Development and validation of a dried blood spot LC-MS/MS assay to quantify ranitidine in paediatric samples.

A novel approach has been developed to determine ranitidine in paediatric samples using dried blood spots (DBS) on Guthrie cards (Whatman 903). A selective and sensitive HPLC-MS/MS assay has been developed and validated using small volumes of blood (30µl). A 6mm disc was punched from each DBS and extracted with methanolic solution of the internal standard (IS) nizatidine. This was further subjected to solid phase extraction (SPE), followed by reversed phase HPLC separation, using a XBridge™ C18 column and mobile phase 10mM ammonium acetate/methanol (98:2 v/v) with a flow rate of 0.3mL/min. This was combined with multiple reaction monitoring (MRM) mass detection using electrospray ionisation (ESI). The calibration curve for ranitidine was found linear over the range 10-500ng/mL (r=0.996). The limit of quantification (LOQ) of the method was validated at 10ng/mL. Accuracy and precision values for within and between days were

Role of nitric oxide in maintenance of basal oral tissue blood flow in anesthetized cats

Experiments were undertaken to determine if nitric oxide (NO) plays a role in regulation of basal blood flow in the oral cavity of pentobarbital anesthetized cats and, if so, to quantify this effect using dose-response relationships. Blood flow was continuously measured from the surface of the tongue and mandibular gingiva (laser-Doppler flowmetry) and from the lingual artery (ultrasonic flowmetry). Cardiovascular parameters also were recorded. Administration of the nonselective inhibitor of nitric oxide synthase (NOS), L-NAME (0.08-20 mg/kg i.v.), produced a dose-related increase of blood pressure associated with decreases of blood flow at all three measurement sites. Maximal blood flow depression of 50-60% was seen 30-60 min after administration of 1.25 mg/kg of L-NAME. D-NAME (1.25 mg/kg i.v.) was inactive at all sites. Subsequent administration of L-arginine partially reversed effects of L-NAME in the lingual artery and tongue, but not in the gingival circulation. The neuronally selective NOS inhibitor, 7-nitroindazole (7-NI, 30 mg/kg i.p.), was devoid of effect on any of the measured parameters. These results suggest that endothelial (but not neuronally derived) NO plays an important role in control of basal blood flow in oral tissues of the cat.

The relationship of clinical and inflammatory markers to outcome in stable patients with cystic fibrosis

Decreased survival in patients with cystic fibrosis has been related to FEV1, BMI, and infection with Burkholderia cepacia complex (BCC). We have assessed the relationship of blood, sputum, and urine inflammatory markers to lung function, BMI, colonization with B cenocepacia (Bc), and patient survival. Thirty-nine stable cystic fibrosis (CF) patients (10 with Bc) were enrolled in a study to determine the effect of alpha-1-antitrypsin on airways inflammation. Pre-treatment measurements were used in this study. Demographics, sputum microbiology, heart rate, oxygen saturation, lung function were recorded. Blood samples were obtained for white blood count (WBC), C-Reactive Protein (CRP), and plasma neutrophil elastase/AAT complexes (pNEC). Neutrophil elastase (NE), neutrophil elastase/AAT complexes (sNEC), interleukin-8 (IL-8), TNF-receptor 1 (sTNFr), and myeloperoxidase (MPO) were measured in sputum and urinary desmosine concentration determined. Patients with Bc had significantly higher levels of pNEC, 332?±?91.4 ng/ml (mean?±?SEM) versus 106?±?18.2 ng/ml (P?=?0.0005) and sNEC, 369?±?76.6 ng/ml versus 197?±?36.0 ng/ml compared to those who were not. Five deaths were reported at the end of 1 year, (four with Bc) (P?=?0.011). Patients who subsequently died had significantly lower lung function FEV1, 1.2?±?0.2 L versus 2.0?±?0.1 L (P?=?0.03) and FVC, 2?±?0.3 L versus 3.1?±?0.2 L (P?=?0.01), compared to those that survived. There was significantly higher NE activity, 3.6?±?1.6 U/ml versus 1.5?±?0.6 U/ml (P?=?0.03), pNEC, 274?±?99 ng/ml versus 142?±?30 ng/ml (P?=?0.05), MPO, 163?±?62 mcg/ml versus 54?±?6.9 mcg/ml (P?=?0.03), and urinary desmosines 108?±?19.9 pM/mg creatinine versus 51.1?±?3.3 pM/mg creatinine (P?=?0.001), in those patients who subsequently died compared to those that survived. These data suggest there is increased neutrophil degranulation in patients infected with Bc and these patients have a poor outcome.

