Development and validation of an HPLC method for the rapid and simultaneous determination of 6-mercaptopurine and four of its metabolites in plasma and red blood cells

An HPLC method has been developed and validated for the rapid determination of mercaptopurine and four of its metabolites; thioguanine, thiouric acid, thioxanthine and methylmercaptopurine in plasma and red blood cells. The method involves a simple treatment procedure based on deproteinisation by perchloric acid followed by acid hydrolysis and heating for 45min at 100 degrees C. The developed method was linear over the concentration range studied with a correlation coefficient >0.994 for all compounds in both plasma and erythrocytes. The lower limits of quantification were 13, 14, 3, 2, 95pmol/8 x 10(8) RBCs and 2, 5, 2, 3, 20ng/ml plasma for thioguanine, thiouric acid, mercaptopurine, thioxanthine and methylmercaptopurine, respectively. The method described is selective and sensitive enough to analyse the different metabolites in a single run under isocratic conditions. Furthermore, it has been shown to be applicable for monitoring these metabolites in paediatric patients due to the low volume requirement (200microl of plasma or erythrocytes) and has been successfully applied for investigating population pharmacokinetics, pharmacogenetics and non-adherence to therapy in these patients.

Potassium canrenoate treatment in paediatric patients:a population pharmacokinetic study using novel dried blood spot sampling

Objective: To characterize the population pharmacokinetics of canrenone following administration of potassium canrenoate (K-canrenoate) in paediatric patients. Methods: Data were collected prospectively from 37 paediatric patients (median weight 2.9 kg, age range 2 days–0.85 years) who received intravenous K-canrenoate for management of retained fluids, for example in heart failure and chronic lung disease. Dried blood spot (DBS) samples (n = 213) from these were analysed for canrenone content and the data subjected to pharmacokinetic analysis using nonlinear mixed-effects modelling. Another group of patients (n = 16) who had 71 matching plasma and DBS samples was analysed separately to compare canrenone pharmacokinetic parameters obtained using the two different matrices. Results: A one-compartment model best described the DBS data. Significant covariates were weight, postmenstrual age (PMA) and gestational age. The final population models for canrenone clearance (CL/F) and volume of distribution (V/F) in DBS were CL/F (l/h) = 12.86 ×  (WT/70.0)0.75 × e [0.066 ×  (PMA - 40]) and V/F (l) = 603.30 ×  (WT/70) × (GA/40)1.89 where weight is in kilograms. The corresponding values of CL/F and V/F in a patient with a median weight of 2.9 kg are 1.11 l/h and 20.48 l, respectively. Estimated half-life of canrenone based on DBS concentrations was similar to that based on matched plasma concentrations (19.99 and 19.37 h, respectively, in 70 kg patient). Conclusion: The range of estimated CL/F in DBS for the study population was 0.12–9.62 l/h; hence, bodyweight-based dosage adjustment of K-canrenoate appears necessary. However, a dosing scheme that takes into consideration both weight and age (PMA/gestational age) of paediatric patients seems more appropriate.

A simple bioanalytical method for the quantification of antiepileptic drugs in dried blood spots

An increasing number of publications on the dried blood spot (DBS) sampling approach for the quantification of drugs and metabolites have been spurred on by the inherent advantages of this sampling technique. In the present research, a selective and sensitive high-performance liquid chromatography method for the concurrent determination of multiple antiepileptic drugs (AEDs) [levetiracetam (LVT), lamotrigine (LTG), phenobarbital (PHB)], carbamazepine (CBZ) and its active metabolite carbamazepine-10,11 epoxide (CBZE)] in a single DBS has been developed and validated. Whole blood was spotted onto Guthrie cards and dried. Using a standard punch (6 mm diameter), a circular disc was punched from the card and extracted with methanol: acetonitrile (3:1, v/v) containing hexobarbital (Internal Standard) and sonicated prior to evaporation. The extract was then dissolved in water and vortex mixed before undergoing solid phase extraction using HLB cartridges. Chromatographic separation of the AEDs was achieved using Waters XBridge™ C18 column with a gradient system. The developed method was linear over the concentration ranges studied with r ≥ 0.995 for all compounds. The lower limits of quantification (LLOQs) were 2, 1, 2, 0.5 and 1 μg/mL for LVT, LTG, PHB, CBZE and CBZ, respectively. Accuracy (%RE) and precision (%CV) values for within and between day were <20% at the LLOQs and <15% at all other concentrations tested. This method was successfully applied to the analysis of the AEDs in DBS samples taken from children with epilepsy for the assessment of their adherence to prescribed treatments.

