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PubMedDate not supplied

Quantitative determination of volatile organic compounds (VOC) in milk by multiple dynamic headspace extraction and GC-MS.

A method for the accurate determination of volatile organic compounds (VOC) in milk samples has been developed and tested. It combines multiple dynamic headspace extraction with GC-MS. Absolute amounts of VOC in the liquid phase are obtained by determining the first order kinetic dependence of the stepwise extraction of the analytes and internal standards from the liquid matrix. Compounds released from milk were collected on a train of traps filled with different solid sorbents to cover all components having a number of carbon atoms ranging from 4 to 15. They were analysed by GC-MS after thermal desorption of VOC from the collecting traps. Quantification of VOC in milk was performed using deuterated compounds as internal standards. The method was used to follow seasonal variations of monoterpenes in goat milk and to detect the impact of air pollution on the quality of milk.

Animals↗

PubMed2003

Validated method for the simultaneous determination of Delta 9-tetrahydrocannabinol (THC), 11-hydroxy-THC and 11-nor-9-carboxy-THC in human plasma using solid phase extraction and gas chromatography-mass spectrometry with positive chemical ionization.

A fully validated, highly sensitive and specific method for the extraction and quantification of Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THCCOOH) in plasma is presented. This method incorporates Escherichia coli beta-glucuronidase hydrolysis to cleave glucuronic acid moieties to capture total analyte concentrations, and simultaneous solid phase extraction (SPE) of the three analytes in a single eluant with separation and quantification on a bench-top positive chemical ionization (PCI) gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode. Quantitation was achieved by the addition of deuterated analogues for each analyte as internal standards (IS). Limits of quantitation (LOQ) were 0.5, 0.5 and 1.0 for THC, 11-OH-THC and THCCOOH, respectively, with linearity ranging up to 50 ng/ml for THC and 11-OH-THC, and 100 ng/ml for THCCOOH. Absolute recoveries ranged from 67.3 to 83.5% for all three analytes. Intra-assay accuracy and precision ranged from 1.2 to 12.2 and 1.4 to 4.7%, respectively. Inter-assay accuracy and precision ranged from 1.4 to 12.2 and 3.1 to 7.3%, respectively. This method was used to analyze plasma samples collected from individuals participating in a controlled oral THC administration study. Statistically significant (P< or =0.05) increases of 40% for 11-OH-THC and 42% for THCCOOH concentrations were found between hydrolyzed and non-hydrolyzed results. This method will be utilized in ongoing controlled cannabinoid administration studies and may be a useful analytical procedure for the fields of forensic toxicology and cannabinoid pharmacology.

Dronabinol↗

PubMed1999

The effectiveness of air bags.

Previous research has shown that the installation of air bags in vehicles significantly reduces crash related deaths, but these analyses have used statistical techniques which have not been capable of controlling for other major determinants of crash survival. This study analysed data from the US FARS database of fatal crashes using conditional logistic regression which is simultaneously able to estimate occupant protection effects for a range of variables. Results of the analysis provided a comparative quantification of both the effect of the air bag as well as other well known determinants of occupant crash survival (age, seat belt use, and gender). When potentially confounding variables were controlled, both the driver and passenger side air bag devices were shown to significantly reduce the probability of death in direct frontal collisions, but the effect size calculated was small compared to the effect of the seat belt. The effect size may also be very small in absolute terms depending on the severity of the crash involved. Given the limited benefit of the air bag, efforts to promote air bags seem particularly difficult to justify in countries such as the United States where the vastly superior occupant protection of the seat belt is under-utilised.

Accident Prevention↗

PubMed1979

A quantitative method for the assessment of the microtopography of human skin.

