Actions of primary health care professionals to reduce maternal mortality in the Brazilian Northeast.

BACKGROUND: Maternal mortality is a global public health problem. Statistics show that in 2013, 289,000 women died from complications during pregnancy, childbirth or the postpartum period worldwide. Between 2010 and 2015, there were 10,075 maternal deaths in Brazil, 3,522of which occurred in the Northeast region. The aim of this study was to investigate the actions taken by primary health care (PHC) professionals to reduce maternal mortality. METHOD: This was a cross-sectional, descriptive field study with a qualitative approach. The sample comprised 81 graduate-level professionals working in PHC in the state of Ceará, Brazil. Data were collected from January to March 2016 using structured interviews, which were digitally audio recorded and transcribed. The results were organized using collective subject discourse and analyzed according with the relevant literature. RESULTS: The PHC professionals took both individual and joint measures to reduce maternal mortality. These activities included home visits, health education, active searches, prenatal care consultations, referrals to specialized care and outreach. The challenges that must be overcome to prevent maternal mortality include poor care and ineffective public policies that are associated with a lack of managerial support. CONCLUSION: Interaction among professionals in the health care network is critical to the development of cross-sectoral projects that improve the quality of women's health care. Prenatal care is a key factor in reducing maternal death and enables the identification and classification of the risks to which pregnant women may be exposed and the implementation of early actions that can ensure a safe and uncomplicated delivery. However, all of these actions require effective public policies and managerial support.

Erratum: Towards a Unified Description of the Rheology of Hard-Particle Suspensions [Phys. Rev. Lett. 115, 088304 (2015)].

This corrects the article DOI: 10.1103/PhysRevLett.115.088304.

Gravitational collapse of depletion-induced colloidal gels.

We study the ageing and ultimate gravitational collapse of colloidal gels in which the interparticle attraction is induced by non-adsorbing polymers via the depletion effect. The gels are formed through arrested spinodal decomposition, whereby the dense phase arrests into an attractive glass. We map the experimental state diagram onto a theoretical one obtained from computer simulations and theoretical calculations. Discrepancies between the experimental and simulated gel regions in the state diagram can be explained by the particle size and density dependence of the boundary below which the gel is not strong enough to resist gravitational stress. Visual observations show that gravitational collapse of the gels falls into two distinct regimes as the colloid and polymer concentrations are varied, with gels at low colloid concentrations showing the onset of rapid collapse after a delay time. Magnetic resonance imaging (MRI) was used to provide quantitative, spatio-temporally resolved measurements of the solid volume fraction in these rapidly collapsing gels. We find that during the delay time, a dense region builds up at the top of the sample. The rapid collapse is initiated when the gel structure is no longer able to support this dense layer.

Towards a Unified Description of the Rheology of Hard-Particle Suspensions.

The rheology of suspensions of Brownian, or colloidal, particles (diameter d≲1 µm) differs markedly from that of larger grains (d≳50 µm). Each of these two regimes has been separately studied, but the flow of suspensions with intermediate particle sizes (1 µm≲d≲50 µm), which occur ubiquitously in applications, remains poorly understood. By measuring the rheology of suspensions of hard spheres with a wide range of sizes, we show experimentally that shear thickening drives the transition from colloidal to granular flow across the intermediate size regime. This insight makes possible a unified description of the (noninertial) rheology of hard spheres over the full size spectrum. Moreover, we are able to test a new theory of friction-induced shear thickening, showing that our data can be well fitted using expressions derived from it.

Transient dynamics during stress overshoots in binary colloidal glasses.

We investigate, using simultaneous rheology and confocal microscopy, the time-dependent stress response and transient single-particle dynamics following a step change in shear rate in binary colloidal glasses with large dynamical asymmetry and different mixing ratios. The transition from solid-like response to flow is characterised by a stress overshoot, whose magnitude is linked to transient superdiffusive dynamics as well as cage compression effects. These and the yield strain at which the overshoot occurs vary with the mixing ratio, and hence the prevailing caging mechanism. The yielding and stress storage are dominated by dynamics on different time and length scales, the short-time in-cage dynamics and the long-time structural relaxation respectively. These time scales and their relation to the characteristic time associated with the applied shear, namely the inverse shear rate, result in two different and distinct regimes of the shear rate dependencies of the yield strain and the magnitude of the stress overshoot.

Associations of preterm birth and neonatal stress exposure with chronic pain in adulthood - Results from the Gutenberg prematurity study.

