SEARCH · PubMed Health

Results for “Absolute quantification”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,009 records · Page 56Linked to original sources

PubMed1990

Measurement of alpha-ketoglutarate dehydrogenase activity in tissue extracts and human platelets using reversed-phase high-performance liquid chromatography.

A new method for the determination of the activity of alpha-ketoglutarate dehydrogenase complex (KGDHC) in mouse brain and liver mitochondria and in human platelets using reversed-phase high-performance liquid chromatography is described. This method is based on the quantification of succinyl-CoA formed in the reaction catalyzed by KGDHC. Succinyl-CoA was separated using a YMC-Pack C8 column employing isocratic elution and detected spectrophotometrically at 254 nm. The detection limit of succinyl-CoA was 0.05 nmol. Succinyl-CoA in the supernatant of the assay mixture was stable for several hours at 4 degrees C and for a week when stored at -20 degrees C. The KGDHC assay showed good linearity with time and added protein, and all tissues demonstrated an absolute requirement for added alpha-ketoglutarate, nicotinamide dinucleotide, and coenzyme A and partial or no requirement for thiamine pyrophosphate, magnesium chloride, and dithiothreitol. The specific activities in liver and brain mitochondria and platelet homogenates determined by the present method were 19.2 +/- 0.9, 18.1 +/- 2.8, and 2.6 +/- 0.3 nmol/min/mg protein, respectively. In human platelets, the present method gives higher specific activity and lower blank values than a prior method using 14CO2 and may be useful in the diagnosis of KGDHC deficiency. This method is simple, rapid, and can be readily employed for the determination of KGDHC activity in various animal tissues and human platelets.

Acyl Coenzyme A↗

PubMed2006

Bacterial fermentation of recombinant major wasp allergen Antigen 5 using oxygen limiting growth conditions improves yield and quality of inclusion bodies.

A process for bacterial expression and purification of the recombinant major wasp allergen Antigen 5 (Ves v 5) was developed to produce protein for diagnostic and therapeutic applications for type 1 allergic diseases. Special attention was focused on medium selection, fermentation conditions, and efficient refolding procedures. A soy based medium was used for fermentation to avoid peptone from animal origin. Animal-derived peptone required the use of isopropyl-beta-D-thiogalactopyranoside (IPTG) for the induction of expression. In the case of soy peptone, a constitutive expression was observed, suggesting the presence of a component that mimics IPTG. Batch cultivation at reduced stirrer speed caused a reduced biomass due to oxygen limitation. However, subsequent purification and processing of inclusion bodies yielded significantly higher amount of product. Furthermore, the protein composition of the inclusion bodies differed. Inclusion bodies were denatured and subjected to diafiltration. Detailed monitoring of diafiltration enabled the determination of the transition point. Final purification was conducted using cation-exchange and size-exclusion chromatography. Purified recombinant Ves v 5 was analyzed by RP-HPLC, CD-spectroscopy, SDS-PAGE, and quantification ELISA. Up to 15 mg highly purified Ves v 5 per litre bioreactor volume were obtained, with endotoxin concentrations less than 20 EU mg(-1) protein and high comparability to the natural counterpart. Analytical results confirm the suitability of the recombinant protein for diagnostic and clinical applications. The results clearly demonstrate that not only biomass, but especially growth conditions play a key role in the production of recombinant Ves v 5. This has an influence on inclusion body formation, which in turn influences the renaturation rate and absolute product yield. This might also be true for other recombinant proteins that accumulate as inclusion bodies in Escherichia coli.

Allergens↗

PubMed2002

High-performance liquid chromatographic analysis of chrysin derivatives on a Nova-Pak C18 column.

A high-performance liquid chromatographic method has been developed for the separation and quantification of chrysin and synthetic chrysin derivatives (12 chrysin alkyl and 7 chrysin acyl derivatives). The chromatography was performed using a Nova-Pak C18 column. A RP-HPLC was performed by using a binary mixture (MeOH-10 mM H3PO4) as a mobile phase, and the column temperature was maintained at room temperature. A flow rate was 1.0 ml/min, and the effluent was monitored at a wavelenth of 280 nm. The retention times for chrysin acyl and alkyl derivatives were within 10 minutes and 20 minutes, respectively. The absolute recovery of samples were all over 96%. The detection limits were 0.1-18 ng at S/N = 3 ratio.

