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.

In vivo vitamin C supplementation increases phosphoinositol transfer protein expression in peripheral blood mononuclear cells from healthy individuals

Ascorbate can act as both a reducing and oxidising agent in vitro depending on its environment. It can modulate the intracellular redox environment of cells and therefore is predicted to modulate thiol-dependent cell signalling and gene expression pathways. Using proteomic analysis of vitamin C-treated T cells in vitro, we have previously reported changes in expression of five functional protein groups associated with signalling, carbohydrate metabolism, apoptosis, transcription and immune function. The increased expression of the signalling molecule phosphatidylinositol transfer protein (PITP) was also confirmed using Western blotting. Herein, we have compared protein changes elicited by ascorbate in vitro, with the effect of ascorbate on plasma potassium levels, on peripheral blood mononuclear cell (PBMC) apoptosis and PITP expression, in patients supplemented with vitamin C (0-2 g/d) for up to 10 weeks to investigate whether in vitro model systems are predictive of in vivo effects. PITP varied in expression widely between subjects at all time-points analysed but was increased by supplementation with 2 g ascorbate/d after 5 and 10 weeks. No effects on plasma potassium levels were observed in supplemented subjects despite a reduction of K+ channel proteins in ascorbate-treated T cells in vitro. Similarly, no effect of vitamin C supplementation on PBMC apoptosis was observed, whilst ascorbate decreased expression of caspase 3 recruitment domain protein in vitro. These data provide one of the first demonstrations that proteomics may be valuable in developing predictive markers of nutrient effects in vivo and may identify novel pathways for studying mechanisms of action in vivo.

Macular pigment optical density is related to blood glutathione levels in healthy individuals

Purpose. To assess the relationship between macular pigment optical density (MPOD) and blood markers for antioxidant defense in otherwise healthy volunteers. Methods. Forty-seven healthy volunteers were subjected to blood analysis to detect the level of circulating glutathione in its reduced (GSH) and oxidized (GSSG) forms. The level of MPOD was measured using heterochromatic flicker photometry. Systemic blood pressure (BP) parameters, heart rate (HR), body mass index (BMI), and plasma levels of total, HDL, and LDL cholesterol and triglycerides (TGs) were also determined. Results. A simple correlation model revealed that the level of MPOD correlated significantly and positively with both GSH (P < 0.001) and t-GSH (P < 0.001) levels but not with those of GSSG (P > 0.05). Age, sex, systemic BP parameters, HR, BMI, and plasma levels of cholesterol and TGs did not have any influence on either MPOD or glutathione levels (all P > 0.05). In addition, a forward stepwise multiple regression analysis showed MPOD to have a significantly and independent correlation with GSH levels (ß = 0.63; P < 0.001). Conclusions. In otherwise healthy older individuals, there is a positive correlation between local and systemic antioxidant defense mechanisms.

Reduction in blood glutathione levels occurs similarly in patients with primary-open angle or normal tension glaucoma

PURPOSE. To investigate in parallel the systemic glutathione levels of patients suffering from primary open angle glaucoma (POAG) or normal tension glaucoma (NTG) with comparable functional loss. METHODS. Thirty-four POAG patients, 30 NTG patients, and 53 controls were subjected to blood analysis to detect the level of circulating glutathione in its reduced (GSH) and oxidized (GSSG) forms. Systemic blood pressure (BP) and ocular perfusion pressure (OPP) parameters were also determined. RESULTS. Independent of age, POAG and NTG patients demonstrated significantly lower GSH and t-GSH levels than age-matched controls (P < 0.001). Additionally, a lower redox index was found, but in POAG patients only, in comparison to both NTG and control groups (P = 0.020). GSSG levels were, however, similar between all study groups (P > 0.05). CONCLUSIONS. This study demonstrates, for the first time, that both POAG and NTG patients exhibit lower GSH and t-GSH levels than age-matched controls, indicating a similar general compromise of the antioxidant defense systems may exist in both conditions. © 2013 The Association for Research in Vision and Ophthalmology, Inc.

Individual variability analysis of fluorescence parameters measured in skin with different levels of nutritive blood flow

Fluorescence spectroscopy has recently become more common in clinical medicine. However, there are still many unresolved issues related to the methodology and implementation of instruments with this technology. In this study, we aimed to assess individual variability of fluorescence parameters of endogenous markers (NADH, FAD, etc.) measured by fluorescent spectroscopy (FS) in situ and to analyse the factors that lead to a significant scatter of results. Most studied fluorophores have an acceptable scatter of values (mostly up to 30%) for diagnostic purposes. Here we provide evidence that the level of blood volume in tissue impacts FS data with a significant inverse correlation. The distribution function of the fluorescence intensity and the fluorescent contrast coefficient values are a function of the normal distribution for most of the studied fluorophores and the redox ratio. The effects of various physiological (different content of skin melanin) and technical (characteristics of optical filters) factors on the measurement results were additionally studied.The data on the variability of the measurement results in FS should be considered when interpreting the diagnostic parameters, as well as when developing new algorithms for data processing and FS devices.

