Urinary di-(2-ethylhexyl) phthalate metabolites for detecting transfusion of autologous blood stored in plasticizer-free bags.

BACKGROUND: Autologous blood transfusion (ABT) efficiently increases sport performance and is the most challenging doping method to detect. Current methods for detecting this practice center on the plasticizer di(2-ethlyhexyl) phthalate (DEHP), which enters the stored blood from blood bags. Quantification of this plasticizer and its metabolites in urine can detect the transfusion of autologous blood stored in these bags. However, DEHP-free blood bags are available on the market, including n-butyryl-tri-(n-hexyl)-citrate (BTHC) blood bags. Athletes may shift to using such bags to avoid the detection of urinary DEHP metabolites. STUDY DESIGN AND METHODS: A clinical randomized double-blinded two-phase study was conducted of healthy male volunteers who underwent ABT using DEHP-containing or BTHC blood bags. All subjects received a saline injection for the control phase and a blood donation followed by ABT 36 days later. Kinetic excretion of five urinary DEHP metabolites was quantified with liquid chromatography coupled with tandem mass spectrometry. RESULTS: Surprisingly, considerable levels of urinary DEHP metabolites were observed up to 1 day after blood transfusion with BTHC blood bags. The long-term metabolites mono-(2-ethyl-5-carboxypentyl) phthalate and mono-(2-carboxymethylhexyl) phthalate were the most sensitive biomarkers to detect ABT with BTHC blood bags. Levels of DEHP were high in BTHC bags (6.6%), the tubing in the transfusion kit (25.2%), and the white blood cell filter (22.3%). CONCLUSIONS: The BTHC bag contained DEHP, despite being labeled DEHP-free. Urinary DEHP metabolite measurement is a cost-effective way to detect ABT in the antidoping field even when BTHC bags are used for blood storage.

CYP17A1 Enzyme activity is linked to ambulatory blood pressure in a family-based population study.

BACKGROUND: Genome-wide association studies have linked CYP17A1 coding for the steroid hormone synthesizing enzyme 17α-hydroxylase (CYP17A1) to blood pressure (BP). We hypothesized that the genetic signal may translate into a correlation of ambulatory BP (ABP) with apparent CYP17A1 activity in a family-based population study and estimated the heritability of CYP17A1 activity. METHODS: In the Swiss Kidney Project on Genes in Hypertension, day and night urinary excretions of steroid hormone metabolites were measured in 518 participants (220 men, 298 women), randomly selected from the general population. CYP17A1 activity was assessed by 2 ratios of urinary steroid metabolites: one estimating the combined 17α-hydroxylase/17,20-lyase activity (ratio 1) and the other predominantly 17α-hydroxylase activity (ratio 2). A mixed linear model was used to investigate the association of ABP with log-transformed CYP17A1 activities exploring effect modification by urinary sodium excretion. RESULTS: Daytime ABP was positively associated with ratio 1 under conditions of high, but not low urinary sodium excretion (P interaction <0.05). Ratio 2 was not associated with ABP. Heritability estimates (SE) for day and night CYP17A1 activities were 0.39 (0.10) and 0.40 (0.09) for ratio 1, and 0.71 (0.09) and 0.55 (0.09) for ratio 2 (P values <0.001). CYP17A1 activities, assessed with ratio 1, were lower in older participants. CONCLUSIONS: Low apparent CYP17A1 activity (assessed with ratio 1) is associated with elevated daytime ABP when salt intake is high. CYP17A1 activity is heritable and diminished in the elderly. These observations highlight the modifying effect of salt intake on the association of CYP17A1 with BP.

Barn owl feathers as biomonitors of mercury: sources of variation in sampling procedures.

