Dramatically decreased high density lipoprotein cholesterol, increased remnant clearance, and insulin hypersensitivity in apolipoprotein A-II knockout mice suggest a complex role for apolipoprotein A-II in atherosclerosis susceptibility

Apolipoprotein (apo) A-II is the second most abundant apolipoprotein in high density lipoprotein (HDL). To study its role in lipoprotein metabolism and atherosclerosis susceptibility, apo A-II knockout mice were created. Homozygous knockout mice had 67% and 52% reductions in HDL cholesterol levels in the fasted and fed states, respectively, and HDL particle size was reduced. Metabolic turnover studies revealed the HDL decrease to be due to both decreased HDL cholesterol ester and apo A-I transport rate and increased HDL cholesterol ester and apo A-I fractional catabolic rate. The apo A-II deficiency trait was bred onto the atherosclerosis-prone apo E-deficient background, which resulted in a surprising 66% decrease in cholesterol levels due primarily to decreased atherogenic lipoprotein remnant particles. Metabolic turnover studies indicated increased remnant clearance in the absence of apo A-II. Finally, apo A-II deficiency was associated with lower free fatty acid, glucose, and insulin levels, suggesting an insulin hypersensitivity state. In summary, apo A-II plays a complex role in lipoprotein metabolism, with some antiatherogenic properties such as the maintenance of a stable HDL pool, and other proatherogenic properties such as decreasing clearance of atherogenic lipoprotein remnants and promotion of insulin resistance.

The use of a change in gentamicin clearance as an early predictor of gentamicin-induced nephrotoxicity

A retrospective review was undertaken in 744 patients who were dose-individualized with gentamicin once daily to evaluate a change in gentamicin clearance as a potential predictor of nephrotoxicity. The definition of nephrotoxicity was chosen to be a change in creatinine clearance greater than 20%. Similarly, a change in gentamicin clearance of greater than 20% was also considered a possible index of nephrotoxicity. Four criteria were developed to assess the usefulness of gentamicin clearance as a predictor of nephrotoxicity. Following the application of the inclusion/exclusion criteria, 132 patients were available for the analysis. The sensitivity, specificity, positive predictive value, and negative predictive value were assessed for each of the criteria. Receiver operating characteristic (ROC) curves were produced to determine if an optimum value in the change of gentamicin clearance could be found to maximize sensitivity and specificity. The overall incidence of nephrotoxicity based on a decrease in creatinine clearance by 20% or more was 3.8%. Women were overrepresented in the nephrotoxic group [71.4% versus 40.1% (P = 0.0025)]. Patients with nephrotoxicity had statistically longer treatment periods, increased cumulative dose, and more dosing predictions (P < 0.05 in each case). The sensitivity of the criteria ranged from 43 to 46%, and specificity ranged from 93 to 99%. The positive and negative predictive values ranged from 63 to 94% and 86 to 89%, respectively. In those patients in whom nephrotoxicity was predicted from a change in gentamicin clearance, this change occurred on average 3 days before the change in creatinine clearance (P < 0.05). A change in gentamicin clearance to predict nephrotoxicity may be a useful addition to current monitoring methods, although it is not the complete answer.

Ion-trapping, microsomal binding, and unbound drug distribution in the hepatic retention of basic drugs