Although the effect of early childhood stress on central nervous pain processing is well known, studies on the association of prematurity and chronic pain are scarce. This study used data from a single-centre retrospective cohort study followed by a prospective clinical examination and pain assessment. The study was based on data from the local birth registry. Newborns born between 1969 and 2002 who had reached adulthood were eligible .. Using a selection algorithm, a study cohort stratified by gestational age (GA) was recruited. Chronic pain conditions were assessed using questionnaire and standardized pain drawings. Data on the pre-, peri- and postnatal clinical course was assessed from medical records. Multivariable logistic regression analyses were conducted to investigate associations between prematurity and chronic pain with adjustment for age, gender, socioeconomic status, and perinatal stress factors. 427 participants born preterm and full-term were included (age 28.5 ± 8.7 years). Chronic pain conditions were similarly common between groups with different levels of prematurity (GA ≥ 37 weeks: 34.5 %, GA33-36 weeks: 37.6 %, GA32-29 weeks: 25.2 %, GA < 29 weeks: 30.4 %, p = 0.20). In multivariable analyses, no association between low GA and the presence of chronic pain was found (OR = 0.99 (CI95 %: 0.94-1.04, p = 0.63); this was also true for a subanalysis of widespread pain. While neither fetal nutritional status nor perinatal stressors were associated with pain, exposure to maternal but not paternal smoking during pregnancy was associated with increased risk to develop pain (OR = 2.77 (CI95 %: 1.31-5.88, p = 0.008) in adults born preterm and full-term. This study suggests that prematurity by itself does not increase the risk of chronic pain later in life, but provides preliminary evidence for maternal smoking during pregnancy as risk factor.

Gastrin-releasing peptide acts via postsynaptic BB2 receptors to modulate inward rectifier K+ and TRPV1-like conductances in rat paraventricular thalamic neurons.

Gastrin-releasing peptide (GRP) is a bombesin-like peptide with a widespread distribution in mammalian CNS, where it has a role in food intake, circadian rhythm generation, fear memory, itch sensation and sexual behaviour. While it has been established that GRP predominantly excites neurons, details of the membrane mechanism involved in this action remain largely undefined. We used perforated patch clamp recording in acute brain slice preparations to investigate GRP-affected receptors and ionic conductances in neurons of the rat paraventricular thalamic nucleus (PVT). PVT is a component of the midline and intralaminar thalamus that participates in arousal, motivational drives and stress responses, and exhibits a prominence of GRP-like immunoreactive fibres. Exposure of PVT neurons to low nanomolar concentrations of GRP induced sustained TTX-resistant membrane depolarizations that could trigger rhythmic burst discharges or tonic firing. Membrane current analyses in voltage clamp revealed an underlying postsynaptic bombesin type 2 receptor-mediated inward current that resulted from the simultaneous suppression of a Ba(2+)-sensitive inward rectifier K(+) conductance and activation of a non-selective cation conductance with biophysical and pharmacological properties reminiscent of transient receptor potential vanilloid (TRPV) 1. A role for a TRPV1-like conductance was further implied by a significant suppressant influence of a TRPV1 antagonist on GRP-induced membrane depolarization and rhythmic burst or tonic firing. The results provide a detailed picture of the receptor and ionic conductances that are involved in GRP's excitatory action in midline thalamus.

Development of a compounded propofol nanoemulsion using multiple non-invasive process analytical technologies.

Propofol is the preferred anaesthetic for induction and maintenance of sedation in critically ill mechanically ventilated COVID-19 patients. However, during the outbreak of the COVID-19 pandemic, regular supply chains could not keep up with the sudden increase in global demand, causing drug shortages. Propofol is formulated as an oil-in-water emulsion which is administered intravenously. This study explores the extemporaneous preparation of a propofol emulsion without specialized manufacturing equipment to temporally alleviate such shortages. A commercially available lipid emulsion (IVLE, SMOFlipid 20 %), intended for parenteral nutrition, was used to create a propofol loaded nanoemulsion via addition of liquid propofol drug substance and subsequent mixing. Critical quality attributes such as mean droplet size and the volume-weighted percentage of large-diameter (>5µm) droplets were studied. The evolution of droplet size and propofol distribution was monitored in situ and non-destructively, maintaining sterility, using Spatially Resolved Dynamic Light Scattering and Near Infrared Spectroscopy, respectively. Using response surface methodology, an optimum was found for a 4 % w/v propofol formulation with a ∼15 min mixing time in a flask shaker at a 40° shaking angle. This study shows that extemporaneous compounding is a viable option for emergency supply of propofol drug product during global drug shortages.

