Increased levels of plasma nucleotides in patients with rheumatoid arthritis.

Novel biomarkers of rheumatoid arthritis (RA), in addition to antibodies against cyclic citrullinated peptides, are required. Metabolome analysis is a promising approach to identify metabolite biomarkers for clinical diagnosis. We adopted a comprehensive non-targeted metabolomics approach combining capillary electrophoresis time-of-flight mass spectrometry (TOFMS) and liquid chromatography TOFMS. We constructed metabolomics profiling of 286 plasma samples of a Japanese population [92 RA patients, 13 systemic lupus erythematosus (SLE) patients and 181 healthy controls). RA case-control association tests showed that seven metabolites exhibited significantly increased levels in RA samples compared with controls (P < 1.0 × 10-4; UTP, ethanolamine phosphate, ATP, GDP, ADP, 6-aminohexanoic acid and taurine), whereas one exhibited a decreased level (xanthine). The plasma levels of these eight metabolites were not significantly different between seropositive and seronegative RA patients (P > 0.05; n = 68 and 24, respectively). The four nucleotide levels (UTP, ATP, GDP and ADP) were significantly higher in the non-treatment patients in comparison between patients with and without treatment (P < 0.014; n = 57 and 35, respectively). Furthermore, we found that none of the four nucleotide levels showed significant differences in SLE case-control association tests (P > 0.2; 13 patients with SLE and the 181 shared controls) and psoriatic arthritis (PsA) case-control association tests (P > 0.11; 42 patients with PsA and 38 healthy controls), indicating disease specificity in RA. In conclusion, our large-scale metabolome analysis demonstrated the increased plasma nucleotide levels in RA patients, which could be used as potential clinical biomarkers of RA, especially for seronegative RA.

Determination of 6-thioguanosine diphosphate and triphosphate and nucleoside diphosphate kinase activity in erythrocytes: novel targets for thiopurine therapy?

6-Thioguanine nucleotides are the sum of 6-thioguanosine 5'-monophosphate (TGMP), -diphosphate (TGDP), and -triphosphate (TGTP) representing essential metabolites involved in drug action of thiopurines. Elevated levels of TGDP have been associated with poor response to azathioprine therapy in patients with inflammatory bowel disease. The conversion of TGDP to TGTP is supposed to be catalyzed by nucleoside diphosphate kinase (NDPK). The aim of this work was to investigate simultaneously individual 6-thioguanosine phosphate levels and NDPK activity in red blood cells (RBCs) of patients on azathioprine therapy. Ion-pair high-performance liquid chromatography methods with fluorescence and ultraviolet detection were applied to quantify individual levels of 6-thioguanosine 5'-phosphates and NDPK activity, respectively, in RBCs. Recombinantly expressed NDPK isoforms A and B were unequivocally identified to catalyze the formation of TGTP (30.6 +/- 3.88 nmol x min x mg for NDPK A versus 41.2 +/- 1.05 nmol x min x mg for NDPK B). Comprehensive analyses on the stability of TGMP, TGDP, and TGTP and the reproducibility of NDPK activity in RBCs were performed to provide a reliable sampling protocol for clinical practice. Of note, isolation of RBCs within 6 hours followed by immediate storage at -80 degrees C is crucial for prevention of degradation of 5'-phosphates. In a clinical study of 37 patients on azathioprine, TGTP was the predominant 6-thioguanosine phosphate in RBCs. In contrast, three patients showed TGTP/(TGDP + TGTP) ratios of 57.2%, 64.3%, and 66% corresponding to elevated TGDP levels. NDPK activity ranged from 4.1 to 11.3 nmol x min x mg hemoglobin. No correlation between NDPK activity and the 6-thioguanosine phosphate levels was found. The question whether interindividual variability of NDPK activity may explain differences in 6-thioguanosine 5'-phosphates levels has to be investigated in a prospective large-scale study.

6-thioguanosine diphosphate and triphosphate levels in red blood cells and response to azathioprine therapy in Crohn's disease.