Differential effects of an anti-oxidant intervention on cardiomyocyte expression of adrenomedullin and intermedin and their receptor components in chronic nitric oxide deficiency

Background: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial oxidative stress and hypertrophic remodeling. Up-regulation of the cardiomyocyte adrenomedullin (AM) / intermedin (IMD) receptor signaling cascade is also apparent in NO-deficient cardiomyocytes: augmented expression of AM and receptor activity modifying proteins RAMP2 and RAMP3 is prevented by blood pressure normalization while that of RAMP1 and intermedin (IMD) is not, indicating that the latter is regulated by a pressure-independent mechanism. Aims: to verify the ability of an anti-oxidant intervention to normalize cardiomyocyte oxidant status and to investigate the influence of such an intervention on expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes. Methods: NO synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 35mg/kg/day) was given to rats for 8 weeks, with/without con-current administration of antioxidants (Vitamin C (25mg/kg/day) and Tempol (25mg/kg/day)). Results: In left ventricular cardiomyocytes isolated from L-NAME treated rats, increased oxidative stress was indicated by augmented (3.6 fold) membrane protein oxidation, enhanced expression of catalytic and regulatory subunits of pro-oxidant NADPH oxidases (NOX1, NOX2) and compensatory increases in expression of anti-oxidant glutathione peroxidase and Cu/Zn superoxide dismutases (SOD1, SOD3). Vitamin C plus Tempol did not reduce systolic blood pressure but normalized augmented plasma levels of IMD, but not of AM, and in cardiomyocytes: (i) abolished increased membrane protein oxidation; (ii) normalized augmented expression of prepro-IMD and RAMP1, but not prepro-AM, RAMP2 and RAMP3; (iii) attenuated (by 42%) increased width and normalized expression of hypertrophic markers, skeletal-�-actin and prepro-endothelin-1 similarly to blood pressure normalization but in contrast to blood pressure normalization did not attenuate augmented brain natriuretic peptide (BNP) expression. Conclusion: normalization specifically of augmented IMD/RAMP1 expression in NO-deficient cardiomyocytes by antioxidant intervention in the absence of blood pressure reduction indicates that these genes are likely to be induced directly by myocardial oxidative stress. Although oxidative stress contributed to cardiomyocyte hypertrophy, induction of IMD and RAMP1 is unlikely to be secondary to cardiomyocyte hypertrophy.

Influence of atenolol and nifedipine on nitric oxide deficient cardiomyocyte hypertrophy and expression of the cardio-endocrine peptide intermedin and its receptor components.

BACKGROUND/AIMS: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of adrenomedullin (AM) and intermedin (IMD) and their receptor activity modifying proteins (RAMPs 1-3) is augmented in cardiomyocytes, indicating that the myocardial AM/ IMD system may be activated in response to pressure loading and ischemic insult. The aim was to examine effects on (i) parameters of cardiomyocyte hypertrophy and on (ii) expression of AM and IMD and their receptor components in NO-deficient cardiomyocytes of an intervention chosen specifically for ability to alleviate pressure loading and ischemic injury concurrently. METHODS: The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 35 mg.kg(-1).day(-1)) was given to rats for 8 weeks, with/ without concurrent administration of beta-adrenoceptor antagonist, atenolol (25 mg.kg(-1).day(-1)) / calcium channel blocker, nifedipine (20mg.kg(-1).day(-1)). RESULTS: In L-NAME treated rats, atenolol / nifedipine abolished increases in systolic blood pressure and plasma AM and IMD levels and in left ventricular cardiomyocytes: (i) normalized increased cell width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP, BNP, ET) genes; (ii) normalized augmented membrane protein oxidation; (iii) normalized mRNA expression of AM, IMD, RAMP1, RAMP2 and RAMP3. CONCLUSIONS: normalization of blood pressure and membrane oxidant status together with prevention of hypertrophy and normalization of the augmented expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes by atenolol / nifedipine supports involvement of both pressure loading and ischemic insult in stimulating cardiomyocyte hypertrophy and induction of these counter-regulatory peptides and their receptor components. Attenuation of augmented expression of IMD in this model cannot however be explained simply by prevention of cardiomyocyte hypertrophy.