Carbon Radioisotopes↗

PubMed2006

Lipoprotein distribution and biological variation of 24S- and 27-hydroxycholesterol in healthy volunteers.

24S- and 27-hydroxycholesterol are obligatory intermediates of cholesterol catabolism and play an important role in the maintenance of whole-body cholesterol homeostasis. Using an HPLC-MS method for oxysterol quantification, the distribution of esterified and unesterified oxysterols in lipoprotein subfractions as well as the influence of daytime, food intake and menstrual cycle on oxysterol concentrations were investigated in healthy volunteers. Moreover, reference intervals for 24S- and 27-hydroxycholesterol in plasma as well as the corresponding levels for 27-hydroxycholesterol in the HDL subfraction were established in 100 healthy volunteers. Both circulating oxysterols are mainly transported in association with HDL and LDL--primarily in the esterified form. No significant diurnal changes and no variations during menstrual cycle of either absolute or cholesterol-related plasma levels were detected. In contrast to 24S-hydroxycholesterol in plasma and 27-hydroxycholesterol in the HDL subfraction, the 95% reference intervals of 27-hydroxycholesterol both in plasma and the non-HDL subfraction were higher in males than in females. The concentrations of 27-hydroxycholesterol in plasma and the non-HDL subfraction showed strong positive correlations with the concentrations of cholesterol, non-HDL cholesterol and triglycerides. Our data on the lipoprotein distribution of oxysterols as well as on their intra- and inter-individual variation set the stage for future clinical studies.

Adult↗

PubMed1998

Influence of lipoprotein(a) plasma concentration on neointimal growth in a monkey model of vascular injury.

Lipoprotein(a) [Lp(a)] has been proposed as a risk factor for both restenosis and coronary heart disease. Recently, we identified Lp(a) in the arterial wall during the initial rapid neointimal growth phase that occurs after balloon injury in cynomolgus monkeys. The purpose of this study was to determine the relationship between circulating Lp(a) levels and the extent of early neointimal formation. Initially, 348 cynomolgus monkeys were screened to identify 15 monkeys that had either high or low circulating Lp(a) levels. In the 15 monkeys, circulating Lp(a) levels were confirmed by two separate measurements over 6 weeks using an immunoturbidimetric assay. Cohorts were identified with plasma Lp(a) levels that differed by four fold. Lp(a) levels expressed as total mass averaged 32 +/- 4 (N = 8) and 136 +/- 12 (N = 7) mg/dl in the low and high groups, respectively. Between the two assays absolute Lp(a) levels differed by less than 6%. Iliac arteries were harvested 14 days after injury induced by expansion of the internal vessel diameter 1.4 times its initial size with an angioplasty balloon. The neointimal area in the high Lp(a) monkeys was 16% greater (0.49 +/- 0.12 mm2, N = 8 versus 0.57 +/- 0.10 mm2, N = 7) than in the low animals; however, this difference was not statistically significant (P = 0.63). Medial areas averaged 1.27 +/- 0.11 and 1.44 +/- 0.20 mm2 (P = 0.48) in these groups, respectively. Tissue Lp(a) quantification, using a mouse monoclonal anti-Lp(a) antibody, indicated that the percent total area staining positive for Lp(a) was 1.7-fold higher in the high versus the low Lp(a) group (2.7 +/- 0.4% versus 1.6 +/- 0.4%, N = 6-8); this difference was not statistical significant (P = 0.28). In summary, a four-fold increase in circulating plasma Lp(a) levels did not result in a statistically significant enhanced neointimal formation at 14 days after balloon injury. This finding suggests that studies of longer duration may be needed to amplify the trend toward increased neointimal growth observed in this study.