BACKGROUND & AIMS: Azathioprine is the gold standard for immunosuppressive therapy in Crohn's disease (CD) and its molecular mechanism of action is caused by the metabolite 6-thioguanosine triphosphate (TGTP). In this study we assessed the impact of TGTP levels for monitoring of azathioprine therapy. METHODS: A novel, highly sensitive assay was established to measure levels of TGTP and its precursors 6-thioguanosine monophosphates and 6-thioguanosine diphosphates (TGDP) in red blood cells from 50 CD patients. The results were correlated with clinical outcome. RESULTS: TGTP levels could be quantified in 47 patients and a subgroup of these patients showed significantly high levels of TGDP. 6-thioguanine nucleotide (6-TGN) levels showed a significant correlation with TGDP plus TGTP concentrations, suggesting that active TGTP and its inactive precursor TGDP are the main metabolites within 6-TGN. Patients with 6-TGN levels higher than 100 pmol/8x10(8) red blood cells showed better response rates, on average, than patients with lower 6-TGN levels. The subgroup of patients with higher 6-TGN and increased TGDP levels showed a worse outcome with lower response rates, more flares, and higher infliximab demand than patients with high 6-TGN, low TGDP, and predominantly detectable TGTP levels. CONCLUSIONS: This study shows that quantification of TGTP levels can be used to monitor azathioprine therapy in inflammatory bowel disease patients. Furthermore, the data suggest that TGDP levels of more than 15% of total 6-TGN levels may be a useful surrogate parameter to predict poor response in a subgroup of azathioprine-treated patients.

Thiopurine therapies: problems, complexities, and progress with monitoring thioguanine nucleotides.

Metabolism of thiopurine drugs--azathioprine, 6-mercaptopurine, and 6-thioguanine--has provided a powerful pharmacogenetic model incorporating polymorphism of the enzyme thiopurine methyltransferase (TPMT) and the primary active metabolite, thioguanine nucleotide (TGN). However, a sense of uncertainty about the usefulness of TGNs and other thiopurine metabolites has appeared. This review critically appraises the basis of thiopurine metabolism and reveals the problems and complexities in TGN research. Erythrocyte TGN is used in transplantation medicine and in chronic inflammatory conditions such as Crohn's disease, as a "surrogate" pharmacokinetic parameter for TGN in the target cells: leukocytes or bone marrow. It is not generally appreciated that erythrocytes do not express the enzyme IMP dehydrogenase and cannot convert mercaptopurine to TGN, which explains some of the confusion in interpretation of erythrocyte TGN measurements. TGN routinely measured in erythrocytes derives from hepatic metabolism. Another concern is that TGN are not generally assayed directly: most methods assay the thiopurine bases. Ion-exchange HPLC and enzymatic conversion of TGNs to nucleosides have been used to overcome this, and may reveal undisclosed roles for an unusual cytotoxic nucleotide, thio-inosine triphosphate, and methylated thiopurines. There appear to be additional interactions between xanthine oxidase and TPMT, and folate and TPMT, that could predict leukopenia. Difficult questions remain to be answered, which may be assisted by technological advances. Prospective TGN studies, long overdue, are at last revealing clearer results.

Rab6 is phosphorylated in thrombin-activated platelets by a protein kinase C-dependent mechanism: effects on GTP/GDP binding and cellular distribution.

In platelets and other secretory cells, protein kinase C (PKC) plays a role in exocytosis stimulated by physiological extracellular signals, although its linkage to the secretory machinery is poorly understood. We investigated whether Rab6, a GTP-binding protein that fractionates with platelet alpha-granules, may be involved in linking these processes. We found that Rab6 contains two PKC consensus phosphorylation sites that are evolutionarily conserved. In platelets metabolically labelled with [(32)P]P(i), Rab6 phosphorylation was induced by phorbol esters or by thrombin. This phosphorylation was blocked by a specific PKC inhibitor (Ro-31-8220), but not by a p38 mitogen-activated protein kinase inhibitor (PD-169316). Physiological stimulation of platelets caused a PKC-dependent translocation of Rab6 from platelet particulate fractions, nearly doubling the fraction of Rab6 in the cytosol. A human Rab6 isoform (Rab6C) that is preferentially expressed in human platelet RNA was cloned and its phosphorylation by PKC was characterized. Rab6C incorporated up to 2 mol of [(32)P]P(i) per mol of active protein. Rab6C bound GDP and GTP with K(d) values of 113+/-12 and 119+/-27 nM respectively, and hydrolysed GTP at a rate of 100+/-15 micromol of GTP/mol of Rab6C per min. PKC phosphorylation of Rab6C increased the affinity for GTP by 3-fold, although it had lesser effects on GDP (1.6-fold). Phosphorylation did not alter the GTPase activity. In summary, thrombin activation of platelets leads to PKC-dependent phosphorylation of Rab6 and a translocation of Rab6 to the cytosol. We suggest that PKC phosphorylation may be an important mechanism through which Rab functional interactions in vesicle trafficking and secretion can be altered in response to an external stimulus.

