Quantitation of anacetrapib, stable-isotope labeled-anacetrapib (microdose), and four metabolites in human plasma using liquid chromatography tandem mass spectrometry.

An ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of (4S,5R)-5-[3,5-bis (trifluoromethyl)phenyl]-3-{[4'-fluoro-5'-isopropyl-2'-methoxy-4-(trifluoromethyl)biphenyl-2-yl] methyl}-4-methyl-1,3-oxazolidin-2-one (anacetrapib, I) and [(13)C5(15)N]-anacetrapib, II in human plasma has been developed to support a clinical study to determine the absolute bioavailability of I. The analytes and the stable-isotope labeled internal standard ([(13)C7(15)N(2)H7]-anacetrapib, III) were extracted from 100µL of human plasma by liquid-liquid extraction using 20/80 isopropyl alcohol/hexane (v/v). The chromatographic separation of the analytes was achieved using Waters BEH Shield RP 18 (50×2.1mm×1.7µm) column and mobile phase gradient of 0.1% formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B) at 0.6mL/min flow rate. The MS/MS detection was performed on AB Sciex 5000 or AB 5500 in positive electrospray ionization mode, operated in selected reaction monitoring mode. The assay was validated in the concentration range 1-2000ng/mL for I; and a lower curve range, 0.025-50ng/mL for II. In addition to the absolute bioavailability determination, it was desired to better elucidate the pharmacokinetic behavior of several hydroxylated metabolites of I. Toward this end, two exploratory assays for the hydroxy metabolites of I were qualified in the concentration range 0.5-500ng/mL. All metabolites were separated on a Supelco Ascentis Express Phenyl-Hexyl (50×2.1mm, 2.7µm) column. Metabolite M4 was analyzed in the negative mode with a mobile phase consisting of a gradient mixture of water (A) and acetonitrile (B). The other three metabolites, M1-M3 were analyzed in the positive mode using a mobile phase gradient of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The assays were utilized to support a clinical study in which a microdosing approach was used to determine the pharmacokinetics of anacetrapib and its metabolites.

Nonlinearity and thresholds in dose-response relationships for carcinogenicity due to sampling variation, logarithmic dose scaling, or small differences in individual susceptibility.

Nonlinear and threshold-like shapes of dose-response curves are often observed in tests for carcinogenicity. Here, we present three examples where an apparent threshold is spurious and can be misleading for low dose extrapolation and human cancer risk assessment. Case #1: For experiments that are not replicated, such as rodent bioassays for carcinogenicity, random variation can lead to misinterpretation of the result. This situation was simulated by 20 random binomial samplings of 50 animals per group, assuming a true linear dose response from 5% to 25% tumor incidence at arbitrary dose levels 0, 0.5, 1, 2, and 4. Linearity was suggested only by 8 of the 20 simulations. Four simulations did not reveal the carcinogenicity at all. Three exhibited thresholds, two showed a nonmonotonic behavior with a decrease at low dose, followed by a significant increase at high dose ("hormesis"). Case #2: Logarithmic representation of the dose axis transforms a straight line into a sublinear (up-bent) curve, which can be misinterpreted to indicate a threshold. This is most pronounced if the dose scale includes a wide low dose range. Linear regression of net tumor incidences and intersection with the dose axis results in an apparent threshold, even with an underlying true linear dose-incidence relationship. Case #3: Nonlinear shapes of dose-cancer incidence curves are rarely seen with epidemiological data in humans. The discrepancy to data in rodents may in part be explained by a wider span of individual susceptibilities for tumor induction in humans due to more diverse genetic background and modulation by co-carcinogenic lifestyle factors. Linear extrapolation of a human cancer risk could therefore be appropriate even if animal bioassays show nonlinearity.

The relationship between spectral characteristics and perceived hypernasality in children.

