Development and validation of LC/MS/MS method for Triciribine and its monophosphate metabolite in plasma and RBC: Application to mice pharmacokinetic studies.

Triciribine (TCN) is a tricyclic nucleoside analog of adenosine and an inhibitor of Akt kinase. Triciribine 5'-monophosphate (TCNP) is a water-soluble analog of Triciribine and has progressed to Phase I and II clinical trials in oncology. TCNP is also an endogenous anabolite of TCN similar to other nucleoside phosphates. Clinical development of TCNP has been hampered by high pharmacokinetic variability due to complex interplay of TCN-TCNP conversion and reconversion in plasma, erythrocytes (RBC) and peripheral organs. TCN has been demonstrated to be an efficacious agent in mice models of acute lung injury at low doses (0.5 mg/kg/day) although its pharmacokinetic-pharmacodynamic (PK/PD) relationship remained unclear. We have developed and validated a sensitive, specific and robust LC/MS/MS assay for quantitation of TCN and TCNP in plasma and RBC. Using a simple protein precipitation method, quantitation of these analytes was accomplished with recoveries exceeding 85% and with a run time of 4 min. This assay was used to determine the pharmacokinetic parameters of TCN and TCNP in mice after single dose intravenous administration at 1, 3 and 10 mg/kg. TCNP accumulates in RBC, has low clearance and a half-life of 18 to 23 h. Unlike other nucleoside phosphates, TCNP was found to be relatively stable in mice plasma serving as a secondary depot. TCN levels were low and with high clearance relative to hepatic blood flow. A combination of sustained levels of TCNP in RBC and plasma serves as a depot for TCN to elicit robust therapeutic activity in acute lung injury mice models.

Effects of intravenous AICAR (5-aminoimidazole-4-carboximide riboside) administration on insulin signaling and resistance in premature baboons, Papio sp.

Premature baboons exhibit peripheral insulin resistance and impaired insulin signaling. 5' AMP-activated protein kinase (AMPK) activation improves insulin sensitivity by enhancing glucose uptake (via increased glucose transporter type 4 [GLUT4] translocation and activation of the extracellular signal-regulated kinase [ERK]/ atypical protein kinase C [aPKC] pathway), and increasing fatty acid oxidation (via inhibition of acetyl-CoA carboxylase 1 [ACC]), while downregulating gluconeogenesis (via induction of small heterodimer partner [SHP] and subsequent downregulation of the gluconeogenic enzymes: phosphoenolpyruvate carboxykinase [PEPCK], glucose 6-phosphatase [G6PASE], fructose- 1,6-bisphosphatase 1 [FBP1], and forkhead box protein 1 [FOXO1]). The purpose of this study was to investigate whether pharmacologic activation of AMPK with AICAR (5-aminoimidazole-4-carboximide riboside) administration improves peripheral insulin sensitivity in preterm baboons. 11 baboons were delivered prematurely at 125±2 days (67%) gestation. 5 animals were randomized to receive 5 days of continuous AICAR infusion at a dose of 0.5 mg·g-1·day-1. 6 animals were in the placebo group. Euglycemic hyperinsulinemic clamps were performed at 5±2 and 14±2 days of life. Key molecules potentially altered by AICAR (AMPK, GLUT4, ACC, PEPCK, G6PASE, FBP1, and FOXO1), and the insulin signaling molecules: insulin receptor (INSR), insulin receptor substrate 1 (IRS-1), protein kinase B (AKT), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were measured using RT-PCR and western blotting. AICAR infusion did not improve whole body insulin-stimulated glucose disposal in preterm baboons (12.8±2.4 vs 12.4±2.0 mg/(kg·min), p = 0.8, placebo vs AICAR). One animal developed complications during treatment. In skeletal muscle, AICAR infusion did not increase phosphorylation of ACC, AKT, or AMPK whereas it increased mRNA expression of ACACA (ACC), AKT, and PPARGC1A (PGC1α). In the liver, INSR, IRS1, G6PC3, AKT, PCK1, FOXO1, and FBP1 were unchanged, whereas PPARGC1A mRNA expression increased after AICAR infusion. This study provides evidence that AICAR does not improve insulin sensitivity in premature euglycemic baboons, and may have adverse effects.

