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PubMed1987

Quantification of peripherally induced reciprocal activation during voluntary muscle contraction.

A new method was developed to compare patterns of coactivation and reciprocal activation of antagonistic muscles during different experimental conditions. Pure coactivation was defined as a muscle activity pattern where there was never a difference between the slope of the agonist EMG record and the slope of the antagonist EMG record. The degree of reciprocal activation was defined as being proportional to the average absolute value of the difference between these two slope values. Use of this method for the comparison of muscle activity patterns during isometric contractions and unexpected movements showed that peripheral input related to agonist unloading and antagonist stretch significantly increased reciprocal activity.

Adult↗
PubMed1989

Quantification of regional V/Q ratios in humans by use of PET. II. Procedure and normal values.

Regional measurements of tissue isotope concentration, made using positron emission tomography (PET), allow tracer models to be used in a quantitative manner to provide topographic distributions of many structural and functional parameters, each derived for the same well-defined lung element. In this paper we describe a technique to measure regional ventilation-perfusion ratios (V/Q), in absolute units, by use of PET and the continuous intravenous infusion of an inert gas isotope, 13N, and report on measurements made in 12 normal subjects (4 smokers). Data were obtained from a single lung section (slice thickness, 1.7 cm full width at half-maximum response to a line source) at the level of the right ventricle in the supine posture during quiet breathing. For the 12 subjects, volume-weighted mean values of V/Q, averaged over individual right and left lung fields, ranged from 0.50 to 1.29. Analysis of these means showed no difference between lungs: right, 0.80 +/- 0.23 SD; left, 0.76 +/- 0.20 SD. Topographically, a systematic fall of V/Q in the ventrodorsal direction was observed in eight of the subjects (mean ventrodorsal difference 0.39, range 0.19-0.90), whereas two showed a clear increase toward dependent lung regions (range 0.16-0.26). Seven of the subjects with a falling ventrodorsal V/Q gradient also exhibited discrete regions of low V/Q at the dorsal lung border. We conclude that, in normal subjects, ventilation and perfusion are generally well matched in the supine posture, but isolated mismatching often occurs in dependent lung regions.

Humans↗
PubMed1997

Absolute and relative bioavailability of the HMG-CoA reductase inhibitor cerivastatin.

To determine the absolute bioavailability of the HMG-CoA reductase inhibitor cerivastatin, 12 healthy young male volunteers received single doses of either 100 micrograms as a 1-minute bolus infusion or 200 micrograms orally as tablets in a controlled, randomized crossover study. In addition, 8 of the 12 subjects participated in a third treatment period in which 200 micrograms cerivastatin were administered as an oral solution as reference for determining the relative bioavailability of the tablet drug formulation. Plasma samples were analyzed for cerivastatin by a specific HPLC assay with fluorescence detection after post-column irradiation of the eluate, with a limit of quantification of 0.1 microgram/l. Following all treatments, cerivastatin was well tolerated and no clinically relevant adverse events or changes in laboratory parameters were observed. Vital signs and ECG remained unchanged. Plasma concentration/time profiles of cerivastatin following intravenous bolus could be described by a 2-compartment model with a distribution half-life of 3-5 min and an elimination half-life of 1.5-2.4 h. For the 2 oral administrations a 1-compartmental pharmacokinetic model with a first-order absorption process was best to describe the plasma concentration/time data. Based on the AUCnorm values of the 7 subjects, valid for complete pharmacokinetic evaluation, the absolute bioavailability of tablet and oral solution was 60.0 and 59.6% (90% confidence intervals 53-68%), respectively. The relative bioavailability of tablet compared with solution was 100.7% (90% confidence interval 89-114%), with tablet and oral solution showing nearly identical in vivo absorption characteristics and almost superimposable plasma concentration/time curves. The tablet formulation, therefore, can be regarded as an optimal oral formulation with respect to galenic aspects.

Administration, Oral↗
PubMed2004

Enantioselective HPLC determination of E-6087, a new COX-2 inhibitor, in human plasma: Validation and pharmacokinetic application.

