A novel dried blood spot-LCMS method for the quantification of methotrexate polyglutamates as a potential marker for methotrexate use in children

Objective: Development and validation of a selective and sensitive LCMS method for the determination of methotrexate polyglutamates in dried blood spots (DBS). Methods: DBS samples [spiked or patient samples] were prepared by applying blood to Guthrie cards which was then dried at room temperature. The method utilised 6-mm disks punched from the DBS samples (equivalent to approximately 12 μl of whole blood). The simple treatment procedure was based on protein precipitation using perchloric acid followed by solid phase extraction using MAX cartridges. The extracted sample was chromatographed using a reversed phase system involving an Atlantis T3-C18 column (3 μm, 2.1x150 mm) preceded by Atlantis guard column of matching chemistry. Analytes were subjected to LCMS analysis using positive electrospray ionization. Key Results: The method was linear over the range 5-400 nmol/L. The limits of detection and quantification were 1.6 and 5 nmol/L for individual polyglutamates and 1.5 and 4.5 nmol/L for total polyglutamates, respectively. The method has been applied successfully to the determination of DBS finger-prick samples from 47 paediatric patients and results confirmed with concentrations measured in matched RBC samples using conventional HPLC-UV technique. Conclusions and Clinical Relevance: The methodology has a potential for application in a range of clinical studies (e.g. pharmacokinetic evaluations or medication adherence assessment) since it is minimally invasive and easy to perform, potentially allowing parents to take blood samples at home. The feasibility of using DBS sampling can be of major value for future clinical trials or clinical care in paediatric rheumatology. © 2014 Hawwa et al.

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.

Direct modulatory effect of C-reactive protein on primary human monocyte adhesion to human endothelial cells

C-reactive protein (CRP) is the prototypic acute phase serum protein in humans. The effects of CRP on primary human monocyte adhesion molecule expression and interaction with the endothelium have not been studied. Herein, we describe an investigation into the phenotypic and functional consequences of CRP binding to peripheral blood monocytes ex vivo. Peripheral whole blood was collected from healthy, non-smoking males. Mononuclear cells (MNC) and monocytes were isolated by differential centrifugation using lymphoprep and Dynal negative isolation kit, respectively. Cells were exposed to CRP from 0 to 250 μg/ml for 0-60 min at 37°C and analysed for (a) CD11b, PECAM-1 (CD31) and CD32 expression by flow cytometry and (b) adhesion to LPS (1 μg/ml; 0-24 h) treated human umbilical vein endothelial cells (HUVEC). CD14+ monocyte expression of CD11b increased significantly up to twofold when exposed to CRP, compared to controls. There was no significant difference in CD32 expression, whereas CD31 expression decreased after exposure to CRP. CRP treatment of monocytes inhibited their adhesion to early LPS-activated HUVEC (0-5 h). However, the adhesion of CRP-treated monocytes to HUVEC was significantly greater to late activation antigens on HUVEC (24 h, LPS) compared to controls. We have shown that CRP can affect monocyte activation ex vivo and induce phenotypic changes that result in an altered recruitment to endothelial cells. This study provides the first evidence for a further role for C-reactive protein in both monocyte activation and adhesion, which may be of importance during an inflammatory event.

The effect of R-plasmid RP1 on the properties of Proteus mirabilis

A clinical isolate of Proteus mirabilis containing R-plasmid RP1 (R+ cells), grown in both iron- and carbon- limited chemically defined media in mixed culture with plasmid-free (R- cells), did not disappear as expected, due to adherence of R+ cells to the wall of the chemostat vessel. Plasmid RP1 promoted adherence to glass and to medical prostheses. The hydrophobicity and surface charge of R+ cells were different from those of R- cells and both factors may contribute to the adherence of R+ cells to surfaces. The mode of cultivation of the cells, whether batch or continuous culture, were also found to affect the result. Antibodies raised against homologous cells increased the surface hydrophobicity of both R+ and R- cells and eliminated the differences between them. Results for surface hydrophobicity varied with the method used for measuring it. R+ cells were more sensitive than R- cells to tbe bacteridical action of normal serum and whole blood and to phagocytosis as measured by chemiluminescence. No clear differences were revealed in the protein antigens of R+ and R- cells by both SDS PAGE gels and immunoblots reacted with homologous antibodies. However, lectins revealed differences in the sugars exposed on the cell surfaces. Chemical analysis of R&43 and R- cells also revealed differences in the content of 2-keto-3-deoxy-D-manno-2-octulosonate, lipopolysaccharide and total fatty acids, when cells were grown in media containing added iron; however, no qualitative differences in the lipopolysaccharide were found. Removal of iron from the medium was found to have considerable effects on the chemical structure of R+ cells but not of R- ones. Adhesion to prostheses and to leucocytes is discussed in the light of the results and the clinical relevance outlined with respect to the initiation of infection and the association of virulence with antibiotic resistance.