Type 1 phosphatase inhibitors reduce the restoration of guanine nucleotide exchange activity of eukaryotic initiation factor 2B inhibited reticulocyte lysates rescued by hemin.

In heme-deficient reticulocyte lysates, the alpha-subunit of eukaryotic initiation factor-2 (eIF-2alpha) is phosphorylated due to the activation of the heme-regulated eIF-2alpha kinase (HRI). Phosphorylation of eIF-2alpha impairs the guanine nucleotide exchange activity of eIF-2B and thereby inhibits or shuts off protein synthesis. Delayed addition of hemin to shut-off lysates inhibits the eIF-2alpha kinase activity of HRI and restores protein synthesis; under those conditions, the endogenous phosphatase of the lysate dephosphorylates phosphorylated eIF-2alpha and restores eIF-2B activity. In this report we present evidence that the restoration of eIF-2B activity is dependent on the concentration of added hemin and is related to HRI activity in lysates. The recovery of eIF-2B activity is not affected by protein synthesis inhibitors such as cycloheximide, pactamycin and puromycin, which do not affect the eIF-2alpha phosphorylation. Also, the functional eIF-2B activity that is available in hemin-supplemented lysates is not affected by phosphatase inhibitors such as okadaic acid and heat-stable inhibitor-2. However, the recovery of eIF-2B activity that is observed by the delayed addition of hemin to inhibited heme-deficient lysates is reduced by inhibitor-2 and high concentrations of okadaic acid. These findings suggest that a type 1 phosphatase is involved in the recovery of eIF-2B activity and protein synthesis upon delayed addition of hemin to heme-deficient lysates.

Liquid-chromatographic study of purine metabolism abnormalities in purine nucleoside phosphorylase deficiency.

Using HPLC methods, we measured the concentrations of nucleosides and nucleotides for a patient with no purine nucleoside phosphorylase (PNP; EC 2.4.2.1) enzymatic activity. Concentrations of inosine and guanosine were abnormally high in urine and plasma, whereas guanosine diphosphate (GDP) and guanosine triphosphate (GTP) concentrations in erythrocytes were depleted. The unusual presence of deoxyribonucleosides (deoxyinosine and deoxyguanosine) and deoxyribonucleotides (dGDP and dGTP) was also notable. Thus, HPLC represents an accurate and useful tool for the study of purine metabolic disorders.

Determination of intracellular levels of 6-mercaptopurine metabolites in erythrocytes utilizing capillary electrophoresis with laser-induced fluorescence detection.

Capillary electrophoresis proved to be a useful technique for the analysis of intracellular levels of 6-thioguanosine mono-, di-, and triphosphate with analysis times of 20 min. Conditions required for baseline separation of the thioguanine nucleotides consisted of a 25 mM KH2PO4 (pH 8.0) buffer and a separation voltage of +28 kV. Laser-induced fluorescence detection (lambda ex = 325 nm, lambda em = 410 nm) of the thioguanine nucleotide metabolites of 6-mercaptopurine (6-MP) was possible following oxidation of the thiol functionality. Tedious extraction procedures involving mercury cellulose resins or phenyl mercury adduct formation, which had been required previously for the selective extraction of thiopurines from erythrocytes, were unnecessary due to the overall specificity of the approach. However, the inclusion of 50 mM EDTA in the sample preparation was required to inhibit the anabolic/catabolic enzymatic activity, which was responsible for the degradation of the analytes. The method demonstrated linearity from 5 to 1700 pmol/100 microliters red blood cells for the three analytes (RSDs < or = 8%). The feasibility of the method was demonstrated for the quantitation of 6-thioguanine nucleotides in patients receiving either oral or intravenous 6-MP therapy.

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)

Regulation of the human neutrophil NADPH oxidase by the Rac GTP-binding proteins.

