioisotopes , Cells, Cultured , Chromatography, High Pressure Liquid/methods , Humans , Kinetics , Models, Biological , Phytohemagglutinins , Purine Nucleosides/blood , Purine Nucleosides/isolation & purification , Purine Nucleotides/blood , Purine Nucleotides/isolation & purification , Pyrimidine Nucleosides/blood , Pyrimidine Nucleosides/isolation & purification , Pyrimidine Nucleotides/blood , Pyrimidine Nucleotides/isolation & purification , Radioisotope Dilution Technique , Reference Values , Ribavirin/pharmacology , Sensitivity and Specificity , T-Lymphocytes/drug effectsABSTRACT
Proliferative defects have been reported at the level of DNA synthesis, even in T-lymphocytes from asymptomatic human immunodeficiency virus type-1+ (HIV-1+) patients. Since purine and pyrimidine ribonucleotide availability is crucial for proliferation, we compared the ability of HIV-1- and HIV-1+ T-lymphocytes (> 95% CD4+ and CD8+) to activate de novo biosynthetic and salvage pathways following phytohemagglutinin stimulation using 14C-labeled precursors. The striking abnormality already detectable in asymptomatic patients' cells was the impaired ability of CTP, UDP-Glc, and UTP pools to expand over 72 h (44-70% of control), although ATP and GTP pools and responses were normal. In symptomatic patients, resting T-cells showed markedly reduced pyrimidine pools (53-74% of control) with no change following activation. Relatively normal ATP, GTP, and NAD pools masked the same impaired response of de novo synthesis to activation, with ATP and GTP being reduced by 50% at 48 h. Purine salvage was more active than the control in unstimulated HIV-1+ cells. This impaired de novo synthesis in HIV-1+ T-lymphocytes severely restricts the availability of ribonucleotides for vital growth-related activities such as membrane expansion and strand break repair as well as DNA and RNA synthesis. The data indicate that resting T-lymphocytes from symptomatic patients survive through enhanced salvage, but the stimulation induces metabolic cell death, and provide an explanation for the activation-associated lymphocyte death seen in HIV-1+ T-lymphocytes.
Subject(s)
Acquired Immunodeficiency Syndrome/blood , Acquired Immunodeficiency Syndrome/immunology , HIV Seropositivity/blood , HIV Seropositivity/immunology , Lymphocyte Activation , Ribonucleotides/blood , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Carbon Radioisotopes , Cells, Cultured , Chromatography, High Pressure Liquid/methods , HIV Infections/blood , HIV Infections/immunology , Humans , Kinetics , Purine Nucleotides/blood , Purine Nucleotides/isolation & purification , Purines/blood , Purines/isolation & purification , Pyrimidine Nucleotides/blood , Pyrimidine Nucleotides/isolation & purification , Pyrimidines/blood , Pyrimidines/isolation & purification , Radioisotope Dilution Technique , Reference Values , Ribonucleotides/isolation & purification , T-Lymphocytes/drug effectsABSTRACT
Biochemical marker in heme and nucleotide metabolisms were reviewed and evaluated in terms of biological monitoring of lead exposure. Analytical methods, reference values, threshold values, dose-effect relationships, validity, applicable range of Pb-B levels, and confounding factors were studied for the biochemical parameters: delta-aminolevulinic acid dehydratase activity (ALA-D), delta-aminolevulinic acid in plasma (ALA-P), delta-aminolevulinic acid in urine (ALA-U), coproporphyrin in urine (CP-U), erythrocyte protoporphyrin (EP) or zinc protoporphyrin (ZP), pyrimidine 5'-nucleotidase (P5N), and pyrimidine nucleotides in blood (PN). The threshold value of Pb-B for ALA-D by CEC method or restoration method was 5 micrograms/dl. The high validity (> 1.8) was found at a wide range of Pb-B levels between 5 and 50 micrograms/dl, while that of ALA-D activity by CEC method was between 20 and 50 micrograms/dl. The threshold value of Pb-B for ALA-P was also as low as 5 micrograms/dl, and that for ALA-U (corrected for creatinine) was between 15 and 30 micrograms/dl or 20 and 40 micrograms/dl in HPLC or colormetric method, respectively. ALA-P showed a high validity (> 1.8) at Pb-B levels of 30 and 60 micrograms/dl, whereas ALA-U did at Pb-B of 60 micrograms/dl alone. EP (ZP) and CP-U were significantly increased in the Pb-B levels higher than 20 to 30 micrograms/dl and 40 micrograms/dl, respectively. The threshold level of Pb-B for P5N activity was less than 10 micrograms/dl, and showed a linear reduction in activity with a rise in Pb-B values between 10 and 60 micrograms/dl. The validity of P5N (1.86 at a cut off of < 10 mumol/h/g Hb) was the highest among biochemical parameters examined at Pb-B values over 40 micrograms/dl. PN was increased in the Pb-B levels higher than 60 micrograms/dl. Individual variation of ALA-U, CP-U, and ZP were relatively larger than those of PN. ALA-P seemed to be one of the most desirable biomarkers for lead exposure, because of simplicity, sensitivity and specificity of analytical method, and of wide range of applicable Pb-B, from normal to intoxication levels. PN might be of diagnostic value for lead intoxication. Lead effect or intoxication could be assessed more exactly by the combination of some specific biomarkers mentioned above.
