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Absolute phasic blood flow measurement in the brain using digital subtraction angiography.

RATIONALE AND OBJECTIVES: In this study, an angiographic method using first-pass distribution analysis was used for quantification of phasic volumetric blood flow. Studies were conducted in an angiographic perfusion phantom and in an animal model (rabbit) after intraarterial injection of contrast material. METHODS: Digital subtraction angiography and first-pass distribution algorithm were used to assess the absolute volumetric flow rates. The method, based on the conservation of contrast material in successive angiographic images, uses the videodensitometric information in the arterial bed. Measurements were made by summing the pixel values in the region of interest, covering the whole perfusion bed. A contrast pass curve was generated for a sequence of images to obtain instantaneous volumetric flow rates. RESULTS: Instantaneous and mean absolute volumetric flow measurements made in the angiographic perfusion phantom and the common carotid artery of the animal models correlated well with validation measurements made using ultrasound flowprobes. The measured (M) and known (K) flow rates in the carotid artery were related by M = 0.87 K + 2.50 mL/minute (r = 0.96, standard error of the estimate = 3.79 mL/minute, n = 25) and M = 0.92 K - 1.00 mL/minute (r = 0.98, standard error of the estimate = 4.04 mL/minute, n = 38) using the videodensitometric and entrance vessel calibration techniques, respectively. Conclusion. Results demonstrate the potential use of the first-pass distribution method in conjunction with digital subtraction angiography for measuring phasic arterial blood flow in vivo.

Algorithms↗

Quantification of 7-dehydrocholesterol in plasma and amniotic fluid by liquid chromatography/particle beam-mass spectrometry as a biochemical diagnostic marker for the Smith-Lemli-Opitz syndrome.

The qualitative and quantitative determination of cholesterol and 7-dehydrocholesterol (7-DHC) in plasma as a biochemical diagnostic marker for the Smith-Lemli-Opitz syndrome by liquid chromatography/particle beam interface-mass spectrometry (LC/PB-MS) is presented. Baseline separation of cholesterol and 7-DHC is achieved on a silica column with hexane+ethanol (99: 1 v/v) as mobile phase within a 10 min analysis. Recoveries of cholesterol and 7-DHC in a simple two-phase extraction system are nearly 100%. The absolute limit of detection using LC/PB-MS is approximately 10 ng. The method presented allows extraction, analysis and quantification of cholesterol and 7-DHC within 15 min without the necessity of sample derivatization.

Amniotic Fluid↗

The effect of averaging cardiac Doppler spectrograms on the reduction of their amplitude variability.

The effect of averaging cardiac Doppler spectrograms on the reduction of their amplitude variability was investigated in 30 patients. Beat-to-beat variations in the amplitude of Doppler spectrograms were also analysed. The quantification of amplitude variability was based on the computation of the area under the absolute value of the derivative function of each spectrum composing mean spectrograms. Fast Fourier transform using a Hanning window was used to compute Doppler spectra. Results obtained over systolic and diastolic periods showed that the reduction of amplitude variability followed an exponentially decreasing curve characterised by the equation f (r) = 100 e-beta(r-1), where r is the number of cardiac cycles, beta the exponentially decreasing rate, and 100 the normalised variability for r = 1. In systole, the decreasing rate beta was 0.165, whereas in diastole it was 0.225. Reductions of the variability in systole for a number of cardiac cycles of 5, 10, 15, and 20 were 48, 77, 90 and 96 per cent, respectively. In diastole, reductions of the variability for the same numbers of cardiac cycles were 59, 87, 96 and 99 per cent, respectively. Based on these results, it can be concluded that no significant improvement in the reduction of amplitude variability may be obtained by averaging more than 20 cardiac cycles.

Echocardiography, Doppler↗

No evidence for parental imprinting of mouse 22q11 gene orthologs.