The effect of treatment of cystic fibrosis pulmonary exacerbations on airways and systemic inflammation

Background: Chronic infection in cystic fibrosis (CF) and airway inflammation leads to progressive lung injury Neutrophils are considered to be responsible for the onset and promotion of the inflammatory response within the CF lung. The relationship between infection and inflammation is complex but circulating inflammatory markers may not truly reflect the local inflammatory response in the lung. The aims of this study were to investigate the change of inflammatory biomarkers and cells within sputum and blood before and after intravenous antibiotics for a pulmonary exacerbation of CF Methods: Assays included neutrophil elastase (NE) and complex, interleukin-8 (IL-8) and soluble intercellular adhesion molecule-1 (sICAM-1), fas ligand (FAS-L), and TNFr-1. Analysis of sputum cell differential and absolute cell counts and immunocytochemistry (CD11b and CD95) on sputum and isolated blood neutrophils were carried out. Results: There were no significant differences in absolute or differential sputum cell counts or sputum sol measurements following antibiotics. There was a significant increase in the percentage of blood neutrophils with minimal CD11b staining, 28 (4.1) mean percentage (SEM) versus41 (2.9) and a decrease in the percentage showing maximal staining 30 (0.5) versus 15 (2.5). There was a significant increase in the percentage of blood neutrophils without CD95 staining, 43 (5.4) mean percentage versus 52 (5.1). Conclusion: These data suggest a modifiable systemic response to IV antibiotics but a local sustained inflammatory response in the lung.

Obesity in People with Learning Disabilities: the impact of nurse led health screenings and health promotion activities

Background. Obesity appears to be more common among people with intellectual disabilities, with few studies focusing on achieving weight reduction. Aim. Firstly, to follow up people identified as overweight and obese following special health screening clinics and to determine the actions taken. Secondly, to evaluate the impact of health promotion classes on participants' weight loss. Methods. A clinic led by two learning disbaility nurses was held for all people aged 10 years and over (n=464) who attended special services within the area of one Health and Social Services Trust in Northern Ireland. In a second study, the nurses organised health promotion classes for 20 people over a 6 - 8 week period. Findings. The health screen identified 64% of adults and 26% of 10 - 19 year olds as being overweight or obese. Moreover, those aged 40 - 49 years who were obese had significantly higher levels of blood pressure. However, information obtained from a follow up questionnaire sent after 3 months suggested that of the 122 people identified for wiehgt reduciton, action had been taken for only 34% of them and only three were reported to have lost weight. The health promotion classes, however, led to a significant reduction in weight and body mass index scores. Conclusion. Health screening per se has limited impact on reducing obesity levels in this client group. Rather, health personnel such as general practitioners, nurses and health promotion staff need to work in partnership with service staff, carers and people with intellectual disabiltieis to create more active lifestyles.

Lys(9) for Glu(9) substitution in glucagon-like peptide-1(7-36)amide confers dipeptidylpeptidase IV resistance with cellular and metabolic actions similar to those of established antagonists glucagon-like peptide-1(9-36)amide and exendin (9-39)

The incretin hormone glucagon-like peptide-1(7-36)amide (GLP-1) has been deemed of considerable importance in the regulation of blood glucose. Its effects, mediated through the regulation of insulin, glucagon, and somatostatin, are glucose-dependent and contribute to the tight control of glucose levels. Much enthusiasm has been assigned to a possible role of GLP-1 in the treatment of type 2 diabetes. GLIP-l's action unfortunately is limited through enzymatic inactivation caused by dipeptidylpeptidase IV (DPP IV). It is now well established that modifying GLP-1 at the N-terminal amino acids, His(7) and Ala(8), can greatly improve resistance to this enzyme. Little research has assessed what effect Glu(9)-substitution has on GLP-1 activity and its degradation by DPP IV. Here, we report that the replacement of Glu(9) of GLP-1 with Lys dramatically increased resistance to DPP IV. This analogue, (Lys(9))GLP-1, exhibited a preserved GLP-1 receptor affinity, but the usual stimulatory effects of GLP-1 were completely eliminated, a trait duplicated by the other established GLP-1-antagonists, exendin (9-39) and GLP-1 (9-36)amide. We investigated the in vivo antagonistic actions of (Lys(9))GLP-1 in comparison with GLP-1(9-36)amide and exendin (9-39) and revealed that this novel analogue may serve as a functional antagonist of the GLP-1 receptor. (C) 2004 Elsevier Inc. All rights reserved.

