Antinociceptive effects, metabolism and disposition of ketamine in ponies under target-controlled drug infusion

Ketamine is widely used as an anesthetic in a variety of drug combinations in human and veterinary medicine. Recently, it gained new interest for use in long-term pain therapy administered in sub-anesthetic doses in humans and animals. The purpose of this study was to develop a physiologically based pharmacokinetic (PBPk) model for ketamine in ponies and to investigate the effect of low-dose ketamine infusion on the amplitude and the duration of the nociceptive withdrawal reflex (NWR). A target-controlled infusion (TCI) of ketamine with a target plasma level of 1 microg/ml S-ketamine over 120 min under isoflurane anesthesia was performed in Shetland ponies. A quantitative electromyographic assessment of the NWR was done before, during and after the TCI. Plasma levels of R-/S-ketamine and R-/S-norketamine were determined by enantioselective capillary electrophoresis. These data and two additional data sets from bolus studies were used to build a PBPk model for ketamine in ponies. The peak-to-peak amplitude and the duration of the NWR decreased significantly during TCI and returned slowly toward baseline values after the end of TCI. The PBPk model provides reliable prediction of plasma and tissue levels of R- and S-ketamine and R- and S-norketamine. Furthermore, biotransformation of ketamine takes place in the liver and in the lung via first-pass metabolism. Plasma concentrations of S-norketamine were higher compared to R-norketamine during TCI at all time points. Analysis of the data suggested identical biotransformation rates from the parent compounds to the principle metabolites (R- and S-norketamine) but different downstream metabolism to further metabolites. The PBPk model can provide predictions of R- and S-ketamine and norketamine concentrations in other clinical settings (e.g. horses).

Capillary electrophoresis contributions to the hydromorphone metabolism in man

CE-ESI multistage IT-MS (CE-MS(n), n < or = 4) and computer simulation of fragmentation are demonstrated to be effective tools to detect and identify phase I and phase II metabolites of hydromorphone (HMOR) in human urine. Using the same CE conditions as previously developed for the analysis of urinary oxycodone and its metabolites, HMOR and its phase I metabolites produced by N-demethylation, 6-keto-reduction and N-oxidation and phase II conjugates of HMOR and its metabolites formed with glucuronic acid, glucose, and sulfuric acid could be detected in urine samples of a patient that were collected during a pharmacotherapy episode with daily ingestion of 48 mg of HMOR chloride. The CE-MS(n) data obtained with the HMOR standard, synthesized hydromorphol and hydromorphone-N-oxide, and CYP3A4 in vitro produced norhydromorphone were employed to identify the metabolites. This approach led to the identification of previously unknown HMOR metabolites, including HMOR-3O-glucide and various N-oxides, structures for which no standard compounds or mass spectra library data were available. Furthermore, the separation of alpha- and beta-hydromorphol, the stereoisomers of 6-keto-reduced HMOR, was achieved by CE in the presence of the single isomer heptakis(2,3-diacetyl-6-sulfato)-beta-CD. The obtained data indicate that the urinary excretion of alpha-hydromorphol is larger than that of beta-hydromorphol.

Capillary electrophoretic investigation of the enantioselective metabolism of propafenone by human cytochrome P-450 SUPERSOMES: Evidence for atypical kinetics by CYP2D6 and CYP3A4

An enantioselective CE method was used to identify the ability of CYP450 enzymes and their stereoselectivity in catalyzing the transformation of propafenone (PPF) to 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF). Using in vitro incubations with single CYP450 enzymes (SUPERSOMES), 5OH-PPF is shown to be selectively produced by CYP2D6 and N-dealkylation is demonstrated to be mediated by CYP2D6, CYP3A4, CYP1A2, and CYP1A1. For the elucidation of kinetic aspects of the metabolism with CYP2D6 and CYP3A4, incubations with individual PPF enantiomers and racemic PPF were investigated. With the exception of the dealkylation in presence of R-PPF only, which can be described by the Michaelis-Menten model, all CYP2D6-induced reactions were found to follow autoactivation kinetics. For CYP3A4, all NOR-PPF enantiomer formation rates as function of PPF enantiomer concentration were determined to follow substrate inhibition kinetics. The formation of NOR-PPF by the different enzymes is stereoselective and is reduced significantly when racemic PPF is incubated. Clearance values obtained for CYP3A4 dealkylation are stereoselective whereas those of CYP2D6 hydroxylation are not. This paper reports the first investigation of the PPF hydroxylation and dealkylation kinetics by the CYP2D6 enzyme and represents the first report in which enantioselective CE data provide the complete in vitro kinetics of metabolic steps of a drug.