Parenteral and enteral nutrition in surgical critical care: Plasma metabolomics demonstrates divergent effects on nitrogen, fatty-acid, ribonucleotide, and oxidative metabolism.

BACKGROUND: Artificial nutrition support is central to the care of critically ill patients and is primarily provided enterally (EN). There are circumstances when parenteral nutrition (PN) is considered necessary. We are uncertain how each of these approaches confer clinical benefits beyond simply providing calories. We sought to better understand how each of these techniques influence metabolism in critically ill patients using a broad-based metabolomics approach. Metabolic responses to EN and PN may differ in ways that could help us understand how to optimize use of these therapies. METHODS: We prospectively enrolled subjects over 7 months in 2015 at an urban, Level I trauma center. Subjects were included before starting either EN or PN during their inpatient admission. Plasma samples were obtained between 1 and 12 hours before initiation of artificial nutrition, and 3 and 7 days later. All samples were analyzed with liquid chromatography/mass spectrometry-based metabolomics. Differences in metabolite concentrations were assessed via principal component analyses and multiple linear regression. RESULTS: We enrolled 30 subjects. Among the critically ill subjects, 10 received EN and 10 received PN. In subjects receiving EN, amino acid and urea cycle metabolites (citrulline, p = 0.04; ornithine, p = 0.05) increased, as did ribonucleic acid metabolites (uridine, p = 0.04; cysteine, 0 = 0.05; oxypurinol, p = 0.04). Oxidative stress decreased over time (increased betaine, p = 0.05; decreased 4-pyridoxic acid, p = 0.04). In subjects receiving PN, amino acid concentrations increased over time (taurine, p = 0.04; phenylalanine, p = 0.05); omega 6 and omega 3 fatty acid concentrations decreased over time (p = 0.05 and 0.03, respectively). CONCLUSION: EN was associated with amino acid repletion, urea cycle upregulation, restoration of antioxidants, and increasing ribonucleic acid synthesis. Parenteral nutrition was associated with increased amino acid concentrations, but did not influence protein metabolism or antioxidant repletion. This suggests that parenteral amino acids are used less effectively than those given enterally. The biomarkers reported in this study may be useful in guiding nutrition therapy for critically ill patients. LEVEL OF EVIDENCE: Therapeutic study, level III; prognostic study, level II.

Mycophenolic acid exposure and complement fraction C3 influence inosine 5'-monophosphate dehydrogenase activity in systemic lupus erythematosus.

Background Mycophenolate mofetil has recently been reported to be effective against systemic lupus erythematosus. The influence of the pharmacokinetics of mycophenolic acid, the active form of mycophenolate mofetil and the major inactive mycophenolic acid phenolic glucuronide on the activity of the target enzyme inosine 5'-monophosphate dehydrogenase, is expected to be revealed. The aim of this study was to identify the factors associated with inosine 5'-monophosphate dehydrogenase activity in systemic lupus erythematosus patients. Methods Fifty systemic lupus erythematosus patients in remission maintenance phase (29 received mycophenolate mofetil [MMF+] and 21 did not [MMF-]) were enrolled. Median and interquartile range of dose of mycophenolate mofetil were 1500 and 1000-1500 mg/day, respectively. Stepwise multiple linear regression analysis was performed to assess the dependence between inosine 5'-monophosphate dehydrogenase activity and 25 predictor values including predose plasma concentrations of free mycophenolic acid and mycophenolic acid phenolic glucuronide. Results Median and interquartile range of predose total plasma concentrations of mycophenolic acid and mycophenolic acid phenolic glucuronide were 2.73 and 1.43-5.73 and 25.5 and 13.1-54.7 µg/mL, respectively. Predose inosine 5'-monophosphate dehydrogenase activity was significantly higher in MMF+ than MMF- patients (median 38.3 and 20.6 nmoL xanthosine 5'-monophosphate/g haemoglobin/h, P<0.01). The plasma concentration of free mycophenolic acid phenolic glucuronide, complement fraction C3 and body weight were significant predictors accounting for interindividual variability in the inosine 5'-monophosphate dehydrogenase activity (adjusted R2 = 0.52, P < 0.01) in a multivariate analysis. Conclusions Predose inosine 5'-monophosphate dehydrogenase activity was higher in systemic lupus erythematosus patients receiving mycophenolate mofetil therapy. Inosine 5'-monophosphate dehydrogenase activity may be determined by mycophenolic acid exposure and complement fraction C3 in systemic lupus erythematosus patients.