Neuropeptide YY1 receptor in human dental pulp cells of noncarious and carious teeth

Aim To determine the distribution of the NPY Y1 receptor in carious and noncarious human dental pulp tissue using immunohistochemistry. A subsidiary aim was to confirm the presence of the NPY Y1 protein product in membrane fractions of dental pulp tissue from carious and noncarious teeth using western blotting. Methodology Twenty two dental pulp samples were collected from carious and noncarious extracted teeth. Ten samples were processed for immunohistochemistry using a specific antibody to the NPY Y1 receptor. Twelve samples were used to obtain membrane extracts which were electrophoresed, blotted onto nitrocellulose and probed with NPY Y1 receptor antibody. Kruskal-Wallis one-way analysis of variance was employed to test for overall statistical differences between NPY Y1 levels in noncarious, moderately carious and grossly carious teeth. Results Neuropeptide Y Y1 receptor immunoreactivity was detected on the walls of blood vessels in pulp tissue from noncarious teeth. In carious teeth NPY Y1 immunoreactvity was observed on nerve fibres, blood vessels and inflammatory cells. Western blotting indicated the presence and confirmed the variability of NPY Y1 receptor protein expression in solubilised membrane preparations of human dental pulp tissue from carious and noncarious teeth. Conclusions Neuropeptide Y Y1 is expressed in human dental pulp tissue with evidence of increased expression in carious compared with noncarious teeth, suggesting a role for NPY Y1 in modulation of caries induced pulpal inflammation. © 2008 International Endodontic Journal.

A simple colorimetric method for the quality control of 1-alkyl-3-methylimidazolium ionic liquid precursors

A simple colorimetric method to monitor the production of ionic liquid precursors is developed, which is based on the determination of 1-methylimidazole with copper(II) chloride. The synthesis of 1-ethyl-3-methylimidazolium chloride, an industrially important ionic liquid precursor, can be followed and the purity of the final product can be readily assessed in a quick and convenient manner.

The Effects of Anti-Hypertensive Drugs Evaluated Using Markov Modelling for Northern Ireland Chronic Kidney Disease Patients

The aim of this paper is to use Markov modelling to
investigate survival for particular types of kidney patients
in relation to their exposure to anti-hypertensive treatment
drugs. In order to monitor kidney function an intuitive three
point assessment is proposed through the collection of blood
samples in relation to Chronic Kidney Disease for Northern
Ireland patients. A five state Markov Model was devised
using specific transition probabilities for males and
females over all age groups. These transition probabilities
were then adjusted appropriately using relative risk scores
for the event death for different subgroups of patients. The
model was built using TreeAge software package in order to
explore the effects of anti-hypertensive drugs on patients.

Adrenomedullin Gene Delivery is Cardio-protective in a Model of Chronic Nitric Oxide Deficiency Combining Pressure Overload, Oxidative Stress and Cardiomyocyte Hypertrophy

BACKGROUND/AIMS:
Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of the vasodilator peptide, adrenomedullin (AM) and its receptors is augmented in cardiomyocytes, indicating that the myocardial AM system may be activated in response to pressure loading and ischemic insult to serve a counter-regulatory, cardio-protective role. The study examined the hypothesis that oxidative stress and hypertrophic remodeling in NO-deficient cardiomyocytes are attenuated by adenoviral vector-mediated delivery of the human adrenomedullin (hAM) gene in vivo.

METHODS:
The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 15mg . kg(-1) . day(-1)) was given to rats for 4 weeks following systemic administration via the tail vein of a single injection of either adenovirus harbouring hAM cDNA under the control of the cytomegalovirus promoter-enhancer (Ad.CMV-hAM-4F2), or for comparison, adenovirus alone (Ad.Null) or saline. Cardiomyocytes were subsequently isolated for assessment of the influence of each intervention on parameters of oxidative stress and hypertrophic remodelling.

RESULTS: Cardiomyocyte expression of the transgene persisted for > or =4 weeks following systemic administration of adenoviral vector. In L-NAME treated rats, relative to Ad.Null or saline administration, Ad.CMV-hAM-4F2 (i) reduced augmented cardiomyocyte membrane protein oxidation and mRNA expression of pro-oxidant (p22phox) and anti-oxidant (SOD-3, GPx) genes; (ii) attenuated increased cardiomyocyte width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP) genes; (iii) did not attenuate hypertension.

CONCLUSIONS: Adenoviral vector mediated delivery of hAM resulted in attenuation of myocardial oxidative stress and hypertrophic remodelling in the absence of blood pressure reduction in this model of chronic NO-deficiency. These findings are consistent with a direct cardio-protective action in the myocardium of locally-derived hAM which is not dependant on NO generation.

