The effect of joint translation constraint on within-participant variability of kinematics and kinetics during running in cerebral palsy.

BACKGROUND: Biomechanical data in cerebral palsy are inherently variable but no optimal model of translational joint constraint has been identified. The primary aim of this study was to determine which model of translational joint constraint resulted in the lowest within-participant variability of lower limb joint angles and moments. The secondary aim was to determine which model best distinguished known functional groups in Cerebral Palsy. METHODS: Three models (three degrees of freedom, six degrees of freedom and six degrees of freedom with specified joint translation constraint) were applied to data from running trials of 40 children with cerebral palsy. FINDINGS: Joint angle standard deviations were largest using the six degrees of freedom model and smallest using the constrained six degrees of freedom model (p < 0.050). For all joints in all planes of motion, joint moment standard deviations were largest using the six degrees of freedom model and smallest using the constrained six degrees of freedom model; standard deviations using the constrained model were smaller than the three degrees of freedom model by 10-30% of moment magnitude (0.01-0.03 Nm/kg; p < 0.001). The six degrees of freedom models distinguished functional subgroups with larger effect size than the three degrees of freedom model only for hip power generation in swing. INTERPRETATION: A model with specified joint constraint minimized within-participant variability during running and was useful for detecting differences in functional capacity in cerebral palsy.

EFFECT OF NITROGEN FEEDING SOURCE ON THE SUPPLY OF NITROGEN FROM ROOT TO SHOOT AND THE SITE OF NITROGEN ASSIMILATION IN MAIZE (ZEA MAYS L. CV. R201).

Maize plants (Zea mays L. cv. R201) were grown to 21 d in pH-controlled gravel culture with 2 mM inorganic N supplied as nitrate alone, ammonium alone or 1:1 nitrate + ammonium. At 21 d, the 14 N feeding solutions were replaced with 15 N solutions, and xylem sap collections were made 4 and 8 h after the commencement of feeding. Leaf and root material was harvested also for in vitro nitrate reductase and glutamine synthetase activity assays. Xylem sap analyses showed that in nitrate-only fed plants the major supply of nitrogen from root to shoot was in the nitrate form (60%) with 35 % carried as amino compounds. However, 93% of 15 N was transported to the shoot as nitrate and only 6% in amino compounds, indicating the more direct routing of newly absorbed nitrogen to the shoot via the former. Leaf NRA was seven-fold greater than that of the root, confirming the shoot as the major site of nitrogen assimilation in plants fed only nitrate. In ammonium-only fed plants, 84% of xylem N was found in organic form (66%16 N), the remainder translocating as ammonium, identifying the root as the major site of ammonium N assimilation. In ammonium + nitrate fed plants, 64% of xylem N was present as organic N (55%16 N), 34% as nitrate (43 %16 N), indicating shared N assimilation between shoot and root, with root assimilation predominating. In plants receiving nitrate, glutamine was the major N compound translocated, in plants receiving only ammonium, asparagine predominated. GS activity was approximately the same in root and shoot and showed no response to N source. The significance of these results is discussed with respect to the reported increased productivity of maize fed a mixed nitrate-ammonium N source.

Characterization of olanzapine (LY170053) in human liver slices by liquid chromatography/tandem mass spectrometry.

Olanzapine metabolism was investigated using incubation of olanzapine with human liver slices. The intent of the investigation was to identify olanzapine metabolites and determine if the human liver slice incubations could potentially produce quantities of the olanzapine glucuronides for future studies. Along with known Phase 1 olanzapine metabolites, N-desmethyl-, 2-hydroxymethyl-, and 4'-N-oxide-, a new hydroxylated species was detected. Detection of Phase 2 metabolites included known N-10-glucuronides, a quaternary glucuronide and a novel glucuronide conjugate. This investigation showed the feasibility of using human liver slices to produce sufficient quantities of olanzapine glucuronides for further studies.

Relation of anterior pelvic tilt during running to clinical and kinematic measures of hip extension.