Subject(s)
Environmental Exposure , Heme/metabolism , Lead Poisoning/diagnosis , Nucleotides/metabolism , 5'-Nucleotidase/blood , Aminolevulinic Acid/blood , Aminolevulinic Acid/urine , Biomarkers/analysis , Coproporphyrins/urine , Environmental Monitoring/methods , Female , Humans , Lead Poisoning/metabolism , Male , Maximum Allowable Concentration , Porphobilinogen Synthase/blood , Protoporphyrins/blood , Pyrimidine Nucleotides/bloodABSTRACT
Nucleotides in erythrocytes of lead-exposed subjects were analyzed by high-performance liquid chromatography (HPLC). Most of the pyrimidine levels correlated well with blood lead concentrations (Pb-B) and pyrimidine 5'-nucleotidase (P5N) activity. Highly significant correlations were found between Pb-B and uridine 5'-diphosphate-glucose (UDPG), cytidine 5'-triphosphate (CTP), or CDP-choline (CDPC). The levels of these compounds were sharply elevated when P5N activity was reduced to levels less than 7 mumole/h.g hemoglobin (Hb), which corresponded to a Pb-B of 60 micrograms/100 g. Therefore, concentration of these nucleotides may provide a useful index of lead poisoning. Adenosine 5'-triphospate (ATP) concentrations were correlated negatively with Pb-B, whereas adenosine 5'-monophosphate (AMP) concentrations were correlated positively with Pb-B. These results suggest that lead affects not only pyrimidine nucleotide metabolism but also purine nucleotide metabolism (energy production system).
Subject(s)
Erythrocytes/chemistry , Lead/blood , Purine Nucleotides/blood , Pyrimidine Nucleotides/blood , 5'-Nucleotidase/blood , Adenosine Monophosphate/blood , Adenosine Triphosphate/blood , Chromatography, High Pressure Liquid , Humans , Lead Poisoning/blood , Occupational Exposure , Purine Nucleotides/metabolism , Pyrimidine Nucleotides/metabolismABSTRACT
An erythrocyte pyrimidine 5'-nucleotidase (P5N) test was performed for 171 workers occupationally exposed to lead. Erythrocyte P5N activity was markedly inhibited by exposure to lead. Among several biological indicators (erythrocyte P5N, delta-aminolevulinic acid dehydratase (ALAD), protoporphyrin (PROTO), urinary delta-aminolevulinic acid (ALA), coproporphyrin (COPRO)), the P5N activity had the highest correlation with the concentration of lead in blood (r = -0.77). A significant inhibition of erythrocyte P5N was found in groups of lead workers with blood-lead levels of more than 10 to 19 micrograms/dl. This P5N inhibition started before any changes occurred in urinary ALA and COPRO. A 45 to 50% inhibition of P5N corresponded to the blood-lead value (50 micrograms/dl) of the BEI recommended by ACGIH. In some lead workers, erythrocyte nucleotides (mainly CTP and UTP) were determined. The data indicated that a marked accumulation of these nucleotides had occurred, and their levels correlated negatively with P5N activity and positively with blood lead.
Subject(s)
5'-Nucleotidase/blood , Erythrocytes/enzymology , Lead/blood , Adult , Chromatography, High Pressure Liquid/methods , Environmental Exposure , Erythrocytes/drug effects , Humans , Male , Middle Aged , Occupational Diseases/enzymology , Pyrimidine Nucleotides/bloodABSTRACT
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.