This study investigated the relative contribution of ion-trapping, microsomal binding, and distribution of unbound drug as determinants in the hepatic retention of basic drugs in the isolated perfused rat liver. The ionophore monensin was used to abolish the vesicular proton gradient and thus allow an estimation of ion-trapping by acidic hepatic vesicles of cationic drugs. In vitro microsomal studies were used to independently estimate microsomal binding and metabolism. Hepatic vesicular ion-trapping, intrinsic elimination clearance, permeability-surface area product, and intracellular binding were derived using a physiologically based pharmacokinetic model. Modeling showed that the ion-trapping was significantly lower after monensin treatment for atenolol and propranolol, but not for antipyrine. However, no changes induced by monensin treatment were observed in intrinsic clearance, permeability, or binding for the three model drugs. Monensin did not affect binding or metabolic activity in vitro for the drugs. The observed ion-trapping was similar to theoretical values estimated using the pHs and fractional volumes of the acidic vesicles and the pK(a) values of drugs. Lipophilicity and pK(a) determined hepatic drug retention: a drug with low pK(a) and low lipophilicity (e.g., antipyrine) distributes as unbound drug, a drug with high pK(a) and low lipophilicity (e.g., atenolol) by ion-trapping, and a drug with a high pK(a) and high lipophilicity (e.g., propranolol) is retained by ion-trapping and intracellular binding. In conclusion, monensin inhibits the ion-trapping of high pK(a) basic drugs, leading to a reduction in hepatic retention but with no effect on hepatic drug extraction.

Using urea dilution to standardise components of pleural and bronchoalveolar lavage fluids in the dog

AIM: To develop a technique to estimate the volume of epithelial lining fluid (ELF) obtained during bronchoalveolar lavage (BAL) and pleural lavage (PL) in the dog, using the urea dilution method. METHODS: BAL and PL fluids were obtained by saline lavage of pulmonary and pleural cavities of nine clinically healthy mixed-breed dogs immediately after euthanasia. Cell counts in the BAL and PL fluids were measured using standard techniques. The concentration of ELF in each lavage fluid was calculated from the relative concentration of urea in plasma and in each type of lavage fluid. Cell counts in ELF were then calculated. RESULTS: There were substantially higher cell counts in ELF compared to BAL or PF fluid. However, nucleated cell counts in ELF could not be predicted from cell counts in BAL or PL fluid. CONCLUSIONS AND CLINICAL RELEVANCE: These results suggest that accurate assessment of cellular or non-cellular components in lavage fluids should include a calculation of the proportion of ELF recovered, using a method such as urea dilution.

Relationship of body condition score and blood urea and ammonia to pregnancy in Italian Mediterranean buffaloes

The relationship of body condition score ( BCS) and blood urea and ammonia to pregnancy outcome was examined in Italian Mediterranean Buffalo cows mated by AI. The study was conducted on 150 buffaloes at 145 +/- 83 days in milk that were fed a diet comprising 14.8% crude protein, 0.9 milk forage units . kg(-1) dry matter and a non- structural carbohydrate/ crude protein ratio of 2.14. The stage of the oestrous cycle was synchronised by the Ovsynch- TAI programme and blood urea and ammonia levels were assessed on the day of AI. Energy corrected milk ( ECM) production and BCS were recorded bi- weekly. The pregnancy risk was 46.7% and was slightly lower in buffaloes with BCS < 6.0 and BCS > 7.5. There were no significant differences in ECM, urea and ammonia between pregnant and non- pregnant buffaloes. However, pregnancy outcome was higher ( P = 0.02) in buffaloes with blood urea < 6.83 mmol . L-1. The likelihood of pregnancy for buffaloes with low urea blood level was 2.6 greater than for high urea level and exposure to a high urea level lowered the probability of pregnancy by about 0.25. The findings indicate that buffaloes are similar to cattle and increased blood levels of urea are associated with reduced fertility when animals are mated by AI.

The effect of different levels of dietary crude protein on urea metabolism of rusa deer (Cervus timorensis)