Given their central role in mercury (Hg) excretion and suitability as reservoirs, bird feathers are useful Hg biomonitors. Nevertheless, the interpretation of Hg concentrations is still questioned as a result of a poor knowledge of feather physiology and mechanisms affecting Hg deposition. Given the constraints of feather availability to ecotoxicological studies, we tested the effect of intra-individual differences in Hg concentrations according to feather type (body vs. flight feathers), position in the wing and size (mass and length) in order to understand how these factors could affect Hg estimates. We measured Hg concentration of 154 feathers from 28 un-moulted barn owls (Tyto alba), collected dead on roadsides. Median Hg concentration was 0.45 (0.076-4.5) mg kg(-1) in body feathers, 0.44 (0.040-4.9) mg kg(-1) in primary and 0.60 (0.042-4.7) mg kg(-1) in secondary feathers, and we found a poor effect of feather type on intra-individual Hg levels. We also found a negative effect of wing feather mass on Hg concentration but not of feather length and of its position in the wing. We hypothesize that differences in feather growth rate may be the main driver of between-feather differences in Hg concentrations, which can have implications in the interpretation of Hg concentrations in feathers. Finally, we recommend that, whenever possible, several feathers from the same individual should be analysed. The five innermost primaries have lowest mean deviations to both between-feather and intra-individual mean Hg concentration and thus should be selected under restrictive sampling scenarios.

Fibroblast growth factor-23 (FGF-23) levels differ across populations by degree of industrialization.

CONTEXT: Compensatory increases in FGF23 with increasing phosphate intake may adversely impact health. However, population and clinical studies examining the link between phosphate intake and FGF23 levels have focused mainly on populations living in highly industrialized societies in which phosphate exposure may be homogenous. OBJECTIVE: Contrast dietary phosphate intake, urinary measures of phosphate excretion and FGF23 levels across populations that differ by level of industrialization. DESIGN: Cross-sectional analysis of three populations Setting: Maywood, IL, U.S., Mah|fe Island, Seychelles, and Kumasi, Ghana Participants: Adults with African ancestry aged 25-45 years Main Outcome: Fibroblast growth factor 23 (FGF23) levels Results: The mean age was 35.1 (6.3) years and 47.9% were male. Mean phosphate intake and fractional excretion of phosphate were significantly higher in the U.S. vs. Ghana while no significant difference in phosphate intake or fractional excretion of phosphate was noted between U.S. and Seychelles for men or women. Overall, median FGF23 values were 57.41 RU/ml (IQR 43.42, 75.09) in U.S., 42.49 RU/ml (IQR 33.06, 55.39) in Seychelles and 33.32 RU/ml (IQR 24.83, 47.36) in Ghana. In the pooled sample, FGF23 levels were significantly and positively correlated with dietary phosphate intake (r=0.11; P < 0.001), and the fractional excretion of phosphate (r=0.13; P < 0.001) but not with plasma phosphate levels (-0.001; P = 0.8). Dietary phosphate intake was significantly and positively associated with the fractional excretion of phosphate (r=0.23; P < 0.001). CONCLUSION: The distribution of FGF23 levels in a given population may be influenced by the level of industrialization, likely due to differences in access to foods preserved with phosphate additives.

Berry polyphenol absorption and the effect of northern berries on metabolism, ectopic fat accumulation, and associated diseases

The prevalence of obesity and type 2 diabetes has increased at an alarming rate in developed countries. It seems in the light of current knowledge that metabolic syndrome may not develop at all without NAFLD, and NAFLD is estimated to be as common as metabolic syndrome in western population (23 % occurrence). Fat in the liver is called ectopic fat, which is triacylglycerols within the cells of non-adipose tissue. Serum alanine aminotransferase (ALT) values correlate positively with liver fat proportions, and increased activity of ALT predicts type 2 diabetes independently from obesity. Berries, high in natural bioactive compounds, have indicated the potential to reduce the risk of obesity-related diseases. Ectopic fat induces common endocrine excretion of adipose tissue resulting in the overproduction of inflammatory markers, which further induce insulin resistance by multiple mechanisms. Insulin resistance inducing hyperinsulinemia and lipolysis in adipocytes increases the concentration of free fatty acids and consequently causes further fat accumulation in hepatocytes. Polyphenolic fractions of berries have been shown to reverse inflammatory reaction cascades in in vitro and animal studies, and moreover to decrease ectopic fat accumulation. The aim of this thesis was to explore the role of northern berries in obesity-related diseases. The absorption and metabolism of selected berry polyphenols, flavonol glycosides and anthocyanins, was investigated in humans, and metabolites of the studied compounds were identified in plasma and urine samples (I, II). Further, the effects of berries on the risk factors of metabolic syndrome were studied in clinical intervention trials (III, IV), and the different fractions of sea buckthorn berry were tested for their ability to reduce postprandial glycemia and insulinemia after high-glucose meal in a postprandial study with humans (V). The marked impact of mixed berries on plasma ALT values (III), as well as indications of the positive effects of sea buckthorn, its fractions and bilberry on omental adiposity and adhesion molecules (IV) were observed. In study V, sea buckthorn and its polyphenol fractions had a promising effect on potprandial metabolism after high-glucose meal. In the literature review, the possible mechanisms behind the observed effects have been discussed with a special emphasis on ectopic fat accumulation. The literature review indicated that especially tannins and flavonoids have shown potential in suppressing diverse reaction cascades related to systemic inflammation, ectopic fat accumulation and insulin resistance development.

