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Results for “Absolute quantification”

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PubMed2006

Direct cancer tissue proteomics: a method to identify candidate cancer biomarkers from formalin-fixed paraffin-embedded archival tissues.

Successful treatment of multiple cancer types requires early detection and identification of reliable biomarkers present in specific cancer tissues. To test the feasibility of identifying proteins from archival cancer tissues, we have developed a methodology, termed direct tissue proteomics (DTP), which can be used to identify proteins directly from formalin-fixed paraffin-embedded prostate cancer tissue samples. Using minute prostate biopsy sections, we demonstrate the identification of 428 prostate-expressed proteins using the shotgun method. Because the DTP method is not quantitative, we employed the absolute quantification method and demonstrate picogram level quantification of prostate-specific antigen. In depth bioinformatics analysis of these expressed proteins affords the categorization of metabolic pathways that may be important for distinct stages of prostate carcinogenesis. Furthermore, we validate Wnt-3 as an upregulated protein in cancerous prostate cells by immunohistochemistry. We propose that this general strategy provides a roadmap for successful identification of critical molecular targets of multiple cancer types.

Amino Acid Sequence↗

PubMed2002

Study of carbon fibres and carbon-carbon composites by scanning thermal microscopy.

Scanning thermal microscopy (SThM) is a relatively new technique based on atomic force microscopy in which the tip is replaced by an ultra-miniature temperature probe. This paper reports on a preliminary investigation of the application of SThM in the characterization of the thermal properties of carbon fibres and carbon-carbon (CC) composites. The technique enabled a comparative study to be made of discrete fibre and matrix thermal properties in a series of model unidirectional composites. The thermal images revealed a marked increase in thermal conductivity of the matrix with increasing temperature of treatment and hence confirmed the development of a highly ordered carbon matrix. The results were in qualitative agreement with previously determined values of thermal conductivity from which the separate values of fibre and matrix thermal conductivity had been derived. The technique was also applied to the characterization of samples of unknown processing history, enabling an estimation to be made of the heat treatment and type of the fibres and matrix present in the composite. It was concluded that SThM promises to be a powerful technique for the study of the thermal properties of CC composites and carbon fibres, as it uniquely enables variations in local thermal conductivity to be detected and resolved. Absolute quantification of the technique remains the key to its future widespread acceptance in materials characterization.

Journal Article↗

PubMed2001

Prolonged vigabatrin treatment modifies developmental changes of GABA(A)-receptor binding in young children with epilepsy.

PURPOSE: To determine whether prolonged treatment with vigabatrin (VGB), an antiepileptic drug (AED) that acts by elevating brain gamma-aminobutyric acid (GABA) levels, interferes with age-related changes of in vivo GABA(A)-receptor binding in children with epilepsy. METHODS: Using [11C]flumazenil (FMZ)-positron emission tomography (PET) imaging, 15 children (aged 1-8 years) with medically intractable epilepsy were studied. Seven of these children were treated with VGB (1,000-2,500 mg/day) for > or =3 months before the FMZ-PET study. The remaining eight patients were medicated with other drugs that are known not to act directly on the GABAergic system. Absolute quantification of PET data was performed by using the volume of distribution (VD) of FMZ in brain tissue representing FMZ ligand binding. RESULTS: After controlling for age, hemispheric FMZ VD values were significantly lower in children treated with VGB as compared with the non-VGB group (p = 0.012). Regional FMZ VD values of the VGB-treated patients were significantly lower in all cortical regions and the cerebellum, whereas the difference was not significant in the thalamus and basal ganglia. No significant drug effect or drug-by-region interaction could be determined when the patients were separated according to treatment with carbamazepine (p = 0.97) or valproate (p = 0.55). CONCLUSIONS: VGB induces a decrease in GABA(A)-receptor binding in the cortex and cerebellum of the developing epileptic brain. A similar effect of other drugs and substances of abuse targeting the GABAergic system may be hypothesized. Because of the important role of the GABAergic system in developmental plasticity, the reversibility and functional consequences of this age-specific drug effect should be further studied.