The purpose of this study was to quantify perceived hypernasality in children. One-third octave spectra of the isolated vowel [i] were obtained from 32 children with cleft palate and 5 children without cleft palate. Four experienced listeners rated the severity of hypernasality of the 37 speech samples using a 6-point equal-appearing interval scale. When the average 1/3-octave spectra from the hypernasal group and the normal resonance group were compared, spectral characteristics of hypernasality were identified as increased amplitudes between F1 and F2 and decreased amplitudes in the region of F2. Based on the findings of the children's speech, 36 speech samples with manipulated spectral characteristics were used to minimize the influences of voice source characteristics on perceived hypernasality. Multiple regression analysis revealed a high correlation (R = 0.84) between the amplitudes of 1/3-octave bands (1 k, 1.6 k, and 2.5 kHz) and the perceptual ratings. Increased amplitudes of bands between F1 and F2 (1 k, 1.6 kHz) and decreased amplitude of the band of F2 (2.5 kHz) was associated with an increasing perceived hypernasality. These results suggest that the amplitudes of the three 1/3-octave bands are appropriate acoustic parameters to quantify hypernasality in the isolated vowel [i].

The influence of acoustic and perceptual factors on perceived hypernasality in the vowel.

The purpose of this study was to examine the influence of acoustic and perceptual factors of speech on listeners' perceived hypernasality in the vowel [i]. The isolated syllable [pi] produced by 22 children with hypernasal speech and 6 noncleft children was rated by 10 listeners. These speech samples were then divided into two groups: (1) the samples (n = 14) that received inconsistent ratings from each listener or variable ratings among listeners (i.e., unreliable ratings) and (2) the samples (n = 14) that received consistent ratings from each listener and similar ratings among listeners (i.e., reliable ratings). These results suggest that the severity of hypernasality was easy to rate in some speech samples and not in others. Voice quality deviation and a particular type of spectral change that related to the severity of hypernasality could be factors that influence perceived hypernasality.

Human responses to propionic acid. II. Quantification of breathing responses and their relationship to perception.

In 20 normal and four anosmic participants, instantaneous inhalation and exhalation flow rates were recorded in response to 15 s stimulations with clean air or propionic acid concentrations (0.16, 1.14, 8.22 and 59.15 p.p.m., v/v) that ranged from peri-threshold for normals to clearly supra-threshold for anosmics. Each odorant/irritant delivery to the face-mask began with an exhalation. This allowed concentration to reach full value before stimulus onset, defined as the point where the participant began to bring the stimulus into the nose by inhalation. Two seconds after this stimulus onset, normals exhibited cumulative inhaled volume (CIV) declines of 39 and 14%, and latencies of 500 and 710 ms, with presentations of 59.15 and 8.22 p.p.m., respectively. With anosmics, 59.15 p.p.m. caused a 19% decline in CIV that began at 730 ms. Examination of the first inhalation after stimulus onset shows that the CIV declines in normals were achieved by a progressive decline in volume (InVol), beginning with a slight drop at 1.14 p.p.m., and a marked decline in duration (InDur) with only the highest concentration. Anosmics exhibited declines in InDur and InVol with only the 59.15 p.p.m. stimulus, and these declines were much more modest than the changes seen in normals. Comparison of these breathing results with perceptual responses from this same experiment demonstrates that: (i) in normals, odor perception rises slightly, but breathing does not change, with the lowest concentration; (ii) the higher breathing sensitivity (declines in InVol) of normals is paralleled by both the higher nasal irritation of these individuals and the presence of odor sensation; (iii) InDur declines in normals only with a stimulus concentration sufficient to cause marked nasal irritation in anosmics; and iv) in anosmics, modest but reliable declines in both InDur and InVol mirror the marked elevation in nasal irritation magnitude seen with only the highest concentration. In view of the failure of prior work to provide evidence that olfactory activation alone can cause any of the breathing changes we observed, we conclude that some breathing parameters are quite useful as rapid and sensitive measures of nasal irritation that arises from activation of nasal trigeminal afferents alone or in combination with the olfactory nerve.

Human responses to propionic acid. I. Quantification of within- and between-participant variation in perception by normosmics and anosmics.