E-6087 is a nonsteroidal anti-inflammatory compound that selectively inhibits cyclooxygenase-2. Because E-6087 has a chiral center, this compound is a racemic mixture of two stereoisomers, (+)-(R)-E-6087 (E-6231) and (-)-(S)-E-6087 (E-6232). A normal-phase liquid-chromatographic method for the enantioselective determination of E-6087 in human plasma was developed and validated. The samples were extracted using solid-phase extraction cartridges containing C(18) sorbent, and the extracts were redissolved in absolute ethanol and injected into the chromatographic system. The enantiomeric separation was achieved on a chiral stationary-phase column of derivatized amylose, and the enantiomers were quantified by fluorescence detection. The method was validated for drug concentrations ranging from 5 to 400 ng/ml for both enantiomers. No peaks interfering with the quantification of enantiomers were observed. The limit of quantification was 5 ng/ml, with precision expressed as a coefficient of variation lower than 10.6% and accuracy expressed as relative error lower than 12.2%. The utility of this method was demonstrated by analysis of plasma samples from healthy volunteers given an oral dose of rac-E-6087. Peak plasma levels of E-6231 were higher than levels obtained for E-6232. Results were consistent with those obtained with a conventional reversed-phase method used for determination of the racemic compound.

Calibration↗
PubMed1998

Influence of Cremophor EL on the quantification of paclitaxel in plasma using high-performance liquid chromatography with solid-phase extraction as sample pretreatment.

For the quantitative determination of paclitaxel in human plasma reversed-phase high-performance liquid chromatographic (HPLC) methods with solid-phase extraction (SPE) as sample pretreatment procedure are frequently used. Recovery problems arose during the quantification of paclitaxel in plasma samples of patients. The major problems were a large batch-to-batch difference in performance of the SPE columns and the effects of the pharmaceutical vehicle Cremophor EL on the performance of the SPE. Cremophor EL concentrations exceeding 1.0% (v/v) had a great impact on the absolute recovery of paclitaxel from human plasma with the SPE procedure. The recoveries decreased approximately 10 to 40% depending on the quality of the batch SPE columns. The problems are avoided by using 2'-methylpaclitaxel as the internal standard. This study points out the importance of including the effects of a pharmaceutical vehicle, like Cremophor EL, in the validation programme of a bioanalytical assay and the use of an internal standard in HPLC paclitaxel assays preceded by SPE as sample pretreatment procedure.

Antineoplastic Agents, Phytogenic↗
PubMed2005

Mass spectrometry-based expression profiling of clinical prostate cancer.

The maturation of MS technologies has provided a rich opportunity to interrogate protein expression patterns in normal and disease states by applying expression protein profiling methods. Major goals of this research strategy include the identification of protein biomarkers that demarcate normal and disease populations, and the identification of therapeutic biomarkers for the treatment of diseases such as cancer (Celis, J. E., and Gromov, P. (2003) Proteomics in translational cancer research: Toward an integrated approach. Cancer Cell 3, 9-151). Prostate cancer is one disease that would greatly benefit from implementing MS-based expression profiling methods because of the need to stratify the disease based on molecular markers. In this review, we will summarize the current MS-based methods to identify and validate biomarkers in human prostate cancer. Lastly, we propose a reverse proteomic approach implementing a quantitative MS research strategy to identify and quantify biomarkers implicated in prostate cancer development. With this approach, the absolute levels of prostate cancer biomarkers will be identified and quantified in normal and diseased samples by measuring the levels of native peptide biomarkers in relation to a chemically identical but isotopically labeled reference peptide. Ultimately, a centralized prostate cancer peptide biomarker expression database could function as a repository for the identification, quantification, and validation of protein biomarker(s) during prostate cancer progression in men.

Biomarkers, Tumor↗
PubMed2005

Quantification of prion gene expression in brain and peripheral organs of golden hamster by real-time RT-PCR.

Determination of tissue-specific expression of cellular prion protein (PrPc) is essential for understanding its poorly explained role in organisms. Herein we report on quantification of PrP mRNA in golden hamsters, a popular experimental model for studying mechanisms of transmissible spongiform encephalopathies (TSE), by real-time RT-PCR. Total RNA was isolated from four different regions of the brain and six peripheral organs of eight golden hamsters. PrP mRNA copy numbers were determined using absolute standard curve method with real-time quantitative PCR instrument. It was found that high mRNA levels were present in all four regions of the brain examined, including obex, neocortex, cerebellum, and thalamus. In peripheral organs examined, inguinal lymph node showed high level of the expression similar to that in overall brain; spleen, heart, liver, and lung showed moderate levels of the expression; and kidney showed the lowest expression. Our result is consistent with the potential involvement of different organs in prion diseases and offers essential data for further study of TSE mechanism in this animal model.

Animals↗
PubMed1983

Thin-layer and gel permeation chromatographic separation of low molecular weight polyethylene glycol oligomers.