Non-Mendelian factors may influence central nervous system (CNS) phenotypes in patients with 22q11 Deletion Syndrome (22q11DS, also known as DiGeorge or Velocardiofacial Syndrome), and similar mechanisms may operate in mice carrying a deletion of one or more 22q11 gene orthologs. Accordingly, we examined the influence of parent of origin on expression of 25 murine 22q11 orthologs in the developing and mature CNS using single nucleotide polymorphism (SNP)-based analysis in interspecific crosses and quantification of mRNA in a murine model of 22q11DS. We found no evidence for absolute genomic imprinting or silencing. All 25 genes are biallelically expressed in the developing and adult brains. Furthermore, if more subtle forms of allelic biasing are present, they are very small in magnitude and most likely beyond the resolution of currently available quantitative approaches. Given the high degree of similarity of human 22q11 and the orthologous region of mmChr16, genomic imprinting most likely cannot explain apparent parent-of-origin effects in 22q11DS.

Alleles↗

Cytofluorometric quantification of cell-surface antigens by indirect immunofluorescence using monoclonal antibodies.

A method has been developed to determine the absolute number of binding sites on lymphoid cells by flow cytometric analysis of indirect immunofluorescence data after saturation the related monoclonal antibodies (mAbs). First, the cell lines RPMI 8402, MOLT-4, CEM and HSB-2 were studied to determine the number of p67, T cell-associated antigen molecules expressed on their membrane, as judged by the binding of radiolabeled T101 (CD5) mAb. Then saturating doses of unlabeled T101 followed by fluorescent anti-mouse reagent were applied to these cell lines in each new experiment in order to build a standard curve relating the mean fluorescence intensity of these known cell populations to the mean number of cell-bound T101 mAb molecules. This internal standard curve was a straight line, and it was used to assess the absolute number of mAb molecules bound to other lymphoid cells. The technique was shown to be applicable to other mAb of IgG class even if used in unpurified form. These biological standards can be routinely used as internal references to establish the quantitative phenotype of lymphoid cells. The present method is referred to as quantitative indirect immunofluorescence assay (QIIF). It can be used with any flow cytometer equipped with a microcomputer.

Antibodies, Monoclonal↗

Expansion of extracellular tracer spaces in the isolated heart perfused with crystalloid solutions: expansion of extracellular space, trans-sarcolemmal leakage, or both?

Comparisons of the distributions of tracers putatively confined to the myocardial extracellular space (ECS) in vivo and in isolated hearts perfused with crystalloid solutions via their coronaries, generally indicate that ECS markedly expands--both absolutely and relative to cellular space (ICS)--over a period of in vitro perfusion. Quantification of the expansion is problematic, however, because of the difficulty of distinguishing between a true increase in anatomical ECS and expansion of tracer space due to penetration of the plasma membrane. This difficulty in turn renders an assessment of the distribution of electrolytes and other substances unreliable. In the present study a combined morphometric-tracer distribution analysis is applied towards distinguishing between ECS expansion and tracer leakage in the rat Langendorff preparation perfused with a modified Krebs-Henseleit solution. The results demonstrate that both ICS and ECS expanded with the first few minutes of perfusion, with the ICS remaining constant thereafter and the ECS increment declining slowly. Although the expansion of the ECS initially is disproportionately greater relative to that of the ICS, this relative difference disappears in about 1 h, i.e., the uptake of water by the myocardium is nearly homogeneous. An apparent increase of ECS relative to ICS, by more than one-third the initial space after 90 min perfusion, was found to be entirely artifactual and apparently due to a non-specific increase in sarcolemmal permeability.

Animals↗

Concurrent quantification of cellular cholesterol, cholesteryl esters and triglycerides in small biological samples. Reevaluation of thin layer chromatography using laser densitometry.

Absolute specificity and high accuracy is required for the quantitation of cholesterol, cholesteryl esters and triglycerides in small biological samples, particularly in a limited number of cells. Both can be achieved through thin-layer chromatography and molybdatophosphoric acid staining, while the shortcomings of traditional spot detection are overcome by laser densitometry. The major advantage of the proposed technique is the concurrent assay of nanogram quantities of cholesterol, cholesteryl esters and triglycerides. Our assay is at least ten-fold more sensitive than common thin-layer chromatography-based techniques and at least four-fold more sensitive than common enzymatic methods. The present low-cost assay is highly reproducible and may be particularly suitable for the routine lipid analysis of a 10% aliquot of relatively small tissue and cell samples, equivalent, for instance, to > or = 10(4) human monocytes.