BACKGROUND: Limited hip extension flexibility due to tight hip flexor musculature or anterior hip capsular and ligamentous structures is a possible cause of increased anterior tilt of the pelvis during running. However, to date, research exploring this relation, as well as the kinematic relation between anterior tilt of the pelvis and peak hip extension range of motion during running, is not available. OBJECTIVE: To assess the relation of anterior pelvic tilt during running to peak hip extension range of motion measured during running and hip extension flexibility measured clinically. METHODS: Hip extension flexibility was assessed using the Thomas test, and the three dimensional kinematic motion of the pelvis and hips were recorded using a VICON motion analysis system with 14 elite athletes running on a treadmill at 20 km/h. RESULTS: Anterior pelvic tilt displayed a significant (p<0.01) correlation with peak hip extension range of motion during running. Anterior pelvic tilt tended to be increased in runners who displayed reduced absolute peak hip extension range of motion during terminal stance. No significant correlation was shown for hip extension flexibility with either anterior pelvic tilt or peak hip extension range of motion during running. CONCLUSIONS: The outcomes of this study indicate that anterior pelvic tilt and hip extension are coordinated movements during running. Static hip extension flexibility measured using the modified Thomas test does not appear to be reflective of these dynamic movements.

Collection, storage, and filtration of in vivo study samples using 96-well filter plates to facilitate automated sample preparation and LC/MS/MS analysis.

The benefits of high-throughput bioanalysis within the pharmaceutical industry are well established. One of the most significant bottlenecks in bioanalysis is transferring in vivo-generated study samples from their collection tubes during sample preparation and extraction. In most cases, the plasma samples must be stored frozen prior to analysis, and the freeze/thaw (F/T) process introduces thrombin clots that are capable of plugging pipets and automated liquid-transfer systems. A new approach to dealing with this problem involves the use of Ansys Captiva 96-well 20-microm polypropylene filter plates to collect, store frozen, and filter plasma samples prior to bioanalysis. The samples are collected from the test subjects, and the corresponding plasma samples are placed directly into the wells of the filter plate. Two Duoseal (patent pending) covers are used to seal the top and bottom of the plate, and the plate is stored at down to -70 degrees C. Prior to sample analysis, the seals are removed and the plate is placed in a 96-well SPE manifold. As the plasma thaws, it passes (by gravity or mild vacuum) through the polypropylene filter into a 96-well collection plate. A multichannel pipet or automated liquid-transfer system is used to transfer sample aliquots without fear of plugging. A significant advantage of this approach is that, unlike other methods, issues related to incomplete pipetting are virtually eliminated. The entire process is rapid since thawing and filtering take place simultaneously, and if a second F/T cycle is required for reanalysis, it is not necessary to refilter the samples (additional clotting was not observed after three F/T cycles). This technique was tested using monkey, rat, and dog plasma and sodium heparin and EDTA anticoagulants. To assess the possibility of nonspecific binding to the polypropylene filter, a variety of drug candidates from diverse drug classes were studied. Validation data generated for two Lilly compounds from distinct classes, before and after filtering, are presented in this paper as practical examples of this technique. While LC/MS/MS is the primary method of bioanalysis in our laboratory, the technique presented in this paper is applicable to other forms of detection as well.

Determination of xanomeline and active metabolite, N-desmethylxanomeline, in human plasma by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

We have developed a method for the determination of xanomeline and its pharmacologically active N-desmethyl metabolite. The validated method uses hexane to extract xanomeline and its N-desmethyl metabolite from basified plasma. The hexane extract is dried, reconstituted, and analyzed using a liquid chromatographic-atmospheric pressure chemical ionization tandem mass spectrometry system. The method was developed to support phase II clinical trials and has proven to be extremely sensitive, fast, and rugged. The method has a limit of quantitation of 75 and 200 pg/ml plasma for xanomeline and the N-desmethyl metabolite, respectively. Sample analysis times were less than 3 min from one injection to the next.