Animals↗

PubMed1987

Variability of central conduction in the course of multiple sclerosis. Serial recordings of evoked potentials in the evaluation of therapy.

In 27 normal subjects evoked potentials (EPs) were serially measured across an interval of 3.5 months. The variation in latencies of the P100 component of the VEP and of the components N13 and N20 of the SEP did not exceed 12% of the absolute values of these latencies on the first recording. The VEP and SEP were also serially recorded over the same interval in 35 patients with 'clinically definite' multiple sclerosis. Changes of the latencies exceeding those of normal subjects were noted in 30% of the cases. The changes in the EPs showed no correlation with the variations in the overall clinical disability. It is argued that EPs are probably more sensitive to changes in function of parts of the white matter than the clinical examination and are useful in the quantification of these changes. As a model for future studies, EPs were used in the 'evaluation' of ACTH therapy. It appeared, that therapy did not have effect on the EPs.

Adrenocorticotropic Hormone↗

PubMed2003

Quantification of the effect of system and object parameters on edge enhancement in phase-contrast radiography.

The purpose of this study was to evaluate the effects of system parameters (focal spot size, tube voltage, geometry, detector resolution, and image noise) and object characteristics (edge gradient/ shape, composition, thickness, and overlying attenuating material) upon the edge enhancement effect in phase-contrast radiography. Each variable of interest was adjusted and images of a 3 mm lucite phantom were obtained with the other variables remaining constant. A microfocus x-ray source coupled to a CCD camera with an intensifying screen was used to acquire the digital images. Two parameters of image analysis were used to quantify the effects. The edge enhancement index (EEI) was used to measure the absolute degree of edge enhancement, while the edge enhancement to noise ratio (EE/N) was used to measure the conspicuity of the edge enhancement relative to image noise. Little effect on EEI was seen from tube voltage, object thickness, overlying attenuating material, while focal spot size and system geometry demonstrated measurable effects upon the degree of edge enhancement. It was also shown that while the edge enhancement effect over straight edges is highly dependent upon how the edge aligns with the x-ray beam, rounded edges, which better model biological objects, do not suffer from this dependence and the EEI reaches its maximal level at any alignment. Decreasing detector resolution diminished the EEI slightly, but even with pixel sizes of 0.360 x 0.360 mm edge enhancement effects were readily visible. The effect of image noise on EE/N was evaluated using different exposure times showing an expected improvement with longer exposure time with EE/N approaching a plateau at 5 min. Many of the parameters that will go into the design of a future PC-R imaging system have been quantified in terms of their effect on the degree of edge enhancement in the acquired image. These results, taken together, indicate that either a specimen or even clinical breast imaging system could be created with currently available technology. The major limitation to a clinical system would be the low x-ray flux from the microfocal x-ray source.

Pattern Recognition, Automated↗

PubMed1987

Sensitive and selective determination of picogram amounts of ciprofloxacin and its metabolites in biological samples using high-performance liquid chromatography and photothermal post-column derivatization.

An analytical method for the detection and quantification of ciprofloxacin and its known metabolites M1, M2, M3 and M4 in urine, serum/plasma, bile, faeces and tissue is described. For the liquid matrices the only sample preparation that is required is dilution. The assay consists of reversed-phase high-performance liquid chromatography and fluorescence detection. For the metabolites M2, M3 and M4 an additional post-column derivatization by successive thermolysis and photolysis is needed. A suitable simple post-column reactor has been constructed. Detection limits for all compounds are between 0.2 and 2.2 ng/ml (absolute detection limits 2-22 pg). Compared with fluorescence detection alone, the derivatization increases the selectivity of detection significantly. The linearity, precision and accuracy of the method were determined.

Bile↗

PubMedDate not supplied

Contribution of the umbilical cord and membranes to untrimmed placental weight.