Cholesterol↗

Quantification of cerebral blood flow by bolus tracking and artery spin tagging methods.

This study deals with perfusion quantification in healthy volunteers using two types of dynamic magnetic resonance imaging (MRI) methods. Absolute cerebral blood flow (CBF) measurements were performed in 11 subjects by applying both bolus tracking of exogenous contrast agent and non-invasive arterial spin labeling MRI techniques. Both methods produced CBF images with good tissue contrast and CBF values are in good agreement with literature data. The correlation between cerebral blood volume (CBV) and CBF is also discussed.

Arteries↗

Uterine rupture.

PURPOSE OF REVIEW: The increasing rates of primary caesarean section are likely to contribute to a rise in the incidence of uterine rupture in developed countries. Uterine rupture continues to be an important cause of maternal death in less and least developed countries. Recent publications adressing the diagnosis, aetiology and management of primary caesarian section are reviewed. RECENT FINDINGS: Uterine rupture is commonly associated with vaginal birth after caesarean section, although the absolute risk varies according to previous obstetric history, gestational age, and induction of labour. Quantification of risk for a range of circumstances allows informed counselling of pregnant women. Imaging techniques may improve the early detection of uterine dehiscence but in the acute setting abnormal fetal heart rate patterns provide early diagnostic information. The scale of the problem in less and least developed countries is sobering and maternal mortality can be determined by the ability to access basic obstetric care. SUMMARY: Uterine rupture remains one of the most frightening complications in obstetric care. The risk of its occurrence is likely to increase for many parous women, placing the emphasis on counselling and early prediction and prevention wherever possible.

Female↗

Quantification in clinical fluorodeoxyglucose positron emission tomography.

Positron emission tomography (PET) is increasingly used clinically to provide functional information on disease processes, especially in oncology using the glucose analogue 2-[18F]fluoro-2-deoxy-D-glucose (F-FDG). In the clinical setting it has become standard practice to use simplified imaging protocols compared to the often complex methods developed for research using PET. This is partly due to scarcity of resources but also for reasons of patient comfort and compliance, and not least expense and patient throughput. Fortunately the resulting loss in information can be justified to some extent on the grounds that in clinical PET it is usually relative rather than absolute metabolic rates that are of interest. Nonetheless, there remain unresolved questions of how best to perform quantification in clinical PET.

Algorithms↗

[Quantitative myocardial perfusion assessment with magnetic resonance imaging in patients with coronary artery disease].

AIM: To elucidate feasibility of the absolute myocardial blood flow (MBF), total coronary resistance (TCR), and myocardial blood flow reserve (MBFR) quantification using MRI in patients with coronary artery disease (CAD). MATERIAL AND METHODS: A total of 19 patients with angiographically documented CAD and 9 healthy subjects were studied by MRI using double-slice saturation-recovery Turbo-FLASH sequence for monitoring myocardial first pass kinetics of Gd-DTPA-BMA at rest and during hyperemia (dipyridamole 0.56 mg/kg). The signal intensity curves were acquired within ROI for perfusion beds of the three main coronary arteries (LAD, LCX and RCA) and left ventricle cavity. Eighty five myocardial segments were included in final analysis (group 1 - supplied by < > coronary arteries, n=26; group 2 - supplied by arteries with non-significant diameter stenoses <50%, n=27; group 3 - supplied by arteries with significant stenoses >/=50%, n=32). Sixteen segments were revascularized subsequently (PCI or CABG). One-compartment model and slope-method were used for flow calculation. Myocardial and blood signal intensities were converted to concentration of Gd-DTPA-BMA according to the < > calibration curve. RESULTS: MBF was similar in groups at baseline (group 1 - 0.98+/-0.54, group 2 - 1.24+/-0.53 and group 3 - 1.28+/-0.48 ml/g/min) but significantly lower in group 3 during hyperemia (2.57+/-1.23, 2.99+/-1.14 vs. 1,79+/-0.94 ml/g/min, p<0.05). MBFR (the ratio of flow during hyperemia to flow at baseline) was significantly lower in group 3 than in groups 2 and 1 (1.4+/-0.7 vs. 2.7+/-1.3 vs. 2.9+/-1.2, respectively, p<0.01). Receiver-operator characteristic analysis of MBFR (value </=1.6) revealed sensitivity and specificity of 81% and 85%, respectively, for detection of CAD as defined by quantitative coronary angiography (stenosis diameter >/=50%). TCR (mean arterial pressure divided by flow) significantly decreased (78.8+/-42.2 vs. 41.3+/-17.3 mm Hg ґ min ґ g/ml, p<0.01), MBF accordingly increased (1.61+/-0.77 vs. 2.58+/-0.91 ml/g/min, p<0.01) during hyperemia and MBFR < > (1.3+/-0.6 vs. 3.0+/-1.3, p<0.00l) in myocardial segments after revascularization. CONCLUSION: Absolute MBF and MBFR calculation by first-pass contrast perfusion MRI are feasible in patients with CAD before and after revascularization.