Subject(s)
Angina Pectoris/metabolism , Myocardial Infarction/metabolism , Purine Nucleotides/blood , Pyrimidine Nucleotides/blood , Adult , Humans , Middle Aged , Myocardium/metabolism , Ulna/blood supply , VeinsABSTRACT
The oxidized form of glutathione transport was studied in human erythrocytes in pyrimidine 5'-nucleotidase (P5N) deficiency, a disorder in which the amounts of CTP and UTP in the erythrocytes are elevated. The inhibition of ATP-requiring oxidized glutathione (GSSG) transport by CTP and UTP is believed to play a role in elevating the levels of the reduced form of glutathione (GSH) in the erythrocytes of patients with P5N deficiency. The current investigation was undertaken to determine if GSSG transport actually decreases in the erythrocytes of such patients. Erythrocytes from a 17-year-old patient and a 13-year-old patient with P5N deficiency hemolytic anemia and from ten normal subjects were used as materials for the experiment. Erythrocytes, which had been previously incubated with [3H]glycine, were incubated at 37 degrees C, and the rate of [3H]GSSG transported by the cells was estimated. The velocity of GSSG transport out of the erythrocytes was quite low in the patients, 3.17-3.65 nmol GSSG/ml erythrocytes/hr at 37 degrees C in one case, and 3.30 nmol GSSG/ml erythrocytes/hr in the other case, vs that in the normal controls (6.00 +/- 0.80 nmol GSSG/ml erythrocytes/hr; mean +/- SD). The activity of gamma-glutamylcysteine synthetase and glutathione synthetase did not decrease in the patients. Decreased transport activity of GSSG in addition to a normal synthesis rate for GSH may explain the increased concentration of erythrocyte GSH in P5N deficiency.
Subject(s)
Erythrocyte Membrane/metabolism , Glutathione/blood , Nucleotidases/deficiency , 5'-Nucleotidase , Adenosine Triphosphate/blood , Adolescent , Biological Transport, Active , Erythrocytes/enzymology , Glutamate-Cysteine Ligase/blood , Glutathione Synthase/blood , Humans , In Vitro Techniques , Male , Pyrimidine Nucleotides/bloodABSTRACT
The effect of high-dose uridine on body temperatures of rabbits and man has been studied in relation to plasma concentrations of uridine and its catabolite uracil. Uridine induced fever in both rabbits and man. High-dose cytidine had no influence on body temperature in rabbits. Plasma concentrations of uridine were between 1 and 1.5 mM at 30 min after an iv bolus injection of 400 mg uridine/kg in rabbits and reached peak levels of 2 mM after a 1-hr infusion of 12 g uridine/m2 in man. The plasma concentration of cytidine in rabbits was about 0.5 mM and that of uridine was 0.30 mM at 30 min after an iv bolus injection of 400 mg cytidine/kg. The mean residence time for uridine in patients and rabbits varied between 80 and 195 min. The area under the plasma concentration-time curve (AUC) for uridine in rabbits was 2.0 mmol.hr/liter, and that for cytidine was 0.6 mmol.hr/liter. A large AUC for uridine indicates a prolonged exposure of tissues to uridine, which might lead to extensive formation of degradation products. The administration of some of these catabolites, dihydrouracil (at 20-40 mg/kg), carbamyl-beta-alanine (at 60 mg/kg), and beta-alanine (at 300-400 mg/kg), resulted in a significant increase in body temperature. It is concluded that the change in body temperature associated with uridine administration was not due to bacterial pyrogens but that one of the degradation products might be involved in thermoregulation.
Subject(s)
Body Temperature Regulation/drug effects , Pyrimidine Nucleotides/pharmacology , Animals , Cytidine/pharmacology , Dose-Response Relationship, Drug , Fever/chemically induced , Humans , Pyrimidine Nucleotides/blood , Pyrimidine Nucleotides/pharmacokinetics , Rabbits , Uracil/analogs & derivatives , Uracil/metabolism , Uracil/pharmacology , Uridine/metabolism , Uridine Phosphorylase/antagonists & inhibitors , beta-Alanine/pharmacologyABSTRACT
The regulation of erythrocyte synthesis of nicotinate and adenine nucleotides has been investigated. Some effectors of the two committed enzymes, nicotinate- and adenine phosphoribosyltransferases, have been identified on crude lysates and on partially purified preparations of the former. Enzyme characteristics have been correlated with the nucleotide synthesis achieved in intact cells incubated in suitable mediums containing (14-C)-nicotinate or adenine. Inorganic phosphate, Mg ions and adenine nucleotides proved to be important effectors of pyridine synthesis, whose products, in turn, do not influence adenine nucleotide production. The production of pyridine nucleotides is little lower than that of adenine nucleotides in intact cells, even if adenine phosphoribosyl-transferase activity appears to be much more limited, inside the cell, than nicotinate phosphoribosyltransferase.