Monoclonal antibody development for acrylamide-adducted human haemoglobin; A biomarker of dietary acrylamide exposure

The spontaneous formation of the neurotoxic carcinogen acrylamide in a wide range of cooked foods has recently been discovered, leading to dietary exposure estimates of 30.8 mu g of acrylamide day(-1) for an average 77 kg human male. This is considerably higher than the European legal limit of acrylamide in drinking water, which is approximately 0.2 mu g of acrylamide person(-1) day(-1). A recent study of 62,573 women over 11.3 years has observed an increased risk of postmenopausal endometrial and ovarian cancer (but not breast cancer) with increasing dietary acrylamide intake, demonstrating significant risk to human health. As individual acrylamide exposure is affected by dietary habits, cooking methods, and cigarette consumption; accurate extrapolation from estimated dietary exposure is extremely difficult. Quantifying biomarkers of acrylamide exposure therefore remains the most effective means of rapidly determining individual exposure to acrylamide, and correlating exposure with lifestyle choices. Current methodologies for the analysis of blood biomarkers of acrylamide are focused on expensive, slower chromatographic techniques such as GC and LC coupled to mass spectrometry. This paper describes the first successful development of two monoclonal antibodies specific to acrylamide-adducted haemoglobin (IC50 of 94 ng ml(-1) and 198 ng ml(-1)), that are suitable for use in a high-throughput biomarker immunoassay to determine individual acrylamide exposure. Further development of acrylamide-haemoglobin standards with defined levels of acrylamide adduction will enable a fully quantitative assay, and allow sensitivity comparisons with alternative chromatographic methods of analysis. (C) 2008 Elsevier B.V. All rights reserved.

Flow-mediated dilatation of the brachial artery is a poorly reproducible indicator of microvascular function in Type I diabetes mellitus

Aim: Two Type I diabetes and control group comparator studies were conducted to assess the reproducibility of FMD and to analyse blood flow data normally discarded during FMD measurement.

Design: The studies were sequential and differed only with regard to operator and ultrasound machine. Seventy-two subjects with diabetes and 71 controls were studied in total.

Methods: Subjects had FMD measured conventionally. Blood velocity waveforms were averaged over 10 pulses post forearm ischaemia and their component frequencies analysed using the wavelet transform, a mathematical tool for waveform analysis. The component frequencies were grouped into 11 bands to facilitate analysis.

Results: Subjects were well-matched between studies. In Study 1, FMD was significantly impaired in subjects with Type I diabetes vs. controls (median 4.35%, interquartile range 3.10-4.80 vs. 6.50, 4.79-9.42, P < 0.001). No differences were detected between groups in Study 2, however. However, analysis of blood velocity waveforms yielded significant differences between groups in two frequency bands in each study.

Conclusions: This report highlights concerns over the reproducibility of FMD measures. Further work is required to fully elucidate the role of analysing velocity waveforms after forearm ischaemia.

Elucidating the molecular physiopathology of acute respiratory distress syndrome in severe acute respiratory syndrome patients

Acute respiratory distress syndrome (ARDS) is a severe form of acute lung injury. It is a response to various diseases of variable etiology, including SARS-CoV infection. To date, a comprehensive study of the genomic physiopathology of ARDS (and SARS) is lacking, primarily due to the difficulty of finding suitable materials to study the disease process at a tissue level (instead of blood, sputa or swaps). Hereby we attempt to provide such study by analyzing autopsy lung samples from patient who died of SARS and showed different degrees of severity of the pulmonary involvement. We performed real-time quantitative PCR analysis of 107 genes with functional roles in inflammation, coagulation, fibrosis and apoptosis: some key genes were confirmed at a protein expression level by immunohistochemistry and correlated to the degree of morphological severity present in the individual samples analyzed. Significant expression levels were identified for ANPEP (a receptor for CoV), as well as inhibition of the STAT1 pathway, IFNs production and CXCL10 (a T-cell recruiter). Other genes unassociated to date with ARDS/SARS include C1Qb, C5R1, CASP3, CASP9, CD14, CD68, FGF7, HLA-DRA, ICF1, IRF3, MALAT-1, MSR1, NFIL3, SLPI, USP33, CLC, GBP1 and TACI. As a result, we proposed to therapeutically target some of these genes with compounds such as ANPEP inhibitors, SLPI and dexamethasone. Ultimately, this study may serve as a model for future, tissue-based analyses of fibroinflammatory conditions affecting the lung. (C) 2009 Elsevier B.V. All rights reserved.