Adenosine In Plasma Research Articles

Probiotic-Derived Ecto-5'-Nucleotidase Produces Anti-Inflammatory Adenosine Metabolites in Treg-Deficient Scurfy Mice.

Probiotic Limosilactobacillus reuteri DSM 17938 (DSM 17938) prolongs the survival of Treg-deficient scurfy (SF) mice and reduces multiorgan inflammation by a process requiring adenosine receptor 2A (A2A) on T cells. We hypothesized that L. reuteri-derived ecto-5'-nucleotidase (ecto-5'NT) activity acts to generate adenosine, which may be a central mediator for L. reuteri protection in SF mice. We evaluated DSM 17938-5'NT activity and the associated adenosine and inosine levels in plasma, gut, and liver of SF mice. We examined orally fed DSM 17938, DSM 17938Δ5NT (with a deleted 5'NT gene), and DSM 32846 (BG-R46) (a naturally selected strain derived from DSM 17938). Results showed that DSM 17938 and BG-R46 produced adenosine while "exhausting" AMP, whereas DSM 17938∆5NT did not generate adenosine in culture. Plasma 5'NT activity was increased by DSM 17938 or BG-R46, but not by DSM 17938Δ5NT in SF mice. BG-R46 increased both adenosine and inosine levels in the cecum of SF mice. DSM 17938 increased adenosine levels, whereas BG-R46 increased inosine levels in the liver. DSM 17938Δ5NT did not significantly change the levels of adenosine or inosine in the GI tract or the liver of SF mice. Although regulatory CD73+CD8+ T cells were decreased in spleen and blood of SF mice, these regulatory T cells could be increased by orally feeding DSM 17938 or BG-R46, but not DSM 17938Δ5NT. In conclusion, probiotic-5'NT may be a central mediator of DSM 17938 protection against autoimmunity. Optimal 5'NT activity from various probiotic strains could be beneficial in treating Treg-associated immune disorders in humans.

Determination of adenosine and its modifications in urine and plasma from breast cancer patients by hydrophilic interaction liquid chromatography-tandem mass spectrometry

RNA modifications have been revealed to be essential in many biological activities, and their disorders are associated with various human diseases, including cancers. 2′-O-methyladenosine (Am), N1-methyladenosine (m1A), N6-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am) and N6,N6-dimethyladenosine (m62A) are important adenosine (A) modifications. The noninvasive collection of urine samples and the diverse contents of metabolites in plasma make them favored biofluids for biomarkers discovery. In this work, we established a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method to quantify these six nucleosides in urine and plasma of healthy controls and breast cancer (BC) patients. The limit of detection (LOD) for A, Am, m1A, m6A, m6Am, and m62A were 0.0025, 0.01, 0.05, 0.005, 0.005, and 0.005 nM. The results showed that the concentrations of Am, m6A, and m6Am were increased, whereas m1A was decreased in the urine of BC patients compared with the healthy controls. We also found that the level ratios of m1A/A, m6A/A, and m6Am/A were all reduced in plasma from BC patients, compared with healthy controls. Interestingly, these ratios of methylated adenosine nucleosides to adenosine in plasma could better discriminate BC patients from healthy controls, compared to the levels of these nucleosides. The present study not only suggests these modified adenosines can act as noninvasive biomarkers of BC but also will contribute to investigating the impacts of RNA methylation on the occurrence and development of BC.

Adenosine-imprinted magnetic core-shell polyvinylbutyral microbeads for quantification of adenosine in plasma

Adenosine is an important molecule in the human body because it participates various biochemical processes, signalling in the physiological processes, and neurological disorders. In the current study, the surface imprinting method was used to prepare adenosine-imprinted magnetic core-shell polyvinylbutyral microbeads. These microbeads were utilized for quantification of adenosine in aqueous solution and control plasma in the range of 1–200 µM. The limit of detection was found to be 1.9 nM, which is quite sensitive compared with to some earlier studies. Fourier transform infrared spectroscopy, scanning electron microscopy, and a Zetasizer (particle size analyzer) were used for characterization of the prepared imprinted microbeads. To determine the efficiency of this method, selectivity experiments were conducted with adenosine-imprinted and non-imprinted magnetic core-shell polyvinylbutyral microbeads and with the competitive nucleosides cytidine, uridine, guanosine, and thymidine. Thermodynamic and kinetic studies were performed to assess adsorption of adenosine onto the adenosine-imprinted magnetic core-shell polyvinylbutyral microbeads from adenosine solution. The efficiency was linked to the specific surface reactivity, polarity and porosity of the imprinted microbeads.