Two experiments were conducted to measure urea recycling and rumen flow dynamics in young rusa deer fed low (LP) or high (HP) protein diets. Pool size and flux rate of labelled urea. into and out of the blood pool were measured using single intravenous (i.v.) injection solutions containing [C-14] - and [N-15]-urea. A curve peeling technique was used to fit the enrichment of N-15 or specific radioactivity (SRA) of C-14 to exponential equations. Body urea-N pool size was significantly greater (P < 0.05) when a HP, compared to a LP diet, was fed. Urea space, expressed as a percent of live weight, total flux rate of urea through the blood pool and the irreversible loss of urea was similar for both diets. The mean (+/- S.E.M.) concentration of plasma urea-N was greater when animals were fed the HP diet compared to the LP diet (2 1.1 +/- 0.3 versus 14.4 +/- 1.4 mg/100 ml, respectively). Voluntary feed intake and digestibility of dietary components were also measured. Daily dry matter intakes were not affected by the crude protein (CP) content of the diet, although apparent DM digestibility was significantly greater for HP diet fed in both experiments. An intraruminal infusion of CrEDTA was used to determine rumen flow dynamics. Ruminal mean retention time, relative net outflow rate of water and passage rate constant (k(w)) were significantly greater (P < 0.05) when the HP diet was fed compared to the LP diet. The extent of urea metabolism and flux rates of urea between the blood and secondary pools appear similar to those previously reported for other ruminants fed diets contrasting in CP content. (c) 2005 Elsevier B.V. All rights reserved.

Investigations of the site and mechanisms of drug-induced reductions in the reabsorption of divalent cations by the kidney

Disturbances in electrolyte homeostasis are a frequent adverse side-effect of the administration of aminoglycoside antibiotics such as gentamicin, and the antineoplastic agent cis-platinum. The aims of this work were to further elucidate the site(s) and mechanism(s) by which these drugs may produce disturbances in the renal reabsorption of calcium and magnesium. These investigations were undertaken using a range of in vivo and in vitro techniques and models. Initially, a series of in vivo studies was conducted to delineate aspects of the acute and chronic effects of both drugs on renal electrolyte handling and to select and evaluate an appropriate animal model: subsequent investigations were focused on gentamicin. In a study of the acute and chronic effects of cis-platinum administration, there were pronounced acute changes in a variety of indices of nephrotoxic injury, including electrolyte excretion. Most effects resolved but there were chronic increases in the urinary excretion of calcium and magnesium. The renal response of three strains of rat (Fischer 344, Sprague-Dawley (SD), and Wistar) to a ranges of doses of gentamicin was also investigated. Drug administration produced substantially different responses between strains, in particular marked differences in calcium and magnesium excretion. The results suggested that the SD rat was an appropriately sensitive strain for use in further investigations. Acute infusion of gentamicin in the anaesthetised SD rat produced rapid, substantial increases in the fractional excretion of calcium and magnesium, while sodium and potassium output were unaffected, confirming previous results of similar experiments using F344 rats. Studies using lithium clearance measurements in the anaesthetised SD rat were undertaken to investigate the effects of gentamicin on proximal tubular calcium reabsorption. Lithium clearance was unaffected by acute gentamicin infusion, suggesting that the site of acute gentamicin-induced hypercalciuria may not be located in the proximal tubule. Inhibition of Ca2+ ATPase activity was investigated as a potential mechanism by which calcium reabsorption could be affected after aminoglycoside administration. In vitro, both Ca2+ ATPase and Na+/K+ ATPase activity could be similarly inhibited by the presence of aminoglycosides, in a dose-related manner. Whilst inhibition of Na+/K+ ATPase could be demonstrated biochemically after in vivo administration of gentamicin, there were no concurrent effects on Ca2+ ATPase activity, suggesting that inhibition of Ca2+ ATPase activity is unlikely to be a primary mechanism of aminoglycoside-induced reductions of calcium reabsorption. Histochemical studies could not discern inhibition of either Na+/K+ ATPase or Ca2+ ATPase activity after in vivo administration of gentamicin. Selection of renal cell lines for further investigative in vitro studies on the mechanisms of altered cation reabsorption was considered using MTT (3-(4,5,-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Neutral Red cytotoxicity assays. The ability of LLC-PK1 and LLC-RK1 cell lines to correctly rank a series of nephrotoxic compounds with their known nephrotoxic potency in vivo was studied. Using these cell lines grown on semi-permeable inserts, alterations in the paracellular transport of 45Ca was investigated as a possible mechanism by which gentamicin could alter calcium reabsorption in vivo. Short term exposure (I h) of LLC-RK1 cells to gentamicin, via both cell surfaces, resulted in a reduction in paracellular permeability to both transepithelial 3H-mannitol and 45Ca fluxes. When LLC-RK1 cells were exposed via the apical surface only, similar dose-related reductions were seen to those observed when cells were exposed to the drug from both sides. Short-term basal exposure to gentamicin appeared to contribute less to the observed reductions in 3H-mannitol and 45Ca fluxes. Experiments investigating transepithelial movement of 45Ca and 3H-mannitol on LLC-PK1 cells after acute gentamicin exposure were inconclusive. Longer exposure (48 h) to gentamicin caused an increase in the permeability of the monolayer and a consequent increase in transepithelial 45Ca flux in the LLC-RK1 cell line; increases in permeability of LLC-PK1 cells to 45Ca and 3H-mannitol were not apparent under the same conditions. The site and mechanism at which gentamicin, in particular, alters calcium reabsorption cannot be definitively described from these studies. However, indirect evidence from lithium clearance studies suggests that the site of the lesion is unlikely to be located in the proximal tubule. The mechanism by which gentamicin exposure alters calcium reabsorption may be by reducing paracellular permeability to calcium rather than by altering active calcium transport processes.