A metabolomic approach to the metabolism of the areca nut alkaloids arecoline and arecaidine in the mouse

The areca alkaloids comprise arecoline, arecaidine, guvacoline, and guvacine. Approximately 600 million users of areca nut products, for example, betel quid chewers, are exposed to these alkaloids, principally arecoline and arecaidine. Metabolism of arecoline (20 mg/kg p.o. and i.p.) and arecaidine (20 mg/kg p.o. and i.p.) was investigated in the mouse using a metabolomic approach employing ultra-performance liquid chromatography-time-of-flight mass spectrometric analysis of urines. Eleven metabolites of arecoline were identified, including arecaidine, arecoline N-oxide, arecaidine N-oxide, N-methylnipecotic acid, N-methylnipecotylglycine, arecaidinylglycine, arecaidinylglycerol, arecaidine mercapturic acid, arecoline mercapturic acid, and arecoline N-oxide mercapturic acid, together with nine unidentified metabolites. Arecaidine shared six of these metabolites with arecoline. Unchanged arecoline comprised 0.3-0.4%, arecaidine 7.1-13.1%, arecoline N-oxide 7.4-19.0%, and N-methylnipecotic acid 13.5-30.3% of the dose excreted in 0-12 h urine after arecoline administration. Unchanged arecaidine comprised 15.1-23.0%, and N-methylnipecotic acid 14.8%-37.7% of the dose excreted in 0-12 h urine after arecaidine administration. The major metabolite of both arecoline and arecaidine, N-methylnipecotic acid, is a novel metabolite arising from carbon-carbon double-bond reduction. Another unusual metabolite found was the monoacylglyceride of arecaidine. What role, if any, that is played by these uncommon metabolites in the toxicology of arecoline and arecaidine is not known. However, the enhanced understanding of the metabolic transformation of arecoline and arecaidine should contribute to further research into the clinical toxicology of the areca alkaloids.

Urinary metabolite profiling reveals CYP1A2-mediated metabolism of NSC686288 (aminoflavone)

NSC686288 [aminoflavone (AF)], a candidate chemotherapeutic agent, possesses a unique antiproliferative profile against tumor cells. Metabolic bioactivation of AF by drug-metabolizing enzymes, especially CYP1A monooxygenases, has been implicated as an underlying mechanism for its selective cytotoxicity in several cell culture-based studies. However, in vivo metabolism of AF has not been investigated in detail. In this study, the structural identities of 13 AF metabolites (12 of which are novel) in mouse urine or from microsomal incubations, including three monohydroxy-AFs, two dihydroxy-AFs and their sulfate and glucuronide conjugates, as well as one N-glucuronide, were determined by accurate mass measurements and liquid chromatography-tandem mass spectrometry fragmentation patterns, and a comprehensive map of the AF metabolic pathways was constructed. Significant differences between wild-type and Cyp1a2-null mice, within the relative composition of urinary metabolites of AF, demonstrated that CYP1A2-mediated regioselective oxidation was a major contributor to the metabolism of AF. Comparisons between wild-type and CYP1A2-humanized mice further revealed interspecies differences in CYP1A2-mediated catalytic activity. Incubation of AF with liver microsomes from all three mouse lines and with pooled human liver microsomes confirmed the observations from urinary metabolite profiling. Results from enzyme kinetic analysis further indicated that in addition to CYP1A P450s, CYP2C P450s may also play some role in the metabolism of AF.