Immunosuppressive adenosine - a novel treatment target for multiple myeloma

Anti-PD1/PDL1 checkpoint therapy has been unsuccessful in myeloma patients. Adenosine is a new therapeutic target for cancer therapy. Two ectoenzymes, CD39 and CD73, catalyse adenosine from extracellular ATP. CD39 converts ATP to AMP and CD73 AMP to adenosine. When investigating the role of adenosine in the immune response patients, we found increased concentration of adenosine in bone marrow plasma from myeloma patients compared with healthy controls. CD39 was expressed on myeloma cells and immune cells whereas CD73 was found on leukocytes and stromal cells in the bone marrow.

Abstract LB-117: Role of ectoenzymes CD39 and CD73 in the immune response to multiple myeloma

Abstract Multiple Myeloma is a cancer of the plasma cells in the bone marrow. Despite from the fact that new drugs have increased survival in the past decade, it is still an incurable cancer. It is therefore important to develop new drugs. Tumor immunosurveillance contributes to the control of cancer growth, but there many different escape mechanisms utilized by tumour to evade anti-tumour immune responses. One of the largely unexplored mechanisms of immune suppression in multiple myeloma is the role of adenosine in the bone marrow microenvironment, which now represents an attractive new therapeutic target for cancer therapy. 2 ectoenzymes, CD39 and CD73 are important for converting extracellular ATP to adenosine. When investigating the role of adenosine in the immune response to multiple myeloma, we found increased concentration of adenosine in the bone marrow plasma from myeloma patients compared with healthy controls. CD39 (ENTPD1) was expressed by myeloma cell lines and on myeloma cells from patients. CD73 was found on leukocytes and stromal cells in the bone marrow. Although very few cells expressed CD73 in the bone marrow, these cells could clearly convert AMP to adenosine. ATP was converted to AMP by CD39+ myeloma cell lines, and CD73+ stromal cells converted AMP to adenosine. Importantly, co-culture of CD39+ myeloma cells and CD73+ stromal cells catalyzed extracellular ATP to immunosuppressive adenosine. Indeed, we found that CD3 T cells stimulated with anti CD3/CD28 beads in the presence of supernatants from co-cultured cells did not proliferate as well as T cell culture without this supernatant. The fact that this proliferation was inhibited by the ZM241385 inhibitor, suggested that the suppression was mediated by the A2AR receptor. The CD39 inhibitor POM1 inhibited adenosine generation in the co-culture system as well as T cell proliferation in vitro. We have recently obtained the CD73 antibody MEDI199447 from MedImmune and are testing the ability of this antibody to prevent adenosine production and T cell proliferation in vitro, as well as the ability to reduce tumor growth in a mouse model of multiple myeloma. The clinical importance of the adenosine pathway in multiple myeloma found when analyzing patient data. Using the publicly available coMMpass database, we found that patients with high expression of CD39 had significantly reduced progression free and overall survival. Citation Format: Rui Yang, Samah Elsaadi, Kristine Misund, Geir Slupphaug, Eline Menu, Carl Hay, Zac Cooper, Karin Vanderkerken, Magne Børset, Anne Marit Sponaas. Role of ectoenzymes CD39 and CD73 in the immune response to multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-117.

Simple Pretreatment and HILIC Separation for LC-ESI-MS/MS Determination of Adenosine in Human Plasma.

A simple pretreatment method and separation mode for the LC-ESI-MS/MS determination of adenosine in human plasma have been developed. Deproteinization by acetonitrile and ultrafiltration followed by chromatographic separation with a hydrophilic interaction chromatographic (HILIC) column give a highly sensitive MS/MS response without ionic suppression caused by the matrix compounds in human plasma. In addition, the presence of ammonium acetate in the mobile phase contributes to high sensitivity in MS/MS detection, facilitating the ionization of adenosine. This method seems to be amenable to the treatment of many samples in clinical practice.