Volatile fatty acid uptake and propionate metabolism in ruminant hepatocytes.

Previous reports have demonstrated that butyrate inhibits metabolism of propionate by liver cells isolated from sheep and goats. Our objectives were to examine some possible mechanisms for this inhibition and to test for this inhibition in the bovine animal. Incorporation of label from 2.5 mM [2-(14)C]propionate into glucose (nmol propionate/mg cell DM/h) in the presence of 0, 1.25, and 2.5 mM butyrate was 107, 66, and 62 by goat hepatocytes and 79, 25, and 29 by calf hepatocytes; therefore, butyrate inhibited propionate metabolism at least as effectively in calves as in goats. In goat hepatocytes 1.25 mM butyrate reduced 1.25 mM propionate uptake to 46% of control, and 1.25 mM [2-(14)C] propionate incorporation into glucose to 44% of control. Propionate had no effect on butyrate uptake. Isovalerate and valerate tended to be cleared from the media to a greater extent than butyrate but had no effect on propionate uptake. Therefore, inhibition of propionate conversion to glucose by butyrate is specific and is not due to a general competition among VFA for metabolism. Butyrate inhibits hepatic propionate utilization generally, not specifically propionate conversion to glucose. Butyrate also inhibited propionate utilization by goat liver homogenates, indicating that butyrate inhibits propionate metabolism at a step subsequent to propionate transport across the hepatocyte plasma membrane.

Determination of xanomeline in human plasma by ion-spray tandem mass spectrometry.

Xanomeline is a muscarinic receptor agonist currently in phase II clinical trials for the treatment of Alzheimer's disease. A fast, sensitive and specific assay has been developed to determine xanomeline plasma concentrations using ion-spray tandem mass spectrometry. Xanomeline and a structural analog, LY282122, were extracted from basifed plasma into hexane. The dried hexane extracts were reconstituted and injected onto a 10 x 1 mm C18 reversed-phase column. A mobile phase of 33 mM ammonium acetate and 0.33% acetic acid in 30/70 (v/v) water-acetonitrile was pumped through the column at 50 microliters min-1. The mobile phase eluant was introduced directly into the ion-spray interface. The mass spectrometer was operated in the positive ion mode for specific detection of the product ions of xanomeline and the internal standard. The method has a linear range of 0.075-5.0 ng xanomeline per milliliter of plasma. Sample run times were 2.5 min from one injection to the next.

Effects of LY295427, a low-density lipoprotein (LDL) receptor up-regulator, on LDL receptor gene transcription and cholesterol metabolism in normal and hypercholesterolemic hamsters.

The action of LY295427 [(3alpha,4alpha, 5alpha)-4-(2-propenylcholestan-3-ol)], a compound that derepresses low-density lipoprotein receptor (LDL-R) expression in a cell-based model, was examined in hamsters. It was found that the compound does not have an effect in normal chow-fed hamsters, in which LDL-R levels are not repressed, but exerts a marked hypocholesterolemic effect (>70% decrease) in cholesterol-coconut oil-fed hamsters, in which LDL-R is repressed. In this model, there is a dose-response for cholesterol lowering with an approximate ED50 value of 40 mg/kg/day and an inverse relationship between serum cholesterol and serum LY295427 levels. LDL-R mRNA is increased (2-fold) and liver cholesterol ester content is decreased (>90%). Unlike the 3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitor lovastatin, the decreased serum cholesterol is confined to the non-high-density lipoprotein fraction. Furthermore, LY295427 does not affect cholesterol biosynthesis, and it does not have a significant effect on cholesterol absorption. These data suggest that LY295427 acts in the hypercholesterolemic hamster by derepressing LDL-R transcription, thereby enhancing cholesterol clearance from the blood. The results with LY295427 suggest that compounds that act to increase LDL-R may represent a novel approach in the pharmacotherapy for hypercholesterolemia.