Effects of colostrum feeding and glucocorticoid administration on insulin-dependent glucose metabolism in neonatal calves

Colostrum feeding and glucocorticoid administration affect glucose metabolism and insulin release in calves. We have tested the hypothesis that dexamethasone as well as colostrum feeding influence insulin-dependent glucose metabolism in neonatal calves using the euglycemic-hyperinsulinemic clamp technique. Newborn calves were fed either colostrum or a milk-based formula (n=14 per group) and in each feeding group, half of the calves were treated with dexamethasone (30 microg/[kg body weight per day]). Preprandial blood samples were taken on days 1, 2, and 4. On day 5, insulin was infused for 3h and plasma glucose concentrations were kept at 5 mmol/L+/-10%. Clamps were combined with [(13)C]-bicarbonate and [6,6-(2)H]-glucose infusions for 5.5h (i.e., from -150 to 180 min, relative to insulin infusion) to determine glucose turnover, glucose appearance rate (Ra), endogenous glucose production (eGP), and gluconeogenesis before and at the end of the clamp. After the clamp liver biopsies were taken to measure mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC). Dexamethasone increased plasma glucose, insulin, and glucagon concentrations in the pre-clamp period thus necessitating a reduction in the rate of glucose infusion to maintain euglycemia during the clamp. Glucose turnover and Ra increased during the clamp and were lower at the end of the clamp in dexamethasone-treated calves. Dexamethasone treatment did not affect basal gluconeogenesis or eGP. At the end of the clamp, dexamethasone reduced eGP and PC mRNA levels, whereas mitochondrial PEPCK mRNA levels increased. In conclusion, insulin increased glucose turnover and dexamethasone impaired insulin-dependent glucose metabolism, and this was independent of different feeding.

Role of proton MR for the study of muscle lipid metabolism

1H-MR spectroscopy (MRS) of intramyocellular lipids (IMCL) became particularly important when it was recognized that IMCL levels are related to insulin sensitivity. While this relation is rather complex and depends on the training status of the subjects, various other influences such as exercise and diet also influence IMCL concentrations. This may open insight into many metabolic interactions; however, it also requires careful planning of studies in order to control all these confounding influences. This review summarizes various historical, methodological, and practical aspects of 1H-MR spectroscopy (MRS) of muscular lipids. That includes a differentiation of bulk magnetic susceptibility effects and residual dipolar coupling that can both be observed in MRS of skeletal muscle, yet affecting different metabolites in a specific way. Fitting of the intra- (IMCL) and extramyocellular (EMCL) signals with complex line shapes and the transformation into absolute concentrations is discussed. Since the determination of IMCL in muscle groups with oblique fiber orientation or in obese subjects is still difficult, potential improvement with high-resolution spectroscopic imaging or at higher field strength is considered. Fat selective imaging is presented as a possible alternative to MRS and the potential of multinuclear MRS is discussed. 1H-MRS of muscle lipids allows non-invasive and repeated studies of muscle metabolism that lead to highly relevant findings in clinics and patho-physiology.

Membrane microdialysis: Evaluation of a new method to assess splanchnic tissue metabolism

OBJECTIVE: Measuring peritoneal lactate concentrations could be useful for detecting splanchnic hypoperfusion. The aims of this study were to evaluate the properties of a new membrane-based microdialyzer in vitro and to assess the ability of the dialyzer to detect a clinically relevant decrease in splanchnic blood flow in vivo. DESIGN: A membrane-based microdialyzer was first validated in vitro. The same device was tested afterward in a randomized, controlled animal experiment. SETTING: University experimental research laboratory. SUBJECTS: Twenty-four Landrace pigs of both genders. INTERVENTIONS: In vitro: Membrane microdialyzers were kept in warmed sodium lactate baths with lactate concentrations between 2 and 8 mmol/L for 10-120 mins, and microdialysis lactate concentrations were measured repeatedly (210 measurements). In vivo: An extracorporeal shunt with blood reservoir and roller pump was inserted between the proximal and distal abdominal aorta, and a microdialyzer was inserted intraperitoneally. In 12 animals, total splanchnic blood flow (measured by transit time ultrasound) was reduced by a median 43% (range, 13% to 72%) by activating the shunt; 12 animals served as controls. MEASUREMENTS AND MAIN RESULTS: In vitro: The fractional lactate recovery was 0.59 (0.32-0.83) after 60 mins and 0.82 (0.71-0.87) after 90 mins, with no further increase thereafter. At 60 and 90 mins, the fractional recovery was independent of the lactate concentration. In vivo: Abdominal blood flow reduction resulted in an increase in peritoneal microdialysis lactate concentration from 1.7 (0.3-3.8) mmol/L to 2.8 (1.3-6.2) mmol/L (p = .006). At the same time, mesenteric venous-arterial lactate gradient increased from 0.1 (-0.2-0.8) mmol/L to 0.3 (-0.3 -1.8) mmol/L (p = .032), and mesenteric venous-arterial Pco2 gradients increased from 12 (8-19) torr to 21 (11-54) torr (p = .005). CONCLUSIONS: Peritoneal membrane microdialysis provides a method for the assessment of splanchnic ischemia, with potential for clinical application.