Development and application of a LC–MS/MS method to quantify basal adenosine concentration in human plasma from patients undergoing on-pump CABG surgery

A sensitive and robust LC–MS/MS method was developed to quantify basal adenosine concentrations in human plasma of patients undergoing on-pump coronary artery bypass grafting (CABG) surgery. A strong cation exchange (SCX) monolithic cartridge was used to enrich analyte, improve robustness, and reduce biological complexity. A simple modifier-free mobile phase was employed to improve sensitivity and reproducibility. This method exhibits consistent precision and accuracy, and the RSDs or REs of all the intraday and interday determinations were within 10%. The calibration curve was linear across the examined dynamic range from 1nM to 500nM (r2=0.996). LOD and LOQ were determined to be 0.257nM and 0.857nM respectively, while LLOQ was below 10nM. This method was used to monitor changes of adenosine levels in patient plasma drawn intraoperatively during on-pump CABG surgery. The analysis of 84 patients revealed that the mean concentration of adenosine in coronary sinus plasma after cardiopulmonary bypass (CPB) is higher than that in coronary sinus before CPB (p=0.0024; two-tailed t-test) and that in radial artery plasma after CPB (p=0.0409; two-tailed t-test). These findings suggest that the equilibrium between adenosine production and elimination has favored the elevation of adenosine basal level during on-pump CABG surgery and the change is specific to heart tissues. Evaluation of adenosine with a sensitive and robust analytical method has important implications on providing consistent results and meaningful insights into adenosine regulation, as well as its steady state and sustained action on the heart. Relating patient characteristics or clinical outcomes with basal adenosine concentration can be used to optimize the CABG–CPB maneuver by regulating adenosine level via pharmacological intervention, and differentiating adenosine's contribution to cardioprotection from other modulatory factors.

Highly sensitive method for the determination of adenosine by LC‐MS/MS‐ESI: method validation and scope of application to a pharmacokinetic/pharmacodynamic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of adenosine in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electro-spray ionization in the positive-ion mode. The assay procedure involves extraction of adenosine and phenacetin (internal standard, IS) from rat plasma with a simple protein precipitation extraction process. The method was validated using rat plasma with extinguished adenosine endogenous levels. Chromatographic separation was achieved using a binary gradient using mobile phase A (acetonitrile) and B (0.2% formic acid in water) at a flow rate of 0.50 mL/min on an Atlantis dC(18) column with a total run time of 4.0 min. The MS/MS ion transitions monitored were 268 → 136 for adenosine and 180 → 110 for IS. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.48 ng/mL and the linearity range extended from 0.48 to 1210 ng/mL. The intra- and inter-day precisions were in the ranges 2.32-12.7 and 4.01-9.40%, respectively.

In vitro stability and metabolism of O2′, O3′, O5′-tri-acetyl-N6-(3-hydroxylaniline) adenosine in rat, dog and human plasma: Chemical hydrolysis and role of plasma esterases

O2′, O3′, O5′-tri-acetyl-N6-(3-hydroxylaniline)adenosine (WS070117), a new structure-type lipid regulator, is being developed in pre-clinical study. In order to monitor drug kinetics it is essential to understand pre-analytical factors that may affect drug assay.In vitro stability and metabolism were investigated using high-performance liquid chromatography (HPLC) method in this study. The hydrolysis products were identified by HPLC–mass spectrometry (MS)/MS method. The esterases involved in WS070117 hydrolysis was assigned via inhibition rate assay.It was found that WS070117 was chemically unstable in alkaline solutions compared to acidic and near neutral solutions. Enzymatic hydrolysis was even more rapid. Hydrolytic rate constants differ between species, being 4.24, 5.96 × 10−3 and 6.85 × 10−2 min−1 in rat, dog and human plasma at 37°C, respectively. The hydrolysis was catalyzed by plasma esterase because NaF (sodium fluoride: a general esterase inhibitor) inhibited WS070117 hydrolysis and metabolite production. Hydrolysis was fast in rat plasma and was catalysed by carboxylesterase and butyrylcholinesterase. In dog plasma, carboxylesterase, butyrylcholinesterase and paraoxonase were mainly responsible. Butyrylcholinesterase was the major esterase involved in WS070117 hydrolysis in human plasma. The WS070117 hydrolysis in plasma proceeded by gradual loss of acetyl groups.The knowledge of in vitro drug stability and metabolic pathways identified in this study will be essential for future pre-clinical and clinical pharmacokinetics studies.