Modified lipids from LDL in the blood during mid-life increase blood brain barrier permeability

Low density lipoprotein levels (LDL) are consistently elevated in cardiovascular disease. It has been suggested that those with high circulating LDL levels in mid-life may be susceptible to develop neurodegenerative diseases in later life. In the circulation, high levels of LDL are associated with increased oxidative modification (oxLDL) and nitration. We have investigated the hypothesis that disruption of blood brain barrier function by oxLDL and their lipids may increase risk of neurodegeneration in later life and that statin intervention in mid-life can mitigate the neurodegenerative effects of hyperlipidaemia. Blood from statin-naïve, normo- and hyperlipidaemic subjects (n=10/group) was collected at baseline. Hyperlipidaemic subjects received statin-intervention whereas normolipidaemic subjects did not prior to a second blood sampling, taken after 3 months. The intervention will be completed in June 2013. Plasma was separated by centrifugation (200g, 30min) and LDL was isolated by potassium bromide density gradient ultracentrifugation. Total homocysteine, LDL cholesterol, 8-isoprostane F2α levels were measured in plasma using commercial kits. LDL were analysed by agarose gel electrophoresis. LDL-lipids were extracted by partitioning in 1:1 chloroform:methanol (v/v) and conjugated to fatty acid free-BSA in serum-free EGM-2 medium (4hrs, 370C) for co-culture with human microvascular endothelial cells (HMVEC). HMVEC were maintained on polycarbonate inserts for two weeks to create a microvascular barrier. Change in barrier permeability was measured by trans-endothelial electrical resistance (TER), FITC-dextran permeability and immunohistochemistry. HMVEC glutathione (GSH) levels were measured after 2 hours by GSH-glo assay. LDL isolated from statin-naïve hyperlipidaemic subjects had higher mobility by agarose gel electrophoresis (Rf;0.53±0.06) and plasma 8-isoprostane F2α (43.5±8.42 pg/ml) compared to control subjects (0.46±0.05 and 24.2±5.37 pg/ml; p<0.05). Compared to HMVEC treatment with the LDL-lipids (5μM) from normolipidaemic subjects, LDL-lipids from hyperlipidaemic subjects increased barrier permeability (103.4±12.5 Ωcm2 v 66.7±7.3 Ωcm2,P<0.01) and decreased GSH (18.5 nmol/mg v 12.3 nmol/mg; untreated cells 26.2±3.6 nmol/mg).

AltitudeOmics: Red Blood Cell metabolic adaptation to high altitude hypoxia

Red blood cells (RBCs) are key players in systemic oxygen transport. RBCs respond to in vitro hypoxia  through  the so-called  oxygen-dependent  metabolic  regulation,  which  involves  the competitive  binding  of  deoxyhemoglobin  and  glycolytic  enzymes  to  the  N-terminal  cytosolic domain  of  band  3.  This  mechanism  promotes  the  accumulation  of  2,3-DPG,  stabilizing  the deoxygenated state of hemoglobin, and cytosol acidification, triggering oxygen off-loading through the  Bohr  effect.  Despite  in  vitro  studies,  in  vivo adaptations  to  hypoxia  have  not  yet  been completely elucidated. Within  the  framework  of  the AltitudeOmics  study,  erythrocytes  were  collected  from  21 healthy volunteers at sea level, after exposure to high altitude (5260m) for 1, 7 and 16days, and following  reascent  after  7days  at 1525m.  UHPLC-MS  metabolomics  results  were  correlated  to physiological and athletic performance parameters. Immediate  metabolic  adaptations  were  noted as early as a few hours from ascending  to >5000m, and maintained for 16 days at high altitude.  Consistent with the mechanisms elucidated in vitro, hypoxia promoted glycolysis and deregulated the pentose phosphate pathway, as well purine catabolism, glutathione homeostasis, arginine/nitric oxide and sulphur/H2S metabolism. Metabolic adaptations were preserved one week after descent, consistently with improved physical performances in comparison to the first ascendance, suggesting a mechanism of metabolic memory.

Balance ability in 7- and 10-year-old children:associations with prenatal lead and cadmium exposure and with blood lead levels in childhood in a prospective birth cohort study

OBJECTIVES: Most studies reporting evidence of adverse effects of lead and cadmium on the ability to balance have been conducted in high-exposure groups or have included adults. The effects of prenatal exposure have not been well studied, nor have the effects in children been directly studied. The aim of the study was to identify the associations of lead (in utero and in childhood) and cadmium (in utero) exposure with the ability to balance in children aged 7 and 10 years. DESIGN: Prospective birth cohort study. PARTICIPANTS: Maternal blood lead (n=4285) and cadmium (n=4286) levels were measured by inductively coupled plasma mass spectrometry in women enrolled in the Avon Longitudinal Study of Parents and Children (ALSPAC) during pregnancy. Child lead levels were measured in a subsample of 582 of ALSPAC children at age 30 months. MAIN OUTCOME MEASURES: Children completed a heel-to-toe walking test at 7 years. At 10 years, the children underwent clinical tests of static and dynamic balance. Statistical analysis using SPSS V.19 included logistic regression modelling, comparing categories of ≥ 5 vs <5 µg/dL for lead, and ≥ 1 vs <1 µg/L for cadmium. RESULTS: Balance at age 7 years was not associated with elevated in utero lead or cadmium exposure (adjusted OR for balance dysfunction: Pb 1.01 (95% CI 0.95 to 1.01), n=1732; Cd 0.95 (0.77 to 1.20), n=1734), or with elevated child blood lead level at age 30 months (adjusted OR 0.98 (0.92 to 1.05), n=354). Similarly, neither measures of static nor dynamic balance at age 10 years were associated with in utero lead or cadmium exposure, or child lead level. CONCLUSIONS: These findings do not provide any evidence of an association of prenatal exposure to lead or cadmium, or lead levels in childhood, on balance ability in children. Confirmation in other cohorts is needed.