Studies on 68Ga-Based Agents for PET Imaging of Cancer and Inflammation

Studies on ⁶⁸Ga-Based Agents for PET Imaging of Cancer and Inflammation Positron emission tomography (PET) is based on the use of radiolabeled agents and facilitates in vivo imaging of biological processes, such as cancer. Because the detection of cancer is demanding and is often obscured by inflammation, there is a demand for better PET imaging agents. The aim was to preliminarily evaluate new PET agents for imaging cancer and inflammation using experimental models. ⁶⁸Ga-chloride and peptides, ⁶⁸Ga-labeled through 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), targeting matrix metalloproteinase-9 (MMP-9) were tested for tumor imaging. In addition, a ⁶⁸Ga-DOTA-conjugated peptide targeting vascular adhesion protein-1 (VAP-1), was tested for inflammation imaging. The ⁶⁸Ga-based imaging agents described here showed potential features by passing the essential in vitro tests, proceeding further to preclinical in vivo evaluation and being able to visualize the target. The target uptake and target-to-background ratios of ⁶⁸Ga-based agents were, however, not optimal. ⁶⁸Ga-chloride showed slow clearance caused by its binding to blood transferrin. In the case of ⁶⁸Ga-DOTA-peptides low in vivo stability and/or low lipophilicity led to too rapid blood clearance and urinary excretion. The properties of ⁶⁸Ga-labeled peptides are modifiable, as shown with matrix metalloproteinase-9 targeting ligands. In the conclusion of this PhD thesis, ⁶⁸Ga-based agents for PET imaging of cancer and inflammation could be applied in the development of drugs, earlier diagnostics and following-up of the efficacy of therapies.

Pharmacokinetics of Oxycodone and Paracetamol in the Elderly. Clinical Pharmacokinetic Study on Orthopaedic Surgical Patients and Healthy Volunteers

The proportion of elderly people over 65 years of age in Finland is expected to grow to over 25% by the 2025. It has been estimated that elderly people today consume nearly 40% of all drugs. Age brings about number of physiological changes that may affect the disposition, metabolism and excretion of drugs. The function of heart, lungs, liver and kidneys decreases even in healthy people, as they get older. The proportion of total body water decreases and the relative fat percentage increases. Also several other factors such as concurrent diseases, concomitant medication and nutritional factors have an effect on drug therapy in elderly. Age increases the risk of adverse drug reactions, which most often are dose-dependent. Despite all this there are not enough studies involving the elderly people and the elderly are most often excluded from clinical trials. Oxycodone is a strong opioid analgesic, which is used to treat moderate or severe pain. Paracetamol is a widely used nonopioid analgesic, which has become popular in the treatment of pain in many patient groups. In this series of studies the pharmacokinetics of oral and intravenous oxicodone as well as intravenous paracetamol in the elderly and young adult patients were investigated. Also a study investigating the interaction of oral antibiotic clarityhromycin, a known cytochrome P450 (CYP) 3A4 inhibitor, with oxycodone pharmacokinetics and pharmacodynamics in elderly and young healthy volunteers was carried out. The pharmacokinetics of oxycodone showed a clear age depency. Patients over 70 years had 50-80% higher mean exposure to oral oxycodone and a twofold greater plasma concentration than young adults 12 h after ingestion of the drug. Elderly patients had 40-80% greater exposure to intravenous oxycodone and patients over 80 years had over twofold greater plasma concentrations 8 h post dose than the young adults. The elderly patients had also greater exposure to intra venous paracetamol compared to young adults. Clarithromycin increased the exposure to oral oxycodone in both young and elderly volunteers. The elderly had marked interindividual variation in the pharmacokinetics and pharmacodynamics when clarithromycin was given concomitantly with oxycodone. Because the pharmacokinetics of oxycodone and intravenous paracetamol depend on the age of the subject, it is important to titrate the analgesic dose individually in the elderly.