Doping control analysis of 46 polar drugs in horse plasma and urine using a 'dilute-and-shoot' ultra high performance liquid chromatography-high resolution mass spectrometry approach.

The high sensitivity of ultra high performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS) allows the identification of many prohibited substances without pre-concentration, leading to the development of simple and fast 'dilute-and-shoot' methods for doping control for human and equine sports. While the detection of polar drugs in plasma and urine is difficult using liquid-liquid or solid-phase extraction as these substances are poorly extracted, the 'dilute-and-shoot' approach is plausible. This paper describes a 'dilute-and-shoot' UHPLC-HRMS screening method to detect 46 polar drugs in equine urine and plasma, including some angiotensin-converting enzyme (ACE) inhibitors, sympathomimetics, anti-epileptics, hemostatics, the new doping agent 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), as well as two threshold substances, namely dimethyl sulfoxide and theobromine. For plasma, the sample (200µL) was protein precipitated using trichloroacetic acid, and the resulting supernatant was diluted using Buffer A with an overall dilution factor of 3. For urine, the sample (20µL) was simply diluted 50-fold with Buffer A. The diluted plasma or urine sample was then analysed using a UHPLC-HRMS system in full-scan ESI mode. The assay was validated for qualitative identification purpose. This straightforward and reliable approach carried out in combination with other screening procedures has increased the efficiency of doping control analysis in the laboratory. Moreover, since the UHPLC-HRMS data were acquired in full-scan mode, the method could theoretically accommodate an unlimited number of existing and new doping agents, and would allow a retrospectively search for drugs that have not been targeted at the time of analysis.

Post-thrombolysis haemostasis changes after rt-PA treatment in acute cerebral infarct. Correlations with cardioembolic aetiology and outcome.

BACKGROUND: Little is known, in man, in the post-thrombolytic molecular dynamics of haemostasis, particularly the effect of rt-PA on antifibrinolytic components such as alpha2 anti-plasmin and Factor XIII. AIMS AND HYPOTHESIS: The purpose of this study was to systematically determine changes in coagulation and fibrinolytic parameters after thrombolysis with rt-PA during 24h. We also aimed to correlate these parameters with different acute ischemic stroke subtypes and global outcome. METHODS: Eighty consecutive patients with cerebral infarcts treated with rt-PA had their plasma levels of fibrinogen, plasminogen, alpha2-antiplasmin, Factor XIII, fibrin(ogen) degradation products (FDP) and D-Dimers measured at baseline (h0), 2 (h2) and 24h (h24) after initiation of thrombolysis. Correlations between the variations of these components were statistically studied, using the Spearman rank test or the Pearson test. These haemostatic parameters were also compared with cardioembolic and non cardioembolic patients, as well as between poor and favourable outcome patients. RESULTS: Between h0 and h2, a decrease in fibrinogen, plasminogen, alpha2-antiplasmin, and factor XIII was observed, while an increase in FDP and D-Dimers took place. These values returned to the initial levels at h24. At 2h, the decrease in fibrinogen was significantly correlated with that of plasminogen (0.48, p=0.01), alpha2-antiplasmin (0.48, p=0.004), and factor XIII (0.44, p=0.01); the decrease in plasminogen was significantly correlated with those of antifibrinolytic components, factor XIII (0.47, p=0.02) and alpha2-antiplasmin (r=0.77, p<0.001). These variations were independent of NIHSS. Cardioembolic infarcts showed a statistically significant greater h0-h2 decrease in plasminogen (p=0.04) and an h0-h2 increase in FDP (p=0.02). Poor outcome was linked to low plasminogen values at 2 and 24h. CONCLUSIONS: Supposed to be fibrin-specific, rt-PA induces a decrease in circulating fibrinogen, significantly linked to a decrease in plasminogen. A collateral increase in antifibrinolytic agents such as factor XIII and alpha2-antiplasmin is also observed. At 2h, a significant decrease in plasminogen and a significant increase in fibrin(ogen) degradation products (FDP) are observed in cardioembolic infarcts, and appear as early independent predictors of this aetiology. A low plasminogen value at 2h is potentially predictive of poor prognosis at 3months.

Antiretroviral therapy-induced mitochondrial toxicity: potential mechanisms beyond polymerase-γ inhibition.