The skin relief influences the exterior aspect of the skin which is very sensitive to aging. It could also be related to the mechanical properties and structure of both dermis and stratum corneum. Consequently, quantitative measurement of the skin surface roughness would seem most useful, as it would permit a quantification of skin aging, an in vivo analysis of mechanical forces acting on the skin structure, and the detection of abnormalities otherwise not visible. The method described comprises three steps: (1) making a silicone rubber negative replica, (2) making an Araldite positive cast, (3) roughness measurement of the cast with a device commonly used in engineering, which provides quantitative parameters: Ra, Rp, Rt, Rmax and others. The reliability of each of these steps was checked, and also the absolute need to locate precisely the site of sampling and to know the angle of the scanning direction with the main axis of the limb or the body. The method seems useful for studying aging, either normal or affected by UV rays and other physiopathological events influencing the skin surface.

Aging↗

PubMed1998

DNA image cytometry and Ag-NORs-staining application in biocompatibility studies on human osteoblast cells in vitro.

We report here the study of the biocompatibility of a bone graft material, the Pyrost, using a previously established in vitro model of human osteoblasts. The effect of this material on cell proliferation was evaluated by the MTS assay. Results indicated the absolute absence of cytotoxic or cytostatic effect of Pyrost on cultured osteoblasts. Viability rate was more than 90% in cells cultured with the material compared to the control. Morphological analysis, undertaken by scanning electron microscopy showed a good adhesion and a spreading of osteoblasts in contact with the material that was colonized by cultured cells. In the second part of this work, we have introduced two methods as complementary biocompatibility tests: DNA image cytometry and interphase Ag-NORs quantification. DNA content was measured in cells cultured with or without Pyrost for 3, 9, 15 and 30 days. The determination of DNA indicated that the majority of osteoblasts population was diploid without aneuploidy. The DNA index and cell distribution profile in DNA histograms were similar in all cell populations. The Ag-NORs amount was used as a parameter for cell kinetic evaluation. We have measured the Ag-NORs index like DNA quantification. The proliferation rate, evaluated by Ag-NORs counts in osteoblasts cultured with or without the material, was identical. However, a decrease in Ag-NORs index was observed from day 3 to day 15 of incubation. These results showed a satisfactory biocompatibility of the Pyrost in human osteoblasts culture. The material did not alter cell viability and had no inducing effect either on proliferation rate or on cell ploidy as demonstrated by DNA image cytometry and Ag-NORs proteins staining.

Aged↗

PubMed2001

Molecular quantification of human beta-glucuronidase levels in a patient with Vohwinkel's syndrome.

The skin must undergo the process of keratinization in order to perform its functions. During the process of differentiation, certain genes are activated while others are repressed, leading to changes in structural proteins and enzymes and in the synthesis of various lipids. An error in any of these steps can ultimately impair the process of keratinization. Vohwinkel's syndrome is the direct result of a defect in keratinization. Patients who have this epidermolytic palmoplantar keratoderma present clinically with hyperkeratosis of the stratum corneum. Hyperkeratosis has been linked to an increase in beta-glucuronidase levels. The authors studied the absolute concentration of human beta-glucuronidase in a patient with Vohwinkel's syndrome as determined through a double-antibody sandwich enzyme-linked immunosorbent assay and a Western blot assay of the blood, urine, and skin of the patient.

Adult↗

PubMed2006

Quantitative age-related differences in human sublingual gland.

OBJECTIVE: To quantify morphological age- and gender-related differences in human sublingual glands (SLG). DESIGN: Ninety right human SLG from necropsies were distributed by gender and age groups. Individuals with ages ranging from 7 months to 92 years were divided into Groups I (0-30 years), II (31-60 years) and III (61-92 years). The glandular volume of each SLG was calculated. The morphometric quantifications of the volume density and total volume of acini, ducts, stroma, adipose tissue and septa were realized under light microscopy using a Zeiss II integration grid with 100 points symmetrically distributed. RESULTS: The glandular volume decreased, on average, 33.78% across the groups. The volume density (V(V)) of acini reduced 60.54% and of septa 34.82%, being replaced partly by an increase of 58.82%, 551.22% and 2,783.33% in the volume density of ducts, stroma and adipose tissue, respectively. In absolute terms, with aging, the total volume (V(T)) of the acini decreased by 54.04% and by 34.84% in the septa. In contrast, the total volume of ducts, stroma and adipose tissue showed increases of 62.53%, 363.25% and 2,308.64%, respectively. Both the V(V) and the V(T) of acini, ducts, stroma and adipose tissue and the V(V) of septa were statistically different with age. No significant difference was observed between genders. CONCLUSION: The decrease of parenchyma accompanied by an increase of stroma observed here in SLG is a common feature of aging in human salivary glands.

Adipose Tissue↗

PubMed2000

The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection.

Hepatitis B virus (HBV) is a noncytopathic virus, and the recognition of infected hepatocytes by HBV-specific CD8 cells has been assumed to be the central mechanism causing both liver damage and virus control. To understand the role of cytotoxic T cells in the pathogenesis of HBV infection, we used functional assays that require T cell expansion in vitro and human histocompatibility leukocyte antigen (HLA)-peptide tetramers that allow direct ex vivo quantification of circulating and liver-infiltrating HBV-specific CD8 cells. Two groups of patients with persistent HBV infection were studied: one without liver inflammation and HBV replication, the other with liver inflammation and a high level of HBV replication. Contrary to expectation, a high frequency of intrahepatic HBV-specific CD8 cells was found in the absence of hepatic immunopathology. In contrast, virus-specific T cells were more diluted among liver infiltrates in viremic patients, but their absolute number was similar because of the massive cellular infiltration. Furthermore, inhibition of HBV replication was associated with the presence of a circulating reservoir of CD8(+) cells able to expand after specific virus recognition that was not detectable in highly viremic patients with liver inflammation. These results show that in the presence of an effective HBV-specific CD8 response, inhibition of virus replication can be independent of liver damage. When the HBV-specific CD8 response is unable to control virus replication, it may contribute to liver pathology not only directly but by causing the recruitment of nonvirus-specific T cells.

CD8-Positive T-Lymphocytes↗

PubMed1983

Variability in the measurement of regional left ventricular wall motion from contrast angiograms.

Four types of variability affecting quantification of regional wall motion from contrast left ventriculograms (LVgrams) were studied. These included beat-to-beat variability in 24 LVgrams, intraobserver and interobserver variability in 20 LVgrams, and study-to-study variability in serial LVgrams of 21 patients with stable coronary artery disease. Motion was measured at 100 equidistant chords perpendicular to a center line drawn midway between the end-diastolic and end-systolic contours and normalized for heart size. Variability was computed as the absolute difference between observations. Beat-to-beat, intraobserver, and interobserver variability at the 100 chords were similar, averaging 14%, 14%, and 17%, respectively, of the mean motion in 64 patients with normal ventriculograms. Study-to-study variability was significantly higher, averaging 30% of mean normal motion, but was reduced when regional motion was calculated as the mean motion of chords within a region of interest. Variability peaked at the apex. Realignment to correct for cardiac rotation significantly increased variability. Investigators whose methods of wall motion analysis rely on identification of the apex as a landmark should be aware of this source of potential variability and error.

Arterial Occlusive Diseases↗

PubMed1989

Quantification and regulation of apolipoprotein E expression in rat Kupffer cells.

Apolipoprotein E (apoE) is synthesized by a wide variety of cells including cells of the monocyte-macrophage lineage. In order to assess the quantitative significance of apoE synthesis in a mature tissue macrophage, apoE synthesis was compared in Kupffer cells and hepatocytes isolated from rat liver. Immunoreactive apoE synthesized by both cell types exhibited identical isoform patterns when examined by high-resolution two-dimensional gel analysis. ApoE synthesis was not detected in hepatic endothelial cells. Northern blot analysis using a rat apoE cDNA probe demonstrated a single mRNA species of approximately 1200 nucleotides in freshly isolated hepatocytes and Kupffer cells. The absolute content of apoE mRNA in each cell type was determined with a DNA-excess solution hybridization assay. The apoE mRNA content (pg/microgram RNA) for Kupffer cells and hepatocytes was 35.7 and 98.8, respectively. Accounting for cellular RNA content and the population size of each cell type in the liver, Kupffer cells were calculated to contain about 0.7% of liver apoE mRNA; hepatocytes account almost quantitatively for the remainder. These results suggest that Kupffer cells are not major contributors to the plasma apoE pool. After intravenous injection of bacterial endotoxin, apoE mRNA was decreased in freshly isolated Kupffer cells whereas whole liver showed no change in apoE mRNA. Endotoxin treatment had no effect on the apoE mRNA content in several peripheral tissues. These results indicate that apoE expression in vivo is differentially regulated by endotoxin in Kupffer cells as compared to hepatocytes or apoE-producing cells in peripheral tissues.

Animals↗

PubMed2001

Factors Controlling the Addition of Carbon-Centered Radicals to Alkenes-An Experimental and Theoretical Perspective.

The successful exploitation of syntheses involving the generation of new carbon-carbon bonds by radical reactions rests on some prior knowledge of the rate constants for the addition of carbon-centered radicals to alkenes and other unsaturated molecules, and of the factors controlling them. Two former classical reviews in Angewandte Chemie by Tedder (1982) and by Giese (1983) provided mechanistic insight and led to various qualitative rules on the complex interplay of enthalpic, polar, and steric effects. In the meantime, the field has experienced very rapid progress: many more experimental absolute rate constants have become available, and there have been major advances in the efficiency and reliability of quantum-chemical methods for the accurate calculation of transition structures, reaction barriers, and reaction enthalpies. Herein we review this progress, recommend suitable experimental and theoretical procedures, and display representative data series for radical additions to alkenes. On this basis, and guided by the pictorial tool of the state-correlation diagram for radical additions, we then offer a new and more stringent quantification of the controlling factors. Our analysis leads to a partial revision of the previous qualitative rules, and it more clearly exhibits the interplay of the reaction enthalpy effects, polar charge-transfer contributions, and steric substituent effects on the reaction energy barrier. The various contributions are cast into the form of new, simple, and physically meaningful but non-linear, predictive equations for the preestimation of rate constants. These equations prove successful in several tests but call for additional theoretical and experimental foundation. The kinetics of related reactions such as polymer propagation, copolymerization, and the addition of radicals to alkynes and aromatic compounds is shown to follow the same principles.

Journal Article↗

PubMed2001

The value of the chloride: sodium ratio in differentiating the aetiology of metabolic acidosis.

OBJECTIVE: Stewart's physicochemical approach to acid-base balance defines the aetiology of a metabolic acidosis by quantifying anions of tissue acids (TA), which consist of unmeasured anions (UMA) and/or lactate. We hypothesised that an increase in TA during metabolic acidosis would lead to a compensatory fall in the plasma chloride (Cl) relative to sodium (Cl:Na ratio) in order to preserve electro-neutrality. Thus, the Cl:Na ratio could be used as a simple alternative to the anion gap in identifying raised TA. PATIENTS: Two hundred and eighty two consecutive patients who were admitted to our Paediatric Intensive Care were enrolled in the study. INTERVENTIONS: We obtained 540 samples (admission n = 282, 24 h n = 258) for analysis of blood chemistry, lactate and quantification of TA and UMA. Samples were subgrouped into those with metabolic acidosis (standard bicarbonate < 22 mmol/l) either with or without increased UMA (> 3 mEq/l). MEASUREMENTS AND RESULTS: Metabolic acidosis occurred in 46% of samples, of which 52.3% (120/230) had increased UMA. The dominant component of TA was UMA rather than lactate, and these two components did not always rise in tandem. Our hypothesis of relative hypochloraemia was supported by a lower Cl:Na ratio (P < 0.0001) but not a lower absolute Cl (P = 0.5) in the acidotic subgroup with raised UMA, and by the inverse relationship between TA and the Cl:Na ratio. (coefficient of determination (r2) = 0.37, P < 0.0001). The best discriminator for the presence of raised TA was the albumin-corrected anion gap (AGcorr), however, this could not track changes in TA with clinical accuracy. The Cl:Na ratio discriminated reasonably well, a ratio of < 0.75 identified TA (positive predictive value (PPV) 88%) with a likelihood ratio (LR) similar to the AG (7.8 vs7.4). Conversely, a high ratio (> 0.79) excluded TA (PPV 81%, LR 4.5). Base deficit (BD) and lactate performed poorly. CONCLUSION: In metabolic acidosis due to TA, plasma Cl concentration decreases relative to sodium. The Cl:Na ratio is a simple alternative to the AG for detecting TA in this setting.

Acidosis↗

PubMed2005

A brightness-area-product-based protocol for the quantitative assessment of antigen abundance in fluorescent immunohistochemistry.

A problem frequently facing researchers examining abundance of expression of a given antigen is measurement. When the antigen is confined to the nucleus, absolute numbers of nuclei or a percentage of nuclei expressing the antigen in a given region can be estimated. When the antigen is localized to cytoplasm, cytoplasmic organelles or processes or membranes, the assessment becomes more difficult. In these settings, an observer/experimenter may assign a density score but intra- and inter-observer agreement using a three-tiered system, and finer resolution than this, is unlikely to be reproducible. Digital image analysis provides an opportunity to minimize observer bias in quantification of immunohistochemical staining. Previously, reported digital methods have mostly employed chromogen-staining methods and often report mean image brightness. We report a method for quantitatively assessing and expressing abundance of expression of an antigen in neural tissue stained with immunofluorescent methods by determining the brightness-area-product (BAP). The described protocol utilizes simple to use commercially available software and calculates BAP rather than mean brightness as a measure more representative of antigen abundance and visual interpretation. Accordingly, we propose this protocol as a useful adjunct to observer interpretation of fluorescent immunohistochemistry and its application to assessment of antigen abundance for varying patterns of antigen localization.

Animals↗

PubMed1999

Defecographic study of rectal evacuation in constipated patients and control subjects.

PURPOSE: To validate a computer-based area calculation method of quantification of rectal evacuation by using defecography videotapes and to use that method to compare evacuation in constipated patients with that in control subjects. MATERIALS AND METHODS: For validation of the method, simultaneous defecography and weight measurements were compared in 36 patients with constipation or incontinence. Evacuation was calculated as the rate of change of the contrast medium-covered rectal area (percentage per second) or of the evacuated amount of contrast medium (percentage per second [relative] and grams per second [absolute]). After method validation, from a series of 215 consecutive constipated patients, individuals with an isolated radiologic diagnosis of intussusception greater than 0.6 cm (n = 27), rectocele greater than 2 cm (n = 19), enterocele (n = 12), or paradoxic puborectal muscle contraction (n = 12) were selected. Rectal evacuation in these groups was compared with that in 30 control subjects. RESULTS: Rectal evacuation rates measured at defecography correlated well with weighed amounts of evacuated contrast medium during the initial and total evacuation periods in 21 patients without contrast medium leak (r = 0.92, P < .001). Constipation overall, a rectocele greater than 2 cm, or paradoxic puborectal muscle contraction were associated with impaired evacuation (P < .001). CONCLUSION: Area calculations of rectal evacuation reflect rectal emptying. A rectocele greater than 2 cm or a paradoxic puborectal muscle contraction may be associated with obstructed defecation.

Adult↗

PubMed1989

[Solvent extraction and high performance liquid chromatography with electrochemical detection for determination of plasma catecholamines].

This paper describes a technique for selectively extracting plasma catecholamines prior to quantification by HPLC-EC. The extraction system was a two-stage process. The first stage involve the complex formation between diphenylbrorate and catechol (diol) groups in alkaline medium. The second stage was a liquid-liquid extraction. The complex combined with tetraoctylammonium bromide to form an ion-pair formation into organic solvent. The catecholamines in turn was extracted with acid. This technique provided a very specific extraction procedure which resulted in chromatograms with few interfering compounds and gave absolute recoveries (100-103%) of norepinephrine, epinephrine and dopamine. Meanwhile, the plasma catecholamines were concentrated and the detective sensitivity was increased. A good linear relationship was found between the concentrations and ratio of peak heights of the catecholamines from 0.125-2 ng. The correlation coefficients ranged from 0.998-0.999. The coefficients of variation of the intra- and inter-assay were within 3 and 6% respectively. The results show that the procedure is very simple and fast. The method is valuable not only for clinical diagnosis but also for laboratory research.

Catecholamines↗

PubMed1990

Determination of serum levels of unesterified lanosterol by isotope dilution-mass spectrometry.

The synthesis of 2H3-labelled lanosterol is described. This compound was used for assay of unesterified lanosterol in serum by isotope dilution-mass spectrometry. After addition of a fixed amount of internal standard (150 ng) to a fixed amount of serum (250 microliters) the steroids were extracted with chloroform and subjected to Lipidex 5000 chromatography. The fraction containing lanosterol was eluted with methanol. This fraction was converted into trimethylsilyl derivative and subjected to mass spectrometric analysis with selected monitoring of the ions at m/z 498 (molecular ion of unlabelled lanosterol) and m/z 501 (molecular ion of 2H3-labelled lanosterol). A standard curve was used for quantification of lanosterol in serum. Under the conditions employed the coefficient of variation was less than 4%. In a recovery experiment the maximal difference between expected and found value was about 5%. Sera from 10 healthy subjects were found to have a mean concentration of lanosterol of 225 ng/ml with a SD of 63 ng/ml. Using a less accurate method for analysis of lanosterol we have shown previously that there is a high correlation between the hepatic HMG CoA reductase and the absolute and relative concentration of lanosterol in serum (concentration of lanosterol relative to cholesterol). When using the same (old) sera as in the previous investigation this could not be confirmed, most probably because of the degradation of lanosterol as a consequence of prolonged storage and/or freezing and thawing. It appears that serum lanosterol must be analysed as soon as possible after the collection of the blood sample in order to obtain accurate results.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed2001

Quantification and development of teratocytes in novel-association host-parasitoid combinations.

We studied the development of teratocytes derived from two congeneric gregarious endoparasitic species, Cotesia chilonis and C. flavipes, parasitizing two congeneric novel hosts, Diatraea saccharalis and D. grandiosella. The host-parasitoid combinations studied allowed us to investigate relationships between host suitability and teratocyte development. D. saccharalis was a suitable host for both parasitoids, whereas D. grandiosella was suitable for C. chilonis development but often encapsulated C. flavipes progeny. Encapsulation of C. flavipes by D. grandiosella commenced around the time of parasitoid egg hatch, when teratocytes were released into the host's hemolymph. The gregarious parasitoids studied here released about 200 teratocytes per egg. Both absolute and normalized (teratocytes/parasitoid) numbers decreased over time. D. saccharalis supported more C. flavipes-derived teratocytes than D. grandiosella, possibly because of the unsuitability of the latter host. On intermediate assay days the number of C. flavipes-derived teratocytes was greater than for C. chilonis. However, C. chilonis-derived teratocytes grew larger than C. flavipes. Teratocytes in all host-parasitoid combinations doubled in size during parasitoid development. Teratocytes generally grew larger in D. grandiosella, which was a less suitable host.

Journal Article↗

PubMed1991

Identification of naturally processed viral nonapeptides allows their quantification in infected cells and suggests an allele-specific T cell epitope forecast.

Virus-specific cytotoxic T lymphocytes (CTL) recognize virus-derived peptides presented by major histocompatibility complex (MHC) class I molecules on virus-infected cells. Such peptides have been isolated from infected cells and were compared to synthetic peptides. We found previously the Kd- or Db-restricted natural influenza nucleoprotein peptides to coelute on reversed phase high performance liquid chromatography columns with certain peptidic by-products present in synthetic peptide preparations. Here we show by extensive biochemical and immunological comparison that the natural peptides in all respects behave as the surmised synthetic nonapeptides, and thus, must be identical to them. The absolute amounts of these natural peptides contained in infected cells could be determined to be between 220 and 540 copies by comparing with defined amounts of pure synthetic nonapeptides. The comparison of the natural Kd-restricted peptide with published synthetic peptides known to contain other Kd-restricted CTL epitopes suggested a new MHC allele-specific T cell epitope forecast method, based on the defined length of nine amino acid residues and on critical amino acid residues at the second and the last position.

Alleles↗