Control of ammonia emissions in naturally ventilated dairy cattle facilities in Portugal

We conducted a study of the processes associated to NH3 emission in naturally ventilated dairy cattle facilities, having described factors that regulate NH3 emission, as well as methodologies for measuring these emissions at these facilities. Appropriate techniques to mitigate NH3 emission in facilities located in regions with warm climates were also identified. The most effective mitigation techniques with simple implementation include strategies associated to: (i) installation design and flooring, which lead to reduced emissions, (ii) excreta pre-excretion, namely the use of diets with optimized crude protein content and increased milk production at farm level; and (iii) excreta post-excretion, particularly by changing the conditions of environmental monitoring within the premises, practice introduction or additive application in the management of excreta deposited on floors.

Comparison of two methods for glucocorticoid evaluation in maned wolves

Analysis of faecal glucocorticoid metabolites provides a powerful noninvasive tool for monitoring adrenocortical activity in wild animals. However, differences regarding the metabolism and excretion of these substances make a validation for each species and sex investigated obligatory. Although maned wolves (Chrysocyon brachyurus) are the biggest canids in South America, their behaviour and physiology are poorly known and they are at risk in the wild. Two methods for measuring glucocorticoid metabolites in maned wolves were validated: a radio- and an enzyme immunoassay. An ACTH challenge was used to demonstrate that changes in adrenal function are reflected in faecal glucocorticoid metabolites. Our results suggest that both methods enable a reliable assessment of stress hormones in maned wolves avoiding short-term rises in glucocorticoid concentrations due to handling and restraint. These methods can be used as a valuable tool in studies of stress and conservation in this wild species.

Identification and application of bile metabolites to assess the exposure of fish to pharmaceuticals in the environment

Thousands of tons of pharmaceuticals are consumed yearly worldwide. Due to the continuous and increasing consumption and their incomplete elimination in wastewater treatment plants (WWTP), pharmaceuticals and their metabolites can be detected in receiving waters, although at low concentrations (ng to low μg/L). As bioactive molecules the presence of pharmaceuticals in the aquatic environment must be considered potentially hazardous for the aquatic organisms. In this thesis, the biotransformation and excretion of pharmaceuticals in fish was studied. The main biotransformation pathways of three anti‐inflammatory drugs, diclofenac, naproxen and ibuprofen, in rainbow trout were glucuronidation and taurine conjugation of the parent compounds and their phase I metabolites. The same metabolites were present in fish bile in aquatic exposures as in fish dosed with intraperitoneal injection. Higher bioconcentration factor in bile (BCFbile) was found for ibuprofen when compared to diclofenac and naproxen. Laboratory exposure studies were followed by a study of uptake of pharmaceuticals in a wild fish population living in lake contaminated with WWTP effluents. Of the analyzed 17 pharmaceuticals and six phase I metabolites, only diclofenac, naproxen and ibuprofen was present in bream and roach bile. It was shown, that diclofenac, naproxen and ibuprofen excreted by the liver can be found in rainbow trout and in two native fish species living in the receiving waters. In the bream and roach bile, the concentrations of diclofenac, naproxen and ibuprofen were roughly 1000 times higher than those found in the lake water, while in the laboratory exposures, the bioconcentration of the compounds and their metabolites in rainbow trout bile were at the same level as in wild fish or an order of magnitude higher. Thus, the parent compounds and their metabolites in fish bile can be used as a reliable biomarker to monitor the exposure of fish to environmental pharmaceuticals present in water receiving discharges from WWTPs.