We hypothesized that competition between nucleotide reverse-transcriptase inhibitor triphosphate and endogenous deoxyribonucleotide triphosphate (dNTP) may lead to depletion of dNTP pools and mitochondrial dysfunction independent of polymerase-γ (pol-γ) inhibition. We collected peripheral blood mononuclear cells from 75 adults (25 cases: HIV-infected patients with mitochondrial toxicity, 25 HIV-infected positive controls, and 25 HIV-negative controls). We observed statistically significant individual and group differences in ribonucleotide (RN) and deoxyribonucleotide (dRN) pools. The median values for the RN pools were 10,062 (interquartile range (IQR): 7,090-12,590), 4,360 (IQR: 3,058-6,838), and 2,968 (IQR: 2,538-4,436) pmol/10(6) cells for negative controls, positive controls, and cases, respectively. Cases had significantly higher absolute mitochondrial DNA copy number as compared with negative controls (P < 0.05). Moreover, cases had significantly higher expression levels of pol-γ, nucleotide transporters, cellular kinases, and adenosine triphosphate (ATP)-binding cassette (ABC) proteins as compared with controls. Antiretroviral therapy (ART) perturbs RN and dRN pools. Depletion of RN and dRN pools may be associated with ART-induced mitochondrial toxicity independent of pol-γ inhibition.

Antiplatelet effect of AMP-activated protein kinase activator and its potentiation by the phosphodiesterase inhibitor dipyridamole.

AMP-activated protein kinase (AMPK) activates endothelial nitric oxide synthase (eNOS) via phosphorylation at the activating site. The eNOS-nitric oxide (NO)/soluble guanylate cyclase (sGC)-cGMP/cGMP-dependent protein kinase (PKG) signaling axis is a major antiaggregatory mechanism residing in platelets. Based on the hypothesis that direct activation of AMPK might be a potential strategy to inhibit platelet aggregation, the antiplatelet effect of AMPK activators was investigated. Treatment of isolated platelets with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) resulted in AMPK activation and a decrease in aggregation, which was abolished by pretreatment with the AMPK inhibitors compound C (CC) and ara-A. Such an AMPK-dependent antiaggregatory effect was also observed with other AMPK activators such as A-769662 and PT1. AICAR induced eNOS activation was followed by NO synthesis, cGMP production, and subsequent phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a PKG substrate. All these events were blocked by CC or ara-A pretreatment, and each event was inhibited by the eNOS inhibitor L-NAME, the sGC inhibitor ODQ, and the PKG inhibitor Rp-8-pCPT-cGMPS. Simultaneous treatment of dipyridamole, a phosphodiesterase (PDE) inhibitor, with AICAR potentiated the antiaggregatory effect by enhancing the cGMP elevation. Administration of AICAR increased platelet cGMP and prolonged FeCl3-induced arterial occlusion time in rats, which further increased in combination with dipyridamole. In conclusion, AMPK activators inhibited platelet aggregation by stimulating the eNOS-NO/sGC-cGMP/PKG signaling pathway. The antiplatelet effect of AMPK activators could be potentiated in combination with a PDE inhibitor through the common mechanism of elevating cGMP. Thus, AMPK may serve as a potential target for antiplatelet therapy.

Quantification of AICAR-ribotide concentrations in red blood cells by means of LC-MS/MS.

AICAR (5-amino-4-imidazolecarboxyamide ribonucleoside) arguably provides performance-enhancing properties even in the absence of physical exercise and, therefore, the substance is banned in elite sports since 2009. Due to the natural presence of AICAR in human blood and urine, uncovering the misuse by direct qualitative analysis is not possible. Entering the circulation, the riboside is immediately incorporated into red blood cells (RBCs) and transformed into the corresponding ribotide (5'-monophosphate) form. Within the present study, an analytical method was developed to determine AICAR-ribotide concentrations in RBC concentrates by means of liquid chromatography-tandem mass spectrometry. The method was validated enabling quantitative result interpretation considering the parameters specificity, precision (intra- and interday), linearity, recovery, accuracy (LOD/LOQ), stability and ion suppression. By analysing 99 RBC samples of young athletes, normal physiological levels of AICAR-ribotide were determined (10-500 ng/mL), and individual levels were found to be stable for several days. Employing in vitro incubation experiments with AICAR riboside in fresh whole blood samples, the ribotide concentrations were observed to increase significantly within 30 min from baseline to 1-10 µg/mL. These levels are considered conserved for the lifetime of the erythrocyte and, thus, the results of the in vitro model strongly support the hypothesis that measuring abnormally high AICAR-ribotide concentrations in RBC of elite athletes has the potential to uncover the misuse of this substance for a long period of time.