Age Factors↗

PubMed2004

Metabolite pattern of Cysticercus cellulosae metacestode from different predilection sites of swine using proton magnetic resonance spectroscopy.

Cysticercosis due to Taenia solium is one of the most common public health problems in various regions of the world. We have performed prolon magnetic resonance spectroscopy (1H MRS) experiments of the fluid aspirated from cysticerci excised from skeletal muscle (n = 16) and brain (n = 9) of infected swine to compare the metabolite pattern of cysticerci in different predilection sites. Perchloric acid extract of cysticercus cysts excised from skeletal muscles (n = 16) was also prepared to ascertain water-soluble, low molecular weight metabolites using 1H MRS. Absolute quantification and statistical analysis of different metabolites was done to look for any significant differences in different locations of cysts. The metabolite pattern of cysticerci was found to be similar in the various predilection sites. Metabolites observed were leucine, valine, alanine, lysine, glycine, lipid contents, lactate, glutamate, acetate, succinate, creatine, choline, and glucose. Concentration of creatine in cysticercus fluid of cysts removed from the muscle was found to be significantly higher (p = 0.001) than the cysts located in the brain. We conclude that the metabolite pattern in the cysticerci is not influenced by the surrounding tissue location; however concentration of certain metabolites may depend upon the tissue location.

Animals↗

PubMed2006

MRI of myocardial perfusion.

An overwhelming number of myocardial perfusion studies are done by nuclear isotope imaging. Magnetic resonance imaging during the first pass of an injected, contrast bolus has some significant advantages for detection of blood flow deficits, namely higher spatial resolution, absence of ionizing radiation, and speed of the test. Previous clinical studies have demonstrated that excellent sensitivity and specificity can be achieved with MR myocardial perfusion imaging for detecting coronary artery disease, and assessment of patients with acute chest pain. Furthermore, an absolute quantification of myocardial blood flow is feasible, as was demonstrated by comparison of MR perfusion imaging, to measurements with isotope labeled microspheres in experimental models. An integrated assessment of perfusion, function, and viability, is thus feasible by MRI to answer important clinical challenges such as the identification of stunned or hibernating, but viable myocardium.

Contrast Media↗

PubMed1999

[The simultaneous determination of the arterial input function for dynamic susceptibility-weighted magnetic resonance tomography of the A. carotis interna and the A. cerebri media].

PURPOSE: The determination of the arterial input function (AIF) is necessary for absolute quantification of the regional cerebral blood volume and blood flow using dynamic susceptibility contrast MRI. The suitability of different vessels (ICA--internal carotid artery, MCA--middle cerebral artery) for AIF determination was compared in this study. METHODS: A standard 1.5 T MR system and a simultaneous dual FLASH sequence (TR/TE1/TE2/alpha = 32/15/25/10 degrees) were used to follow a bolus of contrast agent. Slice I was chosen to cut the ICA perpendicularly. Slice II included the MCA. Seventeen data sets from ten subjects were evaluated. RESULTS: The number of AIF-relevant pixels, the area under the AIF and the maximum concentration were all lower when the AIF was determined from the MCA compared to the ICA. Additionally, the mean transit time (MTT) and the time to maximum concentration (TTM) were longer in the MCA, complicating the computerized identification of AIF-relevant pixels. Data from one subject, who was examined five times, demonstrated that the intraindividual variance of the measured parameters was markedly lower than the interpersonal variance. CONCLUSIONS: It appears to be advantageous to measure the AIF in the ICA rather than the MCA.

Adult↗

PubMed2004

[Contrast-enhanced 3D MR perfusion of the lung: application of parallel imaging technique in healthy subjects].