Pronounced reduction of postprandial glucagon by lixisenatide:a meta-analysis of randomized clinical trials

Glucagon-like peptide-1 (GLP-1) receptor agonists improve islet function and delay gastric emptying in patients with type 2 diabetes mellitus (T2DM). This meta-analysis aimed to investigate the effects of the once-daily prandial GLP-1 receptor agonist lixisenatide on postprandial plasma glucose (PPG), glucagon and insulin levels. Methods: Six randomized, placebo-controlled studies of lixisenatide 20μg once daily were included in this analysis: lixisenatide as monotherapy (GetGoal-Mono), as add-on to oral antidiabetic drugs (OADs; GetGoal-M, GetGoal-S) or in combination with basal insulin (GetGoal-L, GetGoal-Duo-1 and GetGoal-L-Asia). Change in 2-h PPG and glucose excursion were evaluated across six studies. Change in 2-h glucagon and postprandial insulin were evaluated across two studies. A meta-analysis was performed on least square (LS) mean estimates obtained from analysis of covariance (ANCOVA)-based linear regression. Results: Lixisenatide significantly reduced 2-h PPG from baseline (LS mean difference vs. placebo: -4.9mmol/l, p<0.001) and glucose excursion (LS mean difference vs. placebo: -4.5mmol/l, p<0.001). As measured in two studies, lixisenatide also reduced postprandial glucagon (LS mean difference vs. placebo: -19.0ng/l, p<0.001) and insulin (LS mean difference vs. placebo: -64.8 pmol/l, p<0.001). There was a stronger correlation between 2-h postprandial glucagon and 2-h PPG with lixisenatide than with placebo. Conclusions: Lixisenatide significantly reduced 2-h PPG and glucose excursion together with a marked reduction in postprandial glucagon and insulin; thus, lixisenatide appears to have biological effects on blood glucose that are independent of increased insulin secretion. These effects may be, in part, attributed to reduced glucagon secretion. © 2014 John Wiley and Sons Ltd.

Multispectral fundus analysis

Purpose - To generate a reflectance model of the fundus that allows an accurate non-invasive quantification of blood and pigments. Methods - A Monte Carlo simulation was used to produce a mathematical model of light interaction with the fundus at different wavelengths. The model predictions were compared with fundus images from normal volunteers in several spectral bands (peaks at 507, 525, 552, 585, 596 and 611nm). Th e model was then used to calculate the concentration and distribution of the known absorbing components of the fundus. Results - The shape of the statistical distribution of the image data generally corresponded to that of the model data; the model however appears to overestimate the reflectance of the fundus in the longer wavelength region.As the absorption by xanthophyll has no significant eff ect on light transport above 534nm, its distribution in the fundus was quantified: the wavelengths where both shape and distribution of image and model data matched (<553nm) were used to train a neural network which was then applied to every point in the image data. The xanthophyll distribution thus found was in agreement with published literature data in normal subjects. Conclusion - We have developed a method for optimising multi-spectral imaging of the fundus and a computer image analysis capable of estimating information about the structure and properties of the fundus. Th e technique successfully calculates the distribution of xanthophyll in the fundus of healthy volunteers. Further improvement of the model is required to allow the deduction of other parameters from images; investigations in known pathology models are also necessary to establish if this method is of clinical use in detecting early chroido-retinopathies, hence providing a useful screening and diagnostic tool.

Regional variation in peripapillary retinal blood flow in primary open angle glaucoma:a follow-up study

Purpose: To investigate whether regional long-term changes in peripapillary retinal flow, measured by scanning laser Doppler flowmetry (SLDF), occur in patients with primary open angle glaucoma (POAG). Methods: 31 healthy volunteers (mean age: 65 8.3 years) and 33 POAG patients (mean age: 71.2 7.6 years) were followed up every 4 months for 16 months. Using SLDF, three images of the superior and inferior optic nerve head were obtained for each subject. A 1010-pixel frame was used to measure blood flow, volume and velocity in the four quadrants of the peripapillary retina. Central 24-2 visual field testing was carried out at each visit. Repeated measures analysis of covariance was used to assess change over time between the normal and POAG groups for the SLDF parameters. Univariate linear regression analysis for mean deviation and glaucoma change probability (GCP) analysis were used to identify visual field progression. Results: Blood volume, flow and velocity measured in the inferior nasal quadrant of the peripapillary retina decreased significantly over time for the POAG group compared to the normal group (p=0.0073, 0.0097, 0.0095 respectively). Overall, 2 glaucoma patients showed a significantly deteriorating MD slope, while 7 patients showed visual field progression with GPA. All of the patients progressing with GPA, showed change in the superior hemifield and, of those, 14% showed change in the inferior hemifield. Conclusion: Glaucoma patients showed a decrease in blood flow, volume and velocity in the inferior nasal peripapillary retina. A regional variation in microvascular retinal capillary blood flow may provide insight into the pathogenesis of glaucomatous optic neuropathy. Keywords: 331 blood supply • 554 retina • 624 visual fields