A HPLC method for simultaneous determination of 5-aminoimidazole-4-carboxamide riboside and its active metabolite 5-aminoimidazole-4-carboxamide ribotide in tumor-bearing nude mice plasma and its application to pharmacokinetics study.

A HPLC method with on-line solid phase extraction (SPE) and column switching was developed for simultaneous determination of 5-aminoimidazole-4-carboxamide riboside (AICA riboside) and its active metabolite 5-aminoimidazole-4-carboxamide ribotide (AICA ribotide) in nude mice plasma. Plasma sample was deproteinized by adding a half volume of 10% trichloroacetic acid (TCA), and the resulting supernatant was extracted with diethyl ether to remove TCA. 50 µl aqueous fraction was injected onto a WAX-1 SPE column, and AICA ribotide was trapped on the SPE column, while AICA riboside was eluted from the SPE column. The chromatographic separation of AICA riboside was achieved on CG16 column, and separation of AICA ribotide was performed on HILIC-10 and WAX-1 column. The columns temperature was maintained at 40 °C, and the optimal detection wavelength was 268 nm for both AICA riboside and AICA ribotide. The total analytical run time was 40 min. The proposed method was linear over the range of 0.1-500 µg/ml for AICA riboside and 0.03-50 µg/ml for AICA ribotide. The lower limit of quantification (LLOQ) was 100 and 30 ng/ml for AICA riboside and AICA ribotide, respectively. The sensitivity, accuracy and precision of this method were within acceptable limits during validation period. The method was successfully applied to investigate the pharmacokinetics characteristics of AICA riboside and its active metabolite AICA ribotide in nude mice bearing MCF-7 cell xenografts.

Adenylosuccinate lyase deficiency--first British case.

A deficiency of adenylosuccinate lyase (ASDL) is characterised by the accumulation of SAICAriboside (SAICAr) and succinyladenosine (S-Ado) in body fluids. The severity of the clinical presentation correlates with a low S-Ado/SAICAr ratio in body fluids. We report the first British case of ADSL deficiency. The patient presented at 14 days with a progressive neonatal encephalopathy and seizures. There was marked axial and peripheral hypotonia. Brain MRI showed widespread white matter changes. She died at 4 weeks of age. Concentrations of SAICAr and SAdo were markedly elevated in urine, plasma and CSF and the SAdo/SAICAr ratio was low, consistent with the severe phenotype. The patient was compound heterozygous for 2 novel ADSL mutations; c.9 G>C (A3P) and c.572 C>T (R190X).

Significance of a plasma D-dimer test in patients with primary pulmonary hypertension.

BACKGROUND: D-dimer, a degradation product of fibrin, has been increasingly used as a marker or prognostic factor in various thrombotic diseases. OBJECTIVE: To assess the significance of a d-dimer test in patients with primary pulmonary hypertension (PPH). PATIENTS AND METHODS: Fourteen patients with PPH (12 women and 2 men) aged 25 to 68 years (mean +/- SD age, 50 +/- 14 years) entered the study. Plasma d-dimer was determined by Miniquant assay (Biopool International; Venture, CA) 3 +/- 5 months after the disease onset, and patients were followed up for 1 year. We compared the d-dimer levels to the demographic, clinical, and hemodynamic data of the patients. RESULTS: D-dimer levels were positively correlated with New York Heart Association classification (r = 0.59, p = 0.01) and pulmonary artery pressure (r = 0.43, p = 0.03) and were negatively correlated with oxygen saturation (r = - 0.45, p = 0.03) and 6-min walk distance (r = - 0.49, p = 0.04). One-year survival was also negatively correlated with d-dimer (point-biserial r = - 0.71, p = 0.004), with a higher d-dimer value associated with poorer survival. No significant correlations were found between d-dimer values and sex, age, diffusing capacity of the lung for carbon monoxide, or cardiac index. CONCLUSION: D-dimer levels may have a role in the evaluation of patients with PPH. This simple, noninvasive test may be helpful for identifying patients who are at a higher risk for severe disease.