Recent progress in our understanding of the regulation of the phagocyte NADPH oxidase by the Rac GTP-binding protein(s) has provided the first detailed glimpse into the mechanisms of leukocyte regulation by a small GTP-binding protein. Studies over the past year have indicated that the activity of the NADPH oxidase can be modulated by regulation of the GTP/GDP state of Rac. Proteins exist in leukocytes that are able to modify GTP-binding protein function in this manner, and their activity may be regulated by signals generated upon phagocyte stimulation.

Determination of guanine and its nucleosides and nucleotides in human erythrocytes by high-performance liquid chromatography with postcolumn fluorescence derivatization using phenylglyoxal reagent.

A high-performance liquid chromatographic method with on-line postcolumn fluorescence derivatization is described for the simple and sensitive determination of guanine and its nucleosides and nucleotides in human erythrocytes. After deproteinization of the biospecimen, guanine and its nucleosides and nucleotides were separated on a reversed-phase column (TSKgel ODS-120T) by gradient elution with methanol in the aqueous mobile phase consisting of tetra-n-propylammonium phosphate (pH 6.0) and phosphate buffer (pH 6.0). The compounds were then automatically converted into fluorescent derivatives by reaction with phenylglyoxal. This derivatization was selective for guanine-containing compounds. The present method permitted the reliable quantification of GDP and GTP in human erythrocytes. The detection limits (at a signal-to-noise ratio of 3) for guanine and its nucleosides and nucleotides were 3.2-10.0 pmol in a 20-microliters injection volume. The concentrations of GDP and GTP in human erythrocytes were 17.2 +/- 6.2 and 40.2 +/- 5.8 nmol/ml, respectively.

Low structural specificity for nucleoside triphosphates as antagonists of ADP-induced platelet activation.

Although the platelet ADP receptor is thought to exhibit a high degree of structural selectivity, adenosine 5'-O-(thiotriphosphate) (ATP alpha S) is a potent inhibitor of ADP-induced platelet activation and has been recently shown to bind with high affinity (Kd 3 +/- 0.1 nM) to formaldehyde-fixed platelets and to be photoincorporated into an 18-kDa fragment beginning at Tyr-198 of glycoprotein (GP) IIb alpha (Greco, N. J., Yamamoto, N., Jackson, B. W., Tandon, N. N., Moos, M., Jr., and Jamieson, G. A. (1991) J. Biol. Chem. 266, 13627-13633). Further studies have now shown that guanosine 5'-O-(thiotriphosphate) (GTP alpha S) also binds to high affinity sites (Kd 4.7 +/- 0.9 nM; 13,600 +/- 1,140 sites/platelet) and to low affinity sites (Kd 470 +/- 85 nM; 135,900 +/- 19,400 sites/platelet). Competition binding studies showed that all GTP alpha S binding sites were accessible to ADP and vice versa. The corresponding pyrimidine nucleotide cytidine 5'-O-(thiotriphosphate) (CTP alpha S) was found to be similarly effective in competing in the binding of ADP and both 5'-O-(thiotriphosphates) as well as uridine 5'-O-(thiotriphosphate) (UTP alpha S) were potent inhibitors of platelet shape change and aggregation. Ultraviolet irradiation of platelets in the presence of either [35S]GTP alpha S or [35S]UTP alpha S resulted in their specific incorporation into the alpha chain of GPIIb as previously shown with [35S]ATP alpha S. These results show that the structure of the nucleotide base has little influence on its ability to occupy the ADP-binding site on platelets, to function as an inhibitor of ADP-induced activation or to be photoincorporated into GPIIb alpha.

Receptor-regulated formation of GTP[gamma S] with subsequent persistent Gs-protein activation in membranes of human platelets.

Preincubation of human platelet membranes with the ATP analog ATP[gamma S] led to persistent adenylate cyclase activation. This stimulation was increased by copreincubation with PGE1 and obliterated by removing endogenous GDP by the NTP-regenerating system, creatine phosphate plus creatine kinase. PGE1 partially reversed the action of the regenerating system. Control formation of GTP[gamma S] from ATP[gamma S] and GDP in platelet membranes was apparently not stimulated by PGE1. In contrast, in the presence of creatine phosphate plus creatine kinase, which prevented formation of GTP[gamma S], PGE1 stimulated formation of this GTP analog, by partially reversing the action of the NTP-regenerating system. The data indicate that GTP[gamma S] can be formed by a membrane-associated nucleoside diphosphokinase from ATP[gamma S] and GDP, resulting in persistent Gs-protein activation, and that this process can be stimulated by an agonist-activated receptor.