Clinical evaluation of a novel test strip technology for blood glucose monitoring:accuracy at hypoglycaemic glucose levels

Aim: To evaluate OneTouch® Verio™ test strip performance at hypoglycaemic blood glucose (BG) levels (<3.9mmol/L [<70mg/dL]) at seven clinical studies. Methods: Trained clinical staff performed duplicate capillary BG monitoring system tests on 700 individuals with type 1 and type 2 diabetes using blood from a single fingerstick lancing. BG reference values were obtained using a YSI 2300 STAT™ Glucose Analyzer. The number and percentage of BG values within ±0.83. mmol/L (±15. mg/dL) and ±0.56. mmol/L (±10. mg/dL) were calculated at BG concentrations of <3.9. mmol/L (<70. mg/dL), <3.3. mmol/L (<60. mg/dL), and <2.8. mmol/L (<50. mg/dL). Results: At BG concentrations <3.9. mmol/L (<70. mg/dL), 674/674 (100%) of meter results were within ±0.83. mmol/L (±15. mg/dL) and 666/674 (98.8%) were within ±0.56. mmol/L (±10. mg/dL) of reference values. At BG concentrations <3.3. mmol/L (<60. mg/dL), and <2.8. mmol/L (<50. mg/dL), 358/358 (100%) and 270/270 (100%) were within ±0.56. mmol/L (±10. mg/dL) of reference values, respectively. Conclusion: In this analysis of data from seven independent studies, OneTouch Verio test strips provide highly accurate results at hypoglycaemic BG levels. © 2012 Elsevier Ireland Ltd.

Self-monitoring of blood glucose in Black Caribbean and South Asian Canadians with non-insulin treated Type 2 diabetes mellitus:a qualitative study of patients' perspectives

Background: To examine the views and current practice of SMBG among Black Caribbean and South Asian individuals with non-insulin treated Type 2 diabetes mellitus. Methods: Twelve participants completed semi-structured interviews that were guided by the Health Belief Model and analyzed using thematic network analysis. Results: The frequency of monitoring among participants varied from several times a day to once per week. Most participants expressed similar experiences regarding their views and practices of SMBG. Minor differences across gender and culture were observed. All participants understood the benefits, but not all viewed SMBG as beneficial to their personal diabetes management. SMBG can facilitate a better understanding and maintenance of self-care behaviours. However, it can trigger both positive and negative emotional responses, such as a sense of disappointment when high readings are not anticipated, resulting in emotional distress. Health care professionals play a key role in the way SMBG is perceived and used by patients. Conclusion: While the majority of participants value SMBG as a self-management tool, barriers exist that impede its practice, particularly its cost. How individuals cope with these barriers is integral to understanding why some patients adopt SMBG more than others. © 2013 Gucciardi et al.; licensee BioMed Central Ltd.

Multispectral retinal image analysis (MRIA) for the assessment of subretinal fibrosis in neovascular age-related macular degeneration (nAMD)

Purpose: To investigate the use of MRIA for quantitative characterisation of subretinal fibrosis secondary to nAMD. Methods: MRIA images of the posterior pole were acquired over 4 months from 20 eyes including those with inactive subretinal fibrosis and those being treated with ranibizumab for nAMD. Changes in morphology of the macula affected by nAMD were modelled and reflectance spectra at the MRIA acquisition wavelengths (507, 525, 552, 585, 596, 611 and 650nm) were computed using Monte Carlo simulation. Quantitative indicators of fibrosis were derived by matching image spectra to the model spectra of known morphological properties. Results: The model spectra were comparable to the image spectra, both normal and pathological. The key morphological changes that the model associated with nAMD were gliosis of the IS-OS junction, decrease in retinal blood and decrease in RPE melanin. However, these changes were not specific to fibrosis and none of the quantitative indicators showed a unique association with the degree of fibrosis. Moderate correlations were found with the clinical assessment, but not with the treatment program. Conclusion: MRIA can distinguish subretinal fibrosis from healthy tissue. The methods used show high sensitivity but low specificity, being unable to distinguish scarring from other abnormalities like atrophy. Quantification of scarring was not achieved with the wavelengths used due to the complex structural changes to retinal tissues in the process of nAMD. Further studies, incorporating other wavelengths, will establish whether MRIA has a role in the assessment of subretinal fibrosis in the context of retinal and choroidal pathology