Coronary Angiography↗

The absolute bioavailability and effect of food on the pharmacokinetics of zolmitriptan in healthy volunteers.

AIMS: Zolmitriptan (Zomig (formerly 311C90)) is a novel 5-HT1B/1D receptor agonist developed for the acute oral treatment of migraine. A highly sensitive LCMS-MS assay has been developed which allows quantification of plasma concentrations of zolmitriptan and its active metabolite, 183C91, after therapeutic doses. Two studies using this assay method were conducted to investigate the pharmacokinetics, including absolute bioavailability, of 2.5 and 5 mg oral doses of zolmitriptan in men and women, the dose-proportionality of 2.5, 5 and 10 mg doses and the effect of food on the pharmacokinetics of a 5 mg oral dose. METHODS: Two randomized, balanced, open-label, 4-period crossover studies were conducted in a total of 32 healthy volunteers. The first study determined the absolute bioavailability of 2.5 and 5 mg doses of zolmitriptan and compared the pharmacokinetics in men and women. The second study examined the dose-proportionality in pharmacokinetics after fasting doses of 2.5, 5 and 10 mg, and the effect of food on a 5 mg dose. Blood pressure, heart rate, ECG, clinical chemistry, haematology and adverse events were also monitored. RESULTS: The mean (s.d.) absolute oral bioavailability was 0.41 (0.14 and 0.40) 0.09 after 2.5 mg and 0.48+/-0.14 and 0.36+/-0.07 after 5 mg in women and men, respectively. Without adjustment for bodyweight, plasma concentrations of zolmitriptan, but not 183C91, were higher in women than men. Mean (+/-s.d.) AUC was 32.7+/-10.1 and 60.2+/-26.8 ng ml(-1) h after 5 mg in men and women, respectively (95% CI for ratio 0.43-0.77). After 2.5 mg mean (+/-s.d.) AUC was 18.4+/-5.4 and 23.1+/-9.9 ng ml(-1) h in men and women, respectively (95% CI for ratio 0.61-1.09). However, these differences were of no clinical significance. Cmax and AUC of oral zolmitriptan were dose-proportional and there was a 13 and 16% fall in mean zolmitriptan Cmax and AUC, respectively, when administered after food. Adverse effects were minor, predominantly mild and transient, and there were no clinically significant effects on ECG, blood pressure, or laboratory parameters. CONCLUSIONS: At therapeutic doses zolmitriptan has good oral bioavailability in healthy volunteers and has dose-proportional pharmacokinetics that are not affected by food to any clinically relevant extent.

Adolescent↗

Standards for quantification of elements in the otolithic membrane by electron probe X-ray microanalysis: calibration curves and electron beam sensitivity.