Determination of Adenosine and Inosine in Sheep Plasma Using Solid Phase Extraction Followed by Liquid Chromatography with UV Detection

A simple and sensitive liquid chromatography assay following solid phase extraction was developed for simultaneous determination of adenosine and inosine in sheep plasma. The system consisted of a Symmetry C18 column, a mobile phase composed of acetonitrile, 100 mM sodium dihydrogen phosphate and water, and ultraviolet detection at 254 nm. The method showed good sensitivity (limits of detection for adenosine and inosine were 30 and 50 ng/ml, respectively, in the plasma samples), repeatability, and linearity. The developed method was applied to sheep plasma samples from a study examining the cardio active potential of the combination of adenosine and inosine.

Voltammetric determination of adenosine and guanosine using fullerene-C 60-modified glassy carbon electrode

A fullerene-C 60-modified glassy carbon electrode (GCE) is used for the simultaneous determination of adenosine and guanosine by differential pulse voltammetry. Compared to a bare glassy carbon electrode, the modified electrode exhibits an apparent shift of the oxidation potentials in the cathodic direction and a marked enhancement in the voltammetric peak current response for both the biomolecules. Linear calibration curves are obtained over the concentration range 0.5 μM–1.0 mM in 0.1 M phosphate buffer solution at pH 7.2 with a detection limit of 3.02 × 10 −7 M and 1.45 × 10 −7 M for individual determination of adenosine and guanosine, respectively. The interference studies showed that the fullerene-C 60-modified glassy carbon electrode exhibited excellent selectivity in the presence of hypoxanthine, xanthine, uric acid and ascorbic acid. The proposed procedure was successfully applied to detect adenosine and guanosine in human blood plasma and urine, without any preliminary pre-treatment.

Leukocyte count and leukocyte ecto-nucleotidase are major determinants of the effects of adenosine triphosphate and adenosine diphosphate on platelet aggregation in human blood

ADP induces platelet aggregation in human whole blood and platelet-rich plasma (PRP). ATP induces aggregation in whole blood only; this involves leukocytes and is mediated by ADP. Here we studied ATP- and ADP-induced aggregation in patients with raised leukocyte counts (mean 46.2 × 103 leukocytes/µl). Platelet aggregation was measured by platelet counting. ATP, ADP and metabolites were measured by HPLC. Aggregation to ADP (1–10 µM) and ATP (10–100 µM) was markedly reduced, but to ATP (1000 µM) was enhanced (all p < 0.001). Aggregation to ADP in PRP was normal. Increasing the leukocyte count in normal blood reproduced the findings in the patients. Adding leukocytes (either MNLs or PMNLs) to normal PRP enabled a response to ATP and caused marked inhibition of ADP-induced aggregation. Breakdown of ATP or ADP to AMP and adenosine in leukocyte-rich plasma was rapid (t1/2 = 4 min) and far higher than in cell-free plasma or PRP. With ATP there was also formation of ADP, maximal at 4 min. The presence of the ectonucleotidase NTPDase1 (CD39) was demonstrated on MNLs (all of the monocytes and a proportion of the lymphocytes) and all PMNLs by flow cytometry. We conclude that leukocytes provide a means of dephosphorylating ATP which enables ATP-induced aggregation via conversion to ADP, but also convert ADP to AMP and adenosine. Platelet aggregation extent is a balance between these activities, and high white cell counts influence this balance.

Brain to blood efflux transport of adenosine: blood-brain barrier studies in the rat.