PURPOSE: Evaluation of lung perfusion by contrast-enhanced 3D MRI using partial parallel imaging techniques. MATERIALS AND METHODS: Eight healthy volunteers were examined using a contrast-enhanced dynamic FLASH 3D sequence with partial parallel imaging technique at 1.5 T MRI with a TA of 1.5 sec. The whole lung was covered by 36 coronal slices. A ventral, middle and dorsal slice of each lung was manually segmented and signal-to-time curves were computed. For absolute quantification of blood flow through the right and left pulmonary artery, phase-contrast flow measurements were performed. RESULTS: No significant difference was found between the signal intensity in the right (8.9 +/- 2.6) and left (8.0 +/- 3.5) lung, corresponding to a left-to-right signal intensity ratio of 0.9. A significantly higher signal intensity was found in the dorsal regions of the lungs (p = 0.01) compared to the ventral regions. The time to peak of the signal intensity was significantly shorter in the dorsal (15.3 sec) and middle (15.7 sec) regions of the lungs (p = 0.03 and p = 0.04, respectively) than in the ventral regions (16.3 sec). The ratio between blood flow through the left (2.2 L/min) and right (2.7 L/min) lung was 0.84. CONCLUSION: Partial parallel image acquisition can assess the perfusion of the lungs at high temporal resolution. The perfusion is slightly higher on the right than on the left. The signal increases faster and has a higher peak in the dorsal lung regions.

Adolescent↗

PubMed2004

[A software program for quantitative analysis of alveolar oxygen partial pressure (p(A)O(2)) with oxygen-sensitive (3)He-MRI].

PURPOSE: To develop a software tool for quantitative analysis of alveolar oxygen partial pressure (p(A)O(2)) as well as its time course during apnea. MATERIAL AND METHODS: T (1)-relaxation times of hyperpolarized (3)He are reduced by paramagnetic oxygen rendering (3)He-MRI sensitive to oxygen and thus allowing the assessment of the local oxygen partial pressure in the pulmonary airspaces. Oxygen-related relaxation and loss of polarization by RF-excitation can be discriminated by acquiring two image series with varying interscan delay and/or flip angles. Software was developed to calculate the p(A)O(2) and the decay rate in user-defined regions of interest (ROIs) automatically. Moreover, parameter maps can be calculated. In addition to the analysis of 2-dimensional data sets, the software allows the evaluation of 3-dimensional measurements for the first time. Artifacts due to lung motion were reduced by implementing a motion correction algorithm. RESULTS: The software was successfully applied to data sets from healthy volunteers and from patients with various lung diseases. The parameter maps demonstrated a more homogeneous distribution of p(A)O(2) for the volunteers than for the patients. A regional increase in p(A)O(2) was found in a few patients. CONCLUSION: The described software allows the absolute quantification of p(A)O(2) as well as its variation over time. In the future, therefore, the software may gain importance for detecting mismatches between ventilation and perfusion, e. g., in patients with pulmonary embolism or chronic obstructive lung diseases.

Adult↗

PubMed1998

Near-infrared spectrophotometry of the brain in cardiovascular surgery.

Neuropsychological and neurological deficits are still major causes of mortality and morbidity after cardiac operations and are thought to be caused by embolism and cerebral hypoxia. Near-infrared spectrophotometry (NIRS) is a promising method for non-invasive monitoring of cerebral oxygenation and hemodynamics. Different devices provide information on changes of oxygenated (HbO2) and deoxygenated hemoglobin (Hb), oxidized cytochrome aa3 (CytOx) or regional oxygen saturation (rSO2). NIRS has been applied to patients during adult and pediatric cardiovascular surgery with and without deep hypothermic circulatory arrest (DHCA). In most of the studies, significant changes in cerebral oxygenation were detected by NIRS. NIRS measurements were influenced by the cerebral oxygen metabolism and the operative management. However, clinical, experimental, and theoretical issues raise doubts as to the clinical relevance of the hemoglobin saturation (HbO2, Hb, rSO2 signals) during hypothermia and alkalosis, because the oxygen affinity of hemoglobin increases and a high saturation might simply reflect the inadequate oxygen transport into cells. In contrast, recent experiments have proved a high correlation between the CytOx signal and the MRS parameters nucleoside triphosphate and phosphocreatine. Histological damage was significantly related to the lowest CytOx value; in a clinical study it predicted impaired neuropsychological outcome. Therefore, the CytOx signal is of great interest for future studies. NIRS must prove its ability to diagnose cerebral hypoxia consistently during cardiac surgery in a large patient study before this method is brought into routine clinical practice. Absolute quantification and definitions of critical oxygenation margins will be helpful for this goal.