Methylglyoxal, glyoxalses and cell proliferation

The metabolic function of the glyoxalase system was investigated in (a) the differentiation and proliferation of human tumour cells in vitro, (b) the cell-free assembly of microtubules and (c) in the red blood cells during hyperglycaemia associated with Diabetes Mellitus. Chemically-induced differentiation of human promyelocytic HL60 leukaemia cells to neutrophils, and K562 erythroleukaemia cells, was accompanied by a decrease and an increase in the activity of glyoxalase I, respectively. Growth-arrest of Burkitt's lymphoma Raji cells and GM892 lymphoblastoid cells was accompanied by an increase and a decrease in the activity of glyoxalase I respectively. However, differentiation and growth arrest generally proceeded with an increase in the activity of glyoxalase II. Glyoxalase I activity did not consistently correlate with cell differentiation or proliferation status; hence, it is unlikely that glyoxalase I activity is either an indicator or a regulator of cell differentiation or proliferation. Conversely, glyoxalase II activity consistently increased during cell differentiation and growth-arrest and may be both an indicator and regulator of cell differentiation or proliferation. This may be related to the control of cellular microtubule assembly. S-D-Lactoylglutathione potentiated the cell-free, GTP-promoted assembly of microtubules. The effect was dose-related and was inhibited by glyoxalase II. During assembly, S-D-lactoylglutathione was consumed. This suggests that the glyoxalase system, through the influence of S-D-lactoylglutathione, may regulate the assembly of microtubules in cellular systems The whole blood concentrations of methylglyoxal and S-D-lactoylglutathione were increased in Diabetes Mellitus. There was no significant difference between red blood cell glyoxalase activities in diabetics, compared to healthy controls. However, insulin-dependent diabetic patients with retinopathy had a significantly higher glyoxalase I activity and a lower glyoxalase II activity, than patients without retinopathy. Diabetic retinopathy correlated with high glyoxalase I activity and low glyoxalase II activity and suggests the glyoxalase system may be involved in the development of diabetic complications.

Model thrombi formed under flow reveal the role of factor XIII-mediated cross-linking in resistance to fibrinolysis

Background: Activated factor XIII (FXIIIa), a transglutaminase, introduces fibrin-fibrin and fibrin-inhibitor cross-links, resulting in more mechanically stable clots. The impact of cross-linking on resistance to fibrinolysis has proved challenging to evaluate quantitatively. Methods: We used a whole blood model thrombus system to characterize the role of cross-linking in resistance to fibrinolytic degradation. Model thrombi, which mimic arterial thrombi formed in vivo, were prepared with incorporated fluorescently labeled fibrinogen, in order to allow quantification of fibrinolysis as released fluorescence units per minute. Results: A site-specific inhibitor of transglutaminases, added to blood from normal donors, yielded model thrombi that lysed more easily, either spontaneously or by plasminogen activators. This was observed both in the cell/platelet-rich head and fibrin-rich tail. Model thrombi from an FXIII-deficient patient lysed more quickly than normal thrombi; replacement therapy with FXIII concentrate normalized lysis. In vitro addition of purified FXIII to the patient's preprophylaxis blood, but not to normal control blood, resulted in more stable thrombi, indicating no further efficacy of supraphysiologic FXIII. However, addition of tissue transglutaminase, which is synthesized by endothelial cells, generated thrombi that were more resistant to fibrinolysis; this may stabilize mural thrombi in vivo. Conclusions: Model thrombi formed under flow, even those prepared as plasma 'thrombi', reveal the effect of FXIII on fibrinolysis. Although very low levels of FXIII are known to produce mechanical clot stability, and to achieve ?-dimerization, they appear to be suboptimal in conferring full resistance to fibrinolysis.

Population pharmacokinetic and pharmacogenetic analysis of tacrolimus in paediatric liver transplant patients

Aims - To build a population pharmacokinetic model that describes the apparent clearance of tacrolimus and the potential demographic, clinical and genetically controlled factors that could lead to inter-patient pharmacokinetic variability within children following liver transplantation. Methods - The present study retrospectively examined tacrolimus whole blood pre-dose concentrations (n = 628) of 43 children during their first year post-liver transplantation. Population pharmacokinetic analysis was performed using the non-linear mixed effects modelling program (nonmem) to determine the population mean parameter estimate of clearance and influential covariates. Results - The final model identified time post-transplantation and CYP3A5*1 allele as influential covariates on tacrolimus apparent clearance according to the following equation: TVCL = 12.9 x (Weight/13.2)0.35 x EXP (-0.0058 x TPT) x EXP (0.428 x CYP3A5) where TVCL is the typical value for apparent clearance, TPT is time post-transplantation in days and the CYP3A5 is 1 where *1 allele is present and 0 otherwise. The population estimate and inter-individual variability (%CV) of tacrolimus apparent clearance were found to be 0.977 l h−1 kg−1 (95% CI 0.958, 0.996) and 40.0%, respectively, while the residual variability between the observed and predicted concentrations was 35.4%. Conclusion Tacrolimus apparent clearance was influenced by time post-transplantation and CYP3A5 genotypes. The results of this study, once confirmed by a large scale prospective study, can be used in conjunction with therapeutic drug monitoring to recommend tacrolimus dose adjustments that take into account not only body weight but also genetic and time-related changes in tacrolimus clearance.