We describe a gel permeation and a thin-layer chromatographic technique for the complete resolution of oligomers of ethylene glycol up to a chain length of fourteen ethylene oxide [OCH2CH2] units. We employed columns of Bio-Gel P-2 with 0.02 mol/l ammonium acetate as the eluant to prepare milligram quantities of each of the individual oligomers. Thin-layer chromatography on silica gel G plates with an ethyl acetate/methanol/water solvent provides a sensitive and simple method for monitoring multiple specimens. We determined the flame ionization detector response of purified individual ethylene glycol oligomers relative to tetraethylene glycol. These data permit the accurate quantification of the oligomeric profile of commercially available mixtures of low molecular weight polyethylene glycols (PEG). When PEG 400 is administered orally to normal subjects, aged three months through adulthood, they excrete in their urine a mixture of unmetabolized oligomers with a mean molecular weight of 360 +/- 14 daltons. The ability to measure absolute, rather than relative, amounts of ethylene glycol oligomers will permit more accurate studies of passive intestinal permeability in human subjects.

Chromatography, Gas↗
PubMed1983

Segregation index: an orsametric for measuring discrimination against minorities.

Orsametrics, the science of data and measurement appropriate to decision-making models, was proposed by H. M. Wagner in 1971. This paper proposes an orsametric measuring the distance between the current distribution of minorities in the organization and the worst distribution that minorities could have, given a specified proportion of minorities present in the organization. Measurement of this distance via the absolute value norm leads to a constrained model in which a convex, absolute-value functional must be maximized. This is accomplished by means of an algorithm designed to take advantage of the special structural properties of the model. An example is provided to demonstrate how the segregation index can be used to track discrimination levels within an organization through time. (Orsametrics: public sector applications-quantification of discriminatory hiring promotion practices; mathematical modelling-nonlinear, maximizing a convex function subject to linear constraints.

Abstracting and Indexing↗
PubMed2002

T cell receptor excision circle assessment of thymopoiesis in aging mice.

Signal joint T cell receptor delta (TCRD) excision circles (TRECs) are episomal DNA circles generated by the DNA recombination process that is used by T lymphocytes to produce antigen-specific alpha/beta T cell receptors. Measurement of TRECs in thymocytes and peripheral blood T cells has been used to study thymus output in chickens and humans. We have developed a real-time quantitative-PCR assay for the specific detection and quantification of mouse TCRD episomal DNA circles excised from the TCRA locus during TCRA gene rearrangement (mTRECs). We found that the mouse TCRD TRECs detected with this assay were predominantly in naïve phenotype CD4(+) and CD8(+) T cells. In a series of aged mice (range 6-90-week-old) we determined the absolute number of thymocytes and the number of molecules of mTRECs/100,000 thymocytes. We found that the absolute number of thymocytes dramatically decreased with age (P<0.05) and that molecules of mTREC/100,000 thymocytes also declined with mouse age (P<0.05). Splenocytes were isolated from aging mice and the frequency of naïve phenotype CD4 and CD8 cells determined. There was a significant drop in both CD4 and CD8 naïve peripheral T cells in the aged mice over time. mTREC analysis in purified CD4(+) and CD8(+) splenocytes demonstrated a constant level of mTRECs in the CD4 compartment until age 90 weeks, while the mTRECs in the CD8 compartment fell with age (P<0.05). By combining the mouse TREC assay with T cell phenotypic analysis, we demonstrated that IL-7 administration to young mice induced both increased thymopoiesis and peripheral T cell proliferation. In contrast, IL-7 treatment of aged mice did not augment thymopoiesis, nor induce expansion of splenic T cells. Thus, thymus output continues throughout murine adult life, and the thymic atrophy of aging in mice is not reversed by administration of IL-7.

Aging↗
PubMed1996

Effects of high glucose on the production of heparan sulfate proteoglycan by mesangial and epithelial cells.