Subject(s)
Erythrocytes/metabolism , Purine Nucleotides/blood , Pyrimidine Nucleotides/blood , Adenine Nucleotides/blood , Adenine Phosphoribosyltransferase/blood , Humans , In Vitro Techniques , Kinetics , Nicotinamide Mononucleotide/analogs & derivatives , Nicotinamide Mononucleotide/blood , Pentosyltransferases/bloodABSTRACT
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.
Subject(s)
Chromatography, High Pressure Liquid/methods , Purine Nucleosides/analysis , Purine Nucleotides/analysis , Pyrimidine Nucleosides/analysis , Pyrimidine Nucleotides/analysis , Animals , Humans , Mice , Myocardial Infarction/blood , Purine Nucleosides/blood , Purine Nucleotides/blood , Pyrimidine Nucleosides/blood , Pyrimidine Nucleotides/blood , Thymus Gland/analysisABSTRACT
An anion-exchange high-performance liquid chromatography method has been used to quantitate the intracellular purine and pyrimidine nucleotides in extracts of pure lymphocytes, monocytes, neutrophils, eosinophils, erythrocytes, and platelets isolated from the blood of healthy human donors. For accurate and reproducible measurements of the nucleotide profiles in different types of pure leukocytes, the cell suspensions have to be free of platelets and erythrocytes. Incubation of the purified leukocytes for 1 h at 0 degrees C did not alter the nucleotide concentrations but reduced the interdonor variation to 10%. Incubation of purified lymphocytes for 1 h at 37 degrees C caused considerable changes in the relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides. During this incubation the cell viability, the cell number, and the ATP:ADP ratio decreased. Incubation of monocytes and granulocytes for 1 h at 37 degrees C caused considerable loss of cells and/or cell death. For erythrocytes and platelets reproducible nucleotide concentrations were obtained after extraction of freshly isolated cells. During storage of erythrocytes, both at 0 degrees C and at 37 degrees C, a decrease in the ATP:ADP ratio was detected. In all cell types the predominant nucleotides were purine nucleotides, especially adenosine triphosphate. The relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides were very reproducible per cell type and appeared to be characteristic for each cell type. The total nucleotide content was nearly the same for all cell types except erythrocytes, when expressed per microgram of protein. The described methods for purification and storage of blood cells will be useful for comparison of blood cells from healthy donors with those of patients, for example, leukemia patients, in which deviations of the purine and pyrimidine metabolic enzymes have already been described.
Subject(s)
Blood Cells/analysis , Nucleotides/blood , Blood Platelets/analysis , Cell Separation/methods , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Eosinophils/analysis , Erythrocytes/analysis , Humans , Leukocytes/analysis , Lymphocytes/analysis , Monocytes/analysis , Neutrophils/analysis , Purine Nucleotides/blood , Pyrimidine Nucleotides/blood , Specimen HandlingABSTRACT
Uremia causes major increases in the erythrocyte (RBC) purine nucleotides, presumably secondary to phosphate retention, but no previous study has been made of the pyrimidine nucleotides, normally absent from RBC. This investigation was prompted by demonstration of the abnormal presence of RBC pyrimidine nucleotides, primarily cytidine triphosphate (CTP) plus cytidine diphosphate-choline (CDP-C) and cytidine diphosphate-ethanolamine (CDP-E), in two types of congenital hemolytic anemia as well as in lead poisoning. These observations suggested an analogy to the RBC membrane dysfunction of uremia. This is a report of the identification of CDP-C and CDP-E as the predominant abnormal pyrimidine nucleotides in the RBC hemolysates of uremic subjects. High-performance liquid chromatography of hemolysates from uremic adults showed a 50% increase in purine nucleotides and the abnormal presence of pyrimidine nucleotides and diesters at approximately 10% of the concentration of the purine nucleotides. By means of UV spectra and 31P nuclear magnetic resonance, these were identified as CDP-C and CDP-E. The increased purine and abnormal pyrimidine nucleotides of uremic RBC were unrelated to the pre- or posthemodialysis state, allopurinol, levels of blood lead, copper and zinc, or RBC pyrimidine 5'-nucleotidase, the cytosolic enzyme that specifically dephosphorylates the pyrimidine nucleotides. Although the accumulation of CTP, CDP-C and CDP-E may be an epiphenomenon of phosphate retention, it also suggests a common pathway to the accelerated hemolysis of chronic renal insufficiency.