An absolute quantitative standardization technique has been developed to measure Ca and K weight fractions (WF) in the otolithic membrane of the saccule and utricle by scanning electron microscopy and electron probe X-ray analysis using the peak-to-background (P/B) ratio method. Microcrystalline salt standards were used to calibrate Ca and K K alpha P/B or Y = (P/B).Z2/A (Z = atomic number; A = atomic weight) against WF at 10, 15, 20 and 25 kV accelerating voltage. The effect of voltage on the calibration, plotting the coefficient of correlation (r) as a function of voltage, was not dependent on the voltage in the range 10-25 kV for Ca standards. K standards were also independent when P/B was corrected for Z2/A. Background counts in the otoconia (Bo) were obtained at 5, 25, 50, 100, 200 and 500 s and used to test the electron beam sensitivity of saccular and utricular otoconia. Bo was not dependent on the spectra acquisition time, with the exception of Bo under K alpha K peak in the saccule at 10 kV. Ca and K WF were determined at 10, 15, 20 and 25 kV in the saccule and utricle, showing similar values regardless of the voltage used. This method of calibration offers several advantages, such as stability, homogeneity, known composition of the standards, high reproducibility at different voltages even without Z2/A correction and the similarity between the otoconia and crystal standards. We recommend the application of this method for other elements and biomineral systems.

Animals↗

Metabolite ratios to assumed stable creatine level may confound the quantification of proton brain MR spectroscopy.

In localized brain proton MR spectroscopy ((1)H-MRS), metabolites' levels are often expressed as ratios, rather than as absolute concentrations. Frequently, their denominator is the creatine [Cr], which level is explicitly assumed to be stable in normal as well as in many pathologic states. The rationale is that ratios self-correct for imager and localization method differences, gain instabilities, regional susceptibility variations and partial volume effects. The implicit assumption is that these benefits are worth their cost(w)-(w) propagation of the individual variation of each of the ratio's components. To test this hypothesis, absolute levels of N-acetylaspartate [NAA], choline [Cho] and [Cr] were quantified in various regions of the brains of 8 volunteers, using 3-dimensional (3D) (1)H-MRS at 1.5 T. The results show that in over 50% of approximately 2000 voxels examined, [NAA]/[Cr] and [Cho]/[Cr] exhibited higher coefficients of variations (CV) than [NAA] and [Cho] individually. Furthermore, in approximately 33% of these voxels, the ratios' CVs exceeded even the combined constituents' CVs. Consequently, basing metabolite quantification on ratios and assuming stable [Cr] introduces more variability into (1)H-MRS than it prevents. Therefore, its cost exceeds the benefit.

Adult↗

Simultaneous quantitation of rosuvastatin and gemfibrozil in human plasma by high-performance liquid chromatography and its application to a pharmacokinetic study.

A simple, sensitive and specific high-performance liquid chromatography method is described for simultaneous determination of rosuvastatin (RST) and gemfibrozil (GFZ) in human plasma using celecoxib as an internal standard (IS). The assay procedure involved extraction of RST, GFZ and IS from plasma into acetonitrile. Following separation and evaporation of the organic layer the residue was reconstituted in the mobile phase and injected onto an X-Terra C(18) column (4.6 x 150 mm, 5.0 microm). The chromatographic run time was less than 20 min using flow gradient (0.0-1.60 mL/min) with a mobile phase consisting of 0.01 M ammonium acetate:acetonitrile:methanol (50:40:10, v/v/v) and UV detection at 275 nm. Nominal retention times of RST, GFZ and IS were 6.7, 13.9 and 16.4 min, respectively. Absolute recovery of both analytes and IS was greater than 90%. The lower limit of quantification (LLOQ) of RST and GFZ was 0.03 and 0.30 microg/mL, respectively. Linearity was excellent (r(2) = 0.999) in the 0.03-10 microg/mL and 0.3-100 microg/mL ranges for RST and GFZ, respectively. The inter- and intra-day precisions in the measurement of RST quality control (QC) samples 0.03, 0.09, 2.50 and 8.00 microg/mL were in the range 2.37-9.78% relative standard deviation (RSD) and 0.92-10.08% RSD, respectively. Similarly, the inter- and intra-day precisions in the measurement of GFZ quality control (QC) samples 0.30, 0.90, 25.0 and 80.0 microg/mL were in the ranges 2.79-6.27 and 0.96-9.69% RSD, respectively. Accuracies in the measurement of QC samples for RST and GFZ were in the range 85.43-107.23 and 84.98-102.35% respectively, of the nominal values. RST and GFZ were stable in the array of stability studies viz., bench-top, auto-sampler and freeze-thaw cycles. Stability of RST and GFZ was established for 1 month at -80C. The application of the assay in an oral pharmacokinetic study in rats co-administered with RST and GFZ is described.