The blood-brain barrier (BBB) efflux transport of [(14)C] adenosine was studied using the brain efflux index (BEI) technique. BEI increased linearly over the first 2 min after injection, with deviation from linearity thereafter; 90.12 +/- 1.5% of the injected [(14)C] radioactivity remained within the brain after 20 min. The remaining tracer appears to be mainly intracellular, trapped by phosphorylation, as an almost linear increase of BEI over 20 min was observed after intracerebral injection of [(14)C] adenosine together with 5-iodo tubercidin. The BBB efflux clearance of [(14)C] radioactivity was estimated to be 27.62 +/- 5.2 micro L/min/g, almost threefold higher than the BBB influx clearance estimated by the brain uptake index technique. High-performance liquid chromatography (HPLC) analysis of blood plasma collected from the jugular vein after the intracerebral injection revealed metabolic breakdown of [(14)C] adenosine into nucleobases. The BBB efflux transport was saturable with apparent K(m) = 13.22 +/- 1.75 micro m and V(max) = 621.07 +/- 71.22 pmole/min/g, which indicated that BBB efflux in vivo is 6.2-12p mole/min/g, negligible when compared to the reported rate of adenosine uptake into neurones/glia. However, these kinetic parameters also suggest that under conditions of elevated ISF adenosine in hypoxia/ischaemia, BBB efflux transport could increase up to 25% of the uptake into neurones/glia and become an important mechanism to oppose the rise in ISF concentration. HPLC-fluorometry detected 93.6 +/- 5.25 nm of adenosine in rat plasma, which is 17- to 220-fold lower than the reported K(m) of adenosine BBB influx in rat. Together with the observed rapid degradation inside endothelial cells, this indicated negligible BBB influx of intact adenosine under resting conditions. Cross-inhibition studies showed that unlabelled inosine, adenine and hypoxanthine caused a decrease in BBB efflux of [(14)C] radioactivity in a concentration-dependent manner, with K(i) of 16.7 +/- 4.88, 65.1 +/- 14.1 and 71.1 +/- 16.9 micro m, respectively. This could be due to either competition of unlabelled molecules with [(14)C] adenosine or competition with its metabolites hypoxanthine and adenine for the same transport sites.

Characterization of nucleoside transport systems in cultured rat epididymal epithelium.

The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na(+) dependent, while 30% was Na(+) independent. The Na(+)-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na(+)-dependent [(3)H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na(+)-dependent [(3)H]guanosine uptake was inhibited by thymidine by 20% and Na(+)-dependent [(3)H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na(+)-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma.

Simultaneous measurement of adenosine and hypoxanthine in human umbilical cord plasma using reversed-phase high-performance liquid chromatography with photodiode-array detection and on-line validation of peak purity

A new, robust and sensitive reversed-phase high-performance liquid chromatographic method was developed for concomitant measurement of plasma concentrations of the ATP catabolites adenosine and hypoxanthine in human umbilical cord blood. Deproteinized cord plasma was chromatographed on Hypersil C 18 columns, using UV photodiode-array detection, spectral analysis of peaks and on-line confirmation of peak purity. Elution with a gradient of acetonitrile–tetrahydrofuran in ammonium dihydrogen phosphate buffer pH 4.7, yielded sharp, well-resolved peaks of adenosine and hypoxanthine within 16 min. Peak areas were quantified from external calibration curves and converted to plasma concentrations via cord blood hematocrits. In seven deliveries, gestational ages 32–40 weeks, adenosine (range, 0.1–2.1 μ M) was less than hypoxanthine (range, 1.6–18.5 μ M) in the same cord plasma sample. Arteriovenous levels of each purine were similar, except in an abruptio placenta delivery.

Abundance of guanine, guanosine, inosine and adenosine in human seminal plasma.

Guanine, guanosine, inosine and adenosine were found in large amounts in seminal plasma from 145 men, regardless of whether spermatozoa were present or not. The mean guanine level in 61 normozoospermic men was 89.7 +/- 93.1 mumol/l; this was significantly lower in 32 vasectomized men (18.9 +/- 31 mumol/l) suggesting the involvement of the epididymis in its secretion. Guanine and nucleoside levels were significantly higher in the seminal plasma of oligozoo- and azoospermic than normozoospermic men. Guanine and nucleoside levels were consistently inter-related in the seminal plasma of normozoospermic men with the best correlation between guanine and guanosine.

Determination of Plasma Adenosine by High-Performance Liquid Chromatography with Column Switching and Fluorometric Detection

A new method for the rapid determination of plasma adenosine concentrations was developed by using high-performance liquid chromatography with a column switching technique and fluorometric detection. Several "stop solutions" were used to prevent the enzymatic degradation and cellular uptake and release of adenosine in blood samples. Red blood cells and certain denatured proteins were separated by centrifugation. Subsequently, the supernatant was transferred directly into autosample vials and adenosine was reacted with chloroacetaldehyde to form a strong fluorescent, 1-N6-ethenoadenosine. The adenosine derivative was injected directly and separated on a shielded hydrophobic phase column coupled with a C18 reverse-phase column using a column switching valve. Macromolecules and other interfering substances were excluded by the shielded hydrophobic phase column and bypassed to waste. Then, the adenosine derivative and other retained compounds were switched onto the reverse-phase column for further separation and subsequently to the fluorescence detector. The system reduces the analysis time and contamination of the column and hence allows a shorter cleanup time and a longer column lifetime. Adenosine as low as 30 fmol (signal-to-noise ratio, S/N = 3) can be detected by this method. The percentage of recovery of adenosine in plasma treated with adenosine deaminase was above 90%. This method is very rapid (without tedious sample preparation) and sensitive for determining adenosine in canine blood and should prove to be useful in analyzing the effects of ischemia and reperfusion on arterial and coronary venous adenosine concentrations in blood or perfusate samples released from the ischemic or hypoxic myocardium.