Adult↗

PubMed1997

[Results and consequences of frequent quality control of quantitative clinical 1H-MR-spectroscopy].

Different methods exist for an absolute quantification of in vivo proton MR spectra. According to the principle of reciprocity a simple method has been suggested previously, comprising the multiplication of the spectral signals with the unlocalised reference amplitude of the measurement as a correction for the different coil load. Afterwards, the result can be compared to measurements on phantoms in order to obtain in vivo metabolite concentrations. Thus, only one calibration would be sufficient in principle for all measured spectra. However, during a previous serial study on humans and phantoms we observed significant changes in transmitter amplitudes over time, although metabolite peak areas remained quite constant. Hence, the method led to incorrect metabolite concentrations. Since these effects are mostly underestimated or even neglected in clinical spectroscopy, the purpose of the present study was to investigate these instabilities over time in order to find a new correction method for quantitative MRS. According to this new method, the spectra corrected by the coil load have to be modified additionally by dividing by the reference amplitude of a phantom.

Humans↗

PubMed1991

[Modern nuclear medical diagnosis as applied pathophysiology. Basis and outlook].

In their capacity as "image-assisted functional diagnostics", methods of nuclear medicine link morphological patterns of radiology with clinical presentation. Based on pathophysiology they supply an insight into both global and regional parameters, present as basal values or as reserves. Both, single photon emission computed tomography (SPECT) or highly defined positron ECT (PET), enabled computer-assisted topographical overlay and thus an exact comparative evaluation of regional function versus morphology. In addition, PET gives access to a true physiological, absolute quantification employing process specific, carrier-free substrates. Novel tracers (markers of metabolism or perfusion, receptors or transmitter substances, monoclonal antibodies), an intraluminal transport model and the concept of buffer or working reserves aim at conducting early diagnosis, determination of disease severity and parameters that are relevant for therapy, on a level that is pathophysiologically oriented.

Brain Diseases↗

PubMedDate not supplied

Assessment of myocardial viability in dysfunctional myocardium by resting myocardial blood flow determined with oxygen 15 water PET.

BACKGROUND: There is controversy about the role of decreased resting blood flow as the pathophysiologic correlate of hibernating myocardium. The aim of this study was an absolute quantification of volumetric myocardial blood flow (MBFvol) in dysfunctional myocardium with different viability conditions as defined by fluorine 18 deoxyglucose (FDG) positron emission tomography (PET) while taking into consideration the functional recovery after revascularization. The impact of MBFvol in the diagnosis of functional recovery was also investigated. METHODS AND RESULTS: Forty-two patients with severe coronary artery disease and dysfunctional myocardium underwent resting oxygen 15 water PET, as well as FDG PET and technetium 99m tetrofosmin single photon emission computed tomography, all attenuation-corrected. Relative FDG and Tc-99m tetrofosmin uptake (normalized to the segment with 100% Tc-99m tetrofosmin uptake), as well as MBFvol (myocardial blood flow multiplied by the water-perfusable tissue fraction to account for the flow to the entire segment volume), were determined in 18 myocardial segments per patient. Viability in dysfunctional segments (estimated by ventriculography) with reduced Tc-99m tetrofosmin uptake of 70% or lower was classified as viable (FDG >70%, mismatch) or nonviable (FDG < or =70%, match). Fifteen patients underwent revascularization and were followed up. Mismatch segments with improved function were classified as hibernating myocardium. Mean MBFvol in viable myocardium was slightly reduced (0.60 +/- 0.02 mL x min(-1) x mL(-1)) compared with that in normokinetic myocardium (0.64 +/- 0.01 mL x min(-1) x mL(-1)) (P = .036) and was significantly higher than in nonviable myocardium (0.36 +/- 0.01 mL x min(-1) x mL(-1)) (P < .001). Receiver operating characteristic analysis confirmed an FDG uptake greater than 70% as the optimal threshold to predict functional recovery (diagnostic accuracy [ACC], 76%). MBFvol in hibernating myocardium (0.62 +/- 0.04 mL x min(-1) x mL(-1)) was not significantly reduced compared with that in normokinetic myocardium (0.66 +/- 0.02 mL x min(-1) x mL(-1)) and was significantly higher than in persistently dysfunctional myocardium (0.51 +/- 0.04 mL x min(-1) x mL(-1)) (P < .05). The ACC of MBFvol greater than 0.40 mL x min(-1) x mL(-1) as the threshold to predict functional recovery was 61% but did not improve the accuracy of FDG PET by itself. CONCLUSIONS: In patients with severe coronary artery disease and dysfunctional myocardium, MBFvol as determined with O-15 water differs significantly between viable and nonviable myocardium as determined by FDG PET and is not significantly reduced in hibernating compared with normokinetic myocardium. Therefore chronically reduced resting blood flow appears unlikely to be the pathophysiologic correlate of the functional state of hibernation. However, MBFvol does not improve the ACC of FDG PET by itself.