Animals↗

Quantification of various growth factors in different demineralized bone matrix preparations.

Besides autografts, allografts, and synthetic materials, demineralized bone matrix (DBM) is used for bone defect filling and treatment of non-unions. Different DBM formulations are introduced in clinic since years. However, little is known about the presents and quantities of growth factors in DBM. Aim of the present study was the quantification of eight growth factors important for bone healing in three different "off the shelf" DBM formulations, which are already in human use: DBX putty, Grafton DBM putty, and AlloMatrix putty. All three DBM formulations are produced from human donor tissue but they differ in the substitutes added. From each of the three products 10 different lots were analyzed. Protein was extracted from the samples with Guanidine HCL/EDTA method and human ELISA kits were used for growth factor quantification. Differences between the three different products were seen in total protein contend and the absolute growth factor values but also a large variability between the different lots was found. The order of the growth factors, however, is almost comparable between the materials. In the three investigated materials FGF basic and BMP-4 were not detectable in any analyzed sample. BMP-2 revealed the highest concentration extractable from the samples with approximately 3.6 microg/g tissue without a significant difference between the three DBM formulations. In DBX putty significantly more TGF-beta1 and FGFa were measurable compared to the two other DBMs. IGF-I revealed the significantly highest value in the AlloMatrix and PDGF in Grafton. No differences were accessed for VEGF. Due to the differences in the growth factor concentration between the individual samples, independently from the product formulation, further analyzes are required to optimize the clinical outcome of the used demineralized bone matrix.

Bone Matrix↗

GABA-like immunoreactivity in different cellular populations of cerebellar cortex of rats before and after treatment with amino-oxyacetic acid.

The postembedding immunogold procedure was used to detect changes in the levels of gamma-aminobutyric acid (GABA)-like immunoreactivity at the ultrastructural level in the cerebellar cortex of control rats and rats treated with the GABA transaminase inhibitor, amino-oxyacetic acid (AOAA), in order to increase the levels of GABA. GABA-immunoreactive structures were labelled using an antiserum directed against GABA coupled to bovine serum albumin and a secondary antibody conjugated to colloidal gold. The density of gold particles per square micron of tissue was taken as a measure of GABA-like immunoreactivity. In separate groups of control and AOAA-treated animals, the levels of GABA were assessed biochemically in the cerebellum, the cortex, the ventral mesencephalon and the striatum. Six hours after treatment with AOAA the GABA levels in the cerebellum, the cortex, the ventral mesencephalon and the striatum. Six hours after treatment with GABA immunoreactivity of the Golgi and basket cell terminals was significantly greater than that of mossy fibres, granule cell dendrites and perikarya and glial cells. The value obtained for Golgi terminals was the highest of all the structures examined and was twice that of their perikarya. Six hours after treatment with AOAA the GABA immunoreactivity in Golgi and basket cell terminals and in glial cells was greatly enhanced. The drug treatment slightly enhanced the immunoreactivity in mossy fibres and granule cell dendrites but induced no change in granule cell bodies. Thus, in both control and treated rats, the highest GABA immunoreactivity was present in the terminals of GABAergic cells, and the lowest in putative glutamatergic cells. The results demonstrate that there is a high degree of selectivity in the changes in GABA levels following the inhibition of GABA transaminase in the cerebellum. They also confirm the potential of the use of postembedding methods for the quantification of endogenous amino acid at cellular and subcellular levels, in relative and possibly also absolute terms.

4-Aminobutyrate Transaminase↗

Sensitive method for the determination of baclofen in plasma by means of solid-phase extraction and liquid chromatography-tandem mass spectrometry.

A simple and sensitive method for the determination of baclofen in plasma is described. Baclofen and the internal standard, KM 08205, were isolated from plasma by solid-phase extraction using C18 material. After separation by reversed-phase liquid chromatography, the analytes were detected with tandem mass spectrometry. The extraction procedure was optimised regarding the solid-phase extraction material, the pH in the conditioning solution and the washing volume. The method was proven to be selective and sensitive with an absolute recovery of about 95%, a relative standard deviation below 5% and a limit of quantification of 10 nmol/1.

Baclofen↗