The extent of placental trimming before weighing varies within and between obstetric units. Quantification of the contribution of the umbilical cord and membranes to placental weight is required to enable measurements to be compared across populations. In a sample of 50 neonates born in Southampton, placentae of liveborn singletons were weighed three times; after removing any obvious blood clots, after cutting the umbilical cord, and after stripping both the foetal and maternal membranes. The correlation between untrimmed and trimmed placental weight was 0.98. Since the combined weight of the cord and membranes increased with increasing trimmed weight (correlation=0.54), the percentage rather than absolute difference between untrimmed and trimmed weight was calculated. The median difference between untrimmed and trimmed weight was 16 per cent. No association was found between the percentage difference and infant sex, duration of gestation, birthweight, maternal age, labour onset and duration, and presentation at delivery. However, the mode of delivery had a significant effect on this difference; medians for vaginal and Caesarean section deliveries were 19 per cent and 14 per cent respectively.

Adult↗

PubMed2001

Quantification of splice variants using real-time PCR.

A reliable and robust method for measuring the expression of alternatively spliced transcripts is an important step in investigating the significance of each variant. So far, accurate quantification of splice variants has been laborious and difficult due to the intrinsic limitations of conventional methods. The many advantages of real-time PCR have made this technique attractive to study its application in quantification of splice isoforms. We use skipping of exon 37 in the NF1 gene as a model to compare and evaluate the different strategies for quantitating splice variants using real-time PCR. An overview of three different possibilities for detecting alternative transcripts is given. We propose the use of a boundary-spanning primer to quantify isoforms that differ greatly in abundance. We describe here a novel method for creating a reliable standard curve using one plasmid containing both alternative transcripts. In addition, we validate the use of an absolute standard curve based on a dilution series of fluorometrically quantified PCR products.

Alternative Splicing↗

PubMed1999

Immunological and nutritional composition of human milk in relation to prematurity and mother's parity during the first 2 weeks of lactation.

BACKGROUND: To investigate the effect of prematurity and parity on the dynamics of the major immunologic and nutritional proteins of human milk over the first 2 weeks of lactation. METHODS: Microparticle-enhanced nephelometric immunoassays were developed for the quantification of alpha-lactalbumin, beta-casein, serum albumin, lactoferrin, and lysozyme in human milk. These components, immunoglobulin A, and total proteins were assayed in 368 individual samples collected from 74 mothers. RESULTS: The dynamics of the major immunologic and nutritional proteins in early lactation presented similar patterns in preterm and term human milks. In comparison with term milk, preterm milk was globally characterized by higher concentrations of immune proteins and lower concentrations of nutritive proteins. These differences were increased by the degree of prematurity, which, however, influenced the absolute and relative protein concentrations differently, depending on the stage of lactation. The protein composition of term milk was similar, whatever the mother's parity. Conversely, the influence of prematurity on the levels of milk proteins during the first days of lactation was even greater in primiparous mothers. CONCLUSIONS: This precise description of the composition of preterm and term milk, regarding the main nutritional and immunologic proteins, confirms the influence of both prematurity and parity on milk components and demonstrates the combined effect of these two conditions.

Female↗

PubMed2002

Segmentation of cancellous bone from high-resolution computed tomography images: influence on trabecular bone measurements.

The quantification of cancellous bone network from computed tomography (CT) images requires a segmentation step which is crucial and difficult because of the partial volume effect in CT images. In this paper, we present and evaluate a new approach for segmenting cancellous bone network from high-resolution CT (HRCT) slices. The idea is first to detect a skeleton from the crest lines of the structure and then to thicken it to extract the whole bone structure by satisfying local neighborhood constraints. The segmentation requires the adjustment of relative and not absolute parameters like most methods. We quantified the influence of these parameters on architectural measurements. Results were first validated by using a physical phantom and then examined on a series of 12 HRCT images of human lumbar vertebra of different ages. We demonstrated that the choice of segmentation parameters yielded important variability on architectural measurements (up to 20%), but less variability than a more commonly used approach. This stresses the importance of settle on the segmentation parameters for once, which is possible with the proposed method.

Adult↗

PubMed1983

A colorimetric assay for quantification of defective interfering particles of respiratory syncytial virus.