Adult↗

PubMed2003

A high-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS.

We report here an approach for gene expression analysis by combining competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS. A DNA standard is designed with an artificial single nucleotide polymorphism in the gene of interest. The standard is added to the reverse transcription product before PCR. Subsequently, a base extension reaction is carried out at the single nucleotide polymorphism position, and the products are quantified by matrix-assisted laser desorption ionization time-of-flight MS. The approach is capable of relative and absolute quantification of gene expression; it is extremely sensitive (as few as five copies of DNA were quantified) and highly reproducible. It is also capable of simultaneous quantification of both alleles for heterozygotes and alternatively spliced genes. We have incorporated this technique with the homogeneous Mass Extension system (Sequenom) to create a high-throughput, automated gene expression analysis platform where a few hundred genes from 20-500 different samples can be accurately quantified per day.

Base Sequence↗

PubMed1994

Fine mapping of a replication origin of human DNA.

A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of mammalian cells. The method, which does not require cell synchronization or permeabilization, entails the absolute quantification, by a competitive PCR procedure in newly synthesized DNA samples, of the abundance of neighboring DNA fragments distributed along a given genomic region. This procedure was utilized for mapping the start site of DNA replication in a 13.7-kb region of human chromosome 19 coding for lamin B2, which is replicated immediately after the onset of S phase in HL-60 cells. Within this region, DNA replication initiates in a 474-bp area corresponding to the 3' noncoding end of the lamin B2 gene and the nontranscribed spacer between this gene and the 5' end of another highly transcribed one. This localization was obtained both in aphidicolin-synchronized and in exponentially growing HL-60 cells.

Animals↗

PubMed2005

Differential multisite phosphorylation of the trehalose-6-phosphate synthase gene family in Arabidopsis thaliana: a mass spectrometry-based process for multiparallel peptide library phosphorylation analysis.