Population pharmacokinetic and pharmacogenetic analysis of 6-mercaptopurine in paediatric patients with acute lymphoblastic leukaemia

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • The cytotoxic effects of 6-mercaptopurine (6-MP) were found to be due to drug-derived intracellular metabolites (mainly 6-thioguanine nucleotides and to some extent 6-methylmercaptopurine nucleotides) rather than the drug itself. • Current empirical dosing methods for oral 6-MP result in highly variable drug and metabolite concentrations and hence variability in treatment outcome. WHAT THIS STUDY ADDS • The first population pharmacokinetic model has been developed for 6-MP active metabolites in paediatric patients with acute lymphoblastic leukaemia and the potential demographic and genetically controlled factors that could lead to interpatient pharmacokinetic variability among this population have been assessed. • The model shows a large reduction in interindividual variability of pharmacokinetic parameters when body surface area and thiopurine methyltransferase polymorphism are incorporated into the model as covariates. • The developed model offers a more rational dosing approach for 6-MP than the traditional empirical method (based on body surface area) through combining it with pharmacogenetically guided dosing based on thiopurine methyltransferase genotype. AIMS - To investigate the population pharmacokinetics of 6-mercaptopurine (6-MP) active metabolites in paediatric patients with acute lymphoblastic leukaemia (ALL) and examine the effects of various genetic polymorphisms on the disposition of these metabolites. METHODS - Data were collected prospectively from 19 paediatric patients with ALL (n = 75 samples, 150 concentrations) who received 6-MP maintenance chemotherapy (titrated to a target dose of 75 mg m−2 day−1). All patients were genotyped for polymorphisms in three enzymes involved in 6-MP metabolism. Population pharmacokinetic analysis was performed with the nonlinear mixed effects modelling program (nonmem) to determine the population mean parameter estimate of clearance for the active metabolites. RESULTS - The developed model revealed considerable interindividual variability (IIV) in the clearance of 6-MP active metabolites [6-thioguanine nucleotides (6-TGNs) and 6-methylmercaptopurine nucleotides (6-mMPNs)]. Body surface area explained a significant part of 6-TGNs clearance IIV when incorporated in the model (IIV reduced from 69.9 to 29.3%). The most influential covariate examined, however, was thiopurine methyltransferase (TPMT) genotype, which resulted in the greatest reduction in the model's objective function (P < 0.005) when incorporated as a covariate affecting the fractional metabolic transformation of 6-MP into 6-TGNs. The other genetic covariates tested were not statistically significant and therefore were not included in the final model. CONCLUSIONS - The developed pharmacokinetic model (if successful at external validation) would offer a more rational dosing approach for 6-MP than the traditional empirical method since it combines the current practice of using body surface area in 6-MP dosing with a pharmacogenetically guided dosing based on TPMT genotype.