Effects of cytochrome P450 enzyme inhibitors and inducers on the metabolism of S-ketamine

The human body eliminates foreign compounds primarily by metabolizing them to hydrophilic forms to facilitate effective excretion through the kidneys. Cytochrome P450 (CYP) enzymes in the liver and intestine contribute to the metabolism of many drugs. Pharmacokinetic drugdrug interactions occur if the activity of CYPs are inhibited or induced by another drug. Prescribing multiple drugs to the improve effectiveness of therapy or to treat coexisting diseases is a common practice in clinical medicine. Polypharmacy predisposes patients to adverse effects because of the profound unpredictability in CYP enzymatic-mediated drug metabolism. S-ketamine is a phencyclidine derivative which functions as an antagonist of the N-methyl-Daspartate (NMDA) receptor in the central nervous system. It is a unique anaesthetic producing “dissociative anaesthesia” in high doses and analgesia in low doses. Studies with human liver microsomes suggest that ketamine is metabolized primarily via CYP3A4 and CYP2B6 enzymes. In this thesis, in healthy volunteers, randomized and controlled cross-over studies were conducted to investigate the effects of different CYP inducers and inhibitors on the pharmacokinetics and pharmacodynamics of oral and intravenous S-ketamine. The plasma concentrations of ketamine and its metabolite, norketamine, were determined at different timepoints over a 24 hour period. Other pharmacodynamic variables were examined for 12 hours. Results of these studies showed that the inhibition of the CYP3A4 pathway by clarithromycin or grapefruit juice increased the exposure to oral S-ketamine by 2.6- and 3.0-fold. Unexpectedly, CYP3A4 inhibition by itraconazole caused no significant alterations in the plasma concentrations of oral S-ketamine. CYP3A4 induction by St. John´s wort or rifampicin decreased profoundly the concentrations of oral S-ketamine. However, after rifampicin, there were no significant differences in the plasma concentrations of S-ketamine when it was administered intravenously. This demonstrated that rifampicin inhibited the metabolism of Sketamine at the intestinal level. When CYP2B6 was inhibited by ticlopidine, there was a 2.4- fold increase in the exposure of S-ketamine. These studies demonstrated that low dose oral Sketamine is metabolized both via CYP3A4 and CYP2B6 pathways. The concomitant use of drugs that affect CYP3A4 or CYP2B6, during oral S-ketamine treatment, may cause clinically significant drug-drug interactions.

Influence of surface functionalization on the behavior of silica nanoparticles in biological systems

Personalized nanomedicine has been shown to provide advantages over traditional clinical imaging, diagnosis, and conventional medical treatment. Using nanoparticles can enhance and clarify the clinical targeting and imaging, and lead them exactly to the place in the body that is the goal of treatment. At the same time, one can reduce the side effects that usually occur in the parts of the body that are not targets for treatment. Nanoparticles are of a size that can penetrate into cells. Their surface functionalization offers a way to increase their sensitivity when detecting target molecules. In addition, it increases the potential for flexibility in particle design, their therapeutic function, and variation possibilities in diagnostics. Mesoporous nanoparticles of amorphous silica have attractive physical and chemical characteristics such as particle morphology, controllable pore size, and high surface area and pore volume. Additionally, the surface functionalization of silica nanoparticles is relatively straightforward, which enables optimization of the interaction between the particles and the biological system. The main goal of this study was to prepare traceable and targetable silica nanoparticles for medical applications with a special focus on particle dispersion stability, biocompatibility, and targeting capabilities. Nanoparticle properties are highly particle-size dependent and a good dispersion stability is a prerequisite for active therapeutic and diagnostic agents. In the study it was shown that traceable streptavidin-conjugated silica nanoparticles which exhibit a good dispersibility could be obtained by the suitable choice of a proper surface functionalization route. Theranostic nanoparticles should exhibit sufficient hydrolytic stability to effectively carry the medicine to the target cells after which they should disintegrate and dissolve. Furthermore, the surface groups should stay at the particle surface until the particle has been internalized by the cell in order to optimize cell specificity. Model particles with fluorescently-labeled regions were tested in vitro using light microscopy and image processing technology, which allowed a detailed study of the disintegration and dissolution process. The study showed that nanoparticles degrade more slowly outside, as compared to inside the cell. The main advantage of theranostic agents is their successful targeting in vitro and in vivo. Non-porous nanoparticles using monoclonal antibodies as guiding ligands were tested in vitro in order to follow their targeting ability and internalization. In addition to the targeting that was found successful, a specific internalization route for the particles could be detected. In the last part of the study, the objective was to clarify the feasibility of traceable mesoporous silica nanoparticles, loaded with a hydrophobic cancer drug, being applied for targeted drug delivery in vitro and in vivo. Particles were provided with a small molecular targeting ligand. In the study a significantly higher therapeutic effect could be achieved with nanoparticles compared to free drug. The nanoparticles were biocompatible and stayed in the tumor for a longer time than a free medicine did, before being eliminated by renal excretion. Overall, the results showed that mesoporous silica nanoparticles are biocompatible, biodegradable drug carriers and that cell specificity can be achieved both in vitro and in vivo.