"In situ" characterization of guanine nucleotide-binding properties of erythrocyte membranes.

Unsealed membranes from human erythrocytes bind GTP and GTP analogs according to first order kinetics, a single rate constant being observed. With [35S]GTP gamma S this is 0.15 +/- 0.2 min-1. Treatment of the membranes with detergents decreases binding considerably. Scatchard plots reveal uncomplicated patterns of ligand association, with Kd values of 10.2 +/- 2.3 nM [35S]GTP gamma S, of 18.2 +/- 4.3 nM [alpha-32P]GTP and of 28.6 +/- 3.5 nM [alpha-32P]GDP, respectively. The stoichiometry with the three ligands is strictly comparable, i.e. 65 +/- 7 picomoles/mg of membrane protein. Binding of each labeled nucleotide is competitively inhibited by the other two unlabeled ligands, the inhibition constants being very close to the corresponding Kd values. Metabolic depletion and subsequent repletion of intact erythrocytes result in membrane preparations still active in guanine nucleotide binding, with unmodified Kd values. However, the stoichiometry falls to 35 picomoles/mg protein with the "depleted" erythrocyte membranes and regains higher values (50 picomoles/mg protein) with the "repleted" cell membranes. Accordingly, the "in situ" characterization of guanine nucleotide-binding properties of erythrocyte membranes seems to represent a new tool for monitoring the metabolic state of intact erythrocytes.

[Relation between energy metabolism, Na+ and K+ levels, and Na,K-ATPase activity in erythrocytes and their volume and shape during overheating]. / Sootnoshenie énergeticheskogo obmena, soderzhaniia Na+ i K+, aktivnosti Na,K-ATFazy v éritrotsitakh s ikh ob''emom i formoi v usloviiakh peregrevaniia organizma.

Development of heat stroke led to a decrease in main reactions of energy production and energy potential in erythrocytes, to a decrease in activity of Na+, K+-ATPase and in content of sodium and potassium as well as to alterations in forms and to decrease in volume of these cells.

Erythrocyte hexose monophosphate shunt is intact in healthy aged humans.

The integrity of the erythrocyte (RBC) hexose monophosphate shunt was investigated in a group of 33 healthy elderly individuals by determining their RBC glutathione content, glucose-6-phosphate dehydrogenase activity and glutathione regeneration. When these parameters were compared with those of the controls, 44 young healthy adults, no significant differences were found. This study indicates that the RBC hexose monophosphate shunt in healthy elderly individuals is intact. Factors other than senescence per se should be sought in elderly individuals who exhibit dysfunction of this shunt.

G-protein dissociation, GTP-GDP exchange and GTPase activity in control and PMA treated neutrophils stimulated by fMet-Leu-Phe.

The addition of the chemotactic factor fMet-Leu-Phe to cell homogenates causes a decrease in the pertussis toxin catalyzed ADP-ribosylation of a 41 kDa protein. The fMet-Leu-Phe induced decrease is not abolished in homogenates prepared from phorbol 12-myristate 13-acetate treated neutrophils. This decreased ribosylation probably reflects a dissociation of the GTP-binding protein oligomer that is not followed by association, possibly because of the release of the alpha-subunit into the suspending medium. Furthermore, fMet-Leu-Phe stimulates the binding of radiolabelled guanylylimidodiphosphate to membrane preparations. Again, the stimulated binding of guanylylimidodiphosphate is not affected by treating the intact neutrophils with phorbol 12-myristate 13-acetate. In addition leukotriene B4, platelet activating factor and fMet-Leu-Phe activate a high-affinity GTPase in membrane preparations. The basal level of this GTPase activity is dramatically inhibited in membrane preparations isolated from cells treated with phorbol 12-myristate 13-acetate. On the other hand, the fMet-Leu-Phe stimulated component is only marginally reduced. The present findings suggest that PMA does not prevent receptor G-protein interaction.