Fabrication of large binary colloidal crystals with a NaCl structure.

Binary colloidal crystals offer great potential for tuning material properties for applications in, for example, photonics, semiconductors and spintronics, because they allow the positioning of particles with quite different characteristics on one lattice. For micrometer-sized colloids, it is believed that gravity and slow crystallization rates hinder the formation of high-quality binary crystals. Here, we present methods for growing binary colloidal crystals with a NaCl structure from relatively heavy, hard-sphere-like, micrometer-sized silica particles by exploring the following external fields: electric, gravitational, and dielectrophoretic fields and a structured surface (colloidal epitaxy). Our simulations show that the free-energy difference between the NaCl and NiAs structures, which differ in their stacking of the hexagonal planes of the larger spheres, is very small (approximately 0.002 k(B)T). However, we demonstrate that the fcc stacking of the large spheres, which is crucial for obtaining the pure NaCl structure, can be favored by using a combination of the above-mentioned external fields. In this way, we have successfully fabricated large, 3D, oriented single crystals having a NaCl structure without stacking disorder.

Postsynaptic and presynaptic group II metabotropic glutamate receptor activation reduces neuronal excitability in rat midline paraventricular thalamic nucleus.

Drugs that interact with group II metabotropic glutamate receptors (mGluRs) are presently being evaluated for a role in the treatment of anxiety disorders and symptoms of schizophrenia. Their mechanism of action is believed to involve a reduction in excitatory neurotransmission in limbic and forebrain regions commonly associated with these mental disorders. In rodents, the glutamatergic neurons in the midline paraventricular thalamic nucleus (PVT) provide excitatory inputs to the limbic system and forebrain. PVT also displays a high density of group II mGluRs, predominantly the metabotropic glutamate 2 receptor (mGluR2). Because the role of group II mGluRs in regulating cellular and synaptic excitability in this location has yet to be determined, we used whole-cell patch-clamp recording and acute rat brain slice preparations to evaluate PVT neuron responses to a selective group II mGluR agonist, (1R,4R,5S,6R)-4-amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY 379268). LY 379268 consistently induced membrane hyperpolarization and suppressed firing by postsynaptic receptor-mediated activation of a barium-sensitive background K(+) conductance. This effect could be blocked by (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl)propanoic acid (LY 341495), a selective group II mGluR antagonist. In addition, LY 379268 acted at presynaptic receptors to reduce ionotropic glutamate receptor-mediated excitatory synaptic transmission. An mGluR2-positive allosteric modulator, 2,2,2-trifluoro-N-[4-(2-methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl)ethanesulfonamide hydrochloride (LY 487379), resulted in leftward shifts of the LY 379268 dose-response curve for both postsynaptic and presynaptic actions. The data demonstrate that activation of postsynaptic and presynaptic group II (presumably mGluR2) mGluRs reduces neuronal excitability in midline thalamus, an action that may contribute to the effectiveness of mGluR2-activating drugs in rodent models of anxiety and psychosis.

Self-assembly of a colloidal interstitial solid with tunable sublattice doping.

We determine the phase diagram of a binary mixture of small and large hard spheres with a size ratio of 0.3 using free-energy calculations in Monte Carlo simulations. We find a stable binary fluid phase, a pure face-centered-cubic (fcc) crystal phase of the small spheres, and binary crystal structures with LS and LS(6) stoichiometries. Surprisingly, we demonstrate theoretically and experimentally the stability of a novel interstitial solid solution in binary hard-sphere mixtures, which is constructed by filling the octahedral holes of an fcc crystal of large spheres with small spheres. We find that the fraction of octahedral holes filled with a small sphere can be completely tuned from 0 to 1. Additionally, we study the hopping of the small spheres between neighboring octahedral holes, and interestingly, we find that the diffusion increases upon increasing the density of small spheres.

Mechanisms of burn-induced impairment in gastric slow waves and emptying in rats.