A high-fat meal induces low-grade endotoxemia:evidence of a novel mechanism of postprandial inflammation

Background: Bacterial endotoxin is a potently inflammatory antigen that is abundant in the human gut. Endotoxin circulates at low concentrations in the blood of all healthy individuals, although elevated concentrations are associated with an increased risk of atherosclerosis. Objective: We sought to determine whether a high-fat meal or smoking increases plasma endotoxin concentrations and whether such concentrations are of physiologic relevance. Design: Plasma endotoxin and endotoxin neutralization capacity were measured for 4 h in 12 healthy men after no meal, 3 cigarettes, a high-fat meal, or a high-fat meal with 3 cigarettes by using the limulus assay. Results: Baseline endotoxin concentrations were 8.2 pg/mL (interquartile range: 3.4–13.5 pg/mL) but increased significantly (P < 0.05) by ≈50% after a high-fat meal or after a high-fat meal with cigarettes but not after no meal or cigarettes alone. These results were validated by the observations that a high-fat meal with or without cigarettes, but not no meal or smoking, also significantly (P < 0.05) reduced plasma endotoxin neutralization capacity, which is an indirect measure of endotoxin exposure. Human monocytes, but not aortic endothelial cells, were responsive to transient (30 s) or low-dose (10 pg/mL) exposure to endotoxin. However, plasma from whole blood treated with as little as 10 pg endotoxin/mL increased the endothelial cell expression of E-selectin, at least partly via tumor necrosis factor-α–induced cellular activation. Conclusions: Low-grade endotoxemia may contribute to the postprandial inflammatory state and could represent a novel potential contributor to endothelial activation and the development of atherosclerosis.

Spatial correlations between beta-amyloid (Abeta) deposits and blood vessels in early-onset Alzheimer's disease

In cases of late-onset Alzheimer’s disease (AD), there is a spatial correlation between the classsic ‘cored’ type of Beta-amyloid (Abeta) deposit and the large vertically penetrating arterioles in the cerebral cortex suggesting that blood vessels are involved in the pathogenesis of the classic deposits. In this chapter, the spatial correlations between the diffuse, primitive, and classic Abeta deposits and blood vessels were studied in 10 cases of early-onset AD in the age range 40 – 65 years. Sections of frontal cortex were immunostained with antibodies against Abeta?and with collagen IV to reveal the Abeta deposits and blood vessel profiles. In the early-onset cases as a whole, all types of Abeta? deposit and blood vessel profiles were distributed in clusters. There was a positive spatial correlation between the clusters of the diffuse Abeta deposits and the larger (>10µm) and smaller diameter (<10?m) blood vessel profiles in one and three cases respectively. The primitive and classic Abeta deposits were spatially correlated with larger and smaller blood vessels both in three and four cases respectively. Spatial correlations between the Abeta deposits and blood vessels may be more prevalent in cases expressing amyloid precursor protein (APP) than presenilin 1 (PSEN1) mutations. Apolipoprotein E (Apo E) genotype of the patient did not appear to influence the spatial correlation with blood vessel profiles. The data suggest that the larger diameter blood vessels are less important in the pathogenesis of the classic Abeta deposits in early-onset compared with late-onset AD.

Spatial correlations between beta-amyloid (A beta) deposits and blood vessels in familial Alzheimer's disease

In sporadic Alzheimer’s disease (SAD), the classic (‘dense-cored’) ß-amyloid (Aß) deposits are aggregated around the larger blood vessels in the upper laminae of the cerebral cortex. To determine whether a similar relationship exists in familial AD (FAD), the spatial correlations between the diffuse, primitive, and classic ß-amyloid (Aß deposits and blood vessels were studied in ten FAD cases including cases linked to amyloid precursor protein (APP) and presenilin (PSEN) gene mutations and expressing apolipoprotein E (apo E) allele E4. Sections of frontal cortex were immunolabelled with antibodies against Aß and with collagen IV to reveal the Aß deposits and blood vessel profiles. In the FAD cases as a whole, Aßdeposits were distributed in clusters. There was a positive spatial correlation between the clusters of the diffuse Aßdeposits and the larger (>10 µm) and smaller diameter (<10 µm) blood vessels in one and three cases respectively. The primitive Aß deposits were spatially correlated with larger and smaller blood vessels each in four cases and the classic deposits in three and four cases respectively. Apo E genotype of the patient did not influence spatial correlation with blood vessels. Hence, spatial correlations between the classic deposits and larger diameter blood vessels were significantly less frequent in FAD compared with SAD. It was concluded that both Aß deposit morphology and AD subtype determine spatial correlations with blood vessels in AD.