Influence of diurnal hyperosmotic loading on the metabolism and matrix gene expression of a whole-organ intervertebral disc model

It is generally agreed that the mechanical environment of intervertebral disc cells plays an important role in maintaining a balanced matrix metabolism. The precise mechanism by which the signals are transduced into the cells is poorly understood. Osmotic changes in the extracellular matrix (ECM) are thought to be involved. Current in-vitro studies on this topic are mostly short-term and show conflicting data on the reaction of disc cells subjected to osmotic changes which is partially due to the heterogenous and often substantially-reduced culture systems. The aim of the study was therefore to investigate the effects of cyclic osmotic loading for 4 weeks on metabolism and matrix gene expression in a full-organ intervertebral disc culture system. Intervertebral disc/endplate units were isolated from New Zealand White Rabbits and cultured either in iso-osmotic media (335 mosmol/kg) or were diurnally exposed for 8 hours to hyper-osmotic conditions (485 mosmol/kg). Cell viability, metabolic activity, matrix composition and matrix gene expression profile (collagen types I/II and aggrecan) were monitored using Live/Dead cell viability assay, tetrazolium reduction test (WST 8), proteoglycan and DNA quantification assays and quantitative PCR. The results show that diurnal osmotic stimulation did not have significant effects on proteoglycan content, cellularity and disc cell viability after 28 days in culture. However, hyperosmolarity caused increased cell death in the early culture phase and counteracted up-regulation of type I collagen gene expression in nucleus and annulus cells. Moreover, the initially decreased cellular dehydrogenase activity recovered with osmotic stimulation after 4 weeks and aggrecan gene down-regulation was delayed, although the latter was not significant according to our statistical criteria. In contrast, collagen type II did not respond to the osmotic changes and was down-regulated in both groups. In conclusion, diurnal hyper-osmotic stimulation of a whole-organ disc/endplate culture partially inhibits a matrix gene expression profile as encountered in degenerative disc disease and counteracts cellular metabolic hypo-activity.

Labeling the human skeleton with 41Ca to assess changes in bone calcium metabolism

Bone research is limited by the methods available for detecting changes in bone metabolism. While dual X-ray absorptiometry is rather insensitive, biochemical markers are subject to significant intra-individual variation. In the study presented here, we evaluated the isotopic labeling of bone using 41Ca, a long-lived radiotracer, as an alternative approach. After successful labeling of the skeleton, changes in the systematics of urinary 41Ca excretion are expected to directly reflect changes in bone Ca metabolism. A minute amount of 41Ca (100 nCi) was administered orally to 22 postmenopausal women. Kinetics of tracer excretion were assessed by monitoring changes in urinary 41Ca/40Ca isotope ratios up to 700 days post-dosing using accelerator mass spectrometry and resonance ionization mass spectrometry. Isotopic labeling of the skeleton was evaluated by two different approaches: (i) urinary 41Ca data were fitted to an established function consisting of an exponential term and a power law term for each individual; (ii) 41Ca data were analyzed by population pharmacokinetic (NONMEM) analysis to identify a compartmental model that describes urinary 41Ca tracer kinetics. A linear three-compartment model with a central compartment and two sequential peripheral compartments was found to best fit the 41Ca data. Fits based on the use of the combined exponential/power law function describing urinary tracer excretion showed substantially higher deviations between predicted and measured values than fits based on the compartmental modeling approach. By establishing the urinary 41Ca excretion pattern using data points up to day 500 and extrapolating these curves up to day 700, it was found that the calculated 41Ca/40Ca isotope ratios in urine were significantly lower than the observed 41Ca/40Ca isotope ratios for both techniques. Compartmental analysis can overcome this limitation. By identifying relative changes in transfer rates between compartments in response to an intervention, inaccuracies in the underlying model cancel out. Changes in tracer distribution between compartments were modeled based on identified kinetic parameters. While changes in bone formation and resorption can, in principle, be assessed by monitoring urinary 41Ca excretion over the first few weeks post-dosing, assessment of an intervention effect is more reliable approximately 150 days post-dosing when excreted tracer originates mainly from bone.