A spatial pattern analysis of β-amyloid (Aβ) deposition in the temporal lobe in Alzheimer's disease

To determine the factors influencing the distribution of β-amyloid (Aβ) deposits in Alzheimer's disease (AD), the spatial patterns of the diffuse, primitive, and classic Aβ deposits were studied from the superior temporal gyrus (STG) to sector CA4 of the hippocampus in six sporadic cases of the disease. In cortical gyri and in the CA sectors of the hippocampus, the Aβ deposits were distributed either in clusters 200-6400 μm in diameter that were regularly distributed parallel to the tissue boundary or in larger clusters greater than 6400 μm in diameter. In some regions, smaller clusters of Aβ deposits were aggregated into larger 'superclusters'. In many cortical gyri, the density of Aβ deposits was positively correlated with distance below the gyral crest. In the majority of regions, clusters of the diffuse, primitive, and classic deposits were not spatially correlated with each other. In two cases, double immunolabelled to reveal the Aβ deposits and blood vessels, the classic Aβ deposits were clustered around the larger diameter vessels. These results suggest a complex pattern of Aβ deposition in the temporal lobe in sporadic AD. A regular distribution of Aβ deposit clusters may reflect the degeneration of specific cortico-cortical and cortico-hippocampal pathways and the influence of the cerebral blood vessels. Large-scale clustering may reflect the aggregation of deposits in the depths of the sulci and the coalescence of smaller clusters.

Blood Lead Levels in Children and Environmental Lead Contamination in Miami Inner City, Florida

Studies have shown that the environmental conditions of the home are important predictors of health, especially in low-income communities. Understanding the relationship between the environment and health is crucial in the management of certain diseases. One health outcome related to the home environment among urban, minority, and low-income children is childhood lead poisoning. The most common sources of lead exposure for children are lead paint in older, dilapidated housing and contaminated dust and soil produced by accumulated residue of leaded gasoline. Blood lead levels (BLL) as low as 10 μg/dL in children are associated with impaired cognitive function, behavior difficulties, and reduced intelligence. Recently, it is suggested that the standard for intervention be lowered to BLL of 5 μg /dl. The objectives of our report were to assess the prevalence of lead poisoning among children under six years of age and to quantify and test the correlations between BLL in children and lead exposure levels in their environment. This cross-sectional analysis was restricted to 75 children under six years of age who lived in 6 zip code areas of inner city Miami. These locations exhibited unacceptably high levels of lead dust and soil in areas where children live and play. Using the 5 μg/dL as the cutoff point, the prevalence of lead poisoning among the study sample was 13.33%. The study revealed that lead levels in floor dust and window sill samples were positively and significantly correlated with BLL among children (p < 0.05). However, the correlations between BLL and the soil, air, and water samples were not significant. Based on this pilot study, a more comprehensive environmental study in surrounding inner city areas is warranted. Parental education on proper housecleaning techniques may also benefit those living in the high lead-exposed communities of inner city Miami.

Sequence analysis of complementary DNAs encoding C3 in the nurse shark ({\it Ginglymostoma cirratum\/})

Mammalian C3 is a pivotal complement protein, encoded for by a single gene. In some vertebrate species multiple C3 isoforms are products of different C3 genes. The goal of this study was to determine whether multiple genes encode for shark C3. A protocol was developed for the isolation of mRNA from shark blood for the isolation of C3 cDNA clones. RT-PCR amplification of mRNA, using sense (GCGEQNM) and antisense (TWLTAYV) primers encoding conserved regions of human C3, yielded 21 clones. The C3-like clones isolated shared 97% similarity with each other and 40% similarity to human C3. RACE-PCR amplification of shark liver RNA, using gene specific primers, yielded products ranging from 1800bp to 3000bp. Deduced amino acid sequence, corresponding to 408bp of the 1800bp fragment, was obtained which showed 51% similarity to human C3. These results suggest that nurse shark C3 might be encoded for by more than one gene. ^