Multisite protein phosphorylation plays a fundamental role in metabolic regulation. To detect and quantify in vitro kinase phosphorylation activities, we developed a highly selective LC-MS/MS-based method using high resolution multiple reaction monitoring on a triple quadrupole mass spectrometer. This method eliminates the need for stable isotope labeling and enables multiparallel kinase target assays. Using these assays, we made the first observation of in vitro phosphorylation of different trehalose-6-phosphate synthase (TPS) isozymes. TPSs possess putative Ca2+-independent, sucrose non-fermenting 1-related protein kinase 1 (SnRK1) phosphorylation sites. Sixteen synthetic peptides from six different Arabidopsis thaliana TPS isozymes containing the SnRK1 consensus recognition motif were phosphorylated simultaneously in vitro, and their phosphorylation dynamics were determined. We achieved absolute quantification of TPS peptide phosphorylation by tuning the mass spectrometer to the corresponding synthetic standard phosphopeptides. The selectivity of the mass spectrometer in the multiple reaction monitoring mode compensates for the low ionization efficiency of phosphopeptides in the presence of a complex matrix. Results are in close agreement with recent in vivo studies of TPS phosphorylation and regulation and reveal significant differences in the phosphorylation levels of different TPS members within the TPS gene family ranging over 3 orders of magnitude. Substituting EGTA for CaCl2 in the reaction mixture reduced the formation of some of the phospho-TPS peptides drastically, indicating that Ca2+-dependent kinases are active in the presence of Ca2+-independent SnRKs. This agrees with the proposed overlap of the consensus motifs of these kinases and enables delineation between Ca2+-independent and Ca2+-dependent phosphorylation. Results demonstrate that multiparallel kinase target assays are sensitive enough to provide evidence for differential multisite phosphorylation of homologous TPS proteins and their highly conserved putative phosphorylation sites.

Amino Acid Sequence↗

PubMed2005

16S rDNA analyses of the cyanobacterial microbiota through the water-column in a boreal lake with a metalimnic Planktothrix population.

The Planktothrix population in Lake Steinsfjord has attracted particular attention, due to the potential development of toxic blooms. This population is special in the sense that mass developments of Planktothrix occur in the metalimnion. We investigated the distribution of Planktothrix, as well as other cyanobacteria, through the water-column during a Planktothrix mass development at 10-16 m depth. The analyses were done by chlorophyll measurements, microscopy, and by a recently developed 16S rDNA array-based method. These analyses showed that Planktothrix dominated the cyanobacterial community at 11 m, while cyanobacteria belonging to the order Nostocales were predominant at 4 m. The combination of analytical methods presented in this work provides a powerful tool to analyze cyanobacterial communities. We have developed a concept that enables both relative (16S rDNA array analyses) and absolute quantification (chlorophyll a measurements) of cyanobacteria through water-columns. Such approaches will be important in better understanding cyanobacterial microbiota and bloom dynamics.

Anabaena↗

PubMed1984

Ultraviolet detection of synthetic oil contamination of skin.

A study was conducted to evaluate skin contamination occurring on coal liquefaction process workers using quantitative (Spill Spotter) and qualitative (blacklight) ultraviolet fluorescence methods. Other objectives were to determine the effectiveness of showering in removing contamination, and to determine the quantitative relationship between Spill Spotter response and skin contamination concentration. Field measurements showed wide ranges of background skin fluorescence caused by differing skin types. Visible dirt on the skin partially quenched the fluorescence response of the Spill Spotter. Fluorescent skin contamination on selected workers was quantified relative to a calibration curve developed on pigskin using a reference synfuel oil. One group of workers was shown to exhibit a two-to three-fold increase in fluorescence during the work shift. Showering removed skin contamination although residual contamination sometimes remained on workers that exhibited the greatest degree of skin contamination during the workshift. Further refinement of the ultraviolet method for absolute quantification of fluorescent skin contamination is needed.

Baths↗

PubMed2006

Comparative proteomics of clathrin-coated vesicles.

Clathrin-coated vesicles (CCVs) facilitate the transport of cargo between the trans-Golgi network, endosomes, and the plasma membrane. This study presents the first comparative proteomics investigation of CCVs. A CCV-enriched fraction was isolated from HeLa cells and a "mock CCV" fraction from clathrin-depleted cells. We used a combination of 2D difference gel electrophoresis and isobaric tags for relative and absolute quantification (iTRAQ) in conjunction with mass spectrometry to analyze and compare the two fractions. In total, 63 bona fide CCV proteins were identified, including 28 proteins whose association with CCVs had not previously been established. These include numerous post-Golgi SNAREs; subunits of the AP-3, retromer, and BLOC-1 complexes; lysosomal enzymes; CHC22; and five novel proteins of unknown function. The strategy outlined in this paper should be widely applicable as a means of distinguishing genuine organelle components from contaminants.

Adaptor Proteins, Vesicular Transport↗