Delayed gastric emptying is common following severe large cutaneous burns; however, the mechanisms of burn-induced delayed gastric emptying remain unknown. The aim of this study was to explore the possible involvement of hyperglycemia and cyclooxygenase-2 receptors in the burn-induced gastric dysrhythmias. Gastric slow waves and gastric emptying were assessed in rats 6 h following sham or burn injury. Animals were randomized to one sham-burn and seven burn groups: untreated; two groups of saline treated (control); insulin treated (5 IU/kg); cyclooxygenase-2 inhibitor treated (10 mg/kg); ghrelin treated (2 nmol/rat); and gastric electrical stimulation treated. It was found that 1) severe burn injury impaired gastric slow waves postprandially and delayed gastric emptying; 2) the impairment in gastric slow waves included a decrease in the slow-wave frequency and in the percentage of normal slow waves, and an increase in the percentage of bradygastria (P = 0.001, 0.01, and 0.01, respectively vs. preburn values). None of the gastric slow-wave parameters was significantly correlated with gastric emptying; 3) cyclooxygenase-2 inhibitor normalized burn-induced delayed gastric emptying (P = 0.3 vs. sham-burn), but not gastric dysrhythmias (P < 0.002 vs. sham), whereas insulin normalized both gastric emptying (P = 0.4 vs. sham-burn) and gastric dysrhythmias (P = 0.3 vs. sham-burn); 4) both gastric electrical stimulation and ghrelin accelerated burn-induced delayed gastric emptying (P = 0.002 and 0.04, respectively, vs. untreated burn). In conclusion, hyperglycemia alters gastric slow-wave activity and delayed gastric emptying, while cyclooxygenase-2 inhibition delays gastric emptying without altering gastric slow-wave activity.

Crystal nucleation of hard spheres using molecular dynamics, umbrella sampling, and forward flux sampling: a comparison of simulation techniques.

Over the last number of years several simulation methods have been introduced to study rare events such as nucleation. In this paper we examine the crystal nucleation rate of hard spheres using three such numerical techniques: molecular dynamics, forward flux sampling, and a Bennett-Chandler-type theory where the nucleation barrier is determined using umbrella sampling simulations. The resulting nucleation rates are compared with the experimental rates of Harland and van Megen [Phys. Rev. E 55, 3054 (1997)], Sinn et al. [Prog. Colloid Polym. Sci. 118, 266 (2001)], Schätzel and Ackerson [Phys. Rev. E 48, 3766 (1993)], and the predicted rates for monodisperse and 5% polydisperse hard spheres of Auer and Frenkel [Nature 409, 1020 (2001)]. When the rates are examined in units of the long-time diffusion coefficient, we find agreement between all the theoretically predicted nucleation rates, however, the experimental results display a markedly different behavior for low supersaturation. Additionally, we examined the precritical nuclei arising in the molecular dynamics, forward flux sampling, and umbrella sampling simulations. The structure of the nuclei appears independent of the simulation method, and in all cases, the nuclei contains on average significantly more face-centered-cubic ordered particles than hexagonal-close-packed ordered particles.

New unique PAT method and instrument for real-time inline size characterization of concentrated, flowing nanosuspensions.

With the rise of nanotherapeutics -and nano based products in general-, there has been an increasing need for better understanding and control of nano-particle (NP) synthesis and formulation processes. Size characteristics are often primary, if not critical, quality attributes of nanodispersions. Process Analytical Technology (PAT) tools for inline size characterization during dispersion processing are therefore highly desired. Traditional methods for NP sizing -based on Dynamic Light Scattering (DLS) - are typically ill-suited for direct inline application: (i) typical dispersion turbidities in process conditions often exceed by far the application limits for DLS (ii) agitation/flow typical for process conditions is incompatible with standard DLS and (iii) direct and convenient inline application requires a non-invasive PAT tool giving measurements on process relevant time scales. In this article we describe a new non-invasive PAT instrument - the NanoFlowSizer (patent pending)- which provides continuous, real-time, inline size and PSD characterization of concentrated and/or flowing nanodispersions in process environments. The instrument employs Fourier Domain low coherence interferometry, yielding path length resolved dynamic light scattering data of nanodispersions. Particle size characteristics can be analyzed from these data while effects of flow and/or multiple scattering are simultaneously characterized and accounted for. As first application examples we describe (i) real-time monitoring of NP size characteristics by remote measurement of mono and bi-disperse suspensions at different turbidities in a stirred beaker (ii) real-time monitoring of NP size characteristics using an online sampling loop with a micro-flow cell and (iii) real-time inline monitoring of size characteristics of a pharmaceutical nanoemulsion during industrial pilot scale nano-emulsification and for a pharmaceutical NP suspension during circulation, at flowrates ranging up to ~l/min.