Correlations between ANP concentrations in atria, plasma and cerebral structures and sodium chloride preference in Wistar rats

We determined whether ANP (atrial natriuretic peptide) concentrations, measured by radioimmunoassay, in the ANPergic cerebral regions involved in regulation of sodium intake and excretion and pituitary gland correlated with differences in sodium preference among 40 Wistar male rats (180-220 g). Sodium preference was measured as mean spontaneous ingestion of 1.5% NaCl solution during a test period of 12 days. The relevant tissues included the olfactory bulb (OB), the posterior and anterior lobes of the pituitary gland (PP and AP, respectively), the median eminence (ME), the medial basal hypothalamus (MBH), and the region anteroventral to the third ventricle (AV3V). We also measured ANP content in the right (RA) and left atrium (LA) and plasma. The concentrations of ANP in the OB and the AP were correlated with sodium ingestion during the preceding 24 h, since an increase of ANP in these structures was associated with a reduced ingestion and vice-versa (OB: r = -0.3649, P<0.05; AP: r = -0.3291, P<0.05). Moreover, the AP exhibited a correlation between ANP concentration and mean NaCl intake (r = -0.4165, P<0.05), but this was not the case for the OB (r = 0.2422). This suggests that differences in sodium preference among individual male rats can be related to variations of AP ANP level. Earlier studies indicated that the OB is involved in the control of NaCl ingestion. Our data suggest that the OB ANP level may play a role mainly in day-to-day variations of sodium ingestion in the individual rat

Does plasma ANP participate in natriuresis induced by a-MSH?

a-Melanocyte-stimulating hormone (a-MSH; 0.6 and 3 nmol) microinjected into the anteroventral region of the third ventricle (AV3V) induced a significant increase in diuresis without modifying natriuresis or kaliuresis. Intraperitoneal (ip) injection of a-MSH (3 and 9.6 nmol) induced a significant increase in urinary sodium, potassium and water excretion. Intraperitoneal (3 and 4.8 nmol) or iv (3 and 9.6 nmol) administration of a-MSH did not induce any significant changes in plasma atrial natriuretic peptide (ANP), suggesting that the natriuresis, kaliuresis and diuresis induced by the systemic action of a-MSH can be dissociated from the increase in plasma ANP. These preliminary results suggest that a-MSH may be involved in a g-MSH-independent mechanism of regulation of hydromineral metabolism

Calciuria and preeclampsia

Urinary calcium excretion has been reported to be diminished in preeclampsia. The objective of the present study was to determine urinary calcium excretion in pregnant patients with chronic arterial hypertension (CAH) and preeclampsia (PE), and in normotensive patients (N). Forty-four pregnant patients (gestional age, 20-42 weeks; 18 CAH, 17 PE, 9 N) were evaluated for calciuria, proteinuria, plasma uric acid and blood pressure. Patients with PE (82 ± 15.1 mg/24 h) showed significantly lower calciuria (P<0.05) than the group with CAH (147 ± 24.9 mg/24 h) and the N group (317 ± 86.0 mg/24 h) (P<0.05, Student t-test). Plasma uric acid was significantly higher in the PE group (6.1 ± 0.38 mg/dl) than the CAH group (5.0 ± 0.33 mg/dl; P<0.05), which also presented higher proteinuria levels, although the difference was not statistically significant. Diastolic and systolic blood pressure did not differ between the PE (164 ± 105 mmHg) and CAH (164 ± 107 mmHg) groups. Calciuria was significantly lower in the group with preeclampsia than in the group with chronic arterial hypertension. We conclude that calciuria can be a further factor for identifying preeclampsia