The objective of this study was to fully characterize normosmic perception of stimuli expected to cause widely varying degrees of olfactory and nasal trigeminal stimulation and to directly evaluate the possible role of olfactory nerve stimulation in nasal irritation sensitivity. During each of four identical test sessions, four anosmic and 31 normosmic participants were presented with a range of concentrations extending from peri-threshold for normosmics to supra-threshold for anosmics. For each session, odor (O) and nasal irritation (NI) sensitivities were summarized in terms of the concentrations required to produce four sensation levels ('iso-response' concentrations). Within-participant variation in these iso-response concentrations was < 10-fold for 95% of normosmics, for both O and NI. For O but not NI, these apparent fluctuations in sensitivity were largely accounted for by the uncertainty surrounding the iso-response concentrations calculated for each session. Anosmics exhibited minimal within- and between-participant variation in NI and required, for all but the highest perceptual level, a higher concentration than almost all normosmics. Between-participant variation, expressed in terms of 90% confidence interval widths, was approximately 0.5 log units for both O and NI for the highest perceptual level, but increased to approximately 0.8 and 1.8 log units, respectively, for the lowest (peri-threshold) level. Our findings suggest that: (i) most apparent variation over time in O sensitivity is actually a reflection of the uncertainty surrounding estimates of sensitivity obtained for each session; (ii) within- and between-participant variation in O sensitivity is far less than is commonly reported; and (iii) low to moderate levels of NI in normosmics are the result of relatively weak trigeminal stimulation combined with much greater olfactory activation.

Effects of odorants and irritants on respiratory behavior.

A technique that combines psychophysical measurements with continuous recording of nasal patency and respiratory behavior was used to study the psychophysical and respiratory responses of 10 subjects to well-controlled stimulation with three compounds differing in relative stimulatory effectiveness for nasal olfactory and trigeminal chemoreceptors. All four concentrations of acetic acid, amyl acetate, and phenethyl alcohol were well above the odor detection threshold. The magnitudes of both the increase in odor strength and nasal irritation and the decreases in tidal volume were greatest for acetic acid and least for phenethyl alcohol. Among the odorants, differences in nasal irritation were greater than those in odor strength, and tidal volume appeared to have a reasonably close and inverse relationship to nasal irritation.

Assessing the effects of odorants on nasal airway size and breathing.

A technique was developed to obtain continuous measurements of both respiratory behavior and nasal patency in response to well-controlled odorant stimulation. An automated apparatus similar to that described by Walker et al. (27) was used to present precise concentrations of an odorant. The pressure-flow technique (28) was used to continuously measure nasal airway cross-sectional area, nasal airflow rate, air volume and time characteristics associated with breathing before and during odorant stimulation. Immediately following each odorant presentation, subjects entered their psychophysical responses into a microcomputer via an electronic mouse. Respiratory and psychophysical responses of ten normal subjects to four concentrations of acetic acid during eight odorant trials were recorded; eight clean-air trials were also conducted. At the highest concentration, changes in respiratory behavior were observed as early as 200 ms after stimulus onset in some subjects. Inspiratory volumes during odorant presentation were lower than those seen just before stimulation. The magnitude of this decrease was directly related to the concentration of acetic acid and to the perceived intensity of the odor and degree of nasal irritation.

Colour displays and look-up tables: real time modification of digital images.

Image processing in biomedical research has become customary, along with use of colour displays to run image processing packages. The performance of softwares is highly dependent on the device they run on: architecture of colour display, depth of frame buffer, existence of look-up table, etc. Knowledge of such basic features is therefore becoming very important, especially because results can differ from device to device. This introductory paper discusses hardware features and software applications. A general architecture of colour displays is exposed, comparing the features of the most commonly used devices. Basic organisation of memory, electron gun and screen are analysed for each type of display, concluding with a more detailed study of raster scan devices. Frame buffer and look-up table organisation are then analysed in relation with overhead expenses such as time and memory. Relation between image data and displayed images is discussed. By means of examples, the manipulation of colour tables is examined in detail, showing how to improve display of images without altering image data. Finally, the basic operations performed by the look-up table editor developed at University of Geneva are presented.

Development of the Generative Cell Wall in Monotropa uniflora L. Pollen.

During an ultrastructural survey of an achlorophyllus dicotyledonous plant, Monotropa uniflora L., a stage of pollen development was encountered which suggests a relationship between the activity of the rough endoplasmic reticulum and the development of the generative cell wall.