The role of apoptotic cell clearance in a high-lipid environment:links to ageing

Individuals within the aged population show an increased susceptibility to infection, implying a decline in immune function, a phenomenon known as immunosenescence. Paradoxically, an increase in autoimmune disease, such as rheumatoid arthritis, is also associated with ageing, therefore some aspects of the immune system appear to be inappropriately active in the elderly. The above evidence suggests inappropriate control of the immune system as we age. Macrophages, and their precursors monocytes, play a key role in control of the immune system. They play an important role in host defence in the form of phagocytosis, and also link the innate and adaptive immune system via antigen presentation. Macrophages also have a reparative role, as professional phagocytes of dead and dying cells. Clearance of apoptotic cells by macrophages has also been shown to directly influence immune responses in an anti-inflammatory manner. Inappropriate control of macrophage function with regards to dead cell clearance may contribute to pathology as we age. The aims of this study were to assess the impact of lipid treatment, as a model of the aged environment, on the ability of macrophages to interact with, and respond to, apoptotic cells. Using a series of in vitro cell models, responses of macrophages (normal and lipid-loaded) to apoptotic macrophages (normal and lipid-loaded) were investigated. Monocyte recruitment to apoptotic cells, a key process in resolving inflammation, was assessed in addition to cytokine responses. Data here shows, for the first time, that apoptotic macrophages (normal and lipid-loaded) induce inflammation in human monocyte-derived macrophages, a response that could drive inflammation in age-associated pathology e.g. atherosclerosis. Monoclonal antibody inhibition studies suggest the classical chemokine CX3CL1 may be involved in monocyte recruitment to apoptotic macrophages, but not apoptotic foam cells, therefore differential clearance strategies may be employed following lipid-loading. CD14, an important apoptotic cell tethering receptor, was not found to have a prominent role in this process, whilst the role for ICAM-3 remains unclear. Additionally, a small pilot study using macrophages from young (<25) and mid-life (>40) donors was undertaken. Preliminary data was gathered to assess the ability of primary human monocyte-derived macrophages, from young and mid-life donors, to interact with, and respond to, apoptotic cells. MØ from mid-life individuals showed no significant differences in their ability to respond to immune modulation by apoptotic cells compared to MØ from young donors. Larger cohorts would be required to investigate whether immune modulation of MØ by apoptotic cells contribute to inflammatory pathology throughout ageing.

Measurement of apoptotic cell clearance in vitro

The phagocytic clearance of apoptotic cells is a highly efficient and nonphlogistic process in vivo. Research in this area has been limited, at least in part, by technical difficulties associated with the techniques used in the detailed study of apoptotic cell clearance mechanisms. This chapter provides details of methods that may be used to study apoptotic cell clearance in vitro. Such methods have been used successfully to identify phagocyte-associated or apoptotic cell-associated molecular players in the recognition process.

On Integral Means for Fractional Calculus Operators of Multivalent Functions

2000 Mathematics Subject Classification: Primary 30C45, Secondary 26A33, 30C80

Nonexistence Results of Solutions of Semilinear Differential Inequalities with Temperal Fractional Derivative on the Heinsenberg Group

2000 Mathematics Subject Classification: 26A33, 33C60, 44A15, 35K55

Stochastic Solution of a KPP-Type Nonlinear Fractional Differential Equation

Mathematics Subject Classification: 26A33, 76M35, 82B31

Well-Posedness of Diffusion-Wave Problem with Arbitrary Finite Number of Time Fractional Derivatives in Sobolev Spaces H^s

Mathematics Subject Classification 2010: 26A33, 33E12, 35S10, 45K05.

Fractional Derivatives in Spaces of Generalized Functions

MSC 2010: 26A33, 46Fxx, 58C05 Dedicated to 80-th birthday of Prof. Rudolf Gorenflo