Changes in heparan sulfate metabolism may be important in the pathogenesis of diabetic nephropathy. Recent studies performed on renal biopsies from patients with diabetic nephropathy revealed a decrease in heparan sulfate glycosaminoglycan staining in the glomerular basement membrane without changes in staining for heparan sulfate proteoglycan-core protein. To understand this phenomenon at the cellular level, we investigated the effect of high glucose conditions on the synthesis of heparan sulfate proteoglycan by glomerular cells in vitro. Human adult mesangial and glomerular visceral epithelial cells were cultured under normal (5 mM) and high glucose (25 mM) conditions. Immunofluorescence performed on cells cultured in 25 mM glucose confirmed and extended the in vivo histological observations. Using metabolic labeling we observed an altered proteoglycan production under high glucose conditions, with predominantly a decrease in heparan sulfate compared to dermatan sulfate or chondroitin sulfate proteoglycan. N-sulfation analysis of heparan sulfate proteoglycan produced under high glucose conditions revealed less di- and tetrasaccharides compared to larger oligosaccharides, indicating an altered sulfation pattern. Furthermore, with quantification of glomerular basement membrane heparan sulfate by ELISA, a significant decrease was observed when mesangial and visceral epithelial cells were cultured in high glucose conditions. We conclude that high glucose concentration induces a significant alteration of heparan sulfate production by mesangial cells and visceral epithelial cells. Changes in sulfation and changes in absolute quantities are both observed and may explain the earlier in vivo observations. These changes may be of importance for the altered integrity of the glomerular charge-dependent filtration barrier and growth-factor matrix interactions in diabetic nephropathy.

Adult↗
PubMed2002

Characterizing immune reconstitution after long-term highly active antiretroviral therapy in pediatric AIDS.

In this study, we sought to characterize the T lymphocyte recovery in vertically HIV-1-infected children who respond to long-term highly active antiretroviral therapy (HAART). A 3-year longitudinal retrospective study was used to perform a cross-sectional study of 32 children rated according to the time course of CD4(+) T cell percentages in response to antiretroviral therapy and CDC clinical classification: (1) long-term asymptomatic (LTA group): 8 children in A1 during the whole follow-up period; (2) responsive to HAART (Rec group): 13 children in C3 before HAART who achieved CD4(+) T cell counts of > 500 cells/mm(3) after 3 years of HAART; and (3) nonresponsive to HAART (Non-Rec group): 11 children in C3 during the whole follow-up period despite 3 years of HAART. We also studied 17 healthy age-matched uninfected children as controls. Lymphoproliferative responses (LPRs) were evaluated by incorporation of [(3)H]thymidine, identification of T cell subsets by three-color flow cytometry, and determination of thymic production of T cells by quantification of T cell receptor rearrangement excision circles (TRECs). Interestingly, the Rec group showed an increase in percentage of CD4(+) T cells and a decrease in viral load, and recovered LPRs to mitogens and recall antigens, with values similar to those of the LTA group. Moreover, the Rec group produced similar percentages and absolute counts of naive (CD45RA(+)CD62L(+)) CD4(+) and CD8(+) T cells, and TRECs similar to those of the LTA group. In particular, the Rec group produced similar percentages of CD8(+)CD28(-)CD57(+) and CD8(+)CD28(-)CD57(-) T cell subsets compared with controls. Our data indicate that among children who have already progressed to AIDS and severe immunodeficiency but who respond to HAART, the immune system can recover and resemble those of nonprogressors or even uninfected children, in quantitative as well as in functional terms.

Adolescent↗
PubMed2001

Cultivation-independent, semiautomatic determination of absolute bacterial cell numbers in environmental samples by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has found widespread application for analyzing the composition of microbial communities in complex environmental samples. Although bacteria can quickly be detected by FISH, a reliable method to determine absolute numbers of FISH-stained cells in aggregates or biofilms has, to our knowledge, never been published. In this study we developed a semiautomated protocol to measure the concentration of bacteria (in cells per volume) in environmental samples by a combination of FISH, confocal laser scanning microscopy, and digital image analysis. The quantification is based on an internal standard, which is introduced by spiking the samples with known amounts of Escherichia coli cells. This method was initially tested with artificial mixtures of bacterial cultures and subsequently used to determine the concentration of ammonia-oxidizing bacteria in a municipal nitrifying activated sludge. The total number of ammonia oxidizers was found to be 9.8 x 10(7) +/- 1.9 x 10(7) cells ml(-1). Based on this value, the average in situ activity was calculated to be 2.3 fmol of ammonia converted to nitrite per ammonia oxidizer cell per h. This activity is within the previously determined range of activities measured with ammonia oxidizer pure cultures, demonstrating the utility of this quantification method for enumerating bacteria in samples in which cells are not homogeneously distributed.

Ammonia↗
PubMed2006

Quantification of abscisic acid in grapevine leaf (Vitis vinifera) by isotope-dilution liquid chromatography-mass spectrometry.