Blood-brain barrier in vitro model: A tissue engineering approach and validation

This dissertation evaluated the feasibility of using commercially available immortalized cell lines in building a tissue engineered in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Mouse endothelial cell line and rat astrocyte cell lines purchased from American Type Culture Collections (ATCC) were the building blocks of the co-culture model. An astrocyte derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel in vitro biomimetic basement membrane for the endothelial cells to form endothelial tight junctions. Trans-endothelial electrical resistance (TEER) and solute mass transport studies were engaged to quantitatively evaluate the tight junction formation on the in-vitro BBB models. Immuno-fluorescence microscopy and Western Blot analysis were used to qualitatively verify the in vitro expression of occludin, one of the earliest discovered tight junction proteins. Experimental data from a total of 12 experiments conclusively showed that the novel BBB in vitro co-culture model with the astrocyte derived aECM (CO+aECM) was promising in terms of establishing tight junction formation represented by TEER values, transport profiles and tight junction protein expression when compared with traditional co-culture (CO) model setups and endothelial cells cultured alone. Experimental data were also found to be comparable with several existing in vitro BBB models built from various methods. In vitro colorimetric sulforhodamine B (SRB) assay revealed that the co-cultured samples with aECM resulted in less cell loss on the basal sides of the insert membranes than that from traditional co-culture samples. The novel tissue engineering approach using immortalized cell lines with the addition of aECM was proven to be a relevant alternative to the traditional BBB in vitro modeling.

An investigation of the relationship between antemortem and postmortem drug concentrations in blood

In the field of postmortem toxicology, principles from pharmacology and toxicology are combined in order to determine if exogenous substances contributed to ones death. In order to make this determination postmortem and (whenever available) antemortem blood samples may be analyzed. This project focused on evaluating the relationship between postmortem and antemortem blood drug levels, in order to better define an interpretive framework for postmortem toxicology. To do this, it was imperative to evaluate the differences in antemortem and postmortem drug concentrations, determine the role microbial activity and evaluate drug stability. Microbial studies determined that the bacteria Escherichia coli and Pseudomonas aeruginosa could use the carbon structures of drugs as a source of food. This would suggest prior to sample collection, microbial activity could potentially affect drug levels. This process however would stop before toxicologic evaluation, as at autopsy blood samples are stored in tubes containing the antimicrobial agent sodium fluoride. Analysis of preserved blood determined that under the current storage conditions sodium fluoride effectively inhibited microbial growth. Nonetheless, in many instances inconsistent drug concentrations were identified. When comparing antemortem to postmortem results, diphenhydramine, morphine, codeine and methadone, all showed significantly increased postmortem drug levels. In many instances, increased postmortem concentrations correlated with extended postmortem intervals. Other drugs, such as alprazolam, were likely to have concentration discrepancies when short antemortem to death intervals were coupled with extended postmortem intervals. While still others, such as midazolam followed the expected pattern of metabolism and elimination, which often resulted in decreased postmortem concentrations. The importance of drug stability was displayed when reviewing the clonazepam/ 7-aminoclonazepam data, as the parent drug commonly converted to its metabolite even when stored in the presence of a preservative. In instances of decreasing postmortem drug concentrations the effect of refrigerated storage could not be ruled out. A stability experiment, which contained codeine, produced data that indicated concentrations could continue to decline under the current storage conditions. The cumulative data gathered for this experiment was used to identify concentration trends, which subsequently aided in the development of interpretive considerations for the specific analytes examined in the study.

Antibacterial proteins and peptides in nurse shark ( Ginglymostoma cirratum) peripheral blood leukocytes

In many vertebrate and invertebrate species mediators of innate immunity include antimicrobial peptides (AMPs) such as peptide fragments of histones and other proteins with previously ascribed different functions. Shark AMPs have not been described and this research examines the antibacterial activity of nurse shark (Ginglymostoma cirratum) peripheral blood leukocyte lysates. Screening of lysates prepared by homogenizing unstimulated peripheral blood leukocytes identified muramidase (lysozyme-like) and non-muramidase antibacterial activity. Lysates were tested for lysozyme using the lysoplate assays, and antibacterial (AB) activity was assayed for by a microdilution growth assay that was developed using Planococcus citreus as the target bacterium. Fractionation of crude lysates by ion exchange and affinity chromatography was followed by a combination of SDS-PAGE with LC/MS-MS and/or N-terminal sequence analysis of low molecular weight protein bands (<20 kDa). This yielded several peptides with amino acid sequence similarity to lysozyme, ubiquitin, hemoglobin, human histones H2A, H2B and H4 and to antibacterial histone fragments of the catfish and the Asian toad. Not all peptide sequences corresponded to peptides potentially antibacterial. The correlation of a specific protein band in active lysate fractions was accomplished by employing the acid-urea gel overlay assays in which AB activity was seen as zones of growth inhibition on a lawn of P. citreus at a position corresponding to that of the putative AB protein band. This study is the first to describe putative AMPs in the shark and their potential role in innate immunity.^