Seasonal changes in bone metabolism in sheep

There is a great need for animal models of osteoporosis and sheep are a suitable large animal that meets most requirements. Since it is known that bone mass in humans responds to seasonal changes, this study investigated natural bone metabolism in sheep in order to better define the sheep as a model for osteoporosis. Bone mineral density (BMD), trabecular structure, biochemical markers of bone formation and resorption and estrogen were analysed over a period of 18 months. The lowest BMDs, measured by peripheral quantitative computed tomography (pQCT), were observed during winter. Thereafter, a 5.1% increase in BMD was observed during spring and summer (P<0.05). Bone resorption markers showed a variable pattern, with higher values in spring compared to autumn (P<0.001). The physiological estrus phase during autumn was detected by serum estrogen levels. The findings show that it is necessary to take seasonal variations into account if sheep are used to establish an animal model for osteoporosis.

A 4-wk high-fructose diet alters lipid metabolism without affecting insulin sensitivity or ectopic lipids in healthy humans

BACKGROUND: High fructose consumption is suspected to be causally linked to the epidemics of obesity and metabolic disorders. In rodents, fructose leads to insulin resistance and ectopic lipid deposition. In humans, the effects of fructose on insulin sensitivity remain debated, whereas its effect on ectopic lipids has never been investigated. OBJECTIVE: We assessed the effect of moderate fructose supplementation on insulin sensitivity (IS) and ectopic lipids in healthy male volunteers (n = 7). DESIGN: IS, intrahepatocellular lipids (IHCL), and intramyocellular lipids (IMCL) were measured before and after 1 and 4 wk of a high-fructose diet containing 1.5 g fructose . kg body wt(-1) . d(-1). Adipose tissue IS was evaluated from nonesterified fatty acid suppression, hepatic IS from suppression of hepatic glucose output (6,6-2H2-glucose), and muscle IS from the whole-body glucose disposal rate during a 2-step hyperinsulinemic euglycemic clamp. IHCL and IMCL were measured by 1H magnetic resonance spectroscopy. RESULTS: Fructose caused significant (P < 0.05) increases in fasting plasma concentrations of triacylglycerol (36%), VLDL-triacylglycerol (72%), lactate (49%), glucose (5.5%), and leptin (48%) without any significant changes in body weight, IHCL, IMCL, or IS. IHCL were negatively correlated with triacylglycerol after 4 wk of the high-fructose diet (r = -0.78, P < 0.05). CONCLUSION: Moderate fructose supplementation over 4 wk increases plasma triacylglycerol and glucose concentrations without causing ectopic lipid deposition or insulin resistance in healthy humans.

Differential effect of pioglitazone (PGZ) and rosiglitazone (RGZ) on postprandial glucose and lipid metabolism in patients with type 2 diabetes mellitus: a prospective, randomized crossover study

Postprandial metabolism is impaired in patients with type 2 diabetes (T2Dm). Two thiazolidinediones pioglitazone (PGZ) and rosiglitazone (RGZ) have similar effects on glycaemic control but differ in their effects on fasting lipids. This study investigated the effects of RGZ and PGZ on postprandial metabolism in a prospective, randomized crossover trial.