Improved Detection of Ischemia-Induced Increases in Coronary Sinus Adenosine in Patients with Coronary Artery Disease

Attempts to monitor coronary sinus adenosine as a clinical marker of myocardial ischemia in humans have been disappointing. Accordingly, procedures have been developed for detecting adenosine in blood collected from the human coronary sinus. Collection involves using a double-lumen metabolic catheter, which allows blood to be mixed with a stop solution at the catheter tip, thereby minimizing adenosine formation and degradation. A five-component stop solution almost completely arrests adenosine formation and degradation. Adenosine analysis is improved by using both boronate and C18 Sep-Pak columns to purify and concentrate adenosine in human plasma before HPLC. Plasma adenosine in the coronary sinus of patients with and without coronary artery disease, measured before and during peak atrial pacing, showed a twofold atrial pacing-induced increase in adenosine in the patients with coronary artery disease (n = 9, P less than 0.001) but no change in the patients with normal epicardial coronary arteries (n = 6). These preliminary results indicate that coronary sinus adenosine may provide an index of myocardial ischemia in patients with coronary artery disease.

A Simple and Sensitive Radioimmunoassay for Adenosine

We developed a simple and sensitive radioimmunoassay (RIA) for adenosine. The RIA is based on the double antibody method with adenosine 2', 3'-0-disuccinyl-3-[125I]-iodotyrosine methyl ester (diSc-adenosine-[125I]-TME) as a tracer. Anti-adenosine antiserum for the RIA was raised in rabbits immunized with diSc-adenosine conjugated to human serum albumin (diSc-adenosine-HSA). All samples and standards were succinylated prior to assay. The present immunoassay allows detection of 6.25-400 pmol/ml of adenosine in sample. Values obtained by the RIA and by a HPLC analysis showed a high correlation with correlation coefficient of 0.997. In order to determine adenosine in plasmas, blood cells must be separated in the presence of 6 mM EDTA, 0.006% dipyridamole (Dip) and 23 microM 2'-deoxycoformycin (dCF) at 2 degrees C. Adenosine in plasma could be accurately determined by the proposed method even without any pretreatments by deproteinizing. The adenosine levels with or without EDTA-treated normal human plasmas determined were 26.2 +/- 7.26 and 100 +/- 3.62 pmol/ml (mean +/- SEM), respectively.

Improved high-pressure liquid chromatographic-fluorometric assay for measurement of adenosine in plasma

A high-pressure liquid chromatographic system using fluorescence detection was characterized for the determination of adenosine levels in plasma from anesthetized cat, rat, dog, mouse, rabbit, and guinea pig. The optimal concentration of chloroacetaldehyde necessary to convert physiological levels of adenosine to its fluorescent derivative 1,N6-ethenoadenosine (ethenoadenosine) was in excess of 220 mM. As little as 0.2 pmol of ethenoadenosine could be measured, and detection was linear up to 200 pmol. Derivatization of adenosine into ethenoadenosine was highly dependent on temperature and time. However, ethenoadenosine showed thermal instability in that levels dropped sharply after 30 min at 100 degrees C, 2 h at 80 degrees C, and 24 h at 55 degrees C. Adenosine nucleotides were extracted from plasma samples with an efficiency of greater than 91% to prevent adenosine formation from ATP and AMP that would have otherwise occurred during the derivatization procedure. Plasma levels (microM) of adenosine in venous blood were 0.31 in dog, 0.54 in cat, 0.71 in guinea pig, 1.03 in mouse, 1.04 in rat, and 1.68 in rabbit. Plasma levels of adenosine in arterial blood were not significantly different from levels in venous blood. This method can be used to measure even very low levels of adenosine without interference from nucleotide breakdown.