Trastuzumab therapy vs tetracycline controlled ERBB2 downregulation: influence on tumour development in an ERBB2-dependent mouse tumour model.

Trastuzumab (Herceptin) has improved therapy of breast cancer. Only patients overexpressing ERBB2 are treated with trastuzumab, whereas its use in tumours without ERBB2 expression is useless. This led to the concept that the subgroup of trastuzumab-sensitive tumours is 'ERBB2-dependent', meaning that ERBB2 signalling is indispensable for growth of these tumours. We used a mouse model that allows anhydrotetracycline (ATc)-controlled downregulation of ERBB2 in tumour tissue. ERBB2 mRNA and protein expression were downregulated below detection limit leading to a macroscopically complete tumour remission within 14 days. Tumour remission was accompanied by a strong decrease in proliferation, a moderate increase in apoptosis, as well as dephosphorylation of ERK1/2 and AKT/PKB. These data clearly indicate ERBB2 dependence. Therefore, a high sensitivity to trastuzumab may be suspected. Surprisingly, trastuzumab caused a much weaker effect compared to ATc-induced ERBB2 downregulation, although a decrease in ERBB2 membrane localisation was induced. Only a slight decrease in proliferation and a weak transient increase in apoptosis were observed. Interestingly, tumours responded to trastuzumab by a sharp fivefold increase in phosphorylated AKT/PKB as well as a 3.5- and 5.3-fold increase in AKT1 and AKT2 mRNA levels, respectively. In conclusion, 'ERBB2 dependence' is not sufficient to define trastuzumab-responsive tumours. The suboptimal effect of trastuzumab compared to the maximally possible effect induced by ATc demonstrates a high potential for improved ERBB2 blocking therapies.

Role of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) for prognosis in endometrial cancer.

BACKGROUND: Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) contribute to the invasiveness of many carcinomas. Here, we studied a possible association between cytosolic uPA and PA-1 concentrations in tumor tissue with prognosis in patients with endometrial cancer. METHODS: Cytosolic concentrations of uPA and PAI-1 were determined in 69 primary endothelial adenocarcinomas using an enzyme-linked immunoassay (ELISA). A possible influence of uPA and PAI-1 was studied by multivariate Cox regression adjusting for the established clinical prognostic factors FIGO-stage, grading, depth of invasion, diabetes mellitus and age. RESULTS: Both uPA (p=0.011) and PAI-1 (p=0.003) were associated with relapse free time using the multivariate proportional hazards model. Association with overall survival was less pronounced with p=0.021 for uPA and p=0.358 for PAI-1. Concentrations of PAI-1 increased with FIGO stage (p=0.003) and with histological grading (p=0.005). Both uPA and PAI-1 concentrations were negatively correlated with estrogen and progesterone receptor levels. CONCLUSION: The combination of high cytosolic concentrations of uPA (>5 ng/mg total protein) and high PAI-1 (>20 ng/mg total protein) may reveal a group of patients with increased risk of progression.

Primary rat hepatocytes as in vitro system for gene expression studies: comparison of sandwich, Matrigel and 2D cultures.

Recent studies have presented evidence that in vivo obtained gene expression data can be used for carcinogen classification, for instance to differentiate between genotoxic and non-genotoxic carcinogens. However, although primary rat hepatocytes represent a well-established in vitro system for drug metabolism and enzyme induction, they have not yet been systematically optimized for toxicogenomic studies. The latter may be confounded by the fact that cultured hepatocytes show strong spontaneous alterations in gene expression patterns. Therefore, we addressed the following questions: (1) which culture system is optimal, comparing sandwich, Matrigel and 2D cultures, (2) how critical is the impact of culture period on substance-induced alterations in gene expression and (3) do these substance-induced alterations in cultured hepatocytes occur already at in vivo relevant concentrations? For this purpose we analyzed the expression of four genes, namely Abat, Gsk3beta, Myd116 and Sult1a1 that recently have been reported to be influenced by the antihistamine and non-genotoxic carcinogen methapyrilene (MPy). The most reproducible effects of MPy were observed in sandwich cultures. Induction factors of Gsk3beta and Myd116 at 100 microM MPy were 2 and 4 (medians), respectively, whereas expression of Abat and Sult1a1 were inhibited by factors of 7 and 5, respectively. Similar results were observed in hepatocytes maintained for 24 h or 3 weeks in sandwich culture with respect to the influence of MPy on the expression of Abat, Gsk3beta, Myd116 and Sult1a1. To determine whether MPy influences gene expression at in vivo relevant concentrations, 3.5 mg/kg MPy were administered to male Wistar rats intraperitoneally, resulting in plasma concentrations ranging between 1.72 and 0.32 microM 5 and 80 min after injection. Inhibition of Abat and Sult1a1 expression in vitro already occurred at in vivo relevant concentrations of 0.39 microM MPy. Induction of Myd116 was observed at 6.25 microM which is higher but in the same order of magnitude as in vivo relevant concentrations. In conclusion, the presented data strongly suggest that sandwich cultures are most adequate for detection of MPy-induced gene expression alterations and the effect of MPy was detected at in vivo relevant concentrations.