[D-Dimer determination combined with clinical probability for the diagnosis of leg venous thrombosis]. / Association en pratique clinique du dosage des D-Dimères et d'une probabilité clinique pour le diagnostic de thrombose veineuse des membres inférieurs.

OBJECTIVE: To evaluate the results of combination of D-Dimer test and simple clinical model for the diagnosis of deep vein thrombosis (DVT). MATERIALS AND METHODS: Inclusion: clinical suspicion of DVT. Non inclusion criteria were Clinical model performed by the referring physician included probability varying from high to low. D-Dimer test was performed by five different rapid techniques. Standard of reference was Doppler ultrasonography (DU) performed by a senior radiologist. RESULTS: Eight hundred and fifty-four DU were performed on a 14 months time period, including 206 suspicion of pulmonary embolism, 109 postoperative time period, 120 non-included or excluded patients, 278 incomplete observations, 141 complete observations. DVT was present in 33 cases and absent in the other 108 cases (prevalence 23%). Sensitivity and negative predictive value of the five tests were between 82 and 97% and 90 et 97%. The most sensitive test had a specificity of 36% and a positive predictive value of 32%. Combination of clinical model and D-Dimer test did not improve the diagnostic accuracy. CONCLUSION: None of the test evaluated in the present study, even when combined with the clinical model results, did allow the exclusion of DVT.

Effect of methotrexate on blood purine and pyrimidine levels in patients with rheumatoid arthritis.

OBJECTIVE: The mechanism of anti-inflammatory effects of methotrexate (MTX) at low dose may relate to a decrease in availability of the purine precursor or it may depend on accumulation of 5-aminoimidazole-4-carboxamide (AICAR) and the anti-inflammatory nucleoside adenosine. The aim of this study was to evaluate the possible mechanism of action by analysis of changes in blood concentrations of purine and pyrimidine metabolites during MTX treatment. METHODS: Venous blood samples were collected from rheumatoid arthritis patients before and at different times for up to 7 days after the start of MTX treatment. Whole blood concentrations of adenosine, uridine, hypoxanthine, uric acid and erythrocyte nucleotides were measured by HPLC. RESULTS: The initial blood adenosine concentration was 0.073 +/- 0.013 microM and no differences were observed during MTX treatment. However, a decrease in uric acid concentration was observed from 205.5+/-13.5 to 160. 9+/-13.5 microM (P<0.05) within 24 h after MTX administration. The hypoxanthine concentration decreased in parallel with uric acid, while the uridine concentration decreased 48 h after MTX administration. No accumulation of AICAR-triphosphate (ZTP) was observed in the erythrocytes. CONCLUSIONS: MTX decreases circulating purine and pyrimidine concentrations, and their availability for DNA and RNA synthesis, which may affect immune cell proliferation and protein (cytokine) expression. The absence of adenosine concentration changes and lack of ZTP formation is evidence against an AICAR/adenosine mechanism, although localized adenosine concentration changes cannot be excluded.

Simultaneous determination of purine bases, ribonucleosides and ribonucleotides by capillary electrophoresis-electrochemistry with a copper electrode.

Simultaneous determination of purine bases, ribonucleosides and ribonucleotides was achieved by coupling capillary electrophoresis (CE) with wall-jet amperometric detection. A 200 microm diameter copper disk electrode was applied at working potential, +0.65 V vs. saturated calomel electrode. The current response of high sensitivity and stability was obtained in strong basic solutions which were suitable for satisfactory CE separations. The calibration curve was linear over 2-3 orders of magnitude and the limits of detection for adenine, guanine, xanthine, uric acid, adenosine, guanosine, adenosine-5'- monophosphate and guanosine-5'-monophosphate were below 9 fmol (SIN=3). The use of this method for the separation and detection of compounds present in human plasma samples was reported.

Importance of ribonucleotide availability to proliferating T-lymphocytes from healthy humans. Disproportionate expansion of pyrimidine pools and contrasting effects of de novo synthesis inhibitors.

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.

T-lymphocytes from AIDS patients are unable to synthesize ribonucleotides de novo in response to mitogenic stimulation. Impaired pyrimidine responses are already evident at early stages of HIV-1 infection.

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.