A specific, sensitive, precise, and accurate method for the determination of abscisic acid (ABA) in grapevine leaf tissues is described. The method employs high-performance liquid chromatography and electrospray ionization-mass spectrometry (LC-ESI-MS) in selected ion monitoring mode (SIM) to analyze ABA using a stable isotope-labeled ABA as an internal standard. Absolute recoveries ranged from 72% to 79% using methanol/water pH 5.5 (50:50 v/v) as an extraction solvent. The best efficiency was obtained when the chromatographic separation was carried out by using a porous graphitic carbon (PGC) column. The statistical evaluation of the method was satisfactory in the work range. A relative standard deviation (RDS) of < 5.5% and < 6.0% was obtained for intra-batch and inter-batch comparisons, respectively. As for accuracy, the relative error (%Er) was between -2.7 and 4.3%, and the relative recovery ranged from 95% to 107%.

Abscisic Acid↗
PubMed1996

Effect of radiation on blood volume in low-grade astrocytomas and normal brain tissue: quantification with dynamic susceptibility contrast MR imaging.

OBJECTIVE: The purpose of this study was to determine whether it is possible to measure radiation-induced changes in blood volume in low-grade astrocytomas and in normal brain tissue. SUBJECTS AND METHODS: The passage of a bolus of gadopentetate dimeglumine was monitored on a series of 55 T2*-weighted simultaneous dual fast low-angle shot MR images with a standard 1.5-T MR imaging system. Absolute blood volumes were calculated as the area under the tissue concentration-time curve in regions of interest and normalized to the arterial input function. We performed 41 examinations on 19 patients with grade II astrocytomas. For comparison, 13 patients were studied after whole-brain irradiation. RESULTS: A reduction in blood volume (mean +/- SD in milliliters per 100 g) within the tumors from 12.2 +/- 8.7 to 6.5 +/- 5.3 after fractionated conformation radiotherapy was detected, although there was no consistent pattern in different patients. An insignificant reduction was noted in normal gray (9.2 +/- 2.8 to 7.4 +/- 3.2) and white (4.4 +/- 1.9 to 4.1 +/- 2.3) matter outside the target volume. Conversely, we observed a significantly lower blood volume in gray (6.3 +/- 1.2) and white (3.1 +/- 1.0) matter after whole-brain radiotherapy. CONCLUSION: Our results show that a reduction of blood volume in astrocytomas and normal brain tissue after radiotherapy can be quantified by use of dynamic susceptibility contrast MR imaging. Thus, functional monitoring of tumor response and of normal tissue effects becomes possible.

Adolescent↗
PubMed2001

Detection and quantification of infectious hypodermal and hematopoietic necrosis virus and white spot virus in shrimp using real-time quantitative PCR and SYBR Green chemistry.

A rapid and highly sensitive real-time PCR detection and quantification method for infectious hypodermal and hematopoietic necrosis virus (IHHNV), a single-stranded DNA virus, and white spot virus (WSV), a double-stranded DNA (dsDNA) virus infecting penaeid shrimp (Penaeus sp.), was developed using the GeneAmp 5700 sequence detection system coupled with SYBR Green chemistry. The PCR mixture contains a fluorescence dye, SYBR Green, which upon binding to dsDNA exhibits fluorescence enhancement. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. A linear relationship was observed between the amount of input plasmid DNA and cycle threshold (C(T)) values over a range of 1 to 10(5) copies of the viral genome. To control the variation in sampling and processing among samples, the shrimp beta-actin gene was amplified in parallel with the viral DNA. The C(T) values of IHHNV- and WSV-infected samples were used to determine absolute viral copy numbers from the standard C(T) curves of these viruses. For each virus and its beta-actin control, the specificity of amplification was monitored by using the dissociation curve of the amplified product. Using genomic DNA as a template, SYBR Green PCR was found to be 100- to 2000-fold more sensitive than conventional PCR, depending on the virus, for the samples tested. The results demonstrate that SYBR Green PCR can be used as a rapid and highly sensitive detection and quantification method for shrimp viruses and that it is amenable to high-throughout assay.

Animals↗
PubMed2001

Volumetric analysis of osteonecrosis of the femur. Anatomical correlation using MRI.

We tested the accuracy of MRI for the precise quantification of the volume of osteonecrosis in 30 hips (stage III). The values were compared with direct anatomical measurements of the femoral heads obtained after total hip replacement. When the area of osteonecrosis was determined visually, and manually outlined on each slice, the accuracy of the measurement of volume was satisfactory, and the mean absolute deviation between MRI and anatomical measurements was similar to that between two MRI data sets. For ten of the hips which were measured by MRI, both before and after collapse, the volume did not appear to change significantly. Our findings suggest that the volume of osteonecrosis can be determined with accuracy by MRI, both before and after collapse.

Femur Head↗