Acute tension pneumopericardium due to perforated gastric ulcer without diagnostic radiographic findings 72 h before perforation.

INTRODUCTION: Acute tension pneumopericardium due to gastric perforation is a rare and often lethal condition. Only a few case reports have been described in the literature. Diagnosis based on clinical evaluation is difficult and it is usually made incidentally upon computed tomography (CT) or plain radiography of the chest. Since cardiac tamponade caused by pneumopericardium is life-threatening, immediate diagnosis and emergent therapy is vital. CASE REPORT: We report a 75-year-old male with peptic ulcer disease associated with perforation of the pericardium and acute shock. It is the first reported case with a series of two computed tomograms performed during the 72 h preceding the acute onset of tension pneumopericardium. No radiographic evidence of ulcer perforation was present in the three days prior to the acute event. DISCUSSIONS: Gastric ulcer perforation into the pericardium is rare and could not be detected by CT scan prior the deletorious event. Pneumopericardium seems to be fateful and could not be foreseen by clinical or radiological findings..

[Tecnologías de la información en la formación de evaluadores del Programa Hospital Seguro en México]. / Information technologies in the training of evaluators of the Safe Hospital Program in Mexico.

OBJETIVO: Describir el proceso para la formación de evaluadores del Programa Hospital Seguro centrado en el uso de las tecnologías de la información. MÉTODO: Estudio observacional descriptivo en el que se analiza la participación dentro del nuevo curso de evaluadores del Programa Hospital Seguro. RESULTADOS: Se inscribieron 1323 participantes, de los cuales aprobaron 298 (18%); la calificación media fue de 8.85. CONCLUSIONES: La plataforma educativa tipo Moodle fue de utilidad para la capacitación del Programa Hospital Seguro. Se tendrían que mejorar los criterios de participación de los alumnos a fin de incrementar la eficiencia terminal. OBJECTIVE: To describe the process for the training of evaluators of the safe hospital program in the use of ICT. METHOD: Descriptive observational study in which the participation in the new course of appraisers of the safe hospital program is analyzed. RESULTS: 1323 participants registered of which 298 (18%) passed; the average grade was 8.85. CONCLUSIONS: The Moodle type educational platform was useful for the training of the Safe Hospital Program. The criteria for student participation would have to be improved in order to increase terminal efficiency.

Adenosine metabolism and its effect on methylation potential in cultured cells: methodological considerations.

Cell culture models are frequently used to study the role of adenosine in several physiological and pathological processes. In the present study, we have shown that adenosine deaminase activity in medium supplemented with calf serum significantly reduces adenosine concentration in culture medium. In the presence of HepG2 cells, the adenosine concentration in culture medium is decreased much faster, because a large amount of exogenous adenosine is metabolized by cellular enzyme. In order to measure intracellular adenosine, inosine, adenine nucleotides, S-adenosylhomocysteine (AdoHcy) and Sadenosylmethionine (AdoMet) contents, two methods for cell harvesting were compared. First, cells were removed with trypsin/EDTA, second, cells were lysed in cell culture dishes immediately after removing culture medium. Our results show that exact determination of adenosine metabolites requires immediate inactivation of metabolism by cell lysis in culture dishes. Application of adenosine (1mM) resulted in a time-dependent increase in intracellular adenosine, inosine, AMP, ATP, AdoHcy and AdoMet concentration. Since AdoHcy levels increased to a larger extent than AdoMet, the methylation potential, expressed as the ratio of AdoMet/AdoHcy, was reduced from 51.8 (control) to 2.9 (adenosine 1 mM, 2 hrs), suggesting that AdoMet-dependent methylation reactions might be impaired. In conclusion our data demonstrate that extracellular adenosine concentration and intracellular metabolite concentration strongly depend on the methods used to culture and harvest the cells.