Hexose-6-phosphate dehydrogenase modulates 11beta-hydroxysteroid dehydrogenase type 1-dependent metabolism of 7-keto- and 7beta-hydroxy-neurosteroids

BACKGROUND: The role of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in the regulation of energy metabolism and immune system by locally reactivating glucocorticoids has been extensively studied. Experiments determining initial rates of enzyme activity revealed that 11beta-HSD1 can catalyze both the reductase and the dehydrogenase reaction in cell lysates, whereas it predominantly catalyzes the reduction of cortisone to cortisol in intact cells that also express hexose-6-phosphate dehydrogenase (H6PDH), which provides cofactor NADPH. Besides its role in glucocorticoid metabolism, there is evidence that 11beta-HSD1 is involved in the metabolism of 7-keto- and 7-hydroxy-steroids; however the impact of H6PDH on this alternative function of 11beta-HSD1 has not been assessed. METHODOLOGY: We investigated the 11beta-HSD1-dependent metabolism of the neurosteroids 7-keto-, 7alpha-hydroxy- and 7beta-hydroxy-dehydroepiandrosterone (DHEA) and 7-keto- and 7beta-hydroxy-pregnenolone, respectively, in the absence or presence of H6PDH in intact cells. 3D-structural modeling was applied to study the binding of ligands in 11beta-HSD1. PRINCIPAL FINDINGS: We demonstrated that 11beta-HSD1 functions in a reversible way and efficiently catalyzed the interconversion of these 7-keto- and 7-hydroxy-neurosteroids in intact cells. In the presence of H6PDH, 11beta-HSD1 predominantly converted 7-keto-DHEA and 7-ketopregnenolone into their corresponding 7beta-hydroxy metabolites, indicating a role for H6PDH and 11beta-HSD1 in the local generation of 7beta-hydroxy-neurosteroids. 3D-structural modeling offered an explanation for the preferred formation of 7beta-hydroxy-neurosteroids. CONCLUSIONS: Our results from experiments determining the steady state concentrations of glucocorticoids or 7-oxygenated neurosteroids suggested that the equilibrium between cortisone and cortisol and between 7-keto- and 7-hydroxy-neurosteroids is regulated by 11beta-HSD1 and greatly depends on the coexpression with H6PDH. Thus, the impact of H6PDH on 11beta-HSD1 activity has to be considered for understanding both glucocorticoid and neurosteroid action in different tissues.

The influence of local and systemic preconditioning on oxygenation, metabolism and survival in critically ischaemic skin flaps in pigs

Stress proteins represent a group of highly conserved intracellular proteins that provide adaptation against cellular stress. The present study aims to elucidate the stress protein-mediated effects of local hyperthermia and systemic administration of monophosphoryl lipid A (MPL) on oxygenation, metabolism and survival in bilateral porcine random pattern buttock flaps. Preconditioning was achieved 24h prior to surgery by applying a heating blanket on the operative site (n = 5), by intravenous administration of MPL at a dosage of 35 microg/kg body weight (n = 5) or by combining the two (n = 5). The flaps were monitored with laser Doppler flowmetry, polarographic microprobes and microdialysis until 5h postoperatively. Semiquantitative immunohistochemistry was performed for heat shock protein 70 (HSP70), heat shock protein 32 (also termed haem oxygenase-1, HO-1), and inducible nitrc oxide synthase (iNOS). The administration of MPL increased the impaired microcirculatory blood flow in the proximal part of the flap and partial oxygen tension in the the distal part by approximately 100% each (both P<0.05), whereas both variables remained virtually unaffected by local heat preconditioning. Lactate/pyruvate (L/P) ratio and glycerol concentration (representing cell membrane disintegration) in the distal part of the flap gradually increased to values of approximately 500 mmol/l and approximately 350 micromol/l, respectively (both P<0.01), which was substantially attenuated by heat application (P<0.01 for L/P ratio and P<0.05 for glycerol) and combined preconditioning (P<0.01 for both variables), whereas the effect of MPL was less marked (not significant). Flap survival was increased from 56% (untreated animals) to 65% after MPL (not significant), 71% after heat application (P<0.05) and 78% after both methods of preconditioning (P<0.01). iNOS and HO-1 were upregulated after each method of preconditioning (P<0.05), whereas augmented HSP70 staining was only observed after heat application (P<0.05). We conclude that local hyperthermia is more effective in preventing flap necrosis than systemic MPL administration because of enhancing the cellular tolerance to hypoxic stress, which is possibly mediated by HSP70, whereas some benefit may be obtained with MPL due to iNOS and HO-1-mediated improvement in tissue oxygenation.