A colorimetric assay for defective interfering (DI) particles of respiratory syncytial (RS) virus was developed. This quantitative biological assay is based on neutral red dye uptake by DI particle-protected cells that survive standard virus challenge. This assay was more sensitive than the reduction of infectious yield (RIY) assay and was capable of detecting 1 X 10(4) to 2 X 10(4) DI particles/ml. The coefficient of variation for parallel, simultaneous replicates (n = 10) was 23%. Cell-protecting activity in the colorimetric assay appeared simultaneously with activity in the RIY assay on undiluted passage of plaque-purified virus. Both activities were particulate, were inactivated by RS virus antiserum and exhibited similar ultraviolet-inactivation kinetics. The absolute values of the slopes of dilution curves for both assays were similar, and using regression analysis both assays enabled estimation of similar numbers of active particles. These results suggest that both activities are mediated by the same DI particle. The mechanism of cell protection does not appear to involve extracellular interferon because the inclusion of interferon antibody in the assay did not diminish DI particle cell protection. Finally, the colorimetric assay was used to reveal alternating cycles of infectious and DI virus production on serial undiluted passage.

Antigen-Antibody Complex↗

PubMed1995

Quantification of oxidative phosphorylation enzymes after blue native electrophoresis and two-dimensional resolution: normal complex I protein amounts in Parkinson's disease conflict with reduced catalytic activities.

Blue native polyacrylamide gel electrophoresis (BN-PAGE), a method for the isolation of native membrane proteins from biological membranes, was adapted to the isolation of oxidative phosphorylation (OXPHOS) enzymes from milligram amounts of human tissues. Combined with Tricine-sodium dodecyl sulfate (SDS)-PAGE in the second dimension, the protein subunits of OXPHOS complexes could be analyzed and quantified. The characteristics of the technique are described and protocols for processing different tissues are provided. The technique was applied for the analysis of defects of OXPHOS complexes in Parkinson's disease. A significant reduction of complex V was observed in one case. Absolutely normal complex I protein amounts were in contrast to reduced catalytic activities of complex I in Parkinson's disease. This discrepancy can be explained by binding of endogenous complex I inhibitors or by alterations of a protein subunit not affecting the assemblage of the complex but modifying the enzymatic properties.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2000

Helical and single-slice conventional CT versus electron beam CT for the quantification of coronary artery calcification.

OBJECTIVE: We compared electron beam CT with conventional CT to determine the best method for the assessment of the coronary calcium score. We used conventional CT to examine symptomatic and asymptomatic patients suspected of having coronary artery disease. SUBJECTS AND METHODS: One hundred sixty male patients underwent electron beam CT and helical CT with a pitch of 1 (n = 30) and 2 (n = 30) and using a single-slice mode with (n = 50) and without (n = 50) prospective ECG triggering. In another 50 patients, we determined reproducibility for repeated scanning using electron beam CT. For all images, we derived the calcium score according to the Agatston method. We performed regression analysis and determined mean variability. Mean variability was calculated as the ratio of the absolute difference to the mean of the corresponding calcium scores. RESULTS: The correlation coefficients for electron beam CT and all conventional CT modes were very high (range, 0.93-0.98). The mean variability was highest in the helical mode with a pitch of 2 (61.4%) and lowest for the single-slice mode with prospective ECG triggering (25.4%). For repeated electron beam CT, the correlation coefficient and mean variability were 0.99 and 22.1%, respectively. CONCLUSION: ECG-triggered single-slice conventional CT had the best agreement with electron beam CT calcium scores.

Calcinosis↗

PubMed1994

Comparative advantage of 3-day food records over 24-hour recall and 5-day food frequency validated by observation of 9- and 10-year-old girls.

OBJECTIVE: The validity of the 24-hour recall, 3-day food record, and 5-day food frequency was assessed to decide on a dietary assessment method for the National Heart, Lung, and Blood Institute (NHLBI) Growth and Health Study. DESIGN: All subjects were assigned to one of three dietary assessment methods. Unobtrusive observers recorded types and amounts of foods eaten during lunch, and these were compared with the foods reported by the girls in the study. SETTING: School lunchrooms in California and Ohio. SUBJECTS: 58 girls, aged 9 and 10 years. MAIN OUTCOME MEASURES: Reporting errors for dietary assessment methods. STATISTICAL ANALYSES PERFORMED: Descriptive statistics, matched pair t tests, and Spearman correlation coefficients. RESULTS: Comparison of the intakes of energy and selected macronutrients showed different ranges of, and median percentage absolute errors for, each dietary assessment method. Percentage absolute errors ranged between 20 and 33 for the 5-day food frequency method; 19 and 39 for the 24-hour recall; and 12 and 22 for the 3-day food record. The proportion of missing foods (ie, observed food items not reported) and phantom foods (ie, reported food items not observed) by each method were 46% and 40%, respectively, for the 5-day food frequency; 30% and 33%, respectively, for the 24-hour recall; and 25% and 10%, respectively, for the 3-day food record. APPLICATIONS/CONCLUSIONS: Errors in food reporting and quantification can vary with the type of dietary methodology. Agreement between observed and reported intakes from 3-day food records made it the best overall choice. On this basis, it was selected as the method of assessment for the NHLBI Growth and Health Study.

Bias↗

PubMed2006

A technique for analysis of geometric mean renography.

BACKGROUND AND OBJECTIVES: Renography is used routinely to assess relative right to left renal function. Quantification is usually carried out using posterior images. Errors in relative renal function may occur if the kidneys are at different depths. Geometric mean images from combined anterior and posterior views are much less affected by kidney depth and offer the opportunity of more accurate and precise quantification. Background subtraction is a key part of the analysis process and validated protocols for geometric mean imaging have not been devised. This study aims to derive a suitable background subtraction protocol for geometric mean imaging. METHODS: Simultaneous anterior and posterior renography using Tc mercaptoacetyltriglycine (MAG3) was performed on 16 adults. Analysis was carried out using both geometric mean and posterior images. The geometric mean background subtraction protocol was modified to give the same results as a posterior method, which had previously been validated by correlation with measurements of glomerular filtration rate. Absolute and relative uptakes were then obtained from both geometric mean and posterior analyses. For each analysis values were obtained both with and without depth correction. RESULTS: A revised background subtraction protocol for geometric mean renography was devised which operated successfully on all studies. Both absolute renal uptake and relative function values obtained from geometric mean analysis were not systematically different from those obtained using posterior analysis with depth correction. Values of the relative renal function from posterior analysis after depth correction were closer to the geometric mean values than estimates obtained before correction. CONCLUSION: A technique for analysing geometric mean renography data has been developed which gives results consistent with a previously validated posterior-only method.

Adult↗

PubMed1986

Digital angiographic measurement of radiographic contrast material kinetics for estimation of myocardial perfusion.

We studied the use of digital angiography for the quantification of regional myocardial perfusion in the dog using selective left coronary arterial injection of radiographic contrast material as a flow dilution indicator. We developed algorithms for generating time-intensity curves from regions of interest over the proximal coronary artery and the myocardium and for densitometric error correction by subtraction of the intensity curve over a small lead blocker before logarithmic transformation. The resultant myocardial time-density curves were analyzed for time from injection to peak concentration (TPC) and for exponential washout rate (k). A linear correlation was found between absolute coronary arterial blood flow and both k (slope = 0.13, r = .85) and 1/TPC (slope = 0.18, r = .85). Reproducibility of TPC and k for repeated studies was 11% and 16%. Induced hyperemia significantly improved the sensitivity to stenosis by increasing the average difference in TPC and k between regions served by normal and stenotic coronary arteries to 65% and 80%, respectively. By combining selective coronary arterial injection with the left lateral x-ray projection it was possible to avoid most overlap of regional perfusion beds in the dog. This study suggests that contrast dilution measurements made during digital coronary angiography provide a means for assessing the hemodynamic significance of stenoses and the efficacy